INTRODUCTION:

Urolithiasis is one of the oldest and common diseases known to mankind which affects all the age groups. Urinary stone disease epidemiology has grown dramatically, but there are regional differences¹, such as the prevalence in North America (7–13%), Europe (5–9%), and Asia (1–5%).² In India, the lifetime prevalence of urolithiasis was 7.9 % (5.7–10.8 %).³

Diseases such as Urolithiasis place a significant economic burden on the healthcare system, particularly in developed and developing countries where, due to changes in diet and lifestyle, the prevalence of stone disease has significantly increased over the last few decades; unfortunately, this trend is likely to continue for a variety of reasons. Kidney stones are among the most painful urologic conditions. Renal stones affect 5% to 15% of adults. According to epidemiological studies, men are more likely than women to have nephrolithiasis (12%), and both sexes are more likely to have it between the ages of 20 and 40.⁴ The third most common urinary system disorder is urinary calculi. The majority of urinary calculi are caused by a common component of urine, such as calcium oxalate, which accounts for up to 80% of all examined stones. Urinary stones are expected to affect 12% of the population in India, with half of those affected experiencing kidney failure or damage. Furthermore, kidney stones affect nearly 15% of northern India's population.⁵

Crape myrtle is the other name for Lagerstroemia speciosa, is a plant belonging to Lythraceae family. Banaba is the common name for Lagerstroemia speciosa, is a medicinal plant that has traditionally been used as an antidiabetic, which lowers blood glucose levels in the body. Corosolic acid content present in this plant makes it an effective anti-diabetic medication. Banaba is also recommended for bladder disorders, kidney problems and reducing blood pressure. Japanese and Native Indians have used the plant leaves for thousands of years as folkloric medicines for ailments and illness, particularly with antidiabetic property and obesity. Lagerstroemia speciosa has long been used as flock medicine for a variety of medical reasons. According to ongoing research, the plant part has a significant impact on reducing obesity and blood glucose levels. This plant is also used to treat gout, reduce oxidative stress, as a diuretic and also as a decongestant. This plant's main active ingredients are corosolic acid, caffeic acid, p-coumaric acid, ellagic acids and gallic acid. Corosolic acid has been reported to lower blood sugar levels within an hour⁶.

METHODOLOGY:

Chemicals and reagents:

TRIS Buffer was purchased from Merck Ltd., Mumbai, India. Cystone was obtained from Himalaya Health Care Pvt. Ltd. All other chemicals and reagents used were analytical grade and procured from approved chemical suppliers.

Fig 1 Picture of plant Langerstromia speciosa

Collection of Plant Material:

The leaves of Lagerstroemia speciosa belonging to Lythraceae family was collected from Seven Hills College of Pharmacy, Tirupati and authenticated by Dr K. Madhava Chetty, Assistant Professor, Department of Botany, Sri Venkateswara University, Tirupati (India). Fig 1 Picture of plant Langerstromia speciosa.

Determination of physico-chemical parameters:

Determination of physico-chemical parameters such as total ash, acid insoluble ash, water-soluble ash, extractive values, ethanol soluble extractive values and water-soluble extractive values, foreign organic matter and moisture content of the crude drug was determined according to WHO guidelines on quality control methods for medicinal plant materials (WHO, 1992).

Extraction of plant material:

The leaves were washed and dried at room temperature (35°C) for 7 days before being crushed into a coarse powder with a mixer grinder. The coarse powder was separated using sieve number 24. The coarse powder was extracted with 100% ethanol (45-50°C). The coarse powder was wrapped in muslin cloth and extracted using a soxhlet apparatus. The extraction procedure took a total of three hours to complete. On a rotary evaporator, the ethanolic extract was concentrated and the solvent was evaporated until a semisolid mass was obtained. The wet mass was placed in a desiccator to be studied further. The obtained powder was weighed and saved for phytochemical screening and in vitro testing. The Soxhlet extraction method is depicted in Figure 2

Figure 2 Soxhlet Assembly

Qualitative Phytochemical Analysis:

The qualitative phytochemical screening for the plant extract was done according to Sofowara, (1993)⁸, Trease & Evans (1989)⁹ and Harborne (1998)¹⁰.

Separation of Semi permeable Membrane from Eggs:

A sharp device was used to puncture the eggs at the apex in order to empty out the entire contents. The empty eggs were thoroughly washed with distilled water before being placed in a beaker with 4 ml concentrated HCl in 200 ml distilled water. It was left overnight, resulting in complete decalcification of the semipermeable membrane. The semipermeable membranes were carefully removed from egg shells the next day, washed thoroughly with distilled water, placed in ammonia solution to neutralize acid traces, and then rinsed with distilled water. It was kept in the refrigerator, moistened, at a pH of 7-7.4. As a negative control, 10mg of calcium oxalate was suspended in 10ml of distilled water. 5 mL of ethanolic extract of fresh Langerstromia specoisa leaves was taken. Cystone tablets containing 500 mg were placed in absolute ethanol to remove the colour coating, yielding 400 mg. Cystone tablet powder was crushed and dispersed in 100 ml of distilled water before being filtered. Cystone filtrate was used as a positive control for anti-urolithiatic activity in vitro.^11-13Separation of Semi permeable Membrane from Eggs Figure 3.

Figure 3 Separation of Semi permeable Membrane from Eggs

Preparation of calcium oxalate by homogenous precipitation:

Calcium chloride dihydrate 1.47 gm was dissolved in 100 ml distilled water and 1.34 gm of sodium oxalate was dissolved in 100 ml of 2N H2SO4. Both were mixed equally in a beaker to precipitate out calcium oxalate with stirring. The resultant calcium oxalate was freed from traces of sulfuric acid by ammonia solution; washed with distilled water and dried at a temperature 60°C for 2 hours. ^14-16 The microscopical pictogram of calcium oxalate crystal is shown in Figure 4 and the FTIR spectroscopy of calcium oxalate crystal is shown in Figure 5

Fig 4 Microscopical pictogram of Crystals

Fig 5 IR Spectroscopy of Crystals

Method:

Group I: 1ml of calcium oxalate (1mg/ml) + 1ml of distilled water, Group II: 1ml of calcium oxalate (1mg/ml) + 1ml of Cystone solution (400mg/ml), Group III: 1ml of calcium oxalate (1mg/ml) + 1ml of ethanolic extract of Langerstromia specoisa (20mg/ml), Each group was packed in an egg semipermeable membrane tied with thread at one end and suspended in a conical flask containing 150ml of 0.1 M Tris buffer. Another end of the thread is tied by a stick and placed on the mouth of the conical flask, which is covered with aluminium foil.

Egg membrane and titrimetic method

Figure 6 Titrimetric Assay

Figure 7 Titrimetric Assay

All groups were kept in an incubator for three days after being preheated to 37^oC for four hours. Each group's entire content was removed from the sutured semipermeable membrane and transferred into a separate test tube. 4 ml of 1N H2SO4 and 60-80 of 0.02M KMnO4 were mixed together and set aside for 2 hours. After 2 hours, the colour changed from dark pink to colourless.^17-20The Titrimetric assay is depicted in figure 6 and 7

RESULTS AND DISCUSSION:

Table 1: Evaluation of Langerstromia specoisa ethanoloc leaf extract for anti-urolithiatic activity (Calcium oxalate concentration mg/ml) using Titrimetric methods

S. No	% of Dissolution of calcium oxalate	LSEE
1	Blank	0
2	Positive control	87 %
3	Ethanolic extract	81%

Figure 8 Ash value and Extractive value

The physicochemical investigation of Lagerstroemia speciosa leaves showed the following values: total ash content 6.3714 and acid-insoluble ash content 1.4995 % of the dry weight. Ethanol soluble extractive value and water soluble extractive values were 5.2745, and 8.9998 % of the dry weight, respectively. The results of physicochemical parameters are presented in table 3 and Figure 8 shows ash values and extractive values.

Table 2 Physiochemical characteristics

S. No	Physicochemical Parameters	Values
1	Total ash content	6.3714 %
2	Acid insoluble ash content	1.4995 %
3	Ethanol soluble extractive values	5.2745 %
4	Water soluble extractive values	8.9998 %

The preliminary phytochemical investigation of Lagerstroemia speciosa leaf showed that they contained steroids, terpenoids, glycosides, phenolic compounds, α-amino acids, saponins, starch, alkaloids, carbohydrates, organic acids, flavonoids, reducing sugars and tannins.⁷ The results of preliminary phytochemical screening are presented in table 3

Table 3 Preliminary phytochemical screening

S. No	Plant constituents	Test	LSEE
1	Alkaloids	Wagner’s test	+
		Mayer’s test	+
		Hager’s test	+
		Dragendorff’s	+
2	Carbohydrates	Molisch’s test	+
		Benedict’s test	+
		Fehling’s test	+
		Barfoed’s test	+
3	Phenols & Tannins	Ferric chloride	+
		Potassium dichromate	+
		Lead acetate test	+
4	Glycosides	Baljet test	+
		Keller killiani	+
		Legal test	+
5	Proteins	Biuret test	+
6	Flavonoids	Shinoda test	+
7	Saponins	Foam test	+
9	Steroids and Terpenes	Libermann test	+
		Salkowski test	+

This research examined the in vitro antiurolithiatic potential of Lagerstroemia speciosa ethanolic extract. LSEE had the highest percentage of calcium oxalate CaOx dissolution, was 81%. Lagerstroemia speciosa ethanolic extract was found to be equally effective in the dissolution of calcium oxalate as the standard drug cystone. According to the findings of this study the ethanolic extract of Lagerstroemia speciosa had the highest calcium oxalate dissolution. Ethanolic extract was found to be more effective in the dissolution of calcium oxalate. This study established Lagerstroemia speciosa as a plant with nephrolithiatic properties. This in vitro study yielded favourable results, demonstrating that ethanolic extract is quite promising for future investigation and further research in this area.

CONCLUSION:

According to the findings of this study, Lagerstroemia speciosa leaf ethanolic extract has significant anti-urolithiatic activity when compared to the standard (cystone). Lagerstroemia speciosa ethanolic extract (LSEE) showed significant dissolution of calcium oxalate in the titrimetric method in in vitro studies. The current study's findings clearly show that Lagerstroemia speciosa leaf ethanolic extract (LSEE) has antiurolithiatic potential against CaOx urolithiasis in vitro. Although more in vivo and clinical studies are needed to confirm Lagerstroemia speciosa's efficacy as an antiurolithiatic.

ACKNOWLEDGEMENT:

Authors express their sincere gratitude to Seven Hills College of Pharmacy, Tirupati, for continuous motivation, support, and guidance for research activity and for providing all required facilities to accomplish the entitled work.

COMPETING INTEREST:

Authors have declared that no competing interests exist.

REFERENCES:

1. Thongprayoon C, Krambeck AE, Rule AD. Determining the true burden of kidney stone disease. Nat Rev Nephrol. 2020 doi: 10.1038/s41581-020-0320-7.

2. Sorokin I, Mamoulakis C, Miyazawa K, Rodgers A, Talati J, Lotan Y. Epidemiology of stone disease across the world. World J Urol. 2017; 35:1301–20.

3. Lohiya A, Kant S Kapil A, Gupta SK, Misra P, Rai SK. Population-based estimate of urinary stones from Ballabgarh, northern India. Natl Med J India. 2017; 30:198–200.

4. Romero V, Akpinar H and Assimos D G, Kidney stones: A global picture of prevalence, incidence, and associated risk factors, Rev Urol, 2010, 12(2-3), e86–e96.

5. Parmar MS, Kidney stones, BMJ, 2004, 328(7453), 1420–1424.

6. Custer CD, Ranelle RA, Josephine LDY, Amelia SA and Elmer-Rico EM: Hypoglycemic acyivity of irradiated Banaba (Lagerstroemia speciosa Linn.) Leaves, Journal of Applied Science Research 2005; 1(1): 95-98.

7. Mukherjee PK. Quality Control of Herbal Drugs. Business Horizon Pharmaceutical Publications, New Delhi, 2008, 187-191.

8. Sofowora, A. (1993) Phytochemical Screening of Medicinal Plants and Traditional Medicine in Africa Edition. Spectrum Books Ltd., Nigeria, 150-156.

9. Trease, G.E. and Evans, M.D., (1989). A text book of Pharmacognosy. 13th Ed. Builler Trindall and Canssel London. Pp 176-180.

10. Harborne, J.B. (1998) Textbook of Phytochemical Methods. A Guide to Modern Techniques of Plant Analysis. 5th Edition, Chapman and Hall Ltd, London, 21-72.

11. Venkatachalam Sasikala Singanallur Ramu Radha Bavaniamma Vijayakumari In vitro evaluation of Rotula aquatica Lour. for antiurolithiatic activity Journal of Pharmacy Research 2013, 6(3):378-382

12. Dulce María González Mosquera et al. Antiurolithiatic activity of Boldoa purpurascens aqueous extract: An in vitro and in vivo study J Ethnopharmacol. 2020.10; 253:112691.

13. Bawari S, Sah AN, Tewari D. Antiurolithiatic Activity of Daucus carota: An in vitro Study. Pharmacog J. 2018; 10(5):880-4.

14. Yenduri Suvarna, S K. Abdul Rahaman. In Vitro – In Vivo Evaluation of Antiurolithiatic activity of piperine from Piper nigrum. Research J. Pharm. and Tech. 2020; 13(1):63-68.

15. Spandana. K, Shivani. M, Himabindhu. J, Ramanjaneyulu. K. Evaluation of In Vitro Antiurolithiatic Activity of Vigna radiata. Research J. Pharm. and Tech 2018; 11(12): 5455-5457.

16. Neeraja Kamakshi. U, Ganga Rao. B, Venkateswara Rao. B. Comparison of In vitro Antiurolithiatic Activity of Aerva lanata, Sphaeranthus indicus, Merremia emarginata. Research J. Pharm. and Tech. 2017; 10(6): 1653-1656.

17. Kushagra D, Rajiv S, Ruchi G, Neelesh M. In-Vitro Evaluation of Anti-Urolithic Activity of Leaves Extract of Costus igneus. Research J. Pharm. and Tech 2020; 13(3):1289-1292.

18. Sathish Babu. P, Gokula Krishnan, Anand Babu.K, Chitra. K. In silico and In vitro Evaluation of Anti-urolithiatic Activity of Ethanolic Extract of Syzygium cumini Stem Bark. Research J. Pharm. and Tech. 2017; 10(5): 1317-1321.

19. Anshuman Rai, Anamika Gautam, Sakshi Panchal, Ankita Sood, Pankaj Prashar, Narendra Kumar Pandey, Indu Melkani, Bimlesh Kumar. Protective effect of Ethanolic extract of Terminalia arjuna bark against Ethylene Glycol induced Urolithiasis in male rats: In-Vitro and In-Vivo Evaluation. Research J. Pharm. and Tech. 2020; 13(12):6132-6139.

20. Pradnya N. Jagtap, Bhagyashri R. Vyapari, Yashashri H. Nimbalkar, Sitaram V. Kale, Ganesh B. Nigade. Evaluation of Antiurolithiatic activity of Polyherbal formulation (Lithout Tablets) by In-vitro inhibition of Calcium Oxalate Crystallization. Research J. Pharm. and Tech. 2019; 12(11): 5477-5478.

Received on 05.01.2022 Modified on 15.04.2022

Res. J. Pharmacognosy and Phytochem. 2022; 14(3):150-154.

DOI: 10.52711/0975-4385.2022.00028