INTRODUCTION:

The most prevalent disease in modern times is urinary tract infection, which is often brought on by a sedentary lifestyle, poor genital hygiene, and bacteria like E. Coli and other bacteria like Staphylococcus, Lactobacillus, Enterococcus, Klebsiella, Proteus, and Pseudomonas, as well as fungal species like Candida albicans, Non-Albicans Candida, Candida Tropicalis, and Candida Glabrata.¹ The bacteria begin to colonise in the uterus as soon as they pass through the urethra and enter the urinary canal.

This results in the development of symptoms characteristic of bladder cystitis, including lower abdominal discomfort and inflammation, frequent urination, and painful micturition. As the colonisation continues, bacteria ascend into the ureters and then the kidneys, causing pyelonephritis. If left untreated, this condition can progress to acute to chronic renal impairment and renal failure. Prolonged antibiotic medication therapy is necessary for the treatment of UTIs, but it also leads to antibiotic resistance and a host of adverse effects, such as nausea, vomiting, diarrhoea, upset stomach, lack of appetite, hepatomegaly, and renal failure.² Herbal medications may be administered safely for an extended length of time to treat UTIs effectively and safely since they typically do not cause any negative drug effects. Aegle Marmelos, Amomum Subulatum, and Mimosa Pudica exhibit strong antibacterial action, particularly against a variety of UTI-causing microorganisms. Tablets were made up of herbal extracts of Aegle Marmelos leaves, Amomum Subulatum fruits, and Mimosa Pudica leaves and stems. Tablet is a sensible dosage form that is simple to prepare, enables the formulation of a broad variety of dosages with ease, has good patient acceptance, and allows for easy dosage and regimen adjustments with good therapeutic adherence.

MATERIALS AND METHODS:

The Amomum Subulatum, Mimosa Pudica and Aegle Marmelos were purchased from local market, Pune. All chemicals used were of analytical grade. The crude drugs were sun dried and powdered for extraction.

Extraction of crude herbal drugs:

The herbs were initially extracted through a 60minute decoction, followed by an overnight maceration. The mixture was then filtered, concentrated, and fully dried in a 50°C hot air oven. The dried extract was subsequently stored in an airtight glass jar for later use.³

Physicochemical Evaluation:

Moisture content:

Approximately 1gram of the powdered drug was weighed into a pre-weighed flat and thin Petri plate. It was dried in an oven at 100°C or 105°C until two consecutive weighing did not differ by more than 0.5 mg. After drying, the Petri plate was cooled in a desiccator and then weighed again. The loss in weight was recorded as the moisture content.⁴

Ash value:

A flat, thin porcelain dish or a tared silica crucible was weighed and ignited. Approximately 2grams of the powdered drug were added into the dish or crucible. The dish was supported on a pipe-clay triangle placed on a ring of a retort stand. It was heated with a burner using a flame about 2cm high, and the dish was positioned about 7cm above the flame. Heating was continued until vapours almost ceased to be evolved, after which the dish was lowered and heated more strongly until all the carbon was burnt off. The dish was then cooled in a desiccator. The ash was weighed, and the percentage of total ash was calculated with reference to the air-dried sample of the crude drug.⁵

Extractive value:

Approximately 4grams of the coarsely powdered drug were weighed into a beaker. One hundred milliliter of the selected solvent was added to the beaker and the mixture was stirred for a couple of minutes. The beaker was covered with an aluminium foil sheet and set aside for 24hours with frequent stirring. After 24hours, the menstruum was filtered and the filtrate was separated. The obtained menstruum was evaporated in a porcelain dish using a water bath. It was cooled in a desiccator for 10minutes, then weighed immediately, and the percentage extractive value was calculated.⁶

Determination of volatile oil content:

Ten grams of the powdered drug were taken and placed in a round-bottom flask with 250–300ml of water, and a few pieces of porcelain were added to avoid bumping. The flask was attached to a Clevenger apparatus, and a condenser was connected to the Clevenger apparatus. Heating was started using a heating mantle and continued until no more oil was collected. The heating was then turned off, and the apparatus was allowed to stand for 10–15minutes to permit the condensed fluid to settle. The amount of oil obtained was measured, and the results were calculated.⁷

Measurement of fibre length and width of powdered drug:

First, the eyepiece micrometre was calibrated with the stage micrometre to obtain the factor for calculation. A small quantity of the powdered drug was then taken in a test tube and boiled with chloral hydrate solution. The drug fibres were transferred to a watch glass and stained with phloroglucinol and HCl in a 1:1 ratio. The stained powder was mounted in glycerine water and observed under a low-power microscope. The fibres were focused, and their length and width were measured in divisions by rotating the eyepiece micrometre, ensuring that at least 25 fibres were observed. Each reading was multiplied by the calculated factor to obtain the results in microns.⁸

Isolation of E. Coli:

Soil sample was collected and inoculated on the sterilised nutrient agar plate and was incubated for 24 hrs to obtain bacterial colonies and then each colonies was tested for peroxidase test by adding hydrogen peroxide on bacterial colonies and the colonies producing effervescence was confirmed to be colonies of E.coli bacteria.⁹

Microbial assay of herbal extract:

Microbial assay of herbal extract and polyherbal tablet was carried out by paper diffusion method, the concentrate of herbal drug extract was diluted to get the strength solution of 2mg/ml each and was infused in paper disc and was assayed against the bacteria E.coli isolated out from the soil and was incubated for 24hrs and was noted for zone of inhibition.¹⁰

Microbial assay of herbal drug combinations:

Combination of herbal extract of Mimosa Pudica, Amomum Subulatum, Aegle Marmelos at four different ratios 3:6:2, 4:5:2, 5:4:2, 6:3:2mg/ml was made and then infused in paper disc and was tested against the E,coli bacteria for 24hrs of incubation and then was noted for zone of inhibition.^11,12,13

Microbial assay of tablets:

Polyherbal tablet of strength 450mg and Amoxicillin capsule of strength 500mg was powdered and both were dissolved in 10ml of water to obtain polyherbal tablet solution strength of 45mg/ml and amoxicillin strength of 50mg/ml and was tested for antimicrobial activity against E. coli bacteria and was incubated for 24hrs, amoxicillin was used as standard drug.¹⁴

Table 1. Formulation table for tablet

Sr. No	Ingredient	Quantity (mg)	Role
1	Aegle Marmelos Extract	73.10	API
2	Mimosa Pudica Extract	183.25	API
3	Amomum Subulatum Extract	146.6	API
4	MCC	51.61	Binder
5	PVP	20.16	Binder
6	Cross Povidone	15.32	Disintegrant
7	Aerosil	4.8	Glidant
8	Mg Stereate	4.8	Lubricant
9	Distilled Water	q.s	Solvent

Evaluation of granules:

Bulk Density:

Weigh an empty graduated cylinder and note its weight as a starting point. Next, delicately add 10grams of sample do not press them into the cylinder into it. Note the cylinder containing the granules' weight. Bulk density was determined by measuring the volume that the granules in the cylinder occupied, which is known as the bulk volume.¹⁵

Tapped Density:

Measuring the tapped density comes after figuring out the bulk density. Put the granule-filled cylinder on a tapped density tester. There are 100 taps on the cylinder. Once the granules are tapped, note the final volume the granules occupy this is known as the tapped volume. Thus, the tapped density was determined.¹⁶

Angle of Repose:

Setting up a funnel over a level surface at a predetermined height is how you measure the angle of repose. Until the granules form a sturdy conical pile, let them pass freely through the funnel. Calculate the base's radius (r) and the pile's height (h).¹⁷

Moisture content:

Initially start the mains button on tare the weight of the balance by adjusting the knob and then add the sample on the sensitive pan until the red pointer attains zero reading then start the heating process by turning on the IR lamp and continue heating until constant reading is attained note the final reading by adjusting the knob until red pointer matches the indicated line.¹⁸

Evaluation of tablet:

Physical Appearance:

Tablets should have uniform color, shape, size, and a smooth surface without cracks or chips. Any deviation could indicate issues in the manufacturing process.¹⁹

Weight Variation test:

Ensuring individual tablets have a consistent weight within a specified range helps guarantee dosing accuracy. Weight 20 tablets individually and then calculate average tablet weight.²⁰

Thickness test:

Tablet thickness can be measured by micrometer or by other device .Tablet thickness should be controlled within ±5% variation of standered value.²¹

Hardness test:

Tablets should meet specific dimensional parameters and exhibit adequate hardness to withstand handling and transportation without breaking or crumbling. Hardness was measured by Monsanto tester.²²

Friability test:

This measures the tablet's tendency to chip or break when handled. A friability test involves rotating tablets in a drum and assessing the percentage of weight loss due to abrasion. Generally Roche friabilator was used in which 20 tablets are dropped at distance of 6 inches and rotated at 25rpm for 100 revolutions.²³

Disintegration test:

Put one tablet in each tube of a disintegration equipment and submerge the tubes in a beaker of a chosen medium at 37°C to conduct a disintegration test on tablets. Turn on the device so that the basket may rise and fall. Keep an eye on the tablets and record how long it takes for each to disintegrate into smaller pieces with no solid core left.²⁴

Dissolution test:

To perform a dissolution test for tablet testing, start by placing the tablet in a dissolution apparatus filled with a specified volume of dissolution medium, typically maintained at 37°C. Set the apparatus to a defined rotation speed or paddle movement. At predetermined intervals, withdraw samples of the dissolution medium, ensuring the volume is replaced with fresh medium to maintain consistency. Filter the samples to remove any undissolved particles, then analyze them using an appropriate method, such as UV spectrophotometry, to determine the amount of active drug released over time. This process helps assess the tablet's release profile and ensures consistent drug delivery.²⁴

Table 2. Phytochemical screening of drug extract

Extract	Tan	Ster	Flav	Carb	Gly	Sap	Prot	Phen	Alk
M. pudica (Leaf)	+	+	+	+	−	−	+	−	−
M. pudica (Stem)	+	−	+	−	+	+	−	+	+
A. subulatum (Fruit)	+	−	+	+	+	+	−	−	+
A. marmelos (Leaves)	+	−	+	−	−	+	−	−	+

Key: Tan-Tannins, Ster-Steroids, Flav-Flavonoids, Carb-Carbohydrates, Gly-Glycosides, Sap-Saponins, Prot-Proteins, Phen-Phenols, Alk-Alkaloids

RESULTS:

Phytochemical screening of drug extract:

Physicochemical Evaluation:

Table 3. Physiochemical screening of drug extract

Sr. No.	Physicochemical Evaluation Parameter		Result
1	Moisture Content	Amomum Subulatum	31%
		Aegle Marmelos	11%
		Mimosa Pudica	14%
2	Ash value	Amomum Subulatum	6.5%
		Aegle Marmelos	9%
		Mimosa Pudica	10%
4	Volatile oil content	Amomum Subulatum	3%
5	Fibre length of crude drug	Amomum Subulatum	30
		Aegle Marmelos	20
		Mimosa Pudica	100
6	Fibre width of crude drug	Amomum Subulatum	35
		Aegle Marmelos	15
		Mimosa Pudica	40

Microbial assay:

The antimicrobial activity of Mimosa pudica, Amomum subulatum, and Aegle marmelos was evaluated using ethanol, water, and a hydroethanolic mixture as solvents at a concentration of 2mg/ml. In ethanol extracts, Mimosa pudica showed a zone of inhibition of 12mm, while Amomum subulatum and Aegle marmelos exhibited inhibition zones of 11 mm and 13mm, respectively. In water extracts, Mimosa pudica demonstrated an inhibition zone of 10mm, whereas Amomum subulatum and Aegle marmelos showed zones of 10.5mm and 12mm, respectively. The hydroethanolic extracts showed the highest antimicrobial activity, with inhibition zones of 14mm for Mimosa pudica, 12 mm for Amomum subulatum, and 14mm for Aegle marmelos. The antimicrobial activity of different herbal extract combinations containing these three drugs was also evaluated based on their zones of inhibition. Four combinations with varying proportions were tested: Combination 1 containing 3mg of Mimosa pudica, 6 mg of Amomum subulatum, and 2mg of Aegle marmelos showed an inhibition zone of 7mm; Combination 2 containing 4mg of Mimosa pudica, 5mg of Amomum subulatum, and 2mg of Aegle marmelos exhibited a zone of 9 mm; Combination 3 containing 5mg of Mimosa pudica, 4 mg of Amomum subulatum, and 2mg of Aegle marmelos demonstrated the highest antimicrobial activity with a 12mm inhibition zone; and Combination 4 containing 6mg of Mimosa pudica, 3mg of Amomum subulatum, and 2mg of Aegle marmelos showed an inhibition zone of 6.5mm. These results indicate that the antimicrobial efficacy of the herbal extract combinations varied depending on the proportion of each constituent, with Combination 3 being the most potent. The antimicrobial activity of the formulated tablet was further evaluated and compared with a standard antibiotic, Almox-500, using the zone of inhibition method. The test tablet prepared in water at a concentration of 45mg produced an inhibition zone of 26mm, while the standard drug Almox-500 (Amoxicillin) at a concentration of 30mg also showed an inhibition zone of 26mm, indicating that the test tablet exhibited antimicrobial activity comparable to the standard antibiotic and highlighting its potential as an alternative antimicrobial formulation

Figure 1. Results for dose combination of herbal extract

Figure 2. Results of microbial assay of herbal tablet

Evaluation of Granules:

Table 4. Evaluation of granules

Sr. No.	Parameter	Sieve no.	Result
1	Bulk Density of Granules	BSS 24	0.3125
1	Bulk Density of Granules	BSS 44	0.3448
2	Tapped Density of Granules	BSS 24	0.3448
2	Tapped Density of Granules	BSS 44	0.3571
3	Carr’s Index	BSS 24	0.0936
3	Carr’s Index	BSS 44	0.034
4	Hausner’s ratio	BSS 24	1.10
4	Hausner’s ratio	BSS 44	1.03
5	Angle of Repose	BSS 24	27.32
5	Angle of Repose	BSS 44	28.98

Evaluation of Tablet:

Table 5. Evaluation of Tablet

Sr. No	Test parameter	Attribute	Results
1	Weight variation test	Percent weight variation in 20 tablets	0.2222 %
2	Hardness test	Hardness of tablets in kg/cm³	8
			7
			8
			6
			8
3	Friability test	Percent weight loss of 20 tablets	2.2 %
4	Disintegration test	Disintegration time of 6 tablets	7 min. 55 sec.
			7 min. 55 sec.
			7 min. 55 sec.
			8 min. 20 sec.
			8 min. 30 sec.
			8 min. 30 sec.
5	Dissolution test (Uncoated tablets)	Percent drug release at 45 minutes	99.16

In silico studies:

The in silico activity was done by docking the active phytoconstituent form the crude drug extract with the specific targeted proteins of the E.coli which on inhibiting gives antimicrobial activity against the E.coli bacteria. For performing the molecular docking RCSB, Pub Chem database was used to get the structure of the required macromolecules and ligands. For molecular docking PyRx and Autodock vina softwares were used and the data was generated.²⁵

In silico activity of Mimosine:

The DnaB helicase protein on of the cell constituent of E. Coli which when inhibited shows antibacterial activity against the E. Coli bacterial strains. Thus the DnaB helicase protein with pdb id 1B79 was used as targeted protein for the insilico studies of phytoconstituent mimosine from Mimosa pudica.

Table 6. Docking results of Mimosine on protein DnaB helicase

Ligand	Binding Affinity	rmsd/ub	rmsd/lb
protein_ligand	-5.7	0	0
protein_ligand	-5.7	16.344	13.115
protein_ligand	-5.7	3.56	1.842
protein_ligand	-5.6	2.366	1.766
protein_ligand	-5.5	15.125	12.034
protein_ligand	-5.4	9.53	5.461
protein_ligand	-5.4	4.653	2.285
protein_ligand	-5.3	2.555	1.931
protein_ligand	-5.2	9.646	5.798

Figure 3. 2d Diagram for docking of Mimosine on protein DnaB helicase

Figure 4. 3d Diagram for docking of Mimosine on protein DnaB helicase

In silico activity of Sabinene:

The outer membrane lipoprotein of the cell constituent of E. Coli which when inhibited leads to lysis of E. Coli bacterial strains. Thus the membrane lipoprotein with pdb id 2GUS was used as targeted protein for the insilico studies of phytoconstituent Sabinene from Amomum subulatum.

Table 7. Docking results of Sabinene on membrane lipoprotein

Ligand	Binding Affinity	rmsd/ub	rmsd/lb
protein_ligand	-4.9	0	0
protein_ligand	-4.9	4.127	0.823
protein_ligand	-4.7	4.186	1.26
protein_ligand	-4.7	1.872	1.103
protein_ligand	-4.5	12.626	9.986
protein_ligand	-4.5	4.494	1.702
protein_ligand	-4.4	3.287	1.548
protein_ligand	-4.3	4.112	1.54
protein_ligand	-4.3	2.464	1.187

Figure 5. 2d Diagram for docking of Sabinene on membrane lipoprotein of E.coli

Figure 6. 3d Diagram for docking of Sabinene on membrane lipoprotein of E.coli

In silico activity of Luvangetin:

The Dna gyrase B protein of the cell constituent of E. coli which when inhibited shows antibacterial activity against the E. coli bacterial strains. Thus the Dna gyrase B protein with pdb id 1EI1 was used as targeted protein for the insilico studies of phytoconstituent luvangetin from Aegle marmelos.

Table 8. Docking results of Luvangetin on DNA gyrase B

Ligand	Binding Affinity	rmsd/ub	rmsd/lb
protein_ligand	-7.1	0	0
protein_ligand	-7	33.111	31.881
protein_ligand	-6.9	6.422	2.324
protein_ligand	-6.9	23.229	21.637
protein_ligand	-6.9	31.392	30.412
protein_ligand	-6.8	23.429	22.445
protein_ligand	-6.7	27.34	24.627
protein_ligand	-6.7	26.318	24.123
protein_ligand	-6.6	22.674	20.659

Figure 7. 2d Diagram for docking of Luvangetin on DNA gyrase B

Figure 8. 3d Diagram for docking of Luvangetin on DNA gyrase B

DISCUSSION:

This study demonstrates that a polyherbal tablet made from Aegle marmelos, Amomum subulatum, and Mimosa pudica effectively combats urinary tract infections (UTIs), offering a natural alternative amid rising antibiotic resistance. The tablet showed strong antibacterial activity against Escherichia coli, comparable to Amoxicillin, suggesting that the combined action of these plant extracts enhances effectiveness. Phytochemical analysis confirmed the presence of bioactive compounds like flavonoids, tannins, alkaloids, and glycosides, which likely contribute to bacterial inhibition by disrupting cell walls and essential functions. Testing different extract ratios identified the most potent formulation, containing 5 mg Mimosa pudica, 4 mg Amomum subulatum, and 2 mg Aegle marmelos. The tablets met pharmaceutical quality standards, ensuring proper dissolution and bioavailability. Additionally, molecular docking studies revealed that key compounds Mimosine, Sabinene, and Luvangetin bind strongly to bacterial proteins, preventing replication and damaging cell membranes. These findings highlight the potential of polyherbal formulations as effective and safer alternatives to synthetic antibiotics for managing UTIs.

CONCLUSION:

In conclusion, the study on the formulation and evaluation of a polyherbal tablet targeting urinary tract infections (UTIs) highlights the significance of herbal alternatives to conventional antibiotics. The increasing concern over antibiotic resistance and the side effects associated with long-term antibiotic use necessitates the exploration of safer, effective treatments. By employing medicinal plants such as Aegle Marmelos, Amomum Subulatum, and Mimosa Pudica, this research demonstrates the promising antimicrobial activity of these herbs against UTI-causing bacteria like E. coli. The comprehensive evaluation of the herbal extracts through moisture content, ash value, and microbial assays confirms the efficacy of the polyherbal tablet in inhibiting bacterial growth, comparable to standard antibiotics. The granules used for tablet formulation exhibited excellent flow properties, ensuring consistent product quality. Additionally, in silico studies provided molecular insights into the interaction between active phytoconstituents and targeted bacterial proteins, further reinforcing the potential of these herbs in UTI treatment. Overall, the study emphasizes the relevance of polyherbal formulations as a natural, cost-effective, and safe alternative for managing UTIs, particularly in light of the growing challenges posed by antibiotic resistance.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENT:

The authors are thankful to Abhinav Education Society’s College of Pharmacy, Narhe, Pune for encouraging us to carry out this research.

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Received on 17.03.2025 Revised on 10.09.2025

Accepted on 27.01.2026 Published on 01.07.2026

Available online from July 04, 2026

Research J. Pharmacy and Technology. 2026;19(7):3288-3294.

DOI: 10.52711/0974-360X.2026.00468

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Ash value

Fibre length of crude drug