INTRODUCTION:

The presence of several phytochemicals such as polyphenols, terpenes, anthocyanins, flavonoids, alkaloids, and glycosides may be responsible for the fruits' varied health advantages because of its exceptional therapeutic abilities¹. Antioxidant, antimicrobial, antidiabetic, hepato-protective, anti-inflammatory, anti-mutagenic, anti-proliferative, radio-protective, cardio-protective, anti-arthritic, anti-caries, gastrointestinal mobility, and wound healing activity are just a few of the plant's numerous pharmacological and therapeutic qualities².

The prevalence of infectious illnesses has been rising over time. While some of the older infections are still present, new ones appear to be emerging. The majority of infections are brought on by environmental pathogens that may change circumstances to their favor and become opportunistic agents in a host population. A noteworthy illustration of microbial adaptability and the impact of natural selection is the recent emergence of antibiotic-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Pseudomonas aeruginosa, in addition to uropathogenic E. coli (UPEC)³. In addition to creating emergency conditions to identify novel infection-fighting tactics, multidrug-resistant (MDR) bacteria and their strains with decreased antibiotic sensitivity have produced an unsettling scenario for the search for ways to treat such illnesses. In impoverished nations, the leading cause of morbidity and mortality among patients with weakened immune systems is the incidence of potentially fatal illnesses brought on by microorganisms. The common gut flora of both humans and animals is E. coli. The most common cause of UTIs is Uropathogenic E. Coli (UPEC)⁴. UTIs are brought on by ascending colonization or infection by enteric bacteria that live in the bladder, urethra, peri-urethral region, perineum, and sometimes the kidney. The most prevalent urinary pathogen, E. coli, has been found in 50% to 90% of all UTIs. Strong resistance to external stimuli is associated with the formation of biofilms. The diffusion barrier that the exopolysaccharide slime forms limits the flow of molecules through the biofilm^5,6.Since the polysaccharide has a negative charge, positively charged antibiotics cannot enter. Additionally, biofilm shields the bacteria from the antibodies the host immune system produces in reaction to an infection. A major worldwide issue, antibiotic resistance raises health care expenses and causes morbidity and mortality. Antibiotic resistance is sparked by the selection pressure that comes with overusing antibiotics. An organism's resistance is its capacity to proliferate when exposed to high concentrations of antimicrobial agents⁷.

The largest source of medications, including those that are anticancer, antibacterial, antioxidant, and antidiabetic, comes from plants. Numerous plant species have been widely used by tribal people across the world and have been stated to contain antibacterial qualities in the traditional system⁸. Ethno-medicine is the study of traditional medicine that focuses on how people's healthcare and healing practices are influenced by their cultural understanding of health, illness, and disease. People have been studying nature, especially plants, to find new medications since ancient times. As a result, several illnesses are now being treated using medicinal plants that have healing qualities^9,10.

With this in mind, the current study is to assess the phytochemical components of T. chebula and further determine its antimicrobial activity, with a focus on UTI infections.

MATERIAL AND METHOD:

Gathering, Verifying, and Preparing Plant Powder:

The dried fruit sample from Terminalia chebula that was utilized in this study was locally sourced and validated on location. The plant's fruits were cleaned and rinsed to remove any contaminants that could be seen.

Furthermore, all of the different chemicals and reagents used to conduct the current investigation were of analytical research quality. We will purchase these from approved vendors. The devices utilized were from the department of our institute^18-20.

Making a Plant Extract:

Using Soxhlet's equipment, the coarse powder (500g) of the provided sample was extracted using 2.5 liters of 95% ethanol by continuous hot percolation until the extraction was finished. Following the extraction process, the extract was filtered, and a rotary vacuum evaporator was used to evaporate the solvent under low pressure. The residue had a greenish-black hue.

Assessment of Phytochemicals:

Using the usual reagents for each phytochemical, the extracts were tested for the presence of alkaloids, tannins, saponins, phenols, steroids, cardiac glycosides, carbohydrates, amino acids, and monosaccharides¹¹.

Total Phenol Estimation:

The Singleton and Rossi technique was modified to determine the total phenolic contents¹². 100µl of plant extract solution (starting concentrations 1000, 10,000, and 100,000ppm) in 100% methanol was added to a 10 ml volumetric flask containing 6 ml of distilled water. This resulted in final concentrations of 10ppm, 100ppm, and 1000ppm. With vigorous shaking, the Folin-Ciocalteu reagent (500µl 1:10 dilution of 2N solution) was applied right away. After a minute, 1.5ml of a The antioxidant properties of the provided freshly made 20% sodium carbonate aqueous solution was added while being continuously shaken. Flasks were left at room temperature for an hour after the volume was adjusted to 10ml. A spectrophotometer was used to measure absorbance in a 1cm cuvette at 760nm. The standard utilized was gallic acid¹².

Activity of DPPH Radical Scavenging:

BHT was chosen as a positive control due to its well-established antioxidant properties and common use as a reference standard in DPPH assays, ensuring reliable comparison with the extract’s activity. Pure water was used as a negative control to establish a baseline absorbance, as it lacks antioxidant activity and does not interfere with the DPPH reagent. Samples were examined using the DPPH radical scavenging test. Prior to the reactants spending 30 minutes at room temperature in a dark environment, the test solutions (100µl) were treated with 0.9ml of DPPH methanolic solution (2.5 mg/100ml). In contrast to the normal solution, the experiment employed pure water as the sole control solution and BHT at varying doses. After 30 minutes of processing, the spectrophotometer assessed absorbance at 515nm, enabling the extraction radical scavenging activity to be calculated as a percentage value¹³.

Uropathogen Isolation:

Urine samples were collected from UTI patients at nearby hospitals and sent right away to the lab with a cooling pack at a temperature between 4 and 80 degrees Celsius. Ten milliliters of the urine samples were decanted into centrifuge tubes after being thoroughly shaken to re-suspend the organisms. In order to prevent contamination, the tubes were kept closed. For ten minutes, the samples were centrifuged at 2000rpm. While all of the samples were decanted, 0.5ml of the tube's sediment was re-suspended using a sterile wire loop. A loopful of the silt was smeared onto agar plates and inoculated in a nutrient medium tube.

Pathogen Identification:

The incubated plates were used to evaluate colony form, color, and size as part of the cultural observation process. Based on the results of the cultural, microscopic, and microbiological analyses, the chosen colonies were submitted to biochemical analysis (oxidase, catalase, nitrate reduction, indole production, methyl red, voges-proskauer, citrate utilization, urease, etc.) to confirm the presence of the pathogens.

Disc Diffusion Assay for Evaluating for Antimicrobial Sensitivity:

The disc diffusion assay was performed to examine the bacterial isolates' sensitivity to certain antibiotics that are often used to treat uropathogens. Sterile cotton swabs were used to swab the test organisms on the surface of sterile mueller-hinton agar plates after they had been prepared. At room temperature, the cultures were left to dry on the plate for five to ten minutes. To improve contact and facilitate the antibiotics' efficient diffusion into the medium, a variety of antibiotic discs were gently pressed into the agar medium's surface using sterile forceps. The plates were incubated at 37℃ for 16–18 hours while inverted. For the T. chebula ethanolic extract, the procedure was carried out once again.

Result and Discussion:

The department director verified the authenticity of the T. chebula fruits before they were thoroughly cleaned and allowed to dry in the shade. To conduct the intended investigation, they were subsequently ground into a powder.

Phytochemical Screening of the Extracts of T. Chebula Retz:

500g of T. chebula fruit powder produced 12.25% w/w of crude ethanolic extract after phytochemical screening of the extracts. The residue had a greenish-black hue. To qualitatively assess the existence of secondary metabolites that are significant to medicine, the plant extract was screened. When compared to steroids and flavonoids, this study showed that the majority of the extracts included more tannins, phenols, saponins, terpenoids, alkaloids, reducing sugars, and carbohydrates (Table 1), consistent with findings on tannin and phenol prevalence¹⁵, alkaloid and saponin detection¹⁶, and flavonoid quantification¹⁷.

Table 1: Phytochemical analysis of T. chebula Retz extracts

S. No.	Tests	Ethanol
1	Alkaloids	+
2	Tannins	+++
3	Saponins	+
4	Terpenoids	+
5	Steroids	+
6	Phenols	+++
7	Amino acid	+
8	Reducing sugar	+
9	Carbohydrates	++
10	Flavonoids	+++

Qualitative indicators (+, ++, +++) reflect the intensity of color change or precipitate formation in standard phytochemical tests: + = weakly present (faint color/precipitate), ++ = strongly present (moderate color/precipitate), +++ = very strongly present (intense color/precipitate), - = absent.

Antioxidant efficacy In vitro: Calculating Total Phenols:

It is commonly recognized that there is a high link between antioxidant activity and total phenol concentration. The absorbance values of T. chebula crude ethanolic extract at various doses (1000, 10,000, and 1, 00, 000ppm/ml) were compared to a gallic acid standard. Similar to the standard, the absorbance percentage increased as the concentration increased. Consequently, it was determined that the total phenol content of the crude ethanolic extract was 185mg/gram, which is the equivalent of gallic acid. It also demonstrated the presence of dose-dependent active phenol.

Effectiveness of the DPPH Technique in Scavenging free Radicals:

In comparison to reference ascorbic acid, the DPPH radical scavenging capacity of T. chebula crude ethanolic extract from fruit was assessed at five distinct concentrations (500, 400, 300, 200, and 100µg/ml). The extract demonstrated a clear correlation between its impact and concentration. At 500µg/ml extract dose, which matched the 95% inhibition rate of ascorbic acid, the antioxidant activity of the extract at various concentrations reached 18% at the lowest level and 50% at the maximum level, while the inhibition percentage topped 77%. The ability of DPPH to donate hydrogen atoms is what gives it its antioxidant action and radical scavenging power. Standard ascorbic acid's concentration levels rose from 100µg/ml to 500µg/ml, and its antioxidant activity levels reached 98.3%, 95%, 91.6%, 90%, and 86.6%. According to research findings, pyrogallol showed antioxidant activity results of 84%, 75%, 64%, 51%, and 38% (Table 2).

Table 2: DPPH radical hunting by ethanolic T. chebula extract and control pyrogallol

Concentration in µg/ml	500	400	300	200	100
% Of antioxidant activity for pyrogallol	84	75	64	51	38
% Of antioxidant activity for ethanolic extract	77	50	47	22	18
% Of antioxidant activity for ascorbic acid	98.3	95	91.6	90	86.6

Table 3: Biochemical results of isolated uro-pathogens

S. No.	Biochemical tests	*Escherichia sp.*	*Proteus sp.*	*Klebsiella sp.*	*Pseudomonas sp.*	*Staphylococcus sp.*
i.	Gram staining	-ve rod	-ve rod	-ve rod	-ve rod	+ve cocci in clusters
ii.	Motility	motile	motile	motile	motile	non-motile
iii.	Indole	+	+	-	-	-
iv.	Methyl red	+	+	-	+	+
v.	V P	-	-	+	-	-
vi.	Citrate	-	-	+	+	+
vii.	Urease	-	+	+	-	+
viii.	Oxidase	-	-	-	+	-
ix.	Catalase	+	-	+	+	+
x.	TSI	A/A G	K/A	A/A G	K/K NG	-
xi.	Coagulase	-	-	-	-	+
xii.	DNase	-	-	-	-	+

A – Acid; K – Alkali; G – Gas production; NG – No gas

Uropathogen Isolation and Identification from Clinical Specimens:

According to biochemical assays, the main uropathogens found in urine samples from UTI patients were Escherichia sp., Klebsiella sp., Proteus sp., Pseudomonas sp., and Staphylococcus sp. (Fig. 1). As a positive control, ATCC cultures with appropriate reference numbers were employed. According to several biochemical assays, the majority of the bacterial isolates were shown to be multidrug resistant (Table 3).

Fig. 1: Uropathogens on UTI agar; a. Escherichia sp.; b. Pseudomonas sp.; c. Klebsiella sp.; d. Proteus sp

Antibacterial activity measurement using the disc diffusion test:

All of the examined species had strong inhibitory zones for the fruits T. chebula, ethanolic extract, with Escherichia sp. exhibiting the largest zone at 20mm. For the ethanolic extract of T. chebula fruits produced zones of inhibition of 20±4.5mm against Escherichia sp., 16±4.5mm against Klebsiella sp., and 16±4.7mm against Staphylococcus sp., as shown in Figures 2, 3, and 4, respectively.

To examine the pattern of resistance and sensitivity, common medications such tetracycline, gentamycin, ampicillin, and ciprofloxacin were tested against the clinical pathogens (Fig. 5; Table 4). Since no zones of inhibition were seen, Escherichia was determined to be resistant to both ampicillin and ciprofloxacin. It was susceptible to tetracycline and gentamycin. As a negative control, DMSO was employed.

Table 4: Antibiogram pattern of positive and negative control

S. No.	Control		*Escherichia sp.*	*Klebsiella sp.*	*Staphylococcus sp.*
1	Ethanol extract		20±4.3	16±4.5	16±4.7
2.	Reference drugs	AMP (10mcg)	R	R	R
		CIP (5 mcg)	R	R	R
		GEN (10mcg)	18±7.6	14±5.8	22±2.3
		TE (30mcg)	15±5.1	17±3.2	21±3.6
3.	DMSO		R	R	R

Values are mean±SD of triplicates, Antibiogram pattern of positive and negative controls. AMP = Ampicillin, CIP = Ciprofloxacin, GEN = Gentamicin, TE = Tetracycline, DMSO = Dimethyl sulfoxide, R = Resistant (No zone).

Fig. 2: Zones of inhibition for ethanolic T. chebula fruits extract against Escherichia sp.

Fig. 3: Zones of inhibition for ethanolic T. chebula extract against Klebsiella sp.

Fig. 4: Zones of inhibition for ethanolic of T. chebula extract against Staphylococcus sp.

Fig. 5: Zones of inhibition of standard antibiotics against Escherichia a. Ampicillin, b. Ciprofloxacin, c. Gentamycin, d. Tetracyclin

Both ampicillin and ciprofloxacin were ineffective against the clinical pathogens that were isolated from urine. Urinary isolates were shown to be susceptible to gentamycin and tetracyclin. Our results are comparable to those of Patil (2013), who found that 93% of isolates exhibited ampicillin resistance¹⁴. According to a more recent study, E. coli is resistant to ciprofloxacin.

CONCLUSION:

Finding safe treatments is made easier by the rise of antibiotic-resistant uropathogens and the increased incidence of urinary tract infections and their recurrence. Based on initial screening, ethanolic extract was found to be a promising extract against UTI. After screening secondary metabolites, the active ingredient was found. It was discovered that the bacterial isolates taken from UTI patients were resistant to ciprofloxacin and other medicines. Potential antibacterial activity against the chosen uropathogens was shown by the extract. The zones of inhibition for the various T. chebula fruit extracts had diameters ranging from 7 to 24mm. According to the study, ethanol-treated T. chebula Retz fruit extract is efficient in preventing bacterial infections and the spread of cancer. Pyrogallol and T. chebula extract work together to produce the combination antioxidant action. Because both ethanolic extracts restored the levels of kidney and liver marker enzymes, they have natural rejuvenating properties. Although it needs more research, this drug delivery method provides insight into potential drug delivery procedures. It was shown in the study that the ethanolic extract was efficient against a variety of drug-resistant uropathogens.

CONFLICT OF INTEREST STATEMENT:

The authors declare that there is no conflict of interest regarding the publication of this study.

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Received on 14.06.2025 Revised on 25.07.2025

Accepted on 30.08.2025 Published on 11.10.2025

Available online from October 25, 2025

Res.J. Pharmacology and Pharmacodynamics.2025;17(4):269-274.

DOI: 10.52711/2321-5836.2025.00043

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