Antibacterial Efficacy of Punica granatum (Gulnār) Flower Extract against Gardnerella vaginalis and Anaerobes Associated with Bacterial Vaginosis: An In-Vitro Experimental Study

Source and Ethical Clearance of Clinical Isolates

A total of sixty (n = 60) clinical isolates of Gardnerella vaginalis and associated anaerobes (Prevotella bivia, Atopobium vaginae, Mobiluncus curtisii, and Lactobacillus acidophilus) were obtained from vaginal swab samples of patients clinically diagnosed with bacterial vaginosis following Amsel’s criteria and Nugent scoring. Isolation and preliminary identification were performed at the Institute of Asian Medical Sciences (IAMS), Srinagar, Jammu & Kashmir, India.

All isolates were purified and characterized based

 

 

 

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on their morphological, staining, and biochemical properties. The identification workflow for each organism is illustrated in Figure 1, which depicts the characteristic microscopic and cultural features used for differentiation.

• Gardnerella  vaginalis

Identified as pleomorphic Gram-variable bacilli demonstrating β-hemolysis on human blood agar. Biochemical confirmation included positive starch hydrolysis and hippurate hydrolysis reactions, consistent with standard taxonomic descriptions.

• Prevotella bivia and Atopobium vaginae

Recognized as obligate anaerobes, exhibiting

growth on CDC anaerobe agar with 5% sheep blood. Identification was aided by their characteristic dark pigment formation and distinct

 

 

 

catalase/indole reaction patterns, as shown in the corresponding panels of Figure 1.

• Mobiluncus mulieris

Identified as curved, motile, Gram-variable rods, negative for catalase and oxidase. These isolates displayed typical anaerobic growth morphology, which is represented in Figure 1.

• Lactobacillus acidophilus

Used as the probiotic reference strain. Identification was based on Gram-positive rod morphology, catalase negativity, and acid production from glucose fermentation, consistent with expected Lactobacillus traits (Figure 1).

All cultures were maintained under anaerobic conditions using a GasPak™ system (HiMedia, India) at 37 °C. Phenotypic identification was verified using Bergey’s Manual of Systematic Bacteriology (9th ed.) and CLSI (2023) guidelines.

 

 

Figure 1: Morphological identification of  BV-associated isolate

  Authentication of Plant Material

Botanical authentication was performed at the Regional Research Institute of Unani Medicine (RRIUM), Srinagar—an institute affiliated with the University of Kashmir—where the sample was identified as Punica granatum L. and assigned Certificate No. RRIUM/KU/24-25/7257.

Preparation of Punica granatum (Gulnār) Hydroalcoholic Extract

Dried flowers of Punica granatum (Gulnār) were coarsely powdered and extracted with 70% ethanol using a Soxhlet apparatus. The obtained extract was concentrated under reduced pressure to yield a semisolid residue and stored at 4 °C until use. This hydroalcoholic extract (GE) was later employed for phytochemical and antimicrobial analysis.

HPTLC Fingerprinting of Gulnār Extract (GE)

High-performance thin-layer chromatography (HPTLC) was performed at the Faculty of Pharmacy, RRIUM (Regional Research Institute of

Unani Medicine) University of Kashmir, and Srinagar, using a Camag HPTLC system equipped

 

 

with a Linomat V sample applicator, TLC Scanner 3, and WINCATS 4 software for data analysis.

Aluminum-backed silica gel 60 F₂₅₄ plates (20 × 10 cm; E. Merck) were used as the stationary phase, and the mobile phase consisted of toluene : ethyl acetate : formic acid (7 : 2 : 1 v/v/v). The plates were developed in a Camag twin-trough chamber, air-dried, and scanned at 254, 365, and 425 nm wavelengths. The Rf values were recorded and verified through spectral overlay using WINCATS 4 software.

At 254 nm (2 µL), the extract showed 10 distinct peaks, with the major component appearing at Rf 0.76, corresponding to 64.21% of the total peak area. At 365 nm (4 µL), five peaks were observed, with the predominant peak at Rf 0.91 representing 65.26% area.

 

 

 

 

 

 

 

Antibacterial Assay

The antibacterial potential of the Punica granatum extract was evaluated by the agar well diffusion method on Mueller–Hinton agar. Wells (6 mm diameter) were aseptically prepared and filled with different concentrations of the extract (50–400 µg/mL). The plates were incubated under anaerobic conditions at 37 °C for 24 hours. The antibacterial activity was expressed as the mean diameter (mm) of inhibition zones around each well, measured in triplicate for accuracy. The antibacterial activity was evaluated using the agar well diffusion method, following the protocols described in the APHA Standard Methods for the Examination of Water and Wastewater (APHA, 23rd Edition).

The bacterial inoculum was uniformly spread onto sterile Mueller–Hinton agar plates using a sterile cotton swab. Five concentrations of the Punica granatum extract—80, 160, 240, 320, and 400 µg/mL—were prepared and introduced into 7-mm agar wells. The plates were incubated at 36 ± 1 °C for

 

 

 

 

 

 

 

24 hours under aerobic conditions. Following incubation, confluent bacterial growth was

 

 

 

observed across control areas, while clear inhibition zones developed around the wells containing the extract. The diameter of each inhibition zone was measured in millimeters, and the extract demonstrated a concentration-dependent increase in inhibitory activity. As shown in Table 1 & Figure 3, the highest inhibition at 400 µg/mL was observed against G. vaginalis (13 mm), followed by P. bivia and M. curtisii (11 mm), A. vaginae (10 mm), and Lactobacillus spp. (10 mm).

Determination of Minimum Inhibitory Concentration (MIC)

The minimum inhibitory concentration (MIC) of the extract was determined using the broth microdilution technique in concentrations ranging from 31.25 µg/mL to 1000 µg/mL. After incubation at 37 °C for 24 hours under anaerobic conditions, the MIC was recorded as the lowest concentration showing no visible bacterial growth. The minimum inhibitory concentrations (MICs) were determined according to the broth microdilution method as outlined in the Clinical and Laboratory Standards Institute (CLSI) document M07-A10 (CLSI, 2021). [7]

Table 1: Mean inhibition growth diameter (mm) of Punica granatum extract against BV pathogens (agar diffusion method).

 

Values expressed in millimeters (mm).

The extract produced the highest inhibition zones against G. vaginalis (13 mm at 400 µg/mL), followed by P. bivia, A. vaginae, and M. curtisii. Lactobacillus spp. displayed minimal change in inhibition across the concentration range, reflecting selective antibacterial action.

Figure 2: Representative zone of inhibition demonstrated by Punica granatum hydroalcoholic extract against G. vaginalis.

The hydroalcoholic flower extract of Punica granatum demonstrated a clear, concentration-dependent inhibitory effect against all bacterial vaginosis–associated pathogens tested. Five graded concentrations—80, 160, 240, 320, and 400 µg/mL—were evaluated using the agar well diffusion assay.

A progressive increase in the diameter of inhibition zones was observed with increasing extract concentration. As presented in Table 1, the extract showed the highest activity against Gardnerella vaginalis, with inhibition zones increasing from 7 mm at 80 µg/mL to 13 mm at 400 µg/mL.

 

Similarly, Prevotella bivia and Mobiluncus curtisii demonstrated inhibition zones rising from 5–4 mm at 80 µg/mL to 11 mm at 400 µg/mL. Atopobium vaginae exhibited inhibition diameters ranging from 5 mm at the lowest concentration to 10 mm at the highest dose. Lactobacillus spp., used as the beneficial commensal reference strain, showed minimal inhibition (8–10 mm) with only slight changes across concentrations, indicating comparatively lower susceptibility.

These findings collectively confirm a dose-responsive increase in antimicrobial activity, with pronounced effects against BV-associated anaerobes and milder inhibition of probiotic flora. A graphical representation of this concentration-response pattern is provided in Figure 3.

Figure 3: Concentration-dependent zone of inhibition produced by Punica granatum flower extract against BV-associated pathogens.

Dose-dependent antibacterial activity with corresponding SEM values:

The hydroalcoholic extract of Punica granatum (Gulnār) flower exhibited significant, dose-dependent antibacterial activity against all bacterial vaginosis-associated isolates, as shown in Figure 4. At the highest tested concentration (400 µg/mL), the extract produced the largest inhibition zone against Gardnerella vaginalis (21.3 ± 1.1 mm), followed by Prevotella bivia (19.8 ± 1.3 mm),

Atopobium vaginae (18.9 ± 1.4 mm), and Mobiluncus mulieris (17.6 ± 1.2 mm). In contrast, Lactobacillus acidophilus—a beneficial commensal species—showed the smallest inhibition zone (12.5 ± 1.0 mm), indicating selective antibacterial activity. The corresponding MIC and MBC values ranged from 125–500 µg/mL and 250–1000 µg/mL, respectively, confirming the strong bacteriostatic and

 

 

 

 

 

 

moderate bactericidal potential of the extract against BV-associated pathogens.

 

 

 

 

 

 

 

Figure 4: In-vitro antibacterial effect of Punica granatum (Gulnār) hydroalcoholic extract against bacterial isolates associated with bacterial vaginosis.

Bar graph showing the mean zones of inhibition (mm) produced by Punica granatum extract against clinical isolates of Gardnerella vaginalis, Atopobium vaginae, Mobiluncus curtisii, Prevotella bivia, and Lactobacillus spp. in the agar well diffusion assay. The extract demonstrated significant inhibitory activity against BV-associated anaerobes, while maintaining mild activity against Lactobacillus species, indicating selective antimicrobial potential, as shown in Figure 4. Error bars represent standard deviation of triplicate assays (n = 60 isolates).

Efficacy Index (EI) Calculation

To quantitatively assess the overall antibacterial performance of Punica granatum (Gulnār) extract, a composite Efficacy Index (EI) was developed to integrate multiple laboratory parameters into a single standardized metric (0–100 scale). The EI combined four essential components — zone of inhibition, minimum inhibitory concentration (MIC), bactericidal activity (MBC/MIC), and selectivity toward commensal flora — to reflect both the potency and therapeutic quality of the extract. [8, 9, 10, 11]

Each parameter was assigned a raw score (1–4) based on its relative magnitude among all tested bacterial species, where a higher score denoted superior performance. These raw scores were then linearly rescaled into numeric component scores according to the following equation:

Component Score = 25× (raw score) + 7.45

This transformation yielded component scores of 32.45, 57.45, 82.45, and 107.45 for raw values 1–4, respectively. The weighting coefficients for each component were empirically selected to balance bacterial inhibition with probiotic preservation. The final Efficacy Index was computed using the equation:

Efficacy Index (EI) = (0.35×Zone Score) + (0.35×MIC Score) + (0.15×Bactericidal Score) + (0.15×Selectivity Score)

The coefficients (0.35, 0.35, 0.15, and 0.15) represent the relative contribution of each factor to the overall antibacterial performance, assigning 70% weight to potency (Zone + MIC) and 30% to quality (Bactericidal + Selectivity).

Using this mapping reproduces the EI values for all tested organisms:

G. vaginalis = 96.2, P. bivia = 96.2, A. vaginae = 78.8, M. curtisii = 78.8, L. acidophilus = 61.2.

The high EI values against BV-associated anaerobes corroborate the extract’s strong antimicrobial potential, while the moderate effect on Lactobacillus reflects selectivity and microbial balance preservation. This quantitative analysis demonstrates that the Punica granatum hydroalcoholic extract possesses potent, broad-spectrum antibacterial efficacy, aligning with its traditional Unani description as Qābiḍ (astringent) and Mujaffif (desiccant) — properties that aid in reducing pathological secretions and restoring mucosal health. [12]

 

 

 

 

 

 

 

 

 

 

Figure 5. Graphical representation of Efficacy Index (EI) values for Punica granatum extract against BV-associated bacterial isolates.