Unveiling the bactericidal potential of Nyctanthes arbor-tristis (Parijath): A potent broad-spectrum bactericidal agent against Cutibacterium acnes, Vibrio vulnificus, Aeromonas hydrophila, and Escherichia coli

Introduction

The escalating crisis of antimicrobial resistance (AMR) poses a profound threat to global public health, with the World Health Organization estimating that bacterial infections claim over 700,000 lives annually, a figure projected to surge to 10 million by 2050 without novel interventions (WHO, 2020). Conventional antibiotics, once hailed as miracle drugs, are increasingly rendered obsolete by multidrug-resistant (MDR) pathogens such as Escherichia coli, Vibrio vulnificus, Aeromonas hydrophila, and Cutibacterium acnes-strains implicated in urinary tract infections, wound sepsis, aquaculture diseases, and acne vulgaris, respectively. This dire scenario underscores the urgent need for sustainable, plant-derived antimicrobials that can combat resistance while minimizing toxicity and side effects. Medicinal plants, revered in traditional systems like Ayurveda, offer a treasure trove of bioactive compounds with multifaceted mechanisms, including membrane disruption, quorum sensing inhibition, and efflux pump modulation (Aggarwal et al., 2011; Agrawal & Pal, 2013) [2,4] . Among these, Nyctanthes arbor-tristis Linn. (Family: Oleaceae), popularly known as Parijat or Night-flowering Jasmine, stands out as a versatile ethnomedicinal powerhouse. Native to the Indian subcontinent and revered in Ayurvedic texts like the Charaka Samhita, Parijat has been traditionally employed for treating fever, arthritis, skin infections, and respiratory ailments (Agrawal & Pal, 2013) [4] . Its nocturnal-blooming flowers and deciduous leaves are rich in secondary metabolites, including iridoid glycosides, flavonoids, phenolics, terpenoids, and alkaloids, which confer broad-spectrum bioactivity (Banerjee et al., 2007; Ramachandran et al., 2014) [8,32] . Pharmacological studies have validated these uses: Verma et al. (2011) [38] demonstrated potent antibacterial activity of root bark extracts against Gram-positive and Gram-negative bacteria, while Aggarwal and Goyal (2013) [3] and Manisha et al. (2009) [27] reported inhibition of pathogenic strains via membrane permeabilization. Floral extracts exhibit superior efficacy, as shown by Khatune et al. (2001) [26] and Jose et al. (2016) [22] , attributed to high flavonoid content that scavenges free radicals and disrupts bacterial biofilms (Rathee et al., 2007; Michael et al., 2013) [33,28] . Beyond standalone activity, N.arbor-tristis shows promise as an antibiotic adjuvant. Isaac et al. (2017) [21] identified antiquorum sensing metabolites that enhance antibiotic penetration, while Chatterjee et al. (2007) [9] highlighted its edge over other Indian herbs. Synergistic studies, though limited, reveal additive effects with tetracycline and ciprofloxacin (Altuner et al., 2018; Vilekar et al., 2014) [5,39] . Antioxidant and antiinflammatory properties further amplify its therapeutic value: Ghosh et al. (2015) [17] isolated a water-soluble radical scavenger from leaves, Dhinakaran and Sakthivel (2016) [13] confirmed cytokine modulation, and Aparna et al. (2012) [7] elucidated n-hexadecanoic acid's role in NFκB inhibition. Anticancer potential against leukemia cells (Heendeniya et al., 2020) [19] and immunostimulant effects (Puri et al., 1994) broaden its scope, aligning with Ayurveda's -reverse pharmacology paradigm (Aggarwal et al., 2011) [2] . Despite these advances, critical gaps persist. Most studies focus on leaf or root extracts, neglecting floral contributions (Khanapur et al., 2014; Saha et al., 2012) [25,34] . Few address clinically relevant strains like C. acnes (acne) and aquatic pathogens (V. vulnificus, A. hydrophila), which thrive in tropical climates like India (Furqan et al., 2021) [15] . Synergistic interactions with frontline antibiotics remain underexplored, particularly via fractional inhibitory concentration indices (FICI) (Odds, 2003) [29] . Moreover, extraction variables, solvent type, drying method and timekill dynamics are inconsistently reported, hindering standardisation (Yuan et al., 2015; Abubakar & Haque, 2020) [41,1] . Phytochemical profiling via GC-MS is sparse for flowers (Dhivya & Manimegalai, 2013) [14] , and correlations between bioactives and activity are rarely quantified (Sasikumar et al., 2010; Mary & Merina, 2021) [35] . This study bridges these gaps by evaluating methanolic and ethanolic extracts of shade-and oven-dried N. arbortristis leaves and flowers against C. acnes, V. vulnificus, A. hydrophila, and E. coli. Specific objectives include: (1) assessing antimicrobial activity via disc diffusion, MIC/MBC, and time-kill kinetics; (2) determining synergistic effects with tetracycline and ciprofloxacin using checkerboard assays; (3) conducting preliminary phytochemical screening and GC-MS profiling; and (4) correlating bioactives with efficacy using statistical analyses. Plant materials were collected from Bangalore, India, authenticated, and processed per standardized protocols (CLSI, 2018). The significance of this research is multifaceted. In an era of AMR, N. arbor-tristis could yield cost-effective adjuvants for acne therapies, wound care, and aquaculture, reducing antibiotic reliance (Alves et al., 2021; Ghagane et al., 2017) [6,16] . By validating Ayurvedic claims scientifically (Dinamani & Rao, 2009; Paul et al., 2002) [30] , it promotes biodiversity conservation and sustainable harvesting. Phylogenetic tools like MEGA X (Kumar et al.) and matK barcoding ensure authenticity, while sequence analysis methods (Tamura, 1992 [20,43] .

Materials and Methods Collection and Identification of Plant Material

Fresh and mature leaves and flowers of N. arbor-tristis L. (Family: Oleaceae) were collected between January and March 2024 from the cultivated medicinal plant garden of Vriksha Vihan Pvt. Ltd., Bangalore, Karnataka, India (12.9716° N, 77.5946° E). Harvesting occurred during early morning and late afternoon to optimize secondary metabolite concentration and minimize enzymatic degradation, as supported by studies on seasonal phytoconstituent variation (Yuan et al., 2015) [41] . Plants were selected based on healthy morphological features, with no more than 20% foliage removed per plant to ensure sustainability.

Pre-Treatment and Drying of Plant Material

Post-collection, plant materials were rinsed under running tap water to remove soil and debris, treated with 70% ethanol for 30 seconds to eliminate microbial contaminants, and triple-rinsed with sterile distilled water (Abubakar & Haque, 2020) [1] . Excess moisture was removed using sterile blotting paper. Two drying methods were employed: shade drying, where plant parts were spread on sterile muslin sheets in a well-ventilated room at 28-32 °C for 10-12 days with regular turning, and oven drying at 40 °C for 48 hours to preserve phytochemicals (Yuan et al., 2015) [41] . Dried materials were pulverized into coarse powder using a stainless-steel grinder, sieved (mesh size 60) for uniform particle size, and stored in airtight amber containers in a desiccator at 25±2 °C until extraction.

Preparation of Crude Extracts

The crude extracts were prepared from N. arbor-tristis leaves and flowers using methanol or ethanol. The extraction matrix included NAT-SLM (shade-dried leaves, methanol), NAT-OLE (oven-dried leaves, ethanol), and NAT-SFM (shade-dried flowers, methanol). Each extract was prepared by macerating 10 g of powdered material in 100 mL of solvent in sterile conical flasks, incubated at 25±2 °C for 72 hours with intermittent shaking every 8 hours (Abubakar & Haque, 2020) [1] . Extracts were filtered using Whatman No.1 filter paper and concentrated using a rotary vacuum evaporator (Buchi Rotavapor R-300) at 40-45 °C under reduced pressure until semi-solid, with yields determined by weight.

Bacterial Strains and Culture Conditions

Antimicrobial activity was evaluated against four bacterial strains: Cutibacterium acnes (MTCC 1951), Vibrio vulnificus (MTCC 1145), Aeromonas hydrophila subsp. hydrophila (MTCC 1739), and Escherichia coli (MTCC 1687), procured from the Institute of Microbial Technology (IMTECH), Chandigarh, India. Strains were revived in nutrient broth at 37 °C overnight under sterile conditions, with subcultures maintained on slants at 4 °C and reactivated before testing.

Antimicrobial Activity Assessment

The antimicrobial potential of extracts was assessed using a modified Kirby-Bauer disc diffusion method (Choyam et al., 2015) [10] . Mueller-Hinton Agar (MHA) plates were inoculated with 100 µL of 4-hour bacterial cultures adjusted to 0.5 McFarland standard. Sterile 6 mm paper discs were impregnated with 20 µL of extract (100 µg/mL stock concentration) and placed on the agar surface. Plates were pre-incubated at 4 °C for 20 minutes to ensure diffusion, followed by incubation at 37 °C for 24 hours. ZOI were measured in millimetres, with tests conducted in triplicate for reproducibility.

Phytochemical Screening

Preliminary phytochemical screening of the extracts followed standard protocols with modifications (Kancherla et al., 2019) [24] . Tests included: saponins (persistent foam upon shaking in water), terpenoids (reddish-brown ring with chloroform and H₂SO), phenols/tannins (blue-black coloration with 2% ferric chloride; Takó et al., 2020) [36] , flavonoids (yellow-to-colorless shift with NaOH and acidification; Ullah et al., 2020; Hasnat et al., 2024) [37,11] , sterols (violet-green ring with sulfuric acid), quinones (yellow precipitate with HCl), cardiac glycosides (brown-toviolet/green rings with glacial acetic acid and ferric chloride), and proteins (yellow coloration with nitric acid).

MIC and MBC

The MIC and MBC of Nyctanthes arbor-tristis extracts (NAT-SLM, NAT-OLE, NAT-SFM) were determined using the broth microdilution method, following CLSI guidelines (CLSI, 2018). Bacterial strains were cultured in Mueller-Hinton broth (MHB) to a 0.5 McFarland standard (1.5 × 10⁸ CFU/mL). Extracts were dissolved in 5% DMSO to prepare stock solutions (1000 µg/mL), and two-fold serial dilutions (0.1-100 µg/mL) were prepared in 96-well microtiter plates containing 100 µL MHB per well. Each well was inoculated with 100 µL of bacterial suspension, yielding a final volume of 200 µL. Positive (bacteria only) and negative (broth only) controls were included. Plates were incubated at 37 °C for 24 hours. The MIC was defined as the lowest extract concentration preventing visible growth, assessed visually and by optical density at 600 nm using a microplate reader. For MBC determination, 10 µL from wells showing no growth was subcultured onto Mueller-Hinton agar (MHA) plates and incubated at 37 °C for 24 hours. The MBC was defined as the lowest concentration reducing bacterial viability by ≥99.9%. Tests were performed in triplicate to ensure reproducibility.

Time-Kill Kinetics

Time-kill assays were conducted to evaluate the bactericidal or bacteriostatic effects of N. arbor-tristis extracts over time, following CLSI guidelines (CLSI, 1999). Bacterial suspensions (C. acnes, V. vulnificus, A. hydrophila, E. coli) were adjusted to 0.5 McFarland standard in MHB. Extracts were tested at their MIC and 2× MIC concentrations in 10 mL MHB, inoculated with 100 µL of bacterial suspension (final concentration ~10⁶ CFU/mL). Control tubes contained bacteria without extract. Aliquots (100 µL) were sampled at 0, 2, 4, 8, 12, and 24 hours, serially diluted in sterile saline, and plated onto MHA. After incubation at 37 °C for 24 hours, viable colonies were counted, and log₁₀ CFU/mL was plotted against time. Bactericidal activity was defined as a ≥3 log₁₀ reduction in CFU/mL compared to the initial inoculum, while bacteriostatic activity was indicated by <3 log₁₀ reduction. Experiments were conducted in triplicate, and mean values were used to construct time-kill curves.

GC-MS Analysis of Phytochemicals

The ethanol extract of N. arbor-tristis leaves was analyzed via Gas Chromatography-Mass Spectrometry (GC-MS) following Dhivya & Manimegalai (2013) [14] . Five grams of dried leaf powder underwent Soxhlet extraction with 95% ethanol for 6-8 hours, concentrated using a rotary evaporator, and reconstituted in HPLC-grade hexane. Analysis was performed on an Agilent 7890A system with a DB-5MS capillary column (30 m × 0.25 mm × 0.25 μm), using helium as the carrier gas (1 mL/min) in split mode (1:10). The temperature program started at 60 °C for 2 minutes, ramping to 280 °C at 10 °C/min, with the injector at 250 °C. The mass spectrometer operated at 70 eV, scanning 50-600 m/z. Compounds were identified using the NIST Mass Spectral Library.

Results

Antibacterial Activity by Disc Diffusion Assay

The disc diffusion assay clearly (Figure 1) demonstrated that among the three Nyctanthes arbor-tristis extracts, NAT-SFM possessed the most potent antibacterial activity, producing the largest clear zones around the discs. It inhibited C. acnes with a zone of 18.7±0.7 mm, V. vulnificus with 16.8±0.6 mm, A. hydrophila with 15.4±0.5 mm, and E. coli with 14.2±0.5 mm. NAT-SLM displayed moderate activity, forming zones of 16.3±0.6 mm against C. acnes, 13.5±0.5 mm against V. vulnificus, 12.8±0.4 mm against A. hydrophila, and 11.8±0.4 mm against E. coli.

Killing Kinetics and Bactericidal Activity

The 24-hour time-kill study revealed (Figure 3) pronounced bactericidal effects of the extracts, with NAT-SFM emerging as the fastest and most effective killer across all pathogens. Starting from an inoculum of 6.0 log₁₀ CFU/mL, NAT-SFM reduced C. acnes to 1.5 log (4.5-log reduction), V. vulnificus to 2.0 log (4.7-log reduction), A. hydrophila to 1.9 log (4.9-log reduction), and E. coli to 2.5 log (4.4-log reduction) within 24 hours. NAT-OLE showed only bacteriostatic to weakly bactericidal behavior; even at higher concentrations (25-100 µg/mL), viable counts remained relatively high at 24 h (4.3-4.9 log). Untreated control cultures grew slightly to 6.6-6.9 log over the same period, confirming that the observed reductions were solely due to extract activity and not nutrient limitation.

Phytochemical Screening

Qualitative phytochemical analysis indicated a rich and diverse secondary metabolite profile in all extracts (Table1). Saponins, terpenoids, sterols, quinones, cardiac glycosides, and proteins were present in NAT-SLM, NAT-OLE, and NAT-SFM. Notably, phenols/tannins and flavonoids were strongly present in NAT-SFM and NAT-OLE, whereas they were only moderately present in NAT-SLM. This higher abundance of polyphenolic compounds in NAT-SFM and NAT-OLE likely contributes to their enhanced antioxidant and antibacterial potential. Quinones and cardiac glycosides appeared weakly in NAT-OLE but were fully present in the other two fractions, further supporting the superior bioactivity of NAT-SFM.

Determination of MIC and MBC

Determination of minimum inhibitory and bactericidal concentrations confirmed NAT-SFM as the most potent extract. It inhibited and killed C. acnes at the lowest concentrations (MIC 6.25 µg/mL, MBC 12.5 µg/mL), followed by V. vulnificus and A. hydrophila (MIC 12.5 µg/mL, MBC 25 µg/mL each), and E. coli (MIC 25 µg/mL, MBC 50 µg/mL) (Table 2). NAT-SLM required 2-4 times higher concentrations (MIC 12.5-50 µg/mL, MBC 25-100 µg/mL), while NAT-OLE was the least active, needing 100 µg/mL to inhibit E. coli and 200 µg/mL to kill it. Across all strains and extracts, MBC/MIC ratios were ≤4, classifying all preparations as bactericidal rather than merely bacteriostatic.

Phytochemical Characterization Using GC-MS

GC-MS profiling (Table 3.1) of the most active fraction (NAT-SFM) identified tromethamine (18.04%) as the major peak, along with long-chain fatty acids and derivatives: myristic acid (4.57%), palmitic acid (4.48%), hexadecanol (3.08%), 1-chloro-tetradecanamine (3.38%), and stearic acid (0.73%). These compounds are well-documented for their direct antibacterial mechanisms myristic and palmitic acids disrupt bacterial membranes and inhibit fatty acid biosynthesis (FabI), long-chain alcohols increase membrane fluidity and cause leakage (Table 3.2), cationic amines electrostatically disrupt lipopolysaccharide layers in Gramnegative bacteria, and alkyl halides covalently block efflux pumps. The synergistic interplay of these membrane-active and enzyme-inhibiting molecules explains the broadspectrum, rapid bactericidal action observed with NAT-SFM. [41,25,1] . The zone-ofinhibition results ( [26,22,3,27] . Notably, NAT-SFM outperformed NAT-OLE by 28 [3,27,38] . The rapid onset of killing (evident from 4-8 h) suggests direct membrane-targeted mechanisms rather than slower protein-synthesis inhibition a hypothesis strongly supported by GC-MS identification of long-chain fatty acids (myristic, palmitic, and stearic acids) and their derivatives. These compounds are welldocumented for pore formation, membrane fluidisation, and inhibition of bacterial fatty-acid biosynthesis via the FabI pathway (Kabara et al., 1972; Zheng et al., 2005; Hidajati & Setiabudi, 2018) [42,20] . Palmitic acid, in particular, has been shown to disrupt Gram-positive membranes and inhibit enoyl-acyl carrier protein reductase in multiple pathogens (Aparna et al., 2012) [7] . [36,33,28] . The weaker activity of NAT-OLE, despite comparable polyphenolic content, may be attributed to thermal degradation during oven-drying and lower methanol efficiency in extracting certain glycosylated flavonoids (Hasnat et al., 2024; Sasikumar et al., 2010) [35,18] . Although synergistic interaction with conventional antibiotics was originally planned using checkerboard methodology (Odds, 2003) [29] , the standalone bactericidal potency of NAT-SFM at concentrations as low as 6.25-25 µg/mL already approaches or surpasses that of several antibiotics against resistant strains, reducing the immediate necessity for combination therapy. Nevertheless, the presence of cationic amines and alkyl halides (e.g., 1chloro-tetradecanamine) suggests potential efflux-pump blocking capacity that could restore antibiotic efficacy in MDR strains in future studies (Altuner et al., 2018; Isaac et al., 2017) [5,21] . From a translational perspective, the low MIC/MBC values of NAT-SFM against C. acnes (6.25/12.5 µg/mL) are comparable to or better than current topical antiacne agents such as clindamycin or benzoyl peroxide when adjusted for molecular complexity, while offering a natural, non-irritating alternative with additional anti-inflammatory benefits via NFκB inhibition (Aparna et al., 2012; Dhinakaran & Sakthivel, 2016) [7,13] . Similarly, activity against V. vulnificus and A. hydrophila emerging threats in tropical aquaculture and wound infections positions NAT-SFM as a candidate for development of cost-effective topical or aquaculture therapeutics (Ghagane et [16,6,9] . In conclusion, this study successfully bridges traditional Ayurvedic knowledge with modern pharmacological validation by demonstrating that shade-dried floral methanolic extracts of Nyctanthes arbor-tristis constitute a potent, rapid-acting, broad-spectrum antibacterial agent whose activity is driven primarily by membrane-disrupting fatty acids and polyphenolics. These findings strongly support further development of standardised NAT-SFMbased formulations for acne management, wound care, and aquaculture disease control applications that could significantly reduce reliance on conventional antibiotics in the face of escalating antimicrobial resistance (WHO, 2020).

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