Yield percentage of extracts
The percentage yields of the extracts obtained from various plant parts are shown in Figure 1a. Our study found that The crude extraction yields were as follows: TB, 14.5%; TA, 10.75%; AM, 8.25%; and BM, 7.5% (w/w). Previous studies have consistently reported that aqueous extracts from different plant parts tend to yield higher amounts than solvent-based extracts from the same plant.²⁵ This phenomenon can be attributed to the prolonged incubation time and the high polarity of water as the extraction solvent.

Antibacterial potential of extracts
The antimicrobial activities of the extracts against the test organisms displayed varying degrees of inhibition, as summarized in Table 1. All four extracts were effective against the tested organisms, and the zones of inhibition were quantified, excluding the diameter of the well. All extracts demonstrated substantial inhibitory effects at 20 mg/mL. TA extract showed maximum inhibition against E. coli (13.43 ± 0.74 mm) and was less effective against V. alginolyticus (8.40 ± 0.52 mm). TA extract exhibited the highest level of inhibition against E. coli (13.43 ± 0.74 mm) but showed relatively lower effectiveness against V. alginolyticus (8.40 ± 0.52 mm). Similarly, it demonstrated equal inhibitory activity against M. smegmatis and three clinically significant bacterial isolates, S. aureus, P. aeruginosa, and E. coli 385. Previous studies have reported the antibacterial activity of TA (bark) extracts against various clinical isolates, including ear infections, with inhibition zones reported for E. coli (15 mm), S. aureus (17 mm), P. aeruginosa (16 mm), and B. subtilis (11 mm).²⁶

Table (1):
Antimicrobial activity of aqueous extracts (20 mg/ml) of selected plants against bacterial strains and lab isolates

Another study found that TA bark extract (both cold and hot) was effective against S. aureus but lacked inhibitory potential against P. aeruginosa and E. coli.²⁷ Kumar et al. reported similar inhibitory potential of TA bark extract against B. subtilis, E. coli, S. aureus, and P. aeruginosai.⁶ These findings align with the observations of Gupta and Kumar, who investigated the antimicrobial effects of TA against E. coli, P. aeruginosa, and S. aureus, all of which exhibited high sensitivity to the chloroform-methanol extract.²⁸ Furthermore, this antimicrobial effect of TA is consistent with a study conducted by Jaiswal and Kumar, who assessed the antimicrobial effect of the ethyl acetate extract of TA against six pathogenic microbes.²⁹ Numerous other studies support the results of the current study.^27,30,31

The strains of S. aureus, P. aeruginosa, and E. coli 385 used in this study were susceptible to TB. The aqueous extract of TB exhibited significant inhibition of E. coli 385 (19.51 ± 0.61 mm), followed closely by BM (16.10 ± 0.31 mm), while TA extract was less effective against E. coli 385. These findings are consistent with the results of a study conducted by Aqil et al, who investigated the antimicrobial effect of a TB ethanolic extract against methicillin-sensitive and methicillin-resistant S. aureus.³² Devi et al assessed the antimicrobial potential of an aqueous extract of TB in restricting the growth of various microbes, including E. coli and P. aeruginosa, as well as some fungal isolates.³³ Several other researchers have examined the antimicrobial properties of TB, and their findings are consistent with those of the current study.^34,35

As indicated in Table 1, the TB extract displayed the most potent inhibitory effect against all the selected bacterial strains compared to the aqueous extracts of the other plants. Notably, this study used water for phytochemical extraction and the aqueous extract exhibited strong inhibitory effects against the clinical isolates used in this study, which are known to exhibit high levels of drug resistance.

BM also effectively inhibited the growth of the microbes examined in this study. BM exhibited the largest zone of inhibition against S. aureus (20.40 ± 0.31 mm). Previous studies, such as that by TA et al., have assessed the antimicrobial potential of BM against clinical isolates of pathogenic strains, with the methanolic extract displaying a more potent inhibitory effect against K. pneumoniae and S. aureus.³⁶ Haque et al. have described the inhibitory potential of BM against multidrug-resistant urinary tract infections (UTI) and respiratory tract infections (RTI). The diverse array of phytochemicals found in these extracts underscores their pharmaceutical significance.³⁷ Furthermore, Kothari et al., Abhirami et al. and Ganapathy et al. investigated the antimicrobial effects of AM against five bacterial and three fungal strains using various solvents, such as chloroform, petroleum ether, and ethyl acetate.^38-40 The current findings also confirmed the effectiveness of AM against multidrug-resistant E. coli 385.

Minimum inhibitory (MIC) and bactericidal (MBC) concentration
The results of the current study demonstrated that the water extracts efficiently inhibited the growth of both the test strains and laboratory isolates to varying degrees within the wells. The antimicrobial potential of the extracts directly correlated with an increase in extract concentration, as supported by previous studies.⁴¹ All microorganisms used in this study were susceptible to the Minimum Inhibitory Concentration (MIC) of each plant extract, as presented in Table 2. The lowest MIC (0.312 mg/l) and MBC (0.625 mg/ml) values were observed for TB against S. aureus, with an MIC of (0.625 mg/ml) against B. subtilis, V. alginolyticus, P. aeruginosa, and E. coli 385. TB fruit extract has previously been reported to be effective against drug-resistant P. aeruginosa and MRSA at very low concentrations, with MIC values ranging from 0.25 to 0.5 mg/ml.³⁵

Table (2):
Minimum inhibitory concentration (MIC) and MBC of aqueous extracts of plant parts

The TA and BM extracts exhibited MIC values of (2.5 mg/ml) and MBC values of (5.0 mg/ml) against all tested strains, except S. aureus. In a previous study, the MIC value of TA bark aqueous extract was reported to be 25 mg/ml against S. aureus, 12.5 mg/ml against E. coli and B. subtilis, and 6.3 mg/ml against P. aeruginosa.⁶ TA displayed activity at an MIC of (5.0 mg/ml) and an MBC of (10 mg/ml), while BM exhibited an MIC and MBC of 1.25 mg/ml and 2.5 mg/ml, respectively, against S. aureus.

Determination of phytochemical components
Preliminary analysis of the extracts revealed the presence of phenols, sterols, coumarins, tannins, and saponins in the selected plants. Phytochemicals play important roles in the medicinal properties of plants.

Phenols, which were found in all extracts, are well-known secondary metabolites with significant antioxidant potential. They reduce the risk of cancer, inflammation, and cardiovascular diseases.⁴² Coumarins, also identified in the extracts, exert anti-inflammatory and antioxidant effects by blocking the generation of ROS (reactive oxygen species) and downregulating key pathways like NF-κB, TNF-α, and MAPKs, which are associated with inflammation and oxidative stress.⁴³ Tannins, another group of phytochemicals present in plant extracts, are polyphenolic compounds with known antioxidant properties. Traditionally, they have been used to treat a range of health issues, including gastrointestinal, respiratory, and skin problems.⁴⁴ Saponins have various health benefits, including hypocholesterolemic, immune modulation, and anti-inflammatory properties, which have led to their use in traditional pharmaceutical applications.

Quantitative analysis of phytochemicals
Using the FC reagent, the total phenolic content (TPC) of the extracts (TA, TB, AM, and BM) was calculated (Figure 1b) and expressed as gallic acid equivalent (GAE). The results indicated that the extract of AM had the highest TPC with a value of 1.22 ± 0.12 mg/g, followed by TA (1.17 ± 0.23 mg/g), TB (0.66 ± 0.20 mg/g), and BM (0.55 ± 0.21 mg/g). Previous studies have reported much higher concentrations of TPC when different solvents were used for the extraction of phytochemicals, as opposed to water. For example, in the case of TA, TPC values of 96.42, 184.59, and 137.98 mg/g GAE were determined in the methanolic extracts of the leaves, bark, and fruits, respectively.⁴⁵ Meena et al. reported TPC values ranging from 128 to 1228 µg/mL GAE in the distilled water extract of TA bark, which are quite similar to the present study. Kumar et al. found a TPC of 131.9 mg/g GAE in the aqueous fraction of TA bark extract, whereas Kumar et al. reported TPC values of 233.64 mg/g GAE in TA bark and nearly 200 mg/g in the fruit methanolic
extract of TA.^{6, 46,47}

It is well-established that the phenolic content in different solvent extracts is generally higher than that in aqueous extracts.⁴⁸ The high concentration of phenolic compounds in the plant extracts indicated their strong radical-scavenging potential.^49-52 For example, a 2019 study reported nearly 60 mg/g GAE in the aqueous extract of TB fruit.⁵³ The BM contained 67-79 mg/g GAE in the aqueous extract, whereas the methanolic extract contained 37-55 mg/g GAE.⁵⁴ The AM extract to have 31.6 mg/g GAE.⁵⁵ The presence of phenols in AM indicates the antidiabetic potential of plants.

The total tannic acid contents of the extracts (TA, TB, AM, and BM) ranged from 0.775 to 0.952 mg TAE/g (Figure 1b). TA extract contained the highest amount of tannins (0.95 ± 0.22 mg/g), followed by TB (0.89 ± 0.16 mg/g), AM (0.87 ± 0.15 mg/g), and BM (0.77 ± 0.07 mg/g). The tannin contents reported in previous studies were often higher than those observed in the present study. For instance, the fruits of TB are generally reported to be enriched with 23.60–37.36% tannin content.⁵⁶ However, the quantity of phytochemicals in the same plant can vary owing to environmental factors that vary by location.⁵⁷

Radical scavenging and reducing power of plants extracts
The antioxidant potential of the extracts was evaluated using various assays, including DPPH and ABTS radical scavenging assays as well as a reducing power assay. Antioxidants play a crucial role in protecting the body from the damage caused by free radicals by donating hydrogen atoms or electrons to neutralize these harmful molecules. In the DPPH assay, antioxidants reduce DPPH radicals, resulting in a color change from purple to yellow. The effectiveness of antioxidants in scavenging DPPH radicals was indicated by their ability to reduce the absorbance of the radical solution. The lower the EC₅₀ value, the higher the antioxidant capacity.⁵⁸

The results of the present study demonstrated significant scavenging activity of all four extracts (Figure 1 c-f). TA extract exhibited the highest DPPH radical scavenging activity with the lowest EC₅₀ value (1.14 ± 0.06 mg/ml), followed by BM (2.20 ± 0.07 mg/ml), AM (2.34 ± 0.05 mg/ml), and TB (4.15 ± 0.25 mg/ml) (Figure 1c). Comparing these results with those of previous studies, it is evident that the DPPH scavenging potentials of these extracts vary. For example, one study reported an IC₅₀ value of 10.3 µg/ml in the aqueous fraction of TA bark extract. The IC₅₀ values were generally lower for the other solvent extracts, indicating their strong DPPH scavenging potential.⁶

Another study reported an IC₅₀ value of 13.3 µg/ml in the methanolic fraction of TA bark extract.⁴⁵ For BM, the ethanolic extract showed an IC₅₀ value of 90 mg/l for DPPH scavenging ability.⁵⁹

Similar results were obtained by Nampoothiri et al., who found that the methanolic extract of TB showed a higher radical scavenging effect than the aqueous fraction. In the case of methanolic extract, 1.2 µg/ml IC₅₀ value was obtained, while in the case of water extract, 2.43 µg/ml IC₅₀ value was calculated. However, the antioxidant effect of the hexane extract was much lower than that of water.⁶⁰ For the aqueous extract of TB, 3.5 an IC₅₀ value was reported.⁶¹ The methanolic extract of AM exhibited 961.53 µg/ml IC₅₀ value.⁶² Vardhini et al.⁶³ reported that the aqueous AM exhibited 183.58 an IC₅₀ value. The IC₅₀ of the methanolic fraction of the leaf extract was 63.52 µg/mL.⁶⁴

The extract of TA showed the highest activity for ABTS+ with an EC₅₀ (1.02 ± 0.079 mg/ml), BM (1.09 ± 0.027 mg/ml), AM (1.13 ± 0.048 mg/ml) and TB (3.71 ± 0.18 mg/ml) (Figure 1d). In the ABTS assay, 52.6 µg/ml IC₅₀ value for the aqueous extract of TB was reported, which was quite high as compared to solvent extracts, suggesting the low antioxidant potential of water extract.⁶¹ In another study, 10.81 µg/mL IC₅₀ value for the aqueous extract of AM was reported.⁶³ Similar to the DPPH assay, the antioxidant effect of the water extract was quite low compared to that of the other solvent extracts in the ABTS assay of TA.⁴⁶ Leaf extract of TB exhibited 11 µg/ml IC₅₀ value from the aqueous fraction, indicating a better antioxidant effect as compared to the result of the present study.⁶⁵ The inhibition of ABTS with the water fraction of TA was observed with an IC₅₀ value of 251 µg/ml which was quite low as compared to ethanolic extract.⁶⁶

The extracts of the selected plants showed significant electron-donating activity in a concentration-dependent manner by reducing ferric ions to ferrous ions. BM exhibited the highest reducing power was shown by BM followed by TA, AM, and TB (Figure 1e). The reducing power of an antioxidant corresponds to the color change from yellow to green or blue. A higher absorbance indicates a higher ferric-reducing power of the plant extracts. Phenols and tannins in the extracts act as radical scavengers by donating electrons and hydrogen. Researchers have reported that the ethanolic bark extract of TA possesses a strong potential to reduce Fe2+ compared with the distilled water extract. Significantly better FRAP activity was observed in the ethanolic extract than in the distilled water extract, as reported by Meena et al.⁴⁶ This difference in reducing potential may be due to the adequacy of the solvent used for extraction. Other studies have also reported higher FRAP activities in alcoholic extracts.⁶⁷ In this study, the antioxidant potential of TB was significantly lower than that of TA. Similar results were obtained by Ayra et al., where the EC₅₀ value for TA was 232 µg/mL and for TB, it was 265 µg/ml. This improved ability to reduce iron may be due to the high amount of polyphenols with hydroxyl groups.⁶⁸

UV-visible spectroscopy
The qualitative UV-vis profiles of the plant extracts (TA, TB, AM, and BM) were recorded to identify chromophores, aromatic rings, and compounds containing σ and π-bonds. Sharp peaks were observed in the wavelength range of 200–800 nm. The profile showed peaks at 290 and 320 nm with absorptions of 1.3, 1.1, 311.5, and 370 nm with absorptions of 5.256 and 2.046 for TB, and 280 nm with an absorption of 1.45 for AM, and 292.5 nm with an absorption of 1.774 for BM, respectively (Figure 2a, b, c, and d).

FTIR
The FTIR spectroscopic analysis confirmed the presence of various functional groups in the aqueous extracts of the selected plants, as indicated by the following groups: O-H, O=C=O, C=O, C=C, N=O, C-O, C=S, C-H, C-F, C-O, and C-Br (Figure 2e). The presence of carbonyl, amine, and other functional groups in the TA extract has been reported previously.^69,70 The TB extract exhibited peaks corresponding to alkanes, carboxylic acids, and alcohols, as reported by Patil et al.⁷¹ The presence of alcohols, coumarins, tannins, phenols, and aromatic stretching in AM extracts has been confirmed by many studies.^72,73 The BM extract contained alcoholic O-H, carbonyl, carboxylic acid, and aromatic groups.⁷⁴ The groups present in the selected plants are listed in Table 3.

Table (3):
Functional groups obtained through IR spectrum analysis of the aqueous extracts of the plants used in the present study

LC-MS/MS analysis
The present study employed LC-MS/MS to elucidate the chemical profiles of medicinal plant extracts. In particular, the aqueous extract of Aegle marmelos (AM) was found to contain a twenty two compounds. The compounds identified in the AM extract are listed in
Table S1, along with their molecular weight, chemical formula, retention time, and chemical structure. The notable compounds identified included levomefolic acid, commonly used as a folate supplement for food fortification; triamterene, a phenolic compound used in the treatment of edema and liver cirrhosis, and as a diuretic; and balapiravir, an example of a direct-acting antiviral that targets both structural and non-structural viral proteins.^75-77 Additionally, Threonic acid, a phenolic acid, was identified for its potential to inhibit TNF-a, thus potentially offering benefits in memory preservation. Neosaxitoxin, which is known for its local anesthetic properties, and methyldilazine, which exhibits bactericidal activity against Mycobacterium tuberculosis, were also detected. Lucanthone and Remoxipride have emerged as potential antitumor agents with applications in the treatment of conditions such as glioblastoma. Gosogliptin, an anti-diabetic drug, along with Piperoleine B, Altretamine, Azanidazole, and Bortezomib, displays anti-inflammatory and anticancer activities.^78-86 Furthermore, Phenothiazine has been recognized for its antibacterial activity, particularly in the treatment of tract infections. The analysis also revealed the presence of compounds such as 2-Phenylaminoadenosine, Lorcainide, Ebastine, (E)-entacapone, and rivastigmine, each with distinct pharmacological properties.^87-91

In the case of Bacopa monnieri (BM) extract, LC-MS/MS analysis unveiled the presence of 10 compounds (Table S2). Among these, Aminolevulinic acid, which is recognized for its role in treating actinic keratosis, and propicillin, an antibacterial agent, are noteworthy. Promethazine, an antihistamine, and Ciclazindol, an antidepressant and anorectic drug, were also identified. Morclofone, with its antitussive properties, and Pexacerfont, used in the treatment of alcoholism and anxiety disorders, were present. Tenofovir, known for its antiviral activity, and avenanthramides D, which exhibits antineoplastic properties, were detected.^92-97

LC-MS/MS analysis of Terminalia arjuna (TA) extract revealed the presence of five compounds (Table S3). Notably, Thiethylperazine (TEP), known for its anti-nausea properties, was among the identified compounds. TEP is currently undergoing clinical trials (phase 2) for potential applications in Alzheimer’s Disease treatment. Additionally, the extract contains compounds such as oxyphenonium, which is used to treat visceral spasms, and octinoxate, a sunscreen ingredient. Altretamine, which is known for its antineoplastic properties, was also detected.^98,99 (https://pubchem.ncbi.nlm.nih.gov/compound/5749).

In the case of Terminalia bellirica (TB) extract, LC-MS/MS analysis identified approximately nine compounds (Table S4). Notably, Tiletamine, an anesthetic and anticonvulsant agent, and (E)-progabide, which are used to treat epilepsy, were among the identified compounds. Tetraphenylphosphonium, which has potential as an antineoplastic agent, and bergenin, which is known for its antifungal and antiviral properties, were also detected. Cyproterone, recognized for its anti-cancer properties, further expands the potential therapeutic applications of TB extract.^100–105

The use of herbal products for disease control is an innovative alternative to antimicrobial therapies. This approach offers a non-toxic and environmentally friendly means of managing drug-resistant diseases.¹⁰⁶ Owing to the rapid development of resistance against chemotherapeutic drugs (mainly antibiotics), it has become vital to consider any substitute and efficient therapies such as herbs.¹⁰⁷ This study explored the potential of herbal products as antibacterial agents against resistant and susceptible bacteria. These findings support the significant role of plant extracts in the treatment of bacterial infections. The active extracts identified in this study offer valuable insights into the development of new compounds that exhibit enhanced activity and effectiveness against both resistant (MDR, XDR, and PDR) and susceptible bacteria responsible for different infections, surpassing the capabilities of currently available antibiotic agents. The aqueous extracts, namely TA, TB, AM, and BM, exhibited significant antimicrobial activity against various clinically important bacterial strains, including multidrug-resistant Escherichia coli 385. In particular, the TB extract demonstrated high inhibitory activity against E. coli. The minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of these extracts were determined to determine their efficacy against drug-resistant strains. Furthermore, qualitative analysis of these extracts using UV-visible spectroscopy revealed the presence of chromophores and compounds containing various chemical bonds. FTIR spectroscopy confirmed the presence of functional groups that may have contributed to the medicinal properties of the extracts. LC-MS/MS analysis provided insights into the chemical composition of the extracts, identifying various compounds with potential therapeutic applications. These findings highlight the promising antimicrobial and antioxidant properties of these aqueous plant extracts and suggest their potential utility in medicinal and pharmaceutical applications. Further in silico analyses of the retrieved compounds are warranted to explore the specific bioactive components and their mechanisms of action.