2.1. Plant and Extract Preparation

Aerial parts (flower and leaf) of the H. perforatum plant were collected from Belbeis, Elsharkia, Egypt, in March-April 2022, and identified by a botanist in the Faculty of Science at Al-Azhar University using voucher number 946. It was first cleaned with tap water, dried outside, cut into little pieces, and then mechanically pulverized with a blender. After that, it was suspended in methanol for seven days to create the extract. The extracts underwent filtering and evaporation (50°C) before being dried in an oven at 60°C. The entire extract (10 gm of dry powder) was submitted to a bioassay-guided fractionation process that started with water solubilization and progressed via n-hexane, chloroform, and ethyl acetate partitioning in order. Under reduced pressure, each collected fraction was concentrated to produce a black residue [17]. The extraction yield was 31.25, 4.39, 5.71, and 9.83% for methanol, n-hexane, chloroform, and ethyl acetate, respectively.

2.2. Isolation and Detection of Harmful Bacteria

Briefly, clinical samples from patients admitted to Al-Zahraa University Hospital and Cairo Specialized Hospital in Cairo city were used to isolate bacterial strains. The samples were cultivated on appropriate agar media, and VITEK 2 was used to identify the bacterial isolates (Biomerieux, New Delhi, India) [18].

2.3. Antibiotic Susceptibility Assay

The susceptibility to the commercial antibiotics of the bacterial isolates was evaluated using the disc diffusion method. Antibiotics used against Gram-positive bacteria included cefoxitin, benzyl-penicillin, oxacillin, imipenem, gentamicin, ciprofloxacin, moxifloxacin, inducible clindamycin resistance, erythromycin, clindamycin, vancomycin, tetracycline, fusidic acid, and trimethoprim/sulfamethoxazole. On the other hand, antibiotics used against Gram-negative bacteria included temocillin, ampicillin, amoxicillin/clavulanic acid, ticarcillin, ticarcillin/clavulanic acid, piperacillin, piperacillin/tazobactam, cephalothin, cefuroxime, cefotaxime, ceftazidime, ceftriaxone, cefepime, ertapenem, imipenem, meropenem, amikacin, gentamicin, tobramycin, ciprofloxacin, tigecycline, fosfomycin, nitrofurantoin, pefloxacin, minocycline, colistin, and trimethoprim/sulfamethoxazole (Himedia Labs, Mumbai, India) [19].

2.4. Antibacterial Action

To evaluate the extracts’ antibacterial effectiveness against microorganisms, an agar well diffusion experiment was employed [20]. Nutrient agar was the culture medium that was used. Wells with a diameter of 6 mm were drilled into the solid agar. The inocula (1.5 × 10⁸ CFU/ml) were dispensed on nutrient agar plates using sterile swabs, and then, 100 μl of extracts was added. The concentration of each of the used extracts was 0.01 g/ml. The plates were then incubated for a further 24 hours at 37°C. After incubation, the zone of growth inhibition for each extract was measured. The MIC of the active ethyl acetate fraction was reported by Khan et al. [21]. The extracts were serially diluted twice. Each inoculum was made in the proper medium for the broth microdilution procedure, its density was adjusted to 0.5 McFarland standards (10⁸ CFU/ml), and its volume was diluted to 1 : 100. On microtiter plates, the MIC was determined following a 24-hour incubation period at 37°C.

2.5. Ultrastructural Changes Brought on by H. perforatum Whole Extract in K. pneumoniae Cells

A concentration of 10⁶K. pneumoniae cells was treated with 0.25 micrograms per milliliter of total methanol extract from H. perforatum, compared to control cells, and was then left undisturbed for 20 hours to determine their impacts on the ultrastructure. The suspension was rinsed twice with phosphate-buffered saline after being centrifuged down to a pellet. Standard techniques for fixing and embedding biological samples for transmission electron microscopy (TEM) were carried out after these processes, and the JEOL010 instrument was used to analyze the results [22].

2.6. Chromatographic Separation of Phytochemicals

Chromatographic separation was carried out on a 5 m C18 column (50 2.0 mm internal diameter; Bohus, Sweden) fitted with an Agilent LC-ESI-MS System (Agilent Technologies, Palo Alto, CA, USA). The temperature of the column was maintained at 40°C. The whole movie lasted for 70 minutes. The mobile phase, which was acetonitrile-water (50 : 50, v/v) containing 0.1% trifluoroacetic acid, was administered at a flow rate of 0.3 ml/min. When the mass spectrometer was in the positive ion mode, both of its quadrupoles were set to 0.7 full width at half height (FWHM, unit resolution). In addition, the spray voltage was set at 4500 V and the ion tube temperature at 210°C. At 49, 2.0, and 14 arbitrary units, the nitrogen sheath, ion sweep, and auxiliary gases, respectively, were all set. Through infusion experiments, the ESI-MS/MS parameters were altered to create the most deprotonated molecules and the most effective generation of unique fragment ions for all compounds. MS-DIAL V. 3.70 software was used to identify and calculate the relative percentage of molecules [23].

2.7. Antioxidant Assay

2.8. Viability Assay for Cytotoxicity Evaluation

In 96-well plates, the Vero cells were planted with 1 × 10⁴ cells and 100 μl of DMEM growth medium. Confluent cell monolayers were placed into 96-well flat-bottomed microtiter plates (Falcon, Jersey, NJ, USA) using a multichannel pipette after being seeded for 24 hours. New DMEM medium containing various amounts of the samples was then added, followed by successive twofold dilutions of the examined specimen. The microtiter plates were incubated for 48 hours at 37°C in a humid incubator with 5% CO₂. Following crystal violet staining, absorbance was determined at 590 nm [26].

2.9. Statistical Analysis

The t-test was employed by SPSS software for various analyses of the trials, and all tests were conducted in triplicates.

3.1. Antibiotic Susceptibility

Antibiotic susceptibility tests for two Gram-positive and five Gram-negative bacterial isolates were carried out. MRSA strain was resistant to ten of the nineteen used antibiotics while E. faecalis strain was resistant to thirteen of the twenty used antibiotics. Concerning Gram-negative bacterial isolates, for the ratios of the number of resistant antibiotics, the total number of antibiotics was as follows: E. coli (18/23), K. pneumoniae (19/23), P. aeruginosa (16/16), A. baumannii (20/23), Proteus mirabilis (15/23), and S. maltophilia (22/23) (Table 1).

3.2. Susceptibility of MDR Isolates to Different H. perforatum Extracts and MICs

The n-hexane fraction showed no antibacterial activities against all tested strains except for P. mirabilis, while the total extract of H. perforatum and ethyl acetate, chloroform, and aqueous fractions revealed variable antibacterial activities against all tested strains except for A. baumannii. The antibacterial activities of the total extract of H. perforatum and its five solvent fractions were in descending order: total extract > ethyl acetate fraction > chloroform fraction > aqueous fraction > n-hexane fraction. P. mirabilis and S. maltophilia were the lowest susceptible pathogens to the aqueous extract with an inhibition zone of 13.4 mm. The total extract of H. perforatum showed the maximum diameter of the size zone of inhibition against K. pneumoniae (27.9 mm), followed by E. faecalis (25.3 mm), MRSA (23.0 mm), P. aeruginosa (20.4 mm), S. maltophilia (18.3 mm), and P. mirabilis (17.9 mm). The n-hexane showed no antibacterial activity except for Proteus mirabilis (15.3 mm). Ethyl acetate fraction showed the maximum diameter of the size zone of inhibition against K. pneumoniae (24.4 mm), followed by MRSA (22.3 mm), E. faecalis (19.8 mm), P. aeruginosa and P. mirabilis (16.3 mm), and S. maltophilia (15.8 mm). The chloroform fractions of H. perforatum showed more inhibition zone diameter against E. faecalis (20.9 mm), K. pneumoniae and S. maltophilia (18.6 mm), P. aeruginosa (16.35 mm), and MRSA (16.2 mm). Finally, the aqueous fraction showed an inhibition zone against MRSA of 19.3 mm, followed by E. faecalis (18.4 mm), K. pneumoniae (14.8 mm), and P. mirabilis and S. maltophilia (13.4 mm). The antibacterial activities are recorded in Table 2. The MIC values of the total fraction were determined as they revealed the maximum level of antibacterial activity. An MIC value of 125 μg/ml of the total fraction was recorded against P. mirabilis, 62.5 μg/ml was the MIC value against S. maltophilia, 31.25 μg/ml was the MIC value against P. aeruginosa, 15.63 μg/ml was the MIC value against MRSA, 3.9 μg/ml was the MIC value against both E. faecalis and K. pneumoniae, and finally, no MIC value of total fraction was registered against A. baumannii as shown in Table 2.

3.3. TEM Study

In the current study, there were many ultrastructural alterations in K. pneumoniae cells as affected by H. perforatum total methanol extract. In the control sample, TEM micrographs (Figure 1(a)) revealed the healthy, thin, and typical ellipse shape of individual K. pneumoniae cells, which were surrounded by the inner and outer layers without any cellular injury. The cell membrane of H. perforatum-treated K. pneumoniae cells was detached from cells, with misshapen cells with loss of structure. This study showed for the first time the ultrastructure alterations involved in the anti-K. pneumoniae action by H. perforatum total extract (Figure 1(b)).

3.4. Antioxidant Activity

Using the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) tests, the antioxidant activity was assessed as a free radical-scavenging capacity. It has been widely utilized to measure the free radical-scavenging abilities of antioxidants. The DPPH free radical is a stable free radical. The outcomes demonstrated that the studied plant extracts’ DPPH or ABTS radical-scavenging abilities were exerted in a dose-dependent manner (Figure 2). Additionally, H. perforatum’s methanol extract demonstrated half-maximal inhibitory concentrations (IC₅₀) of 98.73 ± 3.59 and 119.86 ± 4.12 μg/ml, respectively. The ethyl acetate extract had exceptional antioxidant potential as indicated by both DPPH and ABTS assays, with IC₅₀ values of 467.18 and 489.32 μg/ml, respectively.

3.5. Cytotoxic Activity

It was demonstrated that the methanol extract from H. perforatum has negligible cytotoxic effects even at high doses (500 μg/ml or less). Under these screening circumstances, the calculated CC₅₀ value was 976.75 μg/ml as depicted in Figure 3.

3.6. LC-MS Outcome

The relative percentages of the separated chemicals were calculated after the H. perforatum methanol extract was chromatographically separated using LC-MS (Table 3, Figure 4). This led to the separation of distinctive fragment ions that were recognized by the system software. According to Table 3, quercetin-3-β-D-galactopyranoside came in second with 8.45% of the total LC-MS chromatogram contents, followed by hypericin as an anthraquinone derivative with 10.89%. As indicated in Figure 5, the most prevalent chemical class in the H. perforatum extract was flavonoids, followed by sugars and polyols, organic acids, anthraquinones, amino acids, esters, thiophene, fatty acids, and phenolics. Additionally, the isolated chemicals were divided into a total of seventeen phytochemical categories. Furthermore, the lipids were exerted by fatty acids and sterols and represented 3.17% of the total contents. On the other hand, deoxyfructosazine as pyrazines (1.83%), alcohols (1.48%), and xanthones (1.26); pentacyclic triterpenoids (olean-12-en-3-ol derivatives) (1.17%); procyanidins as tannins (1.08%); and 1,3,4-trihydro-2-thioxomethyl-2h-isoquinoline as benzopyridines (0.68) and hydrocarbons (0.03%) were also presented as minor contents.