Unveiling phenolic content, antibacterial, and antibiofilm potential of sacha inchi (Plukenetia volubilis L.) seed shell extracts against Staphylococcus aureus

Plant material

Seeds of sacha inchi were purchased from a local supermarket in Riyadh, Saudi Arabia. Seeds were carefully selected, meticulously washed, and dried. The shell of the seeds was manually separated with the help of a small hammer. Dried shells (500 g) were ground to powder using a professional hammer mill set at 25,000 rpm and 5 min running time at 10 min intervals. The grinding was accomplished at 25 °C for 15 min. The ground fine powder was sifted with a vibratory sieve shaker and sieved to obtain a granulometric fraction of 1.5–2.0 µm diameter. It is well accepted that particles with a finer size produce more extract yield.

Chemicals and standards

The analytical grade solvents and chemicals used for extraction and analysis,including ethanol (99.9%, v/v) phosphoric acid, sodium carbonate (>99%), acetonitrile (100%, HPLC grade), sodium hydroxide and Folin–Ciocalteu reagent (2N solution), were procured from Merk (Darmstadt, Germany). Standards of (+)-catechin hydrate, quercetin hydrate, gallic acid–anhydrous, (-)-epicatechin, p-coumaric, protocatechuic, o-coumaric, caffeic, ferulic, p-hydroxybenzoic acid, chlorogenic, procyanidin (B1, B2, B3 and C1), (-)-epigallocatechin gallate, rutin, resveratrol, and gallic acid glucoside were obtained from Sigma-Aldrich (Darmstadt, Germany). 2,2-diphenyl-1-(2,4,6-tri nitrophenyl)-hydrazinyl (DPPH) and 2,2-azino-bis (3-ethylbenzothiazoline-6-sulphonicacid) diammonium salt (ABTS) used for antioxidant assay, were supplied by Sigma-Aldrich.

Preparation of sacha inchi seed shell extracts

The powdered SI seed shells (five g) were extracted individually with 70% of ethanol (3 × 25 mL) and distilled water (3 × 25 mL) for 3 h at ambient temperature and the boiling temperature (60–80 °C range), respectively. The extraction apparatus consisted of a round-bottom flask connected to a condenser, equipped with a magnetic stirrer and a heating mantle. After 3 h, the resulting organic and aqueous extracts were individually centrifuged for 5 min at 10,000 rpm. The supernatant was separated, the solvent was evaporated under vacuum at ±50 °C temperature using a vacuum evaporator, and extraction yields (%) were determined. The extraction yield percentages were computed by applying the formula, Yield% = W₁×100/W₂, where W₁ andW₂ represent the weight of dry extract residue obtained after solvent removal and the weight of the dry seed shell taken. The ethanol and aqueous extract residues were 180.23 and 238.21 mg, respectively. The extract yields were comparable to those of the previously published study (Chirinos et al., 2016). The prepared extracts (ethanol and aqueous) were stored in dark glass vials at 4 °C. Three runs of the extraction process were performed for each extract under similar conditions.

Spectrophotometric determinations

HPLC analysis

The HPLC analysis of ethanolic and aqueous extracts of SI seed shellswas conducted on an LC-20AD Shimadzu HPLC system (Shimadzu, Kyoto, Japan), equipped with a binary solvent delivery module (LC-20AD), a Rheodyne type injector with a 20 µL sample loop and a DAD detector (SPD-M 20 A). An extended guard column was used to perform reverse phase column chromatographic separation (C-18 Pack Capcell column with five µm, 250 mm × 4.6 mm dimensions). The eluent system constitutes methanol-acetonitrile water (MeOH: ACN: H₂O: 40:15:45 v/v) with 1% acetic acid, using isocratic elution over a 30 min period. The DAD was set to operate within a wavelength range of 240 to 800 nm. 20 µL of samples and reference solutions were applied, with flow rate maintained at one mL/min. Software Shimadzu LC solution was employed to collect and analyse the data. The phytocomponents present in the extracts were identified on the basis of UV absorbance (between 240 and 800 nm), retention times for the analytes, and reference standards.

Antioxidant activity

The antioxidant properties of ethanolic and aqueous extract of SI seed shell was elucidated by DPPH assay as described by Katalinic et al. (2006). The antioxidant potentials observed in DPPH radical-scavenging were attributed to the compound’s ability to donate hydrogen atoms. The DPPH^• free radical accepts readily an electron or hydrogen atom to become a stable molecule. The antioxidant activity was assessed by monitoring the reduction in absorbance of the DPPH• radical at 517 nm, which was accompanied by a colour transition from violet to yellow. The ability of SI seed shell extracts to scavenge stable DPPH^• radicals was assessed using spectrophotometric analysis. The solutions of extract were prepared by dissolving 20 mg of dry extract in 40 mL of methanol. Prior to UV measurements, a DPPH^• solution was prepared in methanol (6 × 10⁵ M). After that, three mL of DPPH^• solutions were mixed with varied doses (62.5, 125, 250, 250, 500, 1,000 µg/mL) of each test extract and placed under dark conditions at ambient temperature (25 °C) for 30 min. After 30 min, absorbance at 517 nm was recorded. Ascorbic acid was applied as standardand methanol as control. Triple execution and observation of the experiments were carried out. The radical scavenging percentage was computed by applying following equation. ${\displaystyle \text{Scavenging}\left(\text{\%}\right)=\frac{A_C-A_S}{A_C}\times 100}$

where A_c and A_s denoted absorbance of the control and test sample, respectively.

Antibacterial activity

Evaluation of antibacterial activity

The antibacterial effect of ethanolic and aqueous extracts of SI seed shell was performed using an agar well diffusion assay (Hassan & Ullah, 2019). Briefly, the agar plate surface was inoculated by three strains of S. aureus at a concentration of 1.5 × 10⁸ CFU/ml, over the entire agar surface. Then, eight mm of the circular hole was made aseptically into the agar plate using a sterile cork borer, and a volume of 100 µL of the test sample stock (40 mg/mL) solution was introduced to the well. The positive control employed was the standard antibiotic Gentamycin (10 µg/mL), while water served the negative control. The inhibition zone was subsequently assessed by incubating all of the prepared agar plates for 24 h at 37 °C.

Determination of minimum inhibitory concentration

The minimum inhibitory concentration (MIC) of test samples was ascertained by broth microdilution assay by preparing stock solution (40 mg/mL), conducted in 96-well polystyrene sterile plates towards S. aureus (ATCC29213), S. aureus (clinical) isolated, and MRSA strains. The preparation of microdilution plate included putting in 100 µL of Mueller Hinton Broth (MHB) to wells 2 through 12. The first well contained 200 µL of test sample only and a serial two-fold dilution was performed from wells 2 to 10 by transferring 100 µL from the preceding well to the next, making the total final volume of 100 µL in each of the wells. Lastly, all wells except wells 1 and 12 received 10 µL of bacterial suspension. A negative control with only MHB was designated for well 12, whereas well 11 were retained as a positive control with MHB plus bacterial suspension but no test sample. After that, plates were incubated for 18 to 24 h at 37 °C. After incubation, 40 µL of a freshly prepared 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent (0.5 mg/mL in sterile water) was added to each well (2 to 12), resulting in a total volume of 150 µL in wells 2 to 11, and 140 µL in well 12. The resultant mixture was further incubated (37 °C, 30 min). Following incubation, bacterial growth inhibition was assessed by observing the reduction of yellow MTT to purple formazan crystals by metabolically active cells. The MIC of extract was defined as the minimal extract concentration that inhibited apparent bacterial growth, including that of the standard strains. Gentamycin was employed as positive control and each assay was conducted three times (Xu et al., 2023).

Time kill kinetics

The time-kill kinetics and dose-dependent kinetics test of SI seed shell extracts towards S. aureus (ATCC29213), MRSA, and S. aureus (clinical) strains have been established using a modified time-kill kinetics approach (Appiah, Boakye & Agyare, 2017). Different concentrations (62.5 (1/4MIC), 125 (1/2MIC), 250 (1MIC), 500 (2MIC), and 1,000 (4MIC)) µg/mL) of shell extracts were used against test bacteria by obeying the same protocol as used for evaluating MIC. Microbial growth was estimated by plating 10-fold serial dilutions on MHA after 0, 2, 4, and 8 h of incubation at 37 °C. Three duplicates of each test were conducted. Log10 CFU/mL was applied to express the results.

Antibiofilm assay

The antibiofilm assay was performed by using varying concentrations (250, 500, and 1,000 µg/mL) of ethanolic SI seed shells extract. TSB broth was utilized to culture S. aureus (ATCC29213), MRSA, and S. aureus (clinical) strains for the entire day at 37 °C. To perform microtitration, 96-well polystyrene plates were filled with 200 µL of bacterial suspensions in triplicate, followed by the addition of various doses of SI seed shell extracts (62.5, 125, 250, 500, and 1,000 µg/mL), except for one well which served as an untreated control (UT). Plates were then incubated for a period of 24 h at 37 °C. Suspensions were taken out after incubation, and wells were given three PBS washes (pH 4.0). The wells were treated with 200 µL of glacial acetic acid (30%) for 15 min at ambient temperature after being stained for 30 min with 200 µL of crystal violet (0.1%) solution and then rinsed with PBS. Afterwards, 100 µL of the dispersed crystal violet has been moved to a flat-bottom microtiter dish and absorbance at 595 nm was recorded on ELISA reader. Biofilm inhibition levels were calculated relative to the biofilm formed in untreated wells (set as 100%) and sterile media controls (set as 0%). The results were averaged across three independent biological replicates (Alrashoudi et al., 2025)

Determination of antibiofilm activity by inverted microscope

The antibiofilm activity was detected using the microscopy technique. The three S. aureus strain cultures received individual treatments with varying doses (250, 500, and 1,000 µg/mL) of ethanolic extract of SI seed using a 12-well microtiter plate. One well of a 12-well microtiter plate was left untreated as a control, while Gentamycin treatment served as the positive control. The biofilms were examined using an inverted microscope in a 12-well microtiter plate at 40 × magnification following a 24 h incubation period at 37 °C (Alangari et al., 2022).

Assessment of the cell membrane integrity

Three strains of S. aureus were assessed for cell membrane integrity based on the amount of cell material that leaked into the medium, including proteins and nucleic acids (Nguyen et al., 2024). The cultures of S. aureus were incubated with varied doses of SI seed shell extracts (62.5, 125, 250, 500, and 1,000 µg/mL) for 24 hat 37 °C. The supernatant was separated from the suspensions by centrifuging them for 5 min at 13,000 rpm. The level of liberated proteins and nucleic acids were analysed by using a nano-drop at absorbance of 260 nm and 280 nm on a spectrophotometer, respectively. An untreated sample was retained as a negative control, and Polymyxin B served as the positive control.

Statistical analysis

GraphPad Prism 8.4.3 was applied to execute statistical analyses. Brown-Forsythe ANOVA was utilized to evaluate the significant differences in dose-dependent biofilm inhibition and membrane disruption by SI seed shell extracts across various S. aureus strains. Regression equations derived by plotting the graph between inhibition percentage (%) and concentration were used to determine the IC50 value. The significance threshold of p < 0.05 was established.

Ethical approval

This study was authorized by King Fahad Medical City’s Institutional Review Board in Saudi Arabia and performed in accordance with the Declaration of Helsinki’s standards (IRB log number: 22-026E).

Chemical composition and antioxidant potential of SI seed shell extract

The extraction of bioactive components from the plant material largely depends upon the solvent type used during the extraction process. Ethanol, methanol and water are the solvents most commonly employed in studies investigating the antibacterial properties of plants (Truong et al., 2019). The high extract yields can be achieved only by selecting most suitable extraction solvent/solvent mixture based on the characteristics of herbal material. Two extraction solvents ethanol (70%) and water were used for the extraction of the SI seed shell and the yield of each extract was determined. The results showed that 70% ethanol produced the highest extraction yield at 17.43 ± 0.063%, significantly higher than the 10.27 ± 0.014% yield obtained with water extraction.

To examine the correlation between the polyphenolic attributes of the extract and their antioxidant and antibacterial efficacies, the total polyphenolic content (TPC), total flavonoids (TF), procyanidins (PC), and anthocyanins (AC) content in the prepared extracts were measured by spectrophotometric technique (Table 1). The results exhibited that the 70% ethanolic extract of the SI seed shell had a significantly higher content of total phenolic content (113.21 ± 1.04) and total flavonoid content (43.17 ± 0.94) in contrast to the aqueous extract of the SI seed shell. Similarly, the estimation of procyanidin content revealed that the ethanolic extract of the SI seed shell contained higher amounts of procyanidins (174.12 ± 1.01) than the aqueous extract (152.36 ± 0.39). However, lesser amounts of anthocyanins were found in both extracts. The extraction of phenols, flavonoids, and procyanidins was found to be more significant with ethanol than with water. This might be because the ethanol has better solubility for these phytoconstituents. These findings corroborate previous research demonstrating that alcoholic solvents like ethanol and methanol are more efficient than water at extracting the chemical components from medicinal plants (Liaudanskas et al., 2021; Stanciauskaite et al., 2021).

The individual constituents in the ethanolic and aqueous extract of SI seed shells were identified and quantified by HPLC. The HPLC chromatograph of chemical components and extracts under investigation were depicted in Figs. 1A and 1B. Standard calibration was the technique that was used for quantification. The following order of components was obtained upon elution under the specified HPLC conditions. The amount of individual components in the SI seed shell extracts, as determined by HPLC was presented in Table 2. In the ethanolic extract of SI seed shells, cinnamic acid derivative (12.14 ± 2.36 mg/g) was the most abundant component, followed by hydroxycinnamic acid derivative (10.26 ± 1.03 mg/g), cinnamic acid (7.21 ± 0.92 mg/g), procyanidin B1(6.34 ± 0.94 mg/g), procyanidin B2 (5.69 ± 0.51mg/g) and protocatechuic acid (4.12 ± 1.32 mg/g). In contrast, aqueous extract of SI seed shells contained a high content of procyanidin B1 (8.78 ± 0.8736 mg/g), along with appreciable amounts of hydroxyl cinnamic acid derivative, cinnamic acid derivative, and (+)-catechin. As estimated by HPLC, the ethanolic extract of SI seed shells had the highest TPC compared to aqueous extract. It is evident that there is a strong association between the results of the spectrophotometric and chromatographic techniques. All the quantified phenols in the extracts have therapeutic applications.

Antioxidant potential

The antioxidant capacity of SI seed shell extracts was measured using the DPPH radical scavenging technique. The results of the antioxidant activity of ethanol and aqueous extract of SI seed shells are shown in Table 3. The ethanolic extract of SI seed shells demonstrated significantly higher DPPH radical scavenging capabilities at all tested concentrations compared to the aqueous extract of SI seed shells (P < 0.05, Table 3). The antioxidant potential of SI seed shell extracts was found to be dose-dependent. However, compared to the DPPH^• radical scavenging activities of SI seed shell extracts, the standard (ascorbic acid) showed noticeably greater actions. This study also computed the IC_{5 0} values, representing the doses of the investigated plant extracts required to scavenge 50% of the DPPH radicals. The IC₅₀ values for ethanol and aqueous extract were 31.05 and 66.24 µg/mL, respectively. Conversely, the standard (ascorbic acid) has an IC₅₀ value of 13.32 µg/mL. The higher antioxidant capacity of SI seed shells may be may be attributed to the substantial presence of phenolic and flavonoid components in the ethanolic extract. The variation in the antioxidant capacity of ethanol and aqueous extract may be due to significant variances in their phenolic contents. Also, the extraction solvent has an impact on the extract’s ability to scavenge free radicals. The findings suggest that ethanol would be the best solvent choice for extracting antioxidant components. Overall, the ethanolic extract from SI seed shells showed the highest polyphenol content and antioxidant capacity. The antioxidant action of phenolic components, which allow them to act as hydrogen donors, reducing agents, and singlet oxygen quenchers, are primarily attributed to their redox characteristics. They might also have the ability to chelate metals (Hossain et al., 2011). The main factor influencing the biological action of polyphenols is the presence of at least one phenolic hydroxyl (OH) group in their structure. Thus, key factors affecting antioxidant capacity include the number and position of hydroxyl groups on the aromatic ring, as well as presence of methoxy substituents in the ortho position relative to the hydroxyl group (OH) (Anouar et al., 2013; Rice-Evans, Miller & Paganga, 1996). Additionally, flavonoids also have an antioxidant effect, but this effect varies from among the molecules and is determined by the degree of hydroxylation and the placement of the -OH groups in the B ring. In particular, a B ring with an ortho-hydroxylated structure exhibits enhanced activity because it acts as a preferential binding site for trace metals (Pietta, 2000) or gives the aroxyl radical more stability through the delocalization of electrons (Van Acker et al., 1996).

Antibacterial activity

The characterized extracts (ethanol and aqueous) of SI seed shells were tested for antibacterial against the three strains of S. aureus including S. aureus (ATCC29213), S. aureus clinical, and MRSA. Agar diffusion and a test to ascertain the MIC were the two methods employed for this purpose. The impact of SI seed shell extracts on the growth of S. aureus (ATCC29213), S. aureus (clinical) and MRSA was evaluated. Fig. 2, presents the results of tested bacterial strains treated with ethanol and aqueous extracts of SI seed shells. The antibiotic Gentamycin served as a positive control was examined on a separate Petri dish. The results of the antibacterial effect revealed that the ethanolic extract of SI seed shells exerted strongsensitivity towards S. aureus (clinical) strain with a high (20 mm) zone of inhibition (Fig. 2A), while the significant influence on S. aureus (ATCC29213) (17 mm) and MRSA (17 mm) with a similar zone of inhibition (Figs. 2A and 2B), at 40 mg/mL concentration (Table 4). However, aqueous extract of SI seed shells demonstrated lower inhibition zones and moderate antibacterial activity against all tested strains of S. aureus at the same dose. The antibiotic Gentamicin, at 10 µg/mL dose, demonstrated antibacterial effect comparable to that of the aqueous extract of SI seed shells. The results showed that the ethanolic SI seed shells extract exhibited greater antibacterial potential than both the aqueous extract and the positive control (Gentamicin), effectively targeting all three S. aureus strains studied. The discrepancies in the antibacterial properties of the ethanol and aqueous extracts of SI seed shells observed in this study may be attributed to variances in their phytochemical composition. The enhanced antibacterial action of the ethanolic SI seed shells extract may be due to the substantial presence of phytoconstituents such as phenols, flavonoids, terpenoids, and tannins. These results are consistent with previous studies that reported the antimicrobial potential of plant extracts rich in bioactive compounds, which target a broad spectrum of bacterial strains by disrupting essential bacterial functions. The phenols are reported to interfere with the cytoplasmic membrane function, disrupting energy metabolism and impacting nucleic acid synthesis (Salehi-Sardoei & Khalili, 2022). It has been demonstrated that flavonoids block the bacterial enzymes RNA polymerase, reverse transcriptase, telomerase, and DNA polymerase (Kouadri, 2018). Also, plant extracts have a greater effect on Gram-positive bacteria (S. aureus) than Gram-negative bacteria. Structural differences in their cell walls may make Gram-positive bacteria more vulnerable.Gram-negative bacterial cells possess an additional outer membrane that provides a hydrophilic surface, serving as a barrier to the permeability of many substances, including biological molecules (Tavares et al., 2020).

Minimum inhibitory concentration

The lowest dose of an antimicrobial drug that can prevent the discernible rise in microbial growth following an overnight incubation is known as the minimum inhibitory concentration (MIC). The MIC was determined to evaluate the antibacterial efficacy of the prepared SI seed shell extract at reduced antimicrobial concentrations. Antimicrobials with low MIC values are typically more potent, while those with high MIC values are usually less effective. The MIC value of ethanol and aqueous extract of SI seed shellswas studied and was found to be 250 µg/mL for all the three investigated strains of S. aureus (Table 4). The MIC for all strains (250 µg/mL) further reinforces the efficacy of SI seed extracts as an antibacterial agent, consistent with findings from other studies on plant extracts exhibiting similar MIC values towards Gram-positive bacteria (Hemeg et al., 2020). Thus, the ethanolic extract of SI seed shell with higher antibacterial activity performance and lowest MBC/MIC ratio was examined for the time-kill behaviour and antibiofilm activity.

Time-kill kinetics

The bactericidal activity of ethanolic extract of SI seed shell was estimated by assessing the time course to kill the tested bacteria. The time-kill kinetics of SI seed shell ethanol extract was conducted at five varied doses (62.5–1,000 µg/mL) towards three bacterial strains of S. aureus (ATCC29213), S. aureus (clinical) and MRSA strains over a period of 8 h. Consequently, time-kill graph was plotted between the logarithmic numbers of CFU/mL versus incubation period. The number of surviving bacteria in the extract was determined at the time of sampling using the plate count technique. The percentage decrease and logarithmic reduction in the microbial population at each time point were calculated to represent changes (either reduction or growth) relative to the initial inoculum. The population of test organisms decreased significantly (p ≤ 0.05) at each interval when tested at doses of 1,000 µg/mL and 62.5 µg/mL in the time-kill kinetics investigation against S. aureus, as indicated in Fig. 3. The time kill kinetics assay of SI seed shell ethanol extract showed time and dose-dependent activity against S. aureus strains. The inhibition of S. aureus (ATCC) strain with varying doses (62.5–1,000 µg/mL) of ethanolic SI seed shell extract at successive time intervals (0 h, 2 h, 4 h, and 8 h) showed reduction in Log10 CFU/mL from 9.37 Log10 CFU/mL to 4.37 log10 CFU/mL, indicated dose and time dependent bactericidal activity (Fig. 3A). However, S. aureus clinical and MRSA strainsas demonstrated in Figs. 3B and 3C, displayed a notable decline (p ≤ 0.05) in the test organism population was equally noticed at each time interval when tested atdifferent concentration. In comparison to untreated (UT) S. aureus strains, the S. aureus (clinical) showed an average log reduction in viable cells between 9.146 Log10 and 4.124 Log10 CFU/mL, while, the MRSA strain displayed a 9.367 Log10 to 4.221 Log10 CFU/mL reduction in viable cells. Thus, time-kill kinetics test verified that both time and dose affected thebactericidal activity of SI seed shell ethanol extract. This demonstrates that the SI seed shell extract can both supress bacterial growth and effectively kill the bacteria, which is crucial for therapeutic applications. These findings supported earlier studies that demonstrated the bactericidal effects of plant-derived antimicrobials in a time and dose-dependent manner, indicating that prolonged exposure to bioactive plant components can effectively lower bacterial populations (Techaoei, 2022). Numerous bioactive substances with antibacterial qualities, such as polyphenols, flavonoids, and fatty acids, are present in the ethanolic extract of SI seed shell. The bioactive components are capable of disrupting bacterial cell membranes, allowing internal contents to leak out and limiting cell viability. The flavonoids and polyphenols in the extracts tend to bind to bacterial proteins, leading to their denaturation or inactivation, thereby impairing bacterial survival and function. Additionally, these components may inhibit transpeptidase and glycosyltransferases, two key enzymes involved in peptidoglycan synthesis, thereby weakening the cell wall and leading to cell lysis (Fig. 4) (Vaou et al., 2021).

Antibiofilm activity

Biofilm formation, a significant factor in the persistence of bacterial infections and antibiotic resistance, was effectively inhibited by SI seed shell ethanol extract. The ethanol extract of SI seed shell exhibited significant antibiofilm activity, with biofilm formation decreasing progressively as the extract concentration increased. The antibiofilm activity of treated S. aureus (ATCC29213) strain exerted a significant decline in the antibiofilm activity as the concentration of the extract increased (p ≤ 0.0001) and showed a decline in OD values from 0.878 to 0.451, in contrast to untreated S. aureus (ATCC29213) strain with 1.228 OD value (Fig. 5A). The ethanol extract of SI seed shell efficiently inhibits the biofilm of the other two strains S. aureus (clinical) and MRSA. The extract displayed similar dose-dependent antibiofilm activity with a decrease in OD values from 0.769 to 0.379 for the biofilm of S. aureus clinical (Fig. 5B), whereas extract-treated MRSA biofilm showed a decrease in OD values from 0.671 to 0.319 (Fig. 5C), as compared to untreated S. aureus clinical (OD: 0.781) and untreated MRSA (OD: 0.766). The dose-dependent decrease in biofilm formation, as evidenced by OD reductions for S. aureus (ATCC29213), S. aureus (clinical), and MRSA strains, supported the hypothesis that the extract interferes with biofilm development. This is a significant finding, as biofilms are notoriously resistant to conventional antibiotics treatments. The influence of varied ethanol extract concentrations of SI seed shell on the three tested strains of S. aureus biofilm formation was further validated by the inverted microscopy images. All three of the untreated S. aureus strains showed extensive biofilm formation in their SEM images (Fig. 6A). However, the SEM monographs of extract-treated S. aureus (ATCC29213), S. aureus (clinical), and MRSA strains showed a progressive decrease in biofilm density as the extract concentration increased (Fig. 6B). The most significant decrease in biofilm formation was noted at the highest dose (1,000 µg/mL). Previous studies have provided strong evidence that medicinal plants have the potential to treat a range of viral diseasescaused by numerous pathogens (Manandhar, Luitel & Dahal, 2019). The antibacterial potential of medicinal plants make them effective in curing a variety of diseases (Karygianni et al., 2016). Numerous phytoconstituents found in medicinal plants have been demonstrated to inhibit of quorum sensing molecules and biofilm formation in addition to playing a significant role in preventing microbial pathogenicity (Uc-Cachon et al., 2021). Plant extracts and isolated components have been shown to stop or interfere with the production of biofilms by blocking quorum sensing pathways or directly harming the biofilm matrix; these mechanisms may also be important in the current work with SI seed shell ethanol extract (Uc-Cachon et al., 2021). The ethanolic SI seed shell extract appears to interfere with several of the critical molecular mechanisms that S. aureus uses to build biofilms. One key target is the Agr system, which controls quorum sensing and biofilm development. The Agr system is activated by auto-inducing peptides (AIPs) that bind to the AgrC receptor. This AIP bindingmay be inhibited by the bioactive substances in SI seed shell ethanol extract, which would stop the subsequent processes that promote the production of biofilms. The inhibition of agrA and agrC could result in the suppression of virulence factor expression (Gray, Hall & Gresham, 2013; Cosgriff et al., 2019). Additionally, the synthesis of polysaccharide intercellular adhesin (PIA) is essential for biofilm formation in S. aureus. The ica operon, comprising the genes icaA, icaD, icaB, and icaC, is responsible for producing PIA and other biofilm matrix components, with icaA and icaD being directly involved in PIA biosynthesis (Tamai et al., 2023). The ethanolic extract of SI seed shell may interfere with the expression of these ica genes, particularly icaA and icaD, leading to a reduction in PIA production. By decreasing the expression of these genes and destabilizing the biofilm matrix, the extract may effectively inhibit biofilm formation (Fig. 4) and thereby enhance the effectiveness of antimicrobial agents (Lu et al., 2019). Moreover, the extract may increase S. aureus sensitivity to oxidative stress, potentially by inducing the production ofreactive oxygen species (ROS) that compromisebiofilm integrity and reduce bacterial viability (Guo et al., 2023).

Membrane disruption and nucleic acid leakage

The results of cell membrane integrity measurements showed a gradual increase of protein release over time in the presence of SI seed shell ethanol extract. As the extract concentration increased, the treated S. aureus (clinical) strain exhibited a significantly higher level of protein leakage from the bacterial cells (from 0.022 to 0.058 µg/mL); in contrast, untreated S. aureus clinical strain showed only 0.015 µg/mL of protein in the medium (Fig. 7B). A similar increase in protein leakage level from the bacterial cells of treated strains of S. aureus (ATCC29213) strain (rising from 0.018 to 0.055 µg/mL) and MRSA (rising from 0.015 to 0.054 µg/mL)in the culture medium was observed with increasing concentration of SI seed shell ethanol extract (Figs. 7A and 7C). Additionally, a comparable concentration-dependent effect was noted in thenucleic acid leakage from the bacterial cells of S. aureus (ATCC29213) strain exposed to the SI seed shell ethanol extract. The concentration of SI seed shell extract was found to increase nucleic acid leakage in the treated S. aureus strains, indicating its effectiveness in disrupting bacterial membranes. The highest level of nucleic acid leakage (1.867 mg/mL) in the treated S. aureus (ATCC29213) strain was observed at 1,000 µg/mL concentration of SI seed shell ethanol extract (Fig. 8A), indicating a significant rise in membrane permeability with rising extract doses (p ≤ 0.0001). In comparison, at similar concentrations, the S. aureus (clinical) strain and the antibiotic-resistant strain MRSA exhibited notable nucleic acid leakage of 1.794 mg/mL and 1.523 mg/mL, respectively (Figs. 8B, and 8C), demonstrating the consistent efficacy of SI seed extract against both sensitive and resistant strains. Thus, all the three strains of S. aureus treated with the ethanol extract of SI seed shellshowed significant leakage ofproteins and nucleic acids, suggesting that the extract effectively disrupts bacterial membranes, a key mechanism behind its antibacterial action. This membrane-disruptive effect of SI seed shell extract aligns with the findings of other studies on plant-based antimicrobials that have shown similar capabilities to permeabilize bacterial membranes, leading to cell lysis and death (Lin et al., 2021). Membrane disruption is a critical antibacterial strategy, as it causes the loss of cellular contents and death, which is essential for combating antibiotic-resistant strains like MRSA. These findings underscore the importance of developing therapies that target bacterial membranes, especially in the context of rising antibiotic resistance.