C. madurensis, renowned for its medicinal secondary metabolites like flavonol glycosides, offers a promising avenue for future research in natural product-based therapeutics(Yaquba et al. 2020). The glycoside form of these metabolites enhances their water solubility, reduces toxicity and side effects, and improves targeting, making them particularly valuable(Khodzhaieva et al. 2021). The synthesis of various flavonol glycosides opens a world of possibilities for exploring their medicinal properties. In this context, four compounds, presumed to be flavonol O-glycosides (1–  4), were isolated from the aerial part of C. madurensis using sequential column chromatography. These compounds demonstrated consistent chromatographic characteristics, such as fluorescence in short/long UV light, Rf values, and their changes with NH3 vapors, AlCl3, and FeCl3, highlighting the potential of these compounds and emphasizing the need for ongoing research in this field. On the PC chromatogram, metabolites 1, 3, and 4 were identified as deep purple spots that transitioned to yellow fluorescence when exposed to NH3 vapors and AlCl3. These compounds turned green when treated with FeCl3, suggesting they are likely flavonol 3-O-glycoside structures. Metabolite 2, on the other hand, was seen as a yellow, fluorescent spot under UV light. It turned yellow when exposed to ammonia vapors and green when sprayed with FeCl3 reagent, indicating it is likely a flavonol glycoside with a free C3-OH group. Furthermore, when treated with the Naturstoff spray reagent, their spots changed to orange (for metabolites 1, 2) and greenish-yellow (for metabolites 3, 4), confirming the aglycones as quercetin or kaempferol, respectively (Markham 1982; Agrawal and Olsson 1989).

Given the chromatographic characteristics, it was inferred that metabolite 1 likely possesses a structure similar to that of 3-O-glycosyl quercetin (Markham 1982). NMR studies provided additional structural evidence. The 1H NMR spectrum (refer to Table 1) displayed a unique ABM-spin coupling system for H-2′, 6′, and 5′ at δ7.88 (br s), 7.82 (d, J = 7.6 Hz) and 6.85 (d, J = 7.6 Hz). This is indicative of a 3’,4’-dihydroxy ring B of a quercetin-like aglycone. The absence of H-8 and a singlet signal at δ 6.25, attributable to H-6, suggest that the aglycone is a 3, 5, 7, 8, 3′, 4′ hexahydroxy flavone (gossypetin) (Yaque et al. 2016).

The stereo structure of glycoside moiety was established as β -D-xylopyranoside because of the characteristic δ-values and splitting pattern of its anomeric proton signal at 5.71 (d, J = 6.8 Hz, H-1′′). Also, the presence of a characteristic signal assigned at δH 3.74 which is assignable for the methoxy group. Accordingly, the structure of metabolite 1 was established as quercetin 3-O-4C1- β -D-xylopyranosyl methoxyl ether. Final structure confirmation was achieved from the 13C NMR spectrum (Table 1) which showed 15 C-resonances of quercetin moiety were interpreted including the key signals at ppm 177.9, 148.8, 145.6, and 132.9 of C-4, C-4′, C-3′, and C-3, respectively. In addition, the xylopyranoside moiety was further established by the key 13C-resonances at δ 102.3, 73.7, 76.6, 69.7, and 66.0 for the anomeric carbon, C-2", 3", 4" and 5", respectively(Mizuno et al. 2022). The location of C-3 at 132.9 was a confirmative document for the glycosidation at OH-3. In addition to the downfield shift of C-8 to 126.1 (∆ +  ~ 32 ppm) and upfield shift of C-5, 7, and 9 to 156.0, 156.0, and 149.3, respectively (∆ +  ~ 8 ppm) indicate methoxylation at C-8 which causes upfield effects on 1H and 13C chemical shifts of ortho and para-position of the flavonol(Lee et al. 2008). While the hydroxylation at the A-ring has no effect on the 1H and 13C chemical shift changes at the B-ring, and vice versa (Park et al. 2007). Thus metabolite 1 was established as Gossypetin 8-methoxy, 3-O-β-D-4C1-xylopyranoside.

Metabolite 2 was expected to be a quercetin-type O-glycoside structure. Further confirmation for the proposed structure was obtained from the 1HNMR spectrum (Table 1), which showed an ABM-spin coupling system at ppm 7.89 (br s), 7.83 (d, J = 8.4 Hz), 6.84 (d, J = 8.4 Hz), interpretable for H-6', H-2' and H-5' of 3,4-dihydroxy B-ring. The absence of H-8 and a singlet signal at δ 6.25 assignable to H-6 suggest that the aglycone is 3, 5, 7, 8, 3', 4' hexahydroxy flavone (gossypetin) (Yaque et al. 2016). The stereo structure of glycoside moiety was established as 4C1- β -D-glucopyranoside because of the characteristic δ-values and splitting pattern of its anomeric proton signal at 5.75 (d, J = 7.6 Hz, H-1′′). Accordingly, the structure of metabolite 2 was established as quercetin-8-O-4C1- β -D-glucopyranosyl. 13C NMR spectrum provided the final structural confirmation, as it displayed gossypetin’s characteristic C- resonances, particularly the key ones at δ 177.8, 148.7, 145.5, and 132.9, corresponding to-4, C-4′, C-3′, and C-3, respectively. with upfield chemical shift of C-5 (156.8), 7 (156.8), 9 (149.33, 149.2) as the sugar substitution at C-8 causes upfield effects on 1H and 13C chemical shifts of ortho and para-position of flavonol (Lee et al. 2008) and downfield shift of C-8 (126.2). As well, 6 characteristic resonances agreed with an β-4C1-D-glucopyranosyl structure of the glucoside moiety at 104.8 (C-1"), 77.3 (C-5"), 76.9 (C-3"), 73.7 (C-2"), 70.4 (C-4") and 61.2 (C-6") (Mizuno et al. 2022). Thus metabolite 2 was established as Gossypetin 8-O β-D -glucopyranoside. Metabolite 3 exhibited chromatographic characteristics of flavonol structure(Mabry et al. 1970). On complete acid hydrolysis, it gave kaempferol (organic phase), and hexose (aqueous phase) (Co PC with authentic samples and detection by specific spray reagents). In 1H NMR Table 1 an A2 X2- spin coupling system of two ortho doublets, each of two protons, was assigned at 7.87 and 6.88 with J-value of 8.4 Hz for H-2' /6' and 8.3 Hz for H-3' /5' of 1,4-disubstituted β- ring. Also, two meta doublets were established for H-8 and H-6 at 6.20 and 6.44 respectively, with J-value of 2.2 Hz, of 5, 7-dihydroxy A-ring. In the aliphatic region, showed a ß-anomeric proton signal of hexose moiety at 5.18 (J = 7.7 Hz) (Wang et al. 2015). 13C NMR, Table 1 showed 15 carbon resonances characteristic of kaempferol aglycone among which five key signals at 177.71 (C-4), of C- ring, 160.118 (C-4 ′), 130.72 (C2' /6′), 115.70 (C-3' /5′) of the B-ring and 132.88 (C-3). Also, the presence of six carbon resonances of glucose moiety. Finally, all C-signals of the aglycone and sugar moieties were confirmed by the comparison with those of the structurally related compounds published before (Kazuma et al. 2000). The structure was confirmed by a negative ESI/ MS spectrum that showed m/z 447.0 [M-H]−. Therefore, metabolite 3 was identified as Kaempferol 3-O-ß–D-glucoside (Astragalin). This is the first time to be isolated from this plant. Metabolite 4 displayed chromatographic properties indicative of a kaempferol structure. The 1H NMR data for metabolite 4 revealed three aromatic proton signals at δH 7.87 (H-2′,6′), 6.80 (H-3′,5′), and 6.35 (H-6). The anomeric proton signal of glucose was observed at δH 5.18 (J = 8.6 Hz), suggesting it is in the β-form due to the coupling constants of J = 8.6 Hz. In the 1H NMR data of metabolite 3, two distinct patterns of proton resonances were observed. The first pattern was characteristic of kaempferol, with a broad signal at δH 12.77 ppm for the exchangeable proton of 5-OH with the 4-keto group. According to Table 1, only one carbon resonance was found between δC 90 and 105 at 98.17 ppm. Additionally, carbon resonances at δC 126.15 could only be explained by hydroxylation at position 8. The most up-field peak was present at δC 56.10, and the downfield shift of C-7 to δC 165.78 was assigned to a methoxyl carbon at position 7. This data confirms the structure of metabolite 4 as a herbacetin 7-methyl ether (Hussein et al. 1997). In addition, the presence of a signal at δC 102.27 indicates an anomeric proton of hexose. Furthermore, the signal at δC 61.06 was assigned to the unsubstituted C-6`` of glucose unit. The 13C NMR investigations and literature data were used to characterize the glucose unit, which revealed that it is a glucopyranoside (Mizuno et al. 2022). An explanation that is consistent with the chemical shift values previously mentioned indicates metabolite 4 to be herbacetin 7-methyl ether-3-O-β-D-glucopyranoside.

Flavonol glycosides have both antioxidant and antimicrobial traits. They control reactive oxygen species (ROS) accumulation by scavenging ROS, contributing to their antioxidant activity, which is crucial for plants under various environmental stressors (Dias et al. 2021; Abu El Wafa et al. 2023). So, the antioxidant effect of the isolated metabolites was assessed using various assays, ABTS revealed that metabolites 4, 2, and 3 exhibit significant antioxidant potential, with IC50 values of 12.23 ± 0.08900, 13.90 ± 0.1400, and 7.92 ± 0.1451 µg/ml, respectively. Metabolite 1, with the lowest IC50 value of 6.993 ± 0.05999 µg/mL, outperforms the others with the highest scavenging activity and FRAP ability. While all metabolites show some degree of power reduction, metabolite 1 also has the highest antioxidant capacity. Metabolites 2 and 4 display moderate activity, while metabolite 3 has the least. Metabolite 1, which has emerged as the most potent antioxidant, is a promising candidate for studying anti-MRSA activity. This is in comparison to the total extract of the C. madurensis aerial part. Previous research has shown that alcoholic extracts from the stems and flowers of C. madurensis exhibit stronger antimicrobial effects against S. aureus, P. aeruginosa, and E. coli than leaf extracts and total saponins. The variation in antibacterial activity among the extracts could be attributed to differences in their composition, the structure of their active ingredients, and the way these ingredients interact with the bacterial cell wall (Ibrahim et al. 2017). The influence of flavonoids on cell membranes implies that they can obstruct fatty acid synthesis through enzymatic pathways, leading to the inhibition of cell membrane synthesis. Additionally, flavonoids can hinder the creation of various virulence factors that affect motility, bacterial attachment to the host, and biofilm production (Gallegos et al. 2016) illustrated in (Figure S15, supplementary data).

The relationship between the antioxidant and antimicrobial activities of flavonol glycosides, such as metabolite 1, is complex and can vary depending on the specific compound and the organism it interacts with. Both these properties contribute to their overall health benefits (Tagousop et al. 2018). However, a comprehensive understanding of the interplay between these activities requires further research. This could potentially lead to the development of more effective treatments for MRSA infections. Methicillin-resistant S. aureus (MRSA) is an infection that poses a treatment challenge due to its resistance to antibiotics. While Staphylococcus bacteria are typically harmless, they can lead to severe diseases that can be fatal (Howden et al. 2023). S. aureus is a bacterial species that resides in humans and can cause a range of clinical symptoms (Dayan et al. 2016). If S. aureus enters the body through a skin cut, it can lead to infections ranging from mild to severe, which can be fatal in some instances. A high incidence of skin infection has been recognized as a risk factor for MRSA nasal carriage. Moreover, patients with pathological skin conditions are more prone to disseminate infectious strains (Gordon and Lowy 2008). Antibiotic susceptibility tests are key to finding effective treatments for bacterial infections like MRSA. Most antibiotics tested showed potential effectiveness against MRSA (Qodrati et al. 2022). However, the choice of treatment depends on various factors including the patient’s health, antibiotic side effects, and infection severity. Antibiotic resistance is a major concern, so even if an antibiotic inhibits MRSA in a lab, it must be used wisely in clinical settings to prevent resistance (Dadgostar 2019). The antibiotics, belonging to different groups and operating via different mechanisms, were either susceptible or resistant to MRSA. The inhibition zones’ diameters were ranked in descending order as follows: Levofloxacin LEV-5, Rifaximin RF-30, Streptomycin S-10, and Cefuroxime CXM-30. However, MRSA showed resistance to Ampicillin/Sulbactam SAM-20. Antimicrobial agents are commonly used to treat microbial infections caused by medical conditions, environmental factors, or food poisoning, with MRSA infection being a key example (Enright et al. 2002). Recently, plant flavonoids have attracted significant interest due to their potential to fight pathogenic microbes (Górniak et al. 2019). This encourages us to examine the antibacterial effects of the C. madurensis plant extract and metabolite 1 using the well agar diffusion method. The plant extract and metabolite 1 were tested against MRSA at concentrations of 1.0, 0.5, and 0.25 mg/ml. As shown in Fig. 6, The plant extract and metabolite 1 were both tested for their antibacterial activity against MRSA using a viable cell count test. The plant extract showed substantial antibacterial activity at concentrations of 1.0 and 0.5 mg/ml. However, metabolite 1 demonstrated the most potent antibacterial effect against MRSA at all tested concentrations. Bacterial inhibition was observed within a range of dilutions, with complete inhibition occurring at 10–5 dilutions for 1.0 mg/ml of metabolite 1. As the treatment concentrations increased, the CFU/ml values decreased, indicating a reduction in bacterial viability (Thieme et al. 2021). Metabolite 1 resulted in the lowest CFU/ml at a concentration of 1.0 mg/ml, suggesting it was more effective at inhibiting MRSA growth compared to the plant extract.

Gamma radiation showed a higher total flavonoid content than non-irradiated plants. However, the effects of gamma radiation can vary greatly depending on factors like the plant type, radiation dose, and specific flavonoid glycoside (Alivandi Farkhad and Hosseini 2020). In certain instances, components such as catechin and kaempferol flavonoid glycosides have been observed to notably decrease with gamma radiation (Moghaddam et al. 2011; Breitfellner et al. 2002). The study assessed the effects of gamma irradiation on the antibacterial activity of C. madurensis plant extract and metabolite 1 against MRSA. Metabolite 1 was irradiated at 50 and 100 Gy. The results showed that gamma irradiation did not enhance the inhibitory effect of the substances on MRSA compared to un-irradiated samples. However, the most significant effect was observed at 100 Gy and a concentration of 1.0 mg/ml for both tested samples, with larger inhibition zone diameters indicating stronger antibacterial activity.

FTIR spectroscopy, a crucial tool in antimicrobial research, aids in understanding the molecular interactions between antimicrobial agents and microorganisms and is instrumental in the rapid identification of antibiotic-resistant bacteria (Faghihzadeh et al. 2016; Wang-Wang et al. 2022). Bacterial cells are composed of proteins, fatty acids, carbohydrates, nucleic acids, and lipopolysaccharides and these biochemical molecules can be assigned by FT-IR spectra (Yang et al. 2023). The commonly used region for infrared absorption spectroscopy is 4000 ~ 400 cm−1 because the absorption radiation of most organic compounds and inorganic ions is within this region (Yang et al. 2023). This was exemplified in a study that used FTIR spectroscopy to analyze the spectral changes in MRSA filtrates, both untreated and treated with a plant extract and metabolite 1. The research identified significant disparities between the control and treated spectra. The treatment notably diminished transmittance (%), suggesting a reduction in essential bacterial cell components. This implies that the treatment may have modified the molecular structure of the MRSA cells, potentially impacting their viability. The analysis unveiled considerable differences across the entire spectral range, especially in the membrane amphiphile region (3000–2800 cm−1), the phospholipids DNA-RNA regions (1200 to 1500 cm−1), the proteins and amides I and II regions (1500 to 1700 cm−1), the polysaccharides vibrations regions (900 to 1200 cm−1), and the fingerprint region (600 to 900 cm−1) (Preisner et al. 2010). A vibration band at 1628 cm−1, representative of amide I of the protein’s alpha-helical structure, was significantly diminished while amide II at 1550 cm−1, and amide III at 1260 cm−1. The robust and broadband at 3300 cm−1 could be ascribed to the N–H stretching vibration of nucleic acid constituents such as adenine, cytosine, and/or guanine, and possibly to OH groups potentially bound to nucleic acid (Filip et al. 2009). In addition, in each acyl chain, minimize the CH stretching band intensity value at 2354.92 cm−1 in the spectrum of the treated bacteria indicating a lowering in the methyl group numbers compared to untreated bacteria. Furthermore, in the spectrum of treated bacteria, a decrease in the intensity of the stretching bands indicates a reduction in the number of functional groups compared to untreated bacteria that in turn reflect the role of plant extract and metabolite 1 in reducing the bacterial growth (Kamnev et al. 2002).

Through the process of docking, we can evaluate the antioxidant and antimicrobial properties of molecules by comparing their binding energies with those of known activators or inhibitors. This technique also aids in the development of new molecules with enhanced antioxidant and antimicrobial properties by modifying their chemical structures or adding functional groups. Docking is instrumental in predicting the binding affinity and mode of action of potential drug candidates, and in identifying new targets for drug design. It is particularly useful in studying the antioxidant and antimicrobial activities of molecules, which are key attributes for a multitude of biological processes and diseases (Al-Khaldi et al. 2021). The objective of this study was to elucidate the potential antioxidant mechanisms of isolated metabolites (1–4). Docking simulations were employed to assess the affinity between four metabolites and the binding sites of arachidonate-5-lipoxygenase (PDB: 6n2w) (Ley-Martínez et al. 2022). Due to its inhibitory activity on 5-lipoxygenase (Kahnt et al. 2022), NDGA was chosen as a reference co-ligand for the docking study. The docking scores indicated that all four metabolites had lower binding energy values compared to the co-crystal ligand. Lipoxygenases (LOX), enzymes that catalyze lipid oxidation, are recognized for their role in generating oxidized lipids within atherosclerotic plaques. The enzyme arachidonate 5-lipoxygenase (5-LOX) plays a pivotal role in the synthesis of leukotrienes, and its inhibition provides cellular protection against oxidative stress (Ley-Martínez et al. 2022). The active site of 5-LOX comprises amino acid residues such as Gln557, His372, Gln363, Leu673, Ala410, His432, His-367, His600, and His550 (Li et al. 2011). Four chromen-4-one compounds exhibited a higher docking score of over -12 kcal/mol, compared to the co-crystallized ligand (NDGA) with a score of -11.01 kcal/mol, suggesting a robust affinity for the 5-LOX enzyme. The NDGA molecule establishes four hydrogen bonds with the amino acid residues Ile 406, His 372, His 600, and Arg 596, and forms four hydrophobic contacts with Isoleucine 673, Phenylalanine 359, Alanine 603, and Alanine 410 (Figure S16, Table S1 supplementary data).

The four metabolites (1–4) demonstrated a similar binding mode within the 5-LOX active site to NDGA (Figures S3-S6). Their calculated binding energies were -14.00, -13.54, -13.34, and -12.32 kcal/mol, respectively. The chromen 4-one moiety of the compounds play a significant role in the binding within the pocket, generating hydrogen bonds, hydrophobic interactions, or both. Gossypetin 8-methoxy,3-O-β-D-4C1-xylopyranoside (metabolite 1), the compound with the highest docking score, exhibited twelve hydrogen bonds and formed two Pi-Pi T-shaped bonds with Phe359 and Trp599, and a Pi-Alkyl bond with Ala603 (Figure S17). Gossypetin-8-O-β-D-glucopyranoside (metabolite 2) engaged in hydrogen bonding interactions with several amino acid residues and exhibited four Pi-Pi T-shaped contacts and a Pi-Alkyl bond with Ala603 (Figure S18). Metabolite 3; Kaempferol 3-O-ß–D-glucoside’s chromen-4-one engaged in four hydrophobic contacts and formed five hydrogen bonds with His367, Gln363, and His600. A Pi-Pi T-shaped interaction was observed between the phenolic ring and Try599 (Figure S19). Herbacetin 7-methyl ether-3-O-β-D-glucopyranoside (metabolite 4) had eight hydrogen connections and displayed a Pi-Pi T-shaped interaction with Try599 and two Pi-Alkyl interactions (Figure S6). From the aforementioned results, metabolite 1 exhibited the highest score, leading to its selection for further analysis. The antimicrobial effectiveness of this metabolite implies that its mode of action might involve interactions with key enzymes that play a role in eliminating bacteria. These enzymes include dihydrofolate reductase (PDB ID: 3sr5) (Li et al. 2011), DNA gyrase (PDB ID: 3g75) (Ronkin et al. 2010), penicillin-binding protein (PBP2a) (PDB ID: 1mwt) (Lim and Strynadka 2002), and threonyl-tRNA Synthetase (PDB ID: 1nyq) (Torres-Larios et al. 2003). The co-crystallized ligands were re-docked into their respective enzymes, resulting in a root-mean-square deviation (RMSD) of 0.62 Å (Lipoxygenase enzyme), 0.12 Å (dihydrofolate reductase), 0.08 Å (DNA gyrase), 0.70 Å (PBP2a), and 0.65 Å (threonyl-tRNA synthetase) between the docked and co-crystallized ligand. This outcome validates the docking methodology employed. The study concluded that hydrogen bonds, hydrophobic interactions, and Pi–Pi interactions were the most significant interactions. Dihydrofolate reductase (DHFR) is an enzyme that plays a pivotal role in converting dihydrofolate into tetrahydrofolate. This conversion process is essential for the synthesis of purines, thymidylate, and certain amino acids. DHFR is a significant target in a variety of therapeutic areas, including the treatment of cancer and the development of anti-infective drugs.

The co-crystal ligand Q12 establishes three hydrogen bonds with vital residues and demonstrates π-π stacking with the Phe93 residue. Additionally, it forms seven Pi-Alkyl bonds (refer to Figure S7 and Table S2). Gossypetin 8-methoxy 3-O-β-D-xylopyranoside (also known as metabolite 1), with a docking score of -19.56 kcal/mol, exhibits a stronger affinity compared to Q21, which has a score of -12.28 kcal/mol. Metabolite 1 forms ten hydrogen bonds, displays two instances of π–π stacking, and has five Pi-Alkyl interactions (refer to Figure S8 and Table S2). In the ATP-binding pocket of DNA gyrase B, the compound gossypetin 8-methoxy, 3-O-β-D-xylopyranoside successfully attached to the ATP binding sites of the Staphylococcus aureus gyrase B enzyme. It utilized the same binding strategy as the cocrystal ligand (B48) and achieved a favorable docking score of -15.39 kcal/mol, surpassing B48’s score of -10.51 kcal/mol.

The binding mechanism of B48 involved a single hydrogen bond with the crucial amino acid Asp81 and three pi-interactions with Ile86 (refer to Figure S9). Conversely, gossypetin 8-methoxy, 3-O-β-D-xylopyranoside formed hydrogen bonds with Thr173, Asn54, and Asp81 via its sugar component. Its chromen-5-one ring established hydrogen bonds with Ser129 and Asp57 and participated in three Pi-Alkyl interactions with Ile86 and Ile102 (refer to Figure S10 and supplementary data).

A docking analysis was conducted to comprehend the interaction between gossypetin 8-methoxy, 3-O-β-D-xylopyranoside and the binding site of the Penicillin-Binding Protein 2a (PBP2a) enzyme in Staphylococcus aureus. The co-crystal ligand (PNM) demonstrated significant interactions with specific amino acids within the active site groove, achieving a docking score of -14.18 kcal/mol. It established hydrogen bonds with residues Ser462, Ser598, Ser403, Asn 464, Arg445, and Thr600, and interacted with Met 641 through Pi-sulfur and alkyl bonds, and with Lys597, Lys406, and Lys597 through attractive charge (refer to Figure S11 and Table S2). Gossypetin 8-methoxy, 3-O-β-D-xylopyranoside, on the other hand, achieved a higher docking score of -20.67 kcal/mol. Its chromen-4one moiety formed a Pi-donor hydrogen bond with Thr600, conventional hydrogen bonds with Ser403, Ser462, Ser598, and Met641, carbon-hydrogen bonds with Asn464 and Ser462, a Pi-Alkyl bond with Ala642, and one sulfur bond and two Pi-sulfur bonds with Met641. The sugar ring established two hydrogen connections with Gln613 and Glu602, and the phenolic ring formed three hydrogen bonds with Thr444 and Asn464 (refer to Figure S12 and Table S2). In the active site of Threonyl-tRNA Synthetase (ThrRS), aminoacyl-tRNA synthetases, which facilitate the attachment of amino acids to their corresponding transfer RNAs, are potential targets for the development of antibiotics due to their integral role in protein synthesis and the translation of the genetic code. The co-crystal ligand (TSB) and gossypetin 8-methoxy, 3-O-β-D-xylopyranoside interact with the amino acid residues in the threonyl-binding region. They have docking scores of -18.46 and -19.15 kcal/mol, respectively. TSB forms hydrogen bonds with several amino acids, a Metal-Acceptor link with a zinc atom, and Pi-interactions with Asp385, Leu383, and Met334 residues (refer to Figure S13 and Table S2). Gossypetin 8-methoxy, 3-O-β-D-xylopyranoside demonstrates a similar binding mode, interacting with the adenine- and threonyl-binding regions. It forms hydrogen bonds with Arg518, Ser522, Lys471, and Gly519, a Metal-Acceptor bond with two zinc atoms, and engages in Pi-interactions with Leu383 and Met334 (refer to Figure S14 and Table S2).

A common correlation is often observed between antioxidants and antibacterial properties in various natural extracts. The structure of these antioxidants plays a crucial role in establishing this correlation. Gossypetin 8-methoxy, 3-O-β-D-xylopyranoside (metabolite 1), a flavonol glycoside of C. madurensis, exhibits significant antioxidant properties. Therefore, the antioxidant properties of metabolite 1 could potentially contribute to its effectiveness against methicillin-resistant Staphylococcus aureus (MRSA) ATCC 6538. Both the plant extract and metabolite 1 demonstrate potential antibacterial properties against MRSA, with metabolite 1 showing superior antibacterial activity at all tested concentrations. These findings underscore the potential of plant-derived metabolites in combating antibiotic-resistant bacteria like MRSA. Gamma radiation can significantly affect plant extracts and metabolites, potentially enhancing their medicinal properties. However, the exact impacts can vary greatly depending on numerous factors. While gamma irradiation did not enhance the overall antibacterial activity of the plant extract and metabolite 1 against MRSA, it appeared to have some effect at specific doses and concentrations.

The FTIR study offers valuable insights into the potential antibacterial mechanism of the plant extract and metabolite 1 against MRSA. However, additional studies are needed to fully understand these mechanisms and their implications for the development of new antibacterial agents. Docking analysis was conducted to understand the interaction between metabolite 1 and the binding site of the Penicillin-Binding Protein 2a enzyme in Staphylococcus aureus. This study suggests that the antimicrobial effectiveness of a metabolite could be due to its interactions with key enzymes involved in bacterial eradication. These enzymes include dihydrofolate reductase, DNA gyrase, penicillin-binding protein, and threonyl-tRNA Synthetase. The docking methodology used in the study was validated by the close match between the docked and co-crystallized ligands. Gossypetin 8-methoxy 3-O-β-D-xylopyranoside achieved a higher docking score and formed several bonds and interactions. In conclusion, additional research is necessary to validate these results, fully comprehend these effects, and explore their potential clinical applications.