Antioxidant and inhibitory activities against COX-2 and 5-LOX of different flower extracts

The pharmaceutical industry is transitioning towards nature-derived antioxidants because of the adverse effects associated with synthetic drugs [18]. The capability of various natural constituents to reduce inflammation is supposed to be from the following: firstly, acting as antioxidants; then, interfering with the signaling of free radical species; finally, decreasing the pro-inflammatory signaling transductions [19]. Therefore, the antioxidant (ABTS, FRAP, and DPPH methods), as well as inhibitory activities against COX-2 and 5-LOX of aqueous, methanol, 50% aqueous methanol, methylene chloride, and 50% methylene chloride/methanol extracts of the flowers were assessed using in vitro assays (Fig. 1), in an attempt to understand the ability of these extracts to develop effective intervention for inflammatory disease prevention strategies. Herein, the methanolic extract exhibited the highest antioxidant potential equivalent to 181.5 ± 0.8 µM TE/g (DPPH), 261.1 ± 1.1 µM TE/g (ABTS), and 270.3 ± 2.5 µM TE/g (FRAP), in comparison to ascorbic acid (standard drug). Relative to the other extracts, the methanol extract showed the highest inhibition against COX-2 (IC50 10.6 ± 0.4 µg/mL) compared to Celecoxib (IC50 1.70 ± 0.0 µg/mL), and the highest 5-LOX inhibitory potential (IC50 14.4 ± 1.0 µg/mL) compared to Zileuton as a standard (IC50 5.65 ± 0.4 µg/mL).

Fig. 1

(A) Antioxidant (DPPH ABTS and FRAP methods) and (B) inhibitory activities of T. tipu flowers aqueous, methanolic, water/methanol (1:1), methanol/methylene chloride (1:1) and methylene chloride extracts against COX-2 and 5-LOX

Aq: aqueous extract; H2O/ME: water/methanol (1:1); MC: methylene chloride extract; ME: methanolic extract; ME/MC: methanol/methylene chloride (1:1) extract. Data are represented as mean ± standard deviation of three replicates. Different letters on the bar imply significant differences at P < 0.0001 with Tukey’s test.

Considering the fact that the methanol extract exhibited the highest radical scavenging potential and enzyme inhibitory activities, it is important to further ascertain its anti-inflammatory behavior. Therefore, its inhibitory effects against iNOS enzyme, NO production, and pro-inflammatory cytokines secretion (TNF-α, IL-1β, IL-6, NF-KB, and TNF-R2) in LPS-activated RAW 264.7 macrophages were evaluated.

Cell viability on RAW264.7 macrophages

In MTT assay, T. tipu flowers methanolic extract (ME) did not show cytotoxic activity (up to 500 µg/mL) (Fig. S1) when assayed on RAW264.7 macrophages (after 24 h incubation), indicating ideal safety profile of the methanolic extract so it can be used in alleviating painful disease symptoms and improving human health.

Inhibitory activity of methanolic extract (ME) of T. tipu flowers against iNOS enzyme activity, NO production, and secretion of pro-inflammatory cytokines in LPS stimulated RAW 264.7 macrophages

The excess production of inflammatory mediators in many ailments, like asthma, arthritis, vascular disease, obesity, and dermatitis has become one of the first global morbidity causes [20]. Inflammatory mediators, such as nitric oxide (NO) and pro-inflammatory cytokines [tumor necrosis factor-α (TNF-α), interleukin (IL)- IL-6, and IL-1β] are produced by lipopolysaccharide (LPS, a gram-negative bacteria) through activation of surface receptors, such as tumor necrosis factor receptors (TNF-R2), several protein kinases (MAPKs; extracellular signal-regulated kinase [ERK] and c-Jun N-terminal kinase [JNK]), and transcriptional factors as nuclear factor-κB (NF-κB) in macrophages [21]. In addition, LPS-stimulated macrophages exhibit up-regulation of iNOS expression through the generation of inflammatory cytokines. Therefore, inhibition of iNOS, inflammatory mediators and proinflammatory cytokines in LPS-stimulated macrophages can provide an effective approach for prevention of inflammatory disorders [22].

The ME of T. tipu flowers inhibited iNOS in LPS-induced RAW264.7 macrophages, with IC50 value of 11.1 ± 1.0 µM relative to parthenolide as a standard drug (IC50 2.2 ± 0.0 µM). The results also indicated that the ME of the flowers is an effective inhibitor of LPS-induced NO production in RAW 264.7 cells and decreased the release of TNF-α, IL-β, IL-6, NF-KB, and TNF-R2, compared to standard drugs (Table 1; Fig. 2). These findings imply that ME of the flowers might be utilized as a natural anti-inflammatory resource. This prompted the use of LC-MS metabolic profiling of the ME to enable the preliminary identification of key components that may contribute synergistically to the anti-inflammatory and antioxidant effects, followed by their quantification and verification using computational analyses that could explain the structure-activity relationships.

Table 1 Inhibitory activity of T. tipu flowers methanolic extract (ME) against NO, NF-KB, and TNF-R2 production in LPS stimulated RAW 264.7 macrophagesFig. 2

The inhibitory effect of methanolic extract (ME) of T. tipu flowers on pro-inflammatory cytokines (TNF-α, IL-β, and IL-6) production in LPS stimulated RAW 264.7 macrophages. ME: methanolic extract. * Significant from negative control at P < 0.0001. # Significant from positive control at P < 0.0001 with Tukey’s test

Metabolite profiling

The deficit of reports concerning the chemical profiles of the flowers under investigation motivated the performance of this study to investigate the chemical composition of T. tipu flowers through a non-targeted metabolite profiling of the prepared ME for detecting and identifying large numbers of metabolites using ultra performance liquid chromatography (UPLC) coupled with mass spectrometry (MS). Sixty-two compounds have been tentatively identified in the methanolic extract of T. tipu flowers after careful inspection of MS/MS data, comparison with on line database and reported literature [11, 23]. The compounds belonged to various classes encompassing: 2 sugars, 6 amino acids, 6 organic acids, 14 phenolic acids, 2 coumarins, 12 flavonoids, 8 fatty acids, and 12 phospholipids. Details of the tentatively identified compounds, including retention times, m/z of the detected molecular ions, fragment ions, and molecular formulas, as well as putative identifications are tabulated in Table 2. The base peak chromatogram of the analyzed extract in the negative ionization mode is shown in Fig S2. (supplementary materials). The representative MS/MS spectra of selected compounds from each class are displayed in Figs. S3-11. Interestingly, the main identified compounds were phenolic acids and flavonoids (a total of 26), agreeing with the previous reports for T. tipu leaves [11, 24].

Identification of phenolic acids

Phenolic compounds contribute significantly to antioxidant activity [25]. Fourteen phenolic acids belonged to various classes have been identified from their exact masses and fragmentation patterns [23]. They were mainly benzoic and hydroxycinnamic acid derivatives and were eluted after organic acids in the chromatographic separation. In the MS/MS spectra of phenolic acids, the loss of CO2 group from the carboxylic acid moiety (-44 amu), loss of CO group (-28 amu), as well as the loss of water molecule (-18 amu) led to the formation of characteristic fragment ions [26]. Five free benzoic acids have been identified i.e., benzoic acid, hydroxy benzoic acid, methoxy benzoic acid, protocatechuic acid, and vanillic acid. Two benzoic acid glycosides, protocatechuic acid glucoside and vanillic acid glucoside, were also detected with molecular ions at m/z 315.0721 and 329.0876, respectively, and MS2 spectra due to loss of glucose moiety (-162 amu) and CO2 group. Seven hydroxycinnamic acids were identified caffeic acid glucoside, p-coumaric acid glucoside, chlorogenic acid, cinnamic acid, ferulic acid, coumaric acid pentoside, and dihydrocaffeic acid glucoside. Cinnamic and ferulic acids produced molecular ions (M-H)– at m/z 147.0655 and 193.0494, respectively, and MS2 spectra due to removal of CO2 group from the carboxylic acid function (at m/z 103 and 149, respectively). Chlorogenic acid showed a molecular ion peak at m/z 353.0873 (C16H17O9–) and a base peak at m/z 191 corresponding to the deprotonated quinic acid. Two glycosides of coumaric acid have been identified with molecular ions at m/z 295.0456 (C13H11O8–) and 325.0914 (C7H13O7–). Both showed main fragment ions at m/z 163 and 119 of coumaric acid and its decarboxylated form. Thus, they were identified as coumaric acid pentoside (Fig. S3) and coumaric acid glucoside (Fig. S4), respectively. Caffeic acid glucoside and dihydrocaffeic acid glucoside have been identified from their molecular ions at m/z 341.0879 and 343.1021, respectively. In their MS/MS spectra, they produced fragment ions due to loss of the sugar moiety to give the free acids at m/z 179 and 181, respectively, besides other characteristic ions due to sequential losses of CO, CO2, and H2O groups. The methanol extract demonstrated high phenolic acids content suggesting its antioxidant potential. Additionally, phenolic acids were found to have anti-inflammatory and protective effects against many oxidative stress related diseases viz. cancers, diabetic and cardiovascular disorders [27, 28]. Because of their phenol moiety and resonance-stabilized structure, phenolic acids have antioxidant properties due to electron and H-atom donations and radical quenching mechanisms [27, 29, 30].

Identification of coumarins

Esculetin and its glycoside esculin have been detected in the T. tipu extract for the first time. They displayed molecular ion peaks at m/z 177.0128 and 339.0724, respectively. Esculetin (Fig. S5) showed typical fragmentation pattern of coumarins including successive losses of CO2 (-44 amu) and CO (-28 amu) groups from the molecular ion to produce three main fragment ions at m/z 133 [(M-H)-44]–, 105 [(M-H)-44-28]–, and 89 [(M-H)-44*2]– [31]. While esculin (Fig. S6) gave its base peak at m/z 177 due to the breakdown of the glycosidic linkage and removal of the glucose moiety (-162 amu). Several authors have pointed to the varied range of pharmacological effects of esculetin and esculin, including antioxidant, anti-inflammatory, anticancer, antidiabetic, and neuroprotective [32, 33]. In vivo, esculin is metabolized into esculetin through phase I reaction [32]. Currently, esculetin has been shown in a number of studies to prevent the formation of reactive oxygen species (ROS), decrease the reduction of antioxidant enzymes, and suppress the activation of mitochondria-induced apoptotic pathways, thereby offering protection of cells from oxidative stress-induced apoptosis. It could also inhibit the release of proinflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-lβ, IL-2, and interferon (IFN)-γ, leading to decrease the migration of inflammatory cells. Besides, esculetin inhibited the activation of nuclear factor kappa B (NF-κB), the expression of inducible nitric oxide synthase (iNOS), and the cyclooxygenase-2 protein by blocking the NF-κB pathway and lowering the generation of pro-inflammatory mediators such as nitric oxide (NO) and prostaglandin E2 [32, 34]. It is worth noting that coumarins, i.e., esculin and esculetin are first reported in T. tipu flowers.

Identification of flavonoids

Flavonoids are the most abundant group of plant polyphenolic compounds; they play a vital role in plant physiology [35] and are commonly found as glycosides bound to one or more sugar moieties through C- and/or O-linkage. Increasing scientific evidence suggested that flavonoids could have antioxidant and anti-inflammatory potential by suppressing regulatory enzymes or transcription factors implicated in the control of inflammatory mediators [36]. They exhibited neuroprotective effects against different neurodegenerative diseases, including Alzheimer, Parkinson, multiple sclerosis, and others through inhibiting pro-inflammatory cytokines expression, inflammatory markers, and thus preventing neural damage [37]. Several anti-inflammatory flavonoids have been reported in T. tipu leaves [11, 24]. Interestingly, 12 flavonoids have been tentatively identified in the methanol extract of T. tipu flowers for the first time.

Nine flavonols were detected in the extract of the flowers (Table 2). The identification of these compounds was facilitated by the analysis of fragmentation patterns of their molecular ions and the observation of glycosidic residues [rhamnosyl (-146 amu) and glucosyl (-162 amu)] that were cleaved sequentially and generated the characteristic aglycone, then its fragments, and all were compared to the reported data [23, 38]. Among them, 4 compounds were identified as kaempferol glycosides and 2 were quercetin glycosides, along with the aglycone (quercetin). In addition, two isorhamnetin glycosides were identified. In details, the MS2 spectra of isorhamnetin 3-glucoside [m/z 477.1026, C22H21O12–], quercetin 3-glucoside [m/z 463.0837, C21H19O12–], and kaempferol 3-glucoside [m/z 447.0903, C21H19O11–] showed removal of the attached glucose unit [M-H-162]– and revealed base peaks at m/z 315 (isorhamnetin), 300 (quercetin) and 285 (kaempferol), respectively, confirming O-glycosylated flavonoid. Moreover, the rutinoside glycoside of kaempferol and quercetin have been identified depending on the fragment ions resulted from the consecutive loss of rhamnose and glucose units leading to the base peak at m/z 285 and 300, respectively, corresponding to the free aglycones. It is worth noting that the forementioned flavonol glycosides have been previously isolated from T. tipu leaves and were detected here in the flowers. Kaempferol 3,7-dirhamnoside (kaempferitrin) was identified depending on its molecular ion peak at m/z 577.1569 corresponding to C27H29O14– and its fragmentation behavior. The MS/MS spectrum showed 2 main fragment ions at m/z 431 and 285 (Fig. S7) produced from successive loss of one and two rhamnose units, respectively. Another two flavonol glycoside, narcissin and kaempferol 3-O-rhamnosyl rutinoside were identified depending on their accurate masses, fragmentation patterns, and literature [23, 24].

Two compounds were mono-C-glycosyl flavones producing MS fragmentation patterns characteristic of C-glycosides flavonoids, including cross ring cleavage of the glucose moiety that produced fragmentation pattern of [(M-H)-120]– and [(M-H)-90]– [23]. Orientin and vitexin displayed molecular ions at m/z 447.0907 and 431.0964, respectively. In their MS2 spectra, both exhibited fragmentation pattern of [(M-H)-90]–, [(M-H)-120]–, yielding ions at m/z 357, 327 for orientin, respectively, and m/z 341, 311 for vitexin (Fig. S8), respectively. The lack of a loss of [(M-H)-H2O]–, confirming the sugar substitution in position 8 [38]. As authors are aware, this is the first report of C-glycosylated flavonoids in T. tipu plant.

Identification of organic and fatty acids

Six organic acids were identified in the methanol extract (Table 2). They were coeluted early with amino acids and sugars and observed in the first half of the chromatogram (Fig. S2). Their fragmentation behavior showed remarkable losses of CO2 (-44 amu) and H2O (-18 amu) (Table 2). For instance, succinic acid (Fig. S9) showed (M-H)– at m/z 117.0191 and product ions at m/z 99 [(M-H)-18]– and 73 [(M-H)-44]–. While fatty acids were eluted later and appeared in the last half of the chromatogram (Fig. S2). The LC/MS analysis of the extract revealed many unsaturated fatty acids e.g., linoleic, linolenic acids, hydroxylated fatty acids e.g., trihydroxy octadecatrienoic acid and hydroxy eicosanoic acid, as well as saturated fatty acids e.g., palmitic acid. Figure S10 showed the fragmentation of palmitic acid as a representative of the identified fatty acids. It showed the main ions at m/z 255 [M-H]–, 211 [M-H-CO2]–, and 196 [M-H-CH3]–.

It is well documented that organic acids play an important role as antimicrobial, anti-inflammatory, and antioxidant agents. Furthermore, organic acids are crucial to biological processes because they are intermediate or end products in numerous essential pathways in the metabolism of plants and animals, playing a key role in the citric acid cycle or Krebs cycle [39]. Whereas fatty acids provide energy to the human body and serve as structural material for cell membranes and organ padding. They play a role in vitamin A and D absorption, blood clotting, and immune response. Some of them are prostaglandins and leukotrienes’ chemical precursors. Polyunsaturated fatty acids can aid in lowering blood triglycerides and cholesterol. They lower the risk factors associated with cardiovascular disease, cancer, and diabetes (type 2). These essential fatty acids are required by the body for brain function and cell growth [40].

Identification of phospholipids

Twelve phospholipids (Table 2) have been characterized in the methanol extract including mainly, phosphatidic acids (PA), phosphatidylinositols (PI), and phosphatidylglycerols (PG). Structurally, phospholipids are constituted from the esterification of the fatty acid with phosphoglycerol to produce several derivatives. Sometimes, myoinositol, choline, or ethanolamine, may be attached to the phosphate group resulting in a great variation of phospholipids. In the negative-ion mode, the occurrence of carboxylate ions [RCOO]− recognizes the individual fatty acids e.g., m/z 277 for octadecatrienoic acid (18:3), 279 for octadecadienoic (18:2), and 255 for hexadecanoic acid (16:1). Moreover, the appearance of some diagnostic ions corresponding to specific functional moieties assists in the characterization of the lipid type, i.e., m/z 78 (phosphonate), 152 (phospho-glycerol), and 241 (phospho-myoinositol). For instance, the MS/MS spectrum of PA(18:2/18:2) showed main fragment ions at m/z 279 and 152 corresponding to liberation of 18:2 fatty acid and phospho-glycerol units from the parent ion (Table 2). For PI(18:2/16:0) (Fig. S11), it showed a molecular ion at m/z 833.5167 with a formula of C43H78O13P–, along with the diagnostic ions of phospholipids (171, 152, and 78), other major ions were detected at m/z 279 and 255 corresponding to the fatty acid carboxylate anions liberated from sn1 (less abundant peak) and sn2 (more abundant peak) positions, respectively [41]. Other characteristic ion was detected at m/z 241 corresponding to the inositol-phosphate ion. Following the same fragmentation pattern, the other phospholipids were assigned. Various phospholipids demonstrated antioxidant and anti-inflammatory potentials by suppressing the inflammatory signaling pathways and relieving uncontrolled oxidative stress [42]. To the best of our knowledge, this is the first comprehensive analysis of the metabolites of T. tipu flowers to provide chemical-based evidence for their biological potential. Where most of the identified compounds are reputed for their biological effects on human health as antioxidant and anti-inflammatory, that can help to protect body against oxidative stress and inflammatory processes.

Table 2 Metabolites identified in the methanolic extract of T. tipu flowers using LC–QTOF-MS/MSHPLC-DAD quantification of major identified phenolic compounds in the methanol extract of T. Tipu flowers

HPLC-DAD analysis of the methanolic extract of T. tipu flowers allowed the identification and quantification of 18 phenolic compounds (phenolic acids, coumarins, and flavonoids). The chromatogram is displayed in Fig. S12. The results are presented in Table 3. The flavonoids: orientin and astragalin illustrated relatively the high abundancies of all identified phenolics corresponding to 2078.88 ± 1.08 and 1898.39 ± 0.81 mg /100 g, respectively. Ferulic acid was the most abundant of all phenolic acids representing 1699.91 ± 1.05 mg /100 g. The coumarin esculetin and its glycoside esculin were quantified representing 77.78 ± 0.55 and 341.80 ± 0.44 mg /100 g, respectively. HPLC-DAD technique allowed further quantification of the identified phenolics, representing their abundances in the methanolic extract. It must be noted that this is the first study quantifying the phenolics of T. tipu flowers.

Table 3 Quantification of major identified phenolic compounds in the methanol extract of T. tipu flowers using HPLC-DADIn silico studiesMolecular docking

The results of in-vitro biological study revealed that ME induced inhibitory activities against NO, TNF-α, IL-β, IL-6, NF-KB, and TNF-R2 in LPS-induced RAW264.7 macrophages which was assumed to be associated with its chemical composition. Thus, it is necessary to determine the interactions of the main components of ME of T. tipu flowers with Human NOS, COX-2, and 5-LOX active sites in-silico using molecular docking and dynamic simulations to ascertain the anti-inflammatory activity and to predict the binding modes and interactions of its major compounds with the active sites of investigated enzymes. Based on the LC-MS profiling, the ME of the flowers was enriched in phenolic compounds, 28 compounds out of the total detected compounds, including 14 phenolic acids, 2 coumarins, and 12 flavonoids. Thus, selected compounds from each phenolic class, including 2 phenolic acids (chlorogenic acid and ferulic acid), 2 coumarins (esculetin and esculin), 4 flavonoids (kaempferol-3-glucoside, narcissin, orientin, and vitexin) were subjected to molecular docking study to investigate their affinity towards the studied enzymes. The results are shown in Table 4. As can be observed, all compounds showed high affinity towards COX-2, with docking scores S ranging from − 8.57 to − 5.05 kcal mol–1, 5-LOX (S ranging from − 8.83 to − 5.22 kcal mol–1), and Human NOS (S ranging from − 8.23 to − 4.21 kcal mol–1). In this context, kaempferol-3-glucoside and orientin exerted the highest docking scores against the targeted enzymes (Table 3). Consequently, they were subjected to further molecular dynamic study.

Table 4 Molecular docking data of selected phenolic compounds in the target active sitesMolecular dynamic (MD) simulations

In the study of biological systems, Molecular Dynamic (MD) integration simulations enable investigation of the physical motion of atoms and molecules that cannot be easily accessed by any other means. The insight obtained from performing this simulation offers an extensive viewpoint on the dynamical evolution of biological systems, including changes in conformation and molecular interaction [43]. Therefore, this simulation was run to predict how the selected compounds would behave once they bound to the protein active site, as well as their interaction and stability [44, 45]. For tracing erratic motions and eliminating artefacts that might appear during the simulation, system stability must be validated. This study used Root-Mean-Square Deviation (RMSD) to analyze system stability during the 20 ns simulations. The recorded the average RMSD values for the entire frames of the systems were 1.69 ± 0.25Å, 1.45 ± 0.24Å, and 1.49 ± 0.27Å, for Apo-COX protein, kaempferol-3-glucoside-COX, and orientin–COX complex systems (Fig. 3A), 1.87 ± 0.32Å, 1.46 ± 0.17Å, and 1.79 ± 0.33Å for Apo-LOX protein, kaempferol-3-glucoside-LOX, and orientin–LOX complex systems, respectively (Fig. 4A), and 1.69 ± 0.18Å, 1.65 ± 0.21Å, and 1.68 ± 0.19 Å for Apo-NOS protein, kaempferol-3-glucoside- NOS, and orientin–NOS complex systems, respectively (Fig. 5A).

For the purpose of analyzing residue behavior and its relationship to the ligand during MD simulation, it is essential to evaluate protein structural flexibility upon ligand binding [46]. Using the Root-Mean-Square Fluctuation (RMSF) technique, protein residue variations were assessed to determine the impact of inhibitor binding to the relevant targets across 20 ns of simulations. The computed average RMSF values for the entire frames of the systems were 1.05 ± 0.44Å, 1.01 ± 0.44Å, and 1.05 ± 0.49 Å, for Apo-COX protein, kaempferol-3-glucoside-COX, orientin–COX complex systems (Fig. 3B), 1.18 ± 0.57Å, 1.05 ± 0.44Å, and 1.18 ± 0.54Å for Apo-LOX protein, kaempferol-3-glucoside-LOX, orientin–LOX complex systems, respectively (Fig. 4B), and 1.11 ± 0.66Å,1.05 ± 0.64Å, and 1.07 ± 0.60 Å for Apo-NOS protein, kaempferol-3-glucoside-NOS, and orientin–NOS complex systems, respectively (Fig. 5B). These values revealed that the ligand bound to protein kaempferol-3-glucoside-complex system has a lower residue fluctuation than the other systems.

During an MD simulation, ROG was chosen to assess the stability and overall compactness of the system [47, 48].The average Rg values for the entire frames of the systems were 24.17 ± 0.09Å, 24.02 ± 0.08 Å, and 24.10 ± 0.10 Å, for Apo-COX protein, kaempferol-3-glucoside-COX, and orientin–cox complex systems (Fig. 3C), 27.37 ± 0.17Å, 27.21 ± 0.08Å, and 27.36 ± 0.14Å for Apo-LOX protein, kaempferol-3-glucoside-LOX, and orientin–LOX complex systems, respectively (Fig. 4C). 23.05 ± 0.08Å, 22.92 ± 0.08Å, and 22.98 ± 0.18 Å for Apo-NOS protein, kaempferol-3-glucoside-NOS, orientin–NOS complex systems, respectively (Fig. 5C). According to the observed behavior, kaempferol-3-glucoside- bound complex has a highly stiff structure against three enzymes.

By calculating the protein’s solvent accessible surface area (SASA), the hydrophobic core compactness of the protein was assessed. This was done by measuring the protein’s solvent-visible surface area, which is crucial for biomolecule stability [49]. The average SASA values for the complete frames of the Apo-COX protein, kaempferol-3-glucoside-COX, and orientin-COX complex systems were 23,437, 23,156, and 23243.86, respectively (Fig. 3D). 27152Å, 26451Å, and 26796.51Å, for Apo-LOX protein, kaempferol-3-glucoside-LOX, orientin–LOX complex systems, respectively (Fig. 4D). 20645Å, 20053.87Å, and 20125.54Å for Apo-NOS protein, kaempferol-3-glucoside-NOS, and orientin–NOS complex systems, respectively (Fig. 5D).

When paired with the data from the RMSD, RMSF, and ROG computations, the SASA finding revealed that the kaempferol-3-glucoside and orientin complex systems remain intact inside the catalytic binding site for the three enzymes.

Fig. 3

[A] RMSD of the protein backbone’s Cα atoms. [B] RMSF of each residue of the protein backbone Cα atoms of protein residues (C) ROG of Cα atoms of protein residues; (D) solvent accessible surface area (SASA) of the Cα of the backbone atoms relative (black) to the starting minimized over 20 ns for the catalytic domain binding site of cyclooxygenase-2 enzyme with kaempferol-3-glucoside complex system (red), and orientin complex system (blue)

Fig. 4

[A] RMSD of the protein backbone’s Cα atoms. [B] RMSF of each protein residue’s Cα atom; (c) ROG of each residue’s Cα atom; (d) solvent accessible surface area (SASA) of the backbone atoms relative to the starting minimized over 20 ns for the catalytic domain binding site of 5-lipoxygenase with kaempferol-3-glucoside complex system (red), and orientin complex system (blue)

Fig. 5

[A] RMSD of the protein backbone’s Cα atoms. [B] RMSF of each residue of the protein backbone Cα atoms o