Background

Family Fabaceae is the third largest flowering plants family, exceeded only by the Orchidaceae and Compositae [1]. It comprises about 730 genera and more than 19,400 species. Plants of this family grow in a variety of habitats and climates throughout the world. Many are important food and agricultural plants, including Glycyrrhiza glabra (licorice), Glycine max (soybean), Medicago sativa (alfalfa), Phaseolus (beans) and Pisum sativum (pea) [2]. The Genus Tipuana (Benth.) Benth. (Subfamily Papilionoideae) is represented by Tipuana tipu (Benth.) Lillo species, a South African tree. Also, it is commonly grown in several other countries, such as Egypt, Australia, Argentina, Uruguay, Paraguay, Brazil, and Bolivia [3, 4]. It is an ornamental tree and is also used as a supplementary food to ruminants (e.g., sheep), mostly the leaves which showed high nutritive value [5]. T. tipu was recommended for wound healing, hemorrhoids, gastrointestinal tract disorders, and abdominal and rheumatic pains. It has anti-inflammatory and free-radical scavenging activities, in addition it is used as an astringent for its high tannin content [6, 7]. Ethyl acetate and methylene chloride extracts of the aerial parts of T. tipu were proved to be efficient in inhibiting Listeria monocytogenes and Staphylococcus aureus growth. Moreover, leaf extracts showed promising nephroprotective and antimalarial activities [8]. Few phytochemical studies were performed on the leaf and the bark of T. tipu isolating formononetin, β-sitosterol, β-sitosterol glucoside, lupeol, α-amyrin, 1-nonadecanol, alpinumisoflavone, protocatechualdehyde, protocatechuic acid, and stearic acid [3, 5, 9, 10]. A kaempferol glycoside with marked anti-inflammatory activity, kaempferol 3-O-α-L-rhamnopyranosyl-(1→6)-O-[β-D-glucopyranosyl-(1→2)-4-O-acetyl-α-L-rhamnopyranosyl-(1→2)]-β-D-galactopyranoside, has been isolated from T. tipu (Benth.) Lillo leaves growing in Egypt, with another three flavonol glycosides, kaempferol 3-O-rutinoside, rutin, and kaempferol-3-O-[α-L-rhamnopyranosyl-(1→6)]-[α-L-rhamnopyranosyl-(1→2]-β-glucopyranoside [11].

Despite all progress in synthetic biotechnology and chemistry, herbs are still indispensable sources of medicinal preparations and considered as an important part of healthcare throughout the world [12]. In addition, plant-based medications have emerged as a promising source for develo** new pharmaceuticals and functional items due to the side effects of synthetics [13, 14], along with their higher safety margin and lower cost [15]. Surveying the current literature, all studies mainly focused on the leaves of T. tipu (Benth.) Lillo. Although flowers are considered as rich sources for valuable constituents depending on our phytochemical investigations, few and incomplete studies were traced concerning their contents and antioxidant and anti-inflammatory bioactivities [16]. Accordingly, different solvent extracts of the flowers (aqueous, methanolic, water/methanol (1:1), methanol/methylene chloride (1:1), and methylene chloride) were tested for their antioxidant capacities using different methods, such as DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)), and FRAP (ferric reducing antioxidant power). Furthermore, the in vitro anti-inflammatory (COX-2 and 5-LOX) effects of all extracts were evaluated. Then, the most active extract (methanolic extract) was evaluated for its inhibitory activity against the inducible nitric oxide synthase enzyme (iNOS), nitric oxide production (NO), and the secretion of the pro-inflammatory cytokines (NF-KB, TNF-R2, TNF-α, IL-1β, and IL-6) in LPS-activated RAW 264.7 macrophages. As well as it was of a great interest to provide a comprehensive metabolic profiling of T. tipu flowers using an integrated approach of LC-QTOF-MS/MS and HPLC-DAD analyses. Further, computational study using molecular docking simulations with iNOS, COX-2, and 5-LOX active sites was performed to establish for the first time the correlation between the elucidated chemical profiles and the anti-inflammatory effects of flowers. Thus, this study aims to provide a holistic understanding of the phytochemical composition of T. tipu flowers in relation to their biological potential, as the literature lacks such detailed information. Such an approach is required for future breeding programs alongside quality control purposes that offer chemical-based evidence regarding T. tipu biological potential and health benefits.

Materials and methods

The detailed procedures are described in the Supplementary online resource.

Plant material

Flowers of Tipuana tipu (Benth.) Lillo were collected during Spring season (March 2022) from the Zoo (Giza, Egypt). The taxonomic identity of the plant material was kindly authenticated and verified by Mrs. Teresa Labib, Head of the Taxonomists at Orman Botanic Garden, Egypt. This study complies with local, national, and international guidelines, and no specific consent was required for the collection of the plant material.

Preparation of different flower extracts

The flowers (1.5 kg) were air-dried, grinded, and extracted separately in triplicates (500 g each) with water, methanol, water/methanol (1:1), methanol/methylene chloride (1:1), and methylene chloride (twice, 3 L each) by cold maceration. The extracts were then dried under vacuum in a rotary evaporator at 50 ºC and the residues were kept separately in tight containers until analysis. Finally, the dried residues (30 mg each) were dissolved separately in DMSO for the biological assays and the dried methanolic residue (10 mg) was dissolved in methanol for LC-MS analysis. Three biological replicates were prepared for each solvent type and extracted in parallel under the same conditions (n = 3).

LC/QTOF-MS/MS analysis

T. tipu flowers methanolic extract was subjected to metabolic analysis using a 6530 Q-TOF LC/MS (Agilent Technologies, Santa Clara, CA, USA) outfitted with an autosampler (G7129A), a quaternary pump (G7104C). The extract was separated on a Zorbax RP-18 column (150 mm × 3 mm, dp = 2.7 μm, Agilent Technologies). The flow rate was 0.2 mL/min, and the injection volume was 2 µL. Water (A) and acetonitrile (B) with formic acid (0.1%) in each utilized as the solvents and applied using a gradient elution as follows 5% B linearly increased to 90% B within 30 min, then remained isocratic at 100% B for 2 min before linearly decreasing back to 5% B for the following 5 min. Mass spectra were obtained using ESI in negative ionization mode using the conditions previously described [17]. Briefly, the Q-TOF mass spectrometer was optimized in negative ESI mode with the following parameters: capillary voltage 3500 V, drying gas temperature and flow rate 350 °C and 8 L/min, respectively, nebulizer gas 30 psi, fragmentor voltage 150 V, skimmer voltage 60 V, and OCT 1 RF Vpp voltage 300 V. The auto-MS/MS mode was used in the m/z range of 50-2000, with an MS scan rate of 10 spectra/s and an MS/MS scan rate of 6 spectra/s. The precursor selection criteria were as follows: maximum precursor per cycle 9, absolute threshold 200 counts, relative threshold 0.01%, purity stringency 100%, purity cutoff 30%, precursors sorted by abundance only, and isolation window ∼ 1.3 m/z (narrow). Mass Hunter software (ver. B.06, Agilent Technologies, Inc. 2012) was used for instrument control, data acquisition, and processing of the MS/MS spectra. Metabolites were characterized by their retention times (min), exact molecular ion mass (M-H)–, MS/MS fragment ions relative to accessible databases, such as the Human Metabolome Database (http://www.hmdb.ca/) and lipidMaps (https://www.lipidmaps.org/).

Results and discussion

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
figure 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

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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 macrophages
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Fig. 2
figure 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

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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 [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
figure 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)

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Fig. 4
figure 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)

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Fig. 5
figure 5

[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 Human Nitric oxide synthase with kaempferol-3-glucoside complex system (red), and orientin complex system (blue)

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Binding interaction mechanism based on binding free energy calculation

A well-known method for determining the free binding energies of small molecules to biological macromolecules is the molecular mechanics energy methodology (MM/GBSA), which combines the generalized Born and surface area continuum solvation [50]. The MM-GBSA program in AMBER18 was used to determine the binding free energies using snapshots taken from the systems’ trajectories. Except for Gsolv, all of the estimated energy components (Table 5) showed large negative values indicating favorable interactions.

Table 5 The calculated energy binding for the selected compounds
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Identification of the critical residues responsible for ligands binding

To understand more about key residues involved in the inhibition of the catalytic binding site of COX-2 receptor, the total energy involved when substance contacts these enzymes was further broken down into the participation of specific site residues. From Fig. 6A, the major favorable contribution of kaempferol-3-glucoside to the COX-2 binding site receptor is mainly observed from residues Val57 (-0.154 kcal/mol), Hie58 (-0.463 kcal/mol), Val85 (-1.362 kcal/mol), Leu86 (-0.799 kcal/mol), Arg89 (-0.308 kcal/mol), Gln161 (-1.095 kcal/mol), Ile314 (-0.456 kcal/mol), Tyr317 (-0.24 kcal/mol), Val318 (-2.078 kcal/mol), Leu321 (-1.975 kcal/mol), Ser322 (-1.165 kcal/mol), Tyr 324 (-1.19 kcal/mol), Leu328 (-0.432 kcal/mol), Leu353 (-0.288 kcal/mol), Tyr354 (-1.295 kcal/mol), Ile486 (-0.356 kcal/mol), Phe 487 (-1.81 kcal/mol), Val492 (-2.61 kcal/mol), Gly495 (-1.245 kcal/mol), and Ala496 (-2.36 kcal/mol).

Regarding orientin, the major favorable contribution to the COX-2 binding site receptor is principally observed from residues Hie58 (-0.267 kcal/mol), Met82 (-0.311 kcal/mol), Val 85 (-1.239 kcal/mol), Ile314 (-0.341 kcal/mol), Tyr317 (-0.455 kcal/mol), Val318 (-2.468 kcal/mol), Leu321 (-1.68 kcal/mol), Ser 322 (-2.189 kcal/mol), Tyr324 (-2.357 kcal/mol), Leu328 (-1.015 kcal/mol), Phe350 (-0.348 kcal/mol), Leu353 (-0.427 kcal/mol), Tyr354 (-1.535 kcal/mol), Trp356 (-0.418 kcal/mol), Val492 (-2.354 kcal/mol), Gly495 (-1.08 kcal/mol), Ala496 (-1.794 kcal/mol), and Ser499 (-2.752 kcal/mol) (Fig. 6B).

From Fig. 7A, the major favorable contribution of kaempferol-3-glucoside to the 5-lipoxygenase binding site receptor is predominantly observed from residues Trp141 (-0.652 kcal/mol), Phe145 (-0.387 kcal/mol), Phe 302 (-1.255 kcal/mol), Gln306 ( -2.856 kcal/mol), Thr307 (-0.975 kcal/mol), Hid310 (-0.588 kcal/mol), Leu311 (-2.367 kcal/mol), Hip315 (-1.015 kcal/mol), Ala353 (-1.003 kcal/mol), Arg354 (-0.6 kcal/mol), Leu357 (-2.475 kcal/mol), Ile358 (-1.68 kcal/mol), Gly359 (-0.552 kcal/mol), Hie361 (-0.766 kcal/mol), Pro 498 (-0.405 kcal/mol), Trp528 (-0.637 kcal/mol), Hie529 (-2.488 kcal/mol), Ala532 (-1.369 kcal/mol), Val533 (-0.78 kcal/mol), and Leu356 (-1.372 kcal/mol). Alternatively, the major favorable contribution of orientin to the 5-lipoxygenase binding site receptor is mostly detected from residues Phe302 (-1.036 kcal/mol), Gln306 (-3.975 kcal/mol), Thr307 (-1.105 kcal/mol), Hid310 (-2.021 kcal/mol), Leu311 (-0.822 kcal/mol), Ala353 (-1.617 kcal/mol), Gln356 (-0.585 kcal/mol), Hid479 (-0.338 kcal/mol), Asn483 (-1.139 kcal/mol), Gln486 (-3.03 kcal/mol), Trp528 (-0.507 kcal/mol), Ala532 (-1.011 kcal/mol), Val533 (-1.265 kcal/mol), Leu536 (-3.171 kcal/mol), and Ile602 (-5.123 kcal/mol) (Fig. 7B).

Finally, from Fig. 8A, the major favorable contribution of kaempferol-3-glucoside to the Human Nitric oxide synthase (NOS) synthesize enzymes protein binding site receptor is predominantly observed from residues Met39 (-0.616 kcal/mol), Arg118 (-2.059 kcal/mol), Cys119 (-0.312 kcal/mol), Ile120 (-0.745 kcal/mol), Met293 (-0.613 kcal/mol), Glu296 (-1.238 kcal/mol), Arg300 (-0.455 kcal/mol), Asp301 (-2.298 kcal/mol), Ile381 (-1.47 kcal/mol), Trp382 (-4.6 kcal/mol), Leu383 (-0.527 kcal/mol), and Pro386 (-0.741 kcal/mol). On the other, the major favorable contribution of orientin to the Human Nitric oxide synthase (NOS) protein binding site receptor is predominantly observed from residues Ser37 (-0.468 kcal/mol), Met39 (-2.975 kcal/mol), Gln182 (-2.593 kcal/mol), Asp199 (-0.709 kcal/mol), Trp265 (-0.422 kcal/mol), Tyr266 (-0.782 kcal/mol), Pro269 (-0.259 kcal/mol), Tyr292 (-0.716 kcal/mol), Met293 (-0.249 kcal/mol), Glu296 (-2.435 kcal/mol), Ile297 (-0.42 kcal/mol), Arg300 (-1.125 kcal/mol), Asp301 (-4.969 kcal/mol), Asp304 (-0.265 kcal/mol), Arg307 (-0.26 kcal/mol), Trp382 (-3.375 kcal/mol), and Pro386 (-0.896 kcal/mol) (Fig. 8B). Interestingly, the docking results come in agreement with the previous reports demonstrated the antioxidant and anti-inflammatory activities of orientin and kaempferol-3-glucoside [51, 52].

Fig. 6
figure 6

Per-residue decomposition plots showing the energy contributions to the binding and stabilization of kaempferol-3-glucoside and orientin into the catalytic binding site of Human cyclooxygenase-2 [A], [B] Corresponding inter-molecular interactions are shown [a], [b]

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Fig. 7
figure 7

Per-residue decomposition plots showing the energy contributions to the binding and stabilization of kaempferol-3-glucoside and orientin into the catalytic binding site of Human-5-lipoxygenase [A], [B]. Corresponding inter-molecular interactions are shown [a], [b]

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Fig. 8
figure 8

Per-residue decomposition plots showing the energy contributions to the binding and stabilization of kaempferol-3-glucoside and orientin into the catalytic binding site of Human Nitric oxide synthase (NOS) protein [A], [B]. Corresponding inter-molecular interactions are shown [a], [b]

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Conclusion

This investigation revealed the promising efficiency of T. tipu flowers as antioxidant and anti-inflammatory drug, which is obviously related to its chemical profile and as indicated by the computational analysis. The results suggested the useful incorporation of the flowers’ extract in pharmaceutical and cosmetic industries owing to its potential to suppress the inflammatory key enzymes (iNOS, COX-2, and 5-LOX), as well as scavenge free radicals through its high antioxidant properties. However, it should be noted that these findings are preliminary, and further research with the methanol extract or after purification of specific compounds, followed by more detailed and conclusive in vivo and clinical studies, is strongly advised to fully exploit T. tipu flowers’ potential in the pharmaceutical industry.