Comparative Metabolic Study of Tamarindus indica L.’s Various Organs Based on GC/MS Analysis, In Silico and In Vitro Anti-Inflammatory and Wound Healing Activities

The chemical composition of the n-hexane extract of Tamarindus indica’s various organs—bark, leaves, seeds, and fruits (TIB, TIL, TIS, TIF)—was investigated using gas chromatography-mass spectrometry (GC/MS) analysis. A total of 113 metabolites were identified, accounting for 93.07, 83.17, 84.05, and 85.08 % of the total identified components in TIB, TIL, TIS, and TIF, respectively. Lupeol was the most predominant component in TIB and TIL, accounting for 23.61 and 22.78%, respectively. However, n-Docosanoic acid (10.49%) and methyl tricosanoate (7.09%) were present in a high percentage in TIS. However, α-terpinyl acetate (7.36%) and α-muurolene (7.52%) were the major components of TIF n-hexane extract. By applying a principal component analysis (PCA) and hierarchal cluster analysis (HCA) to GC/MS-based metabolites, a clear differentiation of Tamarindus indica organs was achieved. The anti-inflammatory activity was evaluated in vitro on lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. In addition, the wound healing potential for the n-hexane extract of various plant organs was assessed using the in-vitro wound scratch assay using Human Skin Fibroblast cells. The tested extracts showed considerable anti-inflammatory and wound-healing activities. At a concentration of 10 µg/mL, TIL showed the highest nitric oxide (NO) inhibition by 53.97 ± 5.89%. Regarding the wound healing potential, after 24 h, TIB, TIL, TIS, and TIF n-hexane extracts at 10 g/mL reduced the wound width to 1.09 ± 0.04, 1.12 ± 0.18, 1.09 ± 0.28, and 1.41 ± 0.35 mm, respectively, as compared to the control cells (1.37 ± 0.15 mm). These findings showed that the n-hexane extract of T. indica enhanced wound healing by promoting fibroblast migration. Additionally, a docking study was conducted to assess the major identified phytoconstituents’ affinity for binding to glycogen synthase kinase 3-β (GSK3-β), matrix metalloproteinases-8 (MMP-8), and nitric oxide synthase (iNOS). Lupeol showed the most favourable binding affinity to GSK3-β and iNOS, equal to −12.5 and −13.7 Kcal/mol, respectively, while methyl tricosanoate showed the highest binding affinity with MMP-8 equal to −13.1 Kcal/mol. Accordingly, the n-hexane extract of T. indica’s various organs can be considered a good candidate for the management of wound healing and inflammatory conditions.


Introduction
Tamarindus is a monospecific genus belonging to the Fabaceae family and usually has been identified under the name Tamarind [1]. Tamarindus indica Linn. is a tropical fruit tree that can be found growing wild in many tropical and subtropical areas [2]. Tamarind is a semi-deciduous tree, slow growing but long-lived, that can reach a height of up to 30 m. The leaves are alternate and paripinnate, and its fruit is a legume appearing as indehiscent pods with 1-10 seeds, which represent the main criteria of plants belonging to the Fabaceae family [3][4][5][6]. The fruit was especially well-known and commonly used by the ancient Egyptians and widely consumed and traded in Africa [3]. The fruit has usually been used in food dishes through different applications as a condiment and spice and could be eaten fresh or raw. Tamarind is sold as an entire pod, paste, or concentrate in speciality food stores all throughout the world [7,8]. Its fruit has been reported in the literature for its pharmacological antioxidant, anti-inflammatory, antidiabetic, anti-Alzheimer, and antihyperlipidemic properties [9][10][11]. Tamarind has been commonly used in folk medicine for the treatment of diarrhoea and dysentery, helminth infections, fever, and abdominal pain. It is also used as an anti-malarial and laxative and has a role in wound healing [3]. Traditional Indian and African medicine has made substantial use of different tamarind products, including its leaves, fruit, and seeds [12]. Across many areas of central West Africa, tamarind leaves and bark are used traditionally to alleviate wounds [3,13]. Leaves of Tamarind have nutritional value due to their richness in vitamins and minerals [12]. Its leaves have exhibited potential antioxidant, antibacterial and antifungal, anti-inflammatory, and analgesic activities [14][15][16][17]. Additionally, the leaves of Tamarind have an anti-inflammatory role in Indian folk medicine [18].
Regarding the bark, it is effective as an astringent and tonic agent in lotions or poultices to relieve sores, ulcers, boils, and rashes, owing to its richness with tannins [12]. In addition, the bark showed in vivo antihyperglycemic, anti-inflammatory, and analgesic activities [17,19]. Moreover, leaves and bark have been reported for their anthelmintic activity [20].
Wound healing is a normal complex repair process that requires synchronisation between various biological and immunological systems, including many phases of inflammation, proliferation, and contraction of the wound, with the formation of granulation tissues and remodelling [21][22][23]. Plants and plant products, such as Aloe vera, Sophora flavescens, Punica granatum L., Mimosa pudica L., and Hibiscus rosa sinensis L., have been used in traditional medicine to cure and prevent diseases, especially wounds [24][25][26].
Recently, there has been a great demand for the development of natural products to cure different conditions owing to their safety, availability, versatile biological activities, and unique secondary metabolites [27][28][29][30][31]. Consequently, the exploration of medicinal plants as new candidates for wound healing would be valuable and beneficial, especially medicinal plants that are characterised by biocompatibility, wound healing, and antiinflammatory properties [22,23,32,33]. Many reports have revealed that using n-hexane as an extracting solvent targets non-polar compounds [21,34,35], and many studies have proven the role of n-hexane extract from various plants in anti-inflammatory and wound healing conditions [36][37][38][39].
Thus, the purpose of this present study was to compare and determine the chemical composition of the n-hexane extract of T. indica L.'s various organs using GC/MS analysis. Further, the principal component analysis (PCA) and hierarchical cluster analysis (HCA) were applied to discriminate among various plant organs based on their chemical profiles, which could be used as a tool for the detection of similarities and differences among the four organs. Furthermore, the n-hexane extract obtained from T. indica L.'s various organs were investigated for their anti-inflammatory and wound healing properties for the first time along with a molecular docking study to correlate the chemical composition with the mentioned biological activity. To the best of our knowledge, this is the first study on the wound-healing properties for the various organs of T. indica L. n-hexane extract.

Chemometric Analysis Based on GC/MS Analysis
GC/MS-based metabolites showed both qualitative and quantitative variation among different Tamarindus indica organs. Consequently, chemometric analysis, representing both principal component analysis (PCA) and hierarchal cluster analysis (HCA), was applied to the relative peak areas of all the identified compounds of different T. indica organs to explore the similarities and differences among them. The PCA score plot and loading plot are shown in Figure 3a,b. In the PCA score plot, the percentage of the first two principal components explained 83% of the variance in the data, where PC1 accounted for 63%, while PC2 explained only 20% of the data discrepancies. From the score plot, various T. indica organs were divided into four main groups, and each part was scattered in a separate quadrant. TIL and TIB were positioned on the positive side of PC1, whereas, TIF and TIS were located on the negative side of PC1. An in-depth inspection of the loading plot ( Figure 3b) revealed that lupeol acetate was the major discriminating marker for TIB. Although lupeol was the major identified component in both TIB and TIL, it was ©dentified as the main segregating marker for TIL in addition to lupenone.  Table 1.
Regarding TIF and TIS, γ-Sitosterol and n-Docosanoic acid were the chef compounds responsible for their separation in separate quadrants. By applying HCA, the dendrogram ( Figure 3c) categorised T. indica organs into four main clusters. Cluster I, II, III, and IV displayed TIL, TIB, TIF, and TIS, respectively. The dendrogram showed that TIL and TIB were closely related to each other's when compared to TIF and TIS. Both PCA and HCA successfully discriminated among various T. indica organs proving the effective application of chemometric analysis in combination with GC/MS-based metabolites for the discrimination among various plant organs.

Anti-Inflammatory Activity of Tamarindus indica L. Various Organs
The anti-inflammatory effects of the n-hexane extract of T. indica bark, leaves, seeds, and fruits (TIB, TIL, TIS, and TIF) on lipopolysaccharide (LPS)-induced RAW 264.7 macrophages were represented in ( Table 2). The various organs of T. indica showed promising inhibition of LPS-induced nitric oxide (NO) release in RAW 264.7 macrophages. At a concentration of 10 µg/mL, TIB, TIL, TIS, and TIF n-hexane extracts showed %NO inhibition by 7.53 ± 1.69, 53.97 ± 5.89, 19.54 ± 1.19, and 26.66 ± 3.44%, respectively. Further, at a concentration of 100 µg/mL, TIB, TIL, TIS, and TIF n-hexane extracts showed %NO inhibition by 51.44 ± 1.17, 98.00 ± 1.90, 85.47 ± 0.22, and 83.69 ± 2.39%, respectively as compared to positive control L-N G -nitro arginine methyl ester (L-NAME) (1 mM) that showed 84.64 ± 1.04% NO inhibition. Previous studies revealed that the fruit pulp was applied to inflammations and traditionally used as a gargle for sore throats. Further, the bark decoction was used in cases of eye inflammation [12]. Rimbau et al. revealed the in vivo anti-inflammatory properties of different extracts of tamarind fruit pulp in mice ear oedema induced by arachidonic acid, and rats sub plantar oedema induced by carrageenan [43]. Moreover, the petroleum ether and ethyl acetate extracts of seeds of T. indica showed a significant anti-inflammatory and analgesic potential using carrageenan-induced paw oedema and cotton pellet-induced granuloma models in rats [44]. Among the major compounds in the n-hexane extract of T. indica's various organs were lupenone and sitosterol, which were reported for their anti-inflammatory activity [45]. Additionally, among the major identified compounds, lupeol and lupeol acetate were reported for their anti-inflammatory properties through regulating TNF-α, IL-2, and IL-β specific mRNA, reducing PGE-2 synthesis from macrophage, neutrophil migration, and the number of iNOS cells [46]. Evaluation of the cytotoxicity of the n-hexane extract of T. indica bark, leaves, seeds, and fruits (TIB, TIL, TIS, and TIL) on the HSF cells was necessary to assess the safety of the treatment dose. The cytotoxicity on HSF cells of the n-hexane extract of T. indica's various organs was evaluated using the SRB assay [47]. In our investigation, TIF and TIS (up to 100 µg/mL) had no cytotoxic effect on the HSF cells. While TIB and TIL at a concentration of 10 µg/mL showed HSF cell viability up to 91.04 ± 2.09 and 95.38 ± 0.86%, respectively ( Figure 4). Therefore, a concentration of 10 µg/mL was safe and used further as the treatment dose in the scratch wound assay.

Scratch Wound Assay
In the present study, an in vitro scratch assay using HSF cells was used to assess TIB, TIL, TIS and TIF wound-healing activity. It was evaluated by changes in wound width by measuring the average distance between the borders of the scratches (Figures 5 and 6). The tested plant extracts at a concentration of 10 μg/mL decreased the wound width significantly compared with the control cells (Table 3).  After 24 h, the highest wound healing potential was recorded by TIB and TIS n-hexane extracts with wound widths equal to 1.09 ± 0.04 and 1.09 ± 0.28 mm, respectively. This result was followed by TIL and TIF with wound widths equal to 1.12 ± 0.18 and 1.41 ± 0.35 mm, respectively, as compared to the wound width in the control cells that equalled 1.37 ± 0.15 mm. As the wound width reduced as cell migration was enhanced, our findings revealed that both TIB and TIL n-hexane extracts exhibited almost complete cell migration

Scratch Wound Assay
In the present study, an in vitro scratch assay using HSF cells was used to assess TIB, TIL, TIS and TIF wound-healing activity. It was evaluated by changes in wound width by measuring the average distance between the borders of the scratches (Figures 5 and 6). The tested plant extracts at a concentration of 10 µg/mL decreased the wound width significantly compared with the control cells (Table 3). Table 3. Wound width of the scratched Human Skin Fibroblast cells (HSF) incubated in the absence of the plant extract (negative control) and the presence of n-hexane extract from T. indica: TIB (bark), TIL (leaves), TIS (seeds), and TIF (fruits) (10 µg/mL). after 48 h of observation. Meanwhile, TIS and TIF n-hexane extracts had wound-healing effects on HSF cells, but the time required to conduct wound closure was longer than for the negative control. This revealed that the n-hexane extract of T. indica's various organs exhibited wound-healing potential through fibroblast migration enhancement.  After 24 h, the highest wound healing potential was recorded by TIB and TIS nhexane extracts with wound widths equal to 1.09 ± 0.04 and 1.09 ± 0.28 mm, respectively. This result was followed by TIL and TIF with wound widths equal to 1.12 ± 0.18 and 1.41 ± 0.35 mm, respectively, as compared to the wound width in the control cells that equalled 1.37 ± 0.15 mm. As the wound width reduced as cell migration was enhanced, our findings revealed that both TIB and TIL n-hexane extracts exhibited almost complete cell migration after 48 h of observation. Meanwhile, TIS and TIF n-hexane extracts had woundhealing effects on HSF cells, but the time required to conduct wound closure was longer than for the negative control. This revealed that the n-hexane extract of T. indica's various organs exhibited wound-healing potential through fibroblast migration enhancement.

Time (h)
Previous reports revealed the traditional utilisation of tamarind in eye surgery for conjunctival cell adhesion and corneal wound healing [12]. In a study by Adeniyi et al., the wound-healing activity of T. indica leaves and pulp was investigated in the African catfish, and the results revealed the enhancement of the wound healing significantly in the fish-fed tamarind-fortified diets as a consequence of the elevation of antioxidant enzymes [48]. Attah et al. investigated the wound-healing potential of T. indica fruit paste in adult rabbits, and the results revealed wound closure acceleration and increasing epithelial migration and re-epithelialisation [49]. Plants 2023, 12, x FOR PEER REVIEW 13 of 25 Previous reports revealed the traditional utilisation of tamarind in eye surgery for conjunctival cell adhesion and corneal wound healing [12]. In a study by Adeniyi et al., the wound-healing activity of T. indica leaves and pulp was investigated in the African catfish, and the results revealed the enhancement of the wound healing significantly in the fish-fed tamarind-fortified diets as a consequence of the elevation of antioxidant enzymes [48]. Attah et al. investigated the wound-healing potential of T. indica fruit paste in adult rabbits, and the results revealed wound closure acceleration and increasing epithelial migration and re-epithelialisation [49].
The wound-healing potential of n-hexane extracts of various organs of T. indica can be linked to their chemical components. For example, γ-sitosterol is one of the most famous phytosterols with reported biological activity. Phytosterols were shown to inhibit MMP-1, reduce collagen breakdown, and promote the synthesis of collagen in human keratinocytes [50]. Moreover, they promote keratinocyte migration through the reduction of oxidative stress that effectively accelerates the healing process [51]. In previous studies, lupeol and lupeol acetate, which are the major identified components in the present study, were reported for their wound-healing properties [52][53][54][55][56][57][58]. Lupeol prevents collagen I depletion and restores levels of hydroxyproline, hexosamine, hexuronic acid, and matrix glycosaminoglycans, together with modulating collagen I expression in human fibroblasts during the proliferation phase. Further, it enhances fibroblast proliferation, angiogenesis, and growth factors in wound healing [52][53][54]. Another study reported the potential of lupeol for wound healing in streptozotocin-induced hyperglycaemic rats [56]. Another study by Malinowska et  The wound-healing potential of n-hexane extracts of various organs of T. indica can be linked to their chemical components. For example, γ-sitosterol is one of the most famous phytosterols with reported biological activity. Phytosterols were shown to inhibit MMP-1, reduce collagen breakdown, and promote the synthesis of collagen in human keratinocytes [50]. Moreover, they promote keratinocyte migration through the reduction of oxidative stress that effectively accelerates the healing process [51]. In previous studies, lupeol and lupeol acetate, which are the major identified components in the present study, were reported for their wound-healing properties [52][53][54][55][56][57][58]. Lupeol prevents collagen I depletion and restores levels of hydroxyproline, hexosamine, hexuronic acid, and matrix glycosaminoglycans, together with modulating collagen I expression in human fibroblasts during the proliferation phase. Further, it enhances fibroblast proliferation, angiogenesis, and growth factors in wound healing [52][53][54]. Another study reported the potential of lupeol for wound healing in streptozotocin-induced hyperglycaemic rats [56]. Another study by Malinowska et al. revealed that lupeol acetate was one of the most effective lupeol derivatives in the stimulation of the human skin cell proliferation process [58]. Bopage et al. reported that the presence of a 3-OH group in the lupeol structure is one of the essential features in the lupane skeleton for wound healing activity as compared to lupenone and lupeol acetate [59].
Linolenic acid and squalene are among the major identified constituents in the nhexane extract of T. indica fruits; linolenic acid plays an important role in the wound healing process across its antioxidative and anti-inflammatory properties as well as by encouraging cell proliferation, raising collagen synthesis, promoting dermal reconstruction, and restoring the function of the skin's lipid barrier [60][61][62][63]. Further, squalene is reported to have anti-inflammatory and protective actions against skin damage. It can accelerate the healing of wounds by stimulating the macrophage response to inflammation. Squalene could be helpful during the resolution phase of wound healing [64].
The amounts of lupeol, lupeol acetate, lupenone, and γ-sitosterol show a correlation with the in vitro wound healing potential of the n-hexane extract of various organs of T. indica, and TIB was found to contain the highest amounts of these compounds according to the GC/MS analysis. The present study and the data in the literature show that terpenoids, sterols, and fatty acids play important roles in the wound-healing potential of T. indica. So, T. indica is suggested as a potent natural wound-healing product.

Plant Material
Tamarindus indica L.'s (Fabaceae) various organs (Bark, leaves, seeds, and fruits) were collected from the Zoo Botanical Garden, Giza, Egypt, in October 2021. The various plant organs were kindly identified and authenticated by agricultural engineer Eng. Terase Labib, Consultant of Plant Taxonomy at the Ministry of Agriculture and El-Orman Botanical Garden, Giza, Egypt. Voucher specimens of the various plant organs with codes BUC-PHG-TIL-6, BUC-PHG-TIS-7, BUC-PHG-TIF-8, and BUC-PHG-TIB-9 were kept at the Department of Pharmacognosy, Badr University in Cairo, Cairo, Egypt.

Preparation of the N-Hexane Extracts of Various Organs
The dried samples of the barks, leaves, seeds, and fruits of T. indica (100 g each) were powdered and extracted by cold maceration method with n-hexane (500 mL × 3) separately, followed by filtration for 3 days. The filtrate of each plant material was completely evaporated in vacuo at 45 • C until dry to obtain the dried residue of the n-hexane extract (420 mg, 610 mg, 207 mg, and 312 mg) for barks, leaves, seeds, and fruits, respectively. The dried residue of each plant part was named as follows; T. indica bark (TIB), T. indica leaves (TIL), T. indica seeds (TIS), and T. indica fruits (TIF). All extracts were stored in a tight container at 4 • C in the refrigerator for further analysis [65].

GC/MS Analysis
Gas chromatography coupled with mass spectrometry (GC/MS) analyses were performed on a Shimadzu GCMS-QP 2010 (Shimadzu Corporation, Koyoto, Japan), provided using an Rtx-5MS (30 m × 0.25 mm i.d. × 0.25 µm film thickness) capillary column (Restek, Bellefonte, PA, USA) and attached to a Shimadzu mass spectrometer. The column temperature was initially set at 50 • C for 3 min. Then, the temperature was gradually increased from 50 to 300 • C at a rate of 5 • C/min and then isothermally maintained at 300 • C for 10 min. The temperature of the injector was kept at 280 • C. Helium was used as a carrier gas at a flow rate of 1.37 mL/min. The ion source and the interface were at temperatures of 280 and 220 • C, respectively. An injection of 1 µL of 1% v/v of diluted sample was achieved via a split mode adopting a split ratio of 15:1. Recording of the mass spectrum was performed in EI mode of 70 eV from m/z 35 to 500. Compound quantitation was performed based on the normalisation method, employing the reading of three chromatographic runs.

Identification of the N-Hexane Extract Components
The components of the n-hexane extracts were characterised by comparing their GC/MS spectra, fragmentation patterns, and retention indices with those reported in the literature data [66][67][68][69][70][71][72]. The retention indices were calculated relative to a homologous series of n-alkanes (C8-C28) injected under the same conditions.

Chemometric Analysis
The data obtained from GC-MS were subjected to multivariate analysis. Principal component analysis (PCA) was performed as the first step in data analysis to provide an overview of all observations and samples and to identify and evaluate groupings, trends, and strong outliers [73]. Hierarchal cluster analysis (HCA) was used to allow the clustering of samples. The clustering patterns were constructed by applying the complete linkage method. This presentation was more efficient when the distance between samples (points) was computed using the Euclidean method. Both PCA and HCA were achieved by utilising Unscrambler ® X 10.4 from CAMO (Computer Aided Modeling, Viken, Norway) [74].

Anti-Inflammatory Activity
Murine macrophage RAW264.7 cells (ATCC ® ) were maintained in a complete Dulbecco's Modified Eagle's Medium (DMEM, Corning, NY, USA) supplemented with 10% foetal bovine serum, penicillin (100 U/mL), streptomycin sulphate (100 µg/mL), and 2 mM L-glutamine in a humidified 5% CO 2 incubator. For passaging and treatment, cells were washed with phosphate-buffered saline and scrapped off the flasks using sterile scrappers (SPL, Spain). RAW 264.7 cell stock (0.5 × 106 cells/mL) was seeded into 96-well microwell plates and incubated overnight. The next day, the non-induced triplicate wells received a medium with the sample vehicle (DMSO, 0.1% v/v). The inflammation group of triplicate wells received the inducer of inflammation [lipopolysaccharide (LPS) as 100 ng/mL] in complete culture media containing 0.1% DMSO, v/v. Sample groups of triplicate wells received two screening amounts (10 and 50 µg/mL) of the sample dissolved in DMSO and diluted into culture media containing LPS (Final concentration of DMSO = 0.1%, by volume). L-NAME (L-N G -nitro arginine methyl ester) (1 mM) was used as a standard NOS inhibitor. After 24 h of incubation, a Griess assay [75] was used to determine NO in all wells. Equal volumes of culture supernatants and Griess reagent were mixed and incubated at room temperature for 10 min to form the coloured diazonium salt and read at an absorbance of 520 nm. The NO Inhibition % of the test extract was calculated relative to the LPS-induced inflammation group, normalised to cell viability determined with the Alamar Blue™ reduction assay [76].

Cytotoxicity Assay
The cytotoxicity of the tested plant extract against the HSF (Human Skin Fibroblast) cell line was assessed prior to the wound-healing assay using the sulforhodamine B assay (SRB) [47]. The HSF cell line was obtained from Nawah Scientific Inc. (Mokatam, Cairo, Egypt). Cells were maintained in a DMEM medium supplemented with 100 mg/mL of streptomycin, 100 units/mL of penicillin, and 10% of heat-inactivated foetal bovine serum in a humidified 5% (v/v) CO 2 atmosphere at 37 • C. Aliquots of 100 µL of cell suspension (5 × 10 3 cells) were placed in 96-well plates and incubated in a complete medium for 24 h. Cells were treated with another aliquot of 100 µL medium containing DRSE at various concentrations (0.01, 0.1, 1, 10, and 100 µg/mL). After 72 h of drug exposure, cells were fixed by replacing the medium with 150 µL of 10% TCA and incubated at 4 • C for 1 h. The TCA solution was removed, and the cells were washed five times with distilled water. Aliquots of 70 µL SRB solution (0.4% w/v) were added and incubated in a dark place at room temperature for 10 min. Plates were washed three times with 1% acetic acid and allowed to air-dry overnight. Then, 150 µL of TRIS (10 mM) was added to dissolve the protein-bound SRB stain; the absorbance was measured at 540 nm using a BMGLABTECH ® -FLUO star Omega microplate reader (Ortenberg, Germany).

Scratch Wound Assay
The wound-healing activity of TIB, TIL, TIS, and TIF n-hexane extract was evaluated using in vitro cell migration studies on HSF cells. Cells were plated at a density of 2 × 10 5 /well onto a coated 12-well plate for the scratch wound assay and cultured overnight in 5% FBS-DMEM at 37 • C and 5% CO 2 . On the next day, horizontal scratches were introduced into the confluent monolayer; the plate was washed thoroughly with PBS, control wells were replenished with the fresh medium, and drug wells were treated with fresh media containing the drug. Images were taken using an inverted microscope at the indicated time intervals. The plate was incubated at 37 • C and 5% CO 2 between time points. The experiment was done in triplicate. The acquired images are displayed in Figure 6 and were analysed using MII ImageView software version 3.7. Wound width was calculated as the average distance between the edges of the scratches; the wound width decreases as cell migration is induced. The results are displayed as mean ± standard deviation (Table 3) [77][78][79].

Statistical Analysis
Statistical analyses were done using a one-way analysis of variance (ANOVA) followed by the Tukey-Kramer multiple comparison test (p < 0.05). Statistical analyses were performed using GraphPad Prism 6.01 (GraphPad Inc., La Jolla, CA, USA).

In Silico Molecular Docking Studies
The X-ray 3D structures of glycogen synthase kinase 3-β (GSK3-β), matrix metalloproteinases-8 (MMP-8), and nitric oxide synthase (iNOS) were downloaded from the protein data bank www.pdb.org (accessed on 15 August 2022) using the following IDs: 3F88, 5H8X, and 3N2R, respectively [80][81][82]. All the docking studies were conducted using MOE 2019 [83], which was also used to generate the 2D interaction diagrams between the docked ligands and their potential targets. The identified major compounds were prepared using the default parameters and saved in a single MDB file. The active site for each target was determined from the binding of the corresponding co-crystalised ligand. Finally, the docking was finalised by docking the MDB file containing all the major compounds into the active site of the three enzymes.

Conclusions
The present study investigated the secondary metabolites in the n-hexane extract of T. indica's various organs and their in vitro anti-inflammatory and wound healing properties using the scratch assay with HSF cells. The GC/MS analysis revealed that triterpenoids and steroids were the predominant classes in the T. indica bark and leaf n-hexane extracts. Additionally, the seed and fruit n-hexane extracts showed a high percentage of fatty acids and higher hydrocarbons. PCA and HCA successfully discriminated various T. indica organs based on their GC/MS metabolites. The tested extracts showed promising anti-inflammatory and wound-healing properties. Additionally, the major characterised phytoconstituents achieved promising docking scores in the active sites of glycogen synthase kinase 3-β (GSK3-β), matrix metalloproteinases-8 (MMP-8), and nitric oxide synthase (iNOS), which give the n-hexane extract of the various T. indica organs a chance to be incorporated in the pharmaceutical products for wound healing after further in vivo and clinical trials.