The Antioxidant and In Vitro Wound Healing Activity of Cotyledon orbiculata Aqueous Extract and the Synthesized Biogenic Silver Nanoparticles

The synthesis of silver nanoparticles using biogenic methods, particularly plants, has led to the discovery of several effective nanoparticles. In many instances, plant-derived silver nanoparticles have been shown to have more activity than the plant extract which was used to synthesize the nanoparticles. Silver nanoparticles have been successfully synthesized using the medicinal plant, Cotyledon orbiculata. This is a shrub found in the Western Cape province of South Africa. It has a long history of use in traditional medicine in the treatment of wounds and skin infections. The C. orbiculata synthesized silver nanoparticles (Cotyledon-AgNPs) were reported to have good antimicrobial and anti-inflammatory activities; however, their wound-healing properties have not been determined. This study aimed to determine the wound healing activity of Cotyledon-AgNPs using the scratch assay. Gene expression studies were also done to determine the nanoparticles’ mechanism of action. The Cotyledon-AgNPs showed good antioxidant, growth-promoting and cell migration properties. Gene expression studies showed that the C. orbiculata water extract and Cotyledon-AgNPs promoted wound healing by upregulating genes involved in cell proliferation, migration and growth while downregulating pro-inflammatory genes. This confirms, for the first time that a water extract of C. orbiculata and silver nanoparticles synthesized from this extract are good wound-healing agents.


Introduction
Green nanotechnology is receiving much attention in all scientific research areas worldwide. Its popularity can be attributed to the ability to synthesize biocompatible nanomaterials using simple, eco-friendly, and cost-effective methods. This field of nanotechnology involves the use of biological materials such as plants and microorganisms in nanomaterial synthesis. This significantly reduces energy costs, the use of toxic chemicals and some expensive materials and instruments which are common with the chemical and physical methods [1]. Recently, green nanotechnology has been applied in the form of nanoparticles, nanocomposites, hydrogels and nanofibers for the development of products that are used for wound treatment [2]. Metallic nanoparticles containing silver, gold and zinc are the most studied due to their unique properties most notably their antibacterial activity [3]. In addition to its antibacterial activity, silver promotes wound healing and has been used in wound dressings for the treatment of different types of wounds including burns and wounds [4]. Biogenic silver nanoparticles (AgNPs) have become popular antimicrobial, anti-inflammatory, and good wound healing agents [5]. The wound-healing potential of biogenic nanoparticles in both in vitro and in vivo models has been reported. Phenolic compounds, including flavonols, are not only reducing agents and metal chelators, but they are also good free radical scavengers. Due to their reducing and radical scavenger properties, phenols and flavanols have been reported to possess significant antioxidant activities. The antioxidant activities of the C. orbiculata water extract and Cotyledon-AgNPs were determined using the Ferric Reducing Antioxidant Power (FRAP) and 2 -Azino-Bis-3-Ethylbenzotiazolin-6-Sulfonic Acid (ABTS) assays. The FRAP assay directly measures the amount of antioxidants in a sample by measuring their ferric-reducing activity [36]. However, other antioxidant assays such as ABTS operate on free radical inhibition via hydrogen atom transfer [37]. As shown in Table 1, both the C. orbiculata water extract and the Cotyledon-AgNPs exhibited some antioxidant activity. In the FRAP assay, the C. orbiculata water extract showed more reducing activity than the Cotyledon-AgNPs. The Cotyledon-AgNPs were able to reduce approximately half the amount of ferric ions compared to the C. orbiculata water extract. This finding corresponds to the total polyphenol content (TPC) results, where approximately half of the polyphenols present in the extracts were used in the synthesis of the Cotyledon-AgNPs. Literature reports that the amount of iron reduced in the FRAP assay can be correlated with the amount of antioxidants present [38,39]. This could, therefore, mean that of all the phytochemicals present on the Cotyledon-AgNPs, polyphenols are the ones responsible for the ferric-reducing activity in the FRAP assay. On the other hand, the ABTS assay presented the Cotyledon-AgNPs as more active than the C. orbiculata water extract with values of 134.54 ± 20.59 and 91.14 ± 0.04, respectively. The difference in these results is indicative of the different mechanisms between the FRAP and the ABTS assays. Flavanols, phenolic acids, flavonols and tannins may be responsible for the reported bioactivities of C. orbiculata, as they have been shown to possess antimicrobial and anti-inflammatory activities [40]. These phytochemicals may as well be attributed to the traditional use of the plant. Increased antioxidant activity of AgNPs has been shown to increase their wound healing capabilities [41]. High ROS levels interfere with fibroblast proliferation, and oxidative stress causes damage to cell membranes, proteins and lipids resulting in delayed wound healing [41]. Therefore, antioxidant substances that can scavenge ROS and maintain non-toxic ROS levels in the wound tissues could improve healing [42]. Several biomaterials with antioxidant properties have been developed. In a study by Castro et al. 2015, an antioxidant dressing was prepared and tested using in vitro and in vivo methods. The dressing was shown to have protective effects by reducing ROS levels in fibroblasts; it also regulated the expression of inflammation related genes. In the in vivo studies, the antioxidant dressing promoted faster wound healing compared to the control [43].

Cell Viability and Growth Rate
The cell viability and growth-promoting effects of the C. orbiculata water extract and Cotyledon-AgNPs were assessed on KMST, HaCaT and CHO cells using the WST-1 assay. The viability of cells treated with the C. orbiculata water extract and the Cotyledon-AgNPs were both dose and time-dependent. The Cotyledon-AgNPs reduced the viability of HaCaT and CHO cells at higher concentrations (≥5 µg/mL for HaCaT and ≥20 µg/mL for CHO); however, they were non-toxic to KMST-6 cells at all the concentrations tested. These results correspond to the findings by Zanette et al. 2011, where AgNPs toxicity to HaCaT cells increased with increasing incubation time and concentration [44]. Interestingly, at lower concentrations, the Cotyledon-AgNPs increased the rate of cell growth (Figure 1). At concentrations between 0.1-2.5 µg/mL, Cotyledon-AgNPs increased the growth of KMST-6 and CHO cells at both 24 and 72 h. The Cotyledon-AgNPs also increased the growth rate of HaCaT cells at concentrations between 0.1 and 2.5 and 0.1 and 1.3 µg/mL at 24 and 72 h, respectively.
The C. orbiculata water extract did not reduce the viability of HaCaT and CHO cells at both 24 and 72 h incubation. However, they reduced the viability of KMST-6 cells at concentrations higher than 240 µg/mL. The C. orbiculata water extract promoted cell growth at most of the concentrations tested especially the lower concentrations. Unlike the Cotyledon-AgNPs which decreased cell growth as incubation time increased, the C. orbiculata water extract actually increased the growth of cells with increasing incubation time. The concentrations of 2.5 µg/mL (Cotyledon-AgNPs) and 15 µg/mL (C. orbiculata water extract) were used for the scratch wound healing assay as these showed a stable trend amongst the different cell lines.   Each value represents mean ± standard error of the mean (SEM); statistical significance of the C. orbiculata water extract and Cotyledon-AgNPs-treated cells when compared to the untreated cells is indicated with * for p < 0.05, ** p for <0.01, and *** p < 0.001.

Wound Healing Activity
The wound healing activity of the C. orbiculata water extracts and Cotyledon-AgNPs was tested using the scratch assay. This is a popular, simple method used to determine the wound-healing activities of different compounds [45]. It is related to the proliferation phase of wound healing which involves the migration and proliferation of fibroblasts and keratinocytes. This assay mimics the in vivo migration of cells during wound healing [46]. Both the C. orbiculata water extracts and the Cotyledon-AgNPs were able to close the scratched gap faster than the Negative Control (untreated cells) in all cell lines ( Figure 2). The images show a time-dependent increase in the density of cells in the scratched area until the gap closes. This strongly indicates that the C. orbiculata water extracts and the Cotyledon-AgNPs possess both cell migration and proliferation properties. Therefore, it can be concluded that C. orbiculata promotes wound healing activities via increased migration and proliferation of fibroblasts, keratinocytes and epithelial cells. Our findings support literature that reports that AgNPs promote wound healing through the differentiation of myofibroblasts to fibroblasts, wound contractility and epidermal re-epithelialization through keratinocyte proliferation and migration [2,47]. Similar observations where plantderived AgNPs promoted wound healing via migration of fibroblasts [48,49] and keratinocytes [50] have been previously reported. In HaCaT and CHO cells, Cotyledon-AgNPs (at 2.5 µg/mL) exhibited more activity than the water extract, in-fact at this concentration, Cotyledon-AgNPs had more activity than the Positive Control (treatment with 15 µg/mL allantoin) (Figures 2 and 3). Chinnasamy et al. 2019 reported similar results with Melia azedarach synthesized AgNPs which exhibited increased wound healing activities compared to the M. azedarach extract alone [51]. This might be due to the nanoparticles' small size which increases their surface area to volume ratio thus increasing their activity [2]. It might also be due to phytochemicals present on the nanoparticles (such as polyphenols and flavonols as shown in Table 1) which can increase their bioavailability and enhance their activity [52]. In KMST-6 cells, however, the water extract showed more activity than the Cotyledon-AgNPs. However, considering that a lower concentration of the AgNPs (2.5 µg/mL) was used; the latter statement cannot be confirmed. Although emphCotyledon-AgNPs were more active than the C. orbiculata water extract, the activity of the extract was also significant as it showed good activity at very low concentrations (≤15 µg/mL). These findings show that C. orbiculata most likely contains compounds with highly active wound-healing properties, and therefore, provide some scientific evidence supporting its traditional use. Other medicinal plants were also recorded to have good wound healing activity, these include Plantago australis [53], Spirulina platensis [54], Alternanthera sessilis [55], Commiphora molmol, Aloe vera [56] and Achillea eriophora [57]. Figures 2 and 3 show that all cell lines exhibited different migration rates. It can be noted that complete gap closure of HaCaT, CHO and KMST-6 cells took 48, 72 and 96 h, respectively ( Figure 3). The daily percentage wound closure rates were determined using the equation stated in Section 3.5.3, these rates correspond to the findings from the scratch assay ( Figure 2). After exposure to the C. orbiculata water extract and the Cotyledon-AgNPs, HaCaT and CHO cells responded in a similar pattern, while the response of KMST-6 cells was different. In these cells, the wound closure rates were increased in the first 24 h compared to KMST-6 cells which had a more evenly spread cell migration pattern. The results in Figure 3 where almost complete wound closure was observed in HaCaT cells after day 1 corresponds to the increased growth rate in Cotyledon-AgNPs (2.5 µg/mL) treated HaCaT cells after 24 h as shown in Figure 1. assay ( Figure 2). After exposure to the C. orbiculata water extract and the Cotyledon-AgNPs, HaCaT and CHO cells responded in a similar pattern, while the response of KMST-6 cells was different. In these cells, the wound closure rates were increased in the first 24 h compared to KMST-6 cells which had a more evenly spread cell migration pattern. The results in Figure 3 where almost complete wound closure was observed in HaCaT cells after day 1 corresponds to the increased growth rate in Cotyledon-AgNPs (2.5 µ g/mL) treated Ha-CaT cells after 24 h as shown in Figure 1.

Gene Expression Studies
KMST-6 (fibroblasts) cells were used for gene expression studies. This is because fibroblasts are critical in all stages of wound healing and are involved in key processes such as extracellular matrix production, collagen deposition and wound contraction [58,59]. Gene expression studies investigating the expression levels of 86 wound healing-related genes were conducted on KMST-6 cells. In Cotyledon-AgNPs treated cells, only 17 of the 86 genes were differentially expressed ( Figure 4). Eight (CDH1, COL14A1, EGF, FGA, FGF10, ITGB1, ITGB6, PTGS2) of the differentially expressed genes (DEGs) were upregulated while the other nine (CCL2, CTGF, CXCL2, FGF2, HBEGF, IL6, ITGA2, MMP2, SERPINE1) were downregulated (Figures 4 and 5). Genes with a fold change of ≥±1.5 and p-values of <0.05 were considered DEGs. DAVID and STRING analysis clustered DEGs into three different groups ( Figure 6). Cluster A consisted of five downregulated genes (CCL2, CXCL2, IL6, MMP2, SERPINE1) and one upregulated gene (PTGS2). These five genes are involved in the inflammatory phase of wound healing. Cluster A genes promote inflammation, mostly via leucocyte chemotaxis, cytokine production and prostaglandin production [60][61][62]. Although inflammation plays an important role during the initial stages of wound healing, it is known that extended inflammation can lead to chronic wounds [63]. Based on the gene expression study which shows that most of the genes involved in inflammation are downregulated it can be concluded that the Cotyledon-AgNPs have anti-inflammatory activity. This is in agreement with our previous study which demonstrated the anti-inflammatory activity of Cotyledon-AgNPs in THP-1 macrophages [25]. PINE1) were downregulated (Figures 4 and 5). Genes with a fold change of ≥±1.5 and pvalues of <0.05 were considered DEGs. DAVID and STRING analysis clustered DEGs into three different groups ( Figure 6). Cluster A consisted of five downregulated genes (CCL2, CXCL2, IL6, MMP2, SERPINE1) and one upregulated gene (PTGS2). These five genes are involved in the inflammatory phase of wound healing. Cluster A genes promote inflammation, mostly via leucocyte chemotaxis, cytokine production and prostaglandin production [60][61][62]. Although inflammation plays an important role during the initial stages of wound healing, it is known that extended inflammation can lead to chronic wounds [63]. Based on the gene expression study which shows that most of the genes involved in inflammation are downregulated it can be concluded that the Cotyledon-AgNPs have antiinflammatory activity. This is in agreement with our previous study which demonstrated the anti-inflammatory activity of Cotyledon-AgNPs in THP-1 macrophages [25].
As shown in Table 2, the downregulated genes function in a number of pathways, including TNF signaling and cytokine production, all of which lead to inflammation.      Cluster B contained three (CDH1, EGF, FGF10) upregulated and two (FGF-2, HB-EGF) downregulated genes. Genes in this cluster are mainly involved in epithelial cell proliferation and migration. The upregulated genes stimulate keratinocyte proliferation and migration and also increase the tensile strength of the new skin [64][65][66]. These genes also improve collagen construction and stimulate the formation of the extracellular matrix As shown in Table 2, the downregulated genes function in a number of pathways, including TNF signaling and cytokine production, all of which lead to inflammation. Cluster B contained three (CDH1, EGF, FGF10) upregulated and two (FGF-2, HB-EGF) downregulated genes. Genes in this cluster are mainly involved in epithelial cell proliferation and migration. The upregulated genes stimulate keratinocyte proliferation and migra-tion and also increase the tensile strength of the new skin [64][65][66]. These genes also improve collagen construction and stimulate the formation of the extracellular matrix (ECM) [64,67]. It is possible that the downregulated genes are downstream of the upregulated ones and may, therefore, be activated with increased exposure to the Cotyledon-AgNPs. Although the downregulated genes promote keratinocyte proliferation and migration, they also promote inflammation [68,69]. This might be the reason why they were downregulated. These genes modulate inflammatory cell recruitment and activation, particularly macrophages and fibroblasts [69,70]. Upon activation, fibroblasts produce and secrete pro-inflammatory cytokines (IL6, IL-1B, IL8), chemokines (CCL2) and prostanoids (prostaglandin E2) [71][72][73].
Cluster B genes highly correlate with those in cluster C, these genes function using the same pathways (PI3K-Akt signaling pathway, Regulation of actin cytoskeleton, Rap1 signaling pathway and Focal adhesion) as shown in Table 2. Group C genes are involved in cellular adhesion which regulates cell differentiation and migration [74]. Cell adhesion is the ability of cells to stick to each other or to the ECM through molecules such as collagen, fibronectin, and laminin [75]. It is essential in cell communication and regulation as well as in the development and maintenance of tissues. Cell adhesion is also essential in wound healing as it stimulates signals that regulate important wound-healing processes such as cell differentiation, migration and survival [74]. Integrins are the main mediators of cell attachment to the ECM. They form cellular receptors for the extracellular environment and adjacent cells [76]. Their binding to extracellular ligands such as fibronectin and laminin promotes intracellular signaling which eventually causes cell migration [77]. The upregulated integrin genes ITGB1, ITGB6 and FGA are not only essential in cell adhesion but also in angiogenesis. FGA leads to the formation of an insoluble fibrin matrix. The major function of fibrin is its involvement in the formation of blood clots, therefore, reducing blood loss. Fibrin also forms the temporary ECM in the wounded area and plays important roles in tissue repair and cell migration during angiogenesis [78].
The effects of the C. orbiculata water extract on wound healing genes were also determined. Of the 86 genes tested, only 11 (COL5A3, ACTC1, FGF7, WNT5A, ITGB6, TAGLN, ITGB1, VTN, ITGA4, CSF2, FGF10) were differentially expressed; all of them were upregulated (Figures 7 and 8). Two (FGF10, ITGB1) of these genes were also upregulated in Cotyledon-AgNPs treated cells ( Figure 5), both genes are involved in the proliferation and migration of epithelial cells. Both STRING and the DAVID software were used to analyze these results. These upregulated genes are separated into three clusters all of which promote cell proliferation and migration ( Figure 9). Cluster A genes (particularly FGF10 and WNT5A) are involved in epithelial cell proliferation and migration especially fibroblasts and keratinocytes. The WNT protein family is made up of glycoproteins that regulate cell proliferation, in this family WNT5A was discovered as a key regulator of fibroblast proliferation [79,80].
Cluster B genes are involved in cell adhesion mostly by integrin, leading to increased cell survival. Cluster C consists of two genes (ACTA1 and TAGLN) which are involved in cytoskeleton organization and cell motility [81]. Actin is a structural protein that makes up the cell cytoskeleton while TAGLN is an actin crosslinking protein [81,82]. These genes ultimately support cell differentiation, proliferation, and migration [83]. Genes differentially expressed in response to treatment with C. orbiculata water extract were in similar categories to those expressed by the Cotyledon-AgNPs (cluster B, FGF10 and cluster C, ITGB1), they function using similar pathways as shown in Tables 2 and 3. This shows that the C. orbiculata water extract and the Cotyledon-AgNPs promote wound healing using similar mechanisms which include cell proliferation and migration. This can confirm the results in Table 1 which show that the same phytochemicals present in the extract and responsible for the bioactivities are also present on the nanoparticles. Results obtained using the WST-1 assay (Figure 1) and the scratch assay ( Figure 2) support this finding. migration of epithelial cells. Both STRING and the DAVID software were used to analyze these results. These upregulated genes are separated into three clusters all of which promote cell proliferation and migration (Figure 9). Cluster A genes (particularly FGF10 and WNT5A) are involved in epithelial cell proliferation and migration especially fibroblasts and keratinocytes. The WNT protein family is made up of glycoproteins that regulate cell proliferation, in this family WNT5A was discovered as a key regulator of fibroblast proliferation [79,80].     Cluster B genes are involved in cell adhesion mostly by integrin, leading to increased cell survival. Cluster C consists of two genes (ACTA1 and TAGLN) which are involved in cytoskeleton organization and cell motility [81]. Actin is a structural protein that makes up the cell cytoskeleton while TAGLN is an actin crosslinking protein [81,82]. These genes ultimately support cell differentiation, proliferation, and migration [83]. Genes differentially expressed in response to treatment with C. orbiculata water extract were in similar categories to those expressed by the Cotyledon-AgNPs (cluster B, FGF10 and cluster C, ITGB1), they function using similar pathways as shown in Table 2 and Table 3. This shows that the C. orbiculata water extract and the Cotyledon-AgNPs promote wound healing using similar mechanisms which include cell proliferation and migration. This can confirm the results in Table 1 which show that the same phytochemicals present in the extract and responsible for the bioactivities are also present on the nanoparticles. Results obtained using the WST-1 assay (Figure 1) and the scratch assay ( Figure 2) support this finding.

Plant Extract Preparation and Synthesis of Cotyledon-AgNPs
The plant extracts and Cotyledon-AgNPs used in this study were prepared as described previously [25].

TPC
The TPC of the C. orbiculata water extract and the Cotyledon-AgNPs was determined using the Folin-Ciocalteu method [84]. In a 96-well plate, 25 µL of the samples and standards were mixed with 125 µL of the Folin-Ciocalteu reagent (10%). After 5 min, 100 µL of sodium carbonate was added to the mixture. The plates were incubated for 2 h at room temperature and then read using a spectrophotometer at an absorbance of 765 nm.

Total Flavonoid Content (TFC) The Flavanol Content
The flavanol content was determined using the method by [85] with modifications. In short, 50 µL of the C. orbiculata extract and Cotyledon-AgNPs were mixed with 250 µL of 4-Dimethylamino-cinnamaldehyde. The mixture was incubated for 30 min at room temperature after which the absorbance was read at 640 nm. Results were expressed as milligram catechin equivalent per gram (mg CE/g).

The Flavonol Content
The flavonol content was also determined according to the method in [85], with modifications. Briefly, 12.5 µL of 0.1% HCl in methanol and 225 µL of 2% HCl solutions were added to 12.5 µL of the C. orbiculata extract and Cotyledon-AgNPs. The mixture was incubated for 30 min at room temperature and the absorbance was read at 360 nm. Results were expressed as milligram quercetin equivalent per gram (mg QE/g).

Antioxidant Studies
The antioxidant activity of the C. orbiculata water extract and the Cotyledon-AgNPs was determined using the most common colorimetric assays namely FRAP and ABTS.

FRAP
This method is based on the ability of antioxidants to reduce the ferric ion (Fe 3+ ) to ferrous ion (Fe 2+ ). In the presence of antioxidants at low pH, the ferric tripyridyl triazine complex (colorless) is reduced to its ferrous form which is a blue-colored complex. The ferric-reducing power of the C. orbiculata water extract and Cotyledon-AgNPs was evaluated according to the method developed by Benzi and Strain in 1996 [86]. The FRAP reagent was prepared by mixing 10 mL of acetate buffer (300 mM), 1 mL of 2, 4, 6-tripyridyl-Striazine (TPTZ) (10 mM) and 1 mL of FeCl 3 ·6H 2 O (20mM). In a 96-well plate, the FRAP solution (300 µL) was mixed with 10 µL of the samples (C. orbiculata water extract and Cotyledon-AgNPs) and incubated at 37 • C for 30 min. A spectrophotometer was used to measure the absorbance readings at 593 nm. Ascorbic acid was used in the preparation of the standard solutions. The absorbance of the samples was compared to a standard curve and the values were expressed as µmol AAE/g.

ABTS
The ABTS assay is used to measure the total antioxidant activity of substances. It is based on the ability of antioxidants to scavenge the ABTS (2,2 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) radical cation relative to the Trolox standard. This assay was performed according to the method used by Dube et al. 2017 [84]. Briefly, the ABTS radical mixture was prepared by mixing 5 mL ABTS (7 mM) and 88 µL potassium peroxodisulfate (K 2 S 2 O 8 ) (140 mM). The mixture was incubated in the dark for 24 h at room temperature and was diluted with ethanol to read an absorbance of approximately 2.0 (±0.1) at 734 nm, as the control; 300 µL of the diluted ABTS radical mixture was added to 25 µL of the C. orbiculata extracts, Cotyledon-AgNPs and standard in 96 well plates. The plates were incubated for 30 min at room temperature and absorbance readings were taken at 734 nm. The results were expressed as µmol TE/g.

Cell Culture
HaCaT, KMST-6 and CHO cells were obtained from the DSI/Mintek NIC (Nanotechnology Innovation Centre) laboratory at the University of the Western Cape (South Africa). The HaCaT and KMST-6 cells were cultured in Dulbecco's modified eagle medium (DMEM) supplemented with 10% Fetal bovine serum (FBS) and 1% Penicillin/Streptomycin. The CHO cells were grown in a Hams-F12 medium containing 10% FBS and 1% Penicillin/Streptomycin. All the cells were maintained in a humidi-fied atmosphere of 5% CO 2 in a 37 • C incubator (SL SHEL LAB, Sheldon manufacturing, Cornelius, OR, USA).

Cell Viability and Growth Rate
Both cell viability and growth rate were determined using the WST-1 (Sigma-Aldrich, St. Louis, MO, USA) assay according to the method by [87]. Cells were seeded in 96 well plates at a density of 1 × 10 5 cell/mL and incubated for 24 h. The growth media was replaced with a medium containing C. orbiculata water extract and Cotyledon-AgNPs at different concentrations and the cells were further incubated for 24 to 72 h. After incubation, the conditioned media was replaced with 10% WST-1 reagent (diluted in respective medium) and then plates were incubated for an additional 3 h. The absorbance was measured at 440 nm (reference 630 nm) using a microplate reader (POLARstar Omega plate reader, BMG-Labtech, Ortenberg, Germany). Cell viability was expressed as a percentage of the absorbance of treated cells to control (untreated) cells. The cell growth rate was determined using cell viability; 100% cell viability (untreated cells) was used as the control for growth rate with a value of 1.

Scratch Assay
The scratch assay was determined using a method by [55], with modifications. HaCaT, KMST-6 and CHO cells were seeded in 24 well plates at a density of 2 × 10 5 cell/mL and were incubated for 24 h at 37 • C in 5% CO 2 . After 24 h, a monolayer of confluent cells was observed. To create a scratch, the cell monolayer was scraped in a straight line using a sterile pipette tip. Cellular debris was removed by washing the wells with fresh medium. The cells were incubated at 37 • C in the presence of C. orbiculata water extract and the Cotyledon-AgNPs. The concentrations of C. orbiculata water extract and the Cotyledon-AgNPs were 15 and 2.5 µg/mL, respectively. Allantoin (15 µg/mL) which is a plant-derived commercial drug and known to induce cell growth was used as a positive control [55]. Untreated scratched cells were used as the negative control. All treatments and controls were placed in a medium containing 1% FBS. The assay was conducted in triplicate. Wound closure was monitored at different time intervals using a digital light microscope (EVOS XL Core Cell Imaging System, CA, USA). Images were analyzed using the Image J software. The percentage of wound closure was obtained using the equation, Wound closure (%) = Wound area (0 h) − Wound area (t h) Wound area (0 h) × 100

Gene Expression Studies
KMST-6 cells were seeded at a density of 2 × 10 5 cell/mL in a 25 cm 2 cell culture flask, the flasks were treated with C. orbiculata water extracts (15 µg/mL) and Cotyledon-AgNPs (2.5 µg/mL). Untreated flasks were used as the control. This was conducted in triplicate. The cells were collected into Eppendorf tubes and prepared for RNA extraction. Total RNA was extracted from the untreated and treated cells using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The concentration of the RNA was determined using a Qubit ® 2.0 Fluorometer (Invitrogen by Life Technologies, Carlsbad, CA, USA) according to the manufacturer's protocol. cDNA was synthesized from RNA using the RT2 First Strand Kit (Qiagen, Hilden, Germany) following the manufacturer's protocol. The produced cDNA was used as a template for RT-qPCR which was conducted using the Human wound healing RT2 Profiler PCR Array (Qiagen, Hilden, Germany) following the manufacturer's instructions. The experiment was carried out on the Roche LightCycler 480 (Roche, Basel, Switzerland) in the department of the Institute of Microbial Biotechnology and Metagenomics (IMBM) at the University of the Western Cape. Data collected from the LightCycler 480 was analyzed at GeneGlobe Data Analysis Center under Qiagen.
The relative changes in gene expression were calculated using cycle threshold (CT) values. The CT values were first normalized to those of used housekeeping genes. The fold change was then calculated using the 2 −∆∆CT formula adapted from Livak and Schmittgen [88]. Genes with a fold change of ≥±1.5 and p-values of <0.05 were considered as DEGs and were used for further analysis. The Database for Annotation, Visualization and Integrated Discovery (DAVID; version 6.7) and Search Tool for the Retrieval of Interaction Genes/Proteins (STRING; https://string-db.org/, accessed on 5 November 2022) pathways were used to further analyze the different interactions of the DEGs.

Statistical Analysis
Statistical analysis of the data was conducted using the GraphPad Prism 6 software. All assays were performed in triplicates and the results were expressed as mean ± standard error of the mean (SEM). Statistical significance was indicated as * for p < 0.05, ** p for < 0.01, *** p < 0.001 and **** for p < 0.0001.

Conclusions
It can be concluded that the medicinal plant, C. orbiculata possesses antioxidant and wound-healing activities. Both the C. orbiculata water extract and the Cotyledon-AgNPs showed good antioxidant activities which can increase their wound healing capabilities. They both increased the growth of HaCaT, KMST-6 and CHO cells at low concentrations and promoted cell migration in these cells in the scratch assay. The activity of the Cotyledon-AgNPs can be attributed to their small size which increases their surface area to volume ratio, hence increasing their activity. It might also be due to phytochemicals present on the nanoparticles which can increase their bioavailability and enhance their activity. Gene expression studies confirmed that the C. orbiculata water extract and Cotyledon-AgNPs promote wound healing through the activation of genes involved in the proliferation phase of wound healing. C. orbiculata extract and Cotyledon-AgNPs promote keratinocyte and fibroblast proliferation and migration by upregulating genes such as FGF7 and FGF10. Cotyledon-AgNPs are also involved in hemostasis which promotes clotting thus reducing blood loss. While C. orbiculata has been used in traditional medicine to treat skin conditions and wounds, this study scientifically shows that this plant contains wound-healing properties. We previously demonstrated that Cotyledon-AgNPs have significant antibacterial activity against microorganisms that commonly infect wounds. We also showed previously that these nanoparticles have anti-inflammatory activity. The current study shows that the Cotyledon-AgNPs also promote the growth of keratinocytes and fibroblasts at specific concentrations. We have also shown that these nanoparticles are not toxic at these concentrations. Taken together these findings demonstrate that Cotyledon-AgNPs have immense potential for application as novel wound healing agents.  Data Availability Statement: Supporting data presented in this study are available on request from the corresponding author.