Assessment of Wound Healing Activity of Green Synthesized Titanium Oxide Nanoparticles using Strychnos spinosa and Blighia sapida

The biosynthesis of nanoparticles has been proposed as a cost effective and environmentally friendly alternative to chemical and physical methods. The present work investigates the synthesis of titanium oxide nanoparticles (TiO2 NPs) by green approach using Strychnos spinosa and Blighia sapida leave extracts. The detailed characterization of theTiO2 NPs was carried out using UV-Visible Spectroscopy, Scanning Electron Microscopy (SEM), Xray Diffraction (XRD), and Fourier-Transform Infrared (FTIR) Spectroscopy. The green synthesized TiO2 NPs excitation was confirmed using UV–Vis spectrophotometer at 270 and 290 nm for Strychnos spinosa and Blighia sapida respectively. SEM revealed that the synthesized TiO2NPs were spherical and crystalline in nature. The overall sizes are 40 and 50 nm for Strychnos spinosa and Blighia sapida respectively. FTIR spectroscopic analysisshowed the presence of flavonoids, polyphenols and amide groups likely to be responsible for the green synthesis of titanium oxide nanoparticles usingS. Spinosa and B .sapida aqueous leaf extracts. The XRD pattern showed the characteristic Bragg peaks of (111), (200), (220) and (311) facets of the anatase titanium oxide nanoparticles and confirmed that these nanoparticles were crystalline and spherical in nature. Furthermore, the green synthesized TiO2 NPs wound healing activity was examined in the excision wound model by measuring wound closure, histopathology and protein profiling. This revealed significant wound healing activity in Albino rats. In the present study, topical application ofnanoformulated extracts of B. sapidaa nd S. spinosa significantly accelerated wound healing with 20% nanoformulated ointment having the highest percentage wound contraction ability comparable with gentamicin (a commercially sold antimicrobial agent used in dressing wounds). In conclusion, this work proved the capability of using TiO2 NPs to deliver a novel therapeutic route for wound treatment in clinical practice. DOI: https://dx.doi.org/10.4314/jasem.v24i2.2 Copyright: Copyright © 2020 Hassan et al. This is an open access article distributed under the Creative Commons Attribution License (CCL), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Dates: Received: 16 November 2019; Revised: 11 January 2020; Accepted: 22 February 2020

One of the main objectives in wound healing is restoration in the shortest time with minimal side effects. Today, infection is considered as one of the main causes of mortality owing to wounds, especially after surgery. The management of these wounds is an ongoing problem which places a considerable drain on healthcare systems. Many microbiological studies have reported that S.aureus, streptococci and P. aeruginosa are the most frequently identified aerobic pathogens causing invasive wound infection. (Cho et al., 2005;Naheed et al., 2009;Shiriashi and Hirai 2008). S. aureus is a key human wound-infecting pathogen that interferes with cell functions of the host and causes delayed epithelial closure of the wound, possibly due to its interaction with fibronectin and inhibition of keratinocyte migration (Shah et al., 2008). Antibiotic resistant bacterial strains with different mechanisms are found continually and thus new drugs are required (Bali et al., 2006). Therefore, the finding of new antimicrobial agents with novel mechanisms of action is essential and extensively pursued in antibacterial drug discovery (Coates et al. 2002). Nanotechnology puts together the capabilities to manage the properties of materials by controlling their size range usually from 1-100nm (Prakash et al., 2013). This has motivated carrying out research into numerous potential uses for nanomaterials. An ideal material should be able to protect wounds against microbial interactions. For centuries, metals and metallic oxides including silver, titanium and zinc have been applied as bactericidal and bacteriostatic agents, each with different properties and spectrums of activity. TiO2 nanoparticles have become a new generation of advanced materials due to their novel and interesting optical, dielectric, and photo-catalytic properties from size quantization (Luoet al., 2007).The present study was designed to determine the efficiency of TiO2 NPs against microbial activityon surgically induced full thickness skin wounds inoculated with S. aureus and deep bacterial load of wounds local infection in albino rat. Preparation of the plant extract: The leaves of B. sapida and S. spinosa collected were thoroughly washed and rinsed in distilled water, and then room dried for two weeks. After which hands were used to break them into fine pieces. Then 500 g of each plant was smashed into 1000 ml of sterile distilled water in a gas jar and boiled at 60 o C for 15mins and then filtered through Whatman No.1 filter paper. The percentage yield of the plant extracts are 16% and 15.5% for B. sapida and S. spinosa respectively. The aqueous extracts were stored at 40 o C in an incubator for further experiments (Liu et al., 2013).

Phytochemical screening of plant extracts:
The extracts were subjected to tests for secondary metabolites such as tannins, flavonoids, steroids, glycosides, alkaloids, glycosides and saponins. The tests were carried out using standard methods of analysis (Trease and Evans, 2002).Analyses were done in triplicate.

Synthesis of TiO2 nanoparticles from the extracts:
To prepare an aqueous solution of 1.0M TiCl4, 5.0 ml of TiCl4 was measured using a suction pipette, and mixed with 100 ml of distilled water in a 250 ml Erlenmeyer flask. The content was swirled properly. The mixture was then stored at 40 o C before use. To a 100 ml portion of each of the leaf extracts of B. sapida and S. spinosa, 10 ml of the 1.0 M TiCl4 solution was added in dropson a water bath at a constant temperature of 70 o C for a period of 4 hours with constant stirring at 1000 rpm. The suspension produced was centrifuged at 2000 rpm for 20 mins and the supernatant liquid decanted.The residue was repeatedly washed with de-ionized water. Centrifugation, decantation and washing processes were repeated thrice to remove any impurity from the surface of the titanium oxide nanoparticles (Prakashet al., 2013). The precipitate obtained was dried in an oven at a temperature of 40 o C for 30 mins (Kalyani et al., 2006). The synthesized titanium dioxide nanoparticle samples were then subjected to characterization by UV-Vis, FTIR, XRD and SEM.
Uv-vis spectrophotometry determination: About 10 ml each ofplant extracts, plant extracts mediated titanium oxide nanoparticlesand commercial TiO2 nanoparticles sample in colloidal solutions were separately placed in the cell holder of a UV-Vis spectrophotometer (model UV 1800 Shimadzu, Japan), in order to determine the absorption spectrum of the sample using the range between 200 to 800 nm. Colloidal solution was obtained by mixing warm distilled water with synthesized TiO2 (Liu et al., 2013).
Fourier-transform infrared (FTIR) spectroscopy analysis: The determination of the functional groups on the surface of the plant extract mediated titanium oxide nanoparticles was investigated by using a FTIR spectrophotometer ((Perkin Elmer Spectrum 2, Germany), and the spectra were scanned in the range of 4000 -400 cm −1 at a resolution of 4 cm -1 . The samples were prepared by dispersing each of the biosynthesized titanium oxide NPs and commercial TiO2 nanoparticles uniformly in a matrix of dry KBr, and then compressed to form an almost transparent disc. KBr was used as a standard analyte for the samples (Dhadapani et al., 2012).

Scanning electron microscopy (SEM) of the nanoparticle:
In order to determine the surface morphology of the synthesized nanoparticle, SEM machine (HITACHI Model S-3000H Japan) was used. Thin films of the samples (biosynthesized titanium oxide NPs and commercial TiO2 nanoparticles) were prepared on a carbon coated copper grid by just dropping a very small amount of each sample on the grid,; the film on the SEM grid was allowed to dry and the images of nanoparticles taken (Sundrarajan et al.,2011).
X ray diffraction (XRD) analysis of the nanoparticles: X-raydiffraction measurements of the biosynthesized titanium oxide NPs and commercial TiO2 nanoparticles were recorded on X -ray diffractometer (Philips Analytical). The phase variety, particle size and material identification of the NPs were identified.The samples were taken in lids and put under instrument for analysis (Dhadapani et al., 2012). Creation and contamination of excision wound: The dorsum of rats was shaved and disinfected with methylated spirit. Then, full thickness excision wounds were created following the method described by Morton and Malone (1992).For post wounding, the rats were randomly assigned into 6 groups of 5 animals per group. Then, using sterile Pasteur pipettes, wound on each animal was contaminated by flooding with 1 ml of standardized broth culture of Staphylococcus aureus. The animals were carefully placed in disinfected cages kept in a disinfected, clean and dustfree animal house in the Department of Biochemistry, Federal University of Technology Minna, Niger state, Nigeria. The wounds were not treated for 24 hr post contamination to ensure colonization and establishment of infection. Treatment of animal wound commenced 48 hr post contamination. Four treatment groups consisting of 5 animals per group were treated as follows: groups I and II were treated topically with 10% and 20% B sapida and S. spinosa leave extracts ointment respectively, groups III and IV were treated topically with 10% and 20% nanoformulated B sapida leaf extract mediated TiO2 nanoparticle ointment respectively, groups V and VI were treatedtopically with 10% and 20% commercial TiO2 nanoparticle ointment while groups V11 and VI11 were treated with gentamicin sulphate and sterile soft white paraffin, respectively. Treatment of the animals continued until complete healing occurred.

In vivo (clinical) antimicrobial tests (Wound
Percentage Wound Contraction: The wound diameter of each animal was measured at 3, 7, 10 and 14 days post wounding (dpw) using a transparent meter rule, and the percentage wound contraction calculated using the method of Ezike et al. (2010).
Determination of wound microbial load: At 3 and 14 days post-treatment (dpt), wound swabs from each animal was taken in duplicate using sterile swab sticks. The total viable count of the wound was determined following the standard plate counting method. Briefly, each swab was inoculated into sterile nutrient broth to make stock solutions which was used for double-fold (10 -2 ) dilutions. Using a sterile pipette, 0.1 ml of the 10 -2 dilution was introduced on the surface of sterile nutrient agar and a sterile glass spreader was used for even distribution of the inoculum. The inoculated plates were incubated at 37°C for 24 hours and the colonies were counted (Ezikeet al. 2010).

Phytochemical
Analysis: The preliminary phytochemical analysis was studied (Table1) and results showedthe presence of metabolites.Flavonoids and glycosides were present in both extracts of S. spinosa and B. sapida. While alkaloids and tannins were only present in S. spinosa, saponins and steroids were only found in B. sapida. This revealed pronounced importance of phytochemical compounds because the crude extracts possessed varied composition of secondary metabolites in the form of phytochemicals. Similar results were also reported by Salam et al. (2016) in the latex of Calotropisprocera. The pharmacological action of crude drugs and other therapeutic uses are due to their therapeutically active constituents such as tannins, flavonoids, alkaloids and several other aromatic compounds or secondary metabolites of plants which serve as defense mechanism against predation by many microorganisms, insects and herbivores. So, this phytochemical screening serves as initial step in predicting potential of active compounds.

Physical Properties of Biosynthesized Blighasapida and Strychnosspinosa Mediated Titanium Dioxide:
The biosynthesized B. sapida and S. spinosamediated titanium dioxide nanoparticles were primarily confirmed by the contrast of colour changes of greenish to dark brown and reddish black colour for B. sapida and Strychnosspinosa mediated titanium dioxide nanoparticles respectively, indicating the synthesis of TiO2 NPs. The generation of colour is due to excitation of surface plasmon in the metal nanoparticles. Similar results were also reported earlier for synthesis of titanium dioxide nanoparticles using Azadirachtaindica leaf extracts (Sankar et al., 2015) and also with C. sinensis extract (Bali et al., 2006). TiO2 NPs were observed to be stable in solution (Ahmad et al., 2016).

UV-Visible analysis:
The UV-Vis spectroscopy was used to determine the formation and the stability of the synthesized titanium oxide nanoparticles in aqueous colloidal solution. It is also used to predict the initial phytoconstituents in plant material. The UV spectrum of the prepared biosynthsised TiO2 nanoparticles and commercial titanium oxide Npsarepresented in Figure  1, 2 and 3 indicatingthat they all display maximum absorption in the vicinities of 400-800nm. The spectrum showed the formation of peak in the wavelength of 229 nm for the B. sapidamediated NPs, a peak in the wavelength of 231 nm for the S. spinosamediated counterpart, and a peak of 430 nm for commercial titanium oxide nanoparticles. The UV-Vis spectra of plant extracts of biosynthsised TiO2 nanoparticlesusing S. spinosa and B. sapida had high absorbance of 3.475 and 3.678 respectively compared tocommercial titanium oxide nanoparticles which had1.27. This was because the various metabolites from plant extract introduced to solution made the plasmon band broad and they may be read in the spectrophotometric range with surface plasmon resonance (SPR) which is responsible for exhibiting the absorption of UV-Vis radiation (Kim et al., 2013). These wave lengths arise due to the surface Plasmon resonance of the particle (Joshin et al., 2008). The magnitude of peak, wavelength and spectral bandwidth associated with nanoparticles are dependent on size, shape and material composition (Prakash et al., 2013, Kim et al., 2013. These changes in their properties increased their interacting faces thereby considered as enhancement in terms of absorbance spectrum in the UV-Vis region. Commercial TiO2Nps had the least absorption as indicated in Figure 3.This was similar to the trend obtained by Salam et al.(2016) in the work done on synthesised Citrus paradisi peel extract mediated titanium dioxide nanoparticles andbiological synthesis of TiO2 nanoparticles using extracts of Ananascomosus (Anwar et al., 2010;Wilkinson et al., 2011).  showed characteristic absorption bands at 3220cm -1 (hydroxyl (-OH) group), 1617cm -1 (for (C=O stretching), absorption at 1282 cm -1 (for C-N stretching) and at 1438 cm -1 for alkyl group.  S. spinosamediated TiO2 NPs: The characteristic absorption bands were exhibited at 2920cm -1 (for C-H stretching) and at 1602.8 cm -1 for carbonyl group (C=O), and at 3678.9 cm -1 for Hydroxyl (-OH) group; Amineat 2920cm -1 and 1513 cm -1 for (C=C) stretching vibration.
Commercial TiO2 NPs: Figure 6 indicated that characteristic absorption bands were exhibited at 3712-3768cm -1 for hydroxyl (-OH) group and at 1654 cm -1 for carbonyl group (C=O) (Anwar et al., 2016). From the FTIR results, there were presence of hydroxyl groups of phenols, carbonyl group (C=O), alkyl group. Bali et al.(2006) reported same with amide group; these formedlayers on the nanoparticles and acted as capping agent andprovidedstability on extract of N. tobacuumleaves mediated silver NPs. The HASSAN, H; OMONIYI, KI; OKIBE, FG; NUHU, AA; ECHIOBA, EG; EGWIM, EC functional groups in the FTIR results support the presence of phenolic compounds (flavonoids) in the extracts as evidenced by phytochemical analysis. Raymond et al. (2009) reported that the band at 1742 cm -1 is characteristic of stretching vibrations of the carbonyl functional group in ketones, aldehydes and carboxylic acids in the study on Micrococcamercurialis. Based on these FTIR studies, it can be suggested that the biomolecules present in the plant extract mediated TiO2 Nps play dual role in formation and stabilization to TiO2 nanoparticles.   The green synthesized TiO2 nanoparticles showed monodispersity without aggregation when compared to that of the commercial TiO2 nanoparticles. This is due to the capping of TiO2 nanoparticles with the compounds present in the leave extracts. The particles were found to be spherical with undifferentiated shapes.Previous reports showed that phytochemical compound in nanoparticles are disaggregated and are stable (Nour et al., 2010). Prathna et al. (2010) supports the present findings in the work on Origanumvulgare aqueous leaf extract. It could therefore be speculated that the phytochemicals in these leave extracts coat the surface of the TiO2 nanoparticles thus preventing their aggregation. The SEM images showed average sizes of 50 nm, 40 nm and 72nm for biosynthesized TiO2 nanoparticles obtained using leave extracts of S. spinosa, B. sapida and commercial TiO2 nanoparticles respectively. Titanium dioxide nanoparticles of approximate diameter of 50 and 60 nm have been previously reported (Raymond et al., 2009). The TiO2-NPs of these sizes showed that nanoparticles produced by this method were primarily crystallline (Singhal et al., 2011;Ismagijov et al., 2009). This is in agreement with previous reports fromAhmed et al. (2011).

X-ray Diffractometric analysis (XRD):
The X-ray diffraction was used to confirm the chemical composition and crystalline nature of the synthesized nanoparticles. The XRD pattern of TiO2 nanoparticles obtained using leave extracts of B. sapida, S. spinosa, and commercial TiO2nanoparticles are shown in Figure 10, 11 and 12 respectively. A sharp diffraction peak was observed in Commercial TiO2 nanoparticles, whereas, the intensity of diffraction peak of green synthesized TiO2 nanoparticles are less with slight broadening. The intense sharp peak indicated that the Assessment of Wound Healing Activity of Green Synthesized…..

X-ray Diffractometric analysis (XRD):
The X-ray diffraction was used to confirm the chemical composition and crystalline nature of the synthesized nanoparticles. The XRD pattern of TiO2 nanoparticles obtained using leave extracts of B. sapida, S. spinosa, and commercial TiO2nanoparticles are shown in Figure 10, 11 and 12 respectively. A sharp diffraction peak was observed in Commercial TiO2 nanoparticles, whereas, the intensity of diffraction peak of green synthesized TiO2 nanoparticles are less with slight broadening. The intense sharp peak indicated that the crystalline phase of anatase TiO2 was successfully formed (Ismagijov et al., 2009).  (215) and (224), respectively. The crystallite size for the prepared samples was determined by measuring the broadening of a most intense peak of the phase (main peak) in a diffraction pattern according to Debye-Scherrer equation as follows: Where D is the crystallite size of the particle, θ is the Bragg diffraction angle, β is the full width at halfmaximum (FWHM), λ is the wavelength (0.15418 nm) and k (0.94) is a constant. Theintensity of the diffraction peak of the green synthesised TiO2 nanoparticles was broadened, whereas the peak of commercial TiO2 nanoparticles was comparatively sharp. Ahmed et al. (2016) have reported the correlation between XRD peak broadening and the size reduction during green synthesis protocol. Thus, the broadening of XRD peak of biosynthesized TiO2 nanoparticles observed in the study confirmed the size reduction. The XRD peak of commercial TiO2 nanoparticles is sharp, thus indicating that their size is still larger than the green synthesised TiO2nanoparticles.
Phytochemicals present in the leave extract ofS. spinosa, and B. sapida would have coated the surface of the TiO2 nanoparticles, resulting in decreased intensity in XRD peak. This phytochemical coating may enhance the stability of the nanoparticles, which in turn may enhance their bioavailability, making them suitable for biological applications. Commercial TiO2 nanoparticles on the other hand showed comparatively high intense peaks, clearly indicating that they are uncapped. These results are in agreement with the report of Ahmed et al. (2016) and Singhal et al.(2011).The XRD patterns obtained were matched with the database of JCPDS No: 21-1272; the crystallite size obtained by using this formula ranged between 40 nm and 50 nm for the synthesised TiO2 nanoparticles. The result of this study is similar to that obtained for the synthesis of titanium dioxide nanoparticles mediated with aqueous leaf extract of Ecliptaprostrata that recorded crystallite size in the range of 36 to 68 nm with an average grain size of 49.5nm (Kim et al., 2013).   Table 2. The wound treated with synthesized TiO2NPs indicated no evidence of pus formation, bleeding and microbial infections at any times and wounds heal without any complications whereas the control rats showed persistent inflammation The TiO2 NPs treated wound revealed a significant wound contraction on 4 th day onwards and the upcoming days wound closure was much faster when compared with control animals. On the 14 th day the TiO2 NPs treated animals showed most wound closure (94 %), while control animals exhibited 86 % wound closure. Significant wound closure and reduced wound size in the treated animals demonstrates the efficacy of green synthesized TiO2 NPs on wound healing agent (Bidgoli et al., 2013;Matei et al., 2008). Topically administered TiO2 NPs are effective in faster wound closure which might be due to the large availability, enhanced fibroblasts and collagen action in regenerated wound tissues (Tian et al., 2007;Bidgoli et al., 2013). Myofibroblasts are believed to play a key role in wound contraction by exerting tension on surrounding extracellular matrix and secreting collagen which stabilizes the contraction (Sun et al., 2014).
Furthermore, the high surface area to volume ratio endows nanostructures with unique features contributed to the healing activities.For example, nanoscale particles provide a high probability of interaction with the biological target and an enhanced penetration into the wound site. As a result, nanoparticles have an ability to deliver a sustained and controlled release of therapeutics that result in an accelerated healing process. In view of the above reports, the wound contraction and healing effects of TiO2 NPsusing S. spinosa and B. sapida might be attributed to phytochemical substances present which are known to promote wound healing. The understanding and control of microbial infection of wounds is very important for better healing and its management (Campostrini et al., 2003). Postoperative wounds are usually infected by bacterial organisms (Deshmukh et al., 2009). The significant decreases in wound bacterial load in the treated groups suggest that the extract and TiO2 NPs synthesized has in vivo antibacterial effect against Staphylococcus aureus which is involved in wound contamination. The TiO2 NPs synthesized by the extracts were able to reduce wound bacterial load comparable to the reference drug, gentamicin. Signs of bacterial infection of wounds include delay in the onset of the proliferative and re-modelling phases of wound healing process due to the release of free radical and lytic enzymes at the wound site (Eming et al., 2007).
The absence of irritation and/or pain at wound site during treatment and the significant increase in the rate of wound contraction and wound re-epithelialization is a reflection of good antibacterial potentials of the TiO2 NPs as showed in the in vivo and in vitro antibacterial assay results. The antibacterial activity exhibited by theleave extracts could be attributed to the phytochemical constituents present in these extracts. (Eming et al., 2007).