RETRACTED: A Novel Herbal Hydrogel Formulation of Moringa oleifera for Wound Healing

Treatment of wounds is essential as the wound can also be lethal at some point in time if not healed properly. Ethnomedicinal plants can treat wounds as they have no side effects, whereas, in the case of chemical drugs, the side effects are on the rise. In this study, seeds of Moringa oleifera which is the essential ethnomedicinal plant, were studied for wound healing efficacy. The study was planned for the assessment of in vitro (antioxidant and antimicrobial activities) and in vivo (excision and incision wound healing models) wound healing efficacy of n-hexane extract and hydrogels of Moringa oleifera seeds. The antioxidant and antimicrobial activities were assessed by DPPH free radical scavenging assay and Agar well diffusion method, respectively. In excision and incision wound models, Swiss albino mice were used for wound healing efficacy of hydrogels, i.e., 5% and 10% hexane extracts of Moringa oleifera seeds. The n-hexane extract showed antioxidant as well as antibacterial activities. Moreover, the hydrogels formulated using n-hexane extract of Moringa oleifera seeds showed significant wound healing activity compared to both control and standard until the end of the protocol in both the models. Furthermore, the histopathological investigation confirmed the findings of accelerated regeneration of tissue accompanied by a decrease in inflammatory cells and increased vascularity of the immediate skin. The results (both in vitro and in vivo) claimed conclusively that our n-hexane hydrogel formulation of Moringa oleifera seeds might serve as an alternative therapy in skin restoration during wound healing.


Introduction
Wound healing is defined as an intricate and elaborate biological action initiated in response to an attack on the anatomy and functioning of normal healthy skin.The biopro- cess can be categorized into three major stages viz., inflammatory phase (0-3 days), cellular proliferation (2-12 days), and remodelling phase (3-6 months) [1][2][3].The acute inflammatory responses due to injury results in the necrosis of specialized cells as well as damage to the surrounding matrix, mitigated by substitution of the dead tissue with new healthy cells to aid faster tissue regeneration.However, the healing site is susceptible to microbial infections, a leading cause of delay in wound repair [4], and consequently, the patient's quality of life.The ideal wound healing process must achieve mitigation of tissue damage, ample tissue perfusion (nutrition and oxygenation) with a moist healing environment for the restoration of the anatomy and function of the affected region [5].Ayurveda, known as the Indian traditional system of herbal medicine, has given substantial importance to wound healing and the use of Indian medicinal plants to treat skin damage [6].
Moringa oleifera or horseradish, a medicinally important plant of genus Moringaceae, is mostly found in the sub-Himalayan region of North-Western India and is known for its nutritional and therapeutic ingredients in Ayurveda text to prevent, mitigate or treat any diseases or conditions.Traditionally seeds, fruits, leaves, and roots of this plant are used for the treatment of skin infections, helminthic, abdominal tumours, sores, prostates troubles, scurvy, hysteria, and paralytic attacks [7].M. oleifera has been studied for its antioxidant properties [8] as well as anti-fungal properties and activity against human infection-causing pathogenic microorganisms [9] leading to the development of a potable water purification kit [10].WHO has labelled the consumption of M. oleifera as a good source of food for the treatment of malnutrition due to its antioxidant and antimicrobial properties [11,12].
Phytochemical constituent analysis of the M. oleifera seed shows that this plant consists of all the essential constituents necessary for the wound healing activity [8,9,11,13].Furthermore, the wound healing efficacy of aqueous extract of pulp and seeds of M. oleifera in albino rats has been conducted by Rathi et al. [14].The current study illustrates the use of hexane hydrogel of M. oleifera seeds as an efficient wound healing alternative.

Results
Our study was aimed to evaluate the in vitro antioxidant and antimicrobial efficacy and in vivo wound healing potential of n-hexane extract of M. oleifera seeds and formulated n-hexane hydrogel of M. oleifera seeds, respectively, on Swiss albino mice.

In Vitro Antioxidant Activity
Antioxidant activity of n-hexane extract of M. oleifera seeds in the present study shows the highest scavenging at the concentration of 160 µg/mL and IC50 value of 162.4 as compared to 96.24 of standard ascorbic acid (Figure 1).

In Vitro Antimicrobial Activity
The present work elucidates that n-hexane extract of M. oleifera seeds possesses both gram-positive as well as gram-negative bactericidal potential and thus, can be used as a therapy to treat wound infections.The n-hexane extract shows a minimum zone of inhi-

In Vitro Antimicrobial Activity
The present work elucidates that n-hexane extract of M. oleifera seeds possesses both gram-positive as well as gram-negative bactericidal potential and thus, can be used as a therapy to treat wound infections.The n-hexane extract shows a minimum zone of inhibition of 12 mm against P.aureginosa, 14 mm against S. aureus, and 16 mm against E. coli compared to control (0 mm).The appearance of the zone of inhibition indicated that the n-hexane extract of Moringa oleifera seeds inhibited the growth of test pathogens, thereby validating the antimicrobial activity in n-hexane seed extract.

Excision Wound and Incision Wound Model in Mouse
For the evaluation of wound healing activity, four groups of animals were used.The first group controlled, the second was standard, third and fourth were 5% and 10% hydrogel of n-hexane extract of M. oleifera seeds, respectively.The digital photographs of the wound area of each treatment group taken on days 1, 4, 6, 9, 12, and 14th are presented in Figure 2.  Progress of wound healing was evaluated by measuring the wound closure rate (equation 1) [15].The wound closure rates are shown in Figure 3. Progress of wound healing was evaluated by measuring the wound closure rate (equation 1) [15].The wound closure rates are shown in Figure 3.
In the incision model, tensile breaking strength in grams was used to determine the wound healing efficacy of the M. oleifera n-hexane hydrogel on the 8th day using a tensiometer.The results of tensile strength are presented in Figure 4. Progress of wound healing was evaluated by measuring the wound closure (equation 1) [15].The wound closure rates are shown in Figure 3.In the incision model, tensile breaking strength in grams was used to determine the wound healing efficacy of the M. oleifera n-hexane hydrogel on the 8th day using a tensiometer.The results of tensile strength are presented in Figure 4.

Histopathological Study
The histopathological studies of the tissues of the excision and the incision model were performed.Figure S1 shows the histopathological characters of both excision and incision wound models.

Histopathological Study
The histopathological studies of the tissues of the excision and the incision model were performed.Figure S1 shows the histopathological characters of both excision and incision wound models.

Discussion
Ayurveda, Siddha, and Unani are the classical systems of Indian medicine which consists of various herbal plants for the treatments of skin diseases like cuts, wounds and burns.These medicinal plants have been used for a long time for the treatment of various skin ailments [16].The significant advantage of using ethnomedicinal plants for the wound treatment include no side effects as compared to the chemical drugs which have their side effects on the rise.The wound healing potential, including reduction, oxidative stress, and inflammation has been reported from other plants like Phlomis viscosa Poiret, resveratrol, curcumin, and Spirulina platensis [17][18][19].The present study aimed to evaluate n-hexane extract of M. oleifera seeds for its in vitro antioxidant activity and antimicrobial activity.Furthermore, the present study aimed to check the potential of hydrogel formulated using n-hexane extract of M. oleifera seeds in wound healing.

In Vitro Antioxidant Activity
A wound causes inflammation leading to the production of free radicals by phagocytes.Increased production of these free radicals delays the process of wound healing; thus, their inhibition might be one of the beneficial therapeutic strategies in the action of healing of wounds.Chitra et al. and Shirwaikar et al. have reported many plants that promote wound healing aided by the mechanism of free radical scavenging [20,21].The DPPH method is used for the detection of the free radical scavenging property of plant extracts, including its natural compounds at low concentrations.Antioxidant activity is measured using a concentration-response relationship of scavenging of DPPH free radicals as observed on treatment with hexane extract of M. oleifera seeds.The IC50 value for extract was found comparable with ascorbic acid due to the presence of flavonoids and phenolic constituents in M. oleifera seeds, as reported by Ndhlala et al., 2010 [22].There has been a tremendous interest in deriving antioxidants from natural resources rather than from synthetic sources.Phenolic compounds and tannins present in M. oleifera plant help in decreasing the chance of disease progression, hence associated with the antioxidant compounds [23].Olagbemide et al. and Leone et al. report various phenolic compounds (Gallic acid and flavonoids such as kaempferol, quercetin) in M. oleifera seeds [9,24].
Fitriana et al. and Wright et al. reported antioxidants properties of different extracts of M. oleifera leaves, where IC50 value was relatively higher as compared to IC50 value in the present study [23,25].Moreover, in the study by Wright et al., the IC50 value of all the M. oleifera extracts was found to be higher than the IC50 value of the present study.

In Vitro Antimicrobial Activity
Microbial infection of wound directly affects the wound healing process and may be associated with more than 1 type of bacterial and fungal infections.Undoubtedly, open wounds are prone to infections as broken skin comprises a highly time-variable complex microbiological environment with a mixed flora-an infected wound results in exudates formation and slowing of wound healing.Streptococcus species, S. aureus initially populate the wound before other bacteria such as E.coli take up residence, usually after days or even weeks.The wound left untreated will acquire additional bacterial growth such as of P. aeruginosa.Thus, antimicrobial treatment is a crucial measure in wound healing, and identification of the specific causative pathogen and their antibiotic sensitivity could serve as a valid and influential factor for wound treatment.In the presented study, the Agar well diffusion method was used for the determination of the antimicrobial activity of hexane extract M. oleifera seeds and results depicted inhibition of gram-negative and gram-positive bacteria.The formation of a zone of inhibition showed that the hexane extract of M. oleifera seeds inhibited the growth of test pathogens, thereby validating the antimicrobial activity in hexane seed extract.The hexane extract shows the minimum zone of inhibition of 12 mm against P. aureginosa, 14 mm against S. aureus, and 16 mm against E. coli compared to control (0 mm).
This wound healing efficacy is assumed to be due to phytoconstituents in terpenoids, terpenes, glycosides, saponins, flavonoids, phenols, alkaloids, and tannins in M. oleifera seeds [9,24,26].Previous reports show a direct role of flavonoids, i.e., complex formation with the soluble extracellular proteins and cell walls of bacteria [27].Other studies also validated antibacterial activities against gram-positive and gram-negative bacteria using different extracts of M. oleifera seeds [28,29].The zone of inhibition in various other studies of antibacterial activity of M. oleifera leaves various solvents was found to be less as compared to the reported in the present study with seeds [30].M. oleifera leaves extracts (petroleum, ethanol, methanol, chloroform, and aqueous) have been tested against P. Vulgaris, S. typhi, and S. aureus.All the extracts showed inhibition against these pathogens except petroleum ether extract which showed inhibition against P. aeruginosa.All the pathogens were resistant against the ethanolic extract except E. coli, P. Vulgaris, and S. typhi [31].
However, the present study reports the antibacterial activity in all the pathogens tested using the n-hexane extract of M. oleifera seeds.Thus, for any herb to be regarded as an excellent therapeutic entity for enhancing the process of wound healing, it must possess phytochemical constituents with antioxidant and antimicrobial properties.

Hydrogel Formulated
Hydrogels offer many advantages which included providing a necessary moist environment to the wound area, also acts as an excellent carrier for the topical application of various substrates and their sole release over some time.With all the information and studies on hydrogel formulation, it is noticeable that the hydrogels can be regarded as a suitable candidate to promote wound healing.In the presented study, we have prepared a hydrogel formulation with 5% and 10% hexane extract of M. oleifera seeds.Consequently, the proper hydrogel spreading would assist in the uniform administration of the gel to the skin.Additionally, based on our results, our formulated herbal gel contributed to a faster wound healing compared to the negative control group.Various studies on the formulation of hydrogel using hexane extracted also supported our choice of study [32,33].

Excision Wound and Incision Wound Model in Mouse
Wound restoration or contraction is shrinkage of wound area and mainly depends mostly on the limit and type of damage, essential health, and tissue repairing ability.The presented study evaluates the efficacy of hydrogel of n-hexane extract of M. oleifera seeds for its in vivo wound healing activity using two methods (excision and incision wound model).
The results showed healing of wounds up to 69-74% within four days in case of 10% and 5% hexane hydrogel respectively in comparison to 5% povidone-iodine standard and control by 26% and 18%, at any given point in time.The study also validated that wounds healed up to 97% and 98% on day 12 using 5% and 10% M. oleifera hexane hydrogel as compared to standard and control, which healed by 82% and 62%, respectively.Both the test groups were healed on the 13th day, whereas the standard and control group remained unhealed until the end of the protocol (Figure 3).
The test group observed significant activity as compared to both the controls (p < 0.001) until the end of the protocol, whereas with standard treatment.5% hexane hydrogel showed significant activity until the 12th day (p < 0.001) and day 13th day (p < 0.01).Additionally, 10% of hexane hydrogel showed significant activity until the 12th day (p < 0.001) and 13th day (p < 0.05).Both the test groups of hexane hydrogel of M. oleifera seeds showed equal effectiveness until the end of the protocol.
In the incision wound model both 5% and 10% hexane hydrogel were compared with control and standard, i.e., 5% povidone-iodine and the tensile breaking strength of both 5% hexane hydrogel (152 g) and 10% hexane hydrogel (156 g) were significantly higher in comparison to control (96 g) and standard (115 g) (p < 0.01).An increase in the concentration of collagen and fibre stabilization leads to increased tensile strength.Table 1 gives a brief about all the studies conducted on Moringa oleifera seeds concerning its wound healing activity.

S.No.
A study Conducted on Moringa oleifera Seeds Findings Reference

1.
Evaluation of aqueous extract of pulp and seeds of Moringa oleifera for wound healing in albino rats.The aqueous extract was studied at a dose level of 300 mg/kg body weight using resutured incision; excision and dead space wound models in rats The study included the use of systemically administered Moringa oleifera aqueous pulp and seed extract on the healing of excision, resutured incision and dead space wounds. [14] 2.

Anti-Inflammatory and Healing Activity of Seed Extracts of Moringa Oleifera Harvested In Tamanrasset (Algeria)
This study concluded the efficacy of the anti-inflammatory and healing power of polyphenol and saponins extracts of Moringa oleifera seeds.The study showed anti-inflammatory activity for saponins and polyphenol extracts with respective values of 28.16% and 23.61%.At the end of the study, the wounds treated with the extract of saponins demonstrated wound healing as compared to those treated with the extract of polyphenol.Madecassol ® , used as a reference, showed poor wound healing compared to the wounds of tries.
The saponin extract showed more effective as compared to the extract of polyphenol with significant healing power. [34]

Antipyretic and Wound Healing Activities of Moringa oleifera Lam. in Rats
This study demonstrated significant antipyretic activity in rats using ethanolic, and ethyl acetate extracts of Moringa oleifera seeds and ethyl acetate extract of dried leaves showed significant wound healing activity (10% extracts in the form of ointment) on excision, incision and dead space (granuloma) wound models. [35] 4.
Hemostatic, antibacterial biopolymers from Acacia arabica (Lam.)Willd.and Moringa oleifera (Lam.) as potential wound dressing materials The study presented the potential of the polymeric component of aqueous extracts of gum acacia and the seeds of M. oleifera in wound management.The results revealed that both biopolymers were hemostatic and hasten blood coagulation and showed shortening of activated partial thromboplastin time and prothrombin time and were non-cytotoxic.Both showed antibacterial activity against organisms known to be involved in wound infections with MIC ranging from 500-600 microg mL (−1) for GA and 300-700 microg mL (−1) for MSP. [36]

5.
Evaluation of Moringa oleifera seed biopolymer-PVA composite hydrogel in wound healing dressing Hydrogel composed of polysaccharide polymer from Moringa oleifera seeds and polyvinyl alcohol (MSP/PVA) was synthesized as a wound dressing material which exhibited hemocompatibility, antibacterial activity, bacterial impermeability, antioxidant activity and iron chelation that might help in the healing of chronic wounds as well. [37] In the study conducted by Momoh et al. using M. oleifera leaf extract, the wound took a long time to heal as compared to the present study [38].Furthermore, the study by Kumar et al. examined leaf water extract of M. oleifera on Swiss Albino rats for wound healing showed healing of excision wound on the 14th day whereas incision model was characterized by measurement of breaking strength on the 10th day that was found to be 507.5 g [39].Rathi et al. worked on wound healing activity of M. oleifera seed's and dried pulp's aqueous extract and observed an increase in the rate of closure of wound area, hydroxyproline content, dry granuloma weight, granuloma breaking strength, skinbreaking strength, and decrease in the scar area [14].
More studies were conducted by Coker et al. on the ethyl-acetate extract of M. oleifera and Eyarefe et al. on the wound healing potential of M. oleifera leaves extract by oral administration [35,40,41].These studies also revealed that M. oleifera possesses wound healing activity.
The results of the present study on wound healing activity revealed that hexane hydrogel of M. oleifera seeds significantly increases wound healing in 5% and 10% hydrogel treated groups in both the excision and incision wound models.This is further supported by the evidence that the lesser the rate of wound contraction, the better will be the efficacy of the medication and the higher the rate of wound closing [42].

Histopathological Study
In the excision model, the control group (group 1) showed reduced fibroblast cells, blood vessels, collagen fibres, increased inflammatory cells, a necrotic eschar has formed of coagulated plasma which contains inflammatory cells and colonies of and colonies of bacteria with the presence of diffuse inflammation in the dermis.Standard group (group 2) showed diffused dense inflammation among the layer of hair follicles, including granulomatous inflammation consisting of a cluster of mononuclear histocytes and multicellular giant cells.An increase in fibroblast cells and collagen fibres was also seen.
The test groups, 5% and 10% hexane hydrogel (group 3 and group 4), showed normal epidermis with minimal inflammation in the upper dermis and regeneration of epidermis forming knots of squamous cells.The examination of histopathology revealed that the original regeneration of tissue was much more significant on test group 5% and 10% hexane hydrogel as compared to control as well as standard.
In the incision model, the control group (group 1) showed bacterial colonies with minute ulcers and diffused inflammation in the dermis with decreased production of blood vessels and collagen fibres, while standard group (group 2) showed ulcers with massive inflammation with lower rates of healing reactions and scar over the ulcers had been seen.The test group 5% and 10% hexane hydrogels (groups 3 and 4) showed healing and regeneration of squamous epithelium, including lower inflammation in different sites of the skin.The examination of histopathology revealed that the healing process was much faster in test groups 5% and 10% hexane hydrogel.
Histological evaluation of the wound area displayed that increase in cellular infiltration (measured through staining) in treated samples might be because of the chemotactic effect enhanced by the hexane hydrogel of M. oleifera seeds attracting inflammatory cells towards the wound site [43].

Plant Collection and Phytochemical Constituent Extraction
Collection of M. oleifera seeds was done from M/S Shidh seeds sales Corp., Pand Tiwari, P.o.Premnagar Dehradun 248001.The seeds were crushed to form a powder, and the Soxhlet apparatus with hexane as a solvent was used for the extraction process.Extraction was finished in approximately 42 h, and the used solvent was recovered using Rota evaporator under reduced pressure [44].The extraction procedure is well-reported, and phytoconstituents analysis (both quantitative and qualitative) is exhaustively covered in previous literature [45].

In Vitro Antioxidant Activity
The DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging assay, popularly known as an easy and rapid test for evaluation for the presence of antioxidants and ability to scavenge the oxidative stress producing free radicals in a sample, was performed.DPPH assay was performed following Sakat et al. with minimal modifications.0.5 mL of DPPH was added to 0.5 mL aliquots of standard (ascorbic acid), or test solution in various concentrations: 10, 20, 40, 80, 160 µg/mL [46].0.5 mL of 10% DMSO and 0.5 mL DPPH were loaded in control test tubes.Incubation at 37 • C for 30 min in the dark was provided, and absorbance was recorded at 517 nm.The percentage scavenging by test sample at each concentration was calculated using the formula: IC50 represents the 50% scavenging concentration caused by test or standard samples.

In Vitro Antimicrobial Activity
Various bacterial strains viz., P. aeruginosa, S. aureus, and E. coli were obtained from Molecular and Immuno Parasitology Research Laboratory (MIPL), Shoolini University, Solan HP.

Agar Well Diffusion Method
The antimicrobial study was essentially performed as given in Rojas et al., Kisangau et al. with some modifications.Briefly, preparation and sterilization of nutrient agar plates were done.Sterilized swabs were dipped into standardized bacterial suspension with an inoculums size of 1.5 × 108 cfu/mL, and unneeded culture was removed by turning the swab against the side of the tube.The spread plate method was performed with an evenly spread inoculum over the entire surface of Nutrient agar plates.Plates were allowed to dry for at least 15 min and 6mm diameter wells were made using a sterile cork borer.100 µL (100 mg/mL) of extracts were prepared and introduced into bore agar wells using a sterile dropping pipette.For proper diffusion, plates were placed to cool down for 2 h at room temperature and incubated at 37 • C for 18-24 h [47,48].Antimicrobial activity was determined by measuring the diameter of the zone of inhibition in mm.

Preparation of Test Samples
1 g carbopol was dissolved in 50 mL distilled water at 40-50 • C with 0.2 g propylparaben sodium and 0.5 g methylparaben sodium by stirring.The solution was kept overnight, and the addition of 50 mL of distilled water was done.Stirring was continued with the addition of 10mL of propylene glycol and 5 ml of ethanol.Two to three drops of triethanolamine were added and stirred until the gel was formed at pH 7.0.For the formulation of 5% hydrogel, 5 g of M. oleifera n-hexane seed extract was mixed with 95 g of gel, whereas 10% hydrogel was formulated using 10 g of M. oleifera n-hexane seed extract and 90 g of gel.

Animals
Male Swiss albino mice weighing 20-30 g were procured from a small animal house facility National Institute of Pharmaceutical Experimental Research (NIPER), Mohali, Punjab.Animals were kept at a temperature of 25 ± 2 • C and relative humidity of 45 ± 5 • C during the entire protocol of wound healing in the animal house of Shoolini University, Solan, HP.The animals were provided with food and water ad libidium and were allowed to adapt to the environment for seven days before the start of experimentation.
Animals were assigned into four groups containing four animals in each group, a group I as control: treated with placebo carbopol hydrogel (without Moringa oleifera extract); group II as standard: 5% povidone treated; group III as 5% M. oleifera extract: 5% hydrogel of hexane seeds extract of M. oleifera; group IV as 10% M. oleifera extract: 10% hydrogel of hexane seeds extract of M. oleifera.

Excision Wound Model
For anesthetizing mice, Ketamine hydrochloride (100 mg/kg) I.p and xylazine (10 mg/kg) I.p were used [49].The animals were shaved dorsally with the help of an electric clipper, and an outline was marked around the area of the wound by methylene blue using a circular stainless-steel stencil.The wound of 1cm in width and 0.2 cm depth was created along the markings using a surgical blade, pointed scissors, and toothed forceps [50].Sterile conditions opted for all surgical interventions, and post-operative care was ensured.Animals were treated once daily for 14 days.

Assessment
Digital photographs and wound area measurements in the excision model were taken on 1st, 4th, 6th, 9th, 12th, 13th, and 14th day.Measurement of the healed wound was done using transparent graph paper.The wound healing activities of all the groups were evaluated by measuring the percentage of wound contraction and period of epithelialization.The percentage of wound contraction was calculated as follows [51].

% Wound contraction =
Area of the wound on day 1 − Area of the wound on day n Area of the wound on day 0 × 100% where n= number of days 4th, 6th, 9th, 12th, 13th, and 14th day.

Incision Wound Model
Ketamine hydrochloride (100 mg/kg) and xylazine (10 mg/kg) were used for anaesthetizing mice before and during the wound formation [20].The animal was shaved, and a long incision wound of 1cm length was created on the dorsal side.The parted skin was stitched together using surgical thread (No. 1) and a curved needle (No. 17).All the animals of the groups were treated once daily for eight days.Sutures were removed on the 5th post-wounding day, and the treatment was continued.The skin breaking strength of the healed wound was measured on the 8th day [52].

Histopathological Study
On the 14th day, a tissue sample from the site of the wound was taken from all animals of both excision and incision wound models and was sent for histological study.Samples were fixed in 10% buffered formalin.Further, tissue processing included dehydration, wax impregnation, and preparation of blocks with paraffin.Sectioning was done on a microtome (3-5 micron thick), and hematoxylin and eosin were used for the staining.Epidermis, bacterial colonies, and inflammation were the parameters visualized in all the four groups of both the models, i.e., excision and incision [53].

Statistical Analysis
All the results were presented as Mean±SD and by one way in the incision model and two analyses of variance in excision model (ANOVA) and Bonferroni's multiple comparison test as post-hoc Analysis.p < 0.05 was considered as statistically significant.Graph pad prism software version 5 was used.

Conclusions
The wound healing potential of hexane hydrogel of M. oleifera seeds could be explained using antioxidant, antimicrobial, and wound healing activities of the plant.The hexane extract of M. oleifera seeds possesses antioxidant activity.This work also elucidates that hexane extract of M. oleifera seeds possesses both gram-positive as well as gram-negative bactericidal potential and thus, can be used as a therapy to treat wound infections.In the present study, animals treated with hexane hydrogel of M. oleifera seeds showed significant wound healing activity when compared to control and standard groups in both excision and incision models.In the histopathological study, the increase in cellular proliferation due to the mitogenic activity of the hexane hydrogel of M. oleifera seeds remarkably contributed to the process of wound healing.It was also confirmed by early dermal and epidermal regeneration in the mice treated by test drugs that the hexane hydrogel of M. oleifera seeds had a positive effect on the proliferation of the cells, granular tissue formation, and epithelization.Further, the histopathological observations also confirmed the experimental wound healing study results that were based on the wound area measurement and tensile strength.Thus, it can be concluded that formulated hydrogel by the hexane extracts of M. oleifera seeds could be used as potential herbal wound healing agents, in the management of wounds.
Author Contributions: A.A. and P.G. designed the whole study, nearly executed, analyzed all the experiments, and wrote the manuscript inputs.S.K. has supervised, conceptualized, analyzed, and validated the manuscript.R.G. was involved in the design of the study.P.N., G.K., and X.L. were responsible for analysis, review, and editing.M.V. and K.K. were responsible for review, validation, and funding.All authors have read and agreed to the published version of the manuscript.Informed Consent Statement: "Not applicable" for studies not involving humans.

Figure 1 .
Figure 1.DPPH radical scavenging activity of standard Ascorbic acid and n-hexane extract of M. oleifera seeds.

Figure 2 .
Figure 2. Representation of excision wound healing model of n-hexane extract of M. oleifera seeds ointment.

Figure 2 .
Figure 2. Representation of excision wound healing model of n-hexane extract of M. oleifera seeds ointment.

Figure 2 .
Figure 2. Representation of excision wound healing model of n-hexane extract of M. oleifera seeds ointment.

Figure 4 .
Figure 4. Effect of topical application of ointment of Moringa oleifera n-hexane seeds extracts tensile breaking strength of incision wound.Mean ± SD, analyzed by one-way ANOVA followed by Bonferroni's multiple comparison test post hoc analysis; a*** < 0.01 vs. control, standard, and test drugs 8th day.

Figure 4 .
Figure 4. Effect of topical application of ointment of Moringa oleifera n-hexane seeds extracts tensile breaking strength of incision wound.Mean ± SD, analyzed by one-way ANOVA followed by Bonferroni's multiple comparison test post hoc analysis; a*** < 0.01 vs. control, standard, and test drugs 8th day.

Funding:
The authors would like to acknowledge the funding received from UHK VT2019-2012 and the Ministry of Health of the Czech Republic (FN HK 00179906) and the Charles University in Prague, Czech Republic (PROGRES Q40).Institutional Review Board Statement: All the experiments were conducted as per the Committee for Control and Supervision of Experiments on Animals (CPCSEA) guidelines.The Institutional Animal Ethical Committee approved the experimental protocol (IAEC) (Protocol No. IAEC/SU/17/19).

Table 1 .
Studies on wound healing efficacy of Moringa oleifera seeds.