Electrospun Scaffolds as Antimicrobial Herbal Extract Delivery Vehicles for Wound Healing

Herbal extracts have been used in traditional remedies since the earliest myths. They have excellent antimicrobial, anti-inflammatory, and antioxidant activities owing to various bioactive components in their structure. However, due to their inability to reach a target and low biostability, their use with a delivery vehicle has come into prominence. For this purpose, electrospun nanofibrous scaffolds have been widely preferred for the delivery and release of antimicrobial herbal extracts due to the flexibility and operational versatility of the electrospinning technique. Herein, we briefly reviewed the electrospun nanofibrous scaffolds as delivery systems for herbal extracts with a particular focus on the preclinical studies for wound-healing applications that have been published in the last five years. We also discussed the indirect effects of herbal extracts on wound healing by altering the characteristics of electrospun mats.


Introduction
A wound is the damage of a living tissue [1] which is caused intentionally (e.g., gunshots [2]) or unintentionally (e.g., skin cut, animal bite, traumas [3]) [4].Although the human body has an excellent capability for healing wounds through a cascade of simultaneous phases, imperfect repair of damaged skin may cause vital damage, in particular the emergence of an infection, commonly in chronic wounds [5,6].In contravention of the existence of various types of bacteria in the skin microbiota, biofilm formation or threshold value of substantial bacteria may block the wound-healing process [7].The most common pathogenic strains that infect the wound site were revealed to be the Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa (P.aeruginosa), and methicillin-resistant S. aureus [8].Antibiotics are generally the first option for the treatment of infections, especially local ones [9].However, overuse and maladministration of antibiotics [10][11][12] give rise to systemic toxicity and generation of antibiotic-resistant microorganisms.Therefore, due to the stated crucial concerns, alternative, unconventional, and non-antibiotic natural-based therapeutics (e.g., herbal extracts and essential oils) have come into prominence among most scientists as well as companies.
Herbal extracts have been used for traditional treatment purposes, e.g., burn wounds, bone fractures, and intestinal problems, since the earliest myths [13].They demonstrate an excellent antimicrobial, antioxidant, anti-inflammatory, and anticarcinogenic activity owing to the bioactive compounds (e.g., polyphenols, vitamins) in their structure [14,15].Almost 70% of people worldwide believe the primary health benefit of herbal compounds, according to the report by the World Health Organization [16].Herbs present limitless sources to develop alternative, safe, and renewable therapeutics.For instance, among over two hundred and fifty thousand vascular plants, only around 17% of them have been researched for medicinal purposes [13].Even though herbal extracts have been known for their excellent biological activities, some shortfalls still appear, such as poor biostability and the inability to reach the target [17].Hence, there has been a need to develop engineered carrier and delivery vehicle systems (e.g., electrospun nanofibers, hydrogels) to increase the treatment and targeting efficacy.
Electrospinning is a simple and effective technique to fabricate various size ranges of nanofibers using electric force to pull charged threads of polymer solutions either with or without including any herbal extract [18].Moreover, electrospinning can provide the sustained and targeted release of therapeutics [19].One of the promising advantages of this technique is its operational versatility and flexibility to achieve desirable surface characteristics like large surface-to-volume ratio, and desired porosity [20,21].It is a favorable method for the spinning of several kinds of materials such as natural, synthetic, or blended polymers.Nevertheless, several health concerns related to the usage of synthetic materials started to appear [18].Consequently, the perspective shifted from a preference for synthetic to one for natural materials.In this manner, herbal extracts have become rising stars as attractive sources of electrospun biomaterials for many applications [22].Herein, we briefly describe the role of antimicrobial herbal extracts incorporated in electrospun scaffold delivery systems (Figure 1), with a particular focus on preclinical studies for wound-healing applications that have been published in the last five years.
anticarcinogenic activity owing to the bioactive compounds (e.g., polyphenols, vitamins) in their structure [14,15].Almost 70% of people worldwide believe the primary health benefit of herbal compounds, according to the report by the World Health Organization [16].Herbs present limitless sources to develop alternative, safe, and renewable therapeutics.For instance, among over two hundred and fifty thousand vascular plants, only around 17% of them have been researched for medicinal purposes [13].Even though herbal extracts have been known for their excellent biological activities, some shortfalls still appear, such as poor biostability and the inability to reach the target [17].Hence, there has been a need to develop engineered carrier and delivery vehicle systems (e.g., electrospun nanofibers, hydrogels) to increase the treatment and targeting efficacy.
Electrospinning is a simple and effective technique to fabricate various size ranges of nanofibers using electric force to pull charged threads of polymer solutions either with or without including any herbal extract [18].Moreover, electrospinning can provide the sustained and targeted release of therapeutics [19].One of the promising advantages of this technique is its operational versatility and flexibility to achieve desirable surface characteristics like large surface-to-volume ratio, and desired porosity [20,21].It is a favorable method for the spinning of several kinds of materials such as natural, synthetic, or blended polymers.Nevertheless, several health concerns related to the usage of synthetic materials started to appear [18].Consequently, the perspective shifted from a preference for synthetic to one for natural materials.In this manner, herbal extracts have become rising stars as attractive sources of electrospun biomaterials for many applications [22].Herein, we briefly describe the role of antimicrobial herbal extracts incorporated in electrospun scaffold delivery systems (Figure 1), with a particular focus on preclinical studies for woundhealing applications that have been published in the last five years.

Direct Effect of Herbal Extracts on Wound Healing
Herbal extracts are outstanding and alternative antimicrobial compounds, as opposed to common, traditional therapeutic agents (e.g., antibiotics), owing to their excellent biological properties, such as antimicrobial, antioxidant, anticarcinogenic, and anti-inflammatory activities, arising from various bioactive phytochemicals in their structure.Herbal extracts can regenerate damaged tissue in the wound area and fasten the healing process thanks to these bioactive components [23,24].Furthermore, these phytochemicals can exert their antimicrobial action by damaging the bacterial cell wall and membrane, blocking or disrupting the synthesis of crucial bacterial proteins, as well as inhibiting the

Direct Effect of Herbal Extracts on Wound Healing
Herbal extracts are outstanding and alternative antimicrobial compounds, as opposed to common, traditional therapeutic agents (e.g., antibiotics), owing to their excellent biological properties, such as antimicrobial, antioxidant, anticarcinogenic, and anti-inflammatory activities, arising from various bioactive phytochemicals in their structure.Herbal extracts can regenerate damaged tissue in the wound area and fasten the healing process thanks to these bioactive components [23,24].Furthermore, these phytochemicals can exert their antimicrobial action by damaging the bacterial cell wall and membrane, blocking or disrupting the synthesis of crucial bacterial proteins, as well as inhibiting the action of significant bacterial metabolic pathways (e.g., DNA replication) [15,25,26].The incorporation of herbal extracts into electrospun nanofibrous mats has been extensively studied to enhance their biostability, despite their effective bioactivity.
The preclinical studies revealed that the treatment of wounds with herbal extract including nanofibers enhances the in vivo wound-healing rate (Table 1).The 20% (w/w) of Malva sylvestris extract incorporated in a polyurethane/carboxymethyl cellulose (PU/CMC) mats yielded a complete wound closure on the 14th post-treatment day, whilst its lower concentrations (5-10% w/w) were not sufficient.Due to the presence of various polysaccharides (e.g., flavonoids, naphthoquinones, and anthocyanins), M. sylvestris displayed good biological activity in a full-thickness diabetic wound model [27].The addition of Calendula officinalis increased the closure rate of a wound treated with a pristine chitosan/polyethylene oxide (Chi/PEO) nanofibrous mat for 14 days from 80% to 90% [28].In a study, peppermint extract and gelatin nanoparticles (NPs) were embedded into a PU/Pluronic F127 nanofibrous membrane to enhance wound healing.While both extractand gelatin NPs-loaded membranes showed a 97% wound recovery rate, the scaffolds that contained only peppermint extract presented an almost 75% wound-closure rate within 21 days [29].The treatment of wounds on a BALB/c mice model with henna extract-loaded gelatin/oxidized starch nanofibers presented a reduction in the number of macrophages, an inflammatory response with a thinner inflammation zone, and enhanced collagen deposition, which might have been due to the existence of various aromatic hydrocarbons in the henna structure [30].The addition of palmatine accelerated the wound-healing ability of the PCL/gelatin nanofibers on the rabbit ear model by decreasing the healing time from 14 to 12 days, as well as inhibited hypertrophic scar formation.However, the highest concentration of palmatine (8 w%) showed cytotoxicity on the L929 fibroblasts [31].In addition, almost 97% of the wound on the Wistar rat recovered due to the synergetic effect of the Achyranthes aspera and Datura metel extracts, which were incorporated into the PCL nanofibrous mats within 9 days post-treatment [32].
According to Table 1, when compared to extracts which were applied in the same concentration units (%, w/w), the addition of 2% of Lawsonia inermis extract [33] led to the almost complete wound closure at day 14, whilst the 15% of Malva sylvestris [27], 1% of curcumin [34], and 15% of peppermint [29] extracts displayed around 95%, 75%, and 65% in vivo wound-closure rates, respectively.Moreover, 2-8% of palmatine addition into the PCL/gelatin nanofibers [31] yielded an almost complete wound closure in 12 days.Additionally, none of the extracts showed any cytotoxic effect.Hence, it may be concluded that palmatine is the most effective extract with its lower concentrations for wound healing, followed by the Lawsonia inermis, Malva sylvestris, curcumin, and peppermint extracts.

Indirect Effect of Herbal Extracts on Wound Healing
In addition to their direct effect on wound healing due to their bioactive compounds, herbal extracts may enhance the wound healing ability of electrospun nanofibers by altering their physicochemical, mechanical, and morphological characteristics.At this point, we will review how herbal extracts affected the hydrophilicity, mechanical strength, average fiber diameter, and porosity of electrospun nanofibrous wound dressings that provide benefits such as improved wound-healing.

Effect of Herbal Extracts on the Hydrophilicity of Electrospun Mats
The water uptake capacity of electrospun mats plays a crucial role in wound healing.The fabricated mats can conserve the moisture and nutrients in the wound area, as well as promote cell adhesion and proliferation with a higher swelling ratio [42,49].The water uptake percentage of the Chi/PEO nanofibers increased from 93% to 119% through the incorporation of 2 w% of hydro-alcoholic henna extract, which was attributed to the hydrophilic functional groups of henna, while wound-closure rates were evaluated to be approximately 85% and 90% for pristine and 2 w% henna-including mats, respectively [33].Similarly, the addition of lawsone raised the water content of the PCL/gelatin nanofibers by almost three-fold in the PBS (pH:7) media.This composite mat displayed a lower number of inflammatory cells and more organized fibroblasts with accelerated wound-healing within 14 days [42].Additionally, enhanced wettability of nanofibrous mats may benefit the diffusion of nutrients to the wound area, absorption of exudate, as well enhance cell binding.For example, the presence of several polar phytochemicals in the Gymnema sylvestre extract achieved a decrease in the water contact angle of the PCL/gelatin nanofibers and a slight increase in the wound recovery percentage on the 32nd day post-injury [42].

Effect of Herbal Extracts on the Mechanical Strength of Electrospun Mats
The adequate mechanical properties of engineered electrospun mats are another important parameter to promote the formation of new dermal tissue and resist biodegradation during the wound-healing process [50].An ideal nanofibrous wound dressing should provide a balance between flexibility and hardness [51] and display a tensile strength in the range of 0.8 to 18 MPa, which is proper for dermal cell culture and skin tissue engineering applications [52,53].Herbal extracts may act as a reinforcement agent [37,41] and increase the tensile strength of nanofibers, which leads to the enhancement of wound recovery.To exemplify, the incorporation of Acalypha indica, Aristolochia bracteolate, Thespesia populnea, and henna extracts together increased the tensile strength of the PVA/Guar gum nanofibers because of the crosslinking impact of A. bracteolate and A. indica extracts resulting from their increased nonpolar functional ratio.Although the improved mechanical properties of mats are not the only effective parameter, polyherbal extract-including mats showed a slightly higher wound-closure rate (97%) than pristine mats (93%) within 14 days [37].A similar effect was observed with the addition of the Gymnema sylvestre extract, which improved the ultimate tensile stress of the PCL/gelatin mats from about 1.4 to 4.3 MPa, an effect which may be attributed to the high number of hydrogen-bonding donor molecules in the structure of the extract.In parallel, the presence of an extract in the core/shell nanofibers, which are one of the most common nanofiber structures, significantly improved wound-closure percentage with enhanced epidermal cell proliferation [41].However, in some studies, herbal extracts showed a plasticizing effect [20,28,33,42], resulting in the reduction of the tensile strength.Nevertheless, despite the decreased mechanical strength, the wound-healing ability of mats was not negatively affected.

Effect of Herbal Extracts on the Average Fiber Diameter of Electrospun Mats
The morphology of nanofibrous dressings plays a significant role in wound-healing applications, since the random orientation of nanofibers can mimic the nature of the extracellular matrix (ECM) [54].As a general trend, the incorporation of herbal extract reduced the viscosity of the polymeric spinning solution with the increase in the conductivity, which induces the formation of smaller fibers since it acts as a plasticizer when added to a polymer blend [28,35,36,39,44,48].Smaller-diameter nanofibers have a supportive effect on the wound-healing process due to their greater protein-absorption capacity.In other words, cell adhesion on smaller-diameter fibers is promoted due to their larger specific surface areas [55].To illustrate, while the average diameter of the gelatin/PVA/Chi nanofibers was reduced almost two-fold by the incorporation of 400 µg/mL of curcumin, the curcumin-including composite dressings displayed greater wound recovery on the 14th day of treatment when compared to gauze control [39].In contrast, it was reported that the addition of the palmatine [31] and Melilotus officinalis [45] extracts raised the average nanofiber diameters, which might be explained by the decreased electrical conductivity of the polymeric spinning solution.Nonetheless, the higher nanofiber diameters did not show an inhibitory effect on wound healing; even the acceleration of wound-healing rates [31] and more collagen deposition [45] were revealed.In summary, it can be concluded that, even though the general view is that of decreasing the nanofiber diameter by adding an herbal extract into the formulation, some studies showed opposite outcomes.

Effect of Herbal Extracts on the Porosity of Electrospun Mats
Besides the nanofiber dimension, porosity is accepted as another outstanding parameter for fibrous scaffolds [56].Since the proper porosity allows the permeation of oxygen through the wound bed, it can benefit the acceleration of wound healing [57] by improving the proliferation of fibroblasts and keratinocytes.The resulting proliferating environment can lead to the reepithelization and formation of granulation tissue, advancing the secretion of wound-healing mediators (e.g., angiogenic factors, growth factors, and collagen) [47,54].Case in point, the addition of 5% (w/v) of Cordia myxa ethanolic fruit extract increased the porosity of the PVA nanofibers by 11.8%, as well as provided the proper reepithelization, more collagen deposition, and a 33.6% smaller wound within 14 days [44].Similarly, the incorporation of Aloe vera [56] and Nigella sativa oil [48] enhanced the porosity of the PLGA/PVA and PU nanofibrous scaffolds, respectively, with improved proliferation and wound-healing activity.The increased porosity might be explained by the thinner nanofibers produced through the addition of herbal extracts.Instead, the presence of various concentrations of Calendula officinalis did not affect the porosity of the Chi/PEO nanofibers [28].

Future Aspects
Electrospinning is one of the biomaterial fabrication processes that use the highvoltage-electric field to draw charged polymer melts/solutions through the collector to obtain nano-sized structures.Electrospinning has come into prominence when compared to traditional fabrication methods since it is a simple and user-friendly process that leads to control over the porosity and/or morphology of nanofibers by altering the fabrication parameters (e.g., flowrate, voltage of electric fields, and nozzle diameter).Moreover, electrospun nanofibers display various advantages for tissue engineering applications thanks to the possibility of adjusting the hydrophilicity and stimuli-responsive capacity of the fabricated materials.In particular, electrospun nanofibers are an excellent candidate for a wound dressing due to their high surface area to volume ratio, adjustable and high porosity, good biocompatibility, and mechanical properties.All these outstanding features favor cell attachment, growth, and proliferation, as well as wound moisturizing.In addition, due to growing health concerns regarding the use of synthetic molecules, nature-inspired molecules such as herbal extracts are constantly attracting attention for advancing greener and non-toxic products for wound-healing treatment.Therefore, we believe that electrospinning is a promising technology for developing wound dressings that incorporate natural therapeutics in the formulation of the nanofibers.

Conclusions
Wound care is a problem that has always concerned human health, from the beginning of humanity.In the quest for the treatment of infected wounds, herbal extracts have been slighted when compared to antibiotics, despite the fact that they have been a primary source of traditional remedies since ancient times.However, in the perspective of the development of alternative wound dressings, the study of herbal extract-incorporated delivery systems has recently begun to gain an important place in the literature to overcome the drawbacks of antibiotic usage, e.g., antimicrobial resistance and biofilm formation.Furthermore, electrospun scaffolds are one of the most prominent biomaterial forms owing to their highly porous, nano-sized structures.Through the incorporation of herbal extracts instead of synthetics, the reduction of several health concerns caused by the use of synthetics has been achieved.This review clearly exemplifies both the direct and indirect effects of herbal extracts on wound healing and gives countenance to the advancing of natural, herbal-based nanofibrous delivery systems for effective wound care and infectious treatment.

Figure 1 .
Figure 1.Direct and indirect effects of the incorporation of herbal extracts into electrospun biomaterials.This figure was created using BioRender.com.

Figure 1 .
Figure 1.Direct and indirect effects of the incorporation of herbal extracts into electrospun biomaterials.This figure was created using BioRender.com.