The wound healing effect of botanicals and pure natural substances used in in vivo models

Repairing the wound is a multistep process that includes the spatial and temporal synchronization of a different range of cell types to increase the speed of wound contraction, the proliferation of epithelial cells, and collagen formation. The need for proper management of acute wounds to be cured and not turned into chronic wounds is a significant clinical challenge. The traditional practice of medicinal plants in many regions of the world has been used in wound healing since ancient times. Recent scientific research introduced evidence of the efficacy of medicinal plants, their phyto-components, and the mechanisms underlying their wound-repairing activity. This review aims to briefly highlight the wound-curing effect of different plant extracts and purely natural substances in excision, incision, and burn experimental animal models with or without infection of mice, rats (diabetic and nondiabetic), and rabbits in the last 5 years. The in vivo studies represented reliable evidence of how powerful natural products are in healing wounds properly. They have good scavenging activity against Reactive oxygen species (ROS) and anti-inflammatory and antimicrobial effects that help in the process of wound healing. It is evident that incorporating bioactive natural products into wound dressings of bio- or synthetic polymers in nanofiber, hydrogel, film, scaffold, and sponge forms showed promising results in different phases of the wound-curing process of haemostasis, inflammation, growth, re-epithelialization, and remodelling.


Introduction
The human body includes different organs. One of them is the skin which occupies a large body area. It represents the outermost defensive covering of the body and an immunological barrier that regularly faces different external factors. It fortifies against mechanical pressure, microbial contagion, and septicity and maintains normal body temperature. It is responsible for the sensation of touch, heat, and cold (Richmond and Harris 2014; Kwiecien et al. 2019;Kumar P and Kothari 2021).
The antimicrobial protective role of different skin layers was evidenced through different previous studies. An external layer displays the composition of human skin outside the epidermis called microbiota, epidermis, dermis, adipose tissue, glands (sweat and sebaceous), and hair follicles (Kwiecien et al. 2019).
Epidermis is composed of keratinocytes, melanocytes, Langerhans' cells, and Merkel cells. Keratinocytes are a significant type of cells that has a role in vitamin D formation and produce keratin and lipids to form a water barrier. Keratinocytes could act against chemical and biochemical toxins by creating pro-inflammatory cytokines, e.g., interleukins: IL-1α, IL-1β, IL-3, and IL-6, interferons-alpha and beta, transforming growth factors, tumour necrosis factors, and others (Blume-Peytavi et al. 2016). Melanocytes are responsible for skin pigmentation. The first line of protectors of the skin is represented by Langerhans cells. They transport antigens in the skin to the lymph node. The membranes of Merkel cells interact with free nerve endings in the skin, so they have a sensory function. The dermis layer includes the sweat glands, blood vessels, muscles, and sensory neurons (Yousef et al. 2017). Symbiotic microorganisms of bacteria and fungi are recognized as skin colonies with harmless and vital effects in protecting the skin. They are inside hair follicles, sweat and sebaceous glands to protect the skin against invasive and microbial pathogens. Among them, species of Staphylococcus, Malassezia, Demodex folliculorum, and Demodex brevis were the most important (Grice and Segre 2011;Ibrahim et al. 2020).
Wounds have happened due to the loss of histological composition of the skin tissue due to internal or external factors or sequential loss of function in any layer of the skin, which leads to tissue disturbance (Herman and Bordoni 2020). The existence of wounds permits the entrance of different microbial agents as bacteria and viruses or any foreign elements, into the body. Inflammation of skin wounds is happened because of local microbial infections. Also, a generalized systemic infection (septicemia) could be found, a life-threatening condition (Percival 2002). Consequently, more research should be done to find out simple and effective ways of taking care of skin wounds to heal properly. The main goals are to stop bleeding, get rid of microbial infection of wounds, and help wounds to heal effectively without any complications or deformities (Sarabahi et al. 2012;Jones 2015).
Once any damage has occurred to the skin tissue, multiple cellular and extracellular pathways act in a harmonized way, and their functions must be performed in the appropriate order at a suitable time to achieve repair, growth, and tissue regeneration (Richmond and Harris 2014).
Bleeding due to damaged blood vessels must be stopped, which is considered the initial reaction in the process of wound repair, besides platelet stimulation to compose a fibrin clot. Immediately after that, the disturbed tissues discharge growth factors and pro-inflammatory cytokines. Upon controlling the bleeding, many inflammatory cells such as monocytes, macrophages, and neutrophils are gathered at the wound site to provoke the inflammatory response (inflammatory phase). Moreover, the different self and exogenous antigens trigger the immune system to fight against them (Rodrigues et al. 2019;Alotaibi et al. 2021).
Angiogenesis is the following phase, which is parallel to the inflammation phase. The formation of a new blood vessel characterizes this phase. It is then followed by the growth and proliferative phases, which are predominate by fibroblast relocation and propagation, production of the matrix proteins, keratinocyte proliferation, differentiation, and restoration of hair follicles, etc. lastly, the wound healing process is finished with the remodelling of the extracellular matrix (ECM), besides the reordering of granulation tissue to scar tissue. Collagen synthesis and cross-linking afford stability to the healing tissue (Rodrigues et al. 2019). Figure 1 demonstrates the different phases of wound healing, while Table 1 summarizes herbal extracts studied using in vivo wound healing models. Structures of purely natural substances that were investigated using wound healing in vivo models showed in Fig. 2 and Table 2.
Botanical extracts have been extensively utilized in managing wounds in traditional medicine. Therefore, in vitro and in vivo studies have assessed different extracts for their wound-curing characteristics. Their phytochemical content is the purpose of their remedial features in wound repair. Other phytochemicals and plant-derived substances were investigated for their wound-healing activity as flavonols, flavanones, isoflavones, flavanols, flavonolignans, proanthocyanidins (Carvalho et al. 2021), β-glucans (Majtan and Jesenak 2018), bromelain (Fathi et al. 2020), curcumin (Akbik et al. 2014). It was disclosed that different botanicals and medicinal plants are widely used as a topical treatment for wound repairing, such as aloe vera, banana leaves (Sivamani et al. 2012), turmeric, Centella asiatica, Rosmarinus officinalis, Calendula officinalis (Artem Ataide et al. 2018).
Natural products such as plant extracts and other plantderived products and their phytochemicals assist in managing inflammatory diseases, exert antimicrobial effects, and might aid skin tissue regeneration (Alherz et al. 2022;Attallah NG et al. 2022). They could remove oxidative stress and lower inflammation (Shah and Amini-Nik 2017). The wound-repairing ability of different plant extracts and their actives was confirmed in wound-curing animal models. Such plants improved collagen deposition, the proliferation of epithelial cells, and angiogenesis in diabetic and nondiabetic   Fig. 2 Structures of natural pure substances were investigated using wound healing in vivo models (animal models)  Lee et al. 2022 animal models (Binsuwaidan et al. 2022). Different types of plants are widely used in managing wounds and injuries from previous scientific research (Chingwaru et al. 2019). The current review demonstrates and focuses on the latest findings in the last 5 years (2018-2022) regarding the in vivo studies of wound repairing effect of different plant extracts, the derived substances from plants, and pure natural substances as a new frontier in treating wounds.

Methods of collecting data
Data collected in the frame of this work were generated by common research engines such as ScienceDirect, Web of Science, PubMed, SciFinder-n, and Scopus, using the references "natural products", "wound healing" and refining with keywords "animal models", "burns", "biological", "plants" "wound dressings" and "inflammation". A total of 2194 research items were examined out of which 190 fall into the scope of the review, thus, constituting the baseline of the current survey.

Botanicals and pure natural substances in the preclinical studies
The present review provided the research work, which included the preclinical studies (in vivo) of plant extracts and pure natural substances on wound healing in the last 5 years. The preclinical investigation by using animal models is important for acute and chronic wounds, in vitro studies could be used, but they do not assess the complexity of the wound healing process (Dunn et al. 2013;Zindle et al. 2021). Acute wounds occur through known sequential steps (Zindle et al. 2021). but chronic wounds exhibited impaired or delayed healing. The acute wound heals within 2-3 weeks, followed by the remodelling phase in normal healthy people. The normal healing sequence could be interrupted by other diseases such as diabetes, wound infection, foreign bodies, chronic inflammation, and ischemia. Microbial infection is the famous reason for wound-related morbidity (Said et al. 2009;Rajendran et al. 2018). This led to a physiological imbalance in the mechanism of healing. It might get stuck in one of the phases, and the wound then falls into the non-healing chronic type (FrykbergRobert 2015; Rajendran et al. 2018). It was reported that a wound is not healed in more than 6-8 weeks defined as a chronic/ nonhealing wound (Rajendran et al. 2018). The universal goal of all studies about wound healing is to treat acute wounds perfectly in due time, so we avoid conversion into chronic ones and discover the appropriate therapy if the patient suffers from chronic wounds. Patients with chronic wounds suffer from pain, depression due to isolation from the community, and risk of amputation (Ivanková and Belovičová 2020).

Wound healing potentials of various plant extracts
Different studies of the wound-repairing effect of various plant extracts revealed the diversity of actives responsible for this activity. It was suggested that D-pinitol and caffeic acid, the major constituents of Boerhavia diffusa leaf methanol extract, contributed to the wound-healing effect (Juneja et al. 2020). In another study, the fraction contained a high level of polyphenolic compounds, separated from leaves methanol extract of Coccinia grandis showed a remarkable wound repair effect. This effect was due to (rutin), quercetin-3-Oneohesperidin, nicotiflorin, kaempferol-3-O-glucorhamnoside, and astragalin as well as seco-iridoids of oleuropein and ligstroside (Al-Madhagy et al. 2019). HPLC metabolic profiling of the methanol extract of Ephedra ciliata recognized quercetin as a major compound. The antioxidant and antimicrobial activities of quercetin were related to the woundclosure effect of the extract (Yaseen et al. 2020). Biological guided study of E. characias subsp. wulfenii extracts (methanol, n-hexane, and ethyl acetate) of the aerial parts were tested. It was explored that the methanol extract displayed significant wound-repairing activity in circular excision and linear incision wound models, as well as anti-inflammatory effects. This study explored whether quercetin derivatives (quercitrin, hyperoside, and guaijaverin) were responsible for the wound-repairing effect (Özbilgin et al. 2018). Regarding Jacaranda decurrens Cham., metabolic profiling was done to find out ten compounds in the extract of flavonoidal and triterpenoidal nature. It was concluded that these compounds improved the healing of wounds in this study (Serra et al. 2020). Hydroethanolic extract of leaves of Lafoensia pacari A. St.-Hil. was evaluated in accelerating the contraction of wounds. The plant contained punicalagin, ellagic acid, punicalin, kaempferol, quercetin-3-O-xylopyranoside, and quercitrin, which could be related to re-epithelialization, improved cell proliferation, and enhanced remodeling phase of the wounds (Pereira et al. 2018). The mats composed of polyurethane loaded with Nigella sativa oil were studied to assess the in vivo wound-repairing effect (Aras et al. 2021). The essential oil of Nigella sativa seeds contains thymoquinone, which was reported to have wound-healing activity (Haq et al. 2020). Different studies were performed to obtain an effective wound healing process e. g. loaded thymoquinone chitosan-lecithin micelles which keep thymoquinone at the site of wounds with controlled release of the drug (Negi et al. 2020). Hydro-ethanol extract from Vitis labrusca leaves was found to advance the healing of wounds due to the total phenolic and flavonoid content (Santos et al. 2021). Aqueous ethanol extract of Leaves of Curatella americana Linn. exerted remarkable wound healing properties due to its active constituents. Leaves contain compounds known as wound-healing agents, mainly quercetin, kaempferol, glucosides, catechin, and epicatechin (Fujishima et al. 2020).

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A homogenous polysaccharide was separated from the rhizomes of Curcuma zedoaria and tested in the process of healing wounds in diabetic rats. It was added with plateletrich plasma exosomes and loaded to a hydrogel sponge of chitosan and silk. It was found that the previous combination was effective and safe to speed the curing of wounds in the case of diabetes (Xu et al. 2018). Methanol extract of Dodonaea viscosa leaves caused accelerated epithelization of excision wounds and increased tensile strength of incision wounds of rats. HPTLC chromatogram showed 10 constituents of flavonoids, tannins, and saponins, including rutin and kaempferol, with reported healing effects (Nayeem et al. 2021).

bio-and synthetic polymers of bioactive substances from natural products
Wound dressings can be created from a combination of bioand synthetic polymers. Loading them with bioactive substances from natural products increased the good features of this combination. The combined bio-and synthetic polymers may have little or no anti-bacterial, anti-inflammatory, and antioxidant effects (Alven et al. 2020). Loading the bioactive natural product to either the combined polymers or to only one of them eliminates this problem. Bioactive materials such as curcumin (Lüer et al. 2012;Tejada et al. 2016), quercetin (Choudhary et al. 2020;Karuppannan et al. 2022), rutin (Zhou et al. 2021), bromelain (Kalalinia et al. 2021), thymoquinone, gentiopicroside (Almukainzi M. et al. 2022;Almukainzi May et al. 2022), hesperidin (Carvalho et al. 2021), and others were reported to enhance wound healing by adding them to bio-or synthetic polymers or both.
Different types of wound dressings have existed as traditional or passive, e.g., plasters and wool dressing which are not favorable nowadays because of the pain and possible re-skin damage. The interactive wound dressing of synthetic or bio-polymers could be represented as hydrogel, foams, sprays, films, and nanofibers, which introduced a moist environment for wound healing and facilitated water vapor transmission but with a limited anti-bacterial effect. Bioactive wound dressings could be represented by the previously mentioned types of interactive wound dressings, which may be composed of synthetic polymers of polyethylene glycol, polyvinyl pyrrolidone polyurethanes, polyhydroxyethyl methacrylate, polyglycolic acid, polylactide, poly-ε-caprolactone, as well as biopolymers of pectin, chitosan, cellulose, dextran, and alginate, collagen, which are loaded with antibiotics or growth factors or vitamins, and/or bioactive natural products (Zahedi et al. 2010;Aderibigbe and Buyana 2018;Alven et al. 2020).
The merits of combining synthetic and bio-polymer with bioactive natural products in wound dressings for better wound healing were confirmed in many studies e.g., curcumin (Sharma et al. 2018), quercetin, and rutin (Zhou et al. 2021). Curcumin is the active substance of the roots of turmeric or Curcuma longa. It exerts strong antioxidant and anti-inflammatory, anti-bacterial effects but with low water solubility and oral bioavailability. Curcumin was loaded into bio-and synthetic polymers to overcome this problem (Alven et al. 2020). The combination between bio-and synthetic polymers could overcome the problem of poor mechanical support of bio-polymers (Aycan et al. 2019), besides overcoming the problem of lacking biocompatibility, biodegradability, and bad patient compliance of synthetic polymers (Mir et al. 2018). Effective wound dressing for skin burns represents a challenge to the healthcare system due to the probability of skin structure damage leading to an increased risk of infection. Quercetin and rutin are flavonoids with strong antioxidant, antimicrobial and anti-inflammatory effects but have limited water solubility. It was revealed that incorporating quercetin and rutin into polycaprolactone and chitosan oligosaccharides to form a new bioactive electrospun nanofiber membrane, exhibited superior efficacy among all nanofiber membranes for burn injuries (Zhou et al. 2021).
Regarding diabetic wounds, new scaffolds formed of polyethylene glycolylated graphene oxide collagen hybrid for nanoscale drug delivery of quercetin were tested. It was found that it provided a new scaffold with the advantages of being superior, stable, the controllable release of quercetin, biodegradable nanomaterial, and biocompatible, which permitted collagen formation and angiogenesis. Besides, the mesenchymal stem cells' proliferation and differentiation potential were promoted via adhesion to this scaffold. These new scaffolds could help in solving issues of deficient collagen hyperplasia and insufficient blood supply in the case of diabetic wounds (Chu et al. 2018).

Conclusions and future direction
The current review clarifies that nature introduces medicinal plants with remarkable wound-healing effects. Scientific evidence obtained in the last 5 years has allowed us to expand our knowledge about herbal medicines on wound healing and the underlying molecular mechanisms. Plants, with their natural actives, have the ability to cure wounds and to be utilized in skin wound care. Mainly due to their anti-inflammatory, antimicrobial, and antioxidant activities (Pazyar et al. 2014).
Recent literature has proved that different natural substances, such as flavonoids, saponins, phenolic compounds, and polysaccharides, can operate at various phases of the process through diverse mechanisms and are primarily responsible for the activity of herbal remedies active in wound healing. Polyphenolic compounds have been confirmed therapeutical agents in wound healing by regulating and modulating inflammatory responses. Numerous phytochemicals in medicinal plants have been revealed to be important regulators of homeostasis, re-epithelialization, and regeneration by encouraging fibroblast proliferation and/ or collagen formation. Scientific research confirmed the powerful impact of medicinal plants and their phytochemicals in wound management through multiple connected mechanisms (Maver et al. 2015;Artem Ataide et al. 2018).
The development of novel wound care techniques that integrate herbal healing agents with modern products and procedures is in line with current trends in wound healing. Nanostructures and nanoformulations have recently shown promise in overcoming the limitations of conventional medications. They control the release of medicines, lower the dosages needed for healing, and enhance the solubility and effectiveness of water-insoluble herbal components in healing wounds. The optimal dressing for wound treatment is made of nanofibers due to their well-controlled porosity and resemblance to skin tissue. The incorporation of natural materials into nanofibrous architectures for wound dressing has been studied. A biocompatible formulation made of natural herbal extracts would give the consumer a "green" option, and almost fewer side effects once put on the skin.
Based on these findings, it is recommended that many therapeutic approaches be employed concurrently in managing wounds, especially chronic wound injuries, to speed up the healing process and prevent complications. Moreover, various problems need to be resolved to improve the efficacy and utilization of natural substances in wound healing. Multidisciplinary efforts are required to confirm the products' safety, look at their adverse effects, and do doubleblind controlled clinical trials. Good production standards and regulatory regulations are equally essential to increase practitioners' use of phytotherapy and encourage its incorporation into national health systems.
Funding Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). This review received no external funding.

Data availability
The authors confirm that the data supporting this study are available within the article.

Conflicts of interest The authors declare no conflict of interest.
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