Phyto-Extracts in Wound Healing

Data generated through systematic investigation, carried out on the evaluation of phyto-extracts on wound healing research during the last 20 years have been compiled. About 450 plant species having wound healing properties have been identified. The present knowledge of the wound healing process comprise coagulation, inflammation, proliferation, formation and accumulation of fibrous tissues, collagen deposition, epithelialization, contraction of wound with formation of granulation tissues, remodeling and maturation. The constituents of the plant extracts modulate one or more of the above stages. It was the endeavor to identify the active constituents responsible for antimicrobial activity, free radical scavenging properties, stimulators of enhanced collagen production and/or angiogenesis promoters with identification of lead scaffold chemical structures. Multiple phytochemicals concentrated and blended in optimal concentrations, are expected to be available in future years to carry out multi-tasking efforts in wound healing as more knowledge about the properties of the key constituents are unveiled. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page. __________________________________________________________________________________________


INTRODUCTION
Wounds can be major causes of physical disabilities and may lead to losses of many productive manhours. Wounds are essentially the disruption of functional continuity of cells and tissues at the site of injury, and can be caused by insults to the tissue sites by physical, chemical, microbiological or immunological process. Humans and all animals have in situ capabilities of healing wounds in their body parts through continuous tissue repair and tissue regeneration. However, such capabilities are impaired by age, stress situation, obesity, sex, habits of the patient(such as smoking , alcoholism etc.), conditions of health and immunity status, severity and types of wounds, patient's medication status , disastrous nature of the assault-environment around the site of the wounds and potentials of serious microbial infection (1). Curing of acute and chronic wounds proceed through common basic phases of hemostatis, inflammation, proliferation, fibroplasias, collagen deposition, epithelialization, contraction, remodeling and maturation.

Phases in Wound Healing
During the wound healing process, a series of events encompass the repair especially through the presence and actions of activated platelets, neutrophils and macrophages. Increased vascular permeability and angiogenesis are the consequences of the healing, where multiple cellular and cytokine-mediated events are recruited. The endothelial cells are up-regulated by the actions of secreted soluble factors from the activated cells which include the fibroblast growth factors, transforming growth factors, epidermal growth factors and vascular endothelial growth factors among others (2 -4). The platelets also get activated by the contents from the vascular wall; the main activators such as fibronectin, fibrillar collagen and other matrix proteins cause the kickoff.

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The inflammed skin tissues at the wound site release several prostaglandins, some of which are considered to be the mediators for platelet activation and functioning (5-7). Once activated, the platelets commence aggregation and adhesion; concomitantly these release several mediators including chemotactic factors as well as adhesive proteins. Each factor has a role in the healing cascade (8-10). The mast cells surrounding the blood vessels at the wound site release histamine proteases, tumor necrosis factor, leukotrins and cytokines. These work as chemotactic signals for the recruitment of white blood cells or leukocytes (11) at the site of the wound. While coagulation of blood and vasoconstriction at the wound site are events completed in minutes, the repair process of polymorphonuclear cell migration manifested through vasodilation and inflammation followed by epithelialization, granulation and new tissue formation take about a week. Usually by the end of the first week, fibroblasts are the main cells accumulating at the wound. These cells are involved in differentiation in the wound healing process (12). During the initial phases usually type III collagen is synthesized and laid down at the wound site; during the later phase the stronger type I collagen gets produced (13). Type III collagen, which were in abundance during the proliferation stage gets degraded and is replaced by type I collagen during the maturation stage (14). The collagen fibers get cross-linked by the action of specific enzymes, properly rearranged and aligned for providing maximum rigidity and toughness (15). The maturation phase can vary from three weeks to two years. If the healing process does not move in a foreseeable manner, then the wound may turn into a chronic wound (16).

Direction of Wound Healing Research using Active Phyto-Extracts
The basic understanding that platelets and the fibrins produced from fibrinogen at the wound site set off several biochemical processes which include collagen synthesis, cell migration, fibroplasias and angiogenesis have been significantly investigated. (17)(18)(19)(20)(21). The events at the wound site, especially those including release of platelet factors and others such as cell adhesion, cell proliferation, mitogenesis, angiogenesis, fibroplasia, epithelialization, wound contraction, maturation and remodeling of the wound site have been researched upon by several investigators. The platelets are the cause of release of more than sixty biologically active substances (9) and that each such factor is involved at specific time and in specific concentrations, contributing to specific activities in the wound healing process cascade, recruiting different cell types and coordinating complex interactions among the different actors during the wound healing process. Several factors appear and disappear at different stages of the healing cascade and their quantification along with the identification of involved cells has not yet been generally possible. The complexity of the situation can be gauged when one realizes that there is considerable variation in the concentration of such factors in healthy individuals; the quantities of transforming growth factor (TGF), vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) as an example, in blood samples obtained from 20 different donors showed substantial variation (9). More understanding of the wound healing process especially in terms of quantification of factors are expected to evolve in the coming years. In this context, quantitative insight into the interaction of the various components of the plant extracts with the particularized substances present at the wound site needs also to be studied to scientifically assess their worth. Such studies would require semiquantitative if not quantitative analysis of the active substances present in different classes of plant extracts. This area is yet to evolve although the usefulness of various kinds of plant extracts in curing wounds of different types is real and many of the plant extracts have been in use in traditional practices for several years in different societies.

Active Plant Parts Studied for Wound Healing
During the last two decades, there has been increased interest to assess the utility of plant extracts in wound healing and to gain more insight into the active constituents that promote or modulate the healing process. We have reviewed the literature for the last 20 years. Table 1 provides information about the plants along with their families, which had shown wound healing properties studied in different models; the table also contains information about those wound healing plants that are extensively used in folk medicine.
Based on the information furnished in the literature, the main effects of the active constituents of the plant extracts towards wound healing are summarized as under: 1) Phyto-chemical constituents contributing to antimicrobial activity 2) Phyto-chemical constituents working as antioxidants and as free radical scavengers 3) Active components having enhanced mitogenic activity (contributing to increased cell proliferation), angiogenesis, enhanced collagen production and increased DNA synthesis. Ideally, active substances present in the plant extracts are anticipated to interfere with one or more phases of the wound healing process in a positive manner in proper sequence and at the right time frame to show improved efficacy. There should also be minimization of substances that deteriorate the healing process. Since in actual experiments and usage, all the plant products as cited in the table have shown efficacious results, there are increased needs to isolate and investigate each active ingredient that has a positive role in the healing process. Unfortunately, such data presently are not plentiful. The active ingredients obtained from the plant materials have been analyzed for the presence of alkaloids, carbohydrates, glycosides, terpenoids, diterpenes, sesquiturpenes and phytosterols, phenolic compounds and multiple kinds of tannins, proteins, flavonoids, saponins, lignins, alkaloids and essential oils. (29,32,37,60,98,99 ,139 ,155,357,389,457,500). The identification of secondary metabolites in plant extracts that could bind to cellular receptors at wound site to initiate modulation of wound healing process was recently reviwed (501). In a couple of investigations, the principal active ingredients have been isolated to study especially their antimicrobial properties e.g. terpenes and terpenoids like gentiopicroside, sweroside and swertiamarine from Gentiana lutea; certain pentacyclic triterpenes (502-503) ; essential oil containing concentrates of eucalyptol (28) ; flavones such as kaempferol and quercetin and their derivatives (473); phenylpropanoid glycosides like verbascoside and teupolioside (504); cyanogenic glycosides such as sambunigrin as well as gallic acid and its derivatives (496).Wound healing substances isolated from Termalia arjuna were tannins (457); oleanoic acid from Anredra diffusa (70); polysaccharides from Opuntia ficus-indica (365); shikonin derivatives including deoxyshikonin, acetylshikonin, 3-hydroxyl isovaleryl shikonin and 5,8-O-dimethylacetylshikonin from Onosma argentatum (361); asiaticoside, asiatic acid and madecassic acid from Centella asiatica (146-147); quercetin , isorhammetin and kempferol from Hippophae rhamnoides (261) ; and curcumin from Curcuma longa (168). The list of well-charaterized newer active ingredients is increasing with a galloping speed.
Wound repair process follows a set of biochemical reactions. At the wound site, increased amounts of superoxide anion radicals are produced by activated platelets, neutophils and the macrophases as well as by the fibroblasts, stimulated by the pro-inflammatory cytokines during the inflammation phase. These radicals are part of the inate immune system and are generated to destroy the invading microbes at the wound site. However, the oxidative stress requires careful manipulation and control as increased amounts are detrimental to the surrounding tissues and can cause heavy damage. While the system has its checks and measures in place and utilizes superoxide dismutases, catalases, glutathione peroxidases and peroxiredoxins, secreted by the adjoining cells, the impairment of such cells in certain wounds calls for use of extraneous agents that are more appropriate radical scavengers, working in synergy or independently. Several plants extracts containing proanthocyanidins, polyphenolic flavonoids and polyphenols in such situations are expected to provide enabling support to the healing process initially by the moderation of superoxide anions and later by enhancing the expression of vascular endothelial growth factor (VEGF), thereby enhancing angiogenesis and flow of blood as the repair process advances. The plant components having some such properties are described further below.
Among the soluble compounds in the plant extracts, the flavonoids, quinones, phenolic acids and phenyl propanoids have been found to possess considerable anti-microbial as well as anti-oxidant properties. A large number of flavonoids having the general structure as given in Figure 1 were found to possess antimicrobial and/or antioxidant properties.
Flavonoids are strong scavengers of reactive oxygen species .In wounds there is a tendency for sharp rise in the concentration of reactive oxygen species due to the activation of platelets, neutrophils, macrophages, lymphocytes and fibroblasts at different time points of the healing process. Infection from microbes also adds to the woes. In such situations, plant flavonoids would benefit the healing process by modulating the concentrations of reactive oxygen species. Quantitative information and correlationships are yet inadequate however.
Anthocyanins synthesized in plants via the phenylpropanoid pathway are compounds based on flavylium ion which is a kind of oxonium ion. Anthocyanins have strong radical scavenging properties and many of these compounds also exhibit anti-bacterial properties. Some of the compounds found in wound healing plants are described with general formula as in Figure 2.
Anthocyanins from Black Soyabean seed coat (Glycine max) was found to have enhanced wound healing properties (243). Extracts from Anadenanthera colubrina rich in proanthocyanidins were effective in cutaneous wound healing in rats (62). Caralla brachiata rich in proanthocyanidins is also expected to be useful for such purposes.
Several soluble quinones present in the roots of plants such as Alkanna tinctoria, Arnebia densiflora and Arnebia euchroma and many others were also found to possess antimicrobial propertie (49, 79, 80); these also had some antioxidant properties. The general structures of such soluble quinones are indicated schematically in Figure 3: Structures of several of these compounds with ocurenace and biological properties have been reviewed (503). Emodin from Rheum officinale Baill showed encouraging results of repair of excision wounds in rats (413). Embelin from extracts of leaves of Embelia ribes Burm was effective in healing wound in excision, incision and dead space model on Swiss Albino rats (213).
Terpenoids of different structures including the monocyclic and the multicyclic ones have been identified to possess antimicrobial activity; these compounds are anticipated to manifest their antimicrobial effects through the process of synergy with other compounds present in the plant extract. Besides the monocyclic terpenoids, several dicyclic, tricyclic and the pentacyclic terpenoids of plant origin have been identified which possess considerable antimicrobial activities.  (254)(255), Laurus nobilis (50), Paullinia pinnata (375), Vernonia arborea Hk. (480) etc. are substantially attributed to the presence of a wide range of terpenoids. Since the structures of each group of terpenoids including mono and multicyclic ones vary considerably, a generalized structure could not be assigned to describe a general class of terpenoids having antimicrobial activities. However, assessing the chemical structures of terpenoids present in the above plants for identifying specific mono or multi-cyclic skeletons for in-vitro modification with a view to develop newer compounds is anticipated to be facilitated from the study of the listing of plants.
Phenolics including tannins, substituted cinnamic acids, phenolic acids and phenyl propanoids have also shown antimicrobial as well as antioxidant properties. Tannins from Phyllanthus muellerianus (380), Terminalia arjuna (457), Terminalia avicennioides (458), Terminalia bellirica (459), Terminalia chebula (460) and Terminalia coriacea (461) are reported to promote wound healing. Tannins are polyphenolic compounds containing considerable numbers of hydroxyls, carboxyls and other hydrophilic structures and are considered to be macromolecules. All natural tannins could not be included in one generic structure although there are considerable resemblances in chemical properties among different tannins.
A number of substituted cinnamic acids of the general structure schematically represented in Figure 4, have been isolated from plant extracts having antimicrobial properties.
Caffeic acid, chlorogenic acid and ferulic acid are documented to have wound healing activities (507)(508)(509); these substances also work as free radical scavengers. Plant materials like Buddleja globosa (110) leaves containing caffeic acid derivatives, Scorzonera cana var. jacquiniana and S. eriophora (427) containing chlorogenic acids and Angelica sinensis (510) containing ferulic acid have been found to be effective in wound healing.
Several phenolic acids were also found to possess sound antimicrobial properties. In addition, many phenolic acids also had profound radical scavenging properties. Since the structures of phenolic acids vary considerably, it was not possible to represent all of them with one generic structure. However, plants such as Ageratum conyzoides (44,46), Emblica officinalis (214), Punica granatum (30,404), Salvia hypoleuca (420), Schinus lentiscifolius (423), Strobilanthes crispus (446), Quercus infectoria (407), Ximenia Americana (496) contain tannins and gallic acid and these have excellent wound healing activituies. Various mixed phenolic acids are present in plants such as gall nuts, tea leaves, oak bark etc. (511); although these plant materials have not been used in wound healing, it is anticipated that use of these would have beneficial effects as radical scavengers and therefore could be useful in wound healing.
Phenyl propanoids, especially in the form of glycosides, are natural polyphenols which are widely distributed in the plant kingdom. The roots and aerial parts of the families of Asteraceae, Labiateae, Liliceae, Oleaceae and related ones contain phenylpropanoid glycosides (sometimes also incorporating glucose, galactose and rhamnose in these compounds). Such substances are powerful antioxidants. Utilizing plant cells from Ajuga reptans and Syringa vulgaris two phenylpropanoid glycosides namely teupolioside and verbascoside were producued which had profound antiinflammatory and wound healing properties (504).
Water soluble alkaloids including quinazolines, isoquinazolines, indole derivatives including betalains and eumelanins from a diverse range of plants have been found to possess antioxidant properties and many of these have also antimicrobial characteristics. Adhatoda vasica (36), Adhatoda zeylanica (38), Berberis lycium (512), Catharanthus roseus (139) etc. are rich in certain alkaloids which have antimicrobial properties. These plant extracts are useful in wound healing purposes and are traditionally used by various societies. Since the compounds from such plants considerably vary in chemical structure, a generalized structure for all these alkaloids could not be presented. However, presently much work is being done to synthesize newer antimicrobial compounds utilizing quinazoline and indole backbones (513)(514)(515).
Several natural heteropolysaccharides such as arabinogalactans and rhamnogalacturonans are present in large quantities in certain plants. Hot (364,365), and Parquetina nigrescens (57) containing mainly water soluble polysaccharides have been used in traditional practices for treating external wounds. Although exact structure activity relationships are not yet understood, it is believed that the polysaccharides accelerate the phases of reepithelialization and remodeling by influencing interactions in the cell matrix and by moderating the deposition of laminin (365). Polysaccharides are also believed to exhibit immunomodulatory action on the cells around the wound site (57), which stimulate cell proliferation.
Mitogenic properties are anticipated to enhance healing process as phytochemicals possessing such properties exhibited in a structured manner are expected to enhance cell division. Whole plant extracts from Achyrenthus aspera (33) and 'Calproteins' from Calatropis procera (123) were believed to posssess constituents having mitogenic activities. Extracts of Calendula officinalis flowers have increased proliferation potential for endothelial progenitor cells (518). Extracts of leaves from Datura alba (195) and Euphorbia heterophylla (220) are believed to have strong mitogenic potentials contributing to the healing process. In most of the claims however, the specific compounds responsible for the mitogenic activities have not been identified. Like mitogens, substances promoting angiogenesis would also promote healing process by supply of blood around the wound sites. Extracts of Aloe vera (54), Alternanthera brasiliana (58), mixtures of extracts from Astragali radix and Rehmanniae radix (519) (249), Heliotropium indicum (255), Hyptis suaveolens (273), Indigofera asphalathoides (275), Jasminum sambac (280), Kalanchoe pinnata (289), Leonotis nepetaefolia (299), Martynia annua (315), Moringa oleifera (336), Nigella sativa (347) etc were claimed to be responsible for facilitated healing as evidenced by increased DNA production and total collagen enhancement at wound site with time. However, in all these cases individual specific chemical entities having the properties of enhancing the wound healing process have not been described. Enhanced healing in all these cases probably arises from synchronized action of multiple active ingredients present in the phyto extracts.

CONCLUDING REMARKS
Wound healing is a complex but highly regulated process. Healing of all kinds of wounds follows common steps of recovery. Microbial colonization is often inescapable. Infections of wounds from potentially pathogenic bacteria in most situations of causation of wounds are inevitable. Therefore, the utmost aim is to restore the host-bacterial balance by ensuring that the wound is cleaned up and antimicrobial agents are used with moisture retentive bandages. At the same time as oxidative stress during the initial healing process is high, the next objective is to use agents that scavenge the excess of reactive oxygen anions generated at the wound site and rationalize their concentration. Other objectives are to stimulate the adjoining tissues in the wound so that the processes of cell proliferation, remodeling and maturation are facilitated. The plant kingdom is rich in chemical constituents for mitigating these objectives acting especially as antimicrobial agents as also as the free radical scavengers, and several compounds have since been isolated. The steps of tissue repair involving interactions of neutrophills, macrophages, fibroblasts and other cells at the wound site along with deposition of collagens with proper laying out around the wounds are complex processes and require understaning of multiple interactions with several agents. Concomitantly, formation of new blood vessels through the process of angiogenesis to ensure continuous supply of nutrients and healing supplements also require detailed understaning. In all these processes, several compounds from the plant extracts would work synergistically to provide the desired effect and therefore such phytochemicals concentrated and blended in optimal concentrations from multiple sources are expected to be available in the future years to carry out multi-tasking efforts in wound healing of all kinds as more knowledge about the properties of the key constituents and the healing processes are unveiled.

ACKNOWLEDGEMENT
The assistance of Dhruva Chatterjee, Computer Executive-KEE GAD Biogen Pvt. Ltd, in preparing the Corel Draw figures and in arranging the References systematically is gratefully acknowledged. Authors have no conflict of interest regarding the content of this article.