REVIEW ON WOUND HEALING ACTIVITY OF NATURAL PRODUCTS

For the course of pharmaceutical literature and seminar II (Phar 652)

ADDIS ABABA UNIVERSITY SCHOOL OF GRADUTATE STUDENTS

SCHOOL OF PHARMACY DEPARTMENT OF PHARMACOGNOSY

By: Michael G/ Hiwot

August 2010
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Acknowledgement
I am highly grateful for Dr Kaleab Asres for giving me this seminar work, so that I could make an endeavor to know the different scientific aspects while studying the wound healing activity of natural products.

Table of Contents Contents page

REVIEW ON WOUND HEALING ACTIVITY OF NATURAL PRODUCTS................1 ACKNOWLEDGEMENT .............................................................................................I TABLE OF CONTENTS..............................................................................................II CONTENTS PAGE................................................................................II

LIST OF ABBREVIATIONS ......................................................................................V SUMMARY ................................................................................................................VI 1. INTRODUCTION ....................................................................................................1 2. TYPES OF WOUNDS ...........................................................................................2 3. WOUND HEALING AND THE HEALING CASCADES ........................................4 4. EXISTING THERAPY AIMED FOR WOUND HEALING.......................................6 5. MODELS TO STUDY WOUND HEALING ACTIVITY............................................7
5.1. In vivo models................................................................................................................................................7 5.1.1. Incision wound model ............................................................................................................................7 5.1.2. Excision wound model ...........................................................................................................................7 5.1.3. Dead space analysis ................................................................................................................................7 5.1.4. Burn wound model:.................................................................................................................................8 5.2. In vitro models ..............................................................................................................................................8 5.2.1. In vitro test for fibroblast growth stimulation..........................................................................................9 5.2.2. Chorioallantoic membrane (CAM) model: .............................................................................................9 5.2.3. Antioxidant activity...............................................................................................................................10 5.2.4. Antimicrobial activity ..........................................................................................................................10

6. STUDY PARAMETERS........................................................................................10
6.1. In vivo study parameters............................................................................................................................10 6.1.1. Wound closure ......................................................................................................................................11 6.1.2. Epithelialization period .........................................................................................................................11 6.1.3. Tensile strength:....................................................................................................................................12

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6.1.4. Increase in granulation tissue.................................................................................................................12 6.2. In vitro study parameters...........................................................................................................................13 6.2.1. Antimicrobial activity: ..........................................................................................................................13 6.2.2. Angiogenesis.........................................................................................................................................13

7. PHYTOCHEMICALS RESPONSIBLE FOR WOUND HEALING ACTIVITY.......13

7.1. Polyphenols (flavonoids and tannins)........................................................................................................14 7.1.1. Flavonoids.............................................................................................................................................14 7.1.2. Tannins..................................................................................................................................................17 7.2. Phenolic acids ..............................................................................................................................................18 7.3. Phenyl propanoids ......................................................................................................................................18 7.4. Terpenes and terpenoids.............................................................................................................................19 .............................................................................................................................................................................19 7.5. Alkaloids.......................................................................................................................................................19 .............................................................................................................................................................................20 .............................................................................................................................................................................20 7.6. Saponins........................................................................................................................................................20 7.7. Plant vitamins..............................................................................................................................................20 7.8. Miscellaneous compounds ..........................................................................................................................21

..................................................................................................................................22 8. PLANTS WITH POTENTIAL WOUND HEALING ACTIVITY .............................23

Achillea kellalensis Bioss. & Hausskn. (Asteraceae).......................................................................................23 Ageratum conyzoides L (Asteraceae)...............................................................................................................23 Allium cepa L. (Liliaceae)..................................................................................................................................23 Aloe vera (Asphodelaceae).................................................................................................................................24 Alternanthera brasiliana Kuntz (Amaranthaceae) ........................................................................................24 Anthocleista nobilis G. don. (Loganiaceae)......................................................................................................24 Areca catechu L. (Arecaceae)............................................................................................................................24 Azardica indica (Meleaceae)..............................................................................................................................25 Calotropis gigantea L. (Asclepiadaceae)..........................................................................................................25

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Carica papaya L. (Caricaceae)..........................................................................................................................25 Catharanthus roseus L. (apocyanaceae) ..........................................................................................................25 Centella asiatica (Mackinlayoideae).................................................................................................................26 Cocos nuclifera L. (Arecaceae)..........................................................................................................................26 Cordial dichotoma (Boraginaceae)...................................................................................................................26 Dissotis theifolia (Melastomataceae).................................................................................................................26 Elaeis guineensis Jacq (Mackinlayoideae)........................................................................................................26 Euphorbia heterophylla (Euphorbiaceae)........................................................................................................27 Ficus religiosa (Moraceae).................................................................................................................................27 Ginkgo biloba (Ginkgoaceae)............................................................................................................................27 Helianthus annus L. (Asteraceae).....................................................................................................................27 Hoslundia opposita Vahl (Lamiaceae)..............................................................................................................28 Hydnocarpus wightiana (Flacourtaceae).........................................................................................................28 Hypericum prolificum (Hypericaceae).............................................................................................................28 Jasminum auriculatum (Oleaceae)...................................................................................................................28 Jatropha curcas L. (Euphorbiaceae) ...............................................................................................................29 Lantana camara (Verbenaceae)........................................................................................................................29 Lawsonia inermis L. (Luthraceae)....................................................................................................................29 Mimosa pudica ( Mimosaceae)..........................................................................................................................29 Napoleona imperialis (Lecythidaceae)..............................................................................................................29 Ocimum kilimandscharicum (Lamiaceae) ......................................................................................................30 Ocimum sanctum L. (Labiaceae)......................................................................................................................30 Phyllanthus niruri L. (Euphorbiaceae)............................................................................................................30 Quercus infectoria (Fagaceae) ..........................................................................................................................30 Rubia cordifolia L. (Rubiaceae) .......................................................................................................................31 Tragia involucrata L. (Euphorbiaceae)............................................................................................................31 Trichosanthes dioica (Cucurbitaceae)..............................................................................................................31 Tridax procumbens (Asteraceae)......................................................................................................................32

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Vernonia arborea (Asteraceae).........................................................................................................................32

9. REFERENCE........................................................................................................33

List of abbreviations
CAF: CAPE: CAT: DPPH: EGCG: FBS: HMF: MEM: MIC: NADH: NADPH2: NFkB: ROS: SOD: TBA: Chloramphenicol Caffeic Acid Phenylether Ester Catalase 2, 2-diphenyl- picrylhydrazyl Epigallocatechin gallate Fetal Bovine Serum Hydroxymethylfurfural Minimum Essential Medium Minimum Inhibitory Concentration Nicotinamide adenine dinucleotide Nicotinamide adenine dinucleotide phosphate Nuclear Factor kappa-light-chain-enhancer of activated B cells Reactive Oxygen Species Superoxide Dismutase Thiobarbituric Acid v

TB4: TGF1:

Thymosin beta 4 Transforming Growth factor 1

Summary
A wound is a disruption of the continuity of tissues produced by external force. When considering the manner in which the skin or tissue is broken, there are seven general kinds of wounds: abrasions, incisions, lacerations, punctures, avulsions, amputations and contusions. Because the skin serves as a protective barrier against the outside world, any break in it must be rapidly and efficiently mended. Wound healing involves highly orchestrated sequences of events, which is triggered by tissue injury and ends in either partial or complete regeneration or more commonly by repair. Successful wound healing and tissue regeneration depends on tightly regulated hemostasis, inflammation, matrix synthesis, proliferation, wound contraction and tissue remodeling to restore tissue function and integrity. Wound healing processes are influenced by factors like infections, nutritional status, drugs and hormones, type and sites of wound, and wasting diseases like diabetes. In folklore medicine, medicinal plants have been used widely in facilitating wound healing. Phytochemicals like tannins, flavonoids, polyphenols, alkaloids, terpenes and terpenoids, and ascorbic acid are known to be responsible for wound healing properties of medicinal plants. The high degree of successes of medicinal plants in assisting wound healing has

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inspired many researches, which are aimed at validating the claims and discovering mechanisms, which possibly explains the potentials of these herbs on wound repair processes. While studying the wound healing activity of medicinal plants, there are in vivo and in vitro models. The in vivo model includes: incision wound, excision wound, dead space wound and burn wound models. On the other hand, the in vitro model consists of antioxidant activity testing, anti-microbial activity testing, in vitro test for fibroblast growth stimulation, chorioallantoic membrane (CAM) model and others. The in vivo study parameters are time of epithelialization, wound closure, tensile strength, and increase in granulation tissue. In the case of in vitro models, the study parameters are angiogenesis, antioxidant activity, and antimicrobial activity. In this material, the wound healing activity of fourty (40) different medicinal plant species was reviewed.

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1. Introduction
The wound may be defined as a loss or breaking of cellular and anatomic or functional continuity of living tissues (Nalwaya et al., 2009). Because the skin serves as a protective barrier against the outside world, any break in it must be rapidly and efficiently mended (Martin, 1997). Healing of wounds is an important biological process involving tissue repairs and regeneration (Esimone et al., 2009). It is a complex and dynamic process of restoring cellular structures and tissue layers (Mercandetti and Cohen, 2007). Proper healing of wound is essential for the restoration of disrupted anatomical continuity and disturbed functional status of the skin (Annan and Dickson, 2008). Current estimates indicate that nearly 6 million people suffer from chronic wounds worldwide (Sasidharan et al., 2010). One of the surveys conducted by the WHO reports that more than 80% of the worlds population still depends upon the traditional medicines for various diseases (Patel et al., 2009). Some medicinal plants have been employed in folk medicine for wound care. Some of these plants either possess pro-wound healing activities or exhibit antimicrobial and other related properties that are beneficial in overall wound care (Esimone et al., 2009). Recently, the traditional use of plants for wound healing has received attention by the scientific community. Approximately one-third of all traditional medicines in use are for the treatment of wounds and skin disorders, compared to only 1-3 % of modern drugs (Ghasemi et al., 2010). With a view to the increase in the wide spectrum of medicinal usages, the present day requires a new biologically active ointment which exhibit wound healing activity as local applications (Roy et al., 2009). Wound healing studies are mainly aim to detect various means and factor influencing healing process, so they could be either used or avoid in clinical practice to favorably alter the healing process (Sachin et al., 2009).

2. Types of wounds
When we consider the manner in which the skin or tissue is broken, there are seven general kinds of wounds: abrasions, incisions, lacerations, punctures, avulsions, amputations and contusions. Many wounds, of course, are combinations of two or more of these basic types (http://www.tpub.com/content/medical/10669-c/css/10669-c_101.htm.). Abrasions: Injuries where a superficial layer of tissue is removed

(http://www.medstudentlc.com/page.php?id=65). This kind of wound can become infected quite easily because dirt and germs are usually embedded in the tissues (http://www.tpub.com/content/medical/10669-c/css/10669-c_101.htm.). Incisions: Incision wound is characterized by a clean cut, as by a sharp instrument (http://runonce.msn.com/runonce3.aspx). Incisions tend to bleed freely because the blood vessels are cut cleanly and without ragged edges. Of all classes of wounds, incisions are the least likely to become infected, since the free flow of blood washes out many of the microorganisms that can cause infection (http://www.tpub.com/content/medical/10669c/css/10669-c_101.htm.). Lacerations: These are injuries where by tissue is torn

(http://www.medstudentlc.com/page.php?id=65). A wound made by a dull knife, for instance, is more likely to be a laceration than an incision. Bomb fragments often cause laceration. Lacerations are frequently contaminated with dirt, grease, or other material that is ground into the tissue; they are therefore very likely to become infected (http://www.tpub.com/content/medical/10669-c/css/10669-c_101.htm.). Punctures: Wounds made by nails, needles, wire, and bullets are usually punctures. The possibility of infection is great in all puncture wounds, especially if the penetrating object has tetanus bacteria on it. To prevent anaerobic infections, primary closures are not made in the case of puncture wounds (http://www.tpub.com/content/medical/10669c/css/10669-c_101.htm.).

Avulsions: Injuries where a section of tissue is torn off, either partially or in total. (http://www.medstudentlc.com/page.php?id=65). Bleeding is usually heavy. In certain situations, the torn tissue may be surgically reattached (http://www.tpub.com/content/medical/10669-c/css/10669-c_101.htm.). Amputations: amputation is the removal of the limb from the body. Shock is certain to develop in these cases. The limb can often be successfully reattached (http://www.tpub.com/content/medical/10669-c/css/10669-c_101.htm.). Contusions: Such injuries result from a forceful blow to the skin and soft tissue but, leaving the outer layer of skin intact. These injuries generally require minimal care as there is no open wound. However, an expanding hematoma can damage overlying skin and demands evacuation (http://www.medstudentlc.com/page.php?id=65 ).

3. Wound healing and the healing cascades

Wound healing involves sequences of events, which is triggered by tissue injury and ends in either partial or complete regeneration or more commonly by repair (Ather et al., 2007). The healing cascade begins immediately following injury when the platelets come in contact with exposed collagen (Nayak, 2006). Wound healing can be classified into any of three types: healing by first intention, healing by second intention or healing by third intention, depending on the nature of the edges of the healed wounds (Esimone et al., 2005). Primary wound healing or healing by first intention occurs within hours of repairing a full-thickness surgical incision (Mercandetti and Cohen, 2007). In wounds healed by the first intention, the edges are smoothly closed that no scar is left (Esimone et al., 2005). Wound healing by second intention involves formation of granulation tissues, which fill up the gaps between the wound edges and is associated with significant loss of tissue, leaving little scars (Esimone et al., 2005). In a third type of healing, a full-thickness wound is allowed to close and heal. It results in an inflammatory response that is more intense than with primary wound healing (Mercandetti and Cohen, 2007). Most skin lesions are healed rapidly and efficiently within a week or two. However, the product is neither aesthetically nor functionally perfect (Martin, 1997). The healing process involves four types of phases (Shetty et al., 2006). Clot formation The formation of a clot is the immediate response to any trauma. The clot has two functions; it temporarily protects the uncovered tissues and it serves as a provisional matrix for cell migration (Polimeni et al., 2006). Inflammation Within hours of injury, inflammatory cells populate the clot and cleanse the wound from bacteria and necrotic. Macrophages migrate into the wound area and, in addition to wound debridement, secrete polypeptide mediators targeting cells involved in the wound-healing process (Polimeni et al., 2006). Growth factors and cytokines secreted by macrophages are involved in the proliferation and migration of fibroblasts, endothelial cells, and smooth

muscle cells into the wound area (Polimeni et al., 2006). If the inflammatory phase is prolonged, degradation of collagen will exceed its synthesis (Sasidharan et al., 2010). Granulation The formation of new vasculature requires extracellular matrix and basement membrane degradation followed by migration, mitosis, and maturation of endothelial cells (Mercandetti and Cohen, 2007). Epithelialization of the wound is initiated within hours of injury. Epithelial cells from the basal layer proliferate and migrate through the fibrin clot and eventually the breach in the epithelium is sealed (Polimeni et al., 2006). Remodeling In this phase the wound undergoes contraction resulting in a smaller amount of apparent scar tissue (James and Friday, 2010). Remodeling can last for years after the initial injury occurred. Maximal tensile strength of the wound is achieved by the 12th week, and the ultimate resultant scar has only 80% of the tensile strength of the original skin (Mercandetti and Cohen, 2007). Whether the damaged tissues heal by regeneration or repair depends upon two crucial factors: the availability of cell type(s) needed; and the presence or absence of signals necessary to recruit and stimulate these cells (Polimeni et al., 2006).

Figure1: Phases of cutaneous wound repair. 5

4. Existing therapy aimed for wound healing

Factors like nutritional status, concurrent therapy (such as corticosteroids) and clinical conditions, such as anemia and diabetes affect the wound healing process. Therefore, the objective must be the holistic management of the patient and not just the wound (David, 2008). Topical iodine in the form of Lugols solution regenerates human scar tissue back to normal (David, 2008). Povidone-iodine (5% Betadine) cream is also used for wound healing purpose (Kumar et al., 2009). Topical antimicrobial therapy is one of the most important methods of wound care (Esimone et al., 2009). As an example neomycin-bacitracin powder (Cicatrin), gentamycin ointment, tetracycline ointment, nitrofurazone ointment are among the standard antibiotic used in wound healing (Esimone et al., 2005; Annan and Dickson, 2008; Esimone et al., 2009; Nalwaya et al., 2009).

5. Models to study wound healing activity

5.1. In vivo models
In vivo models of wound healing generally use small rodents such as guinea pigs or rats (Houghton et al., 2005). Granulation, collagen maturation, and scar formation are some of the many phases of wound healing, which run concurrently, but independent of each other (Udupa et al., 2005). As a result, the use of a single model is inadequate as different models provide different information, and there are three in vivo wound study models known so far.

5.1.1. Incision wound model

Wound breaking strength is the important parameter to be studied in incision wound model (Ghasemi et al., 2010). Two longitudinal paravertebral incisions can be made through the skin and cutaneous muscles at a distance 1cm from the midline on either side of the vertebral column of anaesthetized rat (Annan and Dickson, 2008). The parted skin is sutured and the skin breaking strength of the wound is measured after ten days of wound induction (Barua et al., 2009).

5.1.2. Excision wound model

Excision wounds are used to study the rate of wound contraction and epithelialization (Nalwaya et al., 2009). The excision wound is made by excising the full thickness of circular skin from the animal under anaesthesia (Karodi et al., 2009). Then wound contraction is assessed by tracing the wound area first on transparent paper and subsequently transferring to a graph paper (Barua et al., 2009). In excised wound, since the edges are not in contact with each other, contraction and epithelialization are necessary for the repairing process (Ghasemi et al., 2010). Hence, epithelialization and wound contraction are the two parameters to be studied in case of excision wound (Malviya et al., 2009).

5.1.3. Dead space analysis

Dead space wound can be induced by making a pouch through a small cut in the skin of the rat (Paschapur et al., 2009). A polypropylene tube is implanted subcutaneously beneath the skin. The day of the wound creation is considered as day zero (Azeez et al., 2007). On day 10, the animals are sacrificed by overdose of anaesthesia, the polypropylene tube are

carefully removed and dried in an oven at 60C to a constant weight, and the weight is recorded. The level of increase (%) in the weight of granuloma tissue formed is calculated relative to the control (Okoli et al., 2009). The samples are kept at -70C for biochemical analysis until assayed. Regenerated tissues is cut in the form of square pieces along with normal skin on either side of the wound and preserved in 10% buffered formalin for histological studies. Dead space wound is important to study the physical and mechanical changes in the granuloma tissue (Paschapur et al., 2009). In dead space wound; granulation tissue dry weight, breaking strength and hydroxyproline content are the important parameters to be studied (Malviya et al., 2009).

5.1.4. Burn wound model:

The burn makes an extreme damage to the barrier of the skin and triggers a cascade of events such as tissue necrosis and body fluid exudation, creating a perfect medium culture to the bacterium (Feng et al., 2010). Partial thickness burn wound is inflicted upon animals starved overnight and under anaesthesia, but pouring hot molten wax 800C into a metal cylinder with circular opening, placed on the back of the animal. Wound contraction and epithelialization period are the two parameters to be studied in this model (Srivastava and Durgaaprasad, 2008).

5.2. In vitro models

In vitro tests are now widely employed in ethnopharmacological research because of ethical reasons and their usefulness in bioactive-guided fractionation and determination of active compounds. A summary of these tests is shown in table 1 (Houghton et al., 2005).

Table 1: In vitro assays for different activities associated with wound healing. Activity Anti-inflammatory Assay NFkB synthesis inhibition Positive control Actigenin, CAPE (caffeic acid phenylether Eicosanoid synthesis inhibition Fibroblast proliferation Effect on keratinocytes Fibroblast protein expression Collagen lattice formation Antimicrobial activity Antioxidant properties Natural Red uptake by viable cells Involucrin expression Proteomics Collagen lattice contraction Serial dilution to determine MIC ester) Indomethacin for cycloxygenase inhibition 10% foetal serum A23187 Not used Not used Miconazole for fungi and

CAF for bacteria DPPH for free radical scavenging Propyl gallate Malondialdehyde determination Propyl gallate using TBA Ptorection of growing cells challenged oxidant Catalase

5.2.1. In vitro test for fibroblast growth stimulation

Fibroblasts are trypsinized, centrifuged and resuspended in MEM/15%FBS/1% L-glutamine (Annan and Dickson, 2008) Use of trypsin for tissue disaggregation is called trypsinization (Shenoy, 2007). The cells will be seeded at 37C in a humidified incubator of 5%C02. The fibroblast cells will be incubated and assayed after five days using the neutral red assay method to assess the effect of the extracts on the growth of the cells (Annan and Dickson, 2008).

5.2.2. Chorioallantoic membrane (CAM) model:

In this model, embryonated chicken eggs (9 days old) are selected and a small window will be made in the shell (Barua et al., 2009). Albumin is removed on the 4th day after fertilization to drop the embryo away from the shell and to allow the CAM to develop in a

way that was accessible to treatment (Melkonian et al., 2000). Through the window, a sterile disc treated with the extract of interest is placed inside the egg at the junction of two blood vessels. The window is resealed and the egg will be incubated at 37 0c for three days. The window will then be opened and the growth of new capillary will be observed as in figure 2 (Barua et al., 2009).

Figure 2: Emnbryonated chicken egg of 9 days old.

5.2.3. Antioxidant activity

It is believed that reactive oxygen species are deleterious to wound healing due to their harmful effects on cells and tissues (Annan and Dickson, 2008). DPPH radical scavenging activity the easiest method assess the antioxidant activity of natural products. The DPPH scavenging activity of the plant of interest is measured from the bleaching of a purple colored methanol solution of 2, 2- diphenyl - picrylhydrazyl (DPPH) which is used as a reagent in a spectrophotometric assay (Annan and Dickson, 2008).

5.2.4. Antimicrobial activity

Open wounds are particularly prone to infection, especially by bacteria, and provide an entry point for systemic infections. Infected wounds heal less rapidly and often result in the formation of unpleasant exudates and toxins will be produced with concomitant killing of regenerating cells. Antibacterial and antifungal compounds in a traditional remedy may prevent this occurring and may underlie its use in treating wounds (Houghton et al., 2005).

6. Study parameters
6.1. In vivo study parameters

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There are different study parameters while dealing with wound healing in vivo. It includes wound closure, time of epithelialization, tensile strength and scar size.

6.1.1. Wound closure

More than 50% of connective tissue is made up of collagen in case of sutured wounds. Hence lying down and weaving of the collagen material into the healing wound is an important feature. Therefore, it is understandable that substances that influence the collagen turnover or maturation enhance the process of wound healing (Azeez et al., 2007). Wound contraction is a process that occurs throughout the healing process (James and Friday, 2010). It is mainly a part of the proliferative phase of wound healing that occur through the centripetal movement of the tissues surrounding the wound, which is mediated by myofibroblasts (Sasidharan et al., 2010). Although myofibroblasts may be important for long-term wound contraction, scar formation, and matrix remodeling, research a finding suggest that the organized network of cells containing actin filaments at the edge of the normal wounds may initiate wound contraction (Bullard et al., 1999). The presence of myofibroblasts and the apoptosis level can be regulated by both TGF1 and by the extracellular matrix and the tension in the wound bed determines the type of scar at different body sites (Chipev and Simon, 2002). Contractions of wound is studied by tracing the raw wound in excision wound model (Annan and Dickson, 2008), and wound contraction (%) is calculated using the relation below (Okoli et al., 2009).
Wound contractio n (%) = WD 0 WDt 100 WD0 Where:

WD 0 = the wound

diameter on day zero WDt = the wound diameter on day t

6.1.2. Epithelialization period

Epithelialization, which is the process of epithelial renewal after injury, involves the proliferation and migration of epithelial cells towards the center of the wound (Atala, 2008;

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Okoli et al., 2009). The epithelialization time is the time at which a complete scar formation occur (Sachin et al., 2009).

6.1.3. Tensile strength:

Tensile strength is the resistance to breaking under tension. It indicates how much the repaired tissue resists to breaking under tension and may indicate in part the quality of the repaired tissue (Rashed et al., 2003). For this purpose, the newly repaired tissue including scar is excised from treated and control animals and will be loaded between the upper and lower holder of tensile testing machine, in such a way that the effective load bearing size with the wound remaining in the centre (Annan and Dickson, 2008). The total breaking load is calculated the following formula (Kokane et al., 2009).

Figure 2: Tensiometer to measure tensile strength (Rashed et al., 2003).

6.1.4. Increase in granulation tissue

In dead space wound model, increase in granuloma tissue is associated with enhanced collagen maturation and increased protein content as well as angiogenesis in the wound (Okoli et al., 2009). It is a well-accepted fact that wounds in most tissues heal by repair, by laying down non-specific connective tissue, where more than 50% is made up of collagen. Substances that influence the collagen turnover or maturation enhance the process of wound healing. Collagen is a fibrous protein component of the connective tissue consisting of hydroxyproline, hydroxylysine and glycine as principal constituents, among which hydroxyproline is considered a specific aminoacid (Azeez et al., 2007). Hence, the increased hydroxyproline content of the granulation tissue is an indicative for an increase in collagen turnover. Increase in breaking strength of granulation tissue indicates the enhanced collagen maturation by increased cross-linking (Panda and Tripathy, 2009).

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6.2. In vitro study parameters

It is generally acknowledged that in vitro tests are too reductionist to extrapolate their results to provide evidence for clinical efficacy, and that eventually animal testing and clinical trials have to be performed. The effects of pharmacological agents which modulate many of wound healing processes, such as fibroblast proliferation or reduction of oxidative stress, antimicrobial activity can be assessed by in vitro experiments (Houghton et al., 2005).

6.2.1. Antimicrobial activity:

Infection is defined as microbial pathogens proliferating in a wound, causing tissue damage and eliciting a host inflammatory response (Armstrong and Lipsky). A number of microorganisms have been found to infect wounds among which are Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus faecalis, Escherichia coli, Clostridium perfringens, Clostridium tetani, Coliform bacilli and enterococcus (Odimegwu et al., 2008; Odimegwu et al., 2008). As infections being a major cause of morbidity and mortality in wound patients, these herbal extracts may prevent infection that leads to high risk of sepsis, and thereby prevents the prolongation of inflammatory phase (Arnold and Barbul, 2006; Li et al., 2007).

6.2.2. Angiogenesis
Angiogenesis is the formation of new blood vessels; it takes place during embryonic development, wound healing and tumor growth (Hegazy et al., 2009). Angiogenesis during wound repair serves the dual function of providing the nutrients demanded by the healing tissues and contributing to the structural repair through the formation of granulation tissue (Barua et al., 2009). Factors that contribute to angiogenesis include high lactate levels, acidic pH, and, in particular, decreased oxygen tension (Monaco and Lawrence, 2003). Angiogenesis can be studied in chorioallantoic membrane (CAM) model (Melkonian et al., 2000; Barua et al., 2009).

7. Phytochemicals responsible for wound healing activity

Medicinal plants that possess wound healing activity perform their action through their phytochemicals they have in them. Not all phytochemicals have wound healing activity,

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rather the following are the most responsible group of compound that assist wound healing process in many ways.

7.1. Polyphenols (flavonoids and tannins)

These diverse groups of compounds have received much attention as potential natural antioxidant in terms of their ability to act as both efficient radical scavengers and metal chelators (Nagulendran et al., 2007). High correlation coefficients between the phenolic content and antioxidant activities have been reported for various food commodities (Akond et al., 2010). The antioxidant property of honey is well known, because it contains a number of compounds with antioxidant properties such as, flavonoids, phenolic acids, proteins, amino acids, ascorbic acid, HMF, and some enzymes (Makawi et al., 2009). Polyphenols can increase the activity of catalase and glutathione peroxidase, which detoxify H2O2 by converting it to O2 and H2O (Oaka et al., 2005). Mwh69 They are also known to stimulate wound healing (Sasidharan et al., 2010). As an example polyphenols of areca have been stated to promote wound healing of incision and dead space wounds and the period of epithelialization in the excision wounds (Azeez et al., 2007).

7.1.1. Flavonoids
Flavonoids are a large group of natural products widely distributed in nature (Galicka et al., 2007). They are present in fruits, vegetables, chocolates, herbs and beverages, such as wine, tea or beer (table 2) (Callic et al., 2005). The chemical diversity, size, three-dimensional shape, and physical and biochemical properties of flavonoids allow them to interact with targets in different subcellular locations to influence biological activity in plants, animals, and microbes (Buer et al., 2010). They have a C6-C3-C6 backbone, and apart from modifications to this backbone, the marked structural variety of flavonoids is due to their conjugation to sugars at different sites of the molecule (Shieber et al., 2009). Food Compound Subclass Amount (mg/100g)

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Cherries (sweet, raw) Pelargonidin Chocolate (dark) Tea leaves Wine (red) Grapefruit (raw) Celery (raw) Cranberry (raw) Garlic (raw) Orange (raw) Kale (raw) Catechin Epicatechin Catechin Epicatechin Malvidin Catechin Naringenin Apigenin Quercetin Quercetin Myricetin Quercetin Hesperetin Kaempferol

Anthocyanidin Flavan-3-ols Flavan-3-ols Anthocyanidin Flavan-3-ol Flavanone Flavone Flavonol Flavonol Flavonol Flavonol Flavanone Flavonol

0.8 12 41.2 157 293.3 4.2 8.9 78.1 6.1 3.5 14 4.3 22.6 39 14.6

. Table 2: Some food sources for flavonoids (Callic et al., 2005). Any drug that inhibits lipid peroxidation is believed to increase the viability of collagen fibrils by increasing the strength of collagen fibers, preventing the cell damage and by promoting the DNA synthesis (Panda and Tripathy, 2009). Flavonoids have been documented to possess potent antioxidant and free radical scavenging effect, which is believed to be one of the most important components of wound healing (Shenoy et al., 2009). Bioflavonoids are thought to benefit connective tissue by binding to elastin, preventing its degradation by elastases (Galicka et al., 2007). They reduce lipid peroxidation not only by preventing or slowing the onset of cell necrosis but also by improving vascularity (Panda and Tripathy, 2009). The high mobility of the electrons in the benzenoid nucleus of flavonoids accounts for both their antioxidant and free-radical scavenging properties, whereas the structural resemblance between the flavonoid aglycone and many substances inherent to the biochemistry of normal biological cells, e.g., nucleic acid bases, coenzymes, steroid hormones, and neurotransmitters, explains their inhibition of enzymes,

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cytoplasmic/nuclear hormone receptors, and neurotransmitters, as well as gene induction (Havsteen, 2002). Many studies have shown that antimicrobial activities of plants can also be attributed to their flavonoid content (Owoyele et al., 2008); hence, they are helpful in prevention of wound infection. Most of the delay in wound healing is due to insufficient or excessive fibroblast activity. Thus, inhibition of fibroblast growth by flavonoids such as apigenin could be beneficial for the treatment of any skin injury. Quercetin (1), may be useful in healing after renal transplantation mwh new5. Quercitrin (1) isolated from Hypericum perforatum, was able to inhibit the growth of the fungus Fusarium graminearum (Kuster et al., 2009). Flavonoids like rutin (2), naringin (3) and quercetin (1) protect DNA damage induced by ultraviolet (Yeh et al., 2005). Strong antihistamine activity has been shown by thymonin (4) from Mentha spicata var. crispa (Labiatae) mwh new5. Santin (6) may contribute to the well known anti-inflammatory activity of the plant Tanacetum parthenium by inhibiting the cyclo-oxygenase and the 5-lipoxygenase pathways mwh new5.

Quercetin (1)

Rutin (2)

Apigenin (5)

Naringin (3) Thymonin (4)

Santin (6)

16

7.1.2. Tannins
Tannins are phenolic compounds that typically act as astringents and are found in a variety of herbal products used for wound healing. Their astringent and antimicrobial property responsible for wound contraction and increased rate of epithelialization (Panda and Tripathy, 2009). Medicinal plants that are known and/or used for their wound-healing or anti-inflammatory properties tend to have high tannin contents (Arajo et al., 2008). Research results indicated that using the oxidation of linoleic acid as a model system, 3, 4, 5 tri-O-galloylquinic acid (7) displays significantly greater antioxidant properties when compared with ascorbic acid and the commercially used n-propyl gallate as well as gallic acid itself. Resveratrol (8), found in red wine have been suggested to be responsible for health benefits of wine grape through antioxidant mechanism (Yang et al., 2009). Triphenolic stilbene like epigallocatechin gallate (9), inhibited cell death induced by terbutyl hydroperoxide in the presence of ferric ion (Surh, 1999).

Resveratrol (8 )

3,4,5 tri-O-galloylquinic acid (7)

Epigallocatechin gallate (9)

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7.2. Phenolic acids

Phenolics play a beneficial role in protecting tissue from the harmful effects of reactive oxygen species (ROS) through regulation of antioxidant enzyme response through the phenolic-dependent peroxidases with dependency on pentose phosphate pathway but with reduced dependency on SOD and CAT. Oregano being rich in phenolics, as an example rosmarinic acid (10), is an effective direct quencher of free radicals (Randhir et al., 2005).

Rosmarinic acid (10)

7.3. Phenyl propanoids

The phenylpropanoid curcumin (11) and its demethoxy (12) and bisdemethoxy derivatives (13) are known to possess anti-inflammatory and antioxidant activity (Surh, 1999). Together with quercetin, curcumin (diferuloylmethane), may be useful in healing after renal transplantation (Harborne and Williams, 2000). According to the study made in Turkey, a phenylethanoid glycoside verbascoside (14) was found to show significant inhibitory effect on carrageenan-induced hind paw edema in mice was shown to possess a significant wound healing activity in these models mwh new6

R1=R2=OH, R3=R4=OCH3; Curcumin (11) R1=R2=OH, R3= OCH3, R4=H; Demethoxycurcumin (12) R1=R2=OH, R3=R4=H; Bisdemethoxycurcumin (13)

Verbascoside (14)

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7.4. Terpenes and terpenoids

Terpenoids are known to promote the wound healing process, mainly due to their astringent and antimicrobial properties, which seem to be responsible for wound contraction and an increased rate of epithelialization (Sasidharan et al., 2010). Triterpenes are also responsible for promotion of rapid wound healing (Raina et al., 2008). Sesquiterpene lactones are known to possess antioxidant activity property, which may contribute to the wound healing process (Panda and Tripathy, 2009). Four related terpenoidal compounds from Centella asiatica; asiatic acid ( ), madecassic acid ( ), asiaticoside ( ) and madecassoside ( ) known to increase collagen synthesis in dose dependent fashion through modulation of gene expression (Colen et al., 2003). Asiaticoside ( ), a trisaccharide triterpene, has been associated with the healing of wounds and duodenal ulcers of the plant Centella asiatica (Havsteen, 2002).

R=H; Asiatic acid ( ) R=Glc-Glc-Rha; Asiaticoside ( )

R=H; Madecassic acid ( ) R=Glc-Glc-Rha; Madecassoside ( )

7.5. Alkaloids
Alkaloids are known to promote wound healing process due to their antioxidant and antimicrobial activities (Sachin et al., 2009). Extracts from Symphytum asperum and Symphytum caucasicum contain allantoin (figure), claimed to be a cell proliferation stimulating agent responsible for their wound-healing propertiy (Barbakadze et al., 2009). Reportedly the alkaloid fraction of areca enhances the collagen production and hence wound healing. But contrary to the above study, there was no increase in the hydroxyproline content of granulation tissue in arecoline and polyphenol treatments (infact, there was a

19

decrease) and insignificant change in wound breaking strength of the granulation tissue with polyphenol treatment of the dead space wound model (Azeez et al., 2007).

Allantoin

Arecoline

7.6. Saponins
Saponins are known to promote wound healing process due to their antioxidant and antimicrobial activities (Sachin et al., 2009). For example, asiaticoside, a saponin is thought to be one of its active constituents Centella asiatica. A 0.2% asiaticoside solution applied topically twice daily for seven days to punch wounds in guinea pigs resulted in 56% increase in hydroxyproline, 57% increase in tensile strength, increased collagen content, and better epithelialization (MacKay and Miller, 2003). (MacKay and Miller, 2003). Triterpene saponins are also reported to possess immunomodulatory properties (Havsteen, 2002).

Asiaticoside

7.7. Plant vitamins

Vitamin A, C, and E are important in the wound healing process. Vitamin A is required for epithelial and bone tissue development, cellular differentiation, and immune system function. Substantial evidence supports the use of vitamin A as a preoperative nutritional supplement (MacKay and Miller, 2003). Ascorbic acid acts as a cofactor for the synthesis of collagen as well as elastin fibers (Sasidharan et al., 2010). For example the anti-

20

inflammatory property and the presence of vitamin A & proteins in Curcuma longa L. (zingiberaceae) result in the early synthesis of collagen fibers by mimicking fibroblastic activity (Raina et al., 2008). In addition to collagen production, ascorbic acid enhances neutrophil function, increases angiogenesis, and functions as a powerful antioxidant. (MacKay and Miller, 2003). There is a paucity of research to support the hypothesis that vitamin E aids in wound healing; however, many physicians recommend that patients apply vitamin E to surgical sites with the belief that this will improve the cosmetic outcome of the scar (Baumann and Spencer, 1999).

7.8. Miscellaneous compounds

Polyunsaturated fatty acids due to their unsaturation possess anti-oxidative effect, which is related to reacting with reactive oxygen species (ROS) (Zhang et al., 2010). Mwh83 Plant proteins as papain and chymopapain found in the epicarp of papaya are helpful in wound healing due to their antimicrobial and antioxidant activity (Anuar et al., 2008). Research results showed that emodin, an anthraquinone glycoside, promoted repair of rats' excisional wounds via a complex mechanism involving stimulation of tissue regeneration and regulating signaling pathway (Tang et al., 2007). A quinone compound embelin isolated from the leaves of Embelia ribes have signinificant wound healing activity on albino rats (figure) (Swamy et al., 2007). Capsaicin (trans-8-methyl-N-vanillyl-6-nonen- amide); (figure) is a principal pungent ingredient present in hot red and chili pepers that belong to the plant genus Capsicum (Solanaceae) (Surh, 1999). Pungent vanilloids in ginger, like gingerol and paradol are also known to possess antioxidant activity (Surh, 1999). A polyphenolic compound lawsone from Lawsonia inermis were studied for its wound healing activity and gave a significant wound healing (Sakarkar et al., 2004).

Capsaicin

Dihidrocapsaicin

21

Embelin

Lawsone

Emodin

Paradol Gingerol

22

8. Plants with potential wound healing activity

Some medicinal plants have been employed in folk medicine for wound care that either promote direct wound repair or exhibit antimicrobial and other related properties which are beneficial in overall wound care (Odimegwu et al., 2008). Some of the plants used in wound care have also been shown to possess a combination of these properties (Esimone et al., 2009).

Achillea kellalensis Bioss. & Hausskn. (Asteraceae)

Achillea kellalensis is known to contain polyphenols and monoterpenoids like camphor, borneol, -thujone, cineol, bornyl acetate and camphene. In the study done on the extract of Punica granatum, the aqueous and alcoholic extracts showed significant increase in the rate of wound contraction and collagen turnover (Ghasemi et al., 2010).

Ageratum conyzoides L (Asteraceae)

The leaves are applied to the wounds act as septic and heel them quickly. The juice of the fresh plant and extract of dried plant are used to cure allergic rhinitis and sinusitis (Sachin et al., 2009). There are reports that Ageratum conyzoides is used in postpartum recovery in Peninsular Malaysia (Boer and Lamxax, 2009). Phytochemical investigation of different extract showed the presence of alkaloids, and tannins. Research suggests that the ointment of the root extract of Ageratum conyzoides has significant wound healing activity. It may be attributed to antimicrobial and haemostatic action of ageratum attributed to the individual or combined action of phytoconstituents present in it (Sachin et al., 2009).

Allium cepa L. (Liliaceae)

Alcoholic extract of tubers of Allium cepa has shown better wound healing activity in excision, incision and dead space wound models which may be atributed to free radical scavenging action and the antibacterial property of the phytoconstituents (viz; tannins and flavonoids) present in it (Shenoy et al., 2009).

23

Aloe vera (Asphodelaceae)

It has stiff grey to bright green lance-shaped leaves containing clear gel in a central mucilaginous pulp. Recent research has shown that the pharmacologically active agent is concentrated in both the gel and the rind of the Aloe vera leaf (Syed et al., 1996). Topical application and oral administration of Aloe vera to rats with dermal wounds increased the collagen content of the granulation tissue as well as the degree of cross-linkage (MacKay and Miller, 2003). Evidence tends to support that Aloe vera might be an effective interventions used in burn wound healing for first to second degree burns (Maenthaisong et al., 2007). However, in some severe burns, aloe gel may actually impede healing (Raina et al., 2008).

Alternanthera brasiliana Kuntz (Amaranthaceae)

According to research results, topical application of Alternanthera brasiliana has a positive influence on different phases of wound healing including wound contraction, fibroblastic deposition and angiogenesis. Phytochemical screening of Alternanthera brasiliana revealed the presence of alkaloids, steroids and triterpenes (Barua et al., 2009).

Anthocleista nobilis G. don. (Loganiaceae)

On the results of the study conducted in Ghana on invivo and invitro models, Anthocleista nobilis has significant wound healing activity. This could partly be attributed to its antibacterial and antioxidant property as evidenced by its ability to inhibit bacteria growth and protect human fibroblast cells against oxidant injury (Annan and Dickson, 2008).

Areca catechu L. (Arecaceae)

Studies made on Areca catechu indicated that polyphenols and alkaloid fractions have enhanced the healing of incision wounds by increasing the breaking strength of the wounds. The polyphenol fraction especially seems to be more effective where the treatment having a combination of both the alkaloid and polyphenol fractions also has a relatively high wound breaking strength (Azeez et al., 2007).

24

Azardica indica (Meleaceae)

Neem oil contains margosic acid, glycerides of fatty acids, butyric acid and trace valeric acid. Alcoholic extract of neem is useful in eczema, ringworm and scabies. Neem leaf extracts and oil from seeds has proven anti-microbial effect. This keeps any wound or lesion free from secondary infections by microorganisms. Clinical studies have also revealed that neem inhibits inflammation as effectively as cortisone acetate; this effect further accelerates wound healing (Raina et al., 2008).

Calotropis gigantea L. (Asclepiadaceae)

A study was made on Calotropis gigantea on incision wound model and healing by granulation, collagenation, and tensile strength was measured indirectly to assess the collagen content and maturation. The results indicated that latex of Calotropis gigantea significantly promoted collagen (Nalwaya et al., 2009). In incision wound and dead space wound, topical application of Calotropis gigantea increased breaking strength and hydroxyproline of wounds (Deshmukh et al., 2009).

Carica papaya L. (Caricaceae)

The papaya-latex is well known for being a rich source of the four cysteine endopeptidases namely papain, chymopapain, glycyl endopeptidase and caricain and the content may vary in fruit, leaves and roots (Anuar et al., 2008). These antioxidants are considered to be one of the potential contributors to wound healing (Anuar et al., 2008; Gurung and Skalko-Basnet, 2009).

Catharanthus roseus L. (apocyanaceae)

The two classes of active compounds in Vinca are alkaloids and tannins. Researches suggested that the topical administration of ethanol extract of Vinca rosea leaves plays a major role in diabetic wound healing (Nayak, 2006).

25

Centella asiatica (Mackinlayoideae)

The active principles of Centella asiatica are triterpenes and asiaticoside, which are responsible for promotion of rapid wound healing. Aqueous extract of Centella asiatica suspended in 5% propylene glycol promoted wound healing on topical administration in experimentally induced open wounds in rats (Raina et al., 2008).

Cocos nuclifera L. (Arecaceae)

Coconut oil consists of lauric acid, myristic acid, palmitic acid (saturated fatty acid components) and linolic acid, which is the only polyunsaturated fatty acid (Srivastava and Durgaaprasad, 2008). It was reported that Cocos nuclifera has significant improvement in wound contraction and decreased epithelialization period in burn wound model (Srivastava and Durgaaprasad, 2008).

Cordial dichotoma (Boraginaceae)

Researches made on Cordial dichotoma, in three animal models, incision wound, excision wound and dead space wound, revealed that the plant has the potential wound healing activity supporting the traditional claim (Kuppast and Nayak, 2000).

Dissotis theifolia (Melastomataceae)

A study made in Nigeria demonstrated that Dissotis theifolia has antibacterial and wound healing effect when formulated as ointment, on infected excision wound model. Phytochemical studies showed that the crude Dissotis theifolia stem powder and the methanol . extract contain saponins, tannins, glycosides, flavonoids, terpenoids, carbohydrates, alkaloids and steroids (Odimegwu et al., 2008).

Elaeis guineensis Jacq (Mackinlayoideae)

The application of a methanolic extract of Elaeis guineensis was found to improve the different phases of wound repair, including collagen synthesis and maturation, wound contraction, and epithelialization. Phytochemical screening studies showed that the plant has

26

tannin, saponin, alkaloid, flavonoid, steroids, reducing sugar and terpenoid (Sasidharan et al., 2010).

Euphorbia heterophylla (Euphorbiaceae)

Euphorbia heterophylla contains alkaloids, cyanide, tannins, flavonoids and saponins in the order of decreasing concentration. The aqueous and ethanol extracts showed significant wound healing activity when topically administered on rats (James and Friday, 2010).

Ficus religiosa (Moraceae)

Hydro alcoholic leaf extracts ointment of Ficus religiosa showed significant wound healing activity. This was evident by faster rate of wound closure and epithelialization period in excision wound model and significant increase in skin breaking strength in incision wound model (Roy et al., 2009).

Ginkgo biloba (Ginkgoaceae)

Its preparations promote epithelization without altering wound contraction. In case of dead space wounds Ginkgo biloba has increased granulation tissue breaking strength without altering granulation tissue mass weight. However, it did significantly enhance the content of hydroxylproline of granulation tissue. The main constituents of Ginkgo biloba are flavonoids and terpene trilactones and the pro-healing action of the Ginkgo biloba is due to the presence of flavonoids (Raina et al., 2008).

Helianthus annus L. (Asteraceae)

In a study on the alcoholic extract of whole plant of Helianthus annus applied in the form of an ointment on the excised wound of rat led to a significant reduction in total healing period. This has been confirmed by histology where earlier appearances of fibroblasts were seen. Early appearance and higher accumulation of mucopolysaccharides has been stated as indicators of hastened repair (Raina et al., 2008).

27

Hoslundia opposita Vahl (Lamiaceae)

Wound healing activity of Hoslundia opposita could partly be attributed to their antibacterial and antioxidant properties as evidenced in their ability to inhibit bacteria growth and protect human fibroblast cells against oxidant injury. As research indicate, the increase in hydroxyproline content (indication of collagen synthesis) and tensile strength of healing tissue after the administration of the plant extract confirmed the healing potential of the plant (Annan and Dickson, 2008).

Hydnocarpus wightiana (Flacourtaceae)

The wound healing effect of oil of Hydnocarpus spp. was studied with reference to collagenation and the strength of scar tissue. Hydnocarpus wightiana oil administered orally promoted epithelization, but not wound contraction. External application of oil of Hydnocarpus spp. and its paste significantly shortened the epithelization period when compared to control group. Oil may act as adjuvant in healing of wounds and ulcer in leprosy patients and therefore, may be clinically useful (Raina et al., 2008).

Hypericum prolificum (Hypericaceae)

The chemical constituents include anthraquinone derivatives (naphthodianthrones), flavonoids, prenylated phloroglucinols, tannins and volatile oils. Various types of preparations, ointments, creams of Hypericum prolificum have been found to possess wound-healing (Saddiqe et al., 2010). The antibacterial activity of crude extracts can be related to the use of the herb as a wound healer in ancient times (Raina et al., 2008).

Jasminum auriculatum (Oleaceae)

The juice when applied in the form of jelly, locally on linear uniform excised wound in rats is found to promote wound healing This has been assessed by histological, biochemical and contraction rate studies. Fresh juice of the leaves showed an increase and early gain of the tensile strength in the linear wounds in rats. The study indicated that collagenation contributed to improved tensile strength in the early phase of healing (Raina et al., 2008). Mwh13

28

Jatropha curcas L. (Euphorbiaceae)

Research performed on this plant suggest that fresh homogenized crude extract of Jatropha curcas have beneficial influence on various phases of wound healing such as fibroplasia, collagen synthesis and wound contraction resulting in faster healing (Shetty et al., 2006).

Lantana camara (Verbenaceae)

The ethanol extract of L.camara increased the rate of wound contraction in burn wound. The slight reduction in the wound area might be due to the antimicrobial effect of the leaf extract. The phytochemical analysis of the leaf extract by qualitative method showed the presence of triterpenoids and flavonoid (Nayak et al., 2008).

Lawsonia inermis L. (Luthraceae)

Wound healing activity of the plant was studied in excision and incision wound models, the result showing signifi.cant wound healing activities in both models. Flaavonoids, lawsone, tannins, steroids, saponinis were found to be present in the plant (Sakarkar et al., 2004).

Mimosa pudica ( Mimosaceae)

Mimosa pudica has been reported to contain mimosine (an alkaloid), free amino acids, sitosterol, linoleic acid and oleic acid. The drug is also found to be rich in tannins and the total tannin content was reported to be (Kokane et al., 2009). The result of excision wound model of 2% (w/w) methanolic and 2% (w/w) total aqueous extract indicated significant increase in the wound contraction, revealing that the extract has ability to induce cellular proliferation. Increase in tensile strength of the incision wound model also indicates the promotion of collagen fibers (Kokane et al., 2009).

Napoleona imperialis (Lecythidaceae)

The various ointments prepared with Napoleona imperialis exhibited a good wound healing effect comparable to those of Cicatrin, a standard antibiotic used in wound healing. The best activity was observed in the ointment containing Napoleona imperialis in cationic

29

ointment base. This shows that Napoleona imperialis can effectively be employed as a cationic emulsifying ointment in wound healing (Esimone et al., 2005).

Ocimum kilimandscharicum (Lamiaceae)

Aqueous extract of leaves of Ocimum kilimandscharicum possesses a definite pro-healing action. This is demonstrated by a significant increase in the rate of wound contraction and by enhanced epithelization. Significant increase was also observed in skin breaking strength and hydroxyproline content which was a reflection of increased collagen levels by increased cross linking of collagen fibres. In addition, increase in dry granulation tissue weight indicated the presence of higher protein content. Phytochemical screening revealed the presence of tannins, flavonoids and proteins (Paschapur et al., 2009).

Ocimum sanctum L. (Labiaceae)

According to the research result done in India, both the alcoholic and aqueous extract of Ocimum sanctum significantly increased wound breaking strength, hydroxyproline, hexosamines, superoxide dismutase, catalase, and reduced glutathione (Udupa et al., 2005; Shetty et al., 2008). The results suggest that Ocimum sanctum may be useful in the management of abnormal healing and hypertropic scars.

Phyllanthus niruri L. (Euphorbiaceae)

Topical application of methanolic extract caused a significant concentrationrelated reduction in wound diameter and epithelialization period of excision wounds. Several phytochemical constituents of this plant have been isolated. Some of these include the alkaloids, arabinogalactan, ellagic acid, 1-O-galloyl-6-O-luteoyl-alpha-d-glucose, beta-glucogallin, quercetin, beta sitosterol, gallic acid, lignans and prenylated flavanones (Okoli et al., 2009).

Quercus infectoria (Fagaceae)

Phytochemical work reveals that ethanolic extract of galls of Quercus infectoria contains high amount of tannins, presence of gallic acid, ellagic acid, syringic acid, -sitosterol and amentoflavone. In the incision wound model, a significant increase was observed in the skin

30

tensile strength of the ethanol extract-treated group. On dead-space wound model the extract showed a significant increase in dry granuloma weight, granuloma breaking strength and the level of hydroxyproline content. Histological examination revealed that the plant has the potential to increase collagen. Studies on the estimation of antioxidant enzyme reveal that the extract significantly increased the levels of superoxide dismutase and catalase. In studies using the excision wound model, animals treated with the ethanol extract of Quercus infectoria showed a significant decrease in the epithelization period. The extract also facilitated the rate of wound contraction (Umachigia et al., 2008).

Rubia cordifolia L. (Rubiaceae)

Studies made demonstrates the wound healing activity of ethanolic extract and its gel formulation of the roots of the plant Rubia cordifolia and found to be effective in the functional recovery of the healing of wounds and also in histopathological alterations. Phytochemical screening of the ethanolic extract Rubia cordifolia showed the presence of anthraquinone glycosides, saponins, tannins and phytosterols, of which tannins and anthraquinones are the major phytoconstituent present in this plant which may be responsible for wound healing action (Karodi et al., 2009).

Tragia involucrata L. (Euphorbiaceae)

Phytochemical analysis demonstrated the presence of vinyl hexylether, shellsol, 2, 4dimethyl hexane, 2-methylnanone and 2,6-dimethyl heptanes. In vitro antibacterial study and wound healing study on excision wound model indicated that, shellsol and vinyl hexylether contribute to the scientific basis wound healing (Samy et al., 2009). mwh63

Trichosanthes dioica (Cucurbitaceae)

The methanolic extract ointment showed significant increase in the rate of wound contraction and by enhanced epithelialization period. Significant increase in tensile strength, and hydroxyproline content were observed, which was auxiliary supported by histopathological studies. Preliminary phytochemical screening of methanolic extract of

31

Trichosanthes dioica showed the presence of alkaloids, flavonoids and tannins (Shivhare et al., 2010).

Tridax procumbens (Asteraceae)

This juice accelerates two phases of healing namely epithelialization and collagenization, however, it retards scar formation and granulation leaf extracts of this plant also promote wound healing in both normal and immunocompromised (steroid treated) rats in dead space wound model (Raina et al., 2008). Mwh13

Vernonia arborea (Asteraceae)

Research made on this plant showed that aqueous and methanolic bark extract of the plant have significant promotion of wound healing in excision, incision and dead space wound models. Preliminary phytochemical screening of aqueous extract indicated the presence of flavonoids, saponins tannins and glycosides, while the methanolic extract showed additional sesquiterpene and triterpenes (Panda and Tripathy, 2009).

32

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