Home / Pharmacy / Antimicrobial and wound healing properties of leaf extracts, fractions and ointment formulations of spermacoce verticillata linn (family_ rubiaceae)

Antimicrobial and wound healing properties of leaf extracts, fractions and ointment formulations of spermacoce verticillata linn (family_ rubiaceae)

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INTRODUCTION1.1       The human skinThe human skin is the largest organ that covers and protects the internal part of the body from external substances. It is made of three layers -epidermis, dermis and subcutaneous layer and there is a wide variation in the structure of the skin (1).EpidermisThis is the outermost layer of the skin, and consists of four layers, namely; the horny, granular, prickle cells and the basal layer. The basal cells give rise to the prickle cells by mitotic division; then the prickle cells move upwards but as new cells are formed beneath them; they change their polyhedral shape to a flattened shape. As they continue to move upwards they produce a protein, keratin. The granular layer is filled with granules of keratin. The skin releases lytic-enzymes that destroy the cell nucleus and the granules of keratin are distorted, the unbound keratinocytes that are now on top of the skin die and harden into the horny layer.The horny layer is thus formed of dead epidermal cells. It takes about 28 days from the formation of a prickle cell to its loss from the skin surface. In this way, the skin renews itself once every four weeks. Melanin is produced in this layer by certain pigment cells (melanocytes) that protect the skin from UV rays. There are langerhans cells which play some part in the immune function of the skin (1).DermisThis region is thicker than the epidermis. It is made up of the relatively thin papillary layer and a thicker reticular layer. The surface of the papillary layer has many bumps (papillae) that interlock with the base of the epidermal layer. Each papilla is supplied by a capillary vessel. The dermis has a great capacity for retaining water and is a reservoir of body fluid, that contains collagen formed by fibroblast, two fibers- reticulum and elastin, and these three give the dermis its elastic nature. It contains hair follicles, sweat ducts, blood vessels, and nerve endings; the sweat gland is situated deep in the dermis and opens on the skin surface as the sweat duct (1). Figure 1.1 shows the cross section of the human skin.Subcutaneous layerThe layer is below the dermis and consists of connective and fatty tissues. It serves as a fat storage layer and as a padding, shock absorber and insulator for the body (2). 1.1.1   Functions of the human skinThe skin allows man to adapt to a wide variation in the environment. The skin is the largest human organ and it accomplishes a wide variety of tasks. With many nerve endings in the skin, it is able to perceive pain and vibration; the skin can also absorb substances from the environment into the body (medicated creams). The skin prevents germs and pathogens from entering the body and prevents evaporation of tissue fluids. By excreting sweat, it protects the body from overheating (1). The skin is a barrier against cold. On exposure to cold, there is a reduction of its blood flow and this insulates and maintains the body temperature. Increased blood flow and evaporation of sweat enables man to remain cool in hot climates. The presence of pigment in the skin helps to filter out most of the harmful ultraviolet radiations. This functions/integrity of the skin can be compromised by skin diseases and trauma (wounds) (1, 2).      Fig. 1.1:         A Cross section of human skin            1.1.2   Disorders of the skinThe skin is susceptible to diseases which could be as a result of genetic disorders like neurofibromatosis, icthyosis, tuberose, sclerosis and xanthomatasis. Skin diseases/conditions can be caused by hypersensitivity reaction of the skin e.g. dermatitis or eczema. Dermatitis is the inflammation of the epidermis; also skin diseases can be caused by microbial infection of the skin (1). 1.2       MicroorganismsMicroorganisms are ubiquitous in nature that is to say, they can be found almost everywhere, on land, in water, in the air, clothing, manufacturing equipment, in and on man and anywhere that can support its growth. Examples of microorganisms are bacteria, fungi, viruses, protozoa, etc. They are capable of producing diseases in many of its hosts (man inclusive) but they can also at the same time, synthesize useful materials to man. So, some by- products of microbial metabolisms can be useful to man, though there is no consideration as to the usefulness of these by-products to man from the microbe’s point of view. For them, it is just a way for survival in an environment (6). Microorganisms are grown in culture media in the laboratory. The media are designed to supply all the nutrients required to support the growth of the organisms in question. Very small quantities of pure culture of the study organism are aseptically transferred into a sterile liquid or solid medium and incubated at a suitable temperature, which is optimally 25 oC for fungi and 37 oC for bacteria. On solid media, the organisms grow as visible colonies, while in a clear broth it becomes increasingly turbid as the organisms grow in it.It is very important that the organisms used in any research must be pure cultures, so that each organism can be studied as individual species, because the effects of microorganisms growing as a mixed culture cannot be ascribed with certainty to any particular member of the mixture. They are examined carefully for details of their colony sizes, texture and colour (7).1.2.1   Microorganisms and the human skin and skin infectionThe human skin is a natural host for many microorganisms, some of which are normal flora. Some microorganisms that are often encountered on the skin, include Staphylococcus aureus, Streptococcus pyogenes, Corynebacterium spp., Propionibacterium spp., Mycobacterium, spp., yeast -like Candida albicans  and viruse like herpes simplex. Bacteria like Brevibacterium spp., Acinetobacter spp., Neiseria spp., Erysipelothrix insidiosa,  and Haemophilus spp. Others include Helicobacter Pylori, Klebsiella rhinoscleromatis, Pseudomonas aeruginosa, Calymmatobacterium, granulomatis, Bacillus anthracis, Clostridium perferingens, Treponema spp., Mycobacterium spp., Yersinia pestis and even Serratia marcescens. Some of these microorganisms found on the skin are harmless while others are pathogenic depending on the predisposing factors of the host (3).Staphylococci bacteria are a common type of bacteria that live on the skin and mucous membranes (e.g. in nose) of humans. Staphylococcus aureus (S. aureus) is the most important of these bacteria in human disease. Other Staphylococci including S. epidermidis are considered commensals, or normal inhabitants of the skin surface. Staphyloccocal skin infection includes impetigo, ecthyma, cellulits, folliculitis, boils (furuncles and carbuncles) sycosis and Scaled Skin Syndrome (SSS). Staphylococci are becoming increasingly resistant to much commonly used antibiotics including penicillin, macrolides such as erythromycin, tetracycline and amino glycosides (3).Some skin infections have fungal origin, the most popular being the dermatophytes. The three major genera that are recognized to cause fungal infections include the Epidermophyton spp., Microsporum spp., and Trichophyton spp. (3).Dermatophytes are types of fungi that cause skin, hair and nail infections. Infections caused by these fungi are known as “tinea”. They cause diseases such as athlete’s foot and jock itch. Trichophyton rubrum and Trichophyton tonsurans are two common dermatophytes that can be transmitted from person to person, i.e. anthrophillic; others include Microsporum audounii, Trichophyton interdigitale, Trichophyton violaceum, Microsporum ferrugineum, Trichophyton schoenieinii, Trichophyton megninii, Trichophyton sandanense and Trichophyton yaoundei. Other common dermatophytes are transmitted from animals such as cats and dogs to people i.e. zoophillic. They include: Microsporum canis (From cats and dogs) Trichophyton equinum (from horses), Trichophyton erinacei (from hedgehogs), Trichophyton verrucosum (from cattle), Microsporum nanum (from pigs) and Microsporum distortum (a variant of Microsporum canis). The geophillic dermatophytes are transmitted from soil to people; they include: Microsporum gypseum and Microsporum fulvum (4, 5). 1.2.2   Features and classification of test microorganismsMost microorganisms are free-living and can perform activities that are useful to animals and plants but some are capable of causing diseases and are called pathogens such as bacteria, fungi, viruses and protozoa (7).BacteriaBacteria are essentially unicellular although some are chains of cells. They are prokaryote that is, they do not have true nucleus and exhibit a variety of forms, habitat, metabolic path-ways and pathogenicity. They are divided into two groups, namely; Gram-positive and Gram-negative. These are microscopic organisms that are devoid of a well defined nucleus and mitochondria; they have a simple rigid cell wall which allows them to have a more or less independent existence (7).Bacterial cells are divided into two groups, namely; Gram-positive and Gram-negative bacteria. They differ in the strength and structure of their cell walls. The Gram-positive bacteria are nutritionally exacting organisms that take up complex molecules from their environment and because complex molecules are on their own capable of generating considerable osmotic pressure, a higher internal osmotic pressure is required in the Gram-positive cells to create an osmotic gradient along which nutrients could be taken up into their cells. Gram-negative bacteria on the other hand, do not need complex molecules for their nutritional requirement, so there is no need for them to generate high internal osmotic pressure to absorb the simple molecules they survive on. In view of these, Gram-positive bacteria have a very robust cell wall to contend with its high internal osmotic pressure. The cell wall is made up of single layer of repeating units of mucopeptides. Mucopeptides are composed of alternating units of N-acetylmuramic acid and N-acetalyglucoseamine, each strip of mucopeptide is connected to the next by polypeptide cross link. The mucopeptides layers and their polypeptide cross link can be very extensive which is why Gram-positive bacteria possess robust cell walls. The bacterial cell wall functions are for mechanical support and to protect the cells from osmotic damage. The cell wall has no physiological function but a bacteria cell normally cannot survive the loss or malfunction of its cell wall, which is why most antibacterial agents are targeted towards it (6, 7).The cell wall of Gram-negative bacteria in comparison to that of the Gram-positive bacteria is very thin but more sophisticated. The Gram-negative cell walls are made up of lipoprotein, liposaccharide, protein and peptidoglycan. The Gram-negative bacterium, with its less robust cell wall, is capable of giving efficient protection against lethal chemical (7).Gram-stain is the most important staining for identification of bacterial cells. It was described by Christian Gram in 1884, and involves treatment of fixed bacterial smears with gentian violet and methyl violet as primary stains, then Lugol’s iodine. It acts as a mordant by fixing the primary stains to the bacterial cell well. This is followed by discolourising the stain with alcohol or acetone and washing with water, before counter-staining with safranin. Gram-positive bacteria cells will retain the violet colour of the primary stain while the Gram-negative ones will turn purple or red colour of the counter stain (6).In Gram-negative bacteria, alcohol can penetrate the thin cell wall to cause leakage of primary stain-iodine complex, so their cell wall would be free to accept the counter stain hence they take up the red colour of the counter stain (7).Apart from the cell wall, a bacteria cell is made up of cytoplasmic membrane, ribosomes, nucleus (nuclear bodies), mesosome, capsule and flagella. Some Gram-positive bacteria, examples bacilli and clostridia have developed a very effective means of surviving adverse conditions, through the formation of spores. The bacteria of interest here are Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa.1.2.2.1            Staphylococcus aureusThese are non-motile Gram-positive cocci that occur in groups of grape-like clusters (staphylo), hence the name staphylococcus. They are non-capsulated, coagulase positive, DNase positive and catalase positive (8, 9).  1.2.2.2            Bacillus subtilisBacillus is a genus of Gram-positive rod-shaped bacteria and a member of the division Firmicutes. Bacillus can be obligate aerobes or facultative anaerobes, and test positive for the enzyme catalase. Ubiquitous in nature, Bacillus includes both free-living and pathogenic species. Under stressful environmental conditions, the cells produce oval endospores that can stay dormant for extended periods. These characteristics originally defined the genus, but not all such species are closely related, and many have been moved to other genera. Bacillus subtilis is one of the best understood prokaryotes, in terms of molecular biology and cell biology (8). Its superb genetic amenability and relatively large size have provided the powerful tools required to investigate a bacterium from all possible aspects. Two Bacillus species are considered medically significant; Bacillus anthracis, which causes anthrax, and B cereus, which causes a food borne illness similar to that of Staphylococcus aureus (7, 8, 9). A third species, Bacillus thuringiensis, is an important insect pathogen, and is sometimes used to control insect pests. The typed specie is Bacillus subtilis, an important model organism. It is also a notable food spoiler, causing ropiness in bread and related food.<h3>1.2.2.3            ESCHERICHIA COLI</h3><h3>THIS IS GRAM-NEGATIVE ROD SHAPED BACTERIUM, COMMONLY FOUND IN THE LOWER INTESTINE OF WARM-BLOODED ANIMALS, IN SOIL AND IN WATER. THEY ARE SOMETIMES REFERRED TO AS COLIFORMS, AND ARE USUALLY NON-MOTILE BUT CAN BE CAPSULATE. THEY CAN BE IDENTIFIED BIOCHEMICALLY BY THEIR POSITIVE REACTION INDOLE TEST (8, 9).</h3>   1.2.2.4            Pseudomonas aeruginosaIt is Gram-negative, non-sporing motile rod, which can sometimes encapsulate. Pseudomonas aeruginosa is found in the intestinal tract, water, soil, sewage, in hospitals, moist environments such as sinks and buckets. It can equally grow in some eye drops. Many infections are opportunistic hospital–acquired, affecting those in already poor health and immune-suppressed conditions. Pseudomonas aeruginosa is oxidase positive and produces acid from glucose only, with no gas production (8, 9).FungiThey are non-photosynthetic organisms that grow either as singles (yeast) or as colonies of multicellular filaments. They are saprophytic, parasitic or commensal organisms. Fungi are eukaryotes, i.e. their nucleus is enclosed by a nuclear membrane. Their cell wall consists of polysaccharides, polypeptides and chitin, while the cell membrane contains sterols which prevent most antibacterial agents from being effective against them (7).Fungal infection does not cause widespread and dangerous diseases like bacteria but are major causes of individual distress. Fungal infections are called mycoses and based on the site of the body affected, mycoses can be classified as:Systemic mycoses:  This is acquired by inhalation, and may affect the lung to involve other parts of the body (10).Subcutaneous mycoses:  This is acquired when the fungal pathogen gets access into the body through cuts on the skin (8).Superficial mycoses:  Here, the pathogen is confined to the body surfaces like the hair, skin and nails, and does not directly involve living tissues. This class of fungi are called the dermatophytes. When there is a break in the integrity of the skin via wound or trauma, these pathogens access such sites to probably cause secondary infections (8).The fungal pathogens used in this research work were the dermatophytes Microsporum audouini and Trichophyton rubrum and a unicellular fungal Candida albicans.1.2.2.5            Microsporum audouiniiIt grows slowly on Sabouraud agar as gray colony with a radially folded surface. The centre of the colony is reddish on the reverse. It is associated with the disease known as tinea, an infection of keratinzed tissues like epidermis, hair and nails (11).1.2.2.6            Trichophyton rubrumThis causes athletes’ foot and ringworm. It grows slowly in the laboratory. Its texture is waxy, smooth and cottony texture. The colour is bright yellow or red violet. It is the most common dermatophytes that causes finger nail fungus infections, and scalp infections (10).1.2.2.7             Candida albicansThis is the most common causative organism of candidiasis. It occurs as a commensal of the gastrointestinal tract. Skin infections occur too, especially in people whose natural defences are impaired by diseases, wounds and drug therapy. Candida albicans grows well on Sabouraud dextrose agar at 35 – 37 ºC for 2 – 3 days. Its wet preparation (Microscopy) shows budding yeasts and hyphae with buds, they are Gram-positive (8, 12). 1.3                   Microbial infections of the skinThe normal skin is inhabited by some microorganisms called normal flora. These microorganisms grow on intact skin without causing any harm to the host. These same microorganisms can however become opportunistic and cause diseases when the skin integrity is compromised through trauma like wounds, burns, pre-existing skin diseases and poor hygiene (6). Skin infection can be caused by bacteria, fungi, viruses or parasites. 1.3.1               Bacterial infectionsExamples of bacterial skin infections are:1.3.1.1             ImpetigoIt is caused by Streptococcus and/or Staphylococcus species; it is a superficial skin infection mainly involving the surface areas of the skin. Direct contact with the lesions or with exudates from the infected sites is required for transmission. The lesions appear initially as small red spot, which then become vesicles (a small collection of fluid in the epidermis or between the epidermis and dermis) that are filled with an amber fluid. Eruption of the vesicles releases the amber fluid that dries into a brown or yellow crust on the skin surface. Impetigo is very contagious and re-infection of any exposed part of the body is possible if the infection is not controlled. The incidence is most common in children and could increase the risk of glomerular nephritis if left untreated. There is primary impetigo (Impetigo vulgaris) which is caused by the bacteria directly while secondary impetigo (Bockhart’s impetigo) occurs as a secondary infection to other infections   or injuries (13). 1.3.1.2             FolliculitisThis is a bacterial infection of the hair follicles. They may be superficial or deep, and involve the hair shafts. They are caused by S. aureus, although P. aeruginosa is also implicated (13).  1.3.1.3             ErysipelasThis is an infection of the superficial skin caused by Streptococcal species. The infected area is often red and raised with local warmth and edema. It occurs mostly on the face and scalp and is usually accompanied by fever and chill (13). 1.3.1.4             EcthymasIt is caused by the same organisms that cause impetigo i.e. Staphylococcus and / or Streptococcal species, but the lesion of ecthymas is deeper. The legs are most affected. The lesions begin with vesicles that rapidly erode and become crusted, healing with scarring. This condition occurs mostly as a secondary infection to mild trauma or injury/ wound to the skin (13).1.3.1.5             Furuncles and CarbunclesSuperficial infection of the hair follicle is termed folliculitis; a deeper involvement is called a furuncle (small boil). Furuncles are the initial redness and inflammation of the area followed by thinning of the skin around the primary follicle; central ulceration and scarring often occurs. A carbuncle forms when adjacent hair follicles are involved. Both infections are caused by Staphylococcal and Streptococcal organisms (1, 13).1.3.1.6           ParonychiaThis is an infection of the nails caused by Streptococcus and Staphylococcus species. The nails become irregularly shaped and application of mild pressure may exude pus (1, 13). 1.3.2   Fungal InfectionsFungi exist as unicellular organisms called yeast or as multi-cellular filamentous forms called mould; very complex forms which grow into large structures like mushrooms also exist. The basic unit of a mould is the hypha. Hypha is a branching tubular structure and it is of two forms. Some hypha project upwards from the surface of the growth media, and are called the aerial hypha, bearing the reproductive cells, while the other form of hypha penetrates the growth media, and are called the vegetative hypha, concerned with absorption of nutrients. Both the aerial and vegetative hypha can assume certain characteristic features that are used to identify them (7).Yeasts:  They are oval unicellular organisms, though sometimes they seem attached to each other to form chains or pseudo-hypha. The fungal cell wall is made up of N-acetylglucoseamine residues, linked together by B-1-4-glycosidic bonds. Some yeasts of medical importance are Candida albicans, Trichosporon beigeli, and Cryptococcus neoformans (7).Moulds:         The hyphae of many pathogenic moulds are septa that are divided into cells by cross-walls called septa. Hypha without septa are referred to as aseptate. Moulds of medical importance are dermatophytes (7).1.3.2.1             Tinea pedis (Athletes’ foot)This is commonly caused by Trichophyton species and Epidermophyton species. The first signs of Tinea pedis are ulceration, scaling and fissuring on the webs of the little toes. This condition may get mild in the cold weather to recur fully in the warm seasons. As the fungal infections spread, secondary bacterial infection may set in at this stage and the infection sites become purulent and exude an odoriferous serum (1).1.3.2.2            Tinea capitis (Head infection)This is transmitted by direct contact with infected persons or animals. The infection is caused by Microsporum and Trichophyton species. Infection is presented as non-inflamed areas of hair loss to deep, crusted lesions, which may be scarred and with permanent hair loss (1, 2). 1.3.2.3 Tinea cruris (Tinea of the groin)This is caused by Epidermophyton floccosum, Trichophyton rubrum and Trichophyton mentagrophytes. It affects the upper part of the thighs and the pubic area. Tinea cruris is more common in males than females. The margins of the lesions are slightly elevated and more inflamed than its central part. Small vesicles appear at the margins. The lesions are bright red in acute condition and turn brown in chronic cases (13). 1.3.3.4             CandidiasisThis is transmitted by Candida albicans. When it affects the mucous membranes, it is called thrush; at the anus, it is called pruritus ani, while it is vaginal cadidiasis in the vagina. There is Candidia paronychia (nails) that is common in people who routinely immerse their hands in water. Other fungal skin infections include Tinea barbe (of the beard), Tinea manum (hands), Tinea versicolor – where there is partial discolorations of pigmented skin and Tinea unguium in which the nails become hypertrophic, discolored and scaly (2).1.3.4   Viral infectionsThese may occur in or on the skin and may present as warts. Warts are human tumors caused by virus and like other tumors, are due to a group of altered cells that can proliferate uncontrollably. An example is plane warts of the face and back of the hands, plantar warts occur mainly on the soles (1, 13).1.3.4.1             Herpes simplexThis is a viral infection of the skin and mucous membranes. It is caused by herpe virus hominis (HVH) which is made up of two strains. HVH-1causes cold sores on the lips and is transmitted by contact from sufferers while HVH-2 causes genital lesions and is sexually transmitted (1).1.3.4.2             Herpes zosterThis is caused by the same virus that causes chicken pox, Zoster- varicella. Localized and painful shingles are called zosters, and are caused by the activation of chicken pox virus which had lain latent in hosts, years before (1).1.3.4.3             Molluscum contagiosumThis is caused by a DNA- containing pox virus and it is contracted by direct contact with an infected person or formites. The lesions are seen as pinkish nodules with a slight depression on its top. It has a soft core that can be easily squeezed to express a white curd-like substance (1).1.3.5               Parasitic skin infectionsBesides bacteria, fungi (even yeast) and viruses, parasites such as insects or worms   can burrow into the skin, and cause skin infection. Some parasites live in the skin for part of their life cycle, while others for their entire life cycle. Parasitic skin infections frequently cause severe itching and inflammation (14) and include:1.3.5.1             Scabies             This is a mite infestation of the skin that produces tiny reddish bumps and severe itching. Scabies usually spread from infested persons through physical contact. People with scabies have severe itching, even if there are just few mites on the body. Scabies is caused by Sarcoptes scabiei.  1.3.5.2                         JiggersThis is caused by Sandflea (Tunga penetrans), larva migrans (dog hook-worm, Ancylkostoma brasiliensis) Cutaneous larva migrans (creeping eruption) and is a hookworm infection transmitted from warm, moist soil to exposed skin. The hookworm normally inhabits dogs and cats. The eggs of the parasite are deposited on the ground in dog and cat feeler. When bare skin touches the ground, which appears when a person walks barefoot or sun bathes, the hookworm gets into the skin. Starting from the site of infection, usually the feet, legs, buttocks, or back, the hookworm burrows along a haphazard tract, leaving a winding, threadlike, raised, red rash. The eruption itches intensely (14, 15).1.3.6   Non Infectious Skin Diseases: Eczema/ DermatitisDermatitis and eczema are terms which are often used interchangeably to describe an inflammatory condition of the skin produced by a variety of external and endogenous factors of which the characteristic feature is oedema of the epidermis (15, 16). It is regarded as a reaction pattern rather than a specific disease and can have many external or internal causes – Genetic, immunological, infective, vascular, traumatic and emotional factors. Eczema caused by external factors are termed contact dermatitis and those with internal causes are called endogenous eczema (16)Eczematous patches have a poorly defined edge and at various stages may show erythema, oedema, scaling, papules, vesicles, weeping and pustules. Eczema most commonly causes dry, reddened skin that itches or burns, although the appearance of eczema varies from person to person and according to the specific type of eczema. While any region of the body may be affected by eczema, both in children and adult, it typically occurs on the face, neck   and   inside the elbows, knees and ankles. In infant eczema typically occurs on the foreheads, cheek, forearms, legs, scalp and neck.1.3.6.1             DermatitisThis is as a result of acquired sensitization to substances on the skin from outside the body. The sensitizer penetrates the epidermis of the skin through the horny layer, sweat ducts or hair follicles and keratins. In the epidermis, the sensitizer combines with protein to form a stable antigen. The antigen sensitizes lymphocytes to cause a specific cell-mediated reaction to occur, after which any further contact with the sensitizer will be followed by inflammatory reaction of the epidermis. This type of cell-mediated reaction is termed   delayed hypersensitivity (13).There is always a latent interval between the first exposure to a sensitizer and the development of   sensitization. It may be as short as 5 days to months and even years.Hypersensitivity to a sensitizer is confirmed by patch-testing, which consists of application of a small amount of a suspected sensitizer to an area of normal skin. The test is positive if that area develops dermatitis beneath the patch after 24-48 hours. There may be a gradual lessening of the person’s sensitivity to it but most times, sensitivity is life long.The ability of various substances to cause dermatitis varies as the ability of different individuals to same substances varies too.1.3.6.2             Infective DermatitisThis condition is caused by the action of microbial toxins, and not the organism itself. When the skin of susceptible individuals is inoculated with bacterial culture or its filtrate, such conditions develops. The condition responds favorably to systemic and topical antibiotics (1, 13). 1.3.6.3             Endogenous Dermatitis            This is dermatitis caused by unknown internal causes, its symptoms generally lasting longer than those of exogenous dermatitis and examples are atopic and neurodermatitis (13).1.3.6.4             Atopic dermatitisThis skin condition occurs primarily during childhood, around folds of the arms or knees, the symptoms are erythema, scaling and weeping with severe pruritus. Secondary-   associated infections are common. The etiology of the condition is unknown but patients usually have asthma or hay fever in addition. Atopic dermatitis is a chronic skin disease characterized by itchy, inflamed skin and is the most common cause of eczema. The conditions tend to come and go, depending upon exposures to triggers or causative factors. The causative factors include environmental agents like molds, pollen, or pollutants; contact irritants like soaps, detergents, nickel (jewelry), or perfumes; food allergies or other allergies. When it occurs in infancy, it is termed infantile eczema.1.3.6.5             Seborrheic eczemaThis is a form of skin inflammation of unknown cause. The signs and symptoms are patches of inflammation on the skin, on the scalp, face and occasionally other parts of the body. Dandruff and “cradle cap” in infants are examples of seborrheic eczema. It inflames the face on the cheeks and/or the nasal folds, though it is not always associated with itching and runs in families. Emotional stress, oily skin, infrequent shampooing and weather conditions may all increase a person’s risk of developing seborrheic eczema (1).Nummular eczema is characterized by coin-shaped patches of irritated skin, most commonly located on the arms, back, buttocks and lower legs, and may be crusted, scaling and extremely itchy.1.3.6.6             Neurodermatitis (Lichenification)                        Also known as lichen simplex, it is a chronic skin inflammation caused by a scratch – itch cycle that begins with a localized itch (such as an insect bite) that becomes intensively irritated when scratched. This form of eczema results in scale, patches of the skin on the head, lower legs, wrists or forearms (13).The normal treatment of allergy such as avoidance of contact with allergens and administration of antihistamines does not bring relief to the patient. When inflamed eczematous skin markings become exaggerated and the skin becomes thickened and hardened. This is known as lichenified skin and since it is itchier than normal skin, a vicious cycle develops. Emotional stress plays a role in this disorder which is why an alternative name for this disorder is neurodermatitis. Most patients who suffer from it are tense, excitable and the urge to scratch is more of a bad habit like nail biting, than a disease (13).1.3.6.7             Cross sensitization            A person who has reacted to one substance is most likely going to develop reactions to other materials even when the substances are chemically un-related. Sensitivities are usually specific but sometimes the body cannot distinguish chemicals of different structures.Dermatitis can be caused by irritants or by true sensitization. Irritants like detergents remove lipids from keratin and allow the skin to dry excessively and split. This makes the epidermis more permeable to more irritants and sensitivity may occur. When skin eruption occurs, it is important to determine if it was due to exogenous contact or a specific hypersensitivity.History of cause of dermatitis from a person is of very vital importance. It is important to determine the site of onset of the eruption since this will give a lead to the probable cause of dermatitis. A band of erythema round the forehead will of necessity suggest a scarf as the sensitizer. Sensitivity of a person to   a brand of face powder that has been in usage for a very long time is possible, because the user may just have taken years to become sensitive to the powder or the makers of the powder may have changed the constituents of the powder. Some example of areas of body prone to dermatitis and their likely sensitizers are:<ul><li>Scalp-hair dyes and scalp lotions</li><li>Neck-ties, scarves, necklaces, perfume.</li><li>Ears -hair nets, ear clips, hearing aids, ear drops and Glass frames.</li><li>Trunk-clothing</li><li>Genitals -clothing, contraceptives and deodorant.</li><li>Arm-pits -deodorants, shaving powder and shaving sticks</li><li>Thigh -suspender, clothing</li><li>Ankles and feet-socks, stockings, shoe</li></ul>Medicaments applied to the skin are a common cause of sensitization dermatitis and every application is capable of sensitizing someone (13).1.3.6.8             Contact DermatitisThese include irritant dermatitis and allergic dermatitis. Skin diseases are the most common of all reported occupational diseases and the majority of the causes are contact dermatitis, in which the sensitizer irritates the skin on first or multiple exposures; in either case, the result is skin inflammation (13). The clinical feature of contact dermatitis is violent inflammation of the epidermis and oedematous swelling which may stimulate urticaria (13).   1.3.6.9             Irritant Dermatitis Irritant dermatitis can be primary or secondary. A primary irritant such as a strong acid usually causes a response on first exposure, secondary irritants like soap, cosmetics cause an inflammatory response only when the irritant is used repeatedly (1, 2). Primary irritants cause pruritic erythema and ulceration while secondary irritants cause slow grade inflammation that stays for long periods.1.3.6.10           Allergic DermatitisThey could be classified as immediate (anaphylactic), intermediate (arthus) or delayed (tuberculin) the most prominent being delayed hypersensitivity reactions. Allergy cannot occur on first exposure to an allergen, but some people can react abnormally with skin irritations to substances like shellfish on first exposure to them. These are not allergic reactions but idiosyncrasies of such persons.In immediate allergic dermatitis or anaphylactic reaction, the allergen on first contact causes the production of antibodies, which sensitize tissue cells passively, such that subsequent administration of the allergens reaches the sensitized tissue cell, causing their injury and release of endogenous agents like histamines, kinins and prostaglandins and these agents cause further local changes that include contraction of smooth muscles, increased vascular permeability and oedema. The cells injured usually recover, though some may die (1).In intermediate allergic dermatitis or arthus, the antigen combines with the antibodies in tissue spaces or in the circulation, to produce a complex. This causes a primary change which is massive infiltration of the extra vascular tissue. Then a secondary change occurs that changes the tissue and this depends on the composition and strength of the allergen.Delayed (tuberculin) reaction is the major mechanism involved in allergic contact dermatitis. It occurs days after the first contact with an allergen is made; sometimes it may take months or even years to develop. Once the reaction is initiated it builds up in severity. Susceptibility to this type of sensitization may last a life time though it can be overcome in some cases (1, 13).1.4       Factors influencing skin irritationThese include the sensitizer itself, climate and the host. The degree of skin irritation is a function of the intrinsic irritation potential of the test material, its concentration, its ability to remain bound to the skin and the texture of the exposed skin. Environmental conditions play a role in skin texture and its resistance to irritant substances. High humidity allows improved skin hydration and thus faster penetration of irritants; occlusion has same effects as it keeps the skin hydrated. Age and colour of the skin also influence irritant dermatitis. Aged skin is less prone to irritation than youthful skin, possibly because it is more difficult to penetrate an older skin than a younger one. Dark skinned persons seem less susceptible to irritants than lighter skinned individuals (2).Administration of more than one substance promotes skin irritation. A secondary irritant that is not irritating to the skin when applied alone may cause irritation when used as a surfactant or a keratolytic substance. Damaged or traumatized skin encourages skin irritation (1).1.5 Patients attitude to skin infections              Most diseases of man would need subjective and objective information to be diagnosed but the skin is one organ which when diseased or traumatized can be noticed by all without asking. People with skin diseases or conditions are very disturbed by their complaint in comparison with other medical conditions because skin diseases tend to make their victim have a leper like complex, a feeling of disgust and shame as most skin infections are on an organ which can be seen by all, as well as the fear that the contagious diseases may spread to family and friends who might on their own part try to avoid the sufferer. Skin infections are still a serious threat in the developing countries, Nigeria inclusive. This is more so as the issue of drug resistance of many of the causative organisms are on the increase (17).It is a great challenge to treat skin infections as many patients with skin diseases believe that because the lesion is on the surface, it should be easy to cure and it is very difficult, almost impossible to convince a patient into thinking that his compliant has improved when he and others can obviously see it has not (1). A healthy, good looking skin usually implies a healthy person while an un-healthy, sick looking skin is the reverse. Looking good is said to be good business so most people would spend a fortune to keep a healthy radiant skin.1.6       WoundsA wound is a break in the skin, wounds are injuries usually caused by cut or scrapes that disrupts the continuity and integrity of the external surface of the body. This compromises the normal functioning of the skin. Wound healing is a response to the injury that sets into motion a sequence of events. With the exception of bone, all tissues heal with some scarring. The objective of proper wound care is to minimize the possibility of wound infection and it’s scarring (18).Types of WoundsWounds are divided into two types: – open and closed wounds.1.6.1   Open WoundsOpen wounds vary with the type of object that caused it and with the manner in which the skin tissue is broken, there are six kinds of open wounds, incisions, lacerations, punctures, avulsion, abrasions and amputations, sometimes there could be a combination of these six types (18).1.6.1.1             IncisionsIncisions are commonly called cuts, and are wounds caused by shape-edged objects like razor, broken glass, knives, or surgical blades. Incision wound are cut neatly with smooth edges. There is little damage to the surrounding tissue, they are the least most infected wound of all open wound types because the free flow of blood washes away many of the microorganisms that cause infections away from it (18, 19).1.6.1.2             LacerationsThis type of wound are torn rather then cut. The edges are irregular with torn tissues below; such wounds are usually created by blunt objects like blunt knives. Apart from tearing the tissues, they are also crushed. Lacerations are usually contaminated with dirt and other types of foreign materials ground into them so that they are likely to become infected.1.6.1.3             PuncturesPunctures are caused by sharp objects that penetrate the skin and tissue to create a small surface opening. They can be created by nails, needles or bullets; the risk of infection is real in puncture wounds, especially if the penetrating object has tetanus bacteria on it.1.6.1.4             AbrasionsAbrasions are sometimes called grazes, and are superficial wounds caused mostly by a sliding fall on a rough surface in which the top skin is scrapped off. Parts of the body with thin skin like the knees and elbows are most prone to abrasion. This kind of wound can be infected easily because dirt and germs are usually embedded in the tissues from the rough surfaces (18, 19).1.6.1.5             AvulsionAvulsion is tearing away of tissue partially or completely from the body part. Sometimes, the torn tissue may be surgically re-attached to the body part. 1.6.1.6             AmputationThis is the non-surgical removal of a limb from the body. Bleeding is usually heavy and shock may occur. Like in a vulsed tissue, the tissue can be surgically re-attached.1.6.2   Closed Wounds            Closed wounds are also called contusions or bruises; they are caused by a blunt forceful blow/trauma to the skin and soft tissue, leaving the tissue under the skin damaged but the outer layer of skin intact. These injuries may require minimal care as there is no opened wound but hematoma may develop and this demands evacuation.   Hematomas occur when blood vessels are damaged such that it causes blood to gather under the skin (18, 19). 1.6.3   Microbial Contamination of WoundsOpen wounds are prone to infections especially infection by bacteria, these infections may provide an entry point for systemic infections. Microbial infected wounds heal slowly and often result in the production of offensive smelling exudates and toxins that kill regenerating wound cells. Antibacterial and antifungal compounds of natural origin may help prevent this from occurring (20). Infection is the presence of microbial pathogens proliferating in a wound, causing tissue damage and eliciting inflammatory responses (21). A number of microorganisms are found to infect wounds among which are P. aeruginosa, S. aureus, S. faecalis, E.coli, Clostridum perfringes, C. tetran , Coliform bacilli, Herbal enterococcus (18). Use of herbal extracts may prevent infection that may lead to sepsis (22).   1.6.4   Wound Healing    There are stages of the wound healing process.1.6.4.1 Clotting/Inflammation stage/phase This begins with the injury itself. In this phase, is bleeding, immediate narrowing of the blood vessels, clot formation and release of various chemical substances into the wound that will begin the healing process, occur, and specialized cells clear the wound of debris over the course of several days (20, 23).Clotting is the first step in the healing of a wound, prevents any further blood loss. Clotting or coagulation is a rapid response to bleeding that initiates homeostasis to stop excessive loss of blood.  When injury occurs, the vascular integrity of the injury area is broken; there will be extravasation of the blood into the wound site (24). Platelets are the highest number of blood cells at an injury site. When blood from the wound comes into contact with collagen of the torn muscle fibres, the blood platelets adhere to the collagen leading the platelets to secret fibrinogen, which is converted to fibrin by thrombosis. Also released are monocytes which in turn release growth factors and cytokines that are important in the maintenance of the inflammatory reaction and stimulate cell proliferation to enhance wound healing. Thromboxane, histamine, prostacyclines, prostaglandins, serotonin and neutrophils are also released (24).Prostaglandins and thromboxanes cause vasoconstriction of the blood vessel to prevent blood loss but histamine, also in the extravasted blood, can counteract this constriction, and causes vasodilation thus making the blood vessels porous. Blood proteins leak out of the porous blood vessels into extravascular spaces, increase its osmolarity and in a bid to balance this raised osmolar load, draws water into the wound site, hence making it oedematous (20, 23).The neutrophils clean the wound area by secreting enzymes that break down the damaged or injured tissue into wound debris. They also phagocytose the wound debris and contaminating bacteria.Platelets attract monocytes to the wound sites where they mature into macrophages. The macrophages phagocytose bacteria and wound debris, and also release growth factors and cytokines that instill inflammatory reactions and stimulate healing by production of new tissue cells to re-epithelialise the wound.1.6.4.2             Proliferative phaseIn the proliferative phase, a matrix of cell forms. On this matrix, new skin cells and new blood vessels form and it is these new blood vessels known as capillaries that give a healing wound its pink or purple-red appearance. The capillaries supply the rebuilding cells with oxygen and nutrient to sustain the growth of the new wound cells, and also promote the production of the protein- collagen. Collagen acts as the framework upon which the new tissue is built.1.6.4.3             AngiogenesisEndothelial cells that originate from the blood of uninjured wound area migrate through the extracellular matrix to the wound area. They become capillaries that supply the rebuilding wound cells with oxygen and nutrients (25). The endothelial cells are attracted to the wound area by the presence of growth factors and fibrin present and by shortage of O2 (26), the endothelial cells continue to grow and proliferate in the wound area, a process that decreases as O2 supply to the site is increased.1.6.4.4             FibroplasiaAfter the development of new blood capillaries, fibroblasts in the normal tissue adjacent to the wound tissue, proliferate and migrate to the wound site. They mingle with the wound and produce reticular fibres which progress into collagen fibres. Fibroplasia takes about 3-4 days after the injury. After fibroplasia is the granulation process, in this process, the new blood vessels, inflammatory cells, growth factors, endothelia cells and fibroblasts attach and grow on the collagen matrix that had been laid down by fibroblasts (26).1.6.4.5             EpithelialisationThis is the process of laying down new skin or epithelial cells. The skin forms protective barriers between the wound and the environment. Epithelialisation begins within a few hours of the injury to 48 hours in a clean sutured wound; open wounds take longer time because the inflammatory phase is prolonged (27).The epithelial cells originate from keratinocytes of the wound edges, hair follicles and sebaceous glands. The epithelial cells proliferate over and across the wound and when they meet, proliferation stops.1.6.4.6             Re-modeling phaseThis begins after 2-3 weeks or months depending on the type of wound. The collagen frame is more organized as there is continual accumulation of collagen.The blood vessel density becomes less and the wound losses its pinkish colour over time depending on the size of the wound; the wound area increases in strength, and eventually reaches about 50%- 80% of the strength of uninjured wound (28).1.6.5   Factors affecting wound healingFor a wound to heal successfully, its stages of healing- hemostasis, inflammation, proliferation and remodeling must occur in the right sequence at appropriate time frame. Any factor that disrupts this sequence of healing causes improper wound healing or an impaired wound healing. These factors can be local or systemic. Local factors directly influence the characteristics of the wound while systemic factors are the health /diseases status of the individual that affects his/her wound healing ability (28). Local factors1.6.5.1             OxygenationAdequate oxygenation is essential to wound healing, because O2 is necessary for cell metabolism and production of adenosine triphosphate (ATP) which is critical for all wound- healing processes. Oxygen prevents wound infection, induces angiogenesis, increases keratinocytes differentiation and re-epithelialisation. It enhances fibroblast proliferation, collagen synthesis and wound contraction (28). Wound disrupts the vascular distribution in the wound area that subsequently depletes its oxygen content. Depletion of O2 (hypoxia) after injury triggers wound healing, hypoxia induces cytokines and growth factor production from macrophages, keratinocytes and fibroblasts but prolonged hypoxia delays wound healing (28). Wounds on the neck and face which are greatly supplied with blood heal rapidly while those on the extremities heal slowly. Diseases that compromise blood supply/circulation like diabetes slow down healing because proper oxygen level is crucial for optimal wound healing though initial hypoxia at wound areas stimulates wound healing by the release of growth factors and angiogenesis. Oxygen is important for sustenance of the healing process (28).1.6.5.2             InfectionIntact skin usually has microorganisms sequestered on its surface and once the skin is broken by injury or diseases, these microorganisms get access to the underlying tissues to cause contamination. Contamination is the presence of non-replicating organisms on a wound. Replication of organisms in the wound is termed colonization; there is usually no tissue damage at this stage. If the host reaction to the presence of an organism on it is negligible, then the organism is said to be colonizing the wound. Colonized wounds heal without the need for antibiotics as the host immune system can counteract the activities of the organisms (28). When the tissues around/ local to the wound begins to respond to the continuous replication of microorganism by eliciting local damaging tissue responses, there is local infection. Invasive infection is the presence of these replicating organisms within the wound that is accompan

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Antimicrobial and wound healing properties of leaf extracts, fractions and ointment formulations of spermacoce verticillata linn (family_ rubiaceae)

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