CHAPTER ONE

INTRODUCTION

1.1       The human skin

The 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).

Epidermis

This 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).

Dermis

This 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 layer

The 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 skin

The 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 skin

The 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       Microorganisms

Microorganisms 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 infection

The 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 microorganisms

Most 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).

Bacteria

Bacteria 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 aureus

These 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 subtilis

Bacillus 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.

1.2.2.3             ESCHERICHIA COLI

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).

 

 

 

1.2.2.4             Pseudomonas aeruginosa

It 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).

Fungi

They 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 audouinii

It 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 rubrum

This 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 albicans

This 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 skin

The 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 infections

Examples of bacterial skin infections are:

1.3.1.1             Impetigo

It 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             Folliculitis

This 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             Erysipelas

This 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             Ecthymas

It 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 Carbuncles

Superficial 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            Paronychia

This 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 Infections

Fungi 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             Candidiasis

This 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 infections

These 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 simplex

This 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 zoster

This 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 contagiosum

This 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 infections

Besides 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                         Jiggers

This 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/ Dermatitis

Dermatitis 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             Dermatitis

This 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 Dermatitis

This 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 dermatitis

This 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 eczema

This 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:

• Scalp-hair dyes and scalp lotions
• Neck-ties, scarves, necklaces, perfume.
• Ears -hair nets, ear clips, hearing aids, ear drops and Glass frames.
• Trunk-clothing
• Genitals -clothing, contraceptives and deodorant.
• Arm-pits -deodorants, shaving powder and shaving sticks
• Thigh -suspender, clothing
• Ankles and feet-socks, stockings, shoe

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 Dermatitis

These 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 Dermatitis

They 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 irritation

These 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       Wounds

A 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 Wounds

Wounds are divided into two types: – open and closed wounds.

1.6.1    Open Wounds

Open 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             Incisions

Incisions 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             Lacerations

This 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             Punctures

Punctures 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             Abrasions

Abrasions 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             Avulsion

Avulsion 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             Amputation

This 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 Wounds

Open 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 phase

In 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             Angiogenesis

Endothelial 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             Fibroplasia

After 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             Epithelialisation

This 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 phase

This 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 healing

For 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 factors

1.6.5.1             Oxygenation

Adequate 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             Infection

Intact 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 accompanied by a subsequent overall host injury (28).

Wound infection (Local/Invasive) occurs when the virulence factors expressed by the organisms in the wound out -competes the host immune system. This is evidenced by purulent drainage or exudates, erythema and fever.

Wound infection is a problem because the infection stops a wound from healing by prolonging inflammatory phase. The pathogenic microorganisms in the wound will compete with macrophages and fibroblast for the limited nutritional resources available at the wound site.

Wound inflammation is a normal stage of the wound-healing process and is vital for the removal of contaminating microorganisms because when these microorganisms are not effectively removed (decontaminated), inflammation stage is prolonged. Bacteria and their toxins can lead to the prolonged elevation of pro-inflammatory cytokines like iterleukin-1 and this elongates the inflammatory phase. If this continues, the wound may enter a chronic stage and fail to heal. Prolonged inflammation increases the level of matrix metallo proteases, a group of proteases that degrade the intracellular matrix. With the increased protease content, a decreased level of the naturally occurring protease inhibitor occurs. This shift in protease balance causes growth factors that appear in chronic wounds to be rapidly degraded (28). The bacteria in infected wound occur as biofilms. Biofilms formation usually begins with the pioneer cells attaching to the wound surfaces, through adhesion. Once established on the wound, these cells grow and divide to produce micro-colonies which eventually coalesce to produce a bioflim. The resident cells within the biofilm are not exposed to attack by the immune system. Bacteria biofilms are less susceptible to antimicrobial agents, (6). Mature biofilms develop protective microenvironment and are more resistant to conventional antibiotic treatment. Staphylococcus aureus, Pseudomonas aeruginosa, B. hemolytia Streptococci and Escherichia coli are common bacteria in infected wounds (6, 28).

Many chronic ulcers probably do not heal because they have biofilms containing P. aeruginosa that shield them from phagocytic activity of invading polymorphonuclear, neutrophile and antibiotics (28).

1.6.5.3             Age

Aging causes delay in wound healing but not an actual impairment in terms of the quality of wound healing (6, 28). Age delayed wound healing is associated with an altered inflammatory response like delayed T-cell infiltration into the wound area. Delayed re-epithelialisation, collagen synthesis and angiogenesis were observed in animal studies of aged mice as compared to young mice (28).

Every stage of wound healing undergoes characteristic age-related changes like increased secretion of inflammatory mediators, delayed infiltration of macrophages and lymphocytes, impaired  macrophage function, decreased re-epithelization of growth factors, delayed angiogenesis and collagen deposition, reduced collagen turnover and remodeling (6, 28).

1.6.5.4             Wound size

The healing time of a wound is related to its size. A small sized wound heals at a faster rate than a larger one.

1.6.5.5             Depth of wound type

The depth of a wound is proportionate to its healing rate; surface or superficial wounds heal faster than deep wounds. In deep wounds the injury affects much more tissue than a surface wound and this disrupts a larger vascularisation, reducing blood supply and oxygen supply to the wound site, leading to a greater and more prolonged hypoxia. This insufficient perfusion and prolonged hypoxia amplifies the inflammatory stage causing impaired collagen synthesis and inadequate angiogenesis. Accumulation of metabolites in the hypoxic conditions of the wound increases their susceptibility to infection and this also impairs its healing (20).

1.6.5.6             Medication

Medications that interfere with clot formation, inflammatory response and cell proliferation have the ability to affect wound healing, e.g. drugs like glucocorticoid- steroids, non-steroidal anti-inflammatory drugs and chemotherapeutic drugs.

Glucocorticoid Steroids: Glucocorticoid steroids used as anti-inflammatory drugs inhibit wound repair by their anti-inflammatory effects, which suppresses cellular wound responses, fibroblast proliferation and collagen synthesis. Systemic steroids cause wounds to heal with incomplete granulation tissue and reduced wound contraction thereby increasing the risk of wound supra-infection. However, topical application of corticosteroids on wound, accelerates its healing, reduces pain and exudates (20, 28).

Non-Steroidal anti-inflammatory drugs: Non-steroidal anti-inflammatory drugs like Ibuprofen are used for treatment of inflammation and pain management. Animals wound healing studies suggest that systemic Ibuprofen has an anti-proliferative effect on wound healing; it decreased epithelialisation, reduced wound contraction, and impaired angiogenesis. Thus the majority of surgical patients are recommended to discontinue NSAIDS so that they do not have significant NSAID activity at the time of their surgical wound repair; exception are patients on low –dosage of aspirin for cardiovascular diseases (20, 28).

Chemotherapeutic Drug: Most chemotherapeutic drugs inhibit cellular metabolism, rapid cell division and angiogenesis. They also inhibit DNA, RNA, protein synthesis, resulting in decreased fibroblast and vascularization of wounds. They delay cell migration into the wound area, lower collagen formation, reduce proliferations of fibroblast. They do also inhibit contraction of wounds (20).

1.6.5.7             Nutrition

Nutrition affects rates of wound healing. Individuals with non-healing wounds often require special nutrients to improve the wound healing. Nutrients like carbohydrates, proteins, fats, minerals and vitamins affect healing process (28).

Glucose from carbohydrates is the main source of ATP; it provides energy for angiogenesis and deposition of new tissue.

Proteins are needed for capillary formation, fibroblast proliferation, collagen synthesis and wound -remodeling. Deficiency of protein affects all these processes and also affects the immune system, resulting in a decline in leukocytes, phagocytes and increased susceptibility to infection.

The major protein important for wound healing is collagen; it is composed of glycine, proline and hydroxyproline. Collagen is synthesized by the hydroxylation of glycine and proline in the presence of co-factors like ferrous ion and vitamins (29).

Arginine is a precursor to proline, which means that adequate amount of it, will be needed for collagen synthesis (29). Arginine stimulates wound healing by supporting collagen deposition, angiogenesis, wound contraction; it also improves immunity of the host. Another amino acid of importance in wound-healing is glutamine; which stimulates the inflammatory response that occurs in early wound healing (29).

Vitamins: Vitamin C (L-ascorbic acid) is a very powerful antioxidant, that has anti-inflammatory effect too. Vitamin C is needed for collagen synthesis, fibroblast proliferation, angiogenesis and improved capillary fragility. A deficiency of vitamin C affects all these processes that are vital to proper wound healing, and reduces host immunity, thus increasing its susceptibility to wound infection.  Vitamin A is an effective antioxidant that hastens collagen synthesis and proliferation (29).

Vitamin E (tocopherol) is an effective anti-oxidant that helps to maintain the integrity of cellular membranes by providing protection to it against oxidation. It also has anti-inflammatory properties. Topical application of vitamin E prevents scar formation in chronic wounds (29).

Minerals: Minerals are vital for adequate wound healing and their deficiency impairs wound healing. For example, magnesium is a co-factor for many enzymes needed for protein and collagen synthesis; copper is a co-factor for the optimal cross-linking of collagen; zinc is a co-factor for DNA and RNA polymerase, while iron is a co-factor too, involved in the hydroxylation of proline and lysine (29).

Systemic factors

1.6.5.8             Obesity

Obesity increases the risk of many diseases like coronary heart disease, type 2 diabetes, hypertension, dyslipidemia, stroke, respiratory problems. Obese people heal slowly because fat does not have a good supply of oxygen thus wound healing is impaired. Their numerous skin folds harbor microorganisms that contribute to infection and even administered  antibiotics when given, do not help much as there is decreased delivery of the drug as well (29).

1.6.5.9             Host Immunity

Diseases like HIV infection and tuberculosis that compromise host immunity, impair the rate of wound healing. Inflammatory stage of wound healing is un-duly prolonged in such individuals because their body defense mechanisms are unable to accelerate the inflammatory phase of wound healing (30).

1.6.5.10           Health Status of an individual

Diseases that compromise blood supply such as diabetes, slow down wound healing. Diabetic individuals exhibit impaired healing of acute wounds, are prone to develop chronic non-healing diabetic foot ulcers that are often caused by hypoxia. Hypoxia lengthens the inflammatory stage due to increased level of oxygen radicals in the wound that prolong healing. Hypoxia also causes inadequate angiogenesis (30).

1.6.6    Models for the evaluation of wound healing activity

There are two models for studying wound healing namely, in vitro and in vivo models.

1.6.6.1             In vivo models

In vivo models are carried out with small rodents like rats and guinea pigs, and include:

1.6.6.1.1          Excision wound models

They are used to study the rate of wound contraction and epitheliazation (31). The wound is created by excising the full thickness of circular skin from an anaesthetized animal (32). Wound contraction is assessed by measuring the wound diameter using translucent ruler. The edges of excised wounds are not in contact so contraction and epitheliazation are necessary for its healing process (33). This model studies two parameters – contraction and epithelialisation (34).

1.6.6.1.2          Incision wound model

Longitudinal incisions are made on the shaved skin of selected animals under mild anesthesia. The parted skin is brought together by suturing. The skin breaking strength of the wound can be determined (35).

1.6.6.1.3          Dead space analysis

Dead space wound are created by making a pouch through a small opening in the skin of a rat (36). A polypropylene tube is implanted into the pouch beneath the skin and the wound is sutured. After about 10 days, the polypropylene tube is removed and the granulation tissue surrounding it is harvested. These regenerated tissues are cut in the form of squares along with the normal tissues on sides of the wound and both are studied histologically. The physical and mechanical breaking strengths of the tissues are studied (36). Hydroxyproline content of the tissues is studied and histological studies are carried out to examine the pattern of lay down for collagen (37).

1.6.6.1.4          Burn wound model

Burn inflicts extreme damage to the skin, causing tissue necrosis and body fluid exudation, which create a perfect medium for bacterial growth. (39). Partial thickness wound is inflicted upon anesthesied animals, by pouring hot molten wax of about 80^oC into a metal cylinder with circular opening and placing on the back of the animal. Wound contraction and epithelialisation are then studied (37).

1.6.6.2             In vitro models

In vitro models are now widely used in   wound healing research studies because of ethical reason, since they do not involve inflicting pain on live animals and for their usefulness in bioactive guided fractionation and determination of active compounds (20). An example of in vitro parameter studied is antimicrobial activity.

1.6.7    Wound healing study parameters

1.6.7.1 Wound closure

Collagen makes up more than 50% of sutured wounds; hence, any substance that promotes collagen maturation enhances the process of wound healing (39).  Wound closure or contraction is part of the proliferate phase of wound healing which is mediated by mainly fibroblasts (40). Contraction of wounds can be studied by observing the wound healing and wound contraction percentage (%) is calculated using the following formula (41).

WD_o – WD_t x 100       …Equ.  1

WD_o

WD_o= wound diameter on day Zero

WD_t= wound diameter on day t.

1.6.7.2             Epithelialisation period

This is the period of epithelial renewal after injury. It involves the proliferation and migration of epithelial cells towards the center of the wound (41).

1.6.7.3             Tensile strength

This indicates the quality of the repaired tissue and studies how the repaired tissue resists breaking under tension (40, 41). Tissues from the treated and control animals can be loaded between the upper and lower holders of a tensile testing machine and a load is applied that pulls the tissues apart. The load/weight that breaks the tissues is obtained and compared (42).

1.6.7.4 Increase in granulation tissue

Increased granulation tissue is associated with enhanced collagen maturation in dead space wounds (41). These wounds heal by laying down connective tissue, where more than 50% of the connective tissue is made up of collagen. Collagen is a fibrous protein component of connective tissue, and is made up of hydroxyproline mainly hydrolysine and glycine (39). Increased levels of hydroxyproline suggest increased collagen turnover and subsequent increase in granulation tissues (43).

1.6.8    Existing therapy of wound healing

Topical antimicrobial therapy is one of the most important method of wound  care (44). Neomycin- bacitracin powder, (Cicatrin^R), gentamycin ointment, tetracycline ointment and nitrofurazone ointment are among the standard antibiotics used in wound healing (31, 42, 44). Povidone-iodine cream is also used for wound healing purpose (42). Wound healing is not improved/affected by drug usage alone but by factors like nutritional status of the victim and his/ her clinical conditions like diabetes, obesity and anemia. Therefore wound management must involve a holistic approach (44).

 

1.6.9    Types of wound Healing

Once an injury has occurred and platelets from the damaged blood vessels come in contact with exposed collagen, its healing starts (45). Healing of wounds can be of three types- healing by first intention (primary healing), healing by second intention (secondary) and healing by third intention (Tertiary). This classification is based on the nature of the wound edges as it heals (44).

1.6.9.1 Healing by first intention

This occurs when the wound edges close with little or no inflammation resulting into a scar-less healed wound (44), surgical incision is targeted towards this type of healing where little or no post surgical tissue necrosis occurs. Primary healing occurs within hours of repairing a full-thickness surgical incision by firmly suturing the wound edges together, which prevents granulation tissue from being visible, thus leaving little or no scar (28, 43).

1.6.9.2 Healing by secondary intention

There is formation of granulation tissues, which fill up the gap between the wound edges. In this type of healing, significant loss of tissue occurs leaving the wound edge open to heal with scarring (44).

1.6.9.3 Healing by third intention

This occurs when the wound is left open until granulation form and falls before the wound edges are united together, this is more of late closure of a primary wound and it heals with scarring (43).

1.7       Natural products as sources of medicine

Traditional medicine is a major African socio-cultural heritage. It had been in existence for several hundreds of years, and was once believed to be primitive and wrongly challenged with animosity, especially by foreign religion dating back to the colonial days in Africa and subsequently by the conventional or orthodox medical practitioners. Today, traditional medicine has been brought into focus for meeting the goals of a wider coverage of primary health care delivery system, not only in Africa but also to various extents in all countries of the world (46).

Traditional medicine is defined by World Health Organization (47) as the sum total of knowledge or practice whether explicable or inexplicable, used in diagnosing, preventing or eliminating a physical, mental or social disease which may rely exclusively on past experience or observation handed down from generations, verbally or in writing. It also comprises therapeutic practices that have been in existence for hundreds of years before the development of modern medicine and are still in use today without any documented evidence of adverse effects.

The explicable form of traditional medicine can be described as the simplified scientific and direct application of animal or plant materials for healing purposes and which can be investigated, rationalized and explained scientifically. The use of Salia alba the willow plant (containing the Salicylates) for fever and pains which led to the discovery of aspirin belongs to this form of traditional medicine. Herbal medicine is  regarded by WHO as finished and labeled medicinal products that contain, as active ingredients, aerial or underground parts of some plants identified and proven in crude form or as plant preparations. They include plant juice, gums, fatty oils, essential oils e.t.c. (48).

There are several other official modern drugs today which were originally developed like aspirin through traditional medicine e.g. morphine, digoxin, quinine, ergometrine, reserpine, atropine etc, all of which are currently being used by orthodox medicine in modern hospitals all over the world.

The inexplicable form of traditional medicine, on the other hand, is the spiritual, supernatural, magical, occult, mystical or metaphysical form that cannot be easily investigated or explained scientifically e.g. the use of incantations for healing purposes, oracular consultation in diagnosis and treatment of diseases. The explanation is beyond the ordinary scientific, human intelligence or intellectual comprehension (48).

The WHO has since urged developing countries of the world to utilize the resources of traditional medicine for achieving the goals of primary healthcare. This injunction stems from the various advantages of traditional medicine namely: low cost, affordability, ready availability, accessibility, acceptability and low toxicity (49).

Antimicrobial and wound healing activities of traditional medicines have been employed in folk medicine for wound care. Most of these plants exhibit wound healing activities or possess antimicrobial and other related activities that improve the wound care (44). The different plants used for wound care do contain active constituents that are nutritive in action (50).

The use of plants for wound care/healing is gaining a lot of attention and about 1-3 % of traditional medicine in use are for the treatment of skin problems and wounds (33).  Spermacoce verticillata is an example of a local plant that is claimed by herbalists to be useful in the treatment of skin infections, the leaf extracts being used to treat leprous conditions, furuncles, ulcers and gonorrheal sores (51). It has thus become necessary that plants should be formulated into biologically active ointments with wound healing properties for local application at wound sites (52).

1.8       Plants with potential wound healing properties

Medicinal plants have been in use in folk medicine for wound care, as they possess the ability to directly improve wound healing or have its antimicrobial activities that are beneficial to wound care (53). There are certain plants that can improve wound care by a combination of these properties (44).

Ageratum conyzoides L (Asteracege): The leaves when applied to wounds act as antiseptics that aid its quick healing (54). It contains alkaloids and tannins. The root extract ointment showed significant wound healing activity and this activity is attributed to the antimicrobial and haemostatic action of the plant’s individual phytochemical or their combined actions (54).

Allium cepa   L. (Liliaceace): Studies of the alcohol extract of tubes of Allium cepa (onions) showed that it contains tannins and flavonoids that exhibited wound healing action in excision and dead space wound models. Their action was attributed to the presence of free radicals, scavenging and antibacterial action of their phytochemicals (55).

Aloe vera (Asphodelaceae): Topical application and oral administration of Aloe vera gel to rats with dermal wounds increased the collagen content of the granulation tissue (56). It seems that Aloe-vera improves first and second burn wound healing but impairs wound healing of severe burns (57).

Alternanthera brasiliana Kuntz (Amaranthaceae): Photochemical screening of Alternanthere brasilian a revealed the presence of alkaloids, steroids and terpenes. Research has shown that topical application of leaves of this plant improved fibroblastic deposition, angiogenesis and wound contraction (58).

Anthocleista nobilis G. don (Loganiaceae): Studies on the plant show that it had wound healing activities; inhibited bacterial growth and protected the fibroblast cells from oxidants injury (42).

Areca catechu L (Arecaceae): Studies on Areca catechu revealed the presence of alkaloids, and the alkaloidal fraction was shown to improve the healing of incision wounds by increasing the breaking strength of the wound (39).

Azadirachtha indica (Meleaceae): Studies have shown that alcohol leaf extract of Neem (Azadirachtha indica) was useful in treating ring worm, eczemas and scabies. Its leaf extract and oil from its seeds showed antimicrobial activity that kept wound treated with it, free from secondary microbial infections. It also inhibits wound inflammation as effectively as cortisone acetate and this aids wound healing (57).

Calotropis gigantea L. (Asclepiadaceae): A study made on topical application of latex of Calotropis gigantean showed that it promoted collagenation of wound (31). It also increased breaking strength and hydroxyproline of wounds (59).

Carica papaya L. (Caricaeae): The latex of Carica papaya contains cysteine endopeptidases- papain, caricain, chymopapain and endopeptidase. These antioxidants aid wound healing (60, 61).

Catharanthus roseus L (Apocynaceae): Research has shown that ethanol extract of this plant is vital in aiding the wound healing of diabetics (45).

Centella asiatica (Makinlayoideae): Phytochemical analysis of the plant revealed the presence of triterpenes and asiaticoside. Its aqueous extract promoted wound healing when applied topically on open wounds in rats (57).

Cocos nucifera L. (Arecaceae): Cocos nucifera significantly promotes wound contraction and decreases epithelialisation period in burn wound model (45).

Cordial dichotoma (Boraginacea): Studies on this plant showed that it had wound healing potential as claimed traditional medical practitioners (62).

Dissotis theifolia (Melastomataceae): Studies on Dissotis theifolia showed that it possesses antibacterial and wound healing effect when formulated as ointment, on infected excision wound model. Its methanol stem extracts upon phytochemical screening revealed the presence of saponins, tannins, glycosides, flavonoids, terpenoids, carbohydrates, alkaloids and steroids (53).

Other plants of importance in wound healing that have been scientifically proven are Elaeis guineensis Jacg (Mackinlayoidae) that improves the different phases of wound repair (40), Euphorbia heterophylla whose aqueous and ethanol extracts showed significant wound healing upon topical application in rats (63).

Ficus religiosa leaf extracts ointment improved healing of wounds (52). Ginkgo biloba contains flavonoids and terpenes and these constituents provides its wound healing activity (57).

Helianthus annus formulated as ointments, upon application on wounds, hastened its healing (57). Hoslundia opposita has   antibacterial and antioxidant properties which inhibit bacterial growth and protect fibroblast cells against oxidant injury (42).

Studies by Raina et al (57) on Hydrocarpus wightiana paste applied on wounds showed that it hastened epithelialisation period. Raina et al. (57) again confirmed the antibacterial wound healing activity of Hypericum prolificum. The juice of Jasminum auriculatum when applied topically on excised wounds in rats promoted wound healing (57). Research carried out on Jatropha curcas by Shetty et al. (64) showed that it hastened wound healing.

 

The phytochemical analysis of Lantana camara revealed flavonoids and triterpenoids whose antimicrobial effect is thought to increase the rate of burn wound contraction (45). Flavonoids, tannins, steroids and saponins were found in Lawsonia inermis and this plant showed significant wound healing activities on incision and excision wound models (65). Mimosa pudica is found to be rich in tannins and its aqueous extract increases the rate of wound contraction (66). Napoleona imperialis formulated into ointment showed wound healing activity comparable to that of Cicatrin^®, a wound healing antibiotic (44).

Phytochemical screening of Ocimum kilimandscharicum revealed the presence of flavonoids, tannins and proteins and its aqueous leaf extract possesses wound healing property that is attributed to its ability to increase the rate of wound contraction and epithelialisation (36). Studies carried out on alcohol and aqueous extracts of Ocimum sanctum showed that they significantly increased wound breaking strength (67, 68).

Some other plants that possess wound healing activities are Phyllanthus niruri (41), Quercus infectoria (69), Bubia cordiofolia (32), Trichosanthes dioica (70), Tridax procumbens (57), and Verononia arborea (43).

1.8.1    Plant phytochemicals of wound healing importance

Plants with medicinal properties perform these activities through their phytochemical constituents called active principles.  Some phytochemicals shown to possess wound healing activities include:

1.8.1.1 Flavonoids

They are widely distributed in nature (69), occurring in fruits, vegetables, herbs, beverages, tea, beer and chocolates (70). They have a C₆-C₃-C₆ backbone, with many structural varieties due to their conjugation to sugars at different sites of the molecule (68). They are known to possess free radical scavenging effect and a potent antioxidant effect too. These properties are believed to be important components of wound healing (55). The flexibility of the electron in the benzene nucleus of flavonoids accounts for their antioxidant and free radical scavenging properties and the structural resemblance between the flavonoids aglycone and many substances inherent in the biochemistry of human biological cells of nucleic acids coenzymes, steroids, neurotransmitters. This is why they can inhibit receptors, enzymes and neurotransmitters (71).

The antimicrobial activities of many plants have been attributed to their flavonoids content. (71), hence they have the ability to prevent wound infection. Quercetrin isolated from Hypericum perforatum inhibits the growth of microorganisms (72). Santin, a flavonoid from Tanacetum parthenium exhibits anti-inflammatory activity by inhibiting the cyclo-oxygenase and 5-lipoxygenase pathways.

1.8.1.2 Tannins

Plants used for their wound healing and anti-inflammatory properties are known to contain a high amount of tannins (70). Tannins are phenol, i.e. compounds found in most herbal products used for wound healing. They possess antimicrobial and astringent properties which are responsible for wound contraction and epithelialisation (43).

 

1.8.1.3 Terpenes and Terpenoids

Terpenes are known for promotion of rapid wound healing (57). Terpenoids promote wound healing via their astringent and antimicrobial properties that improve wound contraction and increased rate of epithelization (40).

Asiaticoside is a terpene found in the plant, Centell asiatica, and is known to improve wound healing and duodenal ulcers (71). Terpenes are natural products, derived from plants that have medicinal properties and biological activities. They are widespread in nature, mainly in plants as constituents of essential oil, particularly conifers. They are large and varied class of hydrocarbons, but oxygen – containing compounds such as alcohol, aldehydes or ketones (terpenoids) are also found (73). The structure of terpene is repeated isoprene unit (C₅H₈)_n and they are grouped according to the number of such repeated units.

1.8.1.4 Saponins

Saponins possess antioxidant and antimicrobial activities that are known to promote wound healing (54). Triterpene saponin is known to possess immunomodulatory properties (71). The plant Centella asiatica contains asiaticoside a triterpene saponins. When this is applied topically, twice daily, for seven days on wound, 56 % increase in hydroproline resulted. There was increased tensile strength and collagen content with better epithelization (56). Saponins are glycosides with a distinctive foaming characteristic, bitter and acrid taste (56, 71). They are phytochemicals which are found in most vegetables, beans and herbs (74). Saponins are structurally related to steroid hormones and vitamin D. They consist of polycyclic aglycone that is either a choline steroid or triterpenoids attached via C₃ and an ether bond to a sugar side chain. The aglycone is referred to as sapogenin. They are derivable from plants like soap worth (Saponeria spp) and soap berry (Sapindus spp). They are also found in Lobelia inflata, Urginea maritima and Bellis perennis (74).

Saponins are used in sneezing powder, emetics and cough syrups. Some are diuretics while others have the ability to reduce serum cholesterol by preventing its re-absorption after it has been excreted in the bile. They are anti inflammatory and anti cancerous, and have high antimicrobial activities. Saponins can cure eczema (74, 75).

The saponins in official saponin drugs are mainly triterpene derivatives, with a smaller number of steroids. All triterpene saponins possess hemolytic activity, which varies from strong to weak, depending on the type of substitution. Steroid saponins are non-hemolytic. Saponins are detected by exposure to UV-254 nm or Uv-365 nm, but with vanillin – Sulphuric acid reagent, saponins form mainly blue or blue – violet and sometimes yellowish zones (76).

1.8.1.5 Alkaloids

            Alkaloids have antioxidant and antimicrobial properties which are known to promote and improve wound healing process (53). An alkaloid allantion, found in Symphytum asperum and Symphytum caucasicum, is thought to be responsible for their wound healing property (77).

1.8.1.6 Plant vitamins

Vitamins A is necessary for epithelial and bone tissue development, immune defense and cellular differentiation (56). Vitamin C stimulates the synthesis of collagen (40), is an antioxidant, enhances neutrophil function and increases angiogenesis. Vitamin E minimizes/prevents scarring (40, 56).

1.8.1.7 Cardiac glycosides

These are drugs that contain steroids, used in the treatment of congestive heart failure and cardiac arrhythmia (76). They are found as secondary metabolites in several plants and a few animals. The plant sources include Digital purpurea (Foxglove), Digitalis lanata, Strophanthus gratus and Strophanthus kombe (78).

Structurally, glycosides consist of a glucose moiety attached to a steroid component called aglycone. They are structurally derived from the tetra cyclic 10, 13, – dimethylcylopentanoperhydrophnanthrene ring system (76, 78). Before exhibiting their cardio tonic effect, the aglycone a molecule that is bioactive in its free form but inert when conjugated must be detached from the carbohydrate, by the breakage of the glycoside bond by water and enzymes. The cardiotonic agents increase the force of heart muscles contraction without a concomitant increase in oxygen consumption. The myocardium thus becomes a more efficient pump and is better able to meet the demands of the circulating system. Example of cardiac glycosides from natural products includes: quabain, cymarin, oleandrin, theretoxin, digitoxin, tecomin, digitalin and cheiranthin (76).

 

1.9       Antimicrobial agents

These are   compounds that can kill or inhibit the growth of microorganisms, and may have activity against a wide variety of microorganisms like bacteria, fungi, viruses etc. Such an agent is called a broad spectrum antibiotic. A narrow spectrum antibiotic on the other hand, exerts its activity on just few microorganisms and may not be very effective as an antimicrobial agent (7, 79).

The least acceptable effect of any antimicrobial agents is the inhibition of growth of microorganisms. An antimicrobial agent that inhibits growth of microbes is termed a microbiostatic agent while it is called microbiocidal if it kills the microorganisms. Every drug possesses some level of unwanted effects but an ideal antimicrobial agent should have tolerable levels of side effects on man and animal so it is very important that it is selectively toxic to the microorganisms only and not irritant nor sensitize the animal or man it is used on. Many microorganisms have the ability to develop resistance to the cidal or static effects of antimicrobial agents and no antimicrobial agent will be of much use if microorganisms can in- activate it rapidly. Hence, it is desired that an antimicrobial agent must have the capacity to resist the harmful effects of microorganisms.  Medicinal products of natural and chemical origin can sometimes undergo physical and chemical changes such as oxidation, reduction, photolysis, hydrolysis, and these physicochemical changes can bring about degradation of the drug product. It is desired that any ideal antimicrobial agent should retain its physicochemical and antimicrobial properties while in use or on storage (7, 79).

1.9.1       Determination of an antimicrobial agent’s spectrum of activity

An antimicrobial agent can inhibit or kill microorganisms. The extent to which it does this can be estimated by the determination of their spectrum of activity. This involves bringing different microorganisms in contact with the test antimicrobial agent.

The spectrum of activity of the antimicrobial agent is assessed by the number and type of organisms killed or inhibited by it (6). The carpet agar plate and the cup agar plate methods can be used to determine the agents’ spectrum of activity.

 

 

1.9.1.1 Carpet plate method

A known volume of the test organism is used to streak the surface of an already solidified nutrient media in a Petri dish. Sterile paper discs are then impregnated with the antimicrobial agent, placed in the plates and incubated at appropriate temperature and time. Operational conditions used for microbiological techniques are normally specific so that the experiments can be reproduced in any repeat test (8, 80).

The results are taken after a specified time as zones of growth inhibition.

Organisms not susceptible to the antimicrobial agent will grow close to paper while those sensitive to it grow away from it. The distance of the organisms from the paper discs are called inhibition zone diameters (IZDs) and the higher the IZD, the more sensitive is the organism to the antimicrobial agent and vice versa (81).

Organisms can be categorized as sensitive, intermediate or resistant to an antimicrobial agent based on interpretation of IZDs from National Committee for Clinical Laboratory Standards (NCCLS) IZDs guide-lines chart (8).

A regression analysis can be done on the obtained IZDs data and a line of best fit drawn.  Regression equations are formed and from the straight line charts, approximate 1ZDs corresponding to various sample concentration can be estimated.

1.9.1.2 Cup agar plate method

The microorganisms are streaked on already solidified agar or mixed with the molten agar just before it gets cold and poured into plates. Then instead of using paper discs as the reservoir for the antimicrobial agent, a cork borer is used to bore holes in the solidified agar to create cups into which the antimicrobial solutions are filled (79).

In both plate and cup agar methods the antimicrobial agent diffuses from their reservoir into the medium, where it inhibits the growth of sensitive microorganisms. Sensitive microorganisms will have appreciable zones of growth inhibition that are seen as clear zones or areas of no visible microbial growth around the cups/discs, while resistant microorganisms will show no appreciable zone of growth inhibition (8, 79).

1.9.2    Biostatic action of antimicrobial agents

Antimicrobial agents are termed biostatic when they inhibit the growth of microbial cells without necessarily killing the microbes. To assess such antimicrobial agent, a quantitative comparison of the biostatic actions of the agent and a reference agent that involves the determination of the minimum inhibitory concentration of both antimicrobial agents (test and reference) that inhibits the visible growth of a specified test microorganism under identical experimental conditions will be carried out. The assessment parameter for biostatic activity of an antimicrobial agent is the minimum inhibitory concentration (MIC) (7).

1.9.3    Minimum inhibitory concentration (MIC)

This is the least concentration of a particular antimicrobial agent that can inhibit the visible growth of a specific microorganism at specified experimental conditions (79). MIC can be determined by various methods, like

• The broth dilution method
• Agar dilution method.
• Agar diffusion method
• Concentration gradient method

 

1.9.3.1 Broth dilution method

A known concentration of an antimicrobial agent is diluted serially in an arithmetic order called serial doubling dilution. This produces dilutions of antimicrobial agent in decreasing order from the first tube to the last, such that the concentration of antimicrobial agent decreases by half from one tube to the next (79).

A known volume of the microbial test culture is added into the broth/antimicrobial agent tubes and incubated at an ideal temperature for the type of test organism involved.

Microbial growth in the test tube is seen as the presence of turbidity in the incubated tubes, in- contrast to a clear sterile nutrient broth. The concentration of the antimicrobial agent that inhibits growth of the test organism is taken as MIC of that agent on a specific organism under stated experiment condition (79).

1.9.3.2 Agar dilution method

This method is preferred to the broth dilution for assessing herbal extracts and coloured substances which would not be feasible with broth dilution method.

Serial dilutions of the antimicrobial agent are made with sterile water instead of nutrient broth. Each dilution is then mixed with equal volume of double strength sterile molten nutrient agar. By this the final mixture is thus brought back to a normal strength nutrient agar.  They are then poured into Petri dishes and allowed to solidify. Known volume of the test microorganism is then streaked on the surface of the agar and kept for one hour for pre – diffusion before being incubated at appropriate temperature and time conducive for the test organism. Microbial growth is observed as colonies of microbial cells on the surface of the agar. The least concentration of the agar plates that inhibits growth of visible microbial cells is taken as the MIC (7, 8, 79).

1.9.3.3 Concentration gradient technique

Two layers of agar are formed in a plate in the form of wedges. The lower wedge is first formed; containing a known concentration of the test antimicrobial agent. The upper wedge is then formed on the lower wedge free of the antimicrobial agent. After formation of the wedges, a pre – diffusion period is observed so that the agent in the lower wedge can diffuse into the upper wedge to reach its upper surface, because of the slant nature of the lower agar wedge. Varied concentrations of the antimicrobial agent are delivered to the surface of the upper wedge such that a concentration gradient is formed. A known volume of the microbial test suspension is then spread on the surface of the upper wedge and the plate is incubated at appropriate conditions of incubation for that particular test organism. The area of the surface of the plate with sub-inhibitory concentrations of the antimicrobial agent will show growth while part of the inhibitory concentration will show no growth (7).

If a sensitive microorganism is exposed to a concentration gradient of an antimicrobial agent on an agar medium, the zone of microbial growth formed along the concentration gradient will terminate at a point corresponding to the MIC.

XYZ                           ….Equ. 2

Y

 

Where Z= Concentration of the antimicrobial agent in the lower wedge

X= Length of zone of growth

Y= Length of possible zone of growth

Another version of the concentration gradient technique using a performed concentration gradient of an antimicrobial agent on a strip of paper is called the E-test.

The strip is placed on the medium and the antimicrobial agent in it diffuses into the medium to inhibit growth of the microorganisms. The point along the strip where growth terminates is known as the MIC point and can be read directly from the already calibrated concentration strip (7).

1.9.4    Biocidal Activity

This is the measure of the ability of an agent (antimicrobial) to kill all the microbial cells in an enclosed environment (79). The effectiveness of biocidal action can be measured by:

• Cell – killing rate
• D – Value
• Extinction time
• Minimum bactericidal concentration (MBC)

1.9.4.1 Cell- killing rate

This is a measure of the rate at which a known concentration and known volume of an antimicrobial agent kills the microbes in an enclosed environment/area.

Microorganisms do not die in singles but as batches, similar to their growth that occurs in batches and not singles. The number of microorganisms that die or reproduce will always be dependent on the entire number of microorganisms in that system, and this occurs in an exponential pattern (7, 79).

In N_t = In N_o –            kt.       ….Equ. 3

N_o = Population of microbial cells at zero time.

N_t  = Population of microbial cells surviving at time t.

K = Death rate constant.

When a graph of In N_t/N_o is plotted against time (t), a linear graph is obtained. The slope of the graph is the death rate constant (k).

The k_s of different antimicrobial agents can be obtained under same experimental conditions and compared. The higher the killing rate constant, the better is biocidal activity (79).

1.9.4.2 D-value

This is the time it takes to reduce the population of microorganisms in a system by one logarithmic cycle or the time it takes to reduce the population of the microorganisms by 90% of their original population. D–values of different antimicrobial agents can be determined under same experimental conditions and compared. The smaller the D-value, the better the biocidal action.

1.9.4.3 Extinction time

This is the time it would take a known volume and concentration of an antimicrobial agent to kill all the microorganisms in an enclosed environment. The lower the extinction time, the better the microbiocidal effect of an antimicrobial agent. Under standardized experimental conditions, the concentration of the antimicrobial agent C will have an exponential relationship with the extinction time (79).

Cn t = k                                   ….Equ. 4

Taking logarithm of both sides

n log C + log t = log k             ….Equ. 5

Log t = log k- n log C             ….Equ. 6

A plot of log t against log C gives a straight line graph whose slope is n, known as dilution coefficient. Changing the concentration of n by dilution, changes extinction time.  Dilution produces increase in extinction time for antimicrobial agents with high n-value while for antimicrobial agents with small n-values, dilution produces little or no significant changes in their extinction time.

1.9.5    Minimum biocidal (bactericidal) concentration (MBC)

Minimum bactericidal concentration (MBC) is an extension of the MIC; it is the minimum concentration of antimicrobial agent that can kill all the microbial cells in an enclosed environment, while MIC inhibits growth of microbial cells (8).

The MBC of an agent can thus be determined from the broth dilution or agar dilution method of determining MIC. The MIC agar plates or broth tubes that showed no growth are used in determination of MBC. A loopful of the reaction mixture is taken from the broth tubes or a disc of the agar is taken from the agar plates and transferred into fresh nutrient broth without antimicrobial agent and incubated at appropriate experimental conditions for 48 hours. After this period, the tubes are observed for microbial growth or no growth. Turbidity of the broth is seen as growth while a clear broth signifies no growth of microbial cells. The minimum concentration (from the tubes or plates) that produces complete cell death is the minimum biocidal concentration (MBC).

Cultures from MIC tubes or plates that are used for MBC carry along with them some of the antimicrobial agent, and effect of the agent needs to be stopped so that the microbial cells are freed from further action of the antimicrobial agent (79).

Antimicrobial culture in the recovery medium inactivates the antimicrobial agent while for others, specific inactivating agents are required. For example if the antimicrobial agent used is an antibiotic of penicillin origin, then the enzyme penicillinase is used as its in-activator (7, 8, 79).

1.10     The use of antibiotics in managing microbial infections

The term “antimicrobial” is a general term used to refer to all substances that can systematically inhibit (microstatic) or kill (microcidal) microbial cells regardless of their origin (7), while “antimicrobial agent” refers to any chemical substance produced by plants and all other microorganisms either in-vivo (in the body of the host) or in-vitro (outside the host). These antimicrobial substances have least toxicity to the host cells (selective toxicity). They could be in various forms such as antibacterial, antiviral, antifungal, antiprotozoa and antihelmintic. The antibacterial and antifungal activities vary with the species of the plants. Although, hundreds of plant species have been tested for antimicrobial properties, the vast majority has not yet been adequately evaluated (82-84). In recent years, antimicrobial properties of medicinal plants are being increasingly reported from different parts of the world (49, 84).

An ideal antimicrobial drug exhibits selective toxicity (82). This implies that the drug is harmful to the parasite without being harmful to the hosts. The mechanisms of antimicrobial activity include the following;

1. Inhibition of synthesis of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) which are both nucleic acids. Example is trimethoprim/sulphanamides.
2. Inhibition of cell wall synthesis. Example is penicillin/cephalosporins

• Inhibition of protein synthesis. Example is aminoglycosides/chloramphenicol.

1. Alteration in the permeability of cell membrane component leading to leakage of intracellular component of the cell. Example is tetracycline.
2. Inhibition of ergosterol synthesis in the fungal cell membrane. Example is imidazoles.
3. Interaction with ergosterol, a fungal cell membrane that leads to pores formation through which essential fungal cell constituents are lost. Example is amphotericin.

• Interference with microtubule function. Example is griseofulvin.

However, it is possible that antimicrobial agents can act through more than one of the                                        mechanisms mentioned above.

1.10.1  Plant as source of antimicrobials

Plants use as medication is as old as man’s origin. Man through careful observation and use has identified various medicinal plants that can treat and prevent various ailments. Chaulmoogra oil from species of Hydrocarpus gaertrn was one of the earliest records of use of plants for medicinal purpose. It was used for treatment of leprosy and its record was found in the Pharmacopoeia of the Emperor Shen Nung of China between 2700 and 3000 B.C. By this period, man was aware of the medicinal uses and properties of most plants in his environment along with their toxic effects. Hippocrates, a Greek medical doctor was referred to as the father of medicine. He was the first to regard medicine as a science and his Materia medica consisted essentially of herbal recipes (84).

1.11     Antimicrobial resistance

Sometimes microorganisms do not respond to hitherto sensitive antimicrobial agents. This is called resistance. The spread of drug resistance pathogens is one of the most serious threats to the successful treatment of microbial disease (85). Reports have it that bacteria have the ability to evolve defense mechanisms against antibiotics and can become resistant to their effects (86). The more an antibiotic is used, the more likely that bacterium will learn how to evade the affects of antibacterial agents on it (87). Most of the antimicrobial resistance, which is now making it difficult to treat infections, is due to extensive use and misuse of antimicrobial drugs, which have favored the emergence and survival of resistant strains of micro-organisms. Drug resistant strains are common, among which are Gonococci, Pneumococci, Pseudomonas, Meningococci, Staphylococci, Enterococcci, Shigella, Mycobacterium tuberculosis, and Salmonella (8). Remington reported that bacteria resistance to antibiotics has been recognized since the first drugs were introduced in 1935 and approximately 10 years later, 20% of clinical isolates of Neisseria gonorrhea had become resistant to the action of penicillin (86). Penicillin was first introduced in 1941, where less than 1% Staphylococcus aureus isolates was resistant to it. Bacteria become resistant to antimicrobials in several different ways. The type of resistance mechanisms is not confined to a single class of drugs. Different types of bacteria may use different mechanisms to withstand the same chemotherapeutic agent (85). These include;

1. Change in permeability to the drug; Modification of the structured target so that it no longer interacts with the antimicrobial.
2. Development of altered enzymes that still perform their metabolic functions but are much less affected by drugs.

• Development of altered metabolic pathways that by passes the reaction inhibited by the drug.

1. Production of enzymes that destroy the active antimicrobial.

 

Cheesbrough (8) reported that for bacteria to acquire this new property, they must undergo genetic change. Such a change may occur by mutation or by the acquisition of new genetic properties. The transfer of resistance genes (located on plasmids and transposons) from one bacterium to another, requires new genetic material. Some plasmids encode for resistance to several antibiotics and can be transferred between bacteria species, for instance, Escherichia coli to Shigella dysenterae.

 

1.12     Drug delivery systems

Substances with medicinal properties are usually formulated into forms called drug delivery systems before they are administered. The dosage forms can be liquid, solid or semi-solids and their intended routes of administration vary from simple to very complex preparation.

The purpose of drug delivery is to formulate the active drug principle such that it will target exactly areas of need in the body so that the drugs are more efficient and their excipients can solubilise, emulsify, thicken, preserve, impact colour and flavour and also preserve and stabilize the drug products. It is desired that with drug delivery systems, accurate drug dosages can always be reproduced with same therapeutic effects too (88-91,168).

1.12.1. Topical drug delivery systems

Topical drug delivery is the application of a drug-containing formulation on the skin so as to directly place the active principles in the formulation onto the surface of the skin or within the skin. Topical preparations are used for their localized effect at the site of their application. This is by virtue of drug penetration into the underlying layers of skin or mucous membranes. The main advantage of topical delivery system is to bypass first pass metabolism; there is also avoidance of inconveniences and risk of systemic route of drug administration and also the avoidance of pH changes, presence of enzymes and gastric emptying time associated with oral preparations. Most topical formulations are dominated by semi solids but foams, sprays, medicated powers, solutions and medicated adhesives are also used (88, 89)

1.12.1.1           Advantages of topical drug delivery systems

• Avoidance of first pass metabolism
• Convenient and easy to apply
• Avoidance of risks and inconveniences of systemic therapy
• Achievement of efficacy with lower total daily dosage of drug by continuous drug input.
• Avoids fluctuation in drug levels
• Ability to easily terminate the medication, when needed.
• A relatively large area of application in comparison with buccal or nasal cavity.
• Ability to deliver drug more selectively to a specific site
• Avoidance of gastro-intestinal incompatibility
• Improved patient compliance
• Suitable for self-medication.

1.12.1.2           Disadvantages of topical delivery system

• Skin irritation may occur due to the drug and/ or excipients
• Poor permeability of some drugs through the skin
• Possibility of allergic reactions.
• Can be used for only drugs which require very small plasma concentration for action
• Enzymes in epidermis may denature the drugs
• Drugs of large particle sizes are not easy to absorb through the skin.

1.12.1.3           Classification of topical drug delivery systems

Classification of topical drug delivery systems based on physical state: Solids, powders, aerosols, plasters, liquids, lotions, liniments, solutions, emulsions, suspensions, semi-solids, ointments, creams, pastes, jelly, suppositories.

1.12.2  Permeation of topical drugs through the skin

Most topical formulations are meant to be applied on the skin, so a basic knowledge of the skin and its physiology, function and biochemistry is necessary for designing topical preparations. The skin is the largest organ of the body and continues with the mucosal lining of the respiratory, digestive and urogenital tracts to form a capsule, which separates the internal body structure from the external environment. The pH of the skin varies forms between 4.0 to 5.6.  Sweat and fatty acids secreted from sebum, influence the pH of the skin surface. It is thought that acidity of the skin helps in limiting or preventing the growth of pathogens and other organisms (88, 89)

1.13     Routes of drugs adsorption through the skin

There are two routes of absorption through the skin – Transepidermal   and Transfollicular absorption (88, 89).

1.13.1  Transepidermal

This is the principal pathway responsible for diffusion across the skin. Permeation by this route involves partitioning the drug into the Stratum corneum. Diffusion takes places across the Stratum corneum through the intercellular lipoidal route. This route is a tortuous pathway of limited volume. There is another microscopic path though which polar compounds and ions pass. Because their oil-in-water distributing tendencies will not allow them to permeate at rates that are measurable since the epidermis has no direct blood supply, the drug in it is forced to diffuse across it to reach the vasculature immediately beneath (dermis). Permeation through the dermis is through the interlocking channels of the ground substance. Diffusion through the dermis is without molecular selectivity, since gaps between the collagen fibers are far too wide to filter large molecules (88, 89).

1.13.2  Transfollicular (shunt pathway) absorption

The follicular route is an important route for absorption of drugs via the follicular pore. Sebum aids in diffusion of penetrants into sebum, followed by diffusion through the sebum to the depths of the epidermis. Blood vessel serving the hair follicle located in the dermis is the likely point of systemic entry (88, 89).

The driving force of drugs across a membrane is a concentration gradient. The membrane it diffuses through is a diffusional resistor and this resistance (R) is proportional to the thickness of the membrane (h). R is inversely proportional to the diffusive ability/mobility of the drug molecules within the membrane and it is referred to as its diffusion coefficient (D). It is inversely proportional to the fractional area of a route where there is more than one route (F) and inversely proportional to the carrying capacity of a phase (k).

R = h/fDk        ….Equ. 7

 

 

 

 

 

 

 

 

 

 

 

 

Dissolution of drug in vehicle

Diffusion of drug through vehicle to skin surface

__________________

Transepidermal route       Transfollicular route

.

Partitioning into stratum corneum      Partitioning into sebum

 

 

Diffusion through protein-lipid          Diffusion through lipids

Matrix of stratum corneum                 in sebaceous pore

 

 

 

Partitioning through epidermis

Diffusion through dermis

Capillary uptake and systemic dilution

Scheme 1.1:    Kinetics of permeation

 

Knowledge of skin permeation is vital to the successful development of topical formulation. Permeation of a drug involves the following steps

• Absorption by stratum corneum
• Penetration of drug through viable epidermis
• Uptake of the drug by the capillary network in the dermal papillary layer.

This permeation can be possible only if the drug possess certain physicochemical properties. The rate of permeation across the skin (dQ/dt) is given by:

dQ/dt = P_s (Cd-Cr)     ….Equ. 8

Where Cd and Cr are the concentrations of skin penetrant on the surface of the stratum corneum. (donor compartment) and in the body (receptor compartment).

Ps is the overall permeability coefficient of the skin tissues to the penetrant.

Permeability coefficient (Ps) is given by

Ks Dss             ….Equ. 9

Hs

Where Ks is the partition coefficient for the interfacial partition of the penetrant molecule from a solution medium on to the Stratum corneum, Dss is the apparent diffusivity for the steady state diffusion of the drug (penetrant) molecule through a thickness of skin tissues and  Hs is the overall thickness of skin tissues (88, 89).

As Ks, Dss and Hs are constants under given conditions, the permeability coefficient (Ps) for a skin penetrant can be considered to be constant.

The rate of drug permeation can be constant when Cd > Cr, that is, the drug concentration at the surface of the Stratum corneum (Cd) is consistently and substantially greater than the drug concentration in the body (Cr), and the rate of skin permeation dG/Dt is also constant provided the magnitude of Cd remains fairly constant throughout the course of skin permeation. For keeping Cd constant, the drug should be released from the device (drug formulation) at a rate (Rr) that is either constant or greater than the rate of skin uptake (Ra) that is Rr > Ra (88, 89).

1.14     Factors affecting topical permeation

Percutaneous absorption can be improved by chemical or physical enhancer methods. Chemical penetration enhancers are chemicals that increase the skin permeability by altering the nature of the Stratum corneum to reduce its diffusional resistance. They increase the hydration of the Stratum corneum and change the lipids and lipoproteins structures of intercellular channels by denaturation (88, 89). Examples of such chemicals are:

Solvents: Solvents are thought to increase penetration by swelling the polar pathway and, or by fluidizing lipids. Examples include water and alcohols.

Surfactants: These compounds are proposed to enhance transport of drugs across the skin by improved penetration, thought to be as a function of their polar head and hydrocarbon chain length. Examples of surfactants are: anionic surfactant, cationic surfactants and nonionic surfactants (88, 89).

Physicochemical properties of the drug substances: partition coefficient, pH, drug solubility, drug concentration, particle size, polymorphism, molecular weight

 

1.15     Fractionation of leaf extracts

Most of the active principles found in plants are secondary metabolites, which are products of plant metabolism that are secreted or stored in parts of the plant (leaves, backs, lactex, etc). These products are not necessarily required by the plants but are useful as protective mechanisms to them. Some secondary metabolites are toxins. Example, phytoalexins protect against bacterial and fungal attacks (88, 89). Fractionation of plant extracts is believed to optimize the potencies of their secondary metabolites by extending the spectrum of antimicrobial activities of some (88, 89), though the spectrum of activity may be reduced in other plants depending on the active principle isolated by the fractionation process. This has rekindled the interest in drugs of plant origin, especially since drugs of natural origin are easily metabolized, have fewer side effects and less toxicity levels (88, 89).

Accelerated gradient chromatography (AGC) is a medium pressure liquid chromatographic method developed by Peter Baeckstron of the Organic Chemistry Department, Royal Institute of Technology, Stockholm, Sweden. The AGC minimizes the time spent on running preparative columns by the use of continuous accelerating gradients. The gradients were obtained by continuous use of the solvents of different polarities (hexane, ethyl acetate, methanol) to effect separation of the extracts constituents (88, 89).

1.16     Ointments
Ointments are semi-solid preparations that are applied on the skin or mucosa. They are used for medication or emollient effect on the skin and for the protection of skin lesions (92).

 

 

 

1.16.1  Uses of ointments

Medicated ointments can be grouped according to their uses, examples are

Antibiotics ointment (neomycin), antifungal (benzoic acid), acne ointment (sulphur), anti-inflammatory ointment (hydrocortisone), anti-pruritus (benzocaine), antiseptics (zinc oxide), astringents (calamine), eczema ointment (salicylic acid).

1.16.2  Classification of Ointment Bases

There are four main classes of ointment.

Hydrocarbon bases/Oleaginous bases: These are anhydrous, hydrophobic, are insoluble in water and not removable by water. They are earliest ointment bases, which consist of vegetable and animal fat, petroleum hydrocarbon like soft, hard and liquid paraffin. They are almost inert as they consist of saturated hydrocarbon with very few incompatibility and little tendency to rancidity. Instances of skin sensitization are rare and they do not promote the growth of microorganisms in them (92). They are readily available and cheap.

Absorption bases: The term absorption refers to the water absorbing properties of these bases. They are anhydrous but are hydrophilic, so they can absorb several times their own weight of water, to form water-in-oil (w/o) type of ointment. This class of bases can be formulated into ointment with an equal solution of medicated substance added (92).

They fall into two classes; non-emulsified bases and water in oil emulsions

The non – emulsified bases can absorb water and aqueous solution. An example is wool fat, which can absorb upto 50% of its weight in water.

The water in oil emulsion can absorb more water than non-emulsified bases. An example is the hydrous wool fat.

Water miscible bases: Ointments made from water-miscible bases are easily removed from the skin after use unlike the absorption base which though are hydrophilic in nature are rather difficult to wash from the skin (92).

There are three official anhydrous water miscible bases; emulsifying ointment B.P., centrimide emulsifying ointment B.P. and cetomacrogol emulsifying ointment B.P. which are anionic, cationic and non-ionic, respectively. Water-miscible bases also have good miscibility with exudates from lesion, reduce interference with skin functions, have high cosmetic stability, hence there is good patient compliance, are easily removed from hair, unlike hydrocarbon or absorption ointment that are not ideal for scalp condition due to difficulty on their removal.

Water soluble bases: These bases are prepared from mixture of low and high molecular weight polyethylene glycols (Macrogols) which range in their consistency from viscous liquids to waxy solids. The liquids are clear and colorless, with a characteristic odour. The solids are white or creamy hard lump or flakes that are soluble in water and alcohol in the ratio of 1:3 and 1:2, respectively. The macrogols are non-volatile. They are greasy, water soluble and as such can easily be removed from the skin; they are very well absorbed by the skin which can have deleterious effect by exaggerated toxic and side effects. They would not hydrolyze, deteriorate nor support microbial growth (92).

1.16.3  Ideal properties of an ointment base

Ointment bases are vehicles into which a drug is incorporated. An ideal ointment base is expected to have the following attributes (92-93):

• Non-gritty to touch
• Non-greasy to prevent staining of clothes
• Non-irritant to user (on the mucous membranes)
• Has the ability to retain the physicochemical properties of a drug when formulated
• Stable on storage
• Should have the ability to absorb exudates from sites of application if present
• Should have the ability to release the drugs contained in it to desired sites in quantities sufficient enough to elicit therapeutic effects

1.16.4  Preparation of ointment

Ointment can be prepared either by litigation or incorporation or by fusion methods (92, 93).

Preparation of ointment by mechanical incorporation can be achieved by the use of

• Mortar and pestle
• Ointment slab and spatula
• Ointment mill

Preparation of ointment by slab and spatula

The insoluble medicaments are mechanically mixed or triturated with a spatula on a slab. The powder is first mixed with a small quantity of the base to form a concentrated ointment base containing finely divided powder uniformly distributed in it. The concentrated medicament- ointment is gradually diluted with the remaining quantity of the base by titrating with a spatula (81, 92, 93).

Fusion: The ingredients are melted together and stirred to ensure homogeneity (92).

1.16.5  Effective drug release of antimicrobial agents from ointment bases

Antimicrobial ointments must, as a necessity, release their active constituents in order to exert the desired antimicrobial action. The release of these active principles can be measured by agar diffusion technique. Two variations of the test can be used. The agar cup diffusion and the surface plate test (94, 103).

1.16.6  Factors affecting the release and absorption of medicament from ointment bases

The following factors affect the rate and degree of antimicrobial action of medicaments from bases.

1.16.6.1 Factors connected to the antimicrobial agents’ concentration

The rate of antimicrobial action varies directly with concentration of the active constituents. There is a level/concentration of the active principle below which no significant antimicrobial activity is noticed. There is a maximum concentration above in which no significant increase in antimicrobial activity is noticed. Because the receptor site has been saturated with the active constituents so long as the concentration of the ointment falls between these two extremes, a situation exist where the rate of microbial death/inhibition increase with the concentration of the ointment (79).

1.16.6.1.1        Solubility in water

Some active principles depend on electrolytic dissociation for action and this can occur only in the presence of water.

1.16.6.1.2        Ionization constant

Antimicrobial agent can be active in the ionized states i.e. as cations or anions and also in the unionized form. The activity of the agent thus depends on the degree of ionization that is influenced by the pH of the ointment.

 

 

1.16.6.1.3        Lipid/water distribution characteristics

Antimicrobial agents that are formulated as multiphase products distribute themselves between the aqueous and oil phases depending on their partition coefficient (79).

1.16.6.1.4        Inherent Antimicrobial Action

Antimicrobial agents vary considerably in their ability to tackle microorganisms. Broad spectrum antibiotics exert powerful antimicrobial effects on all or most classes of bacteria (Gram positive and Gram negative), while the narrow spectrum antibiotics are effective on a class of bacteria (79).

1.16.6.2           Factors connected to the organisms

Different microorganisms respond differently to the same antimicrobial agent. Some microorganisms may be very sensitive to the action of a particular antimicrobial while others are less positive or even resistant to it. Gram positive bacteria are more sensitive to antimicrobials than Gram-negative ones; also effects of antimicrobials are more drastic on vegetative organisms than on spores (79).

1.16.6.2.1 Microbial density

The more the number of organisms a product is exposed to, the less is the proportion of the active constituent that is made available to the individual microbial cell, and if it falls below the MIC value, it would have no significant bacteriostatic effect on the cell. Also large population of organisms would be heterogeneous. Some of the individual cells may be sensitive to the antimicrobial agent while others will be resistant to it. It then means that the greater the microbial population the higher will be the number of resistant cells (79).

 

1.16.6.2.2 Presence of protective structures

Vegetative cells are more susceptible to antimicrobial agents than bacterial spores. This is because the bacterial spores posses protective structures around them that make them very resistant to antimicrobial agents (79).

1.16.6.2.3 Physiological state of the organisms

The growth stage of organisms affects its responsiveness to an antimicrobial agent. If the antimicrobial agent like the penicillin acts on cells undergoing cell division, then organisms in the stage would be very sensitive to them. If the antimicrobial agent acts by interference with metabolism, then cells that are actively metabolizing would be more rapidly destroyed than dormant cells (spores) (7, 79).

1.16.6.3 Factors connected to the environment

1.16.6.3.1 Temperature

The bactericidal activity of most disinfectants increase with increased temperature. As the temperature is increased in arithmetic progression, the rate of inhibition/killing of the microbial cells increases in geometric progression. The effect of temperature on the rate of antimicrobial activity of an agent, under specified condition, is expressed in a term called temperature coefficient (Q). Q is the change in the rate of an antimicrobial action, say, an ointment per degree rise in temperature of that ointment , a 10^o rise in temperature coefficient is usually employed (Q¹⁰) (79).

Thus, Q¹⁰ values can be calculated by determining the extinction time at two temperatures differing exactly by Q¹⁰

Q¹⁰=   ….Equ. 10

1.16.6.3.2 pH

           This affects the antimicrobial activity of an antimicrobial agent and it influences the type and rate of microbial growth in it. Most bacteria grow best at pH of 6.0 to 8.0 while lower pH values favour growth of yeast and fungal cells. Sometimes the proliferative growth of one type of organisms at its optimal pH can change the pH of its immediate environment to a pH that flavours the growth of secondary organisms. Yeast would thrive well in a medium containing organic reagents which metabolizes these acids in raising the pH of the medium and the now higher pH favours the growth of bacterial cells (79).

pH affects the potency of antimicrobial agents. If the agent is an acid or base its degree of ionization will be dependent on pH of the medium/ointment. Antimicrobials like phenol are effective in their non-ionized states. When present in an alkaline environment that favors the formation of ions, it will have decreased antimicrobial activity. Change in pH of the medium may alter the microbial cell surface electric charge and such a change affects the amount of antimicrobial agent absorbed by the organism. Increasing the external pH renders the microbial cell surface more negatively charged and this enhances the attraction of cationic compounds like chlorhexidine to bind to them, thus eliciting more antimicrobial activity (79).

1.16.6.3.3  Organic matter

Antimicrobial agents are used in practice at sites where organic matter in form of blood, pus, faeces, urine and organic wastes will be present. These contaminants reduce the antimicrobial activity of the agents by adsorbing some of their active principle thus reducing the concentration of the agent made available to the microorganisms themselves or the contaminant may be adsorbed on microorganisms to prevent or reduce the diffusion of the antimicrobial agent into the microbial cells or the contaminant may react with active principle to neutralize its antimicrobial activity (79).

1.16.6.3.4        Surface activity

      The antimicrobial activity of antimicrobials depends also on their surface activity. The higher the surface activity, the more powerful is the microbial cell membranes to the antimicrobial action of the agent. As the concentration of the surfactant increases, the antimicrobial activity  is enhanced  due to increased uptake of the agent by the microbial cell until a concentration at which increasing  surfactant concentration  brings about no increased activity but rather a decrease. This concentration is called the critical micelle concentration.  (79).

1.17     Response of microorganisms to antimicrobial agents

A microorganism be a single cell (unicellular) or a multicellular organism. Microorganisms are very diverse, and include the prokaryotes comprising of the protozoa, fungi and algae. Most microorganisms are microscopic that cannot be seen with un-aided eyes while  some, like the Thiomagarita namibiensis are macroscopic and are visible to the naked eyes (79).

Microorganisms are ubiquitous i.e. they are found everywhere, soil, air, water, on plants and animals. Useful microbes are exploited for production of food and drugs. However there are non-useful microorganisms called pathogens that are harmful to plant and animals as they cause diseases and even death.

An agent can only be used clinically as an antimicrobial agent if it at least possesses the ability to inhibit the growth of microbial cells; though it is preferable that its antimicrobial effect is micro-biocidal i.e. it is capable of killing the entire microbial cell in a system.

The response of microorganisms to antimicrobial is determined when it is brought in contact with the test antimicrobial agent. This must be done under specified experimental conditions. If the microorganisms are inhibited/killed by the agent, they said to be sensitive to that agent and if the organisms are not killed or their growth inhibited, they are termed resistance to the agent. The methods below are employed to determine the response of microorganisms to pharmaceutical products (79).

1.17.1  Agar diffusion method

In this method, agar plate is seeded with organisms that are challenged with known concentration(s) of the antimicrobial agents which have been impregnated into paper disc or filled into holes punched out of the agar. The response of the microorganisms to the test agent is related to the sizes of the zones of inhibition surrounding the paper disc or holes. The more sensitive an organism is to the agent, the further away it grows to the hole or disc while resistant organism grow almost into the hole or disc, with no appreciable zones of inhibition. Different organisms respond differently to different antimicrobial agents and this response varies when the concentration of the agent varies (79).

 

 

 

 

 

 

 

 

 

1.18     Review of study plant: Spermacoce verticillata

1.18.1  Spermacoce verticillata

Fig. 1.2:           The plant Spermacoce verticillata Linn Rubiaceae

This plant is commonly called white head broom; it is also called African borreria, false button weed. It is synonymous to Borreria verticillata Linn (51). It is called Obi-na-ezi by the Igbos, Wawa kage magori/Alkamar tururuwa by the Hausas and the Yorubas call it Irawo ile (95).

Spermacoce verticillata is a woody, bushy, fine stemmed scrambling shrub. It is 1-1.2 m in height, and it has herbaceous or semi woody square stems in the first year which becomes rounded in the following year (96). The brown stem reaches a maximum diameter of about 8  mm; they have solid pith and lack visible annual rings. Spermacoce verticillata produces weak tap roots and a moderate amount of fine roots. The leaves are opposite but appearing with two or more clusters of smaller leave whorls at the nodes. The leaves are sessile or nearly so, linear or linear-lanceolate, 2-6 cm long and pointed at both ends. The tiny white flower grows through the centre of the inflorescence so that the fruits develop at nodes in mid-stem. The capsules are oblong with two carpels, each with one seed. The seeds are ellipsoidal, brown and about 1 mm long. The embryo is either straight or curved. (83, 90, 96 and 97)

1.18.2  Classification of the plant

Plant                            Spermacoce  verticillata

Synonyms                   Borreria podocephala   D.C.

Borreria verticillata Linn

Borreria stricta D.C.

Spermacoce globosa

Kingdom:                   Tracheosbinota (Vascular plants)

Super division:           Spermatophytes (Seed Plants)

Family:                       Rubiaceae

Genus:                        Spermacoce

Species:                       verticillata

Taxonomy:                  Spermacoce verticillata 

Herbarium Specimen – It is deposited in the Forest Research Institute of Nigeria (FRIN) Ibadan with the number 107445.

1.18.3  Common names

Igbo name :                Obi na ezi

Hausa name :             Wawa kaje  magori

Alkamar tururuwa

Karya garma

Yoruba name:           Irawo ile

1.18.4  Geographical distribution

Spermacoce verticillata is a very common tropical plant most common in humid areas and blooms during the wet season. It occurs in agricultural areas, grass lands and urban areas, and is found in Africa but its main origin is uncertain. It grows as a native or naturalized species from Florida through the West Indies and Texas through Central and South America to Argentina and through the moist portions of tropical Africa and Madagascar including Nigeria. It is found in both Northern and Southern Zones of Nigeria e.g.  Jos, Bauchi, Calabar, Nsukka, Lagos, Ile Ife (97, 98)

1.18.5  Bioactive constituents

Spermacoce verticillata   contains certain bioactive Constituents that confer on it its medicinal/therapeutic properties, such as emetine, flavones, irioids, caryophylene, tannins and sesquiterpenes (99).

It also contains an alkaloid called borreverrine which has in vitro antimicrobial actions (100).

The African Borreria contains indole alkaloids like emetine, borrerine, borreverrine and volatile oil (sesquiterpenes and phenolic compounds) (51). The volatile oil, borreverrine has antibacterial activity (101, 102). Iridoids compounds are also isolated from Spermacoce verticillata, and include daphlylloside, asperulocide, feretoside (103, 104). Borreriagenim is another iridoids compound found in Spermacoce verticillata (105).

1.18.6  Medicinal uses of Spermacoce verticillata 

Spermacoce verticillata has some medicinal uses, mostly on skin conditions (51, 83). In Senegal, the roots are used as an anti leprosy agent, anti-paralytic, diuretic, anti bilharzias, and as an abortive agent (106).

The leaf extracts are used to treat leprous conditions, furuncles, ulcers and gonorrheal sores (51). A lotion was prepared with the plant for relief of skin pruritus (97). It is used to treat diarrhoea, also used as a diuretic in the treatment of schistosomiasis (51). It is used to lower blood pressure and as an abortificient. Spermacoce verticillata is used in the treatment of asthma, bronchitis, haemorrhage, diabetes mellitus, cough, dysentery and erysipelas (99)

The volatile oil of Spermacoce verticillata inhibits the growth of Gram positive and Gram negative bacteria (101). This oil inhibits the growth of Staphylococcus aureus and Escherichia coli (51).

Some species of these genera play an important role in traditional medicine in Africa, Asia, Europe, and South America, where they are used in the treatment of malaria, diarrheal and other digestive problems, skin diseases, fever, haemorrhage, urinary and respiratory tract infections, headache, and inflammation of the eye and gums. To date, more than 60 components have been reported from Borreria and Spermacoce species. Studies have confirmed that extracts from Borreria and Spermacoce as well as their isolated compounds possess diverse biological activities, including anti-inflammatory, antitumor, antimicrobial, larvicidal, antioxidant, gastrointestinal, antiulcer, and hepato-protective, with alkaloids and iridoids as the major active principles.

The Rubiaceae family comprises one of the largest angiosperm families, with 650 genera and approximately 13,000 species, distributed not only in tropical and subtropical regions, but also reaching the temperate and cold regions of Europe and Canada (107, 108). In Brazil, this family comprises about 130 genera and 1500 species distributed across different vegetation formations, with great occurrence in the Atlantic forest (108, 109). This family is currently classified into three subfamilies and over 43 tribes (107). The genera Borreria G. F. W. Meyand Spermacoce L., are characterized by a herbaceous habitat, with over 1000 species having mainly pantropic distribution, but a few genera extend into temperate regions, excluding New Zealand (110,111).

Based on their true morphology, they are considered by many authors to be distinct genera, and most others however prefer to combine the two taxa under the generic name Spermacoce (108, 112).

1.18.7  Ethnomedical properties

Borreria and Spermacoce are used medicinally in various manners and are reputed in traditional medicine of Latin America, Asia, Africa and West Indies. The species most used as medicinal are described below:

1. alata (Aubl.) DC. [Syn.: S. alata Aubl.,S. latifolia Aubl., B. latifolia Aubl., K. Schum] is a herbaceous species native to South America (113, 114).
2. articularis commonly known in Brazil as “poaia” is originally native to the temperate and tropical Asia regions and naturalized in Africa and Australia (115). The leaves are used in inflammation of eyes and gums, blindness, cataract, fever, spleen complaints, sore, conjunctivitis, haemorrhage, gallstones, dysentery, and diarrhea (115, 116), and the decoction of the leaves, roots, and seed is used in India for dropsy (117).
3. centranthoides known in Brazil as “sabugueirinho do campo” is a perennial herb originating from fields in southern Brazil, and possibly Uruguay and Argentina. In Brazil, these plants have been used for the treatment of liver ailments (118, 119), kidney disorders (120), and in Argentina as an abortifacient (121).
4. hispida L. is being used as an alternative therapy for diabetes (122). In India, decoction of the plant is used for headache (123) and the seeds as stimulant (124) and for the treatment of internal injuries of nerves and kidneys (125).
5. princeae [(K. Schum.) Verdec], is a scrambling or decumbent perennial herb, native to Africa where it is used for the treatment of skin diseases (101).
6. pusilla Wall, is an annual erect herb native to tropical Africa and Asia. In India, the fresh buds associated with the flowers are used in cuts and wounds (126) and the crushed leaves are applied to the affected areas for bone fractures and scabies and for snake and scorpion bites (127).

S. verticillata L.), known in Brazil as “poaia”, ”poaia preta”, “poaia miuda”, “coroa-de-frade” and “vassourinha”, is a small perennial and erect herb, originating from South and Central Americas and distributed by the Old World, Southern United States to South America (128, 129). In Brazil, the infusion of the leaves is used as antipyretic and analgesic (130, 131), the roots as emetic, and the leaves as antidiarrheal and for treatment of erysipelas and haemorrhoids (132). In West Indies, the decoction of the plant is used for diabetes and dysmenorrhoea, and when prepared with Cuscuta and Zebrina schinizlein is used for amenorrhea (133); while in Senegal, it is used to treat bacterial skin infections and leprosy (100). In Nigeria, fresh aerial part juice is applied for eczema (101) and in Jamaica, the decoction of the endocarp, prepared jointly with Iresine P. Browne, and Desmodium, is used as diuretic and as a remedy for amenorrhea mixed with Cuscuta and Zebrina (134).

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