INTRODUCTION

Maintaining an intact skin is essential for the body’s health considering that it is

the body’s first line of defense. Wounds circumvent this barrier by providing an opening

for bacteria, viruses and other pathogens into the body. Infections are most likely to

happen with improper care. As long as wounds are present there is a certain level of

vulnerability. Also, wounds might cause a certain part of the body to be immobilized.

Shortening the amount of time to achieve healing is beneficial to reduce the dangers that

accompany wounds.

Medicinal plants are widely used by the traditional practitioners for curing various

diseases in their day to day practice. In traditional system of medicine, different parts are

used such as leaves, stems, roots, flowers, or even the whole plant. With that, medicinal

plants are used around the world to treat skin ailments, burns, and in many other herbal

remedies. Aloe Vera for example, is cherished for its wound and pain-relieving effects, its

constituents also have soothing properties. Thus, it has been widely commercialized to

the point where its extracts have been converted into a gel for treating wounds.

Ocimum tenuiflorum is known as the scientific name of the plant holy basil. It is

originally from India and is used in Ayurvedic medicine as an “adaptogen”, a nontoxic

plant extract that is known to increase the body's ability to resist damage so as to counter

life’s stresses. It is considered a sacred plant by the Hindus and is often planted around

Hindu shrines. The Hindu name for holy basil, Tulsi, means "the incomparable one."

Traditional medicine is made from the leaves, stems, and seeds. It is an indigenous plant

in India and Southeast Asia. Numerous ancient systems of medicine value this plant for
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its medicinal properties, including Ayurveda, Greek, Roman, Siddha and Unani1 (Gupta

et al., 2002).

In India it is considered sacred anywhere it is grown (Mondal et al., 2009). It is

the most sacred plant in the Hindu religion. Holy basil is an important part of religious

ceremonies. Like a number of other medicinal herbs from other parts of the world, it is

thought to provide protection for homes where it is cultivated. The smell of the plant is

effective in keeping away insects that typically spread disease, such as mosquitoes and

flies.

There are limited studies regarding the healing effects of O. tenuiflorum, none

have been done in the Philippines since it is not yet popular here in the country.

Antimicrobial properties have also been identified (Singh et al., 2005). This is why the

researchers took interest in the herb. There are many ways on how to extract O.

tenuiflorum (Shetty et al., 2006); this study will be concerned with steam distillation for

the essential oil and rotary evaporation for extract. The two processes will potentially

have varying effects on the yield and quality of phytochemicals (Asoiro et al., 2011).

What the researchers want to find out is which method will yield a product that will have

a greater effect on healing. Determining this will also be a step closer in making products

from O. tenuiflorum in the Philippine market.

This study will be done in collaboration with Herbanext™ Laboratories. They

will supply the O. tenuiflorum needed for the extracts. The two extraction methods to be

compared in the study are in conjunction with the equipment currently present in the

facility.
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Statement of the Problem

This study aims to compare the wound healing effect of Ocimum tenuiflorum

(Tulsi) extracts with a BFAD-approved antibiotic, Trimycin, and to compare the

effectivity of the extracts prepared from two different laboratory methods, namely, steam

distillation and rotary evaporation.

Specific Objectives

Specifically, this study aims to answer the following questions:

1. Is there a significant difference in the mean percentage wound contraction in Mus

musculus with excision wound model during the 15-day observation period

between:

Group A (with application of Ocimum tenuiflorum steam distillation ointment),

Group B (with application of Ocimum tenuiflorum rotary evaporation ointment),

Group C (negative control, without application of topical medicine),

Group D (positive control, with application of Trimycin),

Group E (with application of blank ointment).

2. Is there a significant difference in epithelialization in Mus musculus with excision

wound model during the 15-day observation period between:

Group A, B, C, D, and E.

Statement of Hypotheses
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1. H0: There is no significant difference in the mean percentage wound

contraction in Mus musculus with excision wound model between the groups

during the 15-day observation period.

H1: There is a significant difference in the mean percentage wound contraction

in Mus musculus with excision wound model between the groups during the 15-

day observation period.

2. H0: There is no significant difference in epithelialization in Mus musculus with

excision wound model between the groups during the 15-day observation

period.

H1: There is a significant difference in epithelialization in Mus musculus with

excision wound model between the groups during the 15-day observation period.

Significance of the Study

The results of the study will encourage scientists to dwell on the healing

components of Ocimum tenuiflorum where it will not only be limited to open wounds but

also to other areas of the body where pain occurs.

Since the study primarily focuses on the wound healing potential of Ocimum

tenuiflorum, it can determine if it will be a substantial choice for wound healing besides

other medicinal products. This study is significant to the following:

Advocates of Alternative Medicine

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Alternative medicine is getting more and more popular. Plants most often, if not

always, have lesser side-effects when compared to their synthetic counterparts (Vijaya

and Ananthan, 1997; Dilhuydy and Patients, 2003). Ocimum tenuiflorum is currently not

that common here in the Philippines, but it is relatively easy to cultivate and propagate

due to similar climate conditions in India. Additional and proven knowledge regarding

this plant will help increase awareness and popularity among advocates and might lead to

a future where Ocimum tenuiflorum will be common and well accepted.

Herbal Medicine Companies

Companies are always looking into different kinds of plants to use in their

products. Having and making a product from an exotic plant will give them a competitive

edge. Proving certain properties of a plant will make them more interested in its uses.

Ocimum tenuiflorum products will become more possible. Determining the specific

process that will yield a better product decreases the cost of production for the same

effects for better economic potential.

Teachers, Students, and Future Researchers

This study will be a source of knowledge about said plant which can be used as

topics for research and discussion. For teachers, this study may be used as a tool to

introduce subjects that are related such as Botany, Health, and Chemistry. For students

and future researchers, this may serve as their guide for their projects that are similar to

the study particularly in the field of wound healing. Furthermore, this will become a

standard for future studies that are involved with wound healing potential of a plant.

Assumptions and Limitations

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1. Ocimum tenuiflorum has a wound healing potential that is observable and

measurable in Mus musculus excision wound model.

2. Ocimum tenuiflorum will yield different amounts of oil extract depending on the

extraction method.
3. The extract from steam distillation and the extract from rotary evaporation will

yield different concentrations of the active ingredient of the oil extract.

4. The specimen that will be used for the study is Mus musculus BALB/c albino

strain only.
5. Percentage wound contraction and epithelialization are the only parameters to be

measured.
6. Out of the many methods of extraction, steam distillation and rotary evaporation

are to be used. This is because they are the two main extraction methods present

and available in Herbanext Laboratories.

7. The treatments will be done every day starting from the infliction of excision

wounds until the 15th day.

8. Measurements of percentage wound contraction and epithelialization will be

made every two days starting from the day of infliction of excision wounds until

the 15th day.

Conceptual Framework

Figure 1. Schematic Diagram

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The method of extraction is an independent variable of the study. Steam

distillation and rotary evaporation operate at different temperatures and this will

potentially have an effect on the phytochemicals in the O. tenuiflorum extracts. This will

in turn affect the effectivity of the extracts in healing wounds. The kind of treatment is a

dependent variable. The prescription medicine (Trimycin) and the extracts will be applied

to the excision wound model in Mus musculus. The effect of the prescription medicine

and O. tenuiflorum extracts will be compared with each other. Two parameters will be

measured to determine the effect on the wound: Percent Wound Contraction and

Epithelialization. These two are also the dependent variables of the study.
 REVIEW OF RELATED LITERATURE

Connotation of a Wound

Wound is defined as disruption of cellular, anatomical, and functional continuity

of a living tissue. It may be produced by physical, chemical, thermal, microbial, or

immunological insult to the tissue. When skin is torn, cut, or punctured it is termed as an

open wound and when blunt force trauma causes a contusion, it is called closed wound,

whereas the burn wounds are caused by fire, heat, radiation, chemicals, electricity, or

sunlight (Pathak, 2011). Skin wounds could happen through several causes like physical

injuries resulting in opening and breaking of the skin (Gerard et al., 1999).The most

common symptoms of wounds are bleeding, loss of feeling or function below the wound

site, heat and redness around the wound, painful or throbbing sensation, swelling of tissue

in the area and pus like drainage (Rashed et al., 2003).

In normal skin, the epidermis (outermost layer) and dermis (inner or deeper layer)

exist in steady-state equilibrium and shield from the external environment. When the skin

is broken, the normal (physiologic) process of wound healing begins. Upon injury to the

skin, a set of complex biochemical events takes place in a closely orchestrated cascade to

repair the damage. They result in the loss of continuity of epithelium with or without the

loss of underlying connective tissue (Cevc, 2010).

When a wound occurs, the immune system sends out many healing factors

through the blood vessels to the wound to help it heal. Oxygen we breathe is also sent to

the wound through our blood vessels to help the wound heal. Hence, when the wound

healing process is complete the new skin and/or scar tissue functions as a new covering to

protect us from future injuries (Nguyen et al., 2009).

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Overview of Wound Healing as a Complex Process

Wound healing process is defined by the Wound Healing Society (WHS) as “a

complex and dynamic process that results in restoration of anatomic continuity and

function”. Wound healing, though often taken for granted, is a very dynamic and delicate

process. The wound healing process is a cascade of events, beginning with injury to

tissue (Black and Matassarin-Jacobs, 1997; Silver, 1997). In addition, wound healing is

the interaction of a complex cascade of cellular and biochemical actions leading to the

restoration of structural and functional integrity with regain of strength of injured tissues

(Suntar et al., 2011). It is a complex and dynamic process of replacing devitalized and

missing cellular structures and tissue layers. It involves continuous cell-cell interaction to

proceed in different overlapping phases.

The wound environment is part of a larger ecosystem. If each wound is treated as

part of a macro environment, this will result in sustainable wound repair. Comorbidities,

presence of one or more additional disorders, and other factors that can potentially affect

healing should always be considered. Furthermore, the process of wound healing is

explained as being similar to that of rebuilding a house after it has been damaged for

some reason (Kane, 2007).

Optimum healing of a cutaneous wound requires a well-orchestrated integration

of the complex biological and molecular events (Umadevi et al., 2006). Wound care and

maintenance involves a number of measures including dressing and administration of

painkillers, use of anti-inflammatory agents, topical systemic antimicrobial agents, and

healing promoting drugs (Jain et al., 2009).

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The aim of wound care is to promote wound healing in the shortest time possible

with minimal pain, discomfort, and scarring to the patient and must occur in a

physiological environment, conducive to tissue repair and regeneration (Wu et al., 2007).

The systematic review of in vitro and in vivo experiments could promote closer

collaboration between the research communities and encourage an interative approach to

improving the relevance of various models to clinical trial design.

Kane, in 2007, stated that for wound healing to take place, both the macro- and

microvascular structures must be intact, with adequate cardiac output and flow to perfuse

the wound environment. Adequate nutrition and a well-balanced and functioning immune

system are also important. Without these, white cell debridement, bio-burden control and

wound repair cannot take place, resulting in a non-healing wound.

Phases of Wound Healing

The wound-healing process consists of four highly integrated and overlapping

phases: hemostasis, inflammation, proliferation, and tissue remodeling or resolution

(Gosain and DiPietro, 2004). These phases and their biophysiological functions must

occur in the proper sequence, at a specific time, and continue for a specific duration at an

optimal intensity (Mathieu et al., 2006). The events of each phase must happen in a

precise and regulated manner. Interruptions, aberrancies, or prolongation in the process

can lead to delayed wound healing or a non-healing chronic wound (Gosain and DiPietro,

2004).There are many factors that can affect wound healing which interfere with one or

more phases in this process, thus causing improper or impaired tissue repair.
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The first phase of wound healing is hemostasis. It is initiated by the clotting

cascade. When tissue is first wounded, blood comes in contact with collagen, triggering

blood platelets to begin secreting inflammatory factors. Platelets also express

glycoproteins on their cell membranes that allow them to stick to one another and to

aggregate, forming a mass (fibrin clot) that traps proteins and particles and prevents

further blood loss. This is the main structural support for the wound until collagen is

deposited (Black and Matassarin-Jacobs, 1997; Silver, 1994).

Eventually, the next phase begins. The inflammatory phase begins at the time of

injury and lasts for 24 to 48 hours. This phase begins with hemostasis and leads to

inflammation. Platelets form the initial thrombus release growth factors that induce the

chemotaxis and proliferation of neutrophils and macrophages, which cooperate to remove

necrotic tissue, debris, and bacteria from the wound. Macrophages then become the

prominent cell of this phase and release various growth factors and cytokines that change

the relatively acellular wound into a highly cellular environment (Mustoe et al., 2007)

This is the body’s early defense system against microbial invasion. Neutrophils

are the first and most numerous white blood cells to arrive at the injured site. Their role,

along with macrophages, is to ingest injurious agents, thereby protecting against bacterial

invasion. Monocytes and macrophages are next on the scene (usually about 4 days).

Monocytes can phagocytose foreign material. The macrophages are critical cells in

wound healing because they secrete angiogenesis factor (AGF). AGF stimulates the

formation of new blood vessels. Wound healing is significantly impaired without

macrophages. Eventually, leukocytes and macrophages serve as phagocytes that

recognize foreign protein or damaged tissue, bind to it, engulf it and destroy it. Cell
 12

membranes are disrupted by the release of chemicals, resulting in edema. There are other

mediators of inflammation – the inflammatory response, mast cells, the kinin system, free

radicals and the complement system. Disorders that lead to reduced numbers of

phagocytic cells slow the inflammatory process and make the person more prone to

infection (Black, Matassarin-Jacobs, 1997)

Next, fibroblasts proliferate to become the dominant cell of the

proliferative phase. They produce collagen, which provides structure to the wound and

replaces the fibronectin–fibrin matrix. Angiogenesis of new capillaries occurs to sustain

the fibroblast proliferation. After which, keratinocytes also epithelialize the wound. This

phase contains overlapping of collagen deposits, angiogenesis, granulation, tissue

development and wound contraction. Collagen is secreted reconstructing connective

tissue. Vitamin C, zinc, oxygen and iron are required for this process in which granulation

occurs. Furthermore, collagen, capillaries and cells begin to fill the wound space with

new connective tissue. Granulation tissue is red and bumpy, with a meaty appearance.

The wound contracts as newly formed granulation tissue pulls wound margins inward;

this is caused by the action of myofibroblasts. Eventually, epithelialization occurs as

epithelial cells migrate from surrounding skin. This tissue is very fragile and the skin re-

growth occurs. Moreover, the cells eventually begin to differentiate into various layers of

the epidermis. Consequently, epithelialization can be hastened if a wound is kept moist,

however, the initial scar is bright red, thick and blanches with pressure (Black,

Matassarin-Jacobs, 1997; Silver, 1997)

The remodeling phase begins at about 2 to 3 weeks and can last up to 2 years. At

this time, collagen synthesis and degradation reach equilibrium. Fibroblasts organize and
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cross-link the collagen, wound strength gradually increases, wound contraction occurs,

and the wound loses its pink or purple color as capillary and fibroblast density decrease.

All stages may vary in length because of infection, malnutrition, or other exogenous

factors. Remodeling of the scar continues for approximately 1 year. Scar tissue regains

about two thirds of its original strength. Scar tissue is never as strong as the original

tissue it replaces (Black and Matassarin-Jacobs, 1997; Silver, 1997).

Factors affecting Wound Healing

Multiple factors can lead to impaired wound healing. In general terms, the factors

that influence repair can be categorized into local and systemic. Local factors, such as

oxygenation, are those that directly influence the characteristics of the wound itself while

systemic factors are the overall health or disease state of the individual that affect his or

her ability to heal. Many of these factors are related, and the systemic factors act through

the local effects affecting wound healing (Gosain and DiPietro, 2004).

Local Factors That Influence Healing

Oxygenation

Oxygen is important for cell metabolism, especially energy production by means

of ATP, and is critical for nearly all wound-healing processes. It prevents wounds from

infection, induces angiogenesis, increases keratinocyte differentiation, migration, and re-

epithelialization, enhances fibroblast proliferation and collagen synthesis, and promotes

wound contraction (Bishop, 2008; Rodriguez et al., 2008). In addition, the level of

superoxide production (a key factor for oxidative killing pathogens) by

polymorphonuclear leukocytes is critically dependent on oxygen levels.

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Infections

Once skin is injured, micro-organisms that are normally sequestered at the skin

surface obtain access to the underlying tissues. The state of infection and replication

status of the micro-organisms determines whether the wound is classified as having

contamination, colonization, local infection/critical colonization, and/or spreading

invasive infection. Contamination is the presence of non-replicating organisms on a

wound, while colonization is defined as the presence of replicating micro-organisms on

the wound without tissue damage. Local infection/critical colonization is an intermediate

stage, with micro-organism replication and the beginning of local tissue responses.

Invasive infection is defined as the presence of replicating organisms within a wound

with subsequent host injury (Edwards and Harding, 2004).

A set of experiments Dr. Davis and team members conducted involving the

“Isolation of a Stimulatory System in Aloe Extract.” In this test, the Davis research team

found that the extract decreased inflammation when applied topically by 29.2% and

decreased inflammation by 12.1%. Even more significantly, the wound healing activity

was increased by an average of 47.5. In the original study the extract itself exhibited a

78% anti-inflammatory activity of Aloe Vera, and promised to offer a low-cost, natural

substance that could be used to manage inflammation/infection of wounds in the lower

extremities (Leitner, 2005).

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Systemic Factors That Influence Healing

Age

Many clinical and animal studies at the cellular and molecular level have

examined age-related changes and delays in wound healing. It is commonly recognized

that, in healthy older adults, the effect of aging causes a temporal delay in wound healing,

but not an actual impairment in terms of the quality of healing (Gosain and DiPietro,

2004; Keylock et al., 2008). Delayed wound healing in the aged is associated with an

altered inflammatory response, such as delayed T-cell infiltration into the wound area

with alterations in chemokine production and reduced macrophage phagocytic capacity

(Swift et al., 2001). Delayed re-epithelialization, collagen synthesis, and angiogenesis

have also been observed in aged mice as compared with young mice (Swift et al., 1999).

Overall, there are global differences in wound healing between young and aged

individuals.

Sex Hormones in Aged Individuals

Sex hormones play a role in age-related wound-healing deficits. Compared with

aged females, aged males have been shown to have delayed healing of acute wounds. A

partial explanation for this is that the female estrogens (estrone and 17β-estradiol), male

androgens (testosterone and 5α-dihydrotestosterone, DHT), and their steroid precursor

dehydroepiandrosterone (DHEA) appear to have significant effects on the wound-healing

process (Gilliver et al., 2007). It was recently found that the differences in gene

expression between elderly male and young human wounds are almost exclusively

estrogen-regulated (Hardman and Ashcroft, 2008). Estrogen affects wound healing by

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regulating a variety of genes associated with regeneration, matrix production, protease

inhibition, epidermal function, and the genes primarily associated with inflammation

(Hardman and Ashcroft, 2008). Studies indicate that estrogen can improve the age-

related impairment in healing in both men and women, while androgens regulate

cutaneous wound healing negatively (Gilliver et al., 2007).

Stress

Numerous studies have confirmed that stress-induced disruption of

neuroendocrine immune equilibrium is consequential to health (Glaser and Kiecolt-

Glaser, 2005). The pathophysiology of stress results in the deregulation of the immune

system, mediated primarily through the hypothalamic-pituitary-adrenal (HPA) and

sympathetic-adrenal medullary axes or sympathetic nervous system (SNS; Godbout and

Glaser, 2006; Boyapati and Wang, 2007).

Nutrition

For more than 100 years, nutrition has been recognized as a very important factor

that affects wound healing. Most obvious is that malnutrition or specific nutrient

deficiencies can have a profound impact on wound healing after trauma and surgery.

Patients with chronic or non-healing wounds and experiencing nutrition deficiency often

require special nutrients. Energy, carbohydrate, protein, fat, vitamin, and mineral

metabolism all can affect the healing process (Arnold and Barbul, 2006).

Healing of Wounds in Skin

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The simplest method for evaluating healing in wounds involving the skin is to

examine the wound and determine if it is minimally, acceptably, or ideally healed. An

ideally healed wound of the skin results in return to normal anatomic structure, function,

and appearance that include a fully differentiated and organized dermis and epidermis

with intact barrier function. An acceptably healed wound is characterized by

epithelization capable of sustaining functional integrity during activities of daily living

(Lazarus et al., 1994).

Assessment of Wound Healing

To assess the extent, an accurate assessment of these wounds is based on

observing structures and tissues involved. The assessment of extent includes the

following perimeters: maximum dimensions of length and width, depth area, necrotic

tissue type appearance according to color, consistency and adherence specifically in the

wound bed, determination of tissue condition precisely at the edge of the skin and if

necessary, amount of undermining. The latter refers to the wound being open underneath

the "lip" of the boarder and uses a cotton applicator to assess the wound (Lazarus et al.,

1994).

Assessment of wound status is essential in clinical trials and practice for

monitoring treatment efficacy. Lazarus et al., in 1994, proposed guidelines for the

assessment of wounds where they listed attributes that are clues to the cause,

pathophysiology and status of the wound. Assessment of wound status should begin with

the extent of the wound. Because the extent of the wound changes with time, it requires

periodic assessment. There are several techniques that may be employed to assess wound
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extent. To be clinically acceptable, the assessment of chronic wound healing has to be

noninvasive, inexpensive and practical enough to be regularly used by clinicians.

Wound contraction begins almost concurrently with collagen synthesis.

Contraction, defined as the centripetal movement of wound edges that facilitates closure

of a wound defect, is maximal 5-15 days after injury. Contraction results in a decrease in

wound size, appreciated from end to end along an incision; a 2-cm incision may measure

1.8 cm after contraction. The maximal rate of contraction is 0.75 mm/day and depends on

the degree of tissue laxity, also known as degree of loose tissue, and shape of the wound.

Loose tissues contract more than tissues with poor laxity, and square wounds tend to

contract more than circular wounds. This indicates that square wounds have an earlier

disappearance of myofibroblasts and earlier scarring in the angles of the square wound.

Wound contraction depends on the myofibroblast located at the periphery of the wound,

its connection to components of the extracellular matrix, and myofibroblast proliferation

(Romo, 2012).

Recent studies with significant findings for wound healing characteristic of some

medicinal plants are emphasized in the pharmacological field. For example, the herbal

extracts of Allamanda cathartica, Alternanthera brasiliana, Anogeissus

latifolia, Carallia brachiata, and Cynodon dactylon were analyzed using incision wound

model for the determination of their wound healing potential. In the dead space wound

model studies, the herbal extract showed significant increase in collagen deposition

showing fewer macrophages and fibroblasts and increase in the dry weight of granulation

tissue in EGA (experimental group animals) as compared to CGA (control group animals)

with more aggregation of macrophages with few collagen fibers (Nithya, 2011).
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Wound contraction, which contributes to wound closure, is expressed as a

reduction in percentage of the original wound size is studied starting from the day of

operation until the day of complete epithelialization and evaluated to calculate the degree

of wound healing. The progressive reduction in the wound area is monitored

planimetrically by tracing the raw wound boundaries initially on a sterilized transparency

paper sheet in mm2 without causing any damage to the wound area, and then, the wound

area recorded is measured using a graph paper on every 2–4 days interval (Pathak, 2011).

Epithelialization is the formation of epithelium over a denuded surface. It

involves the migration of cells at the wound edges over a distance of less than 1 mm,

from one side of the incision to the other. Incisional wounds are epithelialized within 24-

48 hours after injury. This epithelial layer provides a seal between the underlying wound

and the environment.

The process begins within hours of tissue injury. Epidermal cells at the wound

edges undergo structural changes, allowing them to detach from their connections to

other epidermal cells and to their basement membrane. Intracellular actin microfilaments

are formed, allowing the epidermal cells to creep across the wound surface. As the cells

migrate, they dissect the wound and separate the overlying eschar from the underlying

viable tissue. In superficial wounds (eg, wounds due to laser resurfacing, dermabrasion,

chemical peel treatments) adnexal structures (eg, sebaceous glands, hair follicles)

contribute to reepithelialization.

Epidermal cells secrete collagenases that break down collagen and plasminogen

activator, which stimulates the production of plasmin. Plasmin promotes clot dissolution

along the path of epithelial cell migration. Migrating epithelial cells interact with a
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provisional matrix of fibrin cross-linked to fibronectin and collagen. In particular,

fibronectin seems to promote keratinocyte adhesion to guide these cells across the wound

base.

Wounds in a moist environment demonstrate a faster and more direct course of

epithelialization. Occlusive and semiocclusive dressings applied in the first 48 hours after

injury may maintain tissue humidity and optimize epithelialization.

When epithelialization is complete, the epidermal cell assumes its original form,

and new desmosomal linkages to other epidermal cells and hemidesmosomal linkages to

the basement membrane are restored (Molnar, 2006).

Role of Plants as Medication

The therapeutic efficacies of many indigenous plants, for various diseases have

been described by traditional herbal medicine practitioners (Kiran et al., 2008). A large

number of plants are used by tribal and folklore in many countries for the treatment of

wounds and burns (Edwards and Harding, 2004).Natural products are a source of

synthetic and traditional herbal medicine. They have the immense potential for the

management and treatment of wounds. These natural agents induce healing and

regeneration of the lost tissue by multiple mechanisms. Still, they are the primary health

care system in some parts of the world.

Natural products, such as plants extract, either as pure compounds or as

standardized extracts, provide unlimited opportunities for new drug discoveries because

of the unmatched availability of chemical diversity (Cosa et al., 2006). According to the

World Health Organization (WHO), more than 80% of the world's population relies on

traditional medicine for their primary healthcare needs. The use of herbal medicines in
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Asia represents a long history of human interactions with the environment. Plants used

for traditional medicine contain a wide range of substances that can be used to treat

chronic as well as infectious diseases (Duraipandiyan et al., 2006).

Traditional medicine has a long history of serving people all over the world.

Traditional medicine has remained as the most affordable and easily accessible source of

treatment in the primary healthcare system of resource poor communities and the local

therapy is the only means of medical treatment for such communities (Davis et al., 1996).

Leaf paste of the plant, Centella asiatica is used in South Orissa, India for

treatment of wounds (Panda and Misra, 2011). Some plants used by the tribal people of

Buldhana district, India to treat wounds include Acacia catechu, Achyranthes aspera,

Cynodon dactylon, and Eclipta alba – these plants being the same as used by the

Kavirajes of Bangladesh (Korpenwar, 2012).

Pharmacological screened plants are those with wound healing potentials. Plants

are more potent healers because they promote the repair mechanism in the natural way.

The healing process can be physically monitored by assessing the rate of contraction of

the wound, period of epithelization, tensile strength, histopathology and weight of

granuloma in different wound models. The healing tissue synthesizes more collagen to

provide tensile strength (Hossan et al., 2009).

The ethnobotany and ubiquitous plants provide a rich resource for natural drug

research and development. In recent years, the use of traditional medicine information on

plant research has received considerable interest. The Western use of such information

has also come under increasing scrutiny and the national and indigenous rights on these

resources have become acknowledged by most academic and industrial researchers.

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Meanwhile, the need for basic scientific investigations on medicinal plants using

indigenous medical systems becomes imminent (Goslen, 1989).

Ocimum tenuiflorum is holy and revered in India that is why the taxonomical

synonym Ocimum sanctum L. is more popular in Indian scientific literature but they are

the same (Blast et al., 2014). Higher content of linoleic acid in O. sanctum L. fixed oil

could contribute towards its antibacterial activity. The oil shows good antibacterial

activity against Staphylococcus aureus, Bacillus pumius and Pseudomonas aeruginosa,

where S. aureus was the most sensitive organism (Singh et al., 2005).

Ethanolic extract of leaves of O. sanctum L. was investigated for normal wound

healing and dexamethasone-depressed healing. The extract significantly increased the

wound breaking strength, wound epithelializes fast and wound contraction was

significantly increased along with increase in wet and dry granulation tissue weight and

granulation tissue breaking strength (Udupa et.al., 2006).

Shetty et al., in 2006, evaluated the wound healing effect of aqueous extract of O.

sanctum L. in rats. Wound-breaking strength in incision wound model, epithelization

period and percent wound concentration in excision wound model were studied owing to

increased per cent wound contraction. Ocimum sanctum L. may be useful in the

management of abnormal healing such as keloids and hypertropic scars.

In another study in 2005, Harshmohan concluded that when the ethanolic extracts of

leaves of Ocimum sanctum were applied externally on male albino rats by topical route

through excision wound model then they showed faster as well as better wound closure
 23

and wound contraction as compared to standard marketed formulation called as Povidone

Iodine ointment.

A study was undertaken to verify the effect of hydro alcoholic extract of Ficus

religiosa leaves on experimentally induced wounds in rats in excision wound, incision

wound and burn wound models. A formulation of leaves extract was prepared in

emulsifying ointment at a concentration of 5% and 10% and applied to the wounds. The

result suggests that leaf extract of Ficus religiosa (both 5% and 10%) applied topically

possess dose dependent wound healing activity. Consequently, the 10% ointment of Ficus

religiosa treated animals showed faster epithelialization of wound than the animals

treated with 5% extract ointment. However, this effect was found to be concentration

related fashion where 10% ointment promotes significant wound-healing activity by

increasing cellular proliferation, formation of granulation tissue, synthesis of collagen

and by increase in the rate of wound contraction as compared to the control animals

(Naira et al., 2009).

Wound healing activity of cold aqueous extract of O. sanctum leaves along with

its effect on tumor necrosis factor-alpha (TNF-alpha) was assessed using excision model

of wound repair in Wistar albino rats. After application of the O. sanctum extract, rate of

epithelization with an increase in wound contraction was observed. In animals, treated

with 10% O. sanctum extract in petroleum jelly, wound healing was faster as compared to

control group which were treated with petroleum jelly alone. During wound healing

phase TNF-alpha level was found to be up regulated by O. sanctum treatment. Early

wound healing may be pronounced due to O. sanctum extract, by elevating TNF-alpha

production (Majumdar, 2005).

 24

Phytoconstituent Analysis of Plant Extract

The past decade has seen considerable change in opinion regarding ethno-

pharmacological therapeutic applications (Natarajan, 2003). The mere presence of

various life sustaining constituents in plants has urged scientists to examine these plants

with a view to determine potential wound healing properties (Jahan, 2003).

Many phytopharmaceutical laboratories are now concentrating their efforts to

identify the active constituents and modes of action of various medicinal plants. The

medicinal value of these plants lies in bioactive phytochemical constituents that produce

definite physiological action on the human body (Farokhzad, 2008). These constituents

include various chemical families like alkaloids, essential oils, flavonoids, tannins,

terpenoids, saponins, and phenolic compounds (Atiyeh et al., 2007).

In a recent study, Catharanthus roseus plant which was a key source of

monoterpenoidindol alkaloid, vincristine and vinblastine was found useful in treatment of

cancer. In a study of ethanolic extract of flower of this plant in a dose of 100 mg/kg/day

demonstrated to possess wound healing property (Sumitra et al., 2010).

The enhanced wound healing potency of various herbal extracts may be attributed

to free radical-scavenging action and the antimicrobial property of the phytoconstituents

present in the extract, and the quicker process of wound healing could be a function of

either the individual or the synergistic effects of bioactive molecules (Inoue et al., 2008).

The screening of herbal extracts has been of great interest to the scientists for the

discovery of new effective drugs (Umadevi et al., 2006). A number of reports concerning

the antibacterial, anti-inflammatory, and wound healing activity of various plants have

appeared in the literature, but the vast majority has yet to be explored.
 25

Research on wound healing drugs is a developing area in modern biomedical

sciences. Scientists who are trying to develop newer drugs from natural resources are

looking toward the Ayurveda, the Indian traditional system of medicine (Rahmatullah et

al., 2010). Most of these drugs are derived from plant origin. These phytomedicine are

not only cheap and affordable but are also safe.

The practice of complementary and alternative medicine is now on the increase in

developing countries in response to World Health Organization directives culminating in

several pre-clinical and clinical studies that have provided the scientific basis for the

efficacy of many plants used in folk medicine (Vijaya and Ananthan, 1997; Dilhuydy and

Patients, 2003).

Role of Temperature in Phytoconstituent Extraction

Extracting essential oil from plants is an intricate process and technically is used

to remove and concentrate the aromatic constituents from plant materials. The effect of

variation in temperature and pressure of oil suggest use of higher pressure while

maintaining a stable yet high ideally temperature range between 60-100 °C in order to

increase solvent density in which solubility depends upon it.

A study conducted by Asoiro et al. (2011) revealed that temperature had

significant effect on Jatropha curcas L. kernel oil yield. As extraction temperature

increased, oil yield rose from 33.35% by weight to a maximum value of 44.41% by

weight at 60°C. Further increase in extraction temperature to 110°C reduced oil yield to

41.5% by weight. Optimum temperature conditions for maximum Jatropha oil yield and

quality was obtained at 60°C.

 26

Steam distillation is employed in the manufacture of essential oils, for use

in perfumes, for example. In this method, steam is passed through the plant material

containing the desired oils. Eucalyptus oil and orange oil are obtained by this method on

the industrial scale. Steam distillation is also sometimes used to separate intermediate or

final products during the synthesis of complex organic compounds. Steam distillation is

also widely used in petroleum refineries and petrochemical plants where it is commonly

referred to as "steam stripping" (Beychok, 1992).

Temperature sensitive materials (such as beta carotene or other plant constituents)

require rotary evaporation distillation to remove solvents from the mixture without

damaging the product. Another reason rotary evaporation is used is that compared

to steam distillation there are lower levels of residue build up. This is important in

commercial applications where heat transfer is produced using heat exchangers (Kolmetz

and Sloley, 2004)

A study by Cacace et al. (2003) demonstrated that it is essential to optimize

systematically the extraction solvent composition, temperature and time for accurate and

reproducible assay of sage polyphenols. This study confirmed that the aqueous solutions

of ethanol or acetone of 30 %, extraction temperature of 60 °C and extraction time of 30

min. were the most efficient for the extraction of polyphenols from dry sage leaves.

Use of Animal Model in Wound Healing

The small mammals have emerged as the model of choice for such studies, which

are beneficial for multiple reasons. They are inexpensive, easily obtainable, require less

space, food, and water, easy to maintain, and can be genetically modified (Edwards and
 27

Harding, 2004).Another advantage of small mammal models is their ability to serve in

experiments where death is an endpoint (Wu, 2007). Additionally, they often have

multiple offspring, which develop quickly allowing experiments to proceed through

multiple generations. Small animals usually have accelerated modes of healing in

comparison to humans, thus experiment duration lasts for days, in contrast to weeks or

months in human experiments (Boyapati et al., 2006).

Animal wound healing models are important biological tools to understand basic

processes of tissue repair and to develop and validate strategies for clinical treatment.

Human wound healing has many unique aspects that relate to the physiology, age, and

environment of the species, but the opportunity to carry out controlled, clinical

experimentation on the mechanism and therapy of wounds is limited.

The majority of laboratory rodents are albinos, due to a common mutation in

tyrosinase gene in all albino laboratory rat strains and in at least some of the albino

mouse strains. Tyrosinase is the rate-limiting enzyme in the production of melanin

pigment. The prevalence of albinism among laboratory rodents is because many of the

earliest established strains were albino, and also albinism is an easy selection marker in

the early days. The albino defect does not affect known metabolic or vital functions. It's

more a cosmetic defect. Remember that rats have 42 chromosomes, that's a lot of genes

not affected by the albino mutation (Kuramoto et al., 2012).

One case study describes Turmeric to have been used for treating wounds in the

rats. The anti-inflammatory property and the presence of vitamin A and proteins in

turmeric result in the early synthesis of collagen fibers by mimicking fibroblastic activity.
 28

Juice of the fresh rhizome is commonly applied to recent wounds, bruises and leech

bites (Jalali et al., 2008).

In general, animal models (with the exception of some transgenic and targeted

gene deletions) attempt to reflect human wound healing problems: dehiscence, ischemia,

ulceration, infection, and scarring. Except for acute wound models, experimentalists must

accept approximations of clinical problems due to the obvious differences in tissue

architecture, immune response, physiology, and other healing responses among species

(Khorshid et al., 2010). They cannot replace the ultimate verification of agents and

actions in human wounds, because of the substantial differences in tissue architecture and

immune response. The experimentalist must consider the merits and disadvantages of

each type of model and species in the context of the data being sought (Gbenga et al.,

2009).

BALB/c is an albino, inbred strain. BALB/c mice have the characteristics of easy

breeding and minimal weight variations between males and females. Much noteworthy is

that the mammary tumor incidence in BALB/c mice is low. This type of mice breed is

used for the standard in immunological purposes (ex. wound healing and lymphocyte

count) as well as cancer based studies. This breed showed profound effects of the genetic

background on the wound healing process. This raises the question whether the genetic

background of mice used in a study is optimal to investigate the effect of a specific

genetic modification or whether some effects remain unnoticed due to the use of a

specific genetic background (Van den Borne et al., 2008).

 29

Local anesthetics such as Lidocaine, Bupivicaine and others may be used to

reduce the perception of pain at the surgical site as local or regional anesthetics.

Lidocaine alters signal conduction in neurons by blocking the fast voltage gated sodium

(Na+) channels in the neuronal cell membrane that are responsible for signal propagation.

In conjunction with other agents, their use may allow reduced levels of general

anesthetics, which may speed recovery and minimize mortality. When carefully used,

direct injection of a local anesthetic can be a useful adjunct to anesthesia. Although

Lidocaine and Bupivacaine influence local inflammatory and proteolytic factors, they do

not impair the rate of healing in well-established models consequently mimicking normal

human wound healing and impaired age-related healing (Hodgson, 2010).

Synthesis

Wounds are disruptions of the skin, the first line of defense of the body against

pathogenic materials. They expose an organism to harm and it is of utmost importance to

get it repaired as soon as possible. As such, a complex and dynamic process happens to

ensure rapid closure and repair. One could even consider a healing wound as its own

microenvironment, discrete from the rest of the organism. Optimizing this

microenvironment will promote wound healing and complete it in the shortest time

possible. There are different phases of wound healing, hemostasis, inflammatory,

proliferative, and then remodeling. Intervention in the earliest phase will facilitate faster

wound healing and maintenance in the later stages will further hasten it. Local and

systemic factors influence the healing process. Local factors include oxygenation, and

infections while systemic factors include age, sex hormones, and stress, and nutrition. It
 30

is important for these variables to be controlled to ensure proper results in the

experiment.

There are different ways to asses wound healing. One of the best way to asses

wound healing over a period of time is that is has to be noninvasive, inexpensive and

practical enough to be regularly used by clinicians, or researchers. Two parameters that

can be measured externally without need of sacrificing the subject is wound contraction,

where the area of the current day is compared the area just after incision, and

epithelialization, the surface of the wound is assessed base on a scale.

Plants have always been used as medication ever since ancient history. They have

been also used as basis for making many synthetic drugs that are in use today. One aspect

that has been associated with the medicinal properties of plants is wound healing.

Chemicals active in the wound healing process could be generalized as phytochemicals.

One plant that is very rich in phytochemicals is O. sanctum. Many Indian authors have

confirmed this. O. sanctum and O. tenuiflorum are taxonomical synonyms which means

they are the same plant.

The method of extraction has a great effect on the percent yield of various

phytochemical extracts. The commonly used methods are rotary evaporation and steam

distillation. These methods coincide with the fact that the effects of temperature as a

variable would dictate a role in the yield. In one case study, it has been denoted that the

effects of higher heat temperature will denature the phytochemicals present. Thus it will

affect antibacterial and eventually healing properties. In essence, these temperature

 31

sensitive materials usually require a suitable heat temperature of about 60°C to fully

optimize the yield results.

Ideal animals for use in wound models should be small, easy to maintain, and

conducive to the research being conducted. BALB/c Mus musculus is a type of mice

breed that is used for the standard in immunological purposes, in this case wound healing.

Local anesthetics, such as Lidocaine, are required when making the excision wound to

minimize pain felt by the mice.

 MATERIALS AND METHODS

Research Locale

Both extraction methods of Ocimum tenuiflorum were done in the Herbanext™

Laboratories located at DC CRUZ BLDG., National Highway, Taloc, Bago City.

Housing, incision, and applications of medicine were done in a secluded room in the

residence of one of the researchers. Conditions were controlled and maintained at a

certain criteria to prevent unnecessary variation in the environment.

Plant material

The source of extracts were Ocimum tenuiflorum. This plant is not naturally found

in the Philippines but can grow relatively easily because of the same climactic conditions

as from its origin, India. Ocimum tenuiflorum has high oil yield when compared with

other plants. The whole plant, the leaves, stems, and roots, was used for extraction. The

plant was chosen due to its medicinal effects in Indian folk culture and literature, also for

its antibacterial properties (Singh et al., 2005).

Extraction Process

There were two methods of extraction. The first is use of a steam distiller and the

second is via rotary evaporator.

Extraction by Steam Distillation

The Pilot-scale Essential Oil Steam Distiller was used to collect high amounts of

essential oil for the experiment proper. O. tenuiflorum was washed and air dried. 5kg of

the herb was placed in the upper vat while 700L of water was placed in the lower one.
 33

The distillation process was done in 3 hours with temperature being maintained at 1100C

(Beychok, 1992).

a. b. c.
Figure 2. a. Pilot-scale steam distiller in b. collection flask whith thin layer of oil
c. essential oil extracted

Extraction by Rotary Evaporation:

Sample preparation

Before proceeding with using the RotaVap (rotary evaporator) the sample was

first prepared. Leaves were dried until less than 10% moisture remained by using an

industrial oven and were powdered by using an industrial grinder. This was then sieved

with 60 mesh siever. 50g of the powdered O. tenuiflorum was then soaked in a 1L 75%

ethanol solution for 12 hours. After which, the supernatant liquid was sieved with 120

mesh siever and filtered again with filter paper to recover extract. The total recovery

volume after filtration was 815mL. The pH was 5.58 at 26.2°C with a specific gravity of

0.8743g and solid content of 13.48.

 34

a. b. c.

d. e.

Figure 3. a. O. tenuiflorum after drying in oven b. Industrial grinder for powdering dried
plant c. Exact measuring of 50g d. 120-mesh sieve result e. filtration setup
Rotavap Operation

Amount of extract that was loaded in round bottom flask was only 500mL at first

from the 815mL recovery volume after filtration to prevent solution loss by boiling over.

All of the joins were air tight for them to withstand the negative pressure. The power of

the pump, water bath, and the evaporator was turned on while proper water source was

ensured. Temperature was set to 60°C (Kolmetz and Sloley, 2004). Water for heating was

leveled with the extract for uniform distribution of heat. The rpm of the flask was set to

80rpm while the pressure was maintained at 20 kPa. The remaining 315 mL was

gradually added afterwards when evaporation reduced the volume of the solution until

such time that the evaporation halted. It took a total of 4 hours for the complete

evaporation process to be finished. A preliminary operation determined that for every

50g, 6.74g will be extracted.

 35

a. b. c.

Figure 4. a. Water kept level with initial solution b. Setup maintained at 60°C and 20 kPa
c. Progress after 1 hour

Ointment Preparation
The ointment is a formulation of 50% lanolin and 50% petroleum jelly. Due to

highly viscous nature of both components, extra care was taken into measuring the

appropriate masses. The preliminary rotary evaporation extraction yielded 6.74g. To

properly make an ointment with 10% active ingredient, it was calculated that the total

weight of the ointment should be 67.4g with the weight of the base being 60.66g (Goel et

al, 2010; Roy et al., 2009). Equal amounts of 30.33g for both lanolin and petroleum jelly

were used. Due to being more viscous, lanolin was added first into the 100mL beaker.

This permitted an effective method of adjusting to be made to the mass of the

components. The petroleum jelly was added next. The beaker was heated in the boiler

part of the rotavap and the contents were continuously stirred until the solution was

homogenous. In its liquefied state, the base was poured into the round bottom flask which

contained the extract from rotary evaporation. Rpm was set to 100 while temperature was

maintained at 60°C. The setup was not pressurized. This continued until the ointment was

homogenous and then it was transferred into a plastic container where it was allowed to
 36

cool and to be stored afterwards. The total weight of the ointment was 90.48g so the

concentration was adjusted to be 7.45%

Essential oil and blank ointments followed the same general procedure. This time

25g of lanolin and 25g of petroleum jelly was used. After the mixture was homogenous, it

was split into two equal parts. The first part was used as the blank ointment. As the name

suggests, there are no additives. It was poured into a plastic container and set aside to

cool. The second part was poured into the round bottom flask of the RotaVap. It had the

same rpm and temperature settings (100, 60°C respectively). To achieve 7.45%

concentration, 1.8g of essential oil was added. The mixture was removed after 30 minutes

to ensure homogeneity.

a. b.

c. d.

Figure 5. a. Heating lanolin and petroleum jelly b. rotary evaporation ointment poured
into plastic container c. ointment base being mixed with essential oil extract d. (from
left) rotary evaporation ointment, blank ointment, essential oil ointment with
corresponding plastic covers at the top
Research Subjects
 37

The research subjects were Mus musculus BALB/c, albino, inbred strain. These

were obtained from a mouse breeder for scientific studies, Jasmin T. Gustilo. She is also

the supplier for a number of pet shops in Bacolod City. Subjects chosen were healthy:

uniform coats with no bald patches, eyes are bright, clear and responsive, active and

running around, good appetite, normal urination (no blood or extremely long interval

between urination), and no mite infestation. Weight was within 20g to 30g and was only

consisted of males. They were fed with standard rodent pellet diet once a day and given

water ad libitum (Harshmohan, 2005). They were all the same age (5 week old) and sex

(male). For every group, there were 8 mice for a total of 40 mice for all the groups. Refer

to Appendix A for handling protocol, and Appendix B for disposal protocol.

Positive Control

Trimycin™ was used for the positive control group because of the antibacterial

properties of three of its four ingredients: Bacitracin, Polymxin B sulfate, and Neomycin.

Previous studies have found that Ocimum tenuiflorum indeed has antibacterial properties

which are quite effective as well. When converted into ointment form, Ocimum

tenuiflorum extracts have the same form of topical application as Trimycin™.

Preparation of Study Site

Plastic microwavable containers were used as cages. They measured 18cm x

12cm x 5cm with holes punched in cover and the bottom of the container. The cover had

7 rows of alternating 3 and 4 holes per row for a total of 24. The distance between each

row was 2.3cm while the distance between individual holes in the same row was 2.8cm.

The bottom had 94 holes.

 38

Figure 6. Hole configuration on each cage

These cages were suspended by a metal stand to avoid contact with the table

surface. There was a total of 5 metal stands, one for each group. They were made with

5mm steel rods welded together and measure 122cm x 17.5 cm x 11cm. They have eight

compartments with one measuring 11cm x 16.5 cm. The compartments are separated

from each other by 4cm to prevent contamination from mice of the same group and are

placed 20 cm away from other groups. Below each metal stand was a plastic cardboard

measuring 132 x 27.5cm with folded up edges to collect the waste material from the

cages. These plastic cardboard were lined with newspapers which were replaced

everyday. Both the cardboard and the newspapers were sterilized first with 95% alcohol

before being used again. All of these are equally elevated from the ground with tables

72cm high. Basic set-up and housing was patterned from the work of Thakur, Jain, 

Pathak, and Sandhu. There were a few modifications. Instead of plastic cages, plastic 

microwavable containers were used with holes punched in them to mimic the same set­

up. The table height was modified to suit the height of the researchers. Plastic cardboard 

with newspaper was used instead of plastic trays.
 39

a. b.

c.

Figure 7. a. Full length of 1 metal stand, with 8 cages b. low angle view of the setup
showing 2 groups c. higher angle view of the same 2 groups.

The cages were sterilized before the mice were put inside them. Each cage housed

individual Mus musculus separately to prevent interaction. There was access to water

(Wilkins distilled water) at all times. They were fed simultaneously with 5g of the

standard rodent pellets (Excel Feeds™) in plastic condiment containers once a day at

9:00 AM. These were placed in an area with proper ventilation and source of sunlight

(cages were not in direct sunlight). Favorable conditions of: 20°C ± 3°C (air-

conditioned), 12 hour light and day cycles, with 40-60% humidity, minimal handling,

minimal vibrations and disturbances was maintained at all times. Mus musculus was

introduced and housed 2 weeks prior to experiment proper for adjustment and prevention
 40

of stress during the experiment. Wearing of surgical gloves (without powder) was always

observed when handling the mice.

a. b.
Figure 8. a. weighing of 5g feeds b. water and feeds inside plastic condiment
container

Infliction of Excision Wounds

Wearing of surgical gloves was observed throughout the procedure. Mus

musculus was anesthetized with a subcutaneous (SC) administration of Lidocaine HCl

2% (diluted first to 0.5% with total dosage calculated with 7mg/kg). The fur on the upper

dorsal part was removed using an electric razor. A template was made with the

measurements 1cmx1cm. Uniform 1cm2 squares were imprinted using a marker

(Tadiparthi et al., 2009). These templates did not have any effect on the wound because

the only contact they had was before the excision. The location of the wound area on the

upper dorsal region was chosen so as to prevent unwanted wounds caused by stretching

and biting from the mouse. The area was then swabbed with a sterile alcohol prep pad

with 70% isopropyl alcohol. Forceps were used to elevate the area to be cut and to

keep the borders of the wound steady. Surgical scissors were used to remove the skin,

subcutaneous tissue, fascia, and fleshy panniculus (full thickness) inside the 1cm2 area.
 41

There was minimal bleeding that was easily managed. The template, scissors, and forceps

were cleansed with 95% alcohol after each use. This experimental protocol was based on 

the work of Tan, Adli, Tumiran, Abdulla, and Yusoff but with minor modifications. A 

different anesthetic (Lidocaine) was used in this experiment and had a different route of 

administration, (SQ injection). Also, Sprauge­Dewly rats were originally used and the 

original incision size was 4cm2 in the works of Thakur, Jain, Pathak, and Sandhu. The

smaller sized mice in this experiment instead had a smaller wound area of 1cm2.

a. b. c.

d. e.

Figure 9. a. Hair removal using electric razor b. (from top left, counter clockwise) needle
holder with ratchet lock, dressing forceps, surgical scissors, hemostatic forceps, marker,
and a vernier caliper c. (from top left, counter clockwise) 70% ethyl alcohol, sterile
alcohol prep pad with 70% isopropyl alcohol and 95% ethyl alcohol d. making the
excision wound e. 1cm2 excision wound

Application of Treatment
 42

All treatment applications were done every day and simultaneously at 10:00AM 

with plastic microspatulas, sterilized and cleaned after each use. In group A, 0.3g of 

steam distillation ointment was applied to the injured area. Application was done as 

evenly and as lightly as possible leaving only a thin layer and not disturbing the wound. 

For group B, rotary evaporation ointment was applied to the injured area in the same 

manner. For group C, there were no applications. Group D received Trimycin™ and

Group E received the blank ointment in the same manner of treatment the earlier two

groups received. A different microspatula was used for every group and they were

sterilized before being used for another mouse of the same group. These were done every 

day starting from the day of excision until 15 days or wound closure, whichever came 

first.

a. b. c.

d. e.
Figure 10. a. Transferring Trimycin™ into plastic microspatula b. Using microspatula to
scoop steam distillation ointment. c. measuring 0.3g of steam distillation ointment d.
spreading treatment steadily and evenly. e. using prep pad to sterilize microspatula
 43

Data Processing and Statistical Design

The subjects need not be sacrificed at the end of the 15 day observation period.  

Examination was done by the researchers. 

Percent wound contraction

The progressive reduction in the wound area was be monitored by measuring the

length and width of the excision wound using calipers and multiplying them yielding

results in measure of cm2. Measurements were made every 2 days until the 15th day or

when the wound completely closed. Percentage wound contraction was calculated by:

Epithelialization

Epithelialization was assessed every 2 days until the 15th day or when the wound

completely closed. It was rated based on the scale:

1 = 100% wound covered, surface intact 

2 = 75% to <100% wound covered and/or epithelial tissue extends >0.5cm into wound 

bed                 

3 = 50% to <75% wound covered and/or epithelial tissue extends to <0.5cm into wound 

bed
 44

4 = 25% to < 50% wound covered 

5 = < 25% wound covered

a. b. c.

d. e.

Figure 11. a. wound with 1 rating b. 2 rating c. 3 rating d.4 rating e. 5 rating

Percent wound contraction calculation was based on the works of Thakur, Jain, 

Pathak, and Sandhu. Wound epithelialization scale was adapted from the Bates­Jensen 

wound assessment tool. Measurements from each day were treated as a separate group of 

data and so each day had a different set of statistical analysis. For percentage wound

contraction, the one-way ANOVA was used to determine if there was a significant

difference between the means of the 5 groups (A, B, C, D, and E). When the p-value of

the one-way ANOVA was less than .05, the null hypothesis was rejected and the

Duncan’s multiple range test was done to determine exactly what groups showed a
 45

significant difference. For epithelialization, the Kruskal Wallis test was used to determine

if there was a significant difference between the groups. When the p-value was below .05,

Mann-Whitney U tests were done do determine what specific groups have a significant

difference.
 RESULTS AND DISCUSSIONS

The research was carried out to compare the two methods of extraction to find out

which one is more effective in wound healing. Furthermore it compared the effect of the

two extracts to negative control, to see if there is a significant improvement; positive

control, to see how it matches up with something already in the market; and blank

ointment, to see if the ointment base has any effect on the healing. The analysis of the

data is presented in the discussions that follow.

 47

Figure 12. Mean percentage wound contraction in 15-Day observation period

An overall view of the data reveals that both the steam distillation essential oil

and rotary evaporation extracts have a higher percentage wound closure compared to the

negative control group. However, it was the rotary evaporation extract that had the closest

wound contraction result in comparison with the positive group (treated with Trymicin).

Subsequently, a period of wound contraction anomaly actually occurred during the first

few days of the treatment. For instance, during days 3 and 5, the positive group had a

lead in terms of wound contraction over the rotary evaporation extract treated group.

However, as the days progressed (specifically days 7 to 15), the rotary evaporation group
 48

had managed to catch up with the positive group in the wound contraction phase. It even

made a greater impact on these days as compared to the said group. The probable cause

of such an anomaly would be synergism. In traditional medicine whole plants or mixtures

of plants are used rather than isolated compounds or pure drugs. There is evidence that

whole plant extracts often have greater in vitro or/and in vivo activity than isolated

constituents at an equivalent dose. Strictly speaking, “synergy” or “potentiation” means

that the effect of the combination is greater than the sum of the individual effects. In a

study conducted by Rasoanaivo, pharmacodynamic synergy has been demonstrated

between the Cinchona alkaloids and between various plant extracts traditionally

combined. Pharmacokinetic interactions occur, for example between constituents of

Artemisia annua tea so that its artemisinin is more rapidly absorbed than the pure drug

(2011). Some plant extracts may have an immunomodulatory effect.

In essence, the standard drug contained three major constituents which were

isolated: neomycin, bacitracin, and polymyxin, antibiotics that work by stopping the

growth of bacteria. On the contrary, the constituents of the plant extract remained as a

whole. Thus, bioavailability and metabolism of medicinal substances can be dramatically

affected by other plant constituents.

Percent Wound Contraction

In all of the days there was no significant difference between the negative group

and the blank ointment, signifying that the ointment formulation has no active effect on

the wound. Refer Appendix C for raw data in mean percentage contraction.

Table 1. Summary of ANOVA results

Day F p-value
 49

3 3.086 0.028**
5 3.063 0.029**
7 3.923 0.010**
9 5.213 0.002**
11 4.725 0.004**
13 4.007 0.009**
15 2.788 0.041**
**
significant difference (P = 0.05)
**
highly significant difference (P = 0.01)

Separate ANOVAs were done for all data sets leading to a total of 7 (one for each

observation date). All of the p-values were below .05. Hence, the first null hypothesis is

not accepted for all the data sets. Beginning from the 7th day the results become highly

significant. This can be possibly attributed to the fact that results become more

pronounced the greater the passage of time (Goel et al., 2010). This high significance is

lost on the 15th day because some of the subjects with fully healed wounds can no longer

have any progress, allowing other groups to catch up. Refer to Appendix D for ANOVA

analysis.

Table 2. Effect of the five different treatment groups on the percentage wound
contraction of the excision wound model in Mus musculus
 50

Different letters in rows denote homogenous subsets and significant differences

according to Duncans’s new multiple range test (P = 0.05)

On day 3 only the positive control group was the only significantly different

treatment compared to the rest. This might be due to the fact that not only does it have 3

antibacterial components it also has Hydrocortisone, a mild topical corticosteroid, which

reduces inflammation. Its effects would definitely show during the early phases of wound

healing where inflammation is very extensive. This would then have diminishing effects

right about day 7. Studies on wound healing are usually done in not less than a 7 days

because results normally do not become significant until such time passes. (Pathak,

2011). Although there was no significant difference, it could be observed the rotary

evaporation group had a greater mean percentage contraction compared to the negative

control group.

The trend for day 5 is generally the same as that of day 3 the positive control

group being significantly different from most of the other treatments. The only difference

is that this time, the rotary evaporation group has begun catching up so much so that it is

no longer significantly different when compared to the positive control group. This

suggests the diminishing effect of the corticosteroid. Although the difference between

steam distillation and rotary evaporation were not significant, it could be observed that

the differences between the means have increased. This goes the same for the work of

Mustoe et al. in 2007 wherein the differences between treatments, although not being

statistically significant, have begun increasing. The rotary evaporation group still did
 51

better compared to the steam distillation group and the negative control group but is

behind the positive control group. The negative control group and the blank ointment

group still has no significant difference between them.

a. b.

Figure 13. a. group C representation b. group D representation (Day 3)

Results became clearer during day 7. At this point in time, there was already a

significant difference between the steam distillation and rotary evaporation groups Its

difference to the negative control group was also statistically significant this time around.

A notable change would also be the fact that this treatment had already begun overtaking

the positive control group. This is probably due to the “synergy” or “potentiation” in the

phytochemicals of the rotary evaporation ointment which give more effect with a greater

passage of time. This is similar to the results in the works of Rasoanaivo in 2011. The

negative and positive control groups are still significantly different and the negative and

blank ointment group also remain not significantly different. The trend of day 11 results

coincide with that of day 7 and 9. These three days, when lumped together, represent the

majority of the 7 data sets and therefore provide the best overview for the results of the

entire experiment. For all of these days, the two experimental groups always had a

significant difference with the rotary evaporation group having the higher mean.
 52

Comparisons to the negative control group always yielded a significant difference while

the comparison to the positive control group were always not significant.

a. b. c. d.

Figure 14. a. group A representation b. group B representation c. group C representation

d. group D representation (Day 9)

Even until day 13, the groups which have consistently high means are the rotary

evaporation and positive control groups. Most subjects from these groups by this time are

nearing complete wound closure and would probably heal completely by day 14 (no data

will be recorded). This started a decline in the lead possessed by rotary evaporation and

steam distillation groups thus causing three homogenous subsets (all the other days only

had two). The most important information from this day was that there was still a

significant difference between the rotary evaporation and positive control groups when

compared with the negative control.

By day 15 most subjects from the rotary evaporation group and positive treatment

group are almost, if not all, completely healed by this time. This allowed other groups to

catch up with their progress. The only significant differences this time are between the

blank control group when compared to both steam distillation and positive control

groups. The rotary evaporation ointment consistently had a higher mean when compared
 53

to the steam distillation ointment. Also, throughout the experiment the percentage wound

contraction of the rotary evaporation and positive control groups was only significant

during day 3. This suggests that the rotary evaporation ointment works just as well as

Trimycin. The negative control and blank ointment groups have not been significantly

different throughout the experiment also. Refer to Appendix E for Duncan’s multiple

range test results, and Appendix H for wound healing closure images of representative

subjects.

a. b. c. d.

Figure 15. a. group A representation b. group B representation c. group C representation

d. group D representation (Day 15)

Epithelialization
Table 3. Summary of Kruskal-
Wallis results
Chi- Asymp.
Day df
square Sig
3 5.484 8 0.241*
5 3.998 8 0.406*
7 12.573 8 0.014*
9 6.872 8 0.143*
11 2.746 8 0.601*
13 2.600 8 0.627*
15 0.000 8 1.000*
 54

**
significant difference (P = 0.05)

The epithelialization only produced a significant difference on the 7 day only. The

non-significant results may probably have been caused by the limited number of subjects

per group (8) in relation to how many groups there are (5) eventually leading to several

overlaps of the same rating just like what happened in the study of Agapito et al. (2013).

This is compounded by the fact that by day 13, only 9 (out of 40) subjects have not

reached full epithelialization and all subjects have reached maximum epithelialization by

day 15. Post-hoc analysis will only be used for the results of day 7. Refer to Appendix C

for the epithelialization scores, and Appendix F for Kruskal-Wallis results.

Post-hoc analysis of the data on the 7th day reveals four pairs which had a

significant difference in epithelialization. These pairs are rotary evaporation compared to

the negative control and blank groups, negative compared to positive control and,
 55

positive control compared to blank. Many studies start showing significant results with

their experimental groups by day 5-7. This is most true when the parameter is assessed

externally (Swift et al., 2001). The ratings given on day 7 were the most varied, paving

the way for a significant difference to be found (Agapito et al., 2013). Once again the

most effective treatments are the rotary evaporation ointment and the positive control.

These two treatments provided the most suitable microenvironment for epithelialization

to be at its most efficient (Bishop, 2008). Furthermore, the potentiation effects are at its

fullest by this day (Rasoanaivo, 2011). This is key to significantly increasing the extent of

epithelialization in the wounds. Refer to Appendix G for Mann-Whitney U results.

The significant difference in the two experimental groups is most likely caused by

the difference in temperature maintained throughout their respected procedures. Many

authors, including Asoiro, Kolmetz, Sloley, and Cacace believe that extracting essential

oil from plants is an intricate process and that there are many ways in how the percent

yield and the quality of the extract can be affected in procedures. The most vital aspect is

the temperature. The effect of variation of oil suggest a stable yet ideal temperature range

between 60-100°C. The rotary evaporation process was carried out in temperatures not

exceeding 60°C but steam distillation was maintained at 100°C. The higher temperature

was not very conducive at preserving the phytochemicals responsible for causing the

healing effect in the wound. It makes sense that all throughout the experiment, rotary

evaporation has had a constant higher percentage wound contraction compared to steam

distillation. Also in terms of epithelialization score, subjects that received rotary

evaporation ointment scored better compared to their other counterparts.

 56

The study conducted by Asoiro et al. (2011) revealed that temperature had

significant effect on oil yield with the maximum value being achieved by maintaining

60°C during extraction. Further increase in extraction temperature to 110°C reduced oil

yield. Optimum temperature conditions for maximum oil yield and quality was obtained

at 60°C. The temperature of the two methods of extraction might have affected the

phytochemicals not only in terms of weight, but also concentration. This further denotes

the importance of not exceeding the 60°C temperature mark. Another study that found of

60 °C as the optimum temperature of extraction was that of Cacace et al. (2003). The

polyphenols extracted in this temperature were of significantly higher quantity and

quality.

Rotary evaporation is also the ideal method of extraction for temperature sensitive

materials, in this case the phytochemicals in O. tenuiflorum. It removes solvents from the

mixture without damaging the product (Kolmetz and Sloley, 2004). Another reason why

it is used is because there are lower levels of residue build up and further sample loss.

.
 SUMMARY, CONCLUSION, AND RECOMMENDATIONS

Summary

Percentage wound contraction

During the early parts of the experiment, the positive control group was leading in

terms of mean percentage contraction. Through Duncan’s multiple range test, it was

found out that it was the only group to be significantly different from the other groups.

It was on day 5 that the progress of the rotary evaporation group began catching

up to the positive control group, as evidenced by both of them not being significantly

different from each other anymore. There was still no significant difference between

steam distillation and rotary evaporation despite the former having the greater mean.

Days 7, 9, and 11 have the same results and because they provide the most

reliable source of information. During this time the steam distillation group was

consistently significantly different compared to rotary evaporation group. Not only was it

very easily observable that the rotary evaporation group always had a greater effect on

the wound, it also during these days that it overtook the positive control group.

During day 13, there was a decline in the progress of both the rotary evaporation

and positive control groups. The steam distillation group was no longer significantly

different from the both of them. Most subjects were about to reach completely closed

wounds.

Finally on day 15, the only significant differences were between the blank control

group when compared to both steam distillation and positive control groups.
 58

Throughout the duration of the experiment there was no significant difference

between the negative control group and the blank ointment group.

Epithelialization

From the day 7 data, a Kruskal-Wallis H test showed that there was a significant

difference in epithelialization between the treatment groups, χ2(4)=12.539, p=0.014, with

a mean rank epithelialization score of 19.88 for Group A, 14.31 for Group B, 28.56 for

Group C, 14.00 for Group D and for 25.75 Group E.

Mann-Whitney U tests revealed that there was a significant difference in

epithelialization between group B and group C (U=11.000, Z=-2.348 p=0.019). This

indicates that the rotary evaporation ointment has a positive effect on the wound

epithelialization compared to the negative control group. Also, there was a significant

difference in epithelialization between group B and group E (U=14.000, Z=-2.061

p=0.039). This denotes that the blank ointment group does not have any effect on

epithelialization with it having the same results as the negative control. Furthermore,

there was a significant difference in epithelialization between group C and group D

(U=9.000, Z=-2.688, p=0.007). The positive control group also has a positive effect on

the epithelialization of the wound when compared to the negative control group. Just like

with the previous comparison involving the negative control group, There was a

significant difference in epithelialization between group D and group E (U=12.000, Z=-

2.440, p=0.015).

Conclusion
 59

Given the limitations of this study and in light of the statistical results, it can be

concluded that:

The five different treatment groups, comprised of Ocimum tenuiflorum steam

distillation ointment, Ocimum tenuiflorum rotary evaporation ointment, negative control,

positive control, and blank ointment, had significantly different effects on the excision

wound of Mus musculus in terms of percentage wound contraction and epithelialization.

The differences were most easily observable during days 7, 9 and, 11 of the 15-day

observation period.

Essential oil from steam distillation and extract from rotary evaporation both have

positive effects on the wound healing process but rotary evaporation is a better method of

extraction. Rotary evaporation is more effective at preserving the quality of the extract by

not damaging the temperature sensitive phytochemicals that are responsible for majority

of the healing effects caused by the plants. Furthermore, the positive effects on wound

healing caused by the rotary evaporation ointment are comparable to that of a commercial

antibacterial topical medicine.

Recommendations

It is strongly recommend that future researchers will:

1. Conduct the experiment on various wound models as opposed to a single wound

model in order to verify that such effects are indeed viable. A wide variety of

models have been developed that examine different aspects of the repair response,

both in vitro and in vivo. Advantages and disadvantages of each must be taken
 60

into consideration, along with relevant background information to help guide

decision-making.
2. Further increase the number of subjects during the experimentation process in

order to have a good statistical result as well as a better means of comparing and

contrasting various effects.

3. Conduct supplementary investigation with regards to the phytoconstiuent analysis

of the plant extract as well the identification of the active moieties and

elucidations of the mechanism of action of such constituents.

4. Validate the concentration of the extracts to be produced after the extraction

process. In other words, temperature and other factors must be considered in order

to avoid imbalances in the amount of extracts.

5. Perform experiments on the various by products left after the extraction process.

The analysis of bioactive compounds present in the byproducts of the plant

extracts involving the applications of common phytochemical screening assays

can be helpful in making sure that nothing in the extraction process goes to waste.

In other words, byproducts of plant extraction can be screened to be used in other

medicinal experiments. It is critical in order to be economically efficient and

productive.

`
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 APPENDICES

Appendix A
Handling Protocol

Safety Guidelines

1. Each person working on a protocol should know the details of procedures

approved for that protocol, and any others on which they are working.
2. Approach the rodent with confidence and handle the animals gently, but firmly.
Both too- rough handling and tentative approaches may result in bites or scratches to
the handler or injuries to the animal.
3. When handling animals, there is always the possibility of accidental release or the
animal being dropped. Most of these manipulations are best performed over a work
surface so that if the animal is dropped or escapes, it is not injured and can be easily
recaptured. Follow your institutional policies concerning animals that contact the
floor.
4. Never handle animals by the tip of the tail, as this may result in a degloving injury
of the tail. Be especially careful with large rats or pregnant mice. Always use the
other hand to support the body as you lift by the tail.
5. Sharp needles work best when giving injections. Although needles for laboratory
rodents are sometimes used for multiple injections, this is not advised for a number of
reasons, the least of which is that the small gauge often used means that the needles
dull quickly.
6. Being bitten or scratched is always a possibility when working with animals. If
working with a substance or an infectious agent that may cause injuries to humans,
take extra precautions, such manipulating animals or agents in fume hoods or
biosafety cabinets.
7. Gentle approaches and acclimation to handling before attempting a procedure can
pay off in animals that are less stressed by handling.
8. Practice restraint before attempting compound administration, and practice
administering substances to control animals before experimental animals.
9. Practicing these techniques regularly instills confidence and confidence results in
better handling, less stressed animals, and better scientific results.
10. With any handling technique, if the animal is recalcitrant, try a different technique.
The animal (and handler) may also benefit from putting the animal back in the cage
and trying again later.
 76

Taken from Manual Restraint and Common Compound Administration Routes in

Mice and Rats by Machholz, et al. (2012).

Manual Restraint

I. One-handed mouse restraint

1. Lift a mouse by the base of the tail and place it on the cage lid, wire
bar cage top, or a similar rough surface.
a. One handed mouse restraint is usually performed with the
non-dominant hand, leaving the dominant hand free for
use.
b. An alternative method allows the technician to use their
lab coat or uniform sleeve covering the forearm to position
the animal prior to restraint.

2. Tuck the base of the tail between the 3rd and 4th finger, while gently
pulling back on the tail. This will cause the mouse to grasp the
surface with all four paws and pull forward.
a. Do not grasp mice by the tip of the tail, especially if
suspending their entire bodyweight by their tail. This can
cause a degloving injury in which the skin of the tail slips
off.

3. Next, firmly grasp the mouse by the scruff with the same hand that is
holding the tail. Grasp with the index finger and thumb near the base
of the head and extend the grasp down the mouse's back by
incorporating the middle and ring fingers.
a. Be sure to apply just enough pressure, or firmness, to the
skin around the neck to prevent the mouse from turning or
twisting out of the restraint, but do not pull the skin so
tightly that the animal cannot breathe.
b. Control of the head is crucial. If the mouse can move its
head, it can reach the handler's fingers and may bite. This
may occur when novice handlers grasp the mouse too far
down the back, rather than right behind the skull.

II. Mouse restraint two-handed

 77

1. Lift a mouse by the base of the tail and place on the cage lid, wire bar
lid, or rough surface.
a. An alternative method allows the technician to use their
lab coat or uniform sleeve covering the forearm to position
the animal prior to restraint.

2. Pull gently backwards on the tail and the mouse will grasp the
surface with four paws and pull forward.

3. Next, with the other hand quickly and firmly grasp the mouse by the
scruff of the neck (see one handed restraint above).

4. With the tail in one hand and the scruff in the other, lift the mouse
and tuck the base of the tail between the palm and the 3rd or 4th finger
of the hand holding the scruff.
a. As with the one-handed method, firmly grasp the scruff to
prevent the mouse from twisting or turning while not
grasping so firmly that the animal cannot breathe.
b. If the mouse is resistant to scruffing, gentle pressure on the
mouse's back can allow the hand to move up for a better
grasp.

Subcutaneous (SC, SQ) Injection

1. Restrain the animal as described above. The animal must be restrained loosely
enough so that the skin may be mobilized.
2. If animals are to be handled routinely after SC injection, do not use the scruff
(nape of the neck). Instead, use the skin on the dorsal rump or the flank. If
animals are to receive multiple SC injections, alternate sites of injection.
3. Grasp the skin and gently pull it upwards, making a "tent".
1. If performing the injection solo, insert the needle and gently tent the skin
upwards with the needle to confirm that the needle is in the
subcutaneous space.
 78

4. Using an appropriately-sized syringe and needle, insert the needle at a 30-45°

angle into the tented skin, and inject the material. Inject parallel to and away
from the fingers holding the skin upwards.
5. If the injection is successful, a small swelling under the skin will be seen.
6. After injection, apply gentle pressure to prevent backflow of the material.

All handling protocol are taken from Manual Restraint and Common Compound
Administration Routes in Mice and Rats by Machholz, et al. (2012).

Appendix B
Disposal Protocol

1. All dead animals should be handled only while wearing gloves. There are
several types of gloves to choose from, including leather, rubber, and latex
gloves. Rubber or latex gloves are preferred due to their low cost, wide
availability, and ease of disinfecting (latex gloves are disposable).

2. The carcass should be placed in a plastic body bag and sealed as soon as
possible.

3. Avoid direct contact with the dead animal's body fluids (i.e., blood, urine,
feces). If contact does occur, wash the skin area contacted with soap and
water as soon as possible.

4. Avoid contact with the dead animal's external parasites (i.e., fleas and ticks).
If possible, spray the carcass with a flea & tick spray prior to handling it.

5. Proper disposal of the carcass (incineration, burying, etc.) is critical to

prevent exposure of other wildlife and humans to disease. Three common
effective methods of carcass disposal are: incineration, burying, and
rendering. Incineration is the preferred method to use when the carcass is
diseased; however, it can also be the most expensive. An acceptable
 79

alternative is to bury the carcass. The carcass should be buried at least 4 feet
deep and covered with lime to discourage scavengers from uncovering and
consuming it.

6. Persons who have direct contact with wildlife, especially carnivorous

animals, on a regular basis are highly recommended to receive the rabies
pre-exposure vaccination series. It is also recommended to have a rabies
antibody titer tested every two years to determine the level of protection.

Disposal protocol is patterned from the California Department of Fish and

Wildlife’s protocols for safe handling & disposal of carcasses (2010).

Appendix C
Raw Data
Wound Area (LengthxWidth)

Day 1 Day 9
E.O. R.V. NEG POS Blank E.O. R.V. NEG POS Blank
0.900 1.100 1.200 0.900 1.560 0.432 0.275 0.740 0.200 0.850
1.110 1.000 1.380 1.000 1.200 0.629 0.180 0.615 0.390 0.740
1.568 1.688 1.135 1.920 1.820 0.880 0.739 0.653 0.770 1.100
1.597 1.375 1.690 1.139 1.958 0.567 0.700 0.809 0.490 1.397
0.926 1.755 1.150 1.320 0.900 0.616 0.810 0.555 0.480 0.259
1.500 1.200 1.000 1.523 1.144 0.720 0.455 0.560 0.520 0.735
1.538 1.978 1.732 1.749 1.680 1.000 0.846 1.313 0.950 1.125
1.320 1.650 1.560 2.424 1.339 0.765 0.592 0.928 1.380 0.583

Day 3 Day 11
E.O. R.V. NEG POS Blank E.O. R.V. NEG POS Blank
0.840 1.000 1.100 0.850 1.430 0.300 0.128 0.539 0.060 0.752
1.000 0.950 1.320 0.903 1.100 0.248 0.140 0.520 0.160 0.560
1.554 1.590 1.025 1.440 1.796 0.413 0.315 0.727 0.461 0.718
1.434 1.212 1.582 0.893 1.872 0.300 0.240 0.485 0.390 1.044
0.810 1.560 1.100 1.100 0.810 0.455 0.525 0.390 0.149 0.200
1.440 1.080 0.990 1.400 1.080 0.600 0.360 0.282 0.300 0.540
 80

1.440 1.820 1.680 1.612 1.540 0.820 0.615 0.909 0.720 0.920
1.210 1.544 1.368 2.220 1.260 0.640 0.490 0.795 0.763 0.518

Day 5 Day 13
E.O. R.V. NEG POS Blank E.O. R.V. NEG POS Blank
0.630 0.723 0.980 0.511 1.188 0.179 0.010 0.158 0.030 0.358
0.931 0.698 1.040 0.638 1.029 0.020 0.010 0.238 0.060 0.325
1.370 1.485 1.023 1.265 1.586 0.303 0.200 0.554 0.209 0.569
1.247 1.018 1.358 0.789 1.623 0.075 0.175 0.425 0.250 0.783
0.720 1.380 1.000 1.000 0.723 0.200 0.275 0.158 0.060 0.120
1.265 1.050 0.800 1.100 1.002 0.420 0.140 0.140 0.100 0.270
1.320 1.608 1.620 1.599 1.404 0.632 0.306 0.624 0.328 0.480
1.122 1.350 1.243 2.048 1.117 0.536 0.279 0.578 0.615 0.362

Day 7 Day 15
E.O. R.V. NEG POS Blank E.O. R.V. NEG POS Blank
0.536 0.504 0.902 0.475 1.053 0.000 0.000 0.020 0.000 0.040
0.704 0.588 0.815 0.488 0.855 0.010 0.000 0.020 0.000 0.060
1.080 1.104 0.846 0.963 1.320 0.153 0.010 0.323 0.021 0.341
0.846 0.800 1.094 0.640 1.610 0.000 0.030 0.183 0.176 0.420
0.720 1.020 0.747 0.657 0.511 0.020 0.120 0.010 0.000 0.020
0.960 0.736 0.630 0.900 0.976 0.120 0.060 0.000 0.030 0.120
1.210 1.210 1.430 1.369 1.320 0.098 0.075 0.438 0.020 0.590
1.020 0.900 1.097 1.720 0.765 0.313 0.060 0.451 0.010 0.205

Percentage Contraction

Day 3 Day 11
E.O. R.V. NEG POS Blank E.O. R.V. NEG POS Blank
6.67 9.09 8.33 5.56 8.33 55.08 93.33 51.79 66.67 88.36
9.91 5.00 4.35 9.75 8.33 62.32 84.00 53.33 77.70 86.00
0.84 5.81 9.69 25.00 1.35 35.96 76.00 60.56 73.68 81.33
10.18 11.85 6.39 21.61 4.37 71.33 65.74 46.67 81.21 82.55
12.55 11.11 4.35 16.67 10.00 66.09 88.73 77.78 50.88 70.09
4.00 10.00 1.00 8.05 5.59 71.80 80.30 52.79 60.00 70.00
6.35 7.99 3.02 7.81 8.33 47.53 58.82 45.24 46.68 68.91
8.33 6.44 12.31 8.40 5.90 49.01 68.53 61.31 51.52 70.31
Mean 7.35 8.41 6.18 12.9 6.53 Mean 57.39 76.93 56.19 63.54 77.19

Day 5 Day 13
E.O. R.V. NEG POS Blank E.O. R.V. NEG POS Blank
30.00 34.32 18.33 43.22 23.88 80.16 99.09 86.88 96.67 77.08
16.13 30.25 24.67 36.25 14.25 98.20 99.00 82.78 94.00 72.92
12.58 12.00 9.87 34.11 12.86 80.70 88.15 51.21 89.11 68.71
21.92 26.00 19.64 30.74 17.11 95.30 87.27 74.85 78.04 60.02
22.27 21.37 13.04 24.24 19.72 78.41 84.33 86.30 95.45 86.67
15.67 12.50 20.00 27.75 12.40 72.00 88.33 86.00 93.43 76.40
14.15 18.71 6.49 8.55 16.43 58.90 84.53 63.98 81.24 71.43
15.00 18.18 20.32 15.52 16.62 59.39 83.09 62.95 74.62 72.98
 81

Mean 18.46 21.67 16.55 27.55 16.66 Mean 77.88 89.22 74.37 87.82 73.28

Day 7 Day 15
E.O. R.V. NEG POS Blank E.O. R.V. NEG POS Blank
40.44 54.18 24.80 47.28 32.50 100 100 98.33 100 97.44
36.58 41.20 40.96 51.25 28.75 99.10 100 98.55 100 95.00
31.10 34.59 25.42 49.84 27.47 90.27 99.41 71.52 98.91 81.26
46.98 41.82 35.30 43.79 17.75 100 97.82 89.17 84.54 78.54
22.27 41.88 35.04 50.23 43.22 97.84 93.16 99.13 100 97.78
36.00 38.67 37.00 40.89 14.68 92.00 95.00 100 98.03 89.51
21.31 38.83 17.46 21.72 21.43 93.63 96.21 74.72 98.86 64.90
22.73 45.45 29.67 29.01 42.87 76.31 96.36 71.06 99.59 84.68
Mean 32.18 42.08 30.71 41.75 28.58 93.64 97.25 87.81 97.49 86.14
Day 9
E.O. R.V. NEG POS Blank
52.00 75.00 38.38 77.78 45.51
43.33 82.00 55.43 61.00 38.38
43.86 56.20 42.45 59.92 39.56
64.48 49.09 52.16 56.96 28.63
33.50 53.85 51.74 63.64 71.28
52.00 62.08 44.00 65.85 35.75
34.96 57.23 24.24 45.67 33.04
42.05 64.12 40.50 43.06 56.45
Mean 45.77 62.45 43.61 59.23 43.57
Epithelialization Scores

Day 1 Day 9
E.O. R.V. NEG POS Blank E.O. R.V. NEG POS Blank
5 5 5 5 5 3 2 3 2 3
5 5 5 5 5 2 2 2 2 3
5 5 5 5 5 2 2 4 3 3
5 5 5 5 5 3 2 3 2 3
5 5 5 5 5 3 2 3 2 2
5 5 5 5 5 3 2 2 3 3
5 5 5 5 5 2 3 2 2 2
5 5 5 5 5 2 2 3 2 2

Day 3 Day 11
E.O. R.V. NEG POS Blank E.O. R.V. NEG POS Blank
4 4 5 4 5 1 1 2 1 1
5 4 4 4 4 1 1 1 1 2
4 4 5 5 5 1 2 3 2 3
4 4 5 5 5 2 1 3 2 2
5 5 5 4 4 3 2 3 1 1
4 4 4 4 5 2 2 2 2 3
4 5 4 4 5 1 2 1 1 1
4 4 5 4 4 1 1 1 1 1
 82

Day 5 Day 13
E.O. R.V. NEG POS Blank E.O. R.V. NEG POS Blank
4 3 4 4 4 1 1 1 1 1
3 3 3 3 4 1 1 1 1 1
3 4 4 4 4 1 1 2 1 2
4 4 4 4 4 2 1 2 1 2
4 4 5 3 3 2 1 2 1 1
3 3 3 4 4 1 2 1 2 2
3 4 4 3 4 1 1 1 1 1
3 3 4 4 3 1 1 1 1 1

Day 7 Day 15
E.O. R.V. NEG POS Blank E.O. R.V. NEG POS Blank
4 2 4 3 4 1 1 1 1 1
2 3 3 2 4 1 1 1 1 1
3 3 4 3 4 1 1 1 1 1
4 3 4 3 3 1 1 1 1 1
3 3 5 2 3 1 1 1 1 1
3 2 3 3 4 1 1 1 1 1
3 4 3 3 3 1 1 1 1 1
3 2 4 3 3 1 1 1 1 1

Appendix D
ANOVA

Day 3

ANOVA
D3PercentContraction

Sum of Squares df Mean Square F Sig.

Between Groups 234.449 4 58.612 3.086 .028

Within Groups 664.756 35 18.993
Total 899.206 39
 83

Day 5
ANOVA
D5PercentContraction

Sum of Squares df Mean Square F Sig.

Between Groups 680.341 4 170.085 3.063 .029

Within Groups 1943.234 35 55.521
Total 2623.575 39

Day 7
ANOVA
 84

D7PercentContraction

Sum of Squares df Mean Square F Sig.

Between Groups 1305.849 4 326.462 3.923 .010

Within Groups 2912.375 35 83.211
Total 4218.223 39

Day 9
ANOVA
D9PercentContraction

Sum of Squares df Mean Square F Sig.

Between Groups 2686.709 4 671.677 5.213 .002

Within Groups 4509.796 35 128.851
Total 7196.505 39
 85

Day 11

ANOVA
D11PercentContraction

Sum of Squares df Mean Square F Sig.

Between Groups 3302.520 4 825.630 4.725 .004

Within Groups 6115.165 35 174.719
Total 9417.686 39

Day 13

ANOVA
 86

D13PercentContraction

Sum of Squares df Mean Square F Sig.

Between Groups 1810.490 4 452.623 4.007 .009

Within Groups 3953.443 35 112.956
Total 5763.934 39

Day 15
ANOVA
D15PercentContraction

Sum of Squares df Mean Square F Sig.

Between Groups 889.532 4 222.383 2.788 .041

Within Groups 2791.464 35 79.756
Total 3680.996 39
 87

Appendix E
Duncan’s Multiple Range Test
Day 3
D3PercentContraction
Duncan

Group N Subset for alpha = 0.05

1 2

Negative 8 6.1800
Blank 8 6.5250
Essential Oil 8 7.3538
 88

RotaVap 8 8.4113
Positive 8 12.8563
Sig. .359 1.000

Day 5
D5PercentContraction
Duncan

Group N Subset for alpha = 0.05

1 2

Negative 8 16.5450
Blank 8 16.6588
Essential Oil 8 18.4650
RotaVap 8 21.6663 21.6663
Positive 8 27.5475
Sig. .219 .123

Day 7
D7PercentContraction
Duncan

Group N Subset for alpha = 0.05

1 2

Blank 8 28.5838
Negative 8 30.7063
Essential Oil 8 32.1763
Positive 8 41.7513
RotaVap 8 42.0775
Sig. .464 .943

Day 9
D9PercentContraction
Duncan

Group N Subset for alpha = 0.05

1 2

Blank 8 43.5750
Negative 8 43.6125
Essential Oil 8 45.7725
Positive 8 59.2350
RotaVap 8 62.4463
 89

Sig. .719 .575

Day 11
D11PercentContraction
Duncan

Group N Subset for alpha = 0.05

1 2

Blank 8 56.1838
Negative 8 57.3900
Essential Oil 8 63.5425
Positive 8 76.9313
RotaVap 8 77.1938
Sig. .233 .964

Day 13

D13PercentContraction
Duncan

Group N Subset for alpha = 0.05

1 2 3

Blank 8 73.2763
Negative 8 74.3688
Essential Oil 8 77.8825 77.8825
Positive 8 87.8200 87.8200
RotaVap 8 89.2238
Sig. .421 .070 .793

Day 15
D15PercentContraction
Duncan

Group N Subset for alpha = 0.05

1 2

Blank 8 86.1387
Negative 8 87.8100 87.8100
Essential Oil 8 93.6438 93.6438
RotaVap 8 97.2450
Positive 8 97.4913
Sig. .121 .054
90
 91

Appendix F
Kruskal-Wallis

Day 3
Test Statisticsa,b

D3Epithelializati Day 11
on

Chi-Square 5.484 Test Statisticsa,b

df 4 D11Epithelializati
Asymp. Sig. .241 on

Chi-Square 2.746
df 4
Day 5
Asymp. Sig. .601
Test Statisticsa,b

D5Epithelializati
on Day 13
Chi-Square 3.998 Test Statisticsa,b

df 4 D13Epithelializat
Asymp. Sig. .406 ion

Chi-Square 2.600

Day 7 df 4
Asymp. Sig. .627
a,b
Test Statistics

D7Epithelializati
Day 15
on
Test Statisticsa,b
Chi-Square 12.539
D15Epithelializat
df 4
ion
Asymp. Sig. .014
Chi-Square .000
df 4
Day 9
Asymp. Sig. 1.000
Test Statisticsa,b

D9Epithelializati
on

Chi-Square 6.872
df 4
Asymp. Sig. .143
 92

Appendix G
Mann-Whitney U

A vs E
A vs B
Test Statisticsa
Test Statisticsa
D7Epithelializati
D7Epithelializati
on
on
Mann-Whitney U 22.000
Mann-Whitney U 22.500
Wilcoxon W 58.000
Wilcoxon W 58.500
Z -1.195
Z -1.113
Asymp. Sig. (2-tailed) .232
Asymp. Sig. (2-tailed) .266
Exact Sig. [2*(1-tailed Sig.)] .328b
Exact Sig. [2*(1-tailed Sig.)] .328b

B vs C*
A vs C
Test Statisticsa
a
Test Statistics
D7Epithelializati
D7Epithelializati
on
on
Mann-Whitney U 11.000
Mann-Whitney U 17.500
Wilcoxon W 47.000
Wilcoxon W 53.500
Z -2.348
Z -1.677
Asymp. Sig. (2-tailed) .019
Asymp. Sig. (2-tailed) .094
Exact Sig. [2*(1-tailed Sig.)] .028b
b
Exact Sig. [2*(1-tailed Sig.)] .130

B vs D
A vs D
Test Statisticsa Test Statisticsa
D7Epithelializati D7Epithelializati
on on
Mann-Whitney U 22.000 Mann-Whitney U 31.000
Wilcoxon W 58.000 Wilcoxon W 67.000
Z -1.284 Z -.123
Asymp. Sig. (2-tailed) .199 Asymp. Sig. (2-tailed) .902
Exact Sig. [2*(1-tailed Sig.)] .328b Exact Sig. [2*(1-tailed Sig.)] .959b

B vs E
 93

Test Statisticsa D7Epithelializati

D7Epithelializati on
on Mann-Whitney U 12.000
Mann-Whitney U 14.000 Wilcoxon W 48.000
Wilcoxon W 50.000 Z -2.440
Z -2.061 Asymp. Sig. (2-tailed) .015
Asymp. Sig. (2-tailed) .039 Exact Sig. [2*(1-tailed Sig.)] .038b
Exact Sig. [2*(1-tailed Sig.)] .065b

C vs D*
Test Statisticsa

D7Epithelializati
on

Mann-Whitney U 9.000
Wilcoxon W 45.000
Z -2.688
Asymp. Sig. (2-tailed) .007
Exact Sig. [2*(1-tailed Sig.)] .015b

C vs E

Test Statisticsa

D7Epithelializati
on

Mann-Whitney U 26.000
Wilcoxon W 62.000
Z -.707
Asymp. Sig. (2-tailed) .480
Exact Sig. [2*(1-tailed Sig.)] .574b

D vs E
Test Statisticsa
 94

Appendix H
Wound Healing Closure of Representative Subjects

Group A Group B Group C Group D

Day 3

Day 5

Day 7

Day 9

Day 11

Day 13

Day 15
 95

Appendix I
Pictures

View of Laboratory from National Highway

Conducting and observing Filtration Process

 96

Researchers with Herbanext™ Laboratories personnel

Researchers in study site

 97

Mouse handling technique sample