CHAPTER 1
Introduction
1
Plant has a pivotal role in medicine through out all recorded history,
in virtually all human cultures. Indeed, plants are foundation upon which
effective medicine has been built. Often discoveries about plant medicines
have catalyzed the progress of basic medical research. There has been a
resurgence of interest in plants as sources of medicines in the recent decades.
80% of the world population relies on medicinal plants for their
primary health care. Such herbal medicines that are easily available, cheaper,
time tested and considered safer than most of modern synthetic drugs. Over
50% of the best selling pharmaceuticals in use today were derived from
natural products. By providing scientific information on medicinal plants,
which influence its productivity, enhance its therapeutic efficiency and
competitiveness in the field of medicine and pharmacy.
Over the past decade, herbal medicine has become an item of global
importance both medicinal and economical. Although usage of these herbal
medicines has increased, their quality, safety and efficiency are serious
concerns in industrialized and developing countries. Thus accurate scientific
assessment has become a prerequisite for acceptance of herbal health claims.
Pothos scandens Linn. (Family: Araceae) have a great medicinal value for
its wound and burn healing properties. The plant is seen many parts of South
India especially in wild areas. Traditionally the plant is used by Ayurvedic
physicians of Cheruvathur, Kerala mainly for its burn healing properties. For
natural product discovery the conventional approach of extraction,
identification and characterization of compounds, test for desired biological
activity and finally formulating in a suitable dosage forms. Ayurveda based
drug discovery uses “Reverse Pharmacology” in which drug candidates are
first identified based on large-scale use in population, and then validated in
clinical trials. Experts say this approach can cut time from 12 to 15 years for
drug discovery and are economical.
India is the largest producer of medicinal herbs and is called the
botanical garden of world. There is about approximately 45,000 plant
species in India, with concentrated hot spots in the region of eastern
Himalayas, Western Ghats and Andaman and Nicobar Islands. The officially
documented plants with medicinal potential are about 3000 but traditional
practitioners use more than 6,0001.
2
CHAPTER 1.1
Importance of Phytochemical
Screening
3
Plants and trees had always been a rich source of lead compounds
(e.g. Morphine, Cocaine, Digitalis, Quinine, Tubocurarine, Nicotine,
Muscarnine, and many others). Many lead compounds are useful drugs in
themselves (e.g. Morphine and Quinine), while others have been basis for
synthetic drugs (e.g. local anaesthetics developed from Cocaine). Plants
remain a promising source of new drugs which have recently been isolated
from plants include anticancer agent from Taxol from the yew tree, and the
antimalarial agent from the artemisinin from a Chinese plant.
Plants provide a bank of rich, complex and highly varied structures,
which are unlikely to be synthesized in laboratories. Furthermore, evolution
has already carried out a screening process whereby plants are more likely to
survive if they contain potent compounds, which deter animals or insects
from eating them. These potent compounds are secondary metabolites with
quite complex structures, in which most of them are biologically active
compounds. It is sobering that very few plants were been fully studied and
the vast majorities have not been studied at all.
It is often worthwhile studying the medical folklore of ancient
civilizations depended greatly on local flora and fauna for their survival.
Therefore, study of medical folklore can give clues as to which plants might
be worth studying in more detail. In general, natural products are particularly
good at providing radically new chemical structure which no chemist would
dream of synthesizing. As a preliminary of phytochemical screening,
specific test for secondary metabolites were performed for alkaloids,
glycosides, flavanoids, proteins & amino acids, gum & mucilage,
carbohydrates.
4
CHAPTER 1.2
Gels as topical application
5
Gels as topical application2,3,4
Gels are semisolid system in which a liquid phase is constrained
within a three dimensional polymeric matrix [natural or synthetic gums] in
which a high degree of physical or sometimes chemical cross linking has
been introduced some of these systems are as clear as water in appearance,
aesthetically pleasing as in gelatin deserts and other turbid. Their clarity
ranges from clear to a whitish translucence2.
Gels are divided into inorganic [two-phase system] and organic
[single phase] gels on the basis of nature of the colloidal phase. In a two
phase system, the gel mass consists of a network of small discrete particles
[e.g. Aluminum hydroxide gel]. If the particle size of the dispersed phase is
relatively large, the gel mass sometimes is referred to as magma [e.g.
Bentonite Magma].
Single phase gels consist of organic macromolecules distributed
uniformly throughout a liquid in such a manner that no apparent boundaries
exist between the dispersed macromolecules [e.g. Carbopol] or from natural
gums [e.g. Tragacanth]. Although these gels are commonly aqueous, alcohol
and oils may be used as the continuous phase.
Likewise the nature of the solvent determines whether the gel is a
Hydrogel [i.e. water based] e.g. Bentonite or an Organogel [with a nonaqueous solvent] e.g. Plastibase.
Solid gels with low solvent concentration are known as Xerogels.
They are often produced by evaporation of the solvent, leaving the gel
framework behind. The can be referred to the gel sate by introduction of an
agent, which on imbibitions, swells the gel matrix. Examples of Xerogels,
include Tragacanth ribbons, acacia tears, dry cellulose and polystyrene
Gels can be prepared from a number of pharmaceutical agents such as
Tragacanth, [2-5%], Sodium alginate [2-10%], Gelatin [2-15%], Methyl
cellulose [2-4%], Sodium carboxy methyl cellulose [2-5%], Carbopol [0.35%] or Polyvinyl alchohol [10-20%]. Other gelling agents include Methyl
hydroxyethyl cellulose, Hydroxymethyl cellulose, Poly oxy ethylene,
Polyoxypropylene [poloxamers] and metallic stearates.
6
Preservatives are to be incorporated into the gels especially for those
prepared from natural sources Preservative should be incorporated into the
gels especially for those prepared from natural sources. Appropriate
preservatives depending upon the use and the gelling agent include the
Parabens [0.2%], Benzoic acid [0.2%] and Chlorocresol [0.1%]
The gels, particularly the single-phase gels, are being used more
frequently in pharmacy and cosmetics because of several properties such as
1. Semisolid state
2. High degree of clarity
3. Ease of application
4. Ease of removal and use.
The gels often provide a faster release of drug substances,
independent of water solubility of the drug as compared to creams and
ointments.
GEL CHARACTERISTICS
Ideally, gelling agents for pharmaceutical and cosmetic use should be
inert, safe and non-reactive to other formulation components. The gelling
agent should a provide responsible solid like nature during storage that can
be broken easily when subjected to the shear forces generated by squeezing
a tube or during topical application. The gel should exhibit little viscosity
change under the temperature variations of normal use and storage. The gel
characteristics should match the intended use. A topical gel should not be
tacky.
FORMULATION CONSIDERATIONS
In the formulation of gel, the efficacy is often dependent on the
composition of the vehicle. The ability of a drug in gel formulation to
penetrate the skin and exert its effect depends on to consecutive physical
events. The drug must first diffuse out of the vehicle to the skin surface and
then, it must penetrate the natural barrier to enter into the site of action.
Many so called ‘vehicle effects’ reported in the literature are the
consequences of this to diffusion processes. Depending on which process is
slower, either event could determine the overall effectiveness of the topical
7
gel dosage form. These two processes are intimately related and are
dependent upon physicochemical properties of the drug, vehicle and barrier.
FORMATION OF GEL
All polymer solutions are possible prone to setting to gels because the
solute consists of long and flexible chains of molecular thickness that
become entangled & attract each other by secondary valency forces. When
the three dimensional polymerization of multifunctional monomers reaches a
given conversion, gelation occurs at a sharp gel point. Cross-linking of
dissolved polymer molecule also causes their solutions to gel, both type of
reaction produce permanent gels held together by primary valence forces.
A gel often contract on standing and some of the interstitial liquid is
squeezed out. This phenomenon called syneresis is due to crystallization or
to the formation of additional contact points between polymer segments on
aging. In the case of irreversible gels formed by 3-dimensional
polymerization, continuing cross-linking or poly condensation reaction
tighten the polymer network and shrink the solid phase.
Even though varieties of structures are associated with a gel networks,
most of the pharmaceutical gels are random coil networks. Random coil
gelation mechanisms are rooted inter polymer – polymer and polymer –
solvent interaction. With a given polymer, the gel net work increases in
strength with increase in polymer concentration. These results in a reduction
of inter particle distance, which subsequently leads to chain entanglement
and further development of cross-links. Continual addition of polymer
strengthens the gel network and results in increased viscoelasticity.
Although the gel network is formed through polymeric interactions,
the nature of the polymer-solvent affinity, actually determines the integrity
of the gel. Classical gel theory is distinguished between three categories of
solvents:
1. Free solvents that are very mobile.
2. Solvent bound as a salvation layer usually through hydrogen
bonding.
3. Solvent entrapped with in the network structure.
8
The ratio of three solvent types in a given gel, are dependent on the polymer
concentration and the solvent affinity for the polymer .solvent affinity
governs the extension of this random coil. The greater the solvent affinity
the more the coil expands and entangles with adjacent coils to form crosslinks.
In a good solvent the polymer chains are interpenetrated by solvent
molecules and the solvation layer is enhanced, which facilitates random
expansion and network formation. In a poor solvent, the polymer chains
contract, to minimize the solvent contact, thereby reducing the effective
number of cross links and weakens the gel network structure.
Gelation theory can be readily applied when formulating gel products
and some of the desirable attributes of the gel formulations are in the
following order. For optimum consumer appeal, the gel should have good
optical clarity and sparkling appearance. To preserve product integrity, the
gel should maintain its viscosity at all temperatures that may be countered
during shipment and storage.
9
CHAPTER 1.3
Skin Characteristics
10
Structure of Skin5
11
Anatomy and Organization of Human Skin5
The skin constitutes one of the largest interfaces between the body
and the environment. One the other hand, the function of human skin is to
protect our body against physical, chemical, microbes, loss of water and
other endogenous substances; on the other hand it is involved in
thermoregulation of the body and serves as an excretory organ. This
bifunctional nature of the skin depends on its highly differentiated structure
of the skin. Understanding the skin absorption process, such as safety
aspects of chemicals, other xenobiotics and cosmetic formulations and
utilizing the opportunity to deliver drug substances to the skin further to
systemic circulation it is essential to study the structure of skin.
Functions
Skin performs the following functions:
1. Protection: an anatomical barrier from pathogens and damage between
the internal and external environment in bodily defense; Langerhans cells
in the skin are part of the adaptive immune system.
2. Sensation: contains a variety of nerve endings that react to heat and cold,
touch, pressure, vibration, and tissue injury; see somatosensory system
and haptics.
3. Heat regulation: the skin contains a blood supply far greater than its
requirements which allows precise control of energy loss by radiation,
convection and conduction. Dilated blood vessels increase perfusion and
heat loss while constricted vessels greatly reduce cutaneous blood flow
and conserve heat. Erector pili muscles are significant in animals.
4. Control of evaporation: the skin provides a relatively dry and
impermeable barrier to fluid loss. Loss of this function contributes to the
massive fluid loss in burns.
5. Aesthetics and communication: others see our skin and can assess our
mood, physical state and attractiveness.
6. Storage and synthesis: acts as a storage center for lipids and water, as
well as a means of synthesis of vitamin D by action of UV on certain
parts of the skin.
12
7. Excretion: sweat contains urea, however its concentration is 1/130th that
of urine, hence excretion by sweating is at most a secondary function to
temperature regulation.
8. Absorption: Oxygen, nitrogen and carbon dioxide can diffuse into the
epidermis in small amounts, some animals using their skin for their sole
respiration organ. In addition, medicine can be administered through the
skin, by ointments or by means of adhesive patch, such as the nicotine
patch or iontophoresis. The skin is an important site of transport in many
other organisms.
9. Water resistance: The skin acts as a water resistant barrier so essential
nutrients aren't washed out of the body.
Components of normal human skin
Human skin consists of a stratified, cellular epidermis and an
underlying dermis of connective tissue. The dermal-epidermal junction is
undulating in section; ridges of the epidermis known as rete ridges, project
into the dermis. The junction provides mechanical support for the epidermis
and acts as a partial barrier against exchange of cells and large molecules
below the dermis is a fatty layer, the panniculus adiposus, usually designate
as‘subcutaneous’. This is separated from the rest of the body by a vestigial
layer of striated muscle, the panniculus carnosus.
There are two main kinds of human skin. Glabrous skin (Non-hairy
skin), found on the palms and soles, is grooved on its surface by
continuously alternating ridges and sulci, in individually unique
configurations known as dermatoglyphics. It is characterized by a thick
epidermis divided into several well marked layers including a compact
stratum corneum, by the presence of encapsulated sense organs within the
dermis, and by a lack of hair follicles and sebaceous glands.
Hair bearing skin on the other hand, has both hair follicles and
sebaceous glands but lacks encapsulated sense organs. There is also wide
variation between different body sites. For example, the scalp, with its large
hair follicles may be contrasted with the forehead, which has only small
vellus-producing follicles with albeit associated with large sebaceous glands.
The axilla is notable because it has apocrine glands in addition to the eccrine
sweat glands, which are found throughout the body.
13
Anatomical Structure of Human Skin
The multitude of different functions of human skin can be achieved by
unique anatomical structure of different skin layers. These are as follows:




Epidermis
Dermis
Subcutaneous tissue
Skin appendages
EPIDERMIS
The epidermis is composed of stratified squamous epithelium and
contains four different types of cells. They are:
1. Keratinocytes
2. Melanocytes
3. Langerhans cells
4. Granstein cells
The keratinocytes of the epidermis are organized into the following
layers from superficial to deepest region. Because of practical reasons the
human epidermis can be divided into:
 Stratum corneum(horny layer)
 Stratum lucidum
 Stratum granulosum (granular layer)
 Stratum spinosum (prickly cell layer)
 Stratum germinativum (basal layer and dermoepidermal
junction).
The multilayer envelope of epidermis varies in thickness, depending
on the cell size and number of layers, ranging from about 0.8mm on the
palms and the soles, down to 0.06mm.
Cells provide epithelial tissue differ from those of all other organs.
The cells that ascend from the proliferative layer of basal cells, they do
change in an ordered fashion from metabolically active dividing cells to
dense, dead, keratinized protein.
14
LAYERS OF EPIDERMIS5
15
Stratum corneum (horny layer)
This is the main barrier function being located in the outermost skin.
It consists of separated, nonviable, cornified, almost non permeable
corneocytes embedded into a continuous lipid bilayer made up of various
classes of lipids, for example: ceramides, cholesterol, cholesterol esters, free
fatty acids and triglycerides. Stratum corneum is crucial for controlling the
percutaneous absorption of dermally applied substances and regulating fluid
homeostasis. The selective permeability of its elegant structure provides a
central theme in many aspects of design of cosmetics. The thickness of
stratum corneum is usually 10-25 micrometer with exceptions in the soles
and palms, and it swells several folds when hydrated. All components of
stratum corneum originate from the basal layer of epidermis, the stratum
germinativum.
Stratum lucidum (prickly cell layer)
These are present only at the palm of the hand and soles of the foot.
The cells are non nuclear. It has an anatomically distinct, poorly staining
hyaline zone forms a thin layer.
Stratum granulosum (granular layer)
The stratum granulosum (or granular layer) is a layer of the epidermis
found between the stratum corneum (and possibly stratum lucidum) and
stratum spinosum. In this layer, keratinocytes are now called granular cells,
and contain keratohyalin and lamellar granules.
Stratum spinosum
The stratum spinosum (or spinous layer) is a layer of the epidermis
found between the stratum granulosum and stratum basale. This layer is also
referred to as the "spinous" or "prickle-cell" layer. Keratinization begins in
the stratum spinosum. Cells that move into the spinosum layer (also called
prickle cell layer) change from being columnar to polygonal. In this layer the
cells start to synthesize keratin.
16
Stratum germinativum
The Stratum germinativum (or basal layer, stratum basale) is the
deepest layer of the epidermis, a continuous layer of cells often described as
one cell thick, though it may be two to three cells thick in glabrous skin and
hyperproliferative epidermis. The basal cells of this layer can be considered
the "stem cells" of the epidermis, undifferentiated, proliferating, and creating
daughter cells that migrate upward, beginning the process of differentiation.
DERMIS
The dermis is a layer of skin between the epidermis and subcutaneous
tissues, and is composed of two layers, the papillary and reticular dermis.
Structural components of the dermis are collagen, elastic fibers, and
extrafibrillar matrix (previously called ground substance). It is about 0.2 to
0.3cm thick. The elasticity of the skin is due to the network or gel structure
of the cells.
The upper papillary layer, contain a thin arrangement of collagen
fibers. The lower reticular layer is made up of thick collagen fibers arranged
parallel.
SUBCUTANEOUS TISSUE
The subcutaneous tissue is a layer of fat that lies between the dermis
of the skin and underlying fascia. Subcutaneous fat insulates the body,
absorbs trauma, and is a reserve energy source. This tissue may be further
divided into two components, the actual fatty layer, or panniculus adiposus,
and a deeper vestigial layer of muscle, the panniculus carnosus. The
subcutaneous layer contains fat and connective tissue that houses larger
blood vessels and nerves. This layer is important is the regulation of
temperature of the skin itself and the body. The size of this layer varies
throughout the body and from person to person.
SKIN APPENDAGES
Skin appendages are appendages that are associated with the skin and
serve a particular function. In humans some of the more common skin
appendages are hairs (sensation, heat loss, filter for breathing, protection),
arrector pilli (smooth muscles that pull hairs straight), sebaceous glands
17
(secrete sebum onto hair follicle to oil the hair), sweat glands (can be sweat
secreted with strong odour (apocrine) or with a faint odour (eccrine)) and
nails (protection).
FACTORS IN SKIN PENETRATION
Factor that influence skin penetration are the physicochemical
properties of drug and vehicle, pH and concentration.
Different physiological variables involve the condition of the skin
i.e. whether intact or injured, the skin age, the area of the skin treated
thickness of the skin barrier phase, the species variation and the skin
moisture content.
The principle physicochemical factor in skin penetration is the
hydration state of stratum corneum, which affect the rate of passage of all
substances that penetrate the skin.
The solubility of a drug determines the concentration presented to the
absorption site and the water/lipid partition coefficient influences the rate of
transport.
An inverse relation ship appears to exist between absorption rate and
molecular weight.
18
CHAPTER 1.4
Wound Healing
19
Introduction5,6
Wound healing is a natural restorative response to tissue injury.
Healing is the interaction of a complex cascade of cellular events that
generates resurfacing, reconstitution, and restoration of the tensile strength
of injured skin. Healing is a systematic process, traditionally explained in
terms of 3 classic phases: inflammation, proliferation, and maturation. A clot
forms and inflammatory cells debride injured tissue during the inflammatory
phase. Epithelialization, fibroplasia, and angiogenesis occur during the
proliferative phase. Meanwhile, granulation tissue forms and the wound
begin to contract. Finally, during the maturation phase, collagen forms tight
cross-links to other collagen and with protein molecules, increasing the
tensile strength of the scar.
For the sake of discussion and understanding, the process of wound
healing may be considered a series of separate events. In actuality, the entire
process is much more complicated, as cellular events that lead to scar
formation occur in tandem. Many aspects of wound healing have yet to be
elucidated. Surgeons should have an understanding of the process of wound
healing to help produce scars that are cosmetically pleasing and do not
impair function.
TYPES OF WOUND HEALING
Wounds may be broadly classified into categories:
 Closed wounds
 Open wounds
Open wounds
Open wounds can be classified according to the object that caused the
wound. The types of open wound are:


Incisions or incised wounds, caused by a clean, sharp-edged object
such as a knife, a razor or a glass splinter.
Lacerations, irregular tear-like wounds caused by some blunt trauma.
The term laceration is commonly misused in reference to incisions.
20




Abrasions (grazes), superficial wounds in which the topmost layer of
the skin (the epidermis) is scraped off. Abrasions are often caused by
a sliding fall onto a rough surface.
Puncture wounds, caused by an object puncturing the skin, such as a
nail or needle.
Penetration wounds, caused by an object such as a knife entering the
body.
Gunshot wounds, caused by a bullet or similar projectile driving into
or through the body. There may be two wounds, one at the site of
entry and one at the site of exit, such is generally known as a throughand-through.
Closed wounds
Closed wounds have fewer categories, but are just as dangerous as open
wounds. The types of closed wounds are:



Contusions, more commonly known as bruises, caused by blunt force
trauma that damage tissue under the skin.
Hematomas, also called blood tumors, caused by damage to a blood
vessel that in turn causes blood to collect under the skin.
Crushing injuries, caused by a great or extreme amount of force
applied over a long period of time.
INFLAMMATORY PHASE
The early events of wound healing are characterized by the
inflammatory phase, a vascular and cellular response to injury. An incision
made through a full thickness of skin causes a disruption of the
microvasculature and immediate hemorrhage. Following incision of the skin,
a 5 to 10 minute period of vasoconstriction ensues, mediated by epinephrine,
norepinephrine,
prostaglandins,
serotonin,
and
thromboxane.
Vasoconstriction causes temporary blanching of the wound and functions to
reduce hemorrhage immediately following tissue injury, aid in platelet
aggregation, and keep healing factors within the wound.
Endothelial cells retract to expose the sub endothelial collagen
surfaces; platelets attach to these surfaces. Adhesion to exposed collagen
surfaces and to other platelets occurs through adhesive glycoproteins:
fibrinogen, fibronectin, thrombospondin, and von Willebrand factor. The
21
aggregation of platelets results in the formation of the primary platelet plug.
Aggregation and attachment to exposed collagen surfaces activates the
platelets. Activation enables platelets to degranulate and release chemotactic
and growth factors, such as platelet-derived growth factor (PDGF),
proteases, and vasoactive agents (eg, serotonin, histamine).
The coagulation cascade occurs via 2 different pathways. The intrinsic
pathway begins with the activation of factor XII (Hageman factor) when
blood is exposed to extravascular surfaces. The extrinsic coagulation
pathway occurs through the activation of tissue factor found in extravascular
cells in the presence of factors VII and VIIa. Both pathways proceed to the
activation of thrombin, which converts fibrinogen to fibrin. The fibrin
product is essential to wound healing and is the primary component of the
wound matrix into which inflammatory cells, platelets, and plasma proteins
migrate. Removal of the fibrin matrix impedes wound healing.
In addition to activation of fibrin, thrombin facilitates migration of
inflammatory cells to the site of injury by increasing vascular permeability.
By this mechanism, factors and cells necessary for healing flow from the
intravascular space and into the extravascular space.
The result of platelet aggregation and the coagulation cascade is clot
formation. Clot formation is limited in duration and to the site of injury. Clot
formation dissipates as its stimuli dissipate. Plasminogen is converted to
plasmin, a potent enzyme that aids in cell lysis. Clot formation is limited to
the site of injury because uninjured nearby endothelial cells produce
prostacyclin, an inhibitor of platelet aggregation. In uninjured adjacent areas,
antithrombin III binds thrombin, and protein C binds factors of the
coagulation cascade, namely, factors V and VII.
The vasoconstriction period is followed by a more persistent period of
vasodilation mediated by histamine, prostaglandins, kinins, and leukotrienes.
Vasodilation is responsible for the erythema, edema, and heat observed after
tissue injury. Vasodilation is an important means by which the wound can be
exposed to increased blood flow, accompanied by the necessary
inflammatory cells and factors that fight infection and debride the wound of
devitalized tissue. Alterations in pH (secondary to tissue and bacterial
degradation), swelling, and tissue hypoxemia at the injury site contribute to
the sensation of wound pain.
22
Following injury, the products of the earliest cellular events activate
intricately related inflammatory pathways that modify subsequent events in
the wound-healing process. For example, Hageman factor activates the kinin
pathway, which produces bradykinin. Bradykinin stimulates vasodilation
and increased vascular permeability. Histamine released from platelets and
circulating mast cells increases vascular permeability and indirectly
stimulates vasodilation through the production of prostaglandins E1 and E2.
Prostaglandins cause vasodilation through the activation of the adenylate
cyclase pathway via the production of cyclic adenosine monophosphate.
Prostaglandins also accumulate at the area of injury through the activation of
phospholipases located on injured cell membranes. Phospholipases stimulate
the release of arachidonic acid, ultimately leading to the production of
prostaglandins, leukotrienes, and other factors.
Hageman factor also activates the classic complement pathway during
the inflammatory phase. Inactive proteins of the complement system (ie, C1C9) are activated by means of a cascade of reactions. These proteins
stimulate important inflammatory events such as chemotaxis, degranulation
of mast cells, and cytolysis. C5a and C567 are chemotactic agents for
neutrophil migration. C3a, C4a, and C5a cause degranulation of mast cells,
which leads to release of histamine and increased vascular permeability. The
membrane attack complex, C567, is responsible for cytolysis. The cellular
aspect of the inflammatory phase occurs within hours of injury. Neutrophils
are the predominant cell type for the first 48 hours after injury but do not
appear essential to the wound-healing process. Neutrophils cleanse the
wound site of bacteria and necrotic matter and release inflammatory
mediators and bactericidal oxygen-free radicals. The absence of Neutrophils
does not prevent healing.
Macrophages are essential to wound healing and perhaps are the most
important cells in the early phase of wound healing. Macrophages
phagocytose debris and bacteria. Macrophages also secrete collagenases and
elastases, which break down injured tissue and release cytokines. In
addition, macrophages release PDGF, an important cytokine that stimulates
the chemotaxis and proliferation of fibroblasts and smooth muscle cells.
Finally, macrophages secrete substances that attract endothelial cells to the
wound and stimulate their proliferation to promote angiogenesis.
Macrophage-derived growth factors play a pivotal role in new tissue
formation, as evidenced by the fact that new tissue formation in
macrophage-depleted animal wounds demonstrates defective repair. In
23
studies in which experimental wounds are rendered monocytopenic,
subsequent stages of fibroplasia and granulation tissue formation are
impaired and the overall rate of wound healing is delayed.
T lymphocytes migrate into the wound during the inflammatory
phase, approximately 72 hours following injury. T lymphocytes are attracted
to the wound by the cellular release of interleukin 1, which also contributes
to the regulation of collagenase. Lymphocytes secrete lymphokines such as
heparin-binding epidermal growth factor and basic fibroblast growth factor.
Lymphocytes also play a role in cellular immunity and antibody production.
PROLIFERATIVE PHASE
Formation of granulation tissue is a central event during the
proliferative phase. Inflammatory cells, fibroblasts, and neovasculature in a
matrix of fibronectin, collagen, glycosaminoglycans, and proteoglycans
comprise the granulation tissue. Granulation tissue formation occurs 3-5
days following injury and overlaps with the preceding inflammatory phase.
Epithelialization
Epithelialization is the formation of epithelium over a denuded
surface. Epithelialization of an incisional wound 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 (e.g. wounds due to laser resurfacing, dermabrasion,
chemical peel treatments) adnexal structures (e.g. 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. The
24
extracellular wound matrix over which epithelial cells migrate has received
increased emphasis in wound-healing research. Migrating epithelial cells
interact with a provisional matrix of fibrin cross-linked to fibronectin and
collagen. The matrix components may be a source of cell signals to facilitate
epithelial cell proliferation and migration. 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.
Fibroplasia
The fibroblast is a critical component of granulation tissue.
Fibroblasts are responsible for the production of collagen, elastin,
fibronectin, glycosaminoglycans, and proteases Fibroblasts grow in the
wound as the number of inflammation cells decrease. The demand for
inflammation disappears as the chemotactic factors that call inflammatory
cells to the wound are no longer produced and as those already present in the
wound are inactivated.
Fibroplasia begins 3-5 days after injury and may last as long as 14
days. Skin fibroblasts and mesenchymal cells differentiate to perform
migratory and contractile capabilities. Fibroblasts migrate and proliferate in
response to fibronectin, platelet-derived growth factor (PDGF), fibroblast
growth factor, transforming growth factor, and C5a. Fibronectin serves as an
anchor for the myofibroblast as it migrates within the wound.
The synthesis and deposition of collagen is a critical event in the
proliferative phase and to wound healing in general. Collagen consists of 3
polypeptide chains, each twisted into a left-handed helix. Three chains of
collagen aggregate by covalent bonds and twist into a right-handed
superhelix, forming the basic collagen unit. A striking structural feature of
collagen is that every third amino acid is glycine. This repeating structural
feature is an absolute requirement for triple-helix formation. Collagen is rich
in hydroxylysine and hydroxyproline moieties, which enable it to form
25
strong cross-links. The hydroxylation of proline and lysine residues depends
on the presence of oxygen, vitamin C, ferrous iron, and -ketoglutarate. A
deficiency of oxygen and vitamin C, in particular, result in
underhydroxylated collagen that is less capable of forming strong cross-links
and, therefore, is more vulnerable to breakdown.
Collagen is secreted to the extracellular space in the form of
procollagen. This form is then cleaved of its terminal segments and called
tropocollagen. Tropocollagen can aggregate with other tropocollagen
molecules to form collagen filaments. Filaments consist of tropocollagen
molecules arrayed in a staggered fashion, joined by intermolecular crosslinks. Filaments aggregate to form fibrils. Collagen fibrils, in turn, aggregate
to form collagen fibers.
Filament, fibril, and fiber formation occur within a matrix gel of
glycosaminoglycans, hyaluronic acid, chondroitin sulfate, dermatan sulfate,
and heparin sulfate produced by fibroblasts. Intermolecular cross-links
within the collagen fiber stabilize it, making it resistant to destruction. Age,
tension, pressure, and stress affect the rate of collagen synthesis. Collagen
synthesis begins approximately 3 days after injury and may continue at a
rapid rate for approximately 2-4 weeks. Collagen synthesis is controlled by
the presence of collagenases and other factors that destroy collagen as new
collagen is made.
Approximately 80% of the collagen in normal skin is type I collagen;
the remaining is mostly type III. In contrast, type III collagen is the primary
component of early granulation tissue and is abundant in embryonic tissue.
Collagen fibers are deposited in a framework of fibronectin. An essential
interaction seems to exist between fibronectin and collagen; experimental
wounds depleted of fibronectin demonstrate decreased collagen
accumulation.
Elastin is also present in the wound in smaller amounts. Elastin is a
structural protein with random coils that allow for stretch and recoil
properties of the skin.
Angiogenesis
A rich blood supply is vital to sustain newly formed tissue and is
appreciated in the erythema of a newly formed scar. These blood vessels
disappear as they become unnecessary, as does the erythema of the scar. The
26
macrophage is essential to the stimulation of angiogenesis and produces
macrophage-derived angiogenic factor in response to low tissue
oxygenation. This factor functions as a chemoattractant for endothelial cells.
Basic fibroblast growth factor secreted by the macrophage and vascular
endothelial growth factor secreted by the epidermal cell are also important to
angiogenesis.
Fibronectin is chemotactic for endothelial cells. Capillaries bud from
existing capillaries in response to these growth factors. Endothelial cells
coalesce and bind fibrin, which adds support to the vessel wall.
Angiogenesis results in greater blood flow to the wound and, consequently,
increased perfusion of healing factors. Angiogenesis ceases as the demand
for new blood vessels ceases. New blood vessels that become unnecessary
disappear by apoptosis.
New blood vessel formation is a complex process that relies on
several angiogenic factors such as vascular endothelial growth factor,
angiogenin, and angiotropin.
Contraction
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/d and depends on the degree of
tissue laxity and shape of the wound. Loose tissues contract more than
tissues with poor laxity, and square wounds tend to contract more than
circular wounds. Wound contraction depends on the myofibroblast located at
the periphery of the wound, its connection to components of the extracellular
matrix, and myofibroblast proliferation.
Radiation and drugs, which inhibit cell division, have been noted to
delay wound contraction. Contraction does not seem to depend on collagen
synthesis. Although the role of the peripheral nervous system in wound
healing is not well delineated, recent studies have suggested that sympathetic
innervation may affect wound contraction and epithelialization through
unknown mechanisms.
27
Contraction must be distinguished from contracture, a pathologic
process of excessive contraction that limits motion of the underlying tissues
and is typically caused by the application of excessive stress to the wound.
MATURATION PHASE
Collagen
Collagen remodeling during the maturation phase depends on
continued collagen synthesis in the presence of collagen destruction.
Collagenases and matrix metalloproteinases in the wound assist removal of
excess collagen while synthesis of new collagen persists. Tissue inhibitors of
metalloproteinases limit these collagenolytic enzymes, so that a balance
exists between formation of new collagen and removal of old collagen.
During remodeling, collagen becomes increasingly organized.
Fibronectin
gradually
disappears,
and
hyaluronic
acid
and
glycosaminoglycans are replaced by proteoglycans. Type III collagen is
replaced by type I collagen. Water is resorbed from the scar. These events
allow collagen fibers to lie closer together, facilitating collagen cross-linking
and ultimately decreasing scar thickness. Intramolecular and intermolecular
collagen cross-links result in increased wound bursting strength. Remodeling
begins approximately 21 days after injury, when the net collagen content of
the wound is stable. Remodeling may continue indefinitely.
The tensile strength of a wound is a measurement of its load capacity
per unit area. The bursting strength of a wound is the force required to break
a wound regardless of its dimension. Bursting strength varies with skin
thickness. Peak tensile strength of a wound occurs approximately 60 days
after injury. A healed wound only reaches approximately 80% of the tensile
strength of unwounded skin.
Cytokines
Cytokines have emerged as important mediators of wound healing
events. By definition, a cytokine is a protein mediator, released from various
cell sources, which binds to cell surface receptors to stimulate a cell
response. Cytokines can reach their target cell by endocrine, paracrine,
28
autocrine, or intracrine routes. Some important cytokines are described as
follows:





Epidermal growth factor was the first cytokine described and is a
potent mitogen for epithelial cells, endothelial cells, and fibroblasts.
Epidermal growth factor stimulates fibronectin synthesis,
angiogenesis, fibroplasia, and collagenase activity.
Fibroblast growth factor is a mitogen for mesenchymal cells and an
important stimulus for angiogenesis. Fibroblast growth factor is a
mitogen for endothelial cells, fibroblasts, keratinocytes, and
myoblasts. This factor also stimulates wound contraction and
epithelialization and production of collagen, fibronectin, and
proteoglycans.
PDGF is released from the alpha granules of platelets and is
responsible for the stimulation of neutrophils and macrophages and
for the production of transforming growth factor. PDGF is a mitogen
and chemotactic agent for fibroblasts and smooth muscle cells and
stimulates angiogenesis, collagen synthesis, and collagenase. Vascular
endothelial growth factor is similar to PDGF but does not bind the
same receptors. Vascular endothelial growth factor is mitogenic for
endothelial cells and plays an important role in angiogenesis.
Transforming growth factor- is released from the alpha granules of
platelets and has been shown to regulate its own production in an
autocrine manner. This factor is an important stimulant for fibroblast
proliferation and the production of proteoglycans, collagen, and fibrin.
The factor also promotes accumulation of the extracellular matrix and
fibrosis. Transforming growth factor- has been demonstrated to
reduce scarring and to reverse the inhibition of wound healing by
glucocorticoids.
Tumor necrosis factor is produced by macrophages and stimulates
angiogenesis and the synthesis of collagen and collagenase. Tumor
necrosis factor is a mitogen for fibroblasts.
29
Overview of involved growth factors
Following are the main growth factors involved in wound healing:
Growth
factor
Abbreviation
Main origins
Effects


Epidermal
EGF
growth factor


Activated
macrophages
Salivary glands
Keratinocytes




Transforming
TGF-α
growth
factor-α


Activated
macrophages
T-lymphocytes
Keratinocytes


Hepatocyte
HGF
growth factor

Mesenchymal
cells


Vascular
endothelial VEGF
growth factor

Platelet
PDGF
derived
growth factor



Mesenchymal
cells
Platelets
Macrophages
Endothelial cells
30


Keratinocyte
and fibroblast
mitogen
Keratinocyte
migration
Granulation
tissue formation
Hepatocyte and
epithelial cell
proliferation
Expression of
antimicrobial
peptides
Epithelial and
endothelial cell
proliferation
Hepatocyte
motility
Vascular
permeability
Endothelial cell
proliferation
Granulocyte,
macrophage,
fibroblast and


Smooth muscle
cells
Keratinocytes









Fibroblast
growth factor FGF-1, -2
1 and 2




Macrophages
Mast cells
T-lymphocytes
Endothelial cells
Fibroblasts




Transforming
TGF-β
growth
factorβ

Platelets
31

smooth muscle
cell chemotaxis
Granulocyte,
macrophage and
fibroblast
activation
Fibroblast,
endothelial cell
and smooth
muscle cell
proliferation
Matrix
metalloproteinas
e, fibronectin
and hyaluronan
production
Angiogenesis
Wound
remodeling
Integrin
expression
regulation
Fibroblast
chemotaxis
Fibroblast and
keratinocyte
proliferation
Keratinocyte
migration
Angiogenesis
Wound
contraction
matrix
deposition
Granulocyte,
macrophage,
lymphocyte,
T-lymphocytes






Macrophages
Endothelial cells
Keratinocytes
Smooth muscle
cells
Fibroblasts




Keratinocyte
KGF
growth factor

Fibroblasts
32
fibroblast and
smooth muscle
cell chemotaxis
Angiogenesis
Fibroplasia
Matrix
metalloproteinas
e production
inhibition
Keratinocyte
proliferation
Keratinocyte
migration,
proliferation and
differentiation
CHAPTER 1.5
Biological Methods for the Study Of
Wound Healing7
33
Biological Methods for the Study of Wound Healing7
Animal models and in-vitro assays have become indispensable tools
for researchers in nearly every scientific discipline. In product development
there is a need for translational research to obtain data that can lead to sound
clinical trials and ultimately, improved wound care. This process is usually
performed in a stepwise fashion starting with in-vitro testing, preclinical,
and then clinical evaluations (Figure 1).
In-vitro studies help determine which concentrations may be
effective in-vivo and determine whether certain products are effective on
various cell types (e.g. fibroblasts and keratinocytes). The next step is to
examine the effect of the product’s use in an animal model(s). This
facilitates investigation of the product in the presence of wound fluid, blood,
immune cells, proteases, etc., which can have an effect on the activity of the
active agent. Many in-vivo animal studies initially investigate the safety
and/or irritancy of the product. It is important to be sure that these agents do
34
not have a toxic effect on tissues. Efficacy animal trials are conducted after
the safety studies are completed. This eventually allows the product to be
evaluated in human trials.
Although definitive studies conducted on human subjects are needed,
such studies present several practical, ethical, and moral concerns. For
example, in order to examine wounds histologically throughout the entire
healing process one must biopsy a human subject at multiple time points,
which is impractical. Furthermore, ethical considerations prevent the
intentional infection of a wound on a human or the use of an untreated
control subject. Some of the practical difficulties lie in obtaining enough
subjects with similar or identical situations to conduct well controlled
studies. Another complication to factor in with human trials is compliance
(e.g. subject’s level of cooperation, ability to understand and follow
instructions). The above difficulties have led researchers to develop multiple
in-vitro and in-vivo models that attempt to mimic or reproduce human
conditions.
[1] In-vitro technique
In-vitro assays are great for examining the effect of agents on
particular cell types. They are relatively inexpensive, fast, and convenient
for the researcher. In addition to providing useful results in a short time, they
possess an obvious humane appeal since they usually do not involve the use
of animals or humans. In-vitro assays are useful in wound healing research
for determining the possible effectiveness of various treatments, particularly
antimicrobial and healing enhancing agents. Another noteworthy attribute of
in-vitro testing is the ability to screen multiple agents or samples
simultaneously. Assays can aid in the early detection of antimicrobial
resistance among pathogens and determination of minimal inhibitory
concentrations (MIC), and allow for highly specific control over the
experimental conditions. However, it is difficult to simulate a “real world”
application. Although some variables such as pH, salinity, and temperature
are easily controlled, in-vitro assays are incapable of completely reproducing
biological conditions (e.g. immune responses, healing) and diseases, such as
diabetes.
In order to approximate in-vivo experiments, in-vitro assays have been
developed that incorporate some variety of cell or tissue system. Wound
closure studies have been conducted on single cell monolayer systems.
35
The principle in vitro technique for studying the skin penetration
evolves the use of variety of diffusion cells in which animal or human skin is
fastened to a holder and the passage of compounds from epidermal surface
to a fluid bath is measured.
Many chemical agents can be used which penetrate in sufficient
concentration to be determined by different physical and chemical analysis.
More recently model systems have been used which do not use membranes.
Solvent such as alcohol –water have been used as models chosen to have
negligible solubility in phase representing the skin, but in which drug is
fairly soluble. A receptor phase like chloroform and isopropyl myristate can
also be used to receive the penetrant.
Important factors influencing release in to receptor phase are
solubility in the vehicle and partition coefficient of the drug between vehicle
and the receptor phase. Optimum release is obtained from vehicle containing
the minimum concentration of solvent required for complete solubilization
of the drug.
[2] In-vivo technique
Small mammal wound healing models. Rodent and small mammal
models of wound healing have emerged as the model of choice for many
researchers. This type of study is beneficial to wound research for multiple
reasons. Small animals are inexpensive, easily obtainable, and require less
space, food, and water. Additionally, they often have multiple offspring,
which develop quickly allowing experiments to proceed through multiple
generations. Small animals usually have accelerated modes of healing
compared to humans, thus experiment duration lasts for days, as opposed to
weeks or months in human experiments. Some small mammals can easily be
altered genetically and provide a wound model capable of approximating
defective human conditions such as diabetes, immunological deficiencies,
and obesity. Another advantage of small mammal models is their ability to
serve in experiments where death is an endpoint, as is some cases of
bacterial or viral infection.
Small animals provide a multitude of model choices for various
human wound conditions. Some models have been developed to investigate
the mechanistic particulars of certain aspects of healing.
36
The major in-vivo methods are histological techniques, use of tracers,
analysis of body fluids and tissues and elicitation of biological response.
Tissue changes in skin following the application of various substances
to the cuetaneous surface can yield information about specific tissues
affected, so that not only absorption is revealed but also the route of
penetration.
For studying the wound in the laboratory, mainly two types of wounds
are produced experimentally. These are excised or open wounds and incised
or sutured wounds. The assessment of healing is made by studying the
regenerating tissue by different parameters.
Following types of wounds are made in laboratory animals for
studying the effect of various drugs.
1) Excised wound or Open Wound
These types of wounds are prepared either on rats or guinea pigs.
Back of each animal is shaved and prepared after washing with spirit for
operation. An area of about 2.0 sq cm is marked out by an Indian market ink
with the help of stencil. The marked area is excised with sharp knife and
scissors under ether anesthesia. After making wound the animals are divided
in two groups. One control group and the other test group and are kept in
isolated cages. Topical application of ointments or lotions are made on is
founds daily. On desired postoperative days this founded animals are
sacrifice and the contraction measured. Biochemical estimation of
granulation tissue and histological examination’s are done.
2)
Incised Or Sutured Skin Wounds
After preparing the animals for operation under aseptic conditions, a
longitudinal cutaneous incision measuring about 3-5c.m was made at the
back or abdomen according to the type of animals selected. Wounds are
closed by interrupted cotton threads stitches, which are placed
approximately at equal distance. The tensile strengths, biochemical and
histological study of the wound are carried out.
37
3) Musculoperitoneal Wounds
To prepare this wound, animals are prepared in the same fashion as
described earlier but their abdomen is opened completely incision measuring
between 2-5c.m according to the size of the animals are made. The wound is
caused in one layer by interrupted linen stitches. Tensile strength and
busting abdomen, by chemical and histological studies are done on this
wounds tissue, after sacrifice the animals on desired post operative days.
4) Burn wounds
The burn is produced under aseptic condition on hair removed areas
of back of rats/guinea pigs with special device cosseting of a square sheet of
an iron piece measuring 4.8sq.cm with wooden handle. It is heated to a red
hot over flying and is placed in contact with the back of the anaesthetized rat
up to ten seconds, with out any pressure. Medication is applied these animals
are sacrificed on desired days and the regenerated tissues are removed for
biochemical and the histological studies etc. their wounds are also measured
for the contraction.
5) Dead Space Wound Method
Subcutaneous implantation of sterilize cotton pellets (10 mg each) and
a plastic road (25-30mm) in the axial are anti groin respectively is done
under ether anesthesia in male albino rabbits. The 10th day old granulomas
are carefully dissected and cleared of the tissues.
38
METHOD OF ASSESSMENT8
1. MACROSCOPIC EXAMINATION OF THE WOUNDS:Gross examination of wound gives some information regarding the
healing. One can easily distinguish the normally healing wounds with that of
a wound with delayed healing by a careful gross examination provided the
different is marked in both the wounds. Gross examination sometimes may
not give much information; hence quantitative methods can be used in such
cases. For this purpose measurement the size of the wounds gives sufficient
information. This can be done using a planimeter or using a graph paper
2. MICROSCOPIC METHOD
This method involves histological examination of tissue.
3. ELECTRON MICROSCOPIC METHOD
This technique is used for the study of details about the cellular
morphology and other alteration at cellular levels during healing and
regeneration.
39
CHAPTER 1.6
Plant Profile9
40
Pothos scandens (Araceae)
41
BOTANICAL INFORMATION9
Botanical Name
: Pothos scandens
Division
: Magnoliophyta
Class
: Liliopsida
Subclass
: Monocots
Order
: Alismatales
Family
: Araceae
Subfamily
: Pothoideae
Tribe
: Potheae
Vernacular Names
Malayalam
Tamil
Kannada
: Annaparuva, Paruvakodi
: Anaparuga
: Adkebiluballi
Botanical Description:
Pothos scandens is the botanical name of the plant. It is a climbing
shrub having adventitious aerial roots. The internodes of the plant are 1.32.5 cms and its leaves are very variable.
The leaves are obovate, elliptic or lanceolate and coriaceous, having a
bright green colour. The apex of the plant is acute, acuminate or apiculate,
with cuneate or rounded base. The petioles of Pothos scandens are semiamplexicaul and broadly winged. They have a length of 2.5-7.5 cms and a
width of 0.6-1.7 cm at the base. The green Spathe is 0.4-0.7 cm long, ovate
and erect, with cuspidate apex. The stipe of the plant is deflexed, to 0.6 cm
long and the spadix is yellow, with an approximate length of 0.5 cm. The
spadix is globose, ovoid or shortly oblong. The fruits or berries of the plant
are oblong and 1.3-1.7 cm long and they are scarlet when ripe.
42
Geographical Source:




AFRICA
Western Indian Ocean: Comoros; Madagascar; Seychelles
ASIA-TEMPERATE
China: China - Yunnan
ASIA-TROPICAL
Indian Subcontinent: Bangladesh; India - Assam, Bihar, Goa,
Karnataka, Kerala, Maharashtra, Meghalaya, Orissa, Tamil Nadu,
Tripura, West Bengal, Andaman and Nicobar.
Indo-China: Cambodia; Laos; Myanmar; Thailand; Vietnam
Indonesia - Java, Kalimantan, Lesser Sunda Islands, Moluccas,
Sumatra;
Ethnobotanical Uses
Pothos scandens has quite a few medicinal properties and usages. The
bruised root of the plant is reportedly applied to promote healing of
abscesses, after being fried in oil. The Indian people use an infusion of the
leaves of this plant as a bath for curing convulsions and epilepsy. Apart from
that, the stem is also reportedly used to treat asthma, after being cut up with
camphor and smoked like tobacco. Traditionally the plant is used by
Ayurvedic physicians of Cheruvathur, Kerala mainly for its burn healing
properties. Other uses include in treatment of vermifuge and small pox.
43
CHAPTER 1.7
Polymer Data10
44
Carbopol 940 Polymer10
Technical Data
General INCI (International Nomenclature for Cosmetic Ingredients)
Name: Carbomer
Appearance: Fluffy, white dry powder
Odour: Slightly Acetic
Safety: 25-year history demonstrating non irritating and non sensitizing
Elegance: Luxurious feel
Efficiency: Forms gel at very low concentration
Viscosity: 40,000-60,000 cps of a 0.5% aqueous dispersion
Moisture Content: Maximum 2.0%
Microorganism Resistance: No support for bacteria fungus and mould
growth
Physical Properties:
The three dimensional nature of these polymers confers some unique
characteristics, such as biological inertness, not found in similar linear
polymers. The Carbopol resins are hydrophilic substances that are not
soluble in water. Rather, these polymers swell when dispersed in water
forming a colloidal, mucilage-like dispersion.
Carbopol polymers are bearing very good water sorption property.
They swell in water up to 1000 times their original volume and 10 times
their original diameter to form a gel when exposed to a pH environment
above 4.0 to 6.0. Because the pKa of these polymers is 6.0 to 0.5, the
carboxylate moiety on the polymer backbone ionizes, resulting in repulsion
between the native charges, which adds to the swelling of the polymer. The
45
glass transition temperature of Carbopol polymers is 105°C (221°F) in
powder form. However, glass transition temperature decreases significantly
as the polymer comes into contact of water. The polymer chains start
gyrating and radius of gyration becomes increasingly larger.
Macroscopically, this phenomenon manifests itself as swelling.
Chemical properties
Carbopol
pentaerythritol.
940
polymer
is
acrylic
acid
cross
linked
with
General Structure of Carbopol Polymers in figure No: 1
Fig. No. 1
Shelf life
Carbopol polymers are stable for years when protected from moisture.
In powder form there is no reason for polymer to degrade.
Storage
Keep in a tightly closed container
Features and Benefits of Carbopol 940 Polymer




Short flow properties
High viscosity
High suspending ability
High clarity
46
Recommended Applications






Hair styling gels
Hydroalcoholic gels
Moisturizing gels
Bath gels
Hand, body and face lotions
Creams
Selecting the Right Carbopol Polymer
Requirement
Recommended
Carbopol Polymer
Clear gels > 3000 cP
Suspensions or emulsions at > 3000 cP
940
934, 940
Suspensions or emulsions at < 3000 cP
941
Higher shear resistance
Better ion resistance
Better thermal stability
934, 940
941
934, 940
47
CHAPTER 2
Review of Literature11
48
Review of Literature 11
S. Ignacimuthu et al studied the traditional knowledge of Kani tribals in
Kouthalai of Tirunelveli hills, Tamil Nadu, India. An ethnobotanical survey
was carried out among the ethnic groups (Kani/Kanikaran) in Southern
Western Ghats of India. Traditional uses of 54 plant species belonging to 26
families are described under this study including Pothos scandens. In this
communication, the information got from the tribal was compared with the
already existing literature on ethnobotany of India. The documented
ethnomedicinal plants were mostly used to cure skin diseases, poison bites,
wounds and rheumatism. The medicinal plants used by kanis are arranged
alphabetically followed by family name, local name, major chemical
constituents, parts used, mode of preparation and medicinal uses.
Christine A. Williams et al studied the Anthocyanin pigments and leaf
flavonoids in the Family: Araceae. The study revealed that Anthocyanins,
variously identified in inflorescence, fruit, leaf or petiole of 59
representative species of the Araceae, are of a simple type.
Mohsin Raza et al studied the anticonvulsant activities of 334 medicinal
plants used for the treatment of epilepsy and convulsive disorders in the
indigenous system of medicine including Pothos scandens.
Geoffrey C. Kite et al studied the Polyhydroxyalkaloids in the Aroid Tribes
Nephthytideae and Aglaonemateae. They conducted a survey of
polyhydroxyalkaloids in species of 52 genera of Araceae revealed the
presence of 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine (DMDP) and αhomonojirimycin (HNJ).
S.A. Salgare et al studied the effect of Ambient Air (from Chembur) on the
Chlorophyll Content of Cultivated Plants. The ambient air from Chambur
inhibited the chlorophyll content of plants collected from polluted zones.
The plants were collected from three different zones i.e. Collector’s colony,
Chembur Colony and Colaba (treated as control). Collections were made in
the winter season. The plants for this study are Malvaviscus arboreus,
Graptophyllun bertense. Ixora cocclnea, Nerlum odorum, Pothos scandens,
Quisqualis Indic, Tanbernae Montana coronarl. Chlorophyll was estimated
using Arnon’s method. Maximum inhibition in the chlorophyll content was
found with plants collected from Collector’s colony.
49
Grewal,-J-S12 investigated the biochemical factors responsible for
susceptibility or resistance of various plants against the scarlet mite,
Brevipalpus phoenicis: I. amino acids analysis. Results are presented of
amino acid analysis by thin layer chromatography for 11 out of 31 plant
species screened for resistance or susceptibility to infestation by Brevipalpus
phoenicis. Species stated to be resistant (Pothos scandens, Bauhinia
variegata, Eucalyptus globulus and maize) contained tryptophan, tyrosine
and hydroxyproline. Plant species lacking dihydroxyphenylalanine (Vicia
feba [faba beans], Dalbergia sissoo and Cestrum nocturnum) did not support
the development of B. phoenicis
Dhanavel,-D13 et al conducted Cytotaxonomical studies in South Indian
Araceae. Studies were carried out in 27 species belonging to 15 genera of
Araceae from Tamil Nadu, India. First record of chromosome numbers were
made in 10 species, namely Alocasia macrorrhiza var. dark pink (2n=28),
Pothos scandens (2n=32), Anthurium cubense (2n=30), A. polyrrhizum [A.
polyrrhizon] (2n=16), Caladium bicolor var. local (2n=24), C. bicolor var.
white stick with red spot (2n=40), Philodendron cymbispathum (2n=36), P.
mello-baretoanum [P. mello-barretoanum = P. bipinnatifidum] (2n=30),
Spathiphyllum wallisii (2n=18) and Dieffenbachia amoena (2n=54). The
somatic chromosome number (2n) ranged from 16 to 54. The primary basic
chromosome number may be 8 and other basic numbers should have
originated by the addition of one or few basic chromosome numbers. The
karyotype analyses show that each genus and species of a particular genus
has a particular combination of different types of chromosomes. Therefore,
karyotype alteration of chromosome play an important role in speciation,
along with aneuploidy, euploidy and higher polyploidy. Hence, the present
study of interrelationship among them will be more useful for future
breeding programmes..
50
CHAPTER 3
Objectives
51
OBJECTIVES
The major objective is to develop this traditional medicinal plant into
scientifically validated drug which is safe and therapeutically active. For the
evaluation, the plant is subjected to:
 Phytochemical screening
 Formulating the extract into conventional dosage form.
 Biological evaluation
52
CHAPTER 4
Plan of Work
53
PLAN OF WORK
PHARMACOGNOSTICAL STUDIES
The Phytochemical investigations of a plant involve the following:
 Authentication of the plant
 Determination of Physicochemical Parameters
 Extraction of the plant
 Isolation and Characterization
PHARMACEUTICAL STUDIES
1. Formulation with Carbopol 940
2. Evaluation of Carbopol 940
 Estimations of drug Content
 Physical Observation
 Extrudability
 pH determination
PHARMACOLOGICAL STUDIES
 Primary Skin Irritation Test
 Wound Healing Studies
54
Chapter 5
Pharmacognostical Studies
55
COLLECTION AND AUTHENTIFICATION OF THE
PLANT
The plant Pothos scandens Linn. (Family: Araceae) was selected for
proposed study and was collected from Perinthalmanna, Malapuram District
Kerala state, India. The collected plant was authenticated by Dr. Jiji Joseph,
Assistant Professor of plant breeding, College of Horticulture, Kerala
Agricultural University, Vellanikara, Thrissur.
PRELIMINARY TREATMENT
The foreign, earthy matter and residual materials were removed
carefully from the leaves and stem parts and then subjected for washing and
stored. The fresh leaves are cut and are subjected for extraction.
PREPARATION OF AQUEOUS AND ALCOHOLIC
EXTRACT
150gms of freshly cut leaves was placed in inside a thimble made
from thick filter paper, which is loaded into the main chamber of the Soxhlet
extractor. The Soxhlet extractor is placed onto a flask containing the
extraction solvent i.e.; using 250ml distilled water and ethanol (90%)
respectively for 80oC & 90oC for 6hrs.
DETERMINATION OF PHYSICOCHEMICAL
PARAMETERS14
The determination of water-soluble or ethanol-soluble extractive is
used as a means of evaluating drugs the constituents of which are not readily
estimated by other means. It indicates the nature of constituents present. The
selected plant leaves are subjected for the following extractive values.
ALCOHOL EXTRACTIVE VALUES
About 5gm of powdered fresh leaves was macerated with 100ml of
90% ethanol in a stoppered conical flask for 24hrs with occasional stirring
during first 6hrs and the first 5ml is discarded. Then 25ml of the filtrate was
56
evaporated on a tarred evaporating dish, and the residue was dried at 105 oC
until a constant weight is obtained.
WATER EXTRACTIVE VALUES
About 5gm of powdered fresh leaves was macerated with 100ml of
distilled water in a stoppered conical flask for 24hrs with occasional stirring
during first 6hrs and the first 5ml is discarded. Then 25ml of the filtrate was
evaporated on a tarred evaporating dish, and the residue was dried at 105 oC
until a constant weight is obtained.
PHYTOCHEMICAL SCREENING OF Pothos scandens
The plant is subjected to preliminary phytochemical screening for the
detection of various plant constituents present. The term qualitative analysis
refers to the establishing and proving the identity of a substance. Systematic
investigation of the plant material for its phytochemical behavior involves
the following stages:
1. Procurement of raw material
2. Qualitative Phytochemical analysis
57
QUALITATIVE PHYTOCHEMICAL
ANALYSIS15,16,17,18,19
The 90% ethanolic and aqueous extract of fresh leaves of
Pothos scandens were subjected to the following chemical test
separately for identification of various constituents.
1. Detection of Alkaloids
a. Mayer’s Test: To 1ml of the extract added to 2ml of Mayer’s
reagent, a dull white precipitate is obtained. Indicates presence
of alkaloid
b. Dragendroff’s Reagent: To 1ml of the extract added to 1ml
Dragendroff’s reagent, an orange red precipitate is obtained.
Indicates presence of alkaloid
c. Hager’s Test: To 1ml of the extract added to 3ml of Mayer’s
reagent, a yellow precipitate is obtained. Indicates presence of
alkaloid
d. Wagner’s Test: To 1ml of the extract added to 2ml of
Wagner’s reagent, a reddish brown precipitate is obtained.
Indicates presence of alkaloid
2. Detection of Carbohydrates
a. Molish Test: To 1ml of the extract added to 1ml of alpha
naphthol solution and concentrated sulphuric acid through sides
of test tubes. Purple or Reddish violet colour at the junction of
two liquid indicates presence of carbohydrate.
b. Iodine Test: To 1ml of the extract added to 3 drops of iodine.
Blue colour indicates presence of starch.
c. Fehling’s Test: To 1ml of the extract added equal quantities of
Fehling’s solution A and Fehling’s solution, upon heating
58
formation of brick red precipitate. Indicates presence of
carbohydrate.
d. Benedict’s test: To 1ml of the extract added Benedict’s
solution, upon heating for 2min, formation of red precipitate.
Indicates presence of carbohydrate.
3. Detection of Glycosides
a. Borntragers Test: A few ml of dilute sulphuric acid was added to
1ml of the extract. Boiled, filtered, cooled and extract the filtrate with
chloroform. The chloroform layer was treated with 1ml of ammonia.
The formation of red colour in the ammoniacal layer indicates
presence of anthraquinone glycoside.
b. Modified Borntragers Test: To a few ml of dilute hydrochloric acid
was added to 1ml of the extract and add few drops ferric chloride
solution. Boiled, filtered, cooled and extract the filtrate with benzene.
The chloroform layer was treated with 1ml of ammonia. The
formation of red colour in the ammoniacal layer indicates presence of
anthraquinone glycoside.
c. Legal Test: The extract was dissolved in pyridine and sodium
nitroprusside solution to make it alkaline. The formation of pink to red
colour shows the presence glycoside.
d. Baljet Test: To 1ml of the test extract was added to 1ml sodium
picrate solution. The formation of yellow to orange colour indicates
presence of glycoside.
4. Test for Saponin Glycosides
a. Foam Test: the extract were diluted with distilled water upto 20 times
and shaken in a graduated cylinder for 15min. formation of 1cm foam
indicates presence of saponins.
59
b. Haemolysis test: a blood smear was prepared and 1drop of extract
was added. Formation of hemolytic zone after 5min indicates presence
of saponins.
5. Test for Steroids
a. Libermann sterol test: To a solution of glycosides or steroidal
aglycones in glacial acetic acid, one drop of concentrated sulphuric
acid was added. A play of colours was observed starting from rose,
red, violet, blue to green.
b. Libermann Buthard Test: The extract was dissolved in 2ml of
chloroform in adry test tube. 10 drops of acetic anhydride and 2drops
of concentrated suphuric acid is added on U.V chamber shows
fluorescence
c. Salkowaski Test: The extract was dissolved in chloroform and equal
volumes of sulphuric acid are added. The formation of bluish red to
cherry red colour in the chloroform layer and green florescence in
acid layer. Indicates presence of steroids.
6. Detection of Amino acids and Proteins
a. Biuret Test: 1ml of extract was added to 1ml of 40% sodium
Hydroxide solution and two drops of 1% copper sulphate solution.
Formation of violet colour indicates presence of protein.
b. Xanthoproteic Test: 1ml of extract was added to 1ml of concentrated
nitric acid. Awhite precipitate is formed; it is then boiled and cooled.
Then 20% sodium hydroxide or ammonia is added. Orange colour
indicates presence of aromatic aminoacid.
c. Ninhydrin Test: 2drops of freshly prepared 0.2% ninhydrin reagent
was added to the extract solution and heat. Development of blue
colour reveals proteins and peptides.
60
7. Detection of Tannins and Phenolics
a. Ferric chloride Test: 1ml of extract was added to 1ml of ferric
chloride .formation of a dark blue or greenish black colour. Indicates
presence of tannins
b. Potassium Dichromate Test: The extract was added to potassium
dichromatic solution, formation of a precipitate shows presence of
tannins and phenolics.
c. Lead Acetate Test: to the extract added 1ml of lead acetate solution.
Formation of white precipitate indicates presence of tannins.
8. Detection of Flavones and Flavonones
a. Aqueous NaOH: To the test solution add few drops of sodium
hydroxide solution, gives an intense yellow colour which turns to
colourless on addition of dilute acid. Indicates presence of flavanoid.
b. Shinoda Test: To the extract add few magnesium turnings and
concentrated hydrochloric acid and boiled. Red colour was produced.
Indicates presence of flavanoid.
9. Detection of Fixed oils
a. Spot test: A small quantity of extract was pressed between two filter
papers. Oil stains indicates presence of fixed oils.
b. Saponification test: To 1ml of the extract was added to a few drops
of 0.5N alcoholic potassium hydroxide along with a drop of
phenolphthalein. The mixture was heated on a water bath for 2hrs.The
formation of soap or partial neutralization indicates the presence of
fixed oils.
61
Chapter 6
Pharmaceutical Studies
62
FORMULATION TRIALS OF Pothos scandens
ALCOHOLIC EXTRACT WITH CARBOPOL 940
Working Formula
INGREDIENDS
A1
%w/w
A2 A3
A4
4
0.5
45
0.1
q.s
100
4
1
45
0.1
q.s
100
4
2
45
0.1
q.s
100
Pothos scandens alcohol extract
Carbopol 940
Polyethylene glycol 400
Methyl paraben
Triethanolamine
Distilled water q.s upto
4
1.5
45
0.1
q.s
100
Procedure
To a beaker accurately weigh Carbopol 940 which is dispersed in
35ml of distilled water with constant stirring using a mechanical stirrer for
30 min at 600-800rpm. To another beaker, add 4ml of the alcoholic extract
in 45gm of PEG 400 and 0.1% methyl paraben in a mechanical stirrer for
30min. Mix the two solutions by constant stirring with adjustment of pH to
neutral using triethanolamine until a clear consistent gel is obtained.
(It is seen that neutralized aqueous gels of Carbopol show maximum
viscosity at pH at 6-9)
63
EVALUATION OF GELS Pothos Scandens Linn
ALCOHOLIC EXTRACT WITH CARBOPOL 940
PHYSICAL OBSERVATIONS:
The gel formulations were observed for their visual appearance and
transparency and homogeneity.
EXTRUDABILITY
The formulations were filled in collapsible tubes after the gels were
set in the container. The Extrudability of formulation is checked.
PH MEASUREMENTS
PH measurements of the gel were carried out using a digital P H meter
by dipping the glass electrode completely in to the gel system to cover the
electrode.
64
Chapter 7
Pharmacological Studies
65
PRIMARY SKIN IRRITATION TEST20
Primary irritation test was done on rats by placing a piece of cotton
wool soaked in a saturated solution of ethanolic extract of Pothos scandens
on a shaved portion of dorsal skin and securing it firmly in place with
adhesive plaster.
This was allowed to remain in close contact with the skin for 24
hours, after which the site of application was examined for irritation with
0.8% formalin as control.
WOUND HEALING STUDIES21,22,23
Healthy male albino rats was selected (150-250), from Kerala
Agriculture University, Mannuthy, Thrissur were used. The animals are kept
in cage for 20 days well fed. The back of the animal was shaved and washed
with spirit. A circular area of 1cm diameter was marked with a marker on
either side of bump region.
The animals were anaesthetized with a combination of ketamine and
xylazine. The back of the animal was shaved and washed with spirit. A
circular area of 1cm diameter was marked with a marker on either side of
bump region. A trichotomy of the back of the rats was performed, sufficient
for 2 perforations are made (test and control). The pieces of tissue were
subsequently excised with aid of scissors, scalpel and a forceps.
The wounds on the left side were filled with gel extract (test wounds)
and on the right side (control wounds) were filled with alcohol. After these
procedures, the animals receive no other treatment until they are fully
recovered. The application was received daily for the next 24 hours post
operative days.
The wound contractions were measured as percentage reduction in
wound area for the 4th, 8th, 10th, and 12th days. The progressive decrease in
the wound area was monitored periodically by tracing the wound margin on
a tracing paper and area is accessed by placing a graph paper over a tracing
paper.
66
Chapter 8
Result and discussion
67
PHARMACOGNOSTICAL STUDIES
1. PHYSICOCHEMICAL PARAMETER
Physicochemical parameter like extractive values were determined for the
selected plant material the result are shown in table No: 1
Table No: 1
Data showing different extractive value for the leaves of
Pothos scandens Linn
Extractive value (%w/v)
Alcohol
Water Soluble
Soluble
S.No Plant material
1
Fresh leaves
scandens
of
Pothos
19.32%
19.22%
2. Preliminary Phytochemical Evaluation
The 90% ethanolic extract and aqueous extract of the fresh leaves of
Pothos scandens was subjected to preliminary phytochemical evaluation.
The results are shown in table No: 2
68
Table No: 2
S.N
1
a.
b.
c.
d.
2
a.
b.
c.
d.
3.
a.
b.
c.
d.
4.
a.
b.
5.
a.
b.
c.
6..
a.
b.
c.
7..
a.
b.
c.
8.
a.
b.
9.
a.
b.
TESTS
90% ethanolic
extract
ALKALOIDS
Dragendroff’s Test
+ve
Wagners test
+ve
Hagers test
+ve
Mayer’s Test
+ve
CARBOHYDRATES
Molish test
-ve
Iodine test
-ve
Fehling’s test
-ve
Benedict’s test
-ve
GLYCOSIDES
Borntrager’s test
-ve
Modified Borntrager’s test
-ve
Legal’s test
-ve
Baljet test
-ve
SAPONIN GLYCOSIDES
Foam Test
-ve
Haemolysis test
-ve
STEROIDS
Libermann sterol test
-ve
Libermann-Butchard test
-ve
Salkowsky test
-ve
AMINO ACIDS & PROTEINS
Biuret test
+ve
Xanthoproteic test
+ve
Ninhydrin test
-ve
TANNINS & PHENOLICS
K2Cr 2O7 test
-ve
FeCl 3
-ve
Lead acetate
-ve
FLAVONES & FLAVONONES
Aqueous NaOH
+ve
Shinoda Test
+ve
FIXED OILS
Spot Test
-ve
Saponification Test
-ve
69
Aqueous
extract
+ve
-ve
+ve
+ve
+ve
-ve
+ve
+ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
+ve
+ve
-ve
-ve
-ve
-ve
+ve
+ve
-ve
-ve
It has been found that the 90% alcoholic extract contain alkaloids, proteins
and flavanoids. Aqueous extract contain alkaloids, carbohydrates, proteins
and flavanoids.
PHARMACEUTICAL STUDIES
FORMULATION TRIAL
The given polymer concentration of 0.5, 1.0, 1.5 and 2.0%, the gel
consistency in 0.5% was less when compared to higher polymer
concentration.
EVALUATION OF GELS Pothos scandens ALCOHOLIC
EXTRACT WITH CARBOPOL 940
PHYSICAL OBSEVATION
TABLE 3
Appearance
PH Measurement
EXTRUDABILITY
A1
A2
A3
A4
A1
A2
A3
A4
A1
A2
A3
A4
Transparent with less gel consistency
Transparent, ,Non greasy gel
Slightly Translucent gel
Translucent gel
7.0
7.2
6.9
7.1
++
+++
+++
+++
++ Good
+++ Excellent
70
PHARMACOLOGICAL STUDIES
PRIMARY SKIN IRRITATION TEST
There was no sign of any kind of reaction, thus the ethanolic extract of
Pothos scandens was found to be safe.
WOUND HEALING STUDIES
Excision wound healing studies showing percentage reduction in
wound size in rats (% closure)
TABLE-4
TEST
DAYS
RAT RBT
RCT
RDT
RET
RFT
AVG
20%
22.42% 17.11% 19.92% 20.46% 18.46% 19.73%
4TH
43.16% 56.93% 30.80% 32.48% 31.96% 28.43% 37.29%
8TH
10TH
12TH
75.53% 87.18% 53.23% 59.49% 53.26% 57.85% 67.42%
83.62% 90.2% 75.29% 80.29% 82.13% 88.76% 83.38%
TABLE-5
CONTROL
DAYS
RAC RBC
RCC
RDC
REC
RFC
AVG
10.23% 9.76% 11.14% 12.28% 10.87% 13.73% 11.34%
4TH
17.87% 16.23% 19.23% 20.71% 16.58% 17.55% 18.03%
8TH
10TH
12TH
29.23% 27.12% 26.78% 27.25% 27.44% 25.43% 27.21%
34.47% 32.21% 35.46% 33.33% 34.86% 84.67% 34.17%
RA , RB , RC , RD , RE - Designation for each rats
C - Control
T - Test
71
Graph 1
Average Percentage contraction of wounds in rats after
treating with Carbopol 940
Avg
%
contraction
of wounds
90
80
70
60
50
40
30
20
10
0
Test
Control
4th
day
8th
day
10th
day
12th
day
On the first day after the excision of skin wounds made at area of
approximately 274mm.sq and its macroscopical studies were performed as
percentage decrease in wound size.
On the forth day, the average percentage decrease in wound size in test was
found to be 19.73% and that of the control 11.34%
On the eight day, the average percentage decrease in wound size in test was
found to be 37.29% and that of the control 18.03%
On the tenth day, the average percentage decrease in wound size in test was
found to be 64.42% and that of the control 27.21%
On the twelth day, the average percentage decrease in wound size in test was
found to be 83.38% and that of the control 37.17%
It was found that complete wound healing for the Test Formulation took
place on 14th day and for the control has taken additional six days for the
complete wound healing. So the given formulation of ethanolic extract of the
plant Pothos scandens was affective in wound healing.
72
Chapter 9
Conclusion
73
Conclusion
The plant Pothos scandens was selected for the study, whose extract was
very useful in the treatment of wounds. Literature survey revealed that this plant
is used traditionally for various ailments, especially for its wound healing
property. Extensive scientific studies were not performed on this plant. Its wound
healing property was not under taken for any scientific study. Hence the present
work is performed.
Form
the
present
study
entitled
“FORMULATION
&
PHARMACOLOGICAL EVALUATION OF HERBAL GEL OF Pothos scandens” the
following conclusions could be drawn.
Physicochemical parameters both alcohol-soluble and water-soluble
extractive values were determined and the results were tabulated in Table No.1
Preliminary phytochemical studies of 90% ethanolic extract were found
to contain alkaloids, protein and flavanoid. Aqueous extract contain
carbohydrate, protein, flavanoid and the results were tabulated in
Table No. 2
Different gel formulations of the ethanolic extract were prepared using
Carbopol 940 in varying proportions of 0.5, 1.0, 1.5 and 2.0%. On physical
evaluation the gel consistency of Formulation A1 was less when compared to
Formulation A2, A3, and A4. Formulation A3 and A4 were found to be
translucent. But formulation A2 was found to be transparent, non greasy and
stable. The PH of the formulation ranges from 6.8 to 7.6 and had an excellent
Extrudability and the results were tabulated in Table No. 3. Hence Formulation
A2 was selected for further study.
Primary Skin Irritation test were performed for Formulation A2 and
there was no signs of irritation. As no relevant data was available regarding the
dose of topical application of the formulation, wound healing studies were
carried out. Wound healing took place on 14th day in case of Test formulation
and control has taken additional six days for complete wound healing. The
results were shown in Table 4 and Table 5.Average % contraction of wounds in
Rats were plotted against no: of days and was shown in Graph 1.Therefore, the
given formulation of ethanolic extract of the plant Pothos scandens was affective
in wound healing.
74
Chapter 10
Suggestions for future work
75
SUGGESIONS FOR FUTURE WORK
In recent years the plant is not subjected to many research works as
we know by the literature review. Different similar species were the subject
of studies in many countries but the studies in Pothos scandens species were
less. This may be due to the less availability of the plant, since it is on the
verge of extinction. So lots of research works can be suggested in bringing
this plant to limelight and its importance. Suggestions are made in the order
of importance for work in pharmaceutical study in a stepwise manner.
PHARMACOGNOSY
 Microscopical characteristics
PHARMACEUTICAL ANALYSIS
The real molecules responsible for the activity are not yet been
identified and separated. In modern medical science the identity of the active
molecule is essential for the further works. The future works in
pharmaceutical analysis may include:
TLC characterization that consists of:
 Preparation of plates
 Separation of components
 Selection of mobile phase
 Nature of substances to be separated
 Nature of the stationary phase
 Chromatographic mode
76
HPLC Characterization
 Schematic representation of HPLC
 The HPLC finger print
Column Chromatographic Isolation
FT-IR Analysis
NMR
PHARMACOLOGICAL STUDIES
 Anti-microbial activity
 Anti-inflammatory
 Anticonvulsant activity
 Anti-asthmatic Activity
 Other activity can be evaluated
PHARMACEUTICAL STUDIES
 Studies on different formulation for its topical applications
 Oral dosage forms
77
Chapter 11
References
78
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