1

UST FACULTY OF PHARMACY

CHAPTER 1
INTRODUCTION

A.
Background of the Study
Wound healing is a complex physiological process that involves a series of
biochemical

reactions

and

cellular

events,

beginning

with

homeostasis,

epithelialization, granulation tissue formation, and remodeling of the extracellular

matrix (Enoch et al., 2005). It is a fundamental response mediated by connective
tissues towards tissue injuries. Collagen and other components of the ground substance
synthesized by the highly vascularized granulation tissue formed within the wound
environment play a critical role in the repair of the damaged tissues (Jurjus, 2006).
Diabetes mellitus (DM) is a group of metabolic disorders characterized by
hyperglycemia resulting from either the insensitivity of the body to insulin or the
inability of pancreatic beta () cells to produce physiological amount of insulin as a
result of autoimmune destruction or genomic DNA mutations (Naveed, 2008). There
are two types of diabetes mellitus. Type I diabetes, which has an early onset, is the
result of acquired underproduction of insulin by the beta cells. A more common type of
the disease also referred to as Type II diabetes is found in adults whose responsiveness
to insulin is abnormally low (Thierer et al., 2007).
Numerous studies have shown that wound healing is compromised in
individuals suffering from diabetes. Such may be the result of the abnormal reduction
in granulation tissue and collagen formation (Yue et al., 1986), the aberrant control of

UST FACULTY OF PHARMACY

cellular apoptosis (Darby et al., 1997), or the decreased growth factor activity in the
wound environment (Bitar et al., 1996).
There is a current interest among the general public for medications that are
derived from nature. Aloe vera also known as Aloe barbadensis Miller is a well-known
medicinal plant frequently used in the cosmetic and health food industries. Different
preparations from the succulent leaves of Aloe vera have been shown to aid tissue
repair. A gel preparation made from its inner leaf fillet has been used topically as a
healing agent for abrasions, burns, dermal ulcers, frostbites, and deep wounds
(Boudreau et al., 2005). In addition to its wound healing property, Aloe vera has also
been shown to have antidiabetic and hypoglycemic properties (Ghannam et al., 1986).
These earlier claims led the authors to investigate the healing properties of the
powdered leaf extract of Aloe vera using diabetic rats as models.
B.
Statement of the Problem
The general objective of the study is to determine the effectiveness of Aloe vera
powder extract as a treatment for dermal wounds of diabetic rats.
Specifically, the researchers seek to answer the following questions:
1.
How much is the percentage yield of the powdered extract?
2.
Determine the physical properties of the powder including the color,
3.

texture, moisture content, and bulk density.

Are the substances chrysophanic acid, barbaloin and the polysaccharide

4.

acemannan present in the powdered extract?

Will there be skin reactions (erythema and edema) upon administration

5.

of Aloe vera powder and standard drug (Iodosorb powder)?

Is there a significant difference on the ratio of collagen contents of the
untreated, Aloe vera treated and Iodosorb treated wounds of diabetic
rats?

UST FACULTY OF PHARMACY

6.

Is there a significant difference on the rate of wound contraction of the

untreated, Aloe vera treated and Iodosorb treated wounds of diabetic
rats?

C.

Significance of the Study

This study will enrich the communitys available knowledge on the wound
healing properties of the powdered Aloe vera extract. Pharmaceutical companies, in
cooperation with the Department of Health and Department of Science and Technology,
will have the opportunity to perform extensive research on the subject and, to this end,
develop a tested product that will benefit patients especially those suffering from
diabetes. This study will also spur the interest of researchers in isolating and
characterizing the plant metabolites that are responsible for the wound healing
properties and, later on, perform rational drug design to improve its potency.
D.

Scope and Limitations

The study will focus on the ability of Aloe vera (Aloe barbadensis Miller)
species as a powder extract in treatment of dermal wounds of diabetic rats. The plant
sample that will be tested will only be collected in a certain area and will not be
compared with other samples taken from other areas. The study will be limited to the
use of the parenchyma of Aloe vera leaves and will determine its physical properties
and its specific components that will be responsible for the healing effect of the plant.
The extracted powder dosage form will only be used to Sprague Dawley rats induced

UST FACULTY OF PHARMACY

with diabetes using streptozotocin. The effectiveness of the powdered extract and the
time of treatment in the wounds will be observed and tested. The effectiveness of the
wound healing will also be tested by the ratio of collagen content and wound contration
rate. In addition, the efficiency of the powdered extract will be compared to Iodosorb
powder as a standard of a wound healing drug using two way ANOVA.
E. Definition of Terms
These are the following terms used in our research:
Abrasions - a scraped spot or area; the result of rubbing or abrading
(http://dictionary.reference.com)
Antidiabetic - a drug used to treat diabetes mellitus (Katzung, 2009)
Autoimmune - relating to the immune response of the body against substance
normally present in the body (Katzung, 2009)
Pancreatic Beta () cells - hormone producing cells of the pancreas (Katzung,
2009)
Collagen - main protein of connective tissue in animals and the most abundant
protein in mammals, making up about 25% of the whole-body protein content
(Marieb, 2005)
Dermis - is a layer of skin beneath the epidermis (Marieb, 2005)
Deoxyribonucleic acid (DNA) - contains the genetic instructions used in
the development and

functioning

some viruses (Marieb, 2005)

of

all

known living

organisms and

UST FACULTY OF PHARMACY

Diabetes mellitus - is a syndrome characterized by disordered metabolism and

abnormally high blood sugar (Katzung, 2009)
Extracellular matrix- is the extracellular part of animal tissue that usually
provides structural support to the cells in addition to performing various other
important functions (Kimball, 2009)
Granulation- new connective tissue and tiny blood vessels that form on the
surfaces of a wound during the healing process (http://www.lexic.us)
Homeostasis maintenance of a stable, constant physiological condition in
living organism (Marieb, 2005)
Hyperglycemia- is a condition in which an excessive amount of blood sugar
Insulin- is a hormone with extensive effects on both metabolism and several
other body systems (Katzung, 2009)
Pancreas is an organ which belongs to the digestive and endocrine system;
secretes digestive enzymes as well as hormones (insulin and glucagon) that
maintains glucose homeostasis (Marieb, 2005)
Parenchyma - the primary tissue of higher plants composed of thin-walled cells
and forming the greater part of leaves, roots, the pulp of fruit, and the pith of
stems (Moore, 2003)
Rind - the outer layer of a fruit (Moore, 2003)
Ulcers (dermal) - a disintegration of the surface of the skin (Marieb, 2009)

UST FACULTY OF PHARMACY

Vascular - relating to the vessels of the body, especially the arteries and veins
that carry blood and lymph (Marieb, 2005)

CHAPTER II
REVIEW OF RELATED LITERATURE

UST FACULTY OF PHARMACY

In this chapter, the researchers will discuss the gathered information that
will support the study regarding the effectiveness of Aloe vera powdered extract in
treatment of the dermal wounds of Diabetic rats.
I.
A.

Aloe vera
Taxonomical Classification (Quisimbing,1978)

Kingdom: Plantae
Phylum: Magnoliophyta
Class: Liliopsida
Order: Asparagales
Family: Asphodelaceae
Genus: Aloe - Linnaeus
Specific epithet: vera - (L.) Burm.f.
Botanical name: - Aloe vera (L.) Burm.f.
B.

Botanical Description

Aloe vera is a stemless succulent plant growing to 60100 cm (2439 inches)

tall, spreading by offsets. The leaves are thick and fleshy, green to grey-green, with

UST FACULTY OF PHARMACY

some varieties showing white flecks on the upper and lower stem surfaces. The margin
of the leaf is serrated and has small white teeth. The flowers are produced in summer
on a spike up to 90 cm (35 in) tall, each flower pendulous, with a yellow tubular corolla
23 cm (0.81.2 in) long. Like other Aloe species, Aloe vera forms arbuscular
mycorrhiza, a symbiosis that allows the plant better access to mineral nutrients in soil.
Aloe vera has a life cycle of perennial (David, 2006).
Aloe vera is also known as the burn plant, true or medicinal aloe
(Graham 2006). Aloe vera originated in the southern half of the Arabian Peninsula,
Northern Africa, the Canary Islands and Cape Verde. Aloe vera grows in arid climates
(David, 2006).
Aloe vera has a long history of cultivation throughout the drier tropical and
subtropical regions of the world, both as an ornamental plant and for herbal medicine.
Aloe vera is relatively easy to care for in cultivation in frost-free climates. The species
requires well-drained sandy potting soil in moderate light. If planted in a pot or other
container, it is important to ensure sufficient drainage with drainage holes. The use of a
good quality commercial potting mix to which extra perlite, granite grit, or coarse sand
is added is recommended. Alternatively, pre-packaged "cacti and succulent mixes" may
also be used. Potted plants should be allowed to completely dry prior to re-watering.
During winter, Aloe vera may become dormant, during which little moisture is
required. In areas that receive frost or snow, the species is best kept indoors or in heated
glasshouses (David, 2006).
C.

Constituents

UST FACULTY OF PHARMACY

The succulent leaves of Aloe vera bear a thick mucilaginous sap. The mucilage,
on hydrolysis, yields glucose, galactose, mannose and galacturonic acid. Aloe vera has
a biological active component which is acetylated mannose, or acemannan, a
polysaccharide (Longe 2005). This substance has been considered to be effective in
stimulating the immune system, including activities against the viruses causing the flu,
measels and AIDS, as well as potency against some veterinary cancers (Longe 2005).
The sap contains numerous compounds, including several athraquinones
glycosides, collectively referred to as aloin and chromones (Levetin, 2006). Aloin is a
bitter, yellow-brown colored compound noted in the exudate of at least 68 Aloe species
at levels from 0.1 to 6.6% of leaf dry weight and in another 17 species at indeterminate
levels. According to W. A. Shenstone, two classes of aloin are to be recognized: (1)
nataloins, which yield picric and oxalic acid (C2O22 or HOOCCOOH) which when
reacted with with nitric acid do not give a red coloration and (2) barbaloins, which
yield aloetic acid (C7H2N3O5), chrysammic acid (C7H2N2O6), picric acid and oxalic acid
upon treatment with nitric acid. Barbaloin can be further classified into -barbaloins,
obtained from Barbados Aloe, reddened in the cold and -barbaloins, obtained from
Socotrine and Zanzibar Aloe, reddened by ordinary nitric acid when warmed or by
fuming acid in the cold. Nataloin (2C 17H13O7H2O) forms bright yellow scales while
barbaloin (C17H18O7) forms prismatic crystals. Aloe species also contain a trace of
volatile oil, to which its odour is due. In addition, the presence of chrysophanic acid
may bring about the extracts healing effect on skin (Levetin, 2006).

UST FACULTY OF PHARMACY

Other species of Aloe, particularly Aloe ferox Mill., A. Africana Mill. and A.
spicata Linn. Provide drugs similar to A. barbadensis. These plants yield lesser
amounts of aloe-emodin but contain about 8% -barbaloin (David, 2006).
D.
Medicinal Uses
Aloe vera has been particularly popular for its medical use. It use is mentioned
in records as early as 1750 B.C.E. and the finding of drawings of Aloe vera on cave
walls in Egypt may reflect its use in Egyptian embalming procedures (Longe, 2005).
In the mid-1930s, Aloe vera leaf gel was used successfully in the treatment of a
women with chronic and severe dermatitis resulting from x-ray treatments, and this
fostered additional trials with others receiving radiation burns (Longe, 2005).
Studies have shown that Aloe sap which contains the chrysophanic acid, when
use topically promotes faster healing with less scarring by stimulating cell growth and
inhibiting bacterial and fungal infection in injuries ranging from deep dermal burns to
radiation burns. Other studies have shown that barbaloin in the sap inhibit pain, itching
and inflammation. The sap also is useful in treating skin and mouth ulcers, eczema,
psoriasis, ringworm, athletes foot and poison ivy rashes (Levetin, 2006).
Aloe sap when taken orally or internally it is used as a powerful purgative for
the relief of constipation. The anthraquinones in the sap apparently initiate the
gastrointestinal tract, resulting in its purgative action. However, the action is drastic and
is, therefore, often used in combination with other drugs to modify the effect. Aloe sap
also shows potency against diabetes. In a recent investigation, dried Aloe sap has been
shown to lower blood glucose levels among a small group of non-insulin dependent
diabetics.
In homeopathic medicine, Aloe is used for hemorrhoids. Fresh leaf juice is

10

UST FACULTY OF PHARMACY

cathartic and also used for eye troubles, spleen and liver ailments, dermatitis and other
skin diseases (David, 2006).
The leaves are also credited with hypocholesteremic activity. Aloe finds
use in menstrual, uterine disorders and stomach pain and as a tonic after pregnancy.
Alocutin A and B are lectins found useful in cancer and inflammation (Longe, 2005).
E.
Pharmaceutical Uses
Gel (slimy polysaccharides dissolved in water) have become popular for their
supposed health properties and nowadays often included in medicinal products and
health tonics. Aloe leaf gel becomes the basis of a very large industry supplying health
drinks and supplements (Levetin, 2006).
F.
Other Uses
1.
Ornamental
Aloe species are frequently cultivated as ornamental plants both in gardens and
in pots. Many Aloe species are highly decorative and are valued by collectors of
succulents (David, 2006).
2. Food Preservative
In Spain have developed a gel based on A. vera that prolongs the conservation
of fresh produce, such as fresh fruit and legumes. This gel is tasteless, colorless and
odorless. This natural product is considered a safe and environmentally friendly
alternative to synthetic preservatives such as sulfur dioxide. The study showed that
grapes at 1C coated with this gel could be preserved for 35 days against 7 days for
untreated grapes (Serrano et al., 2006). According to the researchers, this gel operates
through a combination of mechanics forming a protective layer against the oxygen and
moisture of the air and inhibiting, through its various antibiotic and antifungal
compounds, the action of microorganisms that cause food borne illnesses.

11

UST FACULTY OF PHARMACY

3.

Cosmetic Use

In recent years the cosmetic industry has capitalized on the moisturizing effects
of the sap and it can be found in a variety of skin creams, shampoos, sunscreen, lotions
and bath oils (McMahon, 2006).
Aloe vera is now widely used on face tissues, where it is promoted as a
moisturizer and/or anti-irritant to reduce chafing of the nose of users who suffer hayfever or cold. It has also been suggested that biofuels could be obtained from Aloe vera
seeds. It can also be used to retwist dreadlocked hair, a favourite agent for vegans and
those who prefer natural products. To add Aloe vera is also used for soothing the skin
and keeping the skin moist while eliminating the risk of flaky scalp and skin in harsh
and dry weather (Shukla, 2008).

G.
1.

Current Studies
Local
Antifungal efficacy of Aloe vera in vitro and its use as a preharvest
treatment to maintain postharvest table grape quality - Postharvest
Biology and Technology, Volume 57, Issue 3, September 2010, Pages
183-188 S. Castillo, D. Navarro, P.J. Zapata, F. Guilln, D. Valero,
M. Serrano, D. Martnez-Romero

2.

International

12

UST FACULTY OF PHARMACY

Hypoglycemic and hypolipidemic effects of processed Aloe vera gel

in a mouse model of non-insulin-dependent diabetes mellitus
Phytomedicine, Volume 16, Issue 9, September 2009, Pages 856-863
Kwanghee Kim, Hyunyul Kim, Jeunghak Kwon, Sungwon Lee,
Hyunseok Kong, Sun-A Im, Young-Hee Lee, Young-Ran Lee, SunTack Oh, Tae Hyung Jo, Young In Park, Chong-Kil Lee, Kyungjae
Kim

Possible hypoglycemic effect of Aloe vera L. high molecular weight

fractions on type 2 diabetic patients - Saudi Pharmaceutical Journal,
Volume 17, Issue 3, July 2009, Pages 209-215- Akira Yagi, Sahar
Hegazy, Amal Kabbash, Engy Abd-El Wahab

Protective effect of Aloe vera on polymicrobial sepsis in mice June

2009 (Nari yun, et al)

Effect of Aloe vera polysaccharides on immunity and antioxidant

activities in oral ulcer animal models- January 2009 (ZhanHai Yu, et
al)

Administration of phytosterols isolated from Aloe vera gel reduce

visceral fat mass and improve hyperglycemia in Zucker diabetic
fatty (ZDF) rats - Obesity Research & Clinical Practice, Volume 2,
Issue 4, December 2008, Pages 239-245 - Eriko Misawa, Miyuki

13

UST FACULTY OF PHARMACY

Tanaka, Kouji Nomaguchi, Muneo Yamada, Tomohiro Toida,

Mitsunori Takase, Keiji Iwatsuki, Teruo Kawada

The efficacy of Aloe vera, tea tree oil and saliva as first aid treatment
for partial thickness burn injuries- December 2008, (Leila Cuttle, et
al)

Wound healing and toxicity evaluation of Aloe Vera cream Toxicology Letters, Volume 172, Supplement 1, 7 October 2007,
Page S233- Abdolhossein Moghbel, Aliasghar Hematti, Abdolazim
Ghalambor, Zahra Nazari Khorsgani, Homayoun Agheli, Shahram
Allipanah.

The efficacy of Aloe vera used for burn wound healing: A systematic
review - Burns, Volume 33, Issue 6, September 2007, Pages 713-718
- Ratree Maenthaisong, Nathorn Chaiyakunapruk, Surachet
Niruntraporn, Chuenjid Kongkaew

Antifungal activity of Aloe vera leaves - Fitoterapia, Volume 78,

Issue 3, April 2007, Pages 219-222- Oana Rosca-Casian, Marcel
Parvu, Laurian Vlase, Mircea Tamas

Skin permeation enhancement potential of Aloe vera and a proposed

mechanism of action based upon size exclusion and pull effect International Journal of Pharmaceutics, Volume 333, Issues 1-2, 21
March 2007, Pages 10-16 - Louise Cole, Charles Heard

14

UST FACULTY OF PHARMACY

Effects of heat treatments on the stabilities of polysaccharides

substances and barbaloin in gel juice from Aloe vera Miller Journal of Food Engineering, Volume 75, Issue 2, July 2006, Pages
245-251- Xiu Lian Chang, Changhai Wang, Yongmei Feng, Zhaopu
Liu

Antidiabetic effects of dietary administration of Aloe arborescens

Miller components on multiple low-dose streptozotocin-induced
diabetes in mice: Investigation on hypoglycemic action and systemic
absorption dynamics of aloe components - Journal of
Ethnopharmacology, Volume 103, Issue 3, 20 February 2006, Pages
468-477 Hidehiko Beppu, Kan Shimpo, Takeshi Chihara, Takaaki
Kaneko, Ikuko Tamai, Sachiyo Yamaji, Sayaka Ozaki, Hiroshi
Kuzuya, Shigeru Sonoda

Assessment of Aloe vera (L.) genotoxic potential on Escherichia

coli and plasmid DNA- November 2005 (Alessandra A. Paes-Leme,
et al)

Aloe vera for Preventing Radiation-induced Skin Reactions: A

Systematic Literature Review- September 2005 (J. Richardson, et al)

II.

Diabetes Mellitus
A.
Introduction
Diabetes mellitus is a group of disease in which blood glucose levels are

elevated because of deficient insulin secretion and/or abnormal insulin action (Bishop

15

UST FACULTY OF PHARMACY

et al., 2005). Insulin is a hormone produced in the pancreas, an organ near the stomach.
Insulin is needed to turn sugar and other food into energy. Diabetes is the most
common set of disorders of carbohydrate metabolism, affecting approximately 23.6
million Americans as of 2007 and 83 million Filipinos as of 2005. The prevalence of
diabetes, diagnosed and undiagnosed is increasing with the estimation of 7.8% of the
population have diabetes. This chronic disease is responsible for significant morbidity,
mortality and cost. Diabetes is the leading cause of treated end-stage renal disease, the
most common non-traumatic amputations and the foremost cause of new blindness in
adults ages 20-74. Nerve damage, known as diabetic neuropathy, occurs in 60-70% of
people with diabetes. Most diabetes related deaths, however, are related to the increased
risk of developing atherosclerosis disease. People with diabetes are at least two to four
times most like to have heart disease and cerebrovascular disease than those without
diabetes (McPherson, 2007). In 2007, it was estimated that diabetes in the United States
cost $174 billion, represented by $116 billion in direct costs and $58 billion indirect
costs (National Diabetes Statistics, 2007).
The expert committee on the diagnosis and classification of diabetes
mellitus revised the criteria for the diagnosis of diabetes in 1997, with recent
modifications (American diabetes association, 2004). New recommendations for the
classification and diagnosis of diabetes mellitus include the preferred use of the terms
"type 1" and "type 2" instead of "IDDM" and "NIDDM" to designate the two major
types of diabetes mellitus; simplification of the diagnostic criteria for diabetes mellitus
to two abnormal fasting plasma determinations and a lower cutoff for fasting plasma

16

UST FACULTY OF PHARMACY

glucose (126 mg per dL [7 mmol per L] or higher) to confirm the diagnosis of diabetes
mellitus. These changes provide an easier and more reliable means of diagnosing
persons at risk of complications from hyperglycemia. Risk factors include obesity, firstdegree relatives with diabetes mellitus, hypertension, hypertriglyceridemia or previous
evidence of impaired glucose homeostasis. Earlier detection of diabetes mellitus may
lead to tighter control of blood glucose levels and a reduction in the severity of
complications associated with this disease.
The classification of diabetes was revised with current minor adjustment
(American diabetes association, 2004). There are two types of Diabetes mellitus. It can
occur as Type I diabetes which occurs in childhood and is the result of underproduction
of insulin by the beta cells while Type II diabetes occur only in adulthood whose
responsiveness to insulin is abnormally low (Thierer et al., 2007). Many patients with
type 2 diabetes use insulin therapy, so it is no longer referred to as non-insulindependent diabetes. Uncommon causes of diabetes include genetic defects of beta cell
function and insulin action, pancreatic diseases, endocrinopathies such as Cushings
syndrome, acromegaly and certain drugs, chemicals and infections (McPherson et al.,
2007).
B.

Classification/ Kinds of Diabetes Mellitus

Prediabetes is a condition in which blood sugar levels are too high to be

considered normal but not high enough to be labeled diabetes. People have prediabetes
if their fasting blood sugar level is between 101 mg/dL and 126 mg/dL or if their blood
sugar level 2 hours after glucose tolerance test is between 140 mg/dL and 200 mg/dL.

17

18

UST FACULTY OF PHARMACY

Identifying people with prediabetes is important because the condition carries a higher
risk for future diabetes as well as heart disease. Decreasing body weight by 5 to 10 %
through diet and exercise can significantly reduce the risk of developing future diabetes
(http://www.merckmanuals.com/).
There are two main types of diabetes mellitus, type 1 & type 2, but
several other rare types exist including gestational diabetes mellitus that occurs during
pregnancy. In gestational diabetes, the metabolic abnormality usually disappears after
delivery although women who had this condition are at a higher risk (30 to 60 percent)
of developing diabetes later in life (Gonzales, 2010).
Type 1 diabetes (previously known as insulin-dependent or childhoodonset diabetes) is characterized by a lack of insulin production (WHO).

Type 1

diabetes accounts for about 5 to 10 percent of all cases of diabetes. It is an autoimmune

disease that develops when the bodys defense system (immune system) against
infection and other foreign substances turns awry and attacks and destroys the cells in
the pancreas that produce insulin. It is still unknown what induces the immune system
to attack the cells of the pancreas but genetic and environmental factors (like viruses)
probably play a role. Type 1 diabetes usually arises in childhood or early adulthood
(Gonzales, 2010).
Type 2 diabetes (non-insulin-dependent or adult onset diabetes) is caused by the
bodys ineffective use of insulin. It often results from excess body weight and physical
inactivity (WHO). Type 2 diabetes accounts for about 90 to 95 percent of all the cases
of diabetes. Initially, people with this type of diabetes produce enough insulin, but for

UST FACULTY OF PHARMACY

unknown reasons, the cells do not respond appropriately to it. Subsequently, over a
period of years, insulin production by the pancreas decreases (Gonzales, 2010).
The risk factors for Type 2 diabetes include advancing age, obesity, family
history of diabetes, previous history of gestational diabetes, and a sedentary lifestyle
(Gonzales, 2010).
C.
Causes
Insufficient production of insulin (either absolutely or relative to the body's
needs), production of defective insulin (which is uncommon), or the inability of cells to
use insulin properly and efficiently leads to hyperglycemia and diabetes. This latter
condition affects mostly the cells of muscle and fat tissues, and results in a condition
known as insulin resistance. This is the primary problem in type 2 diabetes. The
absolute lack of insulin, usually secondary to a destructive process affecting the insulin
producing beta cells in the pancreas, is the main disorder in type 1 diabetes. In type 2
diabetes, there also is a steady decline of beta cells that adds to the process of elevated
blood sugars. Essentially, if someone is resistant to insulin, the body can, to some
degree, increase production of insulin and overcome the level of resistance. After time,
if production decreases and insulin cannot be released as vigorously, hyperglycemia
develops (Marks, 2005).
Glucose is a simple sugar found in food. Glucose is an essential nutrient
that provides energy for the proper functioning of the body cells. Carbohydrates are
broken down in the small intestine and the glucose in digested food is then absorbed by
the intestinal cells into the bloodstream, and is carried by the bloodstream to all the
cells in the body where it is utilized. However, glucose cannot enter the cells alone and

19

UST FACULTY OF PHARMACY

needs insulin to aid in its transport into the cells. Without insulin, the cells become
starved of glucose energy despite the presence of abundant glucose in the bloodstream.
In certain types of diabetes, the cells' inability to utilize glucose gives rise to the ironic
situation of "starvation in the midst of plenty". The abundant, unutilized glucose is
wastefully excreted in the urine (Marks, 2005).
Insulin is a hormone that is produced by specialized cells (beta cells) of
the pancreas (It is a deep-seated organ in the abdomen located behind the stomach). In
addition to helping glucose enter the cells, insulin is also important in tightly regulating
the level of glucose in the blood. After a meal, the blood glucose level rises. In response
to the increased glucose level, the pancreas normally releases more insulin into the
bloodstream to help glucose enter the cells and lower blood glucose levels after a meal.
When the blood glucose levels are lowered, the insulin release from the pancreas is
turned down. It is important to note that even in the fasting state there is a low steady
release of insulin than fluctuates a bit and helps to maintain a steady blood sugar level
during fasting. In normal individuals, such a regulatory system helps to keep blood
glucose levels in a tightly controlled range. As outlined above, in patients with diabetes,
the insulin is either absent, relatively insufficient for the body's needs, or not used
properly by the body. All of these factors cause elevated levels of blood glucose known
as hyperglycemia (Marks, 2005).
D.
Signs and Symptoms
The early symptoms of untreated diabetes are related to elevated blood sugar
levels and loss of glucose in the urine. High amounts of glucose in the urine can cause

20

UST FACULTY OF PHARMACY

increased urine output and lead to dehydration. Dehydration causes increased thirst and
water consumption.
The inability of insulin to perform normally has effects on protein, fat and
carbohydrate metabolism. Insulin is an anabolic hormone, that is, one that encourages
storage of fat and protein. A relative or absolute insulin deficiency eventually leads to
weight loss despite an increase in appetite.
The two types of diabetes have very similar symptoms. The first symptoms are
related to the direct effects of high blood sugar levels. When the blood sugar level rises
above 160 to 180 mg/dL, sugar spills into the urine. When the level of sugar in the
urine rises even higher, the kidneys excrete additional water to dilute the large amount
of sugar. Because the kidneys produce excessive urine, people with diabetes urinate
large volumes frequently (polyuria). The excessive urination creates abnormal thirst
(polydipsia). Because excessive calories are lost in the urine, people lose weight. To
compensate, people often feel excessively hungry. Other symptoms include blurred
vision, drowsiness, nausea, and decreased endurance during exercise.
Type 1: In people with type 1 diabetes, the symptoms often begin abruptly and
dramatically. A condition called diabetic ketoacidosis may quickly develop. Without
insulin, most cells cannot use the sugar that is in the blood. Cells still need energy to
survive, and they switch to a back-up mechanism to obtain energy. Fat cells begin to
break down, producing compounds called ketones. Ketones provide some energy to
cells but also make the blood too acidic (ketoacidosis). The initial symptoms of diabetic
ketoacidosis include excessive thirst and urination, weight loss, nausea, vomiting,
fatigue, andparticularly in childrenabdominal pain. Breathing tends to become

21

UST FACULTY OF PHARMACY

deep and rapid as the body attempts to correct the blood's acidity. The breath smells like
nail polish remover, the smell of the ketones escaping into the breath. Without
treatment, diabetic ketoacidosis can progress to coma and death, sometimes within a
few hours (American Diabetes Association, 2007).
Type 2: People with type 2 diabetes may not have any symptoms for years or
decades before they are diagnosed. Symptoms may be subtle. Increased urination and
thirst are mild at first and gradually worsen over weeks or months. Eventually, people
feel extremely fatigued, are likely to develop blurred vision, and may become
dehydrated (Katzung, 2009).
Because people with type 2 diabetes produce some insulin, ketoacidosis does
not usually develop. However, the blood sugar levels can become extremely high (often
exceeding 1,000 mg/dL). Such high levels often happen as the result of some
superimposed stress, such as an infection or drug use. When the blood sugar levels get
very high, people may develop severe dehydration, which may lead to mental
confusion, drowsiness, and seizures, a condition called nonketotic hyperglycemichyperosmolar coma (American Diabetes Association, 2007).
Gestational Diabetes symptoms same with type 1 and 2 like; increased thirst,
increased urination, weight loss in spite of increased appetite, fatigue, nausea and
vomiting, frequent infections including those of the bladder, vagina, and skin and
blurred vision. An oral glucose tolerance test between the 24th and 28th weeks of
pregnancy is the main test for gestational diabetes (American Diabetes Association,
2007).
Complications: Emergency complications include diabetic coma. Long term
complications include: diabetic retinopathy (eye disease), diabetic nephropathy (kidney

22

UST FACULTY OF PHARMACY

disease), diabetic neuropathy (nerve damage), peripheral vascular disease (damage to

blood vessels/circulation), high cholesterol, high blood pressure, atherosclerosis and
coronary artery disease (American Diabetes Association, 2007).
Uncontrolled diabetes can lead to poorly healing wounds. There is skin breaks
and people with uncontrolled diabetes are more likely to have certain skin problems,
including bacterial infections, fungal or yeast infections, dry skin, and itching. Another
is that there is a difficulty in fighting an infection. The immune system helps your body
to fight infection. In diabetes, the immune system does not work as well as in a person
without diabetes. Even small scrapes can result in open, infected sores. Another
symptom would be lack of feeling. This is caused by nerve damage. It most common is
in the feet. People with poor feeling in their feet may not realize that they have cuts, or
that their shoes are causing blisters or wounds. They also may walk abnormally, which
puts a lot of pressure on certain points on their feet. This leads to calluses and wounds
over these pressure points. They may not feel the calluses or the wounds over the
pressure points. Nerve damage can also cause the joints in the foot to change shape.
This change in shape makes walking more abnormal, creating pressure points that can
lead to ulcers. And diabetes could cause clogged arteries. People with diabetes may get
clogged arteries (called atherosclerosis) at younger ages than other people do. If the
arteries in your legs are clogged, you will have poor blood flow to your legs and feet.
People with clogged arteries in their legs are more likely to develop wounds, have
severe wound infections, and have difficulty healing (American Diabetes Association,
2008).
E.

Treatment

23

UST FACULTY OF PHARMACY

The major goal in treating diabetes is to minimize any elevation of blood sugar
(glucose) without causing abnormally low levels of blood sugar. Type 1 diabetes is
treated with insulin, exercise, and a diabetic diet. Type 2 diabetes is treated first with
weight reduction, a diabetic diet, and exercise. When these measures fail to control the
elevated blood sugars, oral medications are used. If oral medications are still
insufficient, treatment with insulin is considered (Mathur, 2005).
Adherence to a diabetic diet is an important aspect of controlling elevated blood
sugar in patients with diabetes. The American Diabetes Association (ADA) has
provided guidelines for a diabetic diet. The ADA diet is a balanced, nutritious diet that
is low in fat, cholesterol, and simple sugars. The total daily calories are evenly divided
into three meals. In the past two years, the ADA has lifted the absolute ban on simple
sugars. Small amounts of simple sugars are allowed when consumed with a complex
meal (Mathur, 2005).
Weight reduction and exercise are important treatments for diabetes. Weight
reduction and exercise increase the body's sensitivity to insulin, thus helping to control
blood sugar elevations (Mathur, 2005).
Medications for type 2 diabetes: Based on what is known, medications for
type 2 diabetes are designed to: increase the insulin output by the pancreas, decrease
the amount of glucose released from the liver, increase the sensitivity (response) of
cells to insulin, decrease the absorption of carbohydrates from the intestine, and slow
emptying of the stomach to delay the presentation of carbohydrates for digestion and
absorption in the small intestine (Shiel, 2005).

24

UST FACULTY OF PHARMACY

When selecting therapy for type 2 diabetes, consideration should be given to:
the magnitude of change in blood sugar control that each medication will provide, other
coexisting medical conditions (high blood pressure, high cholesterol, etc.), adverse
effects of the therapy, contraindications to therapy, issues that may affect compliance
(timing of medication, frequency of dosing) and cost to the patient and the healthcare
system (Shiel, 2005).
This information is not applicable for pregnant women and lactating mothers.
The only recommended way of controlling diabetes in women who are pregnant or
breastfeeding is by diet, exercise and insulin therapy (Shiel, 2005).
III. Wound Healing and Diabetes
The three general phases involved in wound healing are the acute inflammatory
phase, the proliferative phase, and the maturation phase. The initiation and transition of
these phases have no clear-cut boundaries but are descriptors on a continuum of events.
The initial phase, inflammation, involves transient vasoconstriction of local arterioles
and capillaries followed by an influx of inflammatory cells and plasma proteins to
mediate the repair process. The next phase is proliferation, where fibroblastic activity
and angiogenesis by the endothelial cells begin. The maturation phase may last for up
to two years and involves collagen synthesis and breakdown (Than Dinh, 2002).
Diabetic ulcers are chronic wounds that are the result of repetitive trauma in an
insensate foot. Furthermore, the unique characteristics of diabetes results in poor
resistance to infection and peripheral vascular disease that makes treatment of these
wounds difficult. However, adherence to good wound care principles, such as

25

UST FACULTY OF PHARMACY

aggressive debridement of all non-viable tissue, adequate pressure offloading, prompt

infection treatment, use of moist wound dressings, judicious use of advanced wound
care products, and tight glucose control, may provide the best means of treating these
challenging wounds. Finally, it is necessary to note that self-assessment and foot
inspection, especially in patients with diabetes diagnosed with peripheral neuropathy, is
essential in order to detect onset of possible ulceration. Early detection and treatment of
a diabetic ulcer can halt the development of complications and save the foot from
possible amputation (Doupis, 2008).

26

UST FACULTY OF PHARMACY

CHAPTER 3
RESEARCH METHODOLOGY

This chapter dealt with the type of research method and design that was used in
the study of the Effectiveness of Aloe vera powder in treating the dermal wounds of
diabetic rats.
I. Plant collection and Identification
The whole Aloe vera (Aloe barbadensis Miller) plant was obtained from
Malabon, Rizal and was identified and verified by Dr. Rosie S. Madulid, College of
Science, University of Santo Tomas. (See Appendix A)
II. Plant extraction
Full size mature leaves of Aloe vera (Aloe barbadensis) were cut from the plant
and the rind was removed. The colorless parenchyma gel, which was seven (7)
kilograms in weight, was grinded in a blender and centrifuged for 30 minutes at 4C to
remove fibers. The supernatant was freeze dried or lyophilized and stored at room
temperature until use (Panda, 2003). A powder dosage form was obtained. (See
Appendix F)
III. Physical Test
Percentage Yield was determined using the calculation initial mass or the
actual mass obtained and the theoretical mass, using the following formula:
Percentage Yield =

mass of Actual Yield

x 100%
mass of Theoretical Yield

27

UST FACULTY OF PHARMACY

Color and Texture was determined by observation using the extracted Aloe
vera powder.
Bulk density
The cylinder used was tared then was filled with Aloe vera powder extract up
to sixty (60) milliliters and was weighed again. The bulk density was computed using
the formula:
Bulk Density =

Loss on drying or Moisture content

The empty dish was weighed. The powder was added and weighed again. The
evaporationg dish with the powder was placed in the oven at 102C 2C for two (2)
hours. The evaporating dish was cooled and weighed. The evaporating dish was dried
again in the oven at 102C 2C for 1 hour. Cooling and weighing was repeated.
Drying was repeated until weight was constant then the moisture was computed using
the formula (GEA Niro Laboratory, 2006):
% Moisture =

IV. Chemical Test

Chrysophanic Acid
Borntragers test was used in the determination of chrysophanic acid
(anthraquinone). Hexane was added to the sample and was shaken. The upper lipophilic

28

UST FACULTY OF PHARMACY

layer was separated and the lower layer was treated with diluted ammonia. Formation
of violet to pink color indicated the presence of anthraquinone.
Barbaloin
The test was performed according to the European Pharmacopoeia 5th Ed. Main
Volume 5.0, 2005. A small amount of powdered substance was dissolved in boiling
water, cooled, and filtered after mixing with talc. The filtrate was treated with freshly
prepared bromine water to obtain a formation of a brownish-yellow or a violet colored
solution will indicate the presence of Barbaloin.
Carbohydrates
The Molish Test was used to test for carbohydrates. The sample solution was
placed in a test tube and molisch reagent (a solution of -napthol in 95% ethanol) was
added. The solution was then poured slowly into a tube containing concentrated
sulfuric acid which formed two layers. Formation of a purple product at the interface of
the two layers formed will indicate the presence of carbohydrates (GEA Niro
Laboratory, 2006).
In Iodine/ Potassium Iodide Test, iodine solution was added to the sample then
observations were recorded. Formation of a blue-black color was formed will indicate
the presence of polysaccharide (acemannan).

29

UST FACULTY OF PHARMACY

V. Pharmacologic Test
A. Sensitivity Test (Draize Type)
Experimental Animals
Both male and female rabbits from Bioresearch were used to determine the
sensitivity of the obtained powder drug.
The animal model was prepared for this test. The fur was removed from the
dorsal area (back) of the trunk of the test animal by shaving. At least 10% of the area
was clear. The skin was cleaned using 70% alcohol and was divided into half. The left
side was for control and the right side was for treated. These were done for both patch
and scratch test.
Patch Test
For patch test, the test sample was applied at the right side. The back was
covered with sterilized gauze and was kept in place using surgical tapes. The site was
left undisturbed until 24 to 72 hours. The patches were removed and the reactions were
evaluated according to the scores and the back was covered again. This was repeated
after 72 hours.
Scratch Test
For the scratch test, the back of the two rabbits were shaved and scratched using
small needles. The wounded backs were divided into two. Aloe vera powder was
topically added on the first half of the back of the first rabbit leaving the other half
untreated, while Iodosorb was topically administered on the first half of the second

30

UST FACULTY OF PHARMACY

rabbit leaving the other half untreated. Observation was done after 24 and 72 hours
(Yokozeki et al, 2006).
The average of the scores for the patch and scratch test were combined using
the formula to determine the Primary Irritaion Index, which is:
Formula:
Primary Irritation Index =

B. Wound Healing Test

Experimental Animals
Sprague Dawley rats weighing 100-150g obtained from the University of the
Philippines were used for the study. The experimental rats were housed in metal cages
in a well-ventilated room and went on a 12 hour light and dark cycle (Nayak, 2006).
The rats were fed with commercial rat feeds and distilled water ad libitum.
Induction of Diabetes
Diabetes was induced in the rats by a single intraperitoneal injection of
Streptozotocin in citrate buffer. Citrate buffer was made using sodium citrate and was
acidified by the addition of citric acid until it reached pH 4.0. Fasting blood glucose
was checked using Glucose meter (with Glucose oxidase reagent strips) three days after
streptozotocin was injected (Kramer, 2010). Experimental animals with glucose level
greater than 70mg/dL were used for the study (American diabetes association, 2004).
Wound Creation

31

UST FACULTY OF PHARMACY

Wounds were created on the 3rd day after induction of Diabetes. The
experimental animals were caged individually after creation of wounds. The back of
each rat were shaved after anaesthetizing the animal with thiopentone sodium in sterile
water for injection with the strength of 6mg/kg, intraperitoneally. Wounds were made
by cutting from the shaven area using a sterile surgical blade (Gutierrez et al., 2006).
Grouping of Test animals
After wound creation, experimental animals were divided into three groups:
Group I untreated, Group II- wounds treated with Aloe vera powder, and Group IIIwounds treated with Iodosorb (standard).
Administration of the drug
Group of rats received the lyophilized Aloe vera powder twice a day on the
wound surface topically administered. While another set of rats were given Iodosorb
powder twice a day applied topically on the wound surface (Chithra et al. 1997).
Determination of Collagen
Animals were sacrificed on the 8th, 12th and 16th days after wound creation,
and the entire wound on each animal was cut out and stored at 70C until analysis.
The tissues were minced and weighed. Acetone was used to defat the tissues.
After 6 hours or more the acetone was decanted and replaced by fresh acetone which
was allowed to stand for another 6 hours or more. The tissues were extracted with
hexane for 12 to 16 hours. The residue was collected and dried to constant weight at
108 C.

32

UST FACULTY OF PHARMACY

For the extraction of collagen, the dry fat-free tissues were placed in test tubes
with of water. The tubes were stoppered with cotton and gauze which was autoclaved
for 3 hours. After autoclaving, the solution was centrifuged until clear. The residue and
the supernatant liquid were separated. The residue was washed with water and
autoclaved again for 3 hours, while the supernatant in the test tube was evaporated to
dryness by a stream of air into the tube placed in a boiling water bath. The supernatant
and residue from the second autoclaving were separated. The residue was washed twice
with hot water. The supernatant and the two washings were transferred to a test tube
and evaporated to dryness.
To prepare the hydrolysates (hydroxyproline) of the extracted collagen, 6N
hydrochloric acid was added to the extracted collagen. The tubes were sealed and
autoclaved for 3 hours. The hydrolysates were neutralized with sodium hydroxide
solution, diluted to volume and filtered. Hydroxyproline was determined.
The procedure was repeated using the tissue sample on 12th and 16th day.
Wound Contraction Rate
The determination of the rate of wound contraction was done by tracing the
wounds using a transparent paper having a millimeter scale. The change on wound size
was calculated as the percentage of wound area that healed. Observations were done on
the 4th, 8th, 12th and 16th (Nayak et al. 2007).
% Wound closure =

Where n= numbers of days (4th, 8th, 12th and 16th)

33

UST FACULTY OF PHARMACY

VIII. Statistical Analysis

Rats treated with Aloe vera powder extract (Group II) and rats treated with
Iodosorb (GroupIII) were compared with untreated rats (Group I). Comparison of the
percent increase of wound contraction and collagen content of dry granulation in grams
were analyzed using two-way ANOVA from the results taken in various observation
days.

34

35

UST FACULTY OF PHARMACY

CHAPTER 4
RESULTS AND DISCUSSION

This chapter was a representation of the results obtained from the different type
of research method and design performed.

Physical Test
The extracted Aloe vera powder was a white to pinkish white amorphous
powder that has a percent yield of 9.5%, bulk density of 0.145 and moisture content of
3.38%.
Chemical Test
The powder was subjected to various chemical tests. The results obtained were
shown in Table 1.
Table 1 Results of the Chemical Test
Test

Actual Result

Theoretical Result

Inference

Chrysophanic Acid
(Anthraquinone)

Rose pink solution

Rose pink solution

Barbaloin
Acemannan
(Carbohydrates)

Brownish yellow solution Brownish yellow solution

36

UST FACULTY OF PHARMACY

A)Molisch Test

Purple product at the

interface of two layers

Purple product at the

interface of two layers

Blue black solution

Blue black solution

B) Iodine Test

Aloe vera powder showed positive in chrysophanic acid, barbaloin and

carbohydrates (acemannan). Chrysophanic acid and barbaloin are anthraquinones,
which were responsible for wound healing property of Aloe vera, whereas acemannan,
a polysaccharide carbohydrate, was responsible for anti-infective property of Aloe vera.
Sensitivity Test
Table 2 Results of Aloe vera treated wounds of diabetic rats for both patch and scratch test.
(ERY erythema & ED edema)

Animal
No.
1

(+) Aloe vera Treated Patch

After 24 hrs After 72 hrs
ERY ED
ERY ED
0
0
0
0

(+) Aloe vera Treated Scratch

After 24 hrs After 72 hrs
ERY ED
ERY ED
0
0
0
0

Score
0

Table 3 Results of Iodosorb treated wounds of diabetic rats for both patch and scratch test.
(ERY erythema & ED edema)

(+) Iodosorb Treated - Patch

Animal
No.
1

After 24 hrs
ERY ED
0
0

After 72 hrs
ERY ED
0
0

(+) Iodosorb Treated Scratch

After 24 hrs After 72 hrs
ERY ED
ERY ED
0
0
0
0

Score
0

UST FACULTY OF PHARMACY

As shown in Tables 2 and 3, both the Aloe vera powder extract and the standard
drug (Iodosorb) show no dermal irritation in the animal model. This means that Aloe
vera powder is not a primary skin irritant.

The range of the blood glucose level in the experimental animal, rats, before
induction of streptozotocin was 4 to 6 mmol/dL (72 to 108 mg/dL).And the range of blood
glucose level after induction of streptozotocin was 7 to 8 mmol/dL (126 to 144 mg/dL),
indicating that there was an increase in blood glucose level. This means that the rats achieved
hyperglycemia.
Collagen Content

Figure 1. According to post hoc test, The collagen content of Aloe vera treated wounds showed significant
difference than the untreated wounds (Pp value = 0.0000) but showed no significant difference on Iodosorb
treated wounds (P value= 0.3167).

37

UST FACULTY OF PHARMACY

Figure 1, show the collagen content of Group I Untreated, Group II - Aloe

vera treated, and Group III Iodosorb treated (standard drug) of diabetic rats. There
was a steep increase in collagen content observed after 4 days and a steep decrease after
day 8. The levels of collagen content continue to decrease after day 12, with a much
slower rate. The collagen content reached maximum levels 8 days after wound creation.
Aloe vera treated and Iodosorb treated wounds showed large amount of collagen on day
8, as compared to untreated wounds.
Wound Contraction Rate

Figure 2. According to post hoc test, the percent wound contraction of Aloe vera treated wounds showed
significant difference than the untreated wounds but showed no significant difference on Iodosorb treated
wounds. (P value= 0.00 and P value= 0.143)

38

UST FACULTY OF PHARMACY

In Figure 2, it shows the rate of wound contration expressed in terms of

percentage of wound area that healed. It may seen in Group II rats showed 99.34% and
Group III rats showed 100%, where as Group I showed only 86.67%, on the 16th day.

39

UST FACULTY OF PHARMACY

CHAPTER 5
CONCLUSION AND RECOMMENDATION

In this chapter, the researchers of the study finalized the results obtained from
the different type of research method and design performed.

Conclusion
The researchers of the study concluded that the Aloe vera powder obtained was
an amorphous white to pinkish white powder with a moisture content of 3.38%, bulk
density of 0.145 g/mL and percent yield of 9.5% w/w. Barbaloin, acemannan and
chrysophanic acid were found present in the powder extract. The Aloe vera powder and
the standard drug (Iodosorb) showed no dermal irritation in the animal model. Using
two-way ANOVA, the ratio of collagen content and rate of wound contraction of the
Aloe vera treated and Iodosorb treated wounds found to have no significant difference
but showed significant difference with the untreated wounds.
Therefore, wounds treated with Aloe vera powder heal significantly faster than
the untreated wounds, but showed no significant difference with the standard drug
(Iodosorb). As a result, Aloe vera powder may be a potential wound healing drug for
diabetic cases.

40

UST FACULTY OF PHARMACY

Recommendation
Based on the findings and conclusions drawn by the researchers, the following
recommendations are offered:

To conduct stability study on the Aloe vera extract under accelerated

temperatures to qualify the crude extract and characterize the degradation
patterns.

To make further studies on female rats if there is a significant difference on the

efficacy of Aloe vera powder between the male and female rats.

To evaluate the effectiveness of Aloe vera powder in incision wound model

To determine the rate of epithelialization of the wounds

41

UST FACULTY OF PHARMACY

BIBLIOGRAPHY

1. Aloin. Brittish Pharmacopoeia 5th Ed. Main. Volume 5.0. London : Stationery
Office, 2005.
2. Atala, Anthony, Robert Lanza and James A. Thomson. Principles of
Regenerative Medicine. Amsterdam ; Boston : Elsevier/Academic Press. 2008.
3. Barbaloin. European Pharmacopoiea 5th Ed. Main. Volume 5. Strasbourg :
Council of Europe. 2005.
4. Barcroft A,. Myskja, A., (2003). Aloe vera: Natures Silent Healer. London:
BAAM Publishing Ltd. pp. 29-30.
5. British Pharmacopoeia Commission. (2009). British Pharmacopeia 2010.
Norwich, United Kingdom: TSO. 2010.
6. Bryant, Ruth A. and Nix, Denise P. Acute and Chronic Wounds: Current
Management Concepts. 2007. Edinburgh: Elsevier Mosby, 2007.
7. Chithra, P., Sajithlal, G.B., Chandrakasan, G. (1997). Influence of Aloe vera in
Dermal Wounds of Diabetic rats. Journal of Ethnopharmacology, 59, 195-201.
8. Del Rio, Luis Alfonso and Alain Puppo. A Reactive Oxygen Species in Plant
Signaling. Berlin, New York : Springer, 2009.

42

UST FACULTY OF PHARMACY

9. Duggan, Christopher, John B. Watkins and W. Allan Walker. Nutrition in

pediatrics: basic science, clinical applications. Hamilton, Ontario ; Lewiston,
NY : B.C. Decker, 2008.
10. Falanga, V. (2008). Chronic wound: Impaired wound healing and solutions in

the context of wound bed preparation, Blood Cells Mol. Dis, 32, 88.
11. Ford, Ashley M. Focus on Diabetes Mellitus Research. New York : Nova
Biomedical Books, 2006.
12. Fraga, Cesar G. Plant Phenolics and Human Health: Biochemistry, Nutrition,
and Pharmacology. Novartis Foundation. Defining optimal immunotherapies for
type 1 diabetes. Chichester, UK; Hoboken, New Jersey: Wiley-Blackwell,
2008.
13. Frego, Lawrence F. An End to All Disease. Bloomington, IN : Authorhouse,
2006.
14. Gonzales, Ralph and Jean S. Kutner. Current Practice Guidelines in Primary
Care 2011 (LANGE CURRENT Series Current Practice Guidelines in Primary
Care). New York: Lange Medical Books/McGraw-Hill, 2004.
15. Greenhalgh D G. (2003). Wound Healing and Diabetes Mellitus. Clinic Plastic

Surg, 30, 37.

16. Helms, Richard A. and David J. Quan. Textbook of therapeutics: drug and
disease management. Philadelphia, Pa: Lippincott Williams & Wilkins, 2006.

43

UST FACULTY OF PHARMACY

17. Henry, John Bernard, Richard A. McPherson and Matthew R. Pincus. Henry's
Clinical Diagnosis and Management by Laboratory Methods. Philadelphia:
Saunders Elsevier, 2007.
18. Herndon , David N. Total Burn Care. Edinburgh: Saunders Elsevier, 2007.
19. Insel, Paul, Don Ross, Kimberley McMahon and Melissa Bernstein. Nutrition .
New York: McGraw-Hill, 2007.
20. Kamkin, Andre and Irina Kiseleva. Mechanosensitivity of the Heart .
Dordrecht Springer Science+Business Media B.V. 2010.
21. Katsilambros, Nicholas, Eleftherios Dounis, Konstantinos Makrilakis, Nicholas
Tentolouris and Panagiotis Tsapogas. Atlas of the Diabetic Foot. Chichester ;
Hoboken, NJ : Wiley, 2003.
22. Lachapelle, J., & Maibach, H. I., (2003). Patch Testing Prick Testing: Practical
Guide. Retrieved on December 7, 2010 From
http://books.google.com.ph/books?id=Vm5STkGs58C&pg=PA155&dq=Scratch+and+Patch+Test&hl=en&ei=sJBsTc
HmA4TBcdvjvbMF&sa=X&oi=book_result&ct=result&resnum=1&ved=0CC
wQ6AEwAA#v=onepage&q=Scratch%20and%20Patch%20Test&f=false
23. Langley, R. & Moore, J. (2008-2009). AP Chemistry. New York: McGraw Hill.

44

UST FACULTY OF PHARMACY

24. Levetin, Estelle and Karen McMahon. Plants and society. Dubuque, IA :
Wm.C. Brown Publishers, 1996.
25. Majno, Guido and Joris, Isabelle. Cells, tissues, and disease: principles of
general pathology. New York: Oxford University Press, 2004.
26. Marieb , Elaine Nicpon, Jon Mallatt and Patricia Brady Wilhelm. Human
Anatomy. San Francisco: B. Cummings, 2007.
27. Mattson, Mark P., Edward J. Calabrese. Hormesis: A Revolution in Biology,
Toxicology and Medicine. New York : Springer, 2010.
28. Miyachi, Yoshiki. Therapy of Skin Diseases: : A Worldwide Perspective on
Therapeutic Approaches and their Molecular Basis. Dordrecht : Springer, 2010.
29. Mosby Inc. Mosby's medical dictionary. St. Louis, MO : Mosby, 2009.
30. Neuman, R., Logan, M., (1950). The determination of collagen and elastin in
tissue. Journal of Biological Chemistry, 184, 299
31. Nunes, Maria Cecilia N. Color Atlas of Postharvest Quality of Fruits and
Vegetables. Ames, Iowa: Blackwell Pub., 2008.
32. Poretsky, Leonid . Principles of Diabetes Mellitus. New York : Springer Verlag,
2010.
33. Quisimbing, Eduardo A. Medicinal plants of the Philippines. Manila. Katha
Pub. Co., 1951.

45

UST FACULTY OF PHARMACY

34. Regensteiner, Judith. Diabetes and Exercise. New York, NY : Humana Press,
2009.
35. Roden, Michael. Clinical diabetes research: methods and techniques.
Chichester, West Sussex, England ; Hoboken, NJ : John Wiley & Sons, 2007.
36. Ross, Tami. American Dietetic Association Guide to Diabetes Medical Nutrition
Therapy. Chicago, Ill.: American Dietetic Association, 2005.
37. Shai A., Maibach, H., (2005). Wound Healing and Ulcers of the Skin. Retrieved
on December 7, 2010
From:http://books.google.com.phbooksid=p3poHaIN8v8C&pg=PA211&dq=W
ound+healing+aloe+vera&hl=en&ei=Yo9sTYz5NIeycf_syLsF&sa=X&oi=book
_result&ct=result&resnum=1&ved=0CDIQ6AEwAA#v=onepage&q=Wound
%20healing%20aloe20vera&f=false
38. Stashak, Ted S. and Christine Thort. Equine Wound Management. Ames,
Iowa : Wiley-Blackwell : Veterinary Wound Management Society/V.W.M.S.,
2008.
39. Trevor, Anthony J. , Bertram G. Katzung and Susan B. Masters. Pharmacology
Examination and Board Review. New York : McGraw-Hill Medical ; London :
McGraw-Hill [distributor], 2008.
40. Veves , Aristidis and Rayaz A. Malik. Diabetic neuropathy: clinical
management. Totowa, N.J. : Humana Press, 2007.

46

UST FACULTY OF PHARMACY

41. Wen, Hong and Kinam Park. Oral Controlled Release Formulation Design and
Drug Delivery: Theory to Practice. Hong Wen, Kinam Park Hoboken, N.J. :
Wiley, 2010.

47