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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 (β) 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

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in granulation tissue and collagen formation (Yue et al., 1986), the aberrant control of

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,

texture, moisture content, and bulk density.

3. Are the substances chrysophanic acid, barbaloin and the polysaccharide

acemannan present in the powdered extract?

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

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

5. Is there a significant difference on the ratio of collagen contents of the

untreated, Aloe vera treated and Iodosorb treated wounds of diabetic

rats?

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 community’s 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

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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

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 (β) 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)

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Dermis - is a layer of skin beneath the epidermis (Marieb, 2005)

Deoxyribonucleic acid (DNA) - contains the genetic instructions used in

the development and functioning of all known living organisms and

some viruses (Marieb, 2005)

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)

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Rind - the outer layer of a fruit (Moore, 2003)

Ulcers (dermal) - a disintegration of the surface of the skin (Marieb, 2009)

Vascular - relating to the vessels of the body, especially the arteries and veins

that carry blood and lymph (Marieb, 2005)

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CHAPTER II

REVIEW OF RELATED LITERATURE

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. Aloe vera

A. 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.

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B. Botanical Description

Aloe vera is a stemless succulent plant growing to 60–100 cm (24–39 inches)

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

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

2–3 cm (0.8–1.2 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

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required. In areas that receive frost or snow, the species is best kept indoors or in

heated glasshouses (David, 2006).

C. Constituents

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 α-barbaloins,

obtained from Barbados Aloe, reddened in the cold and β-barbaloins, obtained from

Socotrine and Zanzibar Aloe, reddened by ordinary nitric acid when warmed or by

fuming acid in the cold. Nataloin (2C17H13O7·H2O) forms bright yellow scales while

barbaloin (C17H18O7) forms prismatic crystals. Aloe species also contain a trace of

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volatile oil, to which its odour is due. In addition, the presence of chrysophanic acid

may bring about the extract’s healing effect on skin (Levetin, 2006).

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% β-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, athlete’s 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

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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

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

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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 1°C 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.

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 hay-

fever 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).

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G. Current Studies

1. 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. Guillén, D.

Valero, M. Serrano, D. Martínez-Romero

2. International

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, Sun-

Tack 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)

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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

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

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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

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)

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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

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).

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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

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, first-

degree 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-insulin-

dependent diabetes. Uncommon causes of diabetes include genetic defects of beta cell

function and insulin action, pancreatic diseases, endocrinopathies such as Cushing’s

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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.

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 childhood-

onset 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 body’s 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

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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

body’s 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

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

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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

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

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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

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

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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, and—particularly in children—abdominal pain. Breathing tends to become

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

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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 hyperglycemic-

hyperosmolar 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

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

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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

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

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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).

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).

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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

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).

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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 4°C 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                 

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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 102˚C ± 2˚C for two (2)

hours. The evaporating dish was cooled and weighed. The evaporating dish was dried

again in the oven at 102°C ± 2°C 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

Borntrager’s test was used in the determination of chrysophanic acid

(anthraquinone). Hexane was added to the sample and was shaken. The upper lipophilic

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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).

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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

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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

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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 III-

wounds 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 −70°C 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.

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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)

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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.

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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 Brownish yellow solution

Brownish yellow solution

+

Acemannan (Carbohydrates)

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Table 2 Results of Aloe vera treated wounds of diabetic rats for both patch and scratch test. (ERY – erythema & ED – edema)

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A)Molisch Test

B) Iodine Test

Purple product at the interface of two

layers

Blue black solution

Purple product at the interface of two

layers

Blue black solution

+

+

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

Animal No.

(+) Aloe vera Treated - Patch

(+) Aloe vera Treated - Scratch Score

After 24 hrs After 72 hrs After 24 hrs After 72 hrsERY ED ERY ED ERY ED ERY ED

1 0 0 0 0 0 0 0 0 0

2 0 0 0 0 0 0 0 0 0

3 0 0 0 0 0 0 0 0 0

4 0 0 0 0 0 0 0 0 0

Animal No.

(+) Iodosorb Treated - Patch (+) Iodosorb Treated - Scratch Score

After 24 hrs After 72 hrs After 24 hrs After 72 hrsERY ED ERY ED ERY ED ERY ED

1 0 0 0 0 0 0 0 0 0

2 0 0 0 0 0 0 0 0 0

3 0 0 0 0 0 0 0 0 0

4 0 0 0 0 0 0 0 0 0

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

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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).

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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)

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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.

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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.

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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

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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 5 th Ed. Mai n. Volume 5. Strasbourg :

Council of Europe. 2005.

4. Barcroft A,. Myskja, A., (2003). Aloe vera: Nature’s 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=boo

k_result&ct=result&resnum=1&ved=0CDIQ6AEwAA#v=onepage&q=Wound

%20healing%20aloe20vera&f=false

38. Stashak, Ted S. and Christine Théorêt. 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.

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