Effectivity of the Mixture of

Cow Dung and Aloe barbadensis

leaf Exract on Hair Growth Of
Mus Musculus

Researchers:

Bebian Rose M. Flores

Rachel Dawn Acero

Erihca H. Subrabas
 CHAPTER I

INTRODUCTION

A. BACKGROUND OF THE STUDY

Losing hair is a natural and healthy part of the renewal process of the hair.

Experts say that we normally lose about 50 to 100 strands of hair a day. We should worry

when we start experiencing pattern hair loss, temporary hair shedding or even baldness.

Hair loss is a dermatologic disorder that has been recognized for more than 2000 years. It

is common throughout the world and has been estimated to affect nearly 2% of the world

population (Olsen, 1993; Bertolino, 2000). Stress, heredity, aging, lifestyle habits,

nutrition loss and underlying diseases are the common reasons for hair fall. However,

even young people as early as mid 20s encounter hair thinning because of a disrupted hair

growth cycle (Vila, 2016). Thus, people suffering from hair loss or thinning don’t have

healthy hair follicles making the hair strands weak at the base and easy to pluck. In terms

of gender, 50% of men suffer from hair loss caused by the hormone DHT

(Dihydrotestosterone), which is generally known to be responsible for balding but does

not mean women are exempted from it. Hair baldness is used to be a rare case among

women but because of technology uprising, different hair transformation – rebonding,

hair curling, hair coloring and etc,. – have contributed a great risk to women’s hair.

However, there are many things you can do to avoid disrupting your hair cycle or you can
find an alternative treatment it could be cosmetic, medical, and surgical treatment

methods.

Natural products are unequivocally advocated in the cosmetic and hair care industries and

about 1000 plant extracts have been examined for hair care usage. Proanthocynidine from

grape seeds and b-sitosterol in saw palmetto have shown remarkable hair growth–

promoting activity (Takahashi et al., 1998). There are many products available in the

market, which are prepared by combination of one or more herbal drugs and find

acceptability as hair tonic, hair growth promoter, hair conditioner, hair cleansing agent,

antidandruff agents, and for the treatment of alopecia and lice infection (Saraf et al.,

1991; Roy et al., 2007).

Aloe vera (Alo barbadensis) is a thick, short-stemmed plant that stores water in its

leaves and also a popular medicinal plant that is used for cosmetics, pharmaceutical and

food industries. Each leaf is full of a slimy tissue that stores water. This slimy, water-

filled tissue is the “gel” that contains most of the bioactive compounds in the plant,

including vitamins, minerals, amino acids and antioxidants. This aloe vera also contains

proteolytic enzymes which repairs dead skin cells on the scalp that promotes hair growth

(Sravani,2017)

Umesh Soni, founder and CEO of Cowpathy states that in the Indian scriptures,

particularly the vedas, there is mention of cow dung and cow urine having very high

medicinal properties. Ayurveda uses a lot of herbs, but cow dung, with its anti-bacterial

properties, is considered very pure and is part of the ayurveda. It is said that the

Egyptians created a hair tonic made up of a mixture of herbs and cow manure/ dung to
cure hair loss. It is not impossible because cow dung has nutrients that is needed for hair

growth and can make your hair stronger and healthier.

The researchers are able to determine if the mixture of Cow waste and extracted Aloe

vera ( Alo barbadensis) leaf can be used as an alternative treatment for hair growth. This

study was designed to investigate the possible effects of Cow waste and extracted Aloe

vera (Alo barbadensis) leaf mixture on hair growth promotion and rejuvenation of

Albino Mice (Mus Musculus).

B. STATEMENT OF THE PROBLEM

This study aims to examine the effectivity of Cow dung and extracted Aloe vera

(Alo barbadensis) leaf mixture as an alternative treatment on hair growth of Albino mice

(Mus Musculus). Moreover, it specifically seeks to answer the following questions:

1. What are the components of Cow dung and extracted Aloe vera (Alo barbadensis)

leaf mixture that can be used to promote hair growth of Albino Mice (Mus

Musculus)?

2. How does the mixture of Cow dung and Aloe vera (Alo barbadensis) leaf affects the

hair growth of Albino Mice (Mus Musculus)?

3. Is there a significant difference on the hair growth of Albino Mice (Mus Musculus)

before and after treatment?

B.2 OBJECTIVES OF THE STUDY

 The main objective of the study is to ascertain the hair growth of Albino Mice

(Mus Musculus) using Cow dung and extracted Aloe vera (Aloe barbadensis) leaf

mixture.

1. Determine the components of Cow dung and extracted Aloe vera (Alo barbadensis)

leaf mixture that is used for hair growth promotion and rejuvenation of Albino mice

(Mus Musculus).

2. Examine the effectiveness of Cow dung and extracted Aloe vera (Alo barbadensis)

leaf mixture to the hair growth of Albino mice (Mus Musculus).

3. Compare the difference of different concentrations of Cow Dung and extracted Aloe

vera (Alo barbadensis) leaf and a control set-up on the hair growth of Albino Mice

(Mus Musculus).

a) Hair length

b) Hair Follicle number

c) Hair Follicle depth, and

d) Hair Dermal thickness

B.3 RESEARCH HYPOTHESES

Based on the preceding research problems, the researchers formulated the

following hypotheses:

H 1: The mixture of Cow dung and Aloe vera (Alo barbadensis) leaf extract is not

effective on naturally stimulating the hair growth of Albino mice (Mus Musculus).
 H 2: The mixture of Cow dung and Aloe vera (Alo barbadensis) leaf extract is

effective on naturally stimulating the hair growth of Albino mice (Mus Musculus).

H 3: The mixture of Cow dung and Aloe vera (Alo barbadensis) leaf extract is more

effective on naturally stimulating the hair growth of Albino mice (Mus Musculus) if

the concentration is higher.

C. SIGNIFICANCE OF THE STUDY

For years, Aloe Vera has been used as a safe and natural treatment to prevent hair

loss (J. Napoleon, n.d.). And the ancient Egyptians believed that a mixture of herbs and

cow dung could cure hair loss. (E. Lotter, 2016).

By acquiring an in-depth perspective of the contributing factors, this study will be able to

help the following beneficiaries:

People. Help them to know the benefits of the mixture of cow dung and aloe vera leaf as

an alternative treatment that promotes hair growth especially to those people who is/are

experiencing hair fall and hair shedding.

Dermatologist. This study will provide a brilliant idea on formulating a new products in

pharmaceutical out of these mixture. Additionally, this could be amalgamate with the pre
– existing treatment Minoxidil (antihypertensive vasodilator medication that stops hair

loss).

D. SCOPE AND LIMITATION

This study focuses on determining the effects of Cow dung and Aloe vera (Alo

barbadensis) extract in promoting the hair growth of Albino Mice (Mus musculus). The

Aloe vera (Alo barbadensis) plant will be collected at Surigao City and the Cow dung

samples will be collected at Barangay Bonifacio, Surigao City. The "gel" from the Aloe

vera (Alo barbadensis) will be extracted and will be used as the main ingredient same

with the Cow dung which will be decomposed to alleviate the existing pathogents and its

odor. This study will be conducted with a limited number of 15 Albino Mice (Mus

musculus) in different consentrations and a control group consisting of albino mice (Mus

musculus). The following concentration will be: 100% concentration (50% Aloe vera

extract - 50% Cow dung), 75% concentration (37.5% Aloe vera extract - 37.5% Cow

dung), 50 concentration (25% Aloe vera extact - 25% Cow dung) and 25% concentration

(12.5% Aloe vera extract - 12.5% Cow dung). In every trial will contain 3 nuded Albino

Mices (Mus musculus). The experiment will be conducted at P. Reyes, Surigao City
DEFINITION OF TERMS

Alopecia - is commonly known as baldness.

Ayurveda - “ Ayurvedic medicine ”, is one of the world's oldest holistic (“whole-body”)

healing systems. It was developed more than 3,000 years ago in India.

Cowpathy – “ Panchagavya ”, a treatment based on products obtained from cows used

in Ayurvedic medicine and of religious significance for Hindus.

DHT (Dihydrotestosterone) - is thought to cause hair follicles to miniaturize, and

this contributes to male pattern hair loss.

Vedas - are a large body of knowledge texts originating in the ancient Indian

subcontinent.

Albino Mice (Mus Musculus) - The laboratory mouse/ Mus Musculus is a

small mammal of the order Rodentia which is bred and used for scientific research.

Follicle – a tiny hole in the skin from which a hair grows.

Pharmaceutical – substance used in the diagnosis, treatment, or prevention

of disease and for restoring, correcting, or modifying organic functions.

Rejuvenataion – The action or process of making someone or something look or feel

better, younger, or more vital.

Amalgamate – to unite (two or more things, such as two business) into one thing.
Minoxidil – a synthetic drug that is used as a vasodilator in the treatment of

hypertension, and is also used in lotions to promote hair growth.

Aloin – (also known as Aloe latex or Anthraquinones) comes from a thin layer of yellow

sap that separates the outer rind from the inner gel-like portion of the leaf and acts as the

laxative component. In raw form, Aloe vera’s aloin content is around 10,000 parts per

million, which can be harsh on the body.

Biotin - a crystalline, water-soluble vitamin, C10H16O3N2S,

of the vitamin B complex, that is present in all livingcells and functions as

a growth factor and as a catalyst in carboxylation reactions.

Glycoside –a compound formed from a simple sugar and another compound by

replacement of a hydroxyl group in the sugar molecule. Many drugs and poisons derived

from plants are glycosides.

Biogas – a mixture of methane and carbon dioxide produced by the bacterial

decomposition of organic wastes and used as a fuel.

E. REVIEW OF RELATED LITERATURE

Aloe vera

Aloe vera (Aloe barbasensis)is a

tropical plant with succulent leaves has

been used for thousands of years as herbal

medicine. (O. Grundmann,

BPharm,ms,PHD, 2012). Aloe vera is one

such product exhibiting multiple benefits.

Its use in the treatment of oral lesions such as lichen planus and apthous ulcers and also

in the treatment of periodontitis has gained considerable importance in clinical research

(Journal of Indian Society of Periodontology, 2011). The use of aloevera is being

promoted for a large variety of conditions. Aloevera looks like a catus but it isn’t the

plant is a member of the lily family which includes garlic and onion. The aloe plant is

grown in warm tropical areas and cannot survive freezing temperatures. There are many

species of aloe grown around the world but the main medicinal one is Aloevera

Barbadensis. Aloevera a popular herbal remedy used medicinally for thousand years has

multiple dental uses. It has been shown to enhance defence mechanisms, and has variety

of components to help combat periodontal diseases and other oral conditions.

 A. Chemical components of aloe vera:

In 1851 it was discovered that the potency of aloe vera was the result of aloin, a bitter

juice that dried to a yellow powder and functioned as a cathartic medicine. It synonymous

with barbaloin which is a glycoside. Anthraquinone derivatives include anthracenes such

as aloe-emodin, which is 1,8-dihydroxy-3 (hydroxy-methyl)-9,10-anthracenedione. These

water soluble glycosides were separated from the water insoluble resinous material. The

leaves of aloe vera plant contained 99.5% water and 0.0013% protein(Gjerstad, n.d.).

Rowe and Parks also noted fructose, ash, and amylase. Although they did not find

vitamins A or D, Coats and Ahola indicate that aloe contains beta carotene and alpha-

tocopherol. Many other organic compounds and inorganic elements have also been

found.

B. Effects of aloe vera:

Antibacterial/antifungal/antiviral effects of aloevera. Streptoccocus pyogenes and

Streptococcus faecalis are two microorganisms that have been inhibited by aloe vera gel.

(Heggers JP, Pineless GR, Robson MC). Heggers et al in 1995 suggested that the

antibacterial effect of the aloe vera gel in vivo could enhance the wound healing process

by eliminating the bacteria that contributed to inflammation. Aloe vera gel reportedly was

bactericidal against Pseudomonas aeruginosa while acemannan prevented it from

adhering to human lung epithelial cells in a monolayer culture. A processed aloe vera gel

preparation reportedly inhibited the growth of Candida albicans.(J Am Med Technol,

1979) In terms of antiviral effects, acemannan reduced herpes simplex infection in two

cultured target cell lines.(Antiviral Res 1990) In a study Saoo et al noted that fractions of
aloe vera gel containing lectins directly inhibited the cytomegalovirus proliferation in cell

culture, perhaps by interfering with protein synthesis.

Antioxidant effects and free radical scavenging activity of aloevera. Three

relatively recent reports have demonstrated an antioxidant action for some constituents of

aloe vera gel. Three aloesin derivatives from aloe (namely isorabaichromone,

feruoylaloesin, and p- coumaroylaloesin) showed potent free radical and superoxide

anion-scavenging activities in an assay using the lipid peroxidation rat liver microsomal

system as the free radical generator. This lipid peroxidation assay is recognized as a

standard technique for measuring the free radical scavenging effects of anti-oxidants. The

same study noted that aloesin compounds inhibited cyclooxygenase-2 (COX-2) and

thromboxane (Tx) A2 synthase; such actions could explain in part the healing effects of

aloe vera.( A. Yagi , A. Kabash, N. Okamura, H., et. al. Planta Med, 2002)

Cow Dung/Manure and its Uses

In recent years, with the scale and intensive development of cow breeding industry,

environmental pollution caused by mass cow dung has been increasingly emerging. With

over 12 million cows, the daily excretion volume of cow dung can reach up to 0.2-0.3

million tons, especially for large-scale dairy cattle farm, if shortage of efficient

processing means, there would be lots of cow dung that would easily lead to heavy

pollution to soil, source of water and air. What’s worse, mass stacking can also occupy

the limited field region for dairy farm, affecting its normal order of production .

Therefore, along with the promotion of environmental protection , it is necessary and

important to figure out the comprehensive utilization of cow dung. (Compost Turner,

Zhengzhou City)

Cow dung, which is usually a dark brown color (usually combined with soiled bedding

and urine) is often used as manure (agricultural fertilizer). If not recycled into the soil by

species such as earthworms and dung beetles, cow dung can dry out and remain on the

pasture, creating an area of grazing land which is unpalatable to livestock.

As an excellent biomass resource,cow dung also has a great deal of mineral

elements (table 1) and abundant nutrient substance (table 2) as well as other feces of

other livestock poultry. Cow dung has fine texture and more water content with slow

decomposition and low calorific value, which belongs to slow acting fertilizer. (Compost

Turner, Zhengzhou City)

 Cow manure has a soft texture and tends to be deposited in a circular shape,

which gives dung patches their alternate names of cow pies and cow pats. The manure is

used as a rich fertilizer, an efficient fuel and biogas producer, a useful building material, a

raw material for paper making, an insect repellent, and a disinfectant. Cow dung "chips"

are used in throwing contests and cow pie bingo is played as a game. The manure also

plays an essential role in the lives of various animals, plants, and microbes, including

dung beetles and the Pilobolus fungus.

Fuel and Biogas From Cow Manure

Dried cow dung is an excellent fuel. In some cultures dung from domestic cows

or buffalo is routinely collected and dried for fuel, sometimes after being mixed with

straw. Pieces of dung are lit to provide heat and a flame for cooking. Dried dung has lost

its objectionable odor.

Even in North America people are making use of the energy stored in cow dung,

although this is usually done indirectly by making a biogas from the dung. A biogas is a

mixture of gases produced by the anaerobic digestion of organic matter by bacteria. An

"anaerobic" process occurs in the absence of oxygen. The organic matter that is digested

can be animal dung, sewage, plant material, or food waste.

Production and Uses of a Biogas

The general process for making an anaerobic digester for cow dung starts with

placing dung and water in an airtight container. The container must be kept warm and left
undisturbed so that bacteria can do their work. The gas that is produced is withdrawn

through a tube and stored.

Once a biogas has formed, it can be reacted with oxygen to produce energy. The gas can

be used to cook food, heat water in a boiler, and replace conventional fuel in motor

vehicles. In addition, the energy in a biogas can be used to produce electricity.

Using Cow Dung as a Building Material

A mud and cow dung paste is often applied to the floors of rural homes in India

and may be applied to the walls as well. The mixture reportedly forms a waterproof layer

that helps to insulate the house from heat entry or loss and doesn't smell unpleasant. A

relatively new process is to make building bricks from cow dung mixed with straw dust.

The bricks are much lighter than conventional ones. It's been suggested that the manure

residue from biogas production could be used instead of sawdust to make fibreboard. The

manure, which contains fibres, would be sterilized and then mixed with resin to make the

board. Fibreboard has many uses. It's used to manufacture furniture and floors in homes,

for example. The high fibre content of cow dung also enables people to make paper

from the dung. The dung is washed to extract the fibres, which can then be pressed into

paper on a screen. Some people make cow dung paper as a hobby. The paper can also be

bought commercially.

Dung as an Insect Repellent and a Disinfectant

The smoke from burning cow dung has been found to repel insects, including

mosquitoes, leading to the use of cow dung as a pesticide in some areas. Strange as it

may sound, in some cultures cow dung is applied to walls and floors as a disinfectant as
well as an insulator. There may be some value in this seemingly bizarre practice, as the

FAO quote shown below suggests. Unsterilized cow dung may contain microbes that can

infect humans, though, so it's not a good idea to spread dung over a wound. Cow dung

and cow urine possess complex degrading substances and may possess antibacterial

properties (Food and Agriculture Organization of the United Nations).

Cow Manure as a Fertilizer

Removing cow dung from fields is important because the dried pats reduce the

grazing area. In addition, the cow pats give off methane, which acts as a greenhouse gas

in the atmosphere. Water runoff can carry some of the dung into rivers and other bodies

of water, polluting them with excess nutrients. Many people are aware that cow manure

can make a good fertilizer and are reminded of this every time they pass a fertilized and

odoriferous field. Cow manure is rich in minerals, especially nitrogen, phosphorus, and

potassium. It can support the growth of beneficial microorganisms when it's mixed with

soil. Manure can also improve the texture of the soil and help it to maintain moisture.

Often, however, manure is too rich in certain chemicals and needs to be diluted or left to

sit in the soil for a while before crops are planted. (Owlcation, 2018 )

The Hair Growth Cycle

Hair grows from the follicle, or root, underneath the skin. The hair is ‘fed’ by

blood vessels at the base of the follicle, which give it the nourishment it needs to grow.

Between starting to grow and falling out years later, each hair passes through four stages:

anagen, catagen, telogen and exogen. Every hair is at a different stage of the growth
cycle.Over time, the length of the anagen stage decreases. Therefore, the hair may

become weaker and thinner after each cycle. That’s why it’s important to ensure your diet

is rich in specific nutrients to maintain normal, healthy hair growth.

Anagen (Growing Phase).The growing phase lasts two to seven years and

determines the length of our hair. The anagen, or growing, phase is the first part of the

hair growth cycle. During the anagen phase, cells of the bulb divide rapidly, resulting in

new hair growth. Eighty to 90% of hair follicles are in the anagen phase at any given

time.The anagen phase lasts somewhere between two and seven years. The length of the

anagen phase determines the maximum hair length. For example, people who have very
long hair have a very long anagen phase. Eyelashes, eyebrows, and body hair have

shorter growth phases than for the hair on your head, which is why they are much shorter

than scalp hair. There are many factors that influence the length of the anagen phase,

including genetics, nutrition, age and overall health. (Hair Toppiks, 2016)

Catagen (Regression Phase).This stage lasts about ten days. The hair follicle

shrinks and detaches from the dermal papilla. After the hair growth phase enters the

catagen phase. This short, transitional phase only lasts two to three weeks. During the

catagen phase, the hair stops growing and detaches itself from the blood supply. The hair

becomes what is known as a club hair. (Hair Toppiks, 2016)

Telogen (Resting Phase). The resting phase lasts around three months. Around

10-15% of hairs are in this phase. Whilst the old hair is resting, a new hair begins the

growth phase. (Hair Toppiks, 2016) The telogen, or resting, phase follows the catagen

phase. During the telogen phase, the club hair rests while a new hair begins to grow

beneath it. This new hair will eventually take the place of the club hair. The telogen phase

lasts for three months, and 10-15% of all hairs are in this phase at any one time.

Exogen (Shedding Phase).The Exogen represents the period from when a resting

hair reaches its terminal position in the follicle to when it finally detaches. The resting

hair is gradually loosened resulting in shedding of the hair. (Hair Toppiks, 2016) During

the exogen phase, the resting club hair detaches and falls out. Every hair eventually

sheds, and it’s completely normal to lose 50 to 100 hairs each day.In fact, the exogen

phase is why you’ll frequently see hairs in your shower drain or on your hair brush. You

should only be concerned if there’s a sudden change and many more hairs than usual are
falling out. After the exogen phase, the follicle then returns to the anagen phase and the

cycle repeats.

Mus Musculus

Mice

(Mus

musculus)

are an

important research tool for modeling human disease progression and development in the

lab. Despite differences in their size and appearance, mice share a distinct genetic

similarity to humans, and their ability to reproduce and mature quickly make them

efficient and economical candidate mammals for scientific study. Mice (Mus musculus)

are small rodents that breed and sexually mature quickly, making them perfectly suited to

generating large animal colonies for biological research. As compared to other

mammalian species, mice are simple and inexpensive to maintain in the laboratory.

Nevertheless, mouse colonies do have specific husbandry needs that are critical to

preserving animal health and safety as well as experimental reproducibility (The Society

for Mucosal Immunology, 2014). Mice (Mus musculus) are an important research tool for

modeling human disease progression and development in the lab. Despite differences in

their size and appearance, mice share a distinct genetic similarity to humans, and their
ability to reproduce and mature quickly make them efficient and economical candidate

mammals for scientific study. (JoVE, n.d.)

Conceptual Framework

Independent Variables Dependent

Variable

Control Set up

Biotin

(Positive Control)

Hair growth of Albino

Mice (Mus musculus):

 Hair length
 Hair dermal
thickness
 Hair follicle
depth
 Hair follicle
 Eperimental Set up

Mixture of Aloe
vera leaf extract
and Cow dung
100%
75%
50%
25% Chapter II
(Negative Control)
METHODOLOGY

A. MATERIALS/EQUIPMENTS

MATERIALS:

Aloe vera (Alo barbadensis) Hematoxylin and Eosin

(H&E)

Cow Dung/Manure Ethel alcohol

Distilled water

Commercial Product (Biotin)

 EQUIPMENTS:

Lab gown Blender

Gand Gloves Portable weighing scale

Mask Triple beam balance

Test tubes Container

Beaker Spoon

Stirring rod Vernier caliper

Funnel Digital photomicrograph

Knife

Fine Strainer

B. TREATMENT AND GENERAL PROCEDURE

PHYTOCHEMICAL ANALYSIS

The aloe vera (Aloe barbadensin) plant will be air dried within five days. After

the air drying, the leaf of the plant will be cut and is then place in a bottle with ethel

alcohol. Then will be soaked for one day.

The next part is the separation of the extract using the funnel, filter paper and a

beaker. To separate the the ethel alcohol from the pure plant extract , will be place in a

rotary evaporation machine.

Lasty, the pure plant extract will be place in an evaporating dish and now ready

for phytochemical analysis.

STERILIZATION OF COW DUNG

Cow dung/manure will be collected on August of 2018 at Barangay Bonifacio

Surigao City. The researchers will collect and weigh one (1) kilogram manure using a
portable weighing scale. On the sterilization process by composting , the reseachers
followed a set of procedures:

1. Place a compost bin that is expose to the heat of the sun.

2. Fill the bin ¾ full with manure. Place an outdoor thermometer down into the
manure and cover the bin with a tarp.
3. Turn the manure with a small rake every couple of days. This will help
provide oxygen to break down the manure into soil-like compost.
4. Keep an eye on the temperature. When the bin has maintained a temperature
of 165 degrees or higher, remove the sterilized compost.

PREPARATION OF TREATMENTS

A. EPERIMENTAL SET-UP

One kilogram of aloe vera leaf samples will be collected in Barangay Bonifacio

Surigao City and weighed using a triple beam balance. On the extraction process, the leaf

will be properly washed and sliced with a knife from the side and pull it open. Then,

scratch out the content of the leaf with a teaspoon directly into the blender and filtered

using a fine strainer and place it to the container. Next, The sterilized compost undergoes

a process of grinding and sieving using a mortar and pestle. 50ml gel from the Aloe vera
and 50ml sterilized and sieved manure will be mixed and divided into four for different

concentrations as follows:

100% = 40ml (20ml dung, 20ml aloe vera extract)

75% = 30ml (15ml dung, 15ml aloe vera extract) + 10ml distilled water

50% = 20ml (10ml dung, 10ml aloe vera extract) + 20ml distilled water

25% = 10ml (5ml dung, 5ml aloe vera extract) + 30ml distilled water

B. CONTROLLED SET-UP

In order to compare and determine the effectivity of the alternative hair grower

the researchers will be using a commercial product (Biotin) as controlled set-up.

PREPARATION OF MICE CAGE

Five pathogen free cages measuring 6’x 9’x 15’ will be prepared. Each cage

will contain 3 albino mice (Mus Musculus) that will be used for different concentrations

of extract: 1 for 100%, 1 for 75%, 1 for 50%, 1 for 25%, and 1 for the controlled set-up.

Each cage will then be labeled as cage 1, cage 2, cage 3, cage 4, and cage 5.

SELECTION OF PATHOGENS FREE TESTED ORGANISMS

Fifteen pathogen free identical albino mice (Mus Musculus) will be gathered

from WRM Pet Shop. The researchers will consider the following variables: age, weight,
and sex of albino mice (Mus Musculus) should be the same, pathogen free, equal amount

of food, and free from microorganisms. Each of the selected test organism will be shaved

at the back.

APPLICATION OF DIFFERENT CONCENTRATIONS IN THE SUBJECT

The study will utilize fifteen (15) Albino mice (Mus Musculus) as the

experimental subjects. They will be placed in pathogen free cages. Each cage will contain

3 albino mice (Mus Musculus) and will be labeled according to the levels of

concentrations they will receive. Each of the three albino mice (Mus Musculus) in a cage

will be named A, B, C and D to avoid confusion in measuring the hair length, dermal

thickness, follicle depth, and follicle number.

Each albino mice (Mus Musculus) will be given with the extract orally using__

with different levels of concentration accordingly. Three albino mice (Mus Musculus) for

100% pure extract, three albino mice (Mus Musculus) for 75%, three albino mice (Mus

Musculus) for 50%, and another three albino mice (Mus Musculus) for 25%. Three albino

mice (Mus Musculus) will not be given with the extract for they will serve as the

controlled set-up.

DECONTAMINATION AND WASTE DISPOSAL

After the experiment, all materials used will be sterilized at 100˚C for 20

minutes. After the allotted time for decontamination, these materials will be allowed to

cool, washed thoroughly in running water with a liquid detergent, rinsed with distilled
water and stored for next use. The twelve mice will be sold back to the pet shop where it

was taken.

DATA GATHERING TECHNIQUE

DATA COLLECTION

A. Hair Length Determination

Collections of animal hair will be performed on the 2nd week to 4th week. Ten

hairs were collected randomly from the shaving area in mice back and measured with

Vernier caliper carefully.

B.  Number of Follicles

5 sections of mice back skin were stained with hematoxylin and eosin

(H&E). Digital photomicrographs were taken from representative areas at a fixed

magnification of 100x. The H&E dyes were photographed using a digital

photomicrograph and all of the images were cropped in a fixed area of 100 pixels

width. Average hair follicles numbers were counted from representative area
 FLOW CHART

Figure 1

Preparation of Materials

Air-Drying
Alcohol Extraction
Rotary Evaporation

Bioassay (Phytochemical Analysis)

Figure 2

Gathering of Aloe vera Leaves

Extraction of the gel

Gathering of Cow Dung
Composting of Cow Dung
Combining the extract and the compost

Dissolution (Levels of Concentration)

Treatment to the Subject

Assay (Hair Growth)
 RESEARCH DESIGN

Hair Hair Dermal Hair Follicle Hair Follicle

EXPERIMENTAL Lenght Thickness Depth Number
TESTED SET-UP (mm) (mm) (mm) ()
ORGANISM TREATMENT
Concentration (ml) Before After Before After Before After Before After

A1
CAGE 1 B1 100%
C1
A2
CAGE 2 B2 75%
C2
A3
CAGE 3 B3 50%
C3
A4
CAGE 4 B4 25%
C4

COTROL SET-UP

A5
CAGE 5 B5 BIOTIN
C5
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