HAIR GROWTH ACTIVITY OF BAMBUSA SPINOSA AND BAMBUSA

VULGARIS STEM EXTRACT ON ORYCTOLAGUS

CUNICULUS (RABBIT)

A Thesis
Presented to the
Faculty of the College of Pharmacy
University of the Visayas

In Partial Fulfilment
Of the Requirements for the Degree
Bachelor of Science in Pharmacy

By

Kaiye A. Rasonable
Jacel Rhea H. Maranga
Sweet Marjorie A. Genilo

2014

CHAPTER 1
THE PROBLEM AND ITS BACKGROUND
Rationale
The hair grows on almost all skin surfaces of the body except the palms, lips,
nipples, soles and parts of the external reproductive organs. A hair is composed of dead
epidermal cells. The epidermal cells on the scalp and other parts of the body where hair
grows divide and grow. The older cells are pushed toward the surface and become
keratinized and die; thus, the growth of hair. (Shier et al., 2006)
There are 100,000 hairs in an average scalp. Hair grows at a rate of half an inch
every month. Falling hair is just a normal happening. Because after growing it for two to
six years, the hair rests and falls out. An average of 50-100 strands of hair fell every day.
But after falling out, its soon replaced with a new hair and the cycle begins again. But a
lot of people are experiencing an excessive falling out of hair without growing new ones.
The cycle just stopped with falling out of hair until none was left. (Baum, 2012)
Hair loss has been a problem since the ancient times. A lot of people now are
suffering from hair loss disregard the age basis. Statistics shows the number of people
suffering from hair loss, worldwide, seeking treatment is 811, 363. (Relevant Research,
Inc. - International Society of Hair Restoration Surgery, 2014)
As we aged, our hair loses its lustre. By the age of 50, the number of hair follicles
has decreased and continues to drop. Hence hair becomes thin and some cases, baldness.
(Marieb, 2009)

There are lots of drugs or cosmetics brand available in the market for the
treatment of hair loss. One of which is Minoxidil, a topical solution. A 2% minoxidil
(Rogaine) was approved by the FDA for the treatment of baldness in men and women.
But the 5% minoxidil was approved only for men. Another drug used in the treatment of
baldness is Finasteride (Propecia), an oral drug. It is known to be 50% effective in men.
But studies show that its ineffective in older men even with an increased dose of the
drug. (Kee et. al., 2006)
Bamboo is one of the fastest-growing plants in the world. It is part of the grass
family Poacaea. (Wikipedia) The decoction of the roots of Bambusa vulgaris is
traditionally administered in cases of kidney problems; the leaves are used as an
emmenagogue and an anthelmintic; the silicious concentration is said to be stimulant,
astringent, tonic, cooling, an antispasmodic and aphrodisiac, and is useful in coughs,
asthma, etc. Aqueous sap of Bambusa spinosa is regarded by the Filipinos as a phthisis
treatment. (Quisumbing, 1978)
The researchers of this study foresee bamboo as an effective hair grower. Studies
show Bambusa vulgaris could be an effective hair grower. (InsidersHealth.com, 2008)
The researchers were going to test if Bambusa spinosa, another species of bamboo has
the same effect. Since bamboo is abundant and widely grown here in the Philippines,
were going to make use of this natures gift. There are 62 species of bamboo known that
grew in our country, 21 of these are endemic and the rest were introduced. (DOST, 2011)

Conceptual framework

Oryctolagus cuniculus
(rabbit) hair/fur was
shaved

HAIR LOSS, BALDNESS,

OR ALOPECIA

Topical
administration of
Bambusa spinosa
stem extract in
rabbits

Topical
administration of
2% minoxidil in
rabbits

Topical
administration of
Bambusa vulgaris
stem extract in
rabbits

HAIR GROWTH ACTIVITY

Figure 1 Schematic diagram of the process of this study and its possible outcome

Statement of the problem

This study will focus on the hair growth activity of Bambusa spinosa and
Bambusa vulgaris on Oryctolagus cuniculus (rabbit). Specifically, this study aims to:
1. Identify the principal constituent responsible for the regrowth of hair.
2. Compare Bambusa spinosa stem extracts hair growth activity with other species
of bamboo, Bambusa vulgaris stem extract in terms of the following:
1.1 Onset of action
1.2 Length of hair growth in rabbit
3. Find out the significant difference of what species of bamboo exhibit effective
hair growth activity to compare with the commercial minoxidil product.
Hypothesis
Null Hypothesis (H0):
There is no significant difference in the hair growth activity between the bamboo
stem extracts and Minoxidil on Oryctolagus cuniculus.
Alternative Hypothesis (H1):
There is a significant difference in the hair growth activity between the bamboo
stem extracts and Minoxidil on Oryctolagus cuniculus.

Significance of the study

This study will benefit those who have been suffering from hair thinning, hair
loss, baldness and alopecia. The hair is one of the physical factors that give confidence to
a person. When hair starts to lose, the confidence will follow. This study might give hope
to them and that is the target of this research.
This study and its results might serve as an initial study for other researchers. This
will interest them and initiate them to do further research about Bambusa spinosa and
Bambusa vulgaris stem extracts hair growth activity on Oryctolagus cuniculus (rabbit)
which may lead them to another problem and discovery of bamboos constituents and
other uses.
Pharmaceutical companies will also benefit from this study. Research and
Development (R&D) Department of a manufacturing company may use this as an initial
study and develop it to be an effective drug product for human use.
Pharmacists, information seekers and lecturers in the academic field may benefit
from this research accompanied by its results to be used in their lectures and impart the
knowledge to their students the hair growth activity of bamboo stem extracts.
Students, more likely Pharmacy students who have higher probability to read this
study may gain information from this research and help disseminate the information to
their families, friends and colleagues. This will enhance their understanding about the
other use of bamboo; that bamboo is not just used for scaffoldings, building houses and
furniture but it also has its therapeutic uses. This might be an opportunity and encourage
them to conduct further research and make a breakthrough on the field of medical
science.

Scope and limitation

The researchers will only focus on Bamboos hair growth activity on Oryctolagus
cuniculus (rabbit). Twelve (12) rabbits will be used in this experiment with weights
ranging from 600g 1000g. There will be three (3) rabbits in each trial. The rabbits will
be grouped according to the rabbits that will be treated with Bambusa vulgaris stem
extract, Bambusa spinosa stem extract, and minoxidil, and the untreated rabbits.
The researchers will only use the specific species of bamboo which is Bambusa
spinosa and Bambusa vulgaris, authenticated by Department of Environment and Natural
Resources. The plant material will be harvested from a bamboo forest in Bohol and
Pinamungajan, Cebu. The test animals will be purchased from some local pet shops.
The experiment will be conducted in University of the Visayas College of
Pharmacy Laboratory. All equipment and reagents used will be checked by the laboratory
personnel to ensure accuracy of results including the reagents used. The researchers will
also conduct a phytochemical screening of the Bambusa spinosa and Bambusa vulgaris.
Relevant data and results related to the study were recorded and kept. The researchers
will be given one semester to conduct the experiment.

Definition of terms
The following terms were operationally defined for better understanding of the
readers:
Hair growth
The act or process, or a manner of growing hair
Oryctolagus cuniculus
Rabbit; the animal to be used in the experiment
Bambusa spinosa
One species of bamboo which will be the focused of this study; locally known as
kauayan-tinik
Bambusa vulgaris
The other species of bamboo to be used in this study; locally known as kauayankiling
Bambusa spinosa and Bambusa vulgaris stem extract
Extract or juice we obtained from the bamboos stem through decoction process
Onset of action
The duration of time it takes for a drug's effects to come to prominence upon
administration
Minoxidil
Commercial product that is widely used as a hair grower and will be used as a
positive

control

in

this

study

CHAPTER II
REVIEW OF RELATED LITERATURE
Related Legal Basis
In 1966, Laboratory Animal Welfare Act intends to regulate the transport, sale,
handling, and treatment of animals intended for research, exhibition or other purposes.
The animals covered in this Act are dogs, cats, guinea pigs, nonhuman primates, hamsters
and rabbits. (US Animal Welfare Act of 1966)
Humane care and treatment of research and laboratory animals are important
aspects of Animal Welfare Act. The researchers of this study will make sure and
prioritize the safety of the animal were going to use, which is Oryctolagus cuniculus
(rabbit). Since rabbits are covered by the said Act.
Republic Act No. 8485, Section 6 states that it is unlawful for any person to
torture any animal, to neglect to provide adequate care or shelter, or maltreat any animals
or to subject any dog or horse to dogfights or horsefights, kill or cause or procure to be
tortured or deprived of adequate care sustenance or shelter, or maltreat or use the same in
research or experiments not expressly authorized by the Committee on Animal Welfare.
(Animal Welfare Act of 1998)
Based on the foregoing Act, the researchers cannot conduct the experiment
directly without securing first the approval from Philippine Animal Welfare Society.
According to Executive Order No. 879, Section 4, the Department of Agriculture
shall identify not in use and ignored agricultural areas suitable for bamboo plantations.
This should be done without sacrificing areas used for food production. Department of

Agriculture shall assist in establishing bamboo nurseries and in bamboo propagation with
focus on bamboo shoots production. (Executive Order No. 879, s. 2010)
Executive Order No. 879, Section 5 states that Department of Science and
Technology through its appropriate bureaus and offices like the Forest Products Research
and Development Institute (FPRDI) and Food and Nutrition Research Institute (FNRI),
shall undertake the research and transfer of technologies which can reduce production
costs and increase the saleability of bamboo products such as bamboo shoots processing
and packaging, alternative adhesives and finishes, and effective yet affordable treatment
and preservation techniques. (Executive Order No. 879, s. 2010)
Based on the previous legal basis, the researchers expect that this study will be a
help to the Research and Development Division of the government if this will be proven
positive and safe for human use. It needs to be approved first by BFAD before
distribution to the market.
Consumer Act of the Philippines, Article 35, Adulterated cosmetics, states that a
cosmetic shall be deemed to be adulterated: a) if it bears or contains any poisonous or
deleterious substance which may render it injurious to users under the condition of use
prescribed in the labeling thereof, or under the condition of use as are customary or usual:
Provided, That this provision shall not apply to color additive hair dye, the label of which
bears the following legend conspicuously displayed thereon: "Caution: this product
contains ingredients which may cause skin irritation on certain individuals and a
preliminary test according to accompanying directions should first be made. This product
must not be used for dyeing the eyelashes or eyebrows; to do so may cause blindness"
and labeling of which bears adequate directions for such preliminary testing. For

purposes of this paragraph (e) the term "hair dye" shall not include eyelash dyes or
eyebrow dyes. (Republic Act No. 7394, April 13, 1992)
Based on the preceding legal basis, a product for cosmetic use must be approved
by the FDA/BFAD and must contain appropriate labelling and warnings, if ever.
Approved products by the FDA/BFAD are legally safe unless changes to the final rules
and regulations are posted.

Related Legal Literature

The Bambusa spinosa, also known as spiny bamboo, is found throughout the
Philippines. Its basal parts are surrounded by stiff, branched, interlaced, spiny branches.
This bamboo is by far the most commonly used species in the Philippines. It is most
useful for building purposes and for manufacture of furniture and household utensils. The
young shoots are fairly tender and are used for food. Bamboo shoots are only fair sources
of calcium and iron; they contain 1.76 per cent of protein and 4.24 per cent of
carbohydrates. According to Guerrero a decoction of the roots is administered in cases of
anuria. Nadkarni reports that the silicious concretions in the culm contain 90 per cent of
silica or silicum as hydrate if silicic acid, peroxide of iron, potash, lime, aluminia and
vegetable matter. The leaves are used as an emmenagogue and an anthelmintic. The
silicious concentration is said to be stimulant, astringent, tonic, cooling, an antispasmodic
and aphrodisiac, and is useful in coughs, consumption, asthma, etc. He adds that the
young shoots, made into a poultice, are a most efficacious application for dislodgement
of worms from ulcers. The bud of the leaf is also used in leprosy, fevers and haemoptysis.
The root is given as a specific in eruptive affections. (Quisumbing, 1978)

Bambusa vulgaris is found at low and medium altitudes in the settled areas of
most islands and provinces, from northern Luzon to Palawan and Mindanao. This is
probably an introduced plant in the Philippines, being now found throughout the tropics
in cultivation. Unlike Bambusa spinosa, this species is smaller and not supplied with
spiny branches. The stems are also used for houses, bridges, furniture, etc. but are much
inferior to Bambusa spinosa. The young shoots, while edible, are considered inferior to
those of Bambusa spinosa. According to Guerrero, the aqueous sap of this plant is much
esteemed by the Filipinos as a remedy for phthisis. Sulit adds that a cold decoction of the
roots is claimed to relieve kidney trouble. (Quisumbing, 1978)
Based on the two foregoing legal literature, bamboo species are abundant here in
the Philippines. Thus, it is widely used by Filipinos in building houses, utensils, etc.
Filipinos also find ways to discover and learn more about the other uses of bamboo.
Some parts of the bamboo are traditionally used as treatments for specific illnesses. The
researchers of this study targets to discover and prove the other use of bamboo.
Bamboo extracts are rich source of silica. And silica is one of the major components of
hair. Using silica for your hair is good for revitalizing, improving and stimulating
healthier growth of hair and assures a beautiful, shiny and durable hair. It also helps
prevent hair loss and baldness. (InsidersHealth.com, 2008)

Related Legal Studies

Drugs or products that came from natural sources have always been attractive to
consumers than those that were synthesized in the laboratory. Most believe that natural
ingredients, herbs or edible plants are safer as a treatment than any composition based on

synthetic raw materials. Thus, an invention or product extracted from Bambusa vulgaris
was formed. Bamboo is known for its rich nutritious properties. It has been found out that
Bambusa vulgaris extract improves hair condition especially hair shine. (Hermes et. al.,
2006)
Based on the foregoing legal studies, it has been identify that Bambusa vulgaris
have components or constituents that can help and improve the growth of hair. The
researchers of this study would like to determine if the other species of bamboo,
Bambusa spinosa has the same hair growth activity of the former.
Minoxidil was first distributed in the market as an oral tablet for the treatment of
hypertension. It has been found out that one of its side effects is hair growth. And this
enables the use of Minoxidil for hair regrowth purpose that is known and widely accepted
these days. (Abood, 2010)
Minoxidil was the first hair growth drug that was approved by the FDA. It could
be bought without a prescription. Four studies were conducted to test the efficacy of 2%
and 5% concentration of Minoxidil. Evident results for both subjective parameters were
observed. It was considered safe for there were no evidence of photosensitization,
phototoxicity, or contact sensitization in the initial safety study. (U.S. FDA Medical
Review, 1997)

CHAPTER III

MATERIALS AND METHODS

This study will be conducted to determine the hair growth activity of Bambusa
spinosa and Bambusa vulgaris. The respondents of this study are rabbits (Oryctolagus
cuniculus).

Research Method
The proponent will use t-test method to this particular study. The rabbit treated
with 2% minoxidil will be the positive control group while the rabbits treated with
bamboo stem extracts will be the experimental group, and the untreated rabbits will be
the negative control group.

Preparation of the Plant

Fresh stem of the bamboo will be harvested from a bamboo forest in Bohol and
Pinamungajan, Cebu. According to the indigents, it is much preferred to harvest bamboo
during dusk or dawn. Bamboos produce starch or sap at its extent during high noon. It
will be cut to smaller sizes prior to decoction. Its identity will be confirmed by an expert
from Department of Environment and Natural Resources.

Preparation of the Test Solution

The stem of the bamboo (both Bambusa vulgaris and Bambusa spinosa) will be
cut into pieces prior to decoction. Decoction is the process usually used in obtaining
extracts of hard structured plant materials (stem, roots, rhizomes, etc.). The procedure
involves boiling of the plant material in water and letting it stand for five to fifteen

minutes at room temperature. Then, strain it to separate the extract from the plants parts.
(Al-achi, 2008)
Preparation of the Test Animals
Twelve (12) rabbits to be purchased on a local pet shop in the city will be used in
this experiment. It will be provided with clean and proper food and water throughout the
study. The rabbits will be kept at a room temperature environment. The conduct of
experiment will be approved by Department of Animal Welfare.
Approximately 24 hours before conducting the experiment, the rabbits hair or fur
around its dorsal area will be removed by shaving. When shaving the fur, proper handling
and care should be considered to avoid abrading the rabbits skin and hurting them. Only
animals with healthy skin should be used in this experiment. (Derelanko and Auletta,
2014)

Preparation of Positive Control

A healthy rabbit will be treated with minoxidil through topical administration.
Three rabbits will be in this group. The animals will be given the same food and water
given to the experimental group and negative control group. The rabbits fur will also be
shaved.

Phytochemical Testing
Test for Lignin
Weisner test. A solution of Phloroglucinol-HCl will be added to the extract. A
brilliant red color confirms the presence of lignin.
Test for Alkaloids

Wagners reagent. A fraction of extract was treated with 3-5drops of Wagners

reagent [1.27g of iodine and 2g of potassium iodide in
100ml of water] and observed for the formation of reddish brown precipitate (or
colouration).
Test for Carbohydrates
Molischs test. Few drops of Molischs reagent were added to 2ml portion of the
various extracts. This was followed by addition of 2ml of conc. H2SO4 down the side of
the test tube. The mixture was then allowed to stand for two-three minutes. Formation of
a red or dull violet colour at the interphase of the two layers was a positive test.

Test for Cardiac glycosides

Keller Kellianis test. 5ml of each extract was treated with 2ml of glacial acetic
acid in a test tube and a drop of ferric chloride solution was added to it. This was
carefully underlayed with 1ml concentrated sulphuric acid. A brown ring at the interface
indicated the presence of deoxysugar characteristic of cardenolides. A violet ring may
appear below the ring while in the acetic acid layer, a greenish ring may form.
Test for Fats and Oils
Paper Test. The powdered extract will be dissolved in water and drops of it will
be place on a paper. The translucent grease spot on the paper will indicate a positive
result.
Test for Flavonoids
Alkaline reagent test. 2ml of extracts was treated with few drops of 20% sodium
hydroxide solution. Formation of intense yellow colour, which becomes colourless on
addition of dilute hydrochloric acid, indicates the presence of flavonoids.

Test for Phenols

Ferric chloride test. A fraction of the extracts was treated with aqueous 5% ferric
chloride and observed for formation of deep blue or black colour.
Test for Amino acids and Proteins
1% ninhydrin solution in acetone. 2ml of filtrate was treated with 2-5 drops of
ninhydrin solution placed in a boiling water bath for 1-2 minutes and observed for the
formation of purple colour.
Test for Pectin
The extract will be added denatured alcohol (70% rubbing alcohol). The mixture
will be briefly shaken and pour into an evaporating dish. Substance with pectin will form
a jelly-like appearance.
Test for Saponins
Froth test. To 2mls of extract was added 6ml of water in a test tube. The mixture
was shaken vigorously and observed for the formation of persistent foam that confirms
the presence of saponins.
Test for Starch
Iodine test. The aqueous extract 5ml was treated with the reagent of the starch
(iodine). Any shift to blue violet indicates the presence of starch.
Statistical Treatment
The researchers will use T-test to determine the degree of effectiveness of
bamboo stem extract as having hair growth property. The test subjects were evaluated
using several parameters like onset of action and length of hair grown. These variables

were used to determine the T value. The researchers will also conduct the T-Test for the
comparison of the plant extracts with the positive control.
t-Test Formulation
The t-test is used to compare two means. T-test uses means and standard
deviations of two samples to make a comparison. (Broto, 2012)
Arithmetic mean ( and variance (SS) are statistical tools used in getting the tvalue.
Arithmetic mean ( :

Variance:

t-Test Formula:

)(

Where:
t

the t-test

the mean of group 1

the mean of group 2

SS1

the sum of squares of group 1

SS2

the sum of squares of group 2

n1

the number of observations in group 1

n2

the number of observations in group 2

EXTRACTION OF BAMBOO STEM

Collection of Bambusa vulgaris and

Bambusa spinosa (bamboo) stems and cut
into pieces

Boiling the plant material in water

Let it stand at room temperature for five to

fifteen minutes

Strain to obtain the extract and separate it

from the plants parts

Figure 2 Schematic diagram showing the process bamboo stem extraction (decoction)

PARALLEL GROUP DESIGN

POSITIVE
CONTROL GROUP

NEGATIVE
CONTROL GROUP

Administered group
with 2% minoxidil

Untreated with any hair

growing substance

EXPERIMENTAL GROUPS

Administered group
with Bambusa vulgaris
stem extract

Administered group with

Bambusa spinosa stem
extract

Figure 2.1 Schematic diagram of the Parallel group research design