1

CHAPTER 1

THE PROBLEM AND ITS BACKGROUND

Introduction

Scabies is a worldwide skin disease that affects people and animals of all

races and social classes. Scabies can spread rapidly under crowded conditions

where close body and skin contact is frequent. Around 300 million scabies cases

worldwide are reported each year. In the absence of a comprehensive national

registry, the exact figures for the incidence of scabies in the Philippines are not

known but a sizable number of patients have been reported in several Metro

Manila hospitals (Castillo, A. et al., 2014).

Anyone can get scabies which is caused by Sarcoptes scabiei mites, a

highly contagious skin parasite. Different varieties of mites infest a wide range of

mammalian hosts, including dogs (canis), human beings (hominis), cats, cattle,

and horses. In all hosts, mites tunnel in the epidermis and produce an intensely

pruritic dermatitis with hyperkeratosis and alopecia (Currier, R. et al., 2011).

Dogs are common pet of a household. Scabies in dogs are also called

Sarcoptic mange or “galis-aso” in Filipino translation. Pet owners can often see

symptoms on the ear flaps, elbows, hocks, abdomen, and chest of their infested

dogs. Scabies is zoonotic, which means it can be passed from animals to

humans, so pet owners that have close contact with their dogs may develop an

itchy rash (Companion Animal Parasite Council, 2002). However, the animal mite

cannot reproduce on a person and will die on its own in a couple of days.
 2

Although the person does not need to be treated, pet animals should be

treated because its mites can continue to burrow into the person’s skin and

cause symptoms until the animal has been treated successfully (Centers for

Disease Control, 2010).

Sarcoptes scabiei mite in human like warm places such as skin folds, skin

between the fingers, under fingernails, or around the buttock or breast creases.

They can also hide under watch straps, bracelets or rings. It is also possible for it

to be passed on by sharing clothing, towels and bedding with someone who is

infected and usually acquired through prolonged periods of skin-to-skin contact

with an infected person, or through sexual contact (National Health Service, UK,

2009).

There are several medications available such as Permethrin 5% cream,

Lindane 1% cream or lotion, Benzyl benzoate lotion, Crotamiton lotion or

cream, and sulfur-based lotions, ointments, creams, or soaps which are intended

to treat scabies (Walker, 2000). 10% sulfur ointment is usually preferred and is

the oldest scabicidal agent used (Burkhart, C. 2012).The stems of Makabuhay

plant (Tinospora rumphii L., Menispermaceae) could also be scabicidal agent

based on the Filipinos and Malay folkloric practice and they consider this vine as

a universal medicine (Sarbatly, S. et al., 2009).

In this study, researchers would like to prove the scabicidal activity of the

prepared ointment from the crude ethanolic extract of Makabuhay stems

(Tinospora rumphii L., Menispermaceae) in dogs infected with Sarcoptes scabiei

mite and compare it to commercially available 10% Sulfur ointment.

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Background of the Study

Scabies is estimated to have been an infestation for at least 2,500 years.

Until now there are still cases of scabies in different animals, commonly in dogs

and cats, and even in humans. Makabuhay stems (Tinospora rumphii L.,

Menispermaceae) have been used by folklores as a treatment for scabies. The

study was done to prove the scabicidal activity of the prepared ointment from the

crude ethanolic extract of Makabuhay stems (Tinospora rumphii L.,

Menispermaceae) in dogs infected with Sarcoptes scabiei mite. The researchers

hypothesized that the condensed tannins is the one responsible for the scabicidal

activity of Makabuhay stems, because in relation to the other studies, specifically

in plants like Rosa multiflora root and Kakawate leaves, the condensed tannins

was the one that possess the scabicidal activity.

The Makabuhay plant is rarely used in researches and with that, the result

of this study will add to the list of medicinal uses of the plant. The study will also

contribute in lessening the cases of scabies that will benefit both animals and

humans. This is why the researchers would also like to pursue this study to

contribute to the progress of new studies for this skin disease.

Null Hypothesis

a. There is a significant difference between the pre-treatment and post-

treatment of each concentration of the prepared ointment from Makabuhay

stems.
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b. There is no significant difference between the scabicidal activity of the

prepared ointment from Makabuhay stems (Tinospora rumphii L.,

Menispermaceae) when compared to commercially available 10% sulfur

ointment.

Alternative Hypothesis

a. There is no significant difference between the pre-treatment and post-

treatment of each concentration of the prepared ointment from Makabuhay

stems.

b. There is a significant difference between the scabicidal activity of the

prepared ointment from Makabuhay stems (Tinospora rumphii L.,

Menispermaceae) when compared to commercially available 10% sulfur

ointment.

Objective of the Study

The study aims to prove the scabicidal activity of the prepared ointment

from the crude ethanolic extract of Makabuhay stems (Tinospora rumphii L.,

Menispermaceae) in dogs infected with Sarcoptes scabiei mite.

Statement of the Problem

It seeks to answer the following questions:

1. What is the percentage of the total crude ethanolic extract obtained from

Makabuhay stems (Tinospora rumphii L., Menispermaceae)?

2. What are the physical properties of the prepared ointment from

Makabuhay stems (Tinospora rumphii L., Menispermaceae)?

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3. Will the prepared ointment from Makabuhay stems (Tinospora rumphii L.,

Menispermaceae) cause skin irritation in adult male rabbits using Draize

test?

4. At what concentration of the prepared ointment from Makabuhay stems

(Tinospora rumphii L., Menispermaceae) exhibits greatest scabicidal

activity in treating dogs with scabies?

5. Is there a significant difference between the scabicidal activity of the

prepared ointment from Makabuhay stems (Tinospora rumphii L.,

Menispermaceae) when compared to commercially available 10% sulfur

ointment?

Scope and Delimitations of the Study

The study was conducted to determine if the prepared ointment from the

crude ethanolic extract of Makabuhay stems (Tinospora rumphii L.,

Menispermaceae) is effective in the treatment of dogs infected with Sarcoptes

scabiei mite. The researchers conducted organoleptic evaluation to confirm its

identity. The ethanolic extraction of the plant materials was done by maceration.

The ethanolic extract obtained was used in the phytochemical screening to

determine what constituents are present.

The crude ethanolic extract of the stems was used in the preparation of

ointment. The three concentrations of the prepared ointments from Makabuhay

stems (10%, 15%, and 25%) and the normal control (white ointment) were

applied to the divided quadrants of the back portion of five adult male rabbits

weighing 1.5-2.5kg to determine if the prepared ointments cause skin irritation.

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Five dogs infected with scabies from Valenzuela City Pound were used as

experimental subjects. The five dogs were divided into three groups: the normal

control (white ointment), positive control (10% sulfur ointment) and the

experimental group (prepared ointment from Makabuhay stems) that was further

divided into three groups according to different concentrations (10%, 15% and

25%). The study also determined the most effective concentration of the

prepared ointment from Makabuhay stems. The effectiveness of the prepared

ointment from Makabuhay stems (Tinospora rumphii L., Menispermaceae) was

compared with the commercially available 10% Sulfur ointment by using the

Kruskal Wallis Test. The delimitation of the study is the isolation of the specific

constituent that possesses the scabicidal activity.

Significance of the Study

The scabicidal activity of the prepared ointment from the crude ethanolic

extract of Makabuhay stems (Tinospora rumphii L., Menispermaceae) in dogs

infected with Sarcoptes scabiei mite will be beneficial to the following:

 Dogs infected with scabies.

Dogs are the ones commonly infected with the disease that may

transfer to humans who are also manifesting scabies. As a treatment

medication, the study can help to control of the spreading of the disease

by formulating a topical ointment.

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 Pet lovers /owners.

This will help them treat their pets, like dogs if they get infected with

scabies. As pet owners, they are entitled to ensure that their pets are free

from any disease, especially if they are also at risk of getting it.

 Makabuhay plant growers/producers.

They will also benefit from the study because the findings will make

people use the new formulation from Makabuhay plant. This study can be

a big business opportunity for them by planting more Makabuhay to

produce new medicinal products from it.

 Veterinarian and other healthcare professionals.

People with dogs severely infected with scabies go to veterinarian

clinics to treat their pets. The study will produce an additional possible

treatment option that they can be prescribed for their animal patients.

 The future researchers.

Researchers, who will focus on studies concerning scabies, as well

as those who want to conduct further study of the Makabuhay plant, can

make use of the additional knowledge and information gathered from this

study.

Definition of Terms

Alopecia. The partial or complete lack of hairs in areas where they are normally

present (Merck Manual for Pet Health, 2015)

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Callus. Hypertrophy of the epidermis, especially over pressure points. A false

bursa that occurs over bony prominences and pressure points, especially in large

breeds of dogs. Repeated trauma from lying on hard surfaces produces an

inflammatory response, which results in a dense-walled, fluid-filled cavity (Merck

Veterinary Manual, 2015)

Crust. A dried exudate on the skin surface, either serum, blood or pus or a

combination An excessive or abnormal shedding, excessive accumulation of skin

cells, or a loss of the cells' ability to adhere to each other (Royal Veterinary

College, 2016).

Ectoparasite. A parasite that lives on or in the skin but not within the body

(Currier, R., et al., 2011)

Excoriation. Erosion or ulceration caused by scratching, biting or rubbing (Royal

Veterinary College, 2016).

Kruskal Wallis Test. A non-parametric method for one-way analysis of variance

used to determine if three or more samples originate from the same distribution.

Essentially a standard one-way analysis of variance, with ranks assigned to the

data points replacing the data points themselves, and is similar to the Mann-

Whitney U test, but applicable to more than two sample groups (Business

Dictionary, 2016)

Lichenification. An accentuation of the skin markings giving an elephant skin

like appearance (Royal Veterinary College, 2016).

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Macules. Areas of discoloration of the skin, less than 1 cm in diameter. Typically

erythematous, but they may be hyperpigmented (Animal Dermatology Clinic,

2014).

Ointment. A semisolid preparation usually containing medicinal substances

intended for external application (Collins, G. 2000).

Sarcoptic mange. Also known as the itch mite which is the most commonly

diagnosed skin diseases in dogs (Ackerman, L. 1994).

Sarcoptes scabiei mite. An ectoparasite being less than 0.5 mm in length that

causes scabies (Beers, M. 2002).

Scabicidal. An agent that kills itch mites (Fawcett, R. 2003).

Scabies. A type of skin disease which is caused by Sarcoptes scabiei mite that

is characterized by dry, crusted lesions that become pruritic and on excoriation

often develop serous exudates (Chosidow, O. 2006).

Wilcoxon test. Refers to either the Rank Sum test or the Signed Rank test, is a

nonparametric test that compares two paired groups. The test essentially

calculates the difference between each set of pairs and analyzes these

differences (Investopedia, 2010).

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CHAPTER 2

REVIEW OF RELATED LITERATURE

This chapter presents the available literature and studies conducted that

are crucial to the investigation of the scabicidal activity of the prepared ointment

from the crude ethanolic extract of Makabuhay stems (Tinospora rumphii L.,

Menispermaceae) in dogs infected with Sarcoptes scabiei mite.

Plant Material

Retrieved from: http://www.asean.org

Plate 1. Makabuhay Plant (Tinospora rumphii L., Menispermaceae)

The Makabuhay plant is a clinging vine, and the name itself suggests the

primary purpose of such plant. Makabuhay is a Filipino translation of the English

term ‘pro-life or to give life’. It is commonly known as the paliahan in the Visayas

region. The plant belongs to the family of Menispermaceae, and can grow, or

rather climb, up to fifteen meters long. The stems are up to 1 centimeter thick
 11

and somewhat fleshy, with scattered protuberances. It is widely distributed in

nearby towns and in most or all islands of the Philippines (Quisumbing, E. 1978).

Folkloric Use of Makabuhay Stems

Makabuhay stems (Tinospora rumphii L., Menispermaceae) are used in

traditional medicine for pruritus and wounds. The aqueous extract of the stems

is used in the treatment of stomach trouble, indigestion, and diarrhea

(Quisumbing, E. 1987).The decoction of the stems is used for malaria and fever

(Salazar, N., et al., 2012).

The pounded stem, mixed with coconut oil or any cooking oil has been

used for a variety of rheumatic and arthritic complaints. In the Philippines, the

decoction of the Makabuhay stem is also an excellent vulnerary for itches,

ordinary and cancerous wounds (Guerrero, J. 2000). It has been used for the

treatment of skin infections and is believed to treat scabies (Philippine Herbal

Medicine, 2010).

Medicinal Use of Makabuhay Stems

Makabuhay stems (Tinospora rumphii L., Menispermaceae) are used in

the prevention of Atherosclerosis-related cardiovascular disease. The ethanolic

extract of the stems is used as anti-microbial (Cruz, M. 2000) and for the

treatment of hepatic fibrosis (Feria, M. 2007). The aqueous extract of Makabuhay

 12

stem contains many chemicals that are known to have anti-oxidant activities

(Amom, M. et al., 2011).

It is an effective remedy in the treatment of tropical ulcers (Bacon,R.

1997). Father De Sta. Maria includes Makabuhay in his book “Manual de

Medicinas Caseras”, and says that it is given in decoction or powder form as a

febrifuge. A study in the Indira Ghandi Medical College showed it effective in

relieving symptoms of hay fever or allergic rhinitis (Altshul, S. 2007).It is currently

being studied for its possible stimulant effect on the immune system

(Kamarazaman, I., et al., 2012).

Constituents of Makabuhay Stems

Active principle of Makabuhay stems consist of berberine, palmatine,

choline, tinosporine (Kumar, K., et al., 2000).Glycosides present are 18-

norclerodane glycoside, furanoid diterpene glucoside, tinocordiside, syringin

(Ghosal, S.1997). The steroids present are ecdysterone, makisterone A and

giloinsterol (Gangan,V. 1998). The crude ethanolic extract of Makabuhay stems

contains mainly tannins, flavonoids, alkaloids, saponins, carbohydrates and

steroids (Quisumbing, E. 1978).

Condensed Tannins

Proanthocyanidins (condensed tannins) are polymeric flavonoids. These

tannins are derivatives of Flavonoid, catechin, flavonol-3-4-diol. Scabies mites

eat the skin by secreting an enzyme, called Aspartic Protease, which causes the

inflammation, and itching (Mahmood, W. 2013). Tinospora cordifolia, which also

 13

belongs to Menispermaceae family, is positive of condensed tannins, which is

responsible in inhibiting Aspartic proteases (Rege, A. 2014). Major constituent of

Multiflora rose root, 2-(3,4-dihydroxyphenyl)-6-(4-hydroxyphenyl)-8-(2,4-

dihydroxyphenyl)-2,3-trans-6,7-cis-7,8-trans-3,4,7,8-tetrahydro-2H,6Hpyrano

[2,3-f] chromene-3,7,9-triol (RM-3) belongs to condensed tannins, improve atopic

dermatitis-like skin lesions by mite antigen (Park, K. 2011).

Mechanism of Action of the Prepared Ointment of Makabuhay Stems

Makabuhay plants were reported to heal skin wounds or open wounds but

the healing mechanism is still unknown (Dela Cruz, R. 2000).

Pathophysiology of Scabies

Retrieved from http://www.michigan.gov

Plate 2. Microscopic view of Sarcoptes scabiei mite

Scabies is a condition caused by the ectoparasite Sarcoptes scabiei, also

known as the itch mite and related to the mite causing mange in animals. The

scabies mite is an obligate parasite that completes its entire life cycle on

humans. Other variants of the scabies mite can cause infestation in other

mammals, such as dogs, cats, pigs, ferrets, and horses, although they can irritate

human skin as well. However, they are unable to reproduce in humans and can

cause only a transient dermatitis. These mites are very small and not visible to
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the naked eye, being less than 0.5 mm in length. Females are about 0.3 to 0.4

mm while males are 0.25 to 0.35 mm in length and rounded in shape, with tiny

pointed spines on their dorsal surface that assist them in burrowing (Chang, Y.,

et al., 2006).

Sarcoptes scabiei undergoes 4 stages in its life cycle; egg, larva, nymph

and adult (Sargent, S. 1996). The adult female is larger than the male and

following fertilization, the mites burrow into the upper layers of the epidermis

laying thirty to forty eggs that hatch within 3 to 5 days. The larvae excavate and

create new burrows and reach maturity in approximately 4 days where the cycle

is repeated (Chang, Y. et.al., 2006). Mating takes place once, and the female is

fertile for the rest of her life while the male dies soon after mating. The female

makes a serpentine burrow using proteolytic enzymes to dissolve the stratum

corneum of the epidermis, laying eggs in the process then she continues to

lengthen her burrow and lay eggs for the rest of her life, surviving 1-2 months.

Scabies is normally transmitted by either direct skin-to-skin contact with

the infected animal or indirect, through infested fomites. Mites cannot fly or jump

but crawl at the rate of 2.5 cm per minute on warm skin. They can survive for 24

to 36 hours at room temperature and average humidity and remain capable of

infestation and epidermal burrowing.  

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Retrieved from www.stanford.edu

Plate 3. Life Cycle of Sarcoptes scabiei mite

The more parasites present, the greater the likelihood of transmission.

Transmission by means of indirect method is rare with classic scabies but may

occur with crusted scabies in humans. Sexual transmission also occurs

(Chosidow, O. 2006). Scabies mites’ infestation in dogs is commonly known as

mange and is different from Sarcoptes scabiei infestation which is specific to

humans. Mites from mange-infested animals can burrow into human skin but

cannot reproduce, so they die within a few days (Centers for Disease Control,

2010).

Diagnosis of Scabies in Dogs

The standard method of diagnosing canine scabies is to perform a skin

scraping and then identify the mite under the microscope. Most diagnoses are

made based on history and response to treatment for scabies (Burroughs and

Elston, D. 2003).

Sarcoptic mange tends to cause intense itching. It can result in

restlessness and frantic scratching; symptoms that generally appear one week

after exposure. It also can result in hair loss, reddened skin, body sores and

scabs. The most commonly affected areas are a dog’s ears, elbows, face and

legs, but it can rapidly spread to the entire body. When passed to humans,

sarcoptic mange causes a rash of red bumps, similar to mosquito bites

(Ackerman, L. 1994).

Classification of Canine Dermatology Lesions

Level 1
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Normal Skin Structure

A. Epidermis

The epidermis is the outer layer of skin. It provides protection from foreign

substances. It is composed if multiple types of cells, including keratinocytes,

melanocytes, Langerhans cells, and Merkel cells. Each of these cells has special

functions.

Basement Membrane

This layer of skin is located beneath the epidermis and connects the

epidermis to the dermis layer below. It also serves as a protective barrier

between the epidermis and the dermis. Several skin diseases, including a

number of autoimmune conditions, can damage the basement membrane zone.

B. Dermis

The dermis supports and nourishes the epidermis and skin appendages.

The blood vessels, which supply the epidermis with nutrients, are located in the

dermis. Blood vessels also regulate skin and body temperature. Sensory nerves

are located in the dermis and hair follicles. The skin responds to the sensations

of touch, pain, itch, heat, and cold. The dermis secretes the proteins collagen

and elastin, which give support and elasticity to the skin. There are also immune

cells in the dermis that defend against infectious agents that pass through the

epidermis.

C. Subcutis

The subcutis is the innermost layer of the skin. It contains the

subcutaneous fat and muscles. Subcutaneous means beneath the skin. The
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twitch muscle is the major muscle immediately beneath the skin. The

subcutaneous fat provides insulation; a reservoir for fluids, electrolytes, and

energy; and a shock absorber.

Level 2

Primary Lesion

Primary skin lesions are those which develop as a direct result of the

disease process. This classification is naturally artificial. The same lesion type

might be a primary lesion in one disease, but a secondary lesion in another e.g.

alopecia is a primary lesion in canine hypothyroidism (direct consequence if lack

of thyroxine) but secondary lesion in feline flea allergy (caused by the patient;

hair removed but the itchy cat). These lesions are called macule, patch, nodule,

vesicles, bulla follicular cast, alopecia and crust.

Plate 4. Macule Plate 5. Alopecia

Retrieved from www.excellenceindermatology.jp

Plate 6. Crust Plate 7. Vesicles

Retrieved from www.dog-health-handbook.com

Level 3

Secondary Lesion
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Consequences of the dog’s secondary lesion with secondary pyoderma

which implies a bacterial infection that is complicating an underlying skin disease

(common examples in dogs include allergy and demodicosis). But in secondary

pyoderma may present with primary lesions such as papules and pustules.

These lesions in the tertiary level are manifested as epidermal collarettes, scar,

excoriations, erosions, ulcers, fissures, lichenification, hyperpigmentation, and

callus.

Plate 8. Ulcer Plate 9. Excoriation

Retrieved from www.vet.ed.ac.uk

Plate 10. Callus Plate 11. Lichenification

Retrieved from www.vettimes.co.uk

Treatment for Scabies and its Mechanism of Action

The pediculicide and scabicide agents are neurotoxic in lice and scabies,

resulting in paralysis and death. Permethrin is a pediculicide and scabicide

agent. The neurotoxic agents require an intact nervous system of Sarcoptes

scabiei mite to be effective. A second application is often required because

newborn larvae do not have an intact nervous system for several days after

hatching. Action of Permethrin is to interfere sodium channels of insects,

 19

resulting in neurotoxicity and paralysis. Mutations in the sodium transport

proteins have been associated with resistance (Archer, M. 2014).

Sulfur Ointment

Sulfur is the oldest antiscabietic in use. Celsus used sulfur mixed with

liquid pitch for management of scabies as early as 25 AD. Sulfur is used as an

ointment (2%–10%) and usually 10% ointment is preferred. The technique is

very simple: after a preliminary bath, the sulfur ointment is applied and

thoroughly rubbed into the skin over the whole body for two or three consecutive

days. Ointments are more useful than any other preparation (Burkhart, C. 2012).

Topical sulfur ointment is messy, malodorous, stains clothing, and in a hot

and humid climate may lead to irritant dermatitis. It has the advantage of being

cheap and may be the only choice in areas of the world where the need for mass

therapy or economy dictates the choice of scabicide.

Sulfur should be used only in situations where adults cannot tolerate

lindane, permethrin, or ivermectin as it is inferior to all these agents. Sulfur is

recommended as a safe alternative for the treatment of scabies in infants,

children, and pregnant women (Norris Cotton Cancer Center).

Sulfur has germicidal, fungicidal, parasiticidal, and keratolytic actions, and

known to be the oldest scabicidal agent (Karthikeyan, K. 2004). Precipitated

sulfur in petrolatum in concentrations of 5-10% is an inexpensive treatment and

has been used successfully for mass treatments during outbreaks of scabies in

institutional settings (Threinen, D. 2003).

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CHAPTER 3

METHODOLOGY

This chapter presents the methods and procedures that was used for the

determination of the scabicidal activity of prepared ointment from Makabuhay

stems (Tinospora rumphii L., Menispermaceae) in dogs infected with Sarcoptes

scabiei mite.

1. Collection, Authentication and Preparation of the Stems of Makabuhay

(Tinospora rumphii L., Menispermaceae)
Collection and Authentication of Makabuhay stems

The Makabuhay stems were collected from Tondo, Manila. The sample

was submitted to the Botany Division of the National Museum for authentication.

The stems of Makabuhay that were used are fresh and were placed on a tight

container.

Organoleptic Evaluation of Makabuhay Stems (Tinospora rumphii L.,

Menispermaceae)
The appearance, texture and odor excluding the taste of the Makabuhay

stems were determined.

Crude Ethanolic Extraction of Makabuhay Stems (Tinospora rumphii L.,

Menispermaceae) using Maceration Method
The Makabuhay stems were chopped into pieces and were placed in

Erlenmeyer flask. About 150ml of 95% ethanol was used as a solvent to

immerse the 50g of chopped Makabuhay stems and were covered and set aside
 21

for three days with occasional stirring. More ethanol was added to keep the

stems always immersed in the solvent. After three days, the extract was filtered

using cheesecloth. Ethanol was evaporated using a water bath until the presence

of thick concentrated extract.

Determination of Total Crude Extractive and Solubility of the Crude

Ethanolic Extract of Makabuhay Stems
The researchers determined the total crude extractive of the Crude

Ethanolic Extract of Makabuhay Stems by dividing the weight of the extract over

the weight of the fresh sample.

The solubility of the Crude Ethanolic Extract of Makabuhay Stems was

tested using distilled water, 95% Ethanol, Chloroform, Carbon tetrachloride, and

Diethyl ether as solvent.

Phytochemical Analysis of the Ethanolic Extract of Makabuhay Stems

(Tinospora rumphii L., Menispermaceae)
Test for Carbohydrates

The ethanolic extract was dissolved individually in 5 ml distilled water and

filtered. The filtrate was used to test for the presence of carbohydrates. Molisch’s

Test was conducted. The filtrate was treated with 2 drops of alcoholic α-naphthol

solution in a test tube. Formation of the violet ring at the junction indicated the

presence of Carbohydrates.

Test for Flavonoids

The ethanolic extract was hydrolyzed with dilute HCl, and then subjected

to test for flavonoids with Modified Borntrager’s test. The extract was treated with
 22

Ferric chloride solution and immersed in boiling water for about 5 minutes. The

mixture was cooled and extracted with equal volumes of benzene. The benzene

layer was separated and treated with ammonia solution. Formation of rose-pink

color in the ammoniacal layer indicates the presence of anthranol glycosides.

Batesmith and Metcalf Test

The ethanolic extract was treated with 0.5ml of conc. HCl. Any color

change was observed. The mixture was warmed for 15 minutes in a water bath.

Positive result indicates the presence of leukoanthocyanins which is strong red or

violet color.

Test for Tannins

About 15ml of ethanolic extract was evaporated to dryness over a water

bath then cooled. 5 drops of 10% NaCl solution was added. An aqueous solution

of tannic acid was used as control.

Ferric Chloride Test

About 2ml of the extract and the control was treated with 3 drops of ferric

chloride. Positive results were shown by the presence of blue-back color which

indicated the presence of hydrolysable tannins while a brownish-green color

indicated the presence of condensed tannins. Polyphenols also gave colored

products with this test.

Formulation of Ointments from Makabuhay Stems (Tinospora rumphii L.,

Menispermaceae)
Three different concentrations of the prepared ointment from the crude

ethanolic extract of Makabuhay stems was made which only contain basic
 23

excipients of an ointment: the white wax and the white petrolatum (Department of

Health, 2010).

10% Makabuhay Stems Ointment:

Crude Ethanolic Extract of Makabuhay Stems…………….. ......5g

White ointment …………………………………………….…...... 45g

White wax…………………....2.25g
White petrolatum……….….42.75g
To make……………………………………………………… 50g

15% Makabuhay Stems Ointment:

Crude Ethanolic Extract of Makabuhay Stems………….….....7.5g

White ointment …………………………………………...........42.5g

White wax…………………....2.12g
White petrolatum……….….40.38g
To make…………………………………………………………... 50g

25% Makabuhay Stems Ointment:

Crude Ethanolic Extract of Makabuhay Stems………….......12.5g

White ointment ……………………………………………...... 37.5g

White wax…………………..1.88g
White petrolatum……….….35.63g
To make………………………………………………………..… 50g

Preparation of Ointment from the Stems of Makabuhay (Tinospora rumphii

L., Menispermaceae)

The crude ethanolic extract of Makabuhay stems was incorporated in a

250ml beaker containing white ointment. It was stirred until well blended. The

prepared ointment was transferred to a 50g capacity ointment jar.

Physical Properties of the Prepared Ointments from the Stems of

Makabuhay (Tinospora rumphii L., Menispermaceae)
 24

The prepared ointments were evaluated for various parameters such as

color, odor, homogeneity, grittiness (Westenberger, K. 2013).

2. Experimental Subjects

In coordination with the Valenzuela Veterinary Office and their city pound,

the selection of dogs was made through purposive sampling technique. It was

done after the dogs have been tested positive for Sarcoptes scabiei by skin

scraping, regardless of age and sex, with the help of the resident veterinarian

and personnel from the city pound.

The researchers used five dog subjects for the determination of the

efficacy of the prepared ointment from Makabuhay stems, positive control: 10%

sulfur ointment and normal control: white ointment. The five dogs were divided

into three groups: one dog for the normal control (white ointment), one dog for

the positive control (10% sulfur ointment), and three dogs for the experimental

group (prepared ointments from Makabuhay stems). The experimental group

was divided into three groups according to different concentrations (10%, 15%,

and 25%) of the prepared ointment they received.

Skin Irritation Test

The researcher used Draize test, five adult male rabbits weighing about

1.5-2.5 kg was used to test the skin irritation. The area on the back of each

rabbits was shaved prior to the experiment. The shaved areas of skin of each

rabbits were divided into four marked area. The three marked area of respective

animals were used for the topical application of prepared ointment from

Makabuhay stems of different concentrations (10%, 15%, and 25%) and the
 25

remaining marked area considered as a blank sample for testing the skin

irritation as per the method of Draize.

Half a gram of the prepared ointment from Makabuhay stems (10%, 15%,

and 25%) as the test substances, were applied to the shaved area approximately

6cm2 of skin of respective animals. Both the treated sites were covered by gauze

and the back of the rabbit was wrapped with a non-occlusive bandage. The

animals were returned to their cages after. After 24 hours, the bandage and the

test materials were removed and an hour later the sites were examined for skin

irritation. Observation of the sites was done 24 hours after application, and was

repeated after another 48 and 72 hours. The reactions, defined as erythema and

edema, were evaluated according to the scoring system for skin reactions.

Control marked area on animals were also prepared in the same manner

similar to the test animals using white ointment as normal control. The Score of

Primary Irritation (SPI) were calculated for each rabbit as the following: Scores

for erythema and edema at 24, 48 and 72 hours summed and divided by the

number of the observations for the treated sites. The SPI for the control sites was

also calculated in the same fashion as test.

Table 1
Classification System for Skin Reaction
(Retrieved from: www.upjournals.com)

Reaction
Erythema Score
No erythema 0
Very slight erythema 1
Well defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet redness) to eschar formation 4
 26

Edema Score
No edema 0
Very slight edema 1
Well defined edema (edges of the area well defined by define raising) 2
Moderate edema (raising approximately 1 mm) 3
Severe edema (raised more than 1 mm and extended beyond the area
4
of exposure)

Total possible score for primary irritation 8

Erythema∧edema grade at 24 , 48∧72 hrs

SPI for each rabbit = ∑
number of observations

The difference between the summation of SPI scores of five animals from

the treated site and control site were calculated and used for Primary Irritation

Index determination. The Primary Irritation Index (PII) was calculated as the

arithmetical mean of the SPI values of the five rabbits. The irritation degree was

categorized as negligible, or slight, moderate or severe irritation based on the PII

(Draize, et.al., 1944).

PII=
∑ SPI (test )−∑ SPI (base)
Number of animals

Table 2
Response Categories of Irritation in Rabbit
(Retrieved from: www.upjournals.com)

Category Primary Irritation Index (PII)

Negligible 0-0.4
Slight irritation 0.5-1.9
Moderate irritation 2.-4.9
Severe irritation 5-8
Diagnosis of Dogs Infected with Sarcoptes scabiei mite Before and After
the Treatment
 27

The sample of the epidermal tissue was taken for laboratory identification

for the presence of Sarcoptes scabiei infestation. The hair of the test animal was

clipped, and then the skin was pressed prior to scraping to push the mites out of

the depths of the hair follicles. The skin was scraped in the direction of hair

growth with a blade covered with mineral oil, the capillary bleeding was

observed. The scraped material was placed on a glass slide, and then a drop of

mineral oil was added before putting a cover slip and observed under a

microscope, using an eyepiece with 10 x magnifications and a scanner objective

(Lising, S. 2014).

The experimental subjects were also checked for the presence of

bacterial, fungal and other types of diseases by the resident veterinarian for a

complete health certification of the test animal. The acquired data may be of use

in the final assessment of the effectivity of the prepared ointment from

Makabuhay stems. The diagnosis was repeated after the treatment period using

the same procedure from the initial diagnosis.

Application of Prepared Ointments and Normal Control

The subjects were bathed once every week using an ordinary soap. The

ointments were applied to the normal, positive control and experimental group on

the affected area once a day for three consecutive days for three weeks. The

amount of ointments applied was enough to cover the affected area.

The three dogs from the experimental group received the 10%, 15%, 25%

prepared Makabuhay stems ointment respectively. The fourth dog received the

10% sulfur ointment. The fifth dog received the white ointment. The researchers
 28

had a rotational duty on applying the prepared ointments to dogs and were

assisted by the licensed veterinarian and personnel from Valenzuela City pound.

Statistical Analysis

Wilcoxon Test was used to determine the significant difference of the pre-

application of the three prepared ointments from its post-application. Kruskal

Wallis Test was used to determine the significant difference of the 10%, 15% and

25% prepared Makabuhay ointments from the 10% Sulfur Ointment in terms of

its Scabicidal activity. The researchers used the IBM SPSS statistical data editor

program to confirm the data gathered and analyzed.

 29

CHAPTER 4

PRESENTATION, ANALYSIS AND INTERPRETATION OF DATA

This chapter presents the results, analysis and interpretation of data

gathered from the experiments conducted by the researchers that are essential

for the determination of the scabicidal activity of the prepared ointment from the

crude ethanolic extract of Makabuhay stems (Tinospora rumphii L.,

Menispermaceae) in dogs infected with Sarcoptes scabiei mite.

Crude Ethanolic Extraction of Makabuhay Stems

The crude ethanolic extract of Makabuhay stems produced is clear,

brownish green solution and is acidic using pH paper after 3 days of extraction by

maceration. It obtained a total crude extractive of 29.33%.

Table 3
Total Crude Extractive of Crude Ethanolic Extract of Makabuhay Stems
Weight of Weight of Total Crude
Trial
sample extract Extractive
1 333.33g 97.73g 29.32%
2 333.33g 96.60g 28.98%
3 333.33g 98.93g 29.68%
Average Total Crude Extractive 29.33%

Table 3 shows the three trials done to determine the total crude extractive

of the Makabuhay stems with the use of the following formula:

Total Crude Extractive = weight of extract x 100

 30

weight of sample

Table 4
Results of Solubility Test of the Crude Ethanolic Extract
of Makabuhay Stems

Solvent Result
Water Soluble
Ethanol Soluble
Chloroform Insoluble
Carbon tetrachloride Insoluble
Diethyl ether Insoluble
Table 4 presents the results of the solubility test of the crude ethanolic extract of

Makabuhay stems. The data shows that the extract is soluble in polar solvent

and insoluble in organic solvents.

Table 5
Results of Phytochemical Analysis of Crude Ethanolic Extract of
Makabuhay Stems

Constituent Test used Result

Ferric chloride test Positive
Tannins
Gelatin test Negative
Batesmith and Metcalf test Positive
Flavonoids Wilstater “cyanidin” test Positive
Modified Borntrager’s Negative
Carbohydrates Molisch test Positive
Mayer’s Negative
Alkaloids Valser’s Negative
Wagner’s Negative
Plant acids Sodium carbonate test Positive
Fixed oil Stain test Negative
Glycosides Kedde’s test Negative
Positive – presence of the constituent; Negative – absence of the constituent
 31

Table 5 shows that the crude ethanolic extract of Makabuhay stem

contains condensed tannins, leucoanthocyanins, and insoluble carbohydrates

and plant acid constituents.

Physical Properties of the Prepared Ointments from Makabuhay Stems

Plate 12. 10%, 15% and

25% Prepared Ointments from Makabuhay Stems

The prepared ointments from Makabuhay stems are odorless, greasy, and

slightly yellow in color that intensifies as the concentration increases.

Skin Irritation Test

The following data were obtained after conducting the skin irritation test for

the normal control, 10%, 15% and 25% of the prepared Makabuhay stem

ointments using Draize Test in five adult male rabbits weighing 1.5kg to 2.5kg.

Table 6 shows the results for the erythema reaction of the five adult male

rabbits upon testing for skin irritation.

Table 6
Erythema Reaction

Ointment Score
Normal No erythema
 32

10% prepared Makabuhay ointment No erythema

15% prepared Makabuhay ointment No erythema
25% prepared Makabuhay ointment Very slight erythema

The scorings used for the computation of Score of Primary Irritation (SPI)

were based on the Classification System for Skin Reaction provided in Table 1.

Only the 25% of the prepared Makabuhay stem ointment caused a very slight

erythema (Score: 1) while the normal control, 10% and 15% prepared

makabuhay stem ointments presented no erythema (Score: 0).

Table 7
Edema Reaction

Ointment Score of Primary Irritation

Normal No edema
10% prepared Makabuhay
No edema
ointment
15% prepared Makabuhay
No edema
ointment
25% prepared Makabuhay
No edema
ointment

Table 7 shows the results of edema reaction observed on the five adult

male rabbits. The scorings used for the computation of Score of Primary Irritation

(SPI) were also based on the Classification System for Skin Reaction provided in

Table 1. All the prepared Makabuhay stem ointments as well as the normal

control showed no edema (Score: 0).

Results on Tables 6 and 7 were computed using the Score of Primary

Irritation (SPI) formula. The computed SPI of normal control, 10% and 15% of the
 33

prepared Makabuhay ointments were 0, while for the 25% prepared Makabuhay

Ointment Primary Irritation Index

Normal Negligible
10% prepared Makabuhay ointment Negligible
15% prepared Makabuhay ointment Negligible
25% prepared Makabuhay ointment Negligible
ointment, the computed SPI is 0.67.

Table 8
Primary Irritation Index

The computed Primary Irritation Index (PII) for each rabbit in normal

control, 10%, and 15% of the prepared Makabuhay ointments is 0, while for the

25% of the prepared Makabuhay ointment, the computed PII for each rabbit is

0.13.

Therefore, the normal, 10%, 15% and 25% of the prepared Makabuhay

stem ointments fall within the Negligible category which is 0- 0.4. The Primary

Irritation Index (PII) of each oinments were computed using the PII formula and

interpreted using the Response Categories of Irritation in Rabbit showed in

Table 2. Raw data of Tables 6 and 7 is shown in Tables 18 and 19 in Appendix

as well as the computation of SPI of each rabbit and PII.

Localization of Lesions
 34

Table 9
Localization of Skin Lesions Among Dogs Infected with
Sarcoptes scabiei mite
Right Left
Dog Right Left side of side of Right Left Right Left
Group Back
No. ear ear the the arm arm leg leg
body body
10% 1 + + + + +
15% 2 + + + + + +
25% 3 + + + + + +
10%Sulfur 4 + + + + + +
Normal 5 + + + + + + + +

Lesion localizations were variable and were determined in every

experimental subject involved. Primary to secondary lesions were observed on

the ears, arms, legs and back of the dogs.

Diagnosis of Dogs infected with Sarcoptes scabiei mite

Prior to the application of the prepared ointments from the crude ethanolic

extract of Makabuhay stems, a licensed veterinarian from the Valenzuela City

Pound diagnosed the experimental subjects with Sarcoptes scabiei mite for

confirmation.

Table 10
Microscopic Analysis Before and After Treatment

Dog
Group Before After
No.

10% 1
 35

15% 2

25% 3

10% Sulfur 4

Normal 5

Table 11
Presence and Absence of Sarcoptes scabiei mite
during the Treatment Period
Group Dog Day 0 Day 4 Day 7 Day 10
No. Present Absent Present Absent Present Absent Present Absent
10% 1 ✓ X ✓ X ✓ X ✓ ✓
15% 2 ✓ X ✓ X ✓ X ✓ ✓
25% 3 ✓ X ✓ X X ✓ X ✓
10% 4 ✓ X ✓ X ✓ X X ✓
Sulfur
Normal 5 ✓ X ✓ X ✓ X ✓ X
Day 0 – initial diagnosis; Day 4 – after 1st week of treatment; Day 7 – after 2nd week of treatment;
Day 10 – final diagnosis

After the treatment using the 10%, 15% and 25% of the prepared

ointments from Makabuhay stems for three weeks, the skin scraping of the dogs

were re-examined under the microscope. The dog 1 and dog 2 skin scrapings,
 36

which received the 10% and 15% Makabuhay stems ointment respectively,

showed that there were presence of dead and live mites. However, it was

noticeable that the number of dead mites in 10% compared to 15% Makabuhay

stems ointment was lesser. The dog 3 skin scraping, which received the 25%

Makabuhay stems ointment, showed only the remnants of the dead mites. The

dog 4 skin scraping, which received the 10% sulfur ointment, showed the

absence of live mites due to paralysis. The dog 5 skin scraping, which received

the normal control, showed presence of live mites. The results showed that the

cell membrane of the mites was disrupted by the prepared Makabuhay stems

ointment which caused the death of Sarcoptes scabiei mite.

Statistical Analysis

The results obtained in the research were used to identify whether to

accept or to reject the null hypothesis of the study. IBM SPSS statistical data

editor program was used in the research to confirm the data gathered and

analyzed.

Table 12
Hypothesis Test Summary of the Pre-treatment and Post-treatment
of each Experimental Groups

Groups Null Hypothesis Test Sig. Decision

10% Prepared The median of differences
Ointment from between the 10% pre-
Makabuhay treatment and 10% post- 0.046
Stems treatment equals 0.
15% Prepared The median of differences
Ointment from between the 15% pre- Related-
Makabuhay treatment and 15% post- Samples 0.025 Reject the
Stems treatment equals 0. Wilcoxon null
25% Prepared The median of differences Signed hypothesis
 37

Ointment from between the 25% pre- Rank Test .

Makabuhay treatment and 25% post- 0.014
Stems treatment equals 0.
The median of differences
10% Sulfur between the 10% sulfur
Ointment pre-treatment and 10% 0.014
sulfur post-treatment
equals 0.
Asymptotic significances are displayed. The significance level is 0.05.

The null hypothesis of the study is that there is a significant difference

between the pre-treatment and the post-treatment of the each group. Using

Wilcoxon signed rank test, all groups obtained a significance which is lower than

the 0.05 significance level given. These results therefore means that one should

reject the null hypothesis and that there is a significant difference between the

pre-treatment and post-treatment of the each group showing improvement in the

experimental subjects’ condition.

The data obtained shows that the 25% prepared ointment from

Makabuhay stems which has the least significance when compared to the other

prepared ointment from Makabuhay stems concentrations has the same

significance with the sulfur ointment which is 0.014. This means that there is no

significant difference between the scabicidal activity of the 25% ointment

prepared from Makabuhay stems and sulfur ointment.

Table 13
Hypothesis Test Summary of the Scabicidal Activity
Among the Experimental Groups
Null Hypothesis Test Sig. Decision

The distribution of effect is Independent- Failed to reject

the same across categories Samples 0.260 the null
of group. Kruskal-Wallis hypothesis.
Test
 38

Asymptotic significances are displayed. The significance level is 0.05.

Using the Kruskal-Wallis Test, Table 13 shows that the obtained

significance which is 0.260 is higher than the significance level 0.05. The results

show that all ointments used in the study do not have a significant difference

among them and that all possess comparable significant scabicidal activity during

the treatment period.

 39

CHAPTER 5

SUMMARY, CONCLUSION, AND RECOMMENDATION

This chapter presents the summary, conclusion and recommendation

based from the results obtained from the determination of the Scabicidal Activity

of the Crude Ethanolic Extract from Makabuhay Stem (Tinospora rumphii L.,

Menispermaceae) in dogs infected with Sarcoptes scabiei mite.

Objective of the Study

The study aims to prove the scabicidal activity of the prepared ointment

from the crude ethanolic extract of Makabuhay stems (Tinospora rumphii L.,

Menispermaceae) in dogs infected with Sarcoptes scabiei mite.

Statement of the Problem

It seeks to answer the following questions:

1. What is the percentage of the total crude ethanolic extract obtained from

Makabuhay stems (Tinospora rumphii L., Menispermaceae)?

2. What are the physical properties of the prepared ointment from

Makabuhay stems (Tinospora rumphii L., Menispermaceae)?

3. Will the prepared ointment from Makabuhay stems (Tinospora rumphii L.,

Menispermaceae) cause skin irritation in adult male rabbits using Draize

test?
 40

4. At what concentration of the prepared ointment from Makabuhay stems

(Tinospora rumphii L., Menispermaceae) exhibits greatest scabicidal

activity in treating dogs with scabies?

5. Is there a significant difference between the scabicidal activity of the

prepared ointment from Makabuhay stems (Tinospora rumphii L.,

Menispermaceae) when compared to commercially available 10% sulfur

ointment?

Summary of Findings

The study aims to prove the scabicidal activity of the prepared ointment

from the crude ethanolic extract of Makabuhay stems (Tinospora rumphii L.,

Menispermaceae) in dogs infected with Sarcoptes scabiei mite.

1. The total crude ethanolic extract obtained from Makabuhay stems was

29.33%.

2. The physical properties of the prepared ointment from Makabuhay stems

are odorless and slightly yellow in color and intensify as the concentration

increases.

3. Draize Test was conducted to five male adult rabbits to determine skin

irritation. Results showed that 10%, 15% and 25% concentrations of

prepared ointments from Makabuhay Stems were negligible.

4. After the treatment using the 10%, 15% and 25% of the prepared

Makabuhay stems ointments for three weeks, the skin scraping of the

dogs were re-examined under the microscope. The result showed that the
 41

cell membrane of the mite was disrupted. The dog 1 and dog 2 skin

scrapings, which received the 10% and 15% Makabuhay stems ointment

respectively, showed that there were presence of dead and live mites.

However, it was noticeable that the number of dead mites in 10%

compared to 15% Makabuhay stems ointment was lesser. The dog 3 skin

scraping, which received the 25% Makabuhay stems ointment, showed

only the remnants of the dead mites. The dog 4 skin scraping, which

received the 10% sulfur ointment, showed the absence of live mites due to

paralysis. The dog 5 skin scraping, which received the normal control,

showed presence of live mites. The results showed that the cell

membrane of the mites was disrupted by the prepared Makabuhay stems

ointment which caused the death of Sarcoptes scabiei mite.

5. Based on the statistical analysis, all ointments possess a significant

scabicidal activity during the treatment period but 25% prepared ointment

from Makabuhay stem obtained the same significance with 10% sulfur

ointment compared to the other concentrations. This means that the 25%

prepared ointment has a scabicidal activity comparable to the 10% sulfur

ointment.

Conclusion

Based on the summary of findings, the total crude ethanolic extract

obtained from Makabuhay stems was 29.33%. The prepared ointment from

Makabuhay stems are odorless and slightly yellow in color and intensify as the

concentration increases. Draize Test was conducted to five male adult rabbits to
 42

determine skin irritation and the results showed that 10%, 15% and 25%

concentrations of prepared ointments from Makabuhay Stems were negligible.

After the treatment using the 10%, 15% and 25% of the prepared Makabuhay

stems ointments for three weeks, the skin scraping of the dogs were re-examined

under the microscope, results showed that the cell membrane of the mite was

disrupted. The dog 1 and dog 2 skin scrapings, which received the 10% and

15% Makabuhay stems ointment respectively, showed that there were presence

of dead and live mites. However the number of dead mites in 10% compared to

15% Makabuhay stems ointment was lesser. The dog 3 which received the 25%

Makabuhay stems ointment, showed only the remnants of the dead mites. The

dog 4 which received the 10% sulfur ointment, showed the absence of live mites

due to paralysis. The dog 5 which received the normal control, showed presence

of live mites. The results showed that the cell membrane of the mites was

disrupted by the prepared Makabuhay stems ointment which caused the death of

Sarcoptes scabiei mite. Based on the statistical analysis the 25% prepared

ointment has a scabicidal activity comparable to the 10% sulfur ointment,

therefore 25% ointment has no significant difference with the reference drug 10%

sulfur ointment. Lower concentrations (10% and 15%) also possess scabicidal

activity but are significantly different with the reference drug.

Recommendations

Based on the conclusions, the researchers hereby recommend the following:

1. Isolation of the specific constituent that possesses the scabicidal activity to

make the formulation more concentrated with the active constituent.

 43

2. Conduct a stability test study for the prepared Makabuhay stem ointments

to determine its expiry and storage conditions

3. Conduct the skin irritation test in a BAI approved animal institution.

4. Test the prepared ointment from Makabuhay stems to other animals that

can also be infected with scabies.

5. Use a larger population of animals in each test group to provide more

substantial results.

.
 44

BIBLIOGRAPHY

A. Books

“A Primer on Medicinal Plants for Livestock Healthcare”. 2011.

http://crees.org/resources/files/NMC WSARE-Plant Herbal Medicine for Livestock
Booklet ALL.pdf

B. Website

“Excoriation”. 2014.
http://www.rvc.ac.uk/review/dermatology/Lesions/excoriation.htm

“Lichenification”. 2014.
http://www.rvc.ac.uk/review/dermatology/Lesions/lichen.htm

“Mange in Dogs (Canine Scabies)”. 2015. http://www.m.webmd.com

“OECD Guidelines For The Testing Of Chemicals. In Vitro Skin Irritation:

Reconstructed Human Epidermis Test Method”. 2013.
https://ntp.niehs.nih.gov

“Parasitic Diseases”. 2010. http://www.cdc.gov

“Pigmentation Disturbances”. 2014.

http://www.rvc.ac.uk/review/dermatology/Lesions/pigment.htm

“Sarcoptes Scabiei - Scabies”. 2011. http://www.parasitesinhumans.org

“Scabies”. 2000.http://www.petsandparasites.org

“Scabies”. 2011. https://web.stanford.edu

“Skin Diseases”. 2009. http://www.who.int

“Skin Testing”. 2015. http://aadconline.com

“Traditional Medicine & Healing: How to prepare makabuhay

ointment”.2015. http://doh.

“Treatment for Scabies”. 2015. http://www.mayoclinic.org

 45

“UPLB National Institute of Molecular Biology and Biotechnology”.

2014.http://biotech.uplb.edu.ph

“Vesicles and Bullae” (2 of 2). 2014.

http://www.rvc.ac.uk/review/dermatology/Lesions/vesicles2.htm

“What is Kruskal Wallis Test?”. (n.d.).

http://www.businessdictionary.com/definition/Kruskal-Wallis-Test.html

“Wilcoxon Test Definition | Investopedia”. 2010.

http://www.investopedia.com/terms/w/wilcoxon-test.asp

Aiello S. “Mange in Dogs and Cats”.2012.http://www.merckvetmanual.com

Burgess, I. “Infection control fact file”. 2003. http://www.nursingtimes.net

Castillo, E., et al. (2005). “Research information series on ecosystems

Volume 17”. http://erdb.denr.gov.ph

Centers for Disease Control. “Sarcoptic Mange in Dogs | Treatment and

Prognosis”.2015. http://www.cdc.gov

Chosidow, O. “Clinical practice: Scabies”. 2006. http://www.med.illinois.edu

Dweck, A., & Cavin, J. “Andawali (Tinospora rumphii) – a review”. 2015.

http://www.medinacollege-edu.com

Foster & Smith. “Pet Education”. 2015.http://www.peteducation.com

Golant, A., & Levitt, J. “Scabies: A Review of Diagnosis and Management

Based on Mite Biology”. 2012. https://pedclerk.bsd.uchicago.edu

Harr, K. “Tropical medicine rounds: Scabies community prevalence and

mass drug administration in two Fijian villages”. 2003. http://edoc.rki.de

Hipol, R., Cariaga, M.“Anti-Inflammatory Activities of the Aqueous Extract of

the Stem of Tinospora rumphii”. 2012.http://www.journalofnaturestudies.org

Kandárová, H., Hayden, P., & Klausner, M. “Result Filters”. 2009.

http://www.ncbi.nlm.nih.gov

Markell EK, John DT, Krotoski WA. Markell and Voge’s Medical Parasitology,
8th ed. Philadelphia: W.B. Saunders, 2000.

May, A. “Scabies”. 2015. http://www.bhchp.org

 46

Nettleman, M. “Scabies Causes, Symptoms, Treatment”. 2014.

http://www.emedicinehealth.com

Tantiado, R., & Tan, V. “Evaluation of the Angiosuppresive Activity of

Tinospora rumphii Boerl. Stem Extract Using the Chorioallantoic Membrane
Assay in Anas platyrhynchos Embryos”. 2012.http://www.sersc.org

Wendel, K., & Rompalo, A. “Scabies and Pediculosis Pubis: An Update of

Treatment Regimens and General Review”. 2015. http://cid.oxfordjournals.org
 47

APPENDIX 1

Plate 13. The Crude Ethanolic Extraction of Makabuhay Stems

by Maceration

APPENDIX 2

Table 14
Solubility Test Results

APPENDIX 3

Table 15
Phytochemical Analysis Results
Constituent Test used Result Pictures

Ferric
Tannins Condensed tannins
chloride test
 48

Tannins Gelatin test Positive

Table 15
Phytochemical Analysis Results (Continuation)

Batesmith
Flavonoids and Metcalf Leucoanthocyanins
test

Wilstater
Flavonoids Positive
“Cyanidin”

Modified
Flavonoids Borntrager’ Negative
s

Insoluble
Carbohydrates Molisch test
carbohydrates

Alkaloids Mayer’s Negative

 49

Alkaloids Valser’s Negative

Table 15
Phytochemical Analysis Results (Continuation)

Alkaloids Wagner’s Negative

Sodium
Plant Acid Bicarbonate Positive
test

Fixed Oil Stain test Negative

APPENDIX 4

Table 16
Application of Normal Control, 10%, 15%, and 25%
Prepared Makabuhay Stems Ointment
No. of
24hrs 48hrs 72hrs
Rabbits

Rabbit 1
 50

Rabbit 2

Rabbit 3

Table 16
Application of Normal Control, 10%, 15%, and 25%
Prepared Makabuhay Stems Ointment (Continuation)

Rabbit 4

Rabbit 5

Table 17
Results of Skin Irritation Test after 24, 48 and 72 hours

No. of After 72hrs

After 24hrs After 48hrs
Rabbits

Rabbit 1

Rabbit 2
Rabbit 4

Rabbit 3
Rabbit 5
 51

Table 17
Results of Skin Irritation Test
after 24, 48 and 72 hours (Continuation)

Table 18
Erythema Reaction

Concentratio
10% 15% 25% Normal
n of Prepared
Control
Oinment
Observation 24 48 72 24 48 72 24 48 72 24 48 72
hours
0 0 0 0 0 0 0 0 0 0 0 0
Rabbit 1
0 0 0 0 0 0 0 0 0 0 0 0
Rabbit 2
0 0 0 0 0 0 0 0 1 0 0 0
Rabbit 3
0 0 0 0 0 0 0 0 1 0 0 0
Rabbit 4
0 0 0 0 0 0 0 0 0 0 0 0
Rabbit 5
0 0 0 0 0 0 0 0 2 0 0 0
Total
0-No erythema, 1- Very Slight erythema, 2- Well-defined erythema, 3- Moderate- Severe
erythema, 4- Severe erythema to eschar formation

Table 19
Edema Reaction

Concentration
10% 15% 25%
of Prepared
oinment
 52

Observation 24 48 72 24 48 72 24 48 72
hours
0 0 0 0 0 0 0 0 0
Rabbit 1
0 0 0 0 0 0 0 0 0
Rabbit 2
0 0 0 0 0 0 0 0 0
Rabbit 3
0 0 0 0 0 0 0 0 0
Rabbit 4
0 0 0 0 0 0 0 0 0
Rabbit 5
0-No edema, 1- Very slight edema, 2- Well defined edema, 3- Moderate edema, 4- Severe
edema

Erythema∧edema grade at 24 , 48∧72 hrs

SPI for each rabbit = ∑
number of observations

PII=
∑ SPI (test )−∑ SPI ( base)
Number of animals

Table 20
Computations of SPI and PII

10% 15% 25%

SPI for each rabbit
SPI for each rabbit (15%) SPI for each rabbit (10%) 0+ 0+2
0+ 0+0 0+ 0+0 (25%) = ∑
=∑ =0 =∑ =0 3
3 3
= 0.67
PII (10%) = PII (15%) = PII (25%) =
∑ SPI ( 0 )−∑ SPI (0) = ∑ SPI ( 0 )−∑ SPI (0) = ∑ SPI ( 0.67 )−∑ SPI ( 0)
5 5 5
0 0 = 0.13
SPI – Score of Primary Irritation, PII – Primary Irritation Index

APPENDIX 5

Experimental Groups
 53

Plate 14. Normal Control Group

Plate 15. Positive Control Group (10% Sulfur Ointment)

 54

Plate 16. Experimental Group (10% Prepared Makabuhay Ointment)

Plate 17. Experimental Group (15% Prepared Makabuhay Ointment)

Plate 18. Experimental Group (25% Prepared Makabuhay Ointment)

 55

APPENDIX 6

Plate 20. Hypothesis Test Summary of Pre-Treatment and Post-treatment of

the 10% Group

Plate 21.
Hypothesis Test Summary of Pre-Treatment and Post-Treatment of the 15%
Group

Plate 22. Hypothesis Test Summary of Pre-Treatment and Post-Treatment

of the 25% Group
 56

Plate 23. Hypothesis Test Summary of Pre-Treatment and Post-treatment of

the 10% Sulfur Ointment Group

Plate 24. Hypothesis Test Summary of the Scabicidal Activity

among the Experimental Groups

Plate 25. Score of Each Condition in 10% Prepared Makabuhay Stems

Ointment Group
 57

Plate 26. Score of Each Condition in 15% Prepared Makabuhay Stems

Ointment Group

Plate 27. Score Of Each Condition in 25% Prepared Makabuhay Stems

Ointment Group
 58

Plate 28. Score of Each Condition in 10% Sulfur Ointment Group

 59

APPENDIX 7

Plate 29. Certificate of Authentication from the

Botany Division of National Museum
 60

Plate 30. Certificate of Thesis Experimentation from

Valenzuela City Pound
 61

Plate 31. Certificate of Diagnosis Before Treatment

from Valenzuela City Pound
 62

Plate 32. Certificate of Diagnosis After Treatment

from Valenzuela City Pound
 63

Rona Jerise P. Angeles

#40 Pariancillo Street, Polo
Valenzuela City, Philippines 1444
Mobile no.: +639264725923
E-mail: rona_angeles10@yahoo.com

OBJECTIVE

To be able to use my knowledge of medical techniques and medications,

and provide an exceptional health services to patients for the fulfillment of my
responsibility as a highly competent pharmacist.

SUMMARY OF QUALIFICATIONS

 Computer-literate.
 Efficiently handle multiple tasks and projects simultaneously.
 Able to work with minimal supervision and as a cooperative team member.
 Highly reliable and trustworthy.

EDUCATIONAL ATTAINMENT

Tertiary : Manila Central University - Caloocan

Bachelor of Science in Pharmacy
June 2012 - 2016

Secondary : San Diego Parochial School - Valenzuela

June 2008 - March 2012

Elementary : San Diego Parochial School - Valenzuela

June 2002 – March 2008

TRAININGS

• Major Internship at Mercury Drug Corporation

Polo, Valenzuela City
June - November 2015
 64

• Minor Manufacturing Internship at Scheele Laboratories Philippines, Inc.

Dalandanan, Valenzuela City
May - June 2015
• Minor Hospital Internship at Meycauayan Doctors Hospital
Meycauayan, Bulacan
April - May 2014
• Minor Community Internship at Mercury Drug Corporation
South Supermarket, Malinta, Valenzuela City
July - October 2013

SEMINARS ATTENDED

• Implementation of Current Good Manufacturing Practice and

Administrative Order 34, Series 2014
Filemon D. Tanchoco Auditorium
Manila Central University, Caloocan City
December 9, 2015
• This is W.A.R.: Win Against Resistance
Emilio T. Yap Auditorium
University of the Philippines, Manila
November 27, 2015
• Environmental Stewardship: The Role of Filipino Pharmacists
Filemon D. Tanchoco Auditorium
Manila Central University, Caloocan City
January 13, 2015
• Good Customer Service through Effective Communication Skills
Centennial Gymnasium
Manila Central University, Caloocan
September 2013

PERSONAL DATA

Date of Birth : September 10, 1995 Place of Birth: Valenzuela City

Civil Status : Single Gender : Female
Nationality : Filipino Religion : Roman Catholic
Height : 5”6 Weight : 70 kg

I hereby certify that the above information is true and correct.

RONA JERISE P. ANGELES

 65

Sharmaine Rizzel T. Angeles

#355 M. de Castro St., Bagong Barrio
Caloocan City, Philippines 1400
Mobile no.: +63.906.282.1479
E-mail: sharmainerizzelangeles@yahoo.com

OBJECTIVE

To be able to apply my skills and knowledge toward any responsibility that

I will undertake and further enhance my values, develop my talents, and broaden
my capabilities for a continuous career improvement providing adequate service
to patients and assuring optimal drug therapy outcome to make a highly
competent and proficient pharmacist that is a part of the health care team.

SUMMARY OF QUALIFICATIONS

 Computer-literate.
 Efficiently handle multiple tasks and projects.
 Ability to work independently or as a part of a team.
 Highly responsible and dependable.

EDUCATIONAL ATTAINMENT

Tertiary : Manila Central University

Bachelor of Science in Pharmacy
June 2012 - 2016
University of the East - Caloocan
Bachelor of Science in Accounting Technology
June 2011

Secondary : Manila Central University

June 2007 - March 2011

Elementary : Manila Central University

June 2001 – March 2007

TRAININGS AND OTHER ATTAINMENTS

 Manufacturing Pharmacy Internship (Minor)

Growrich Manfacturing Inc.
September 2015 – March 2016
 66

 Hospital Pharmacy Internship (Major)

Veterans Memorial Medical Center
May – August 2015
 Hospital Pharmacy Internship (Minor)
Veterans Memorial Medical Center
July – October 2014
 Community Pharmacy Internship (Minor)
Watsons Your Personal Store
July – October 2013
 Filemon D. Tanchoco Partial Scholar
Manila Central University
2nd Year – 1st Semester
 Tan Yan Kee Scholarship
University of the East - Caloocan
1st year – 2nd Semester

SEMINARS ATTENDED

 Implementation of Current Good Manufacturing Practice and AO 34,

Series 2014
Filemon D. Tanchoco Auditorium, Manila Central University
December 2015
 This is W.A.R.: Win Against Resistance Seminar
Emilio T. Yap Auditorium, UP College of Pharmacy
November 2015
 Environmental Stewardship: The Role of Filipino Pharmacists
Filemon D. Tanchoco Auditorium, Manila Central University
January 2015
 Good Customer Service through Effective Communication Skills
Centennial Gymnasium, Manila Central University
September 2013

PERSONAL DATA

Date of Birth : October 25, 1994 Place of Birth: Manila City

Civil Status : Single Gender : Female
Nationality : Filipino Religion : Born Again
Height : 5’3 Weight : 48 kg

I hereby certify that the above information is true and correct.

SHARMAINE RIZZEL T. ANGELES

 67

Lovely Ann D. Ayapana

Blk 49 Lot 4 Phase 3 F1 Sabalo St. Kaunlaran
Village, Caloocan City, Philippines
Contact no. 09352878555 / 09470148944
E-mail: lovejhayann@gmail.com

OBJECTIVE

To be able to fulfill my solemn oath as a part of the healthcare team

providing excellent service to the people In need wherein I can
continuously learn the essentials of being a pharmacist and improve my
knowledge and understanding on patient care, handling responsibilities
effectively, further develop my talents, broaden my capabilities and skills
to awaken hidden potentials for further career enhancement and growth of
a competent and proficient pharmacist.

SUMMARY OF QUALIFICATIONS

 Computer-literate.
 Efficiently handle multiple tasks and projects simultaneously.
 Able to work with minimal supervision and as a cooperative team member.
 Highly reliable and trustworthy.
 Can speak Filipino, English, Cebuano and Karay-a.

EDUCATIONAL ATTAINMENT

Tertiary : Manila Central University

EDSA, Caloocan City
Bachelor of Science in Pharmacy
2012 – 2016

Secondary : La Consolacion College- Caloocan City

496 A. Mabini Street, Caloocan City
2008 – 2012

Elementary : La Consolacion College-Caloocan City

496 A. Mabini Street, Caloocan City
2007– 2008
Poblacion Santander Central Elementary School
Poblacion Santander Cebu
2002-2007
 68

TRAININGS AND OTHER ATTAINMENTS

• Manufacturing Pharmacy Internship Growrich Manfacturing Inc.

(September 2015 – March 2016)
Novaliches, Caloocan City North, Metro Manila
• Major Hospital Internship: Veterans Memorial Medical Center (May-
August 2015)
Project 6, Diliman, Quezon City, Metro Manila
• Minor Hospital Internship: Veterans Memorial Medical Center (July –
October 2014)
Project 6, Diliman, Quezon City, Metro Manila
• Mercury Drug Corporation Agora, Navotas City Community Pharmacy
Internship Training (July – October 2013)
• Manila Central University: Filemon D. Tanchoco Partial Scholar 2 nd
Year – 1st Semester , 3rd Year – 1st Semester

SEMINARS ATTENDED

• Implementation of Current Good Manufacturing Practice and

Administrative Order 34, Series of 2014 (December 9, 2015)
Tanchoco Auditorium, Manila Central University, Caloocan City
• This is W.A.R.: Win Against Resistance Seminar (November 27, 2015)
Emilio T. Yap Auditorium, UP College of Pharmacy Manila
• Environmental Stewardship: The Role of Filipino Pharmacists (January
2015)
Tanchoco Auditorium, Manila Central University, Caloocan City
• Good Customer Service through Effective Communication Skills
(September 2013)
Centennial Gymnasium, Manila Central University, Caloocan

PERSONAL DATA

Date of Birth : March 25, 1996 Place of Birth: Manila City

Civil Status : Single Gender : Female
Nationality : Filipino Religion : Roman Catholic
Height : 4’11 Weight : 65 kg

I hereby certify that the above information is true and correct.

LOVELY ANN D. AYAPANA

 69

Len Mariz D. Castro

#072 Timog Rd. Caingin
Meycauayan City Bulacan, Philippines 3020
Mobile no.: +63.926.782.3196
E-mail: lenmarizcastro31@gmail.com

OBJECTIVE
To be able to apply the knowledge and skills to work in such an
environment that will enhance my capabilities to provide care that
optimizes medication therapy, patient-centered ability and to manage
healthcare system resources to improve therapeutic outcomes to make a
highly competent and proficient pharmacist that is a part of the health care
team.

SUMMARY OF QUALIFICATIONS

 Hard worker, quick learner, and ability to assume responsibility

 Proficient in the use of computers
 Demonstrated ability to adapt to new equipment & technology
 Enthusiastic, dependable, self-motivated
EDUCATIONAL ATTAINMENT
Tertiary : Manila Central University
Bachelor of Science in Pharmacy
Caloocan City
2012-2016
Secondary : St. Mary’s College of Meycauayan
Meycauayan, Bulacan
2008-2012
Elementary : St. Mary’s College of Meycauayan
Meycauayan, Bulacan
2002-2008

TRAININGS AND OTHER ATTAINMENTS

 Manufacturing Pharmacy Internship (Minor)

 70

Growrich Manfacturing Inc.

September 2015 – March 2016

 Hospital Pharmacy Internship (Major)

Valenzuela Medical Center
May – October 2015
 Hospital Pharmacy Internship (Minor)
Veterans Memorial Medical Center
July – October 2014
 Community Pharmacy Internship (Minor)
o Watsons Your Personal Store
o July – October 2013
 Filemon D. Tanchoco Partial Scholar
o Manila Central University
o 1st year-2nd semester, 2nd year-1st semester, 3rd year-2nd
semester, 4th year- 1st semester

SEMINARS ATTENDED

 Implementation of Current Good Manufacturing Practice and AO 34,

Series 2014
Filemon D. Tanchoco Auditorium, Manila Central University
December 2015
 This is W.A.R.: Win Against Resistance Seminar
Emilio T. Yap Auditorium, UP College of Pharmacy
November 2015
 Environmental Stewardship: The Role of Filipino Pharmacists
Filemon D. Tanchoco Auditorium, Manila Central University
January 2015
 Good Customer Service through Effective Communication Skills
Centennial Gymnasium, Manila Central University
September 2013
PERSONAL DATA

Date of Birth : March 1, 1996 Place of Birth: Valenzuela City

Civil Status : Single Gender : Female
Nationality : Filipino Religion : Catholic
Height : 5’4” Weight : 60kg

I hereby certify that the above information is true and correct.

 71

LEN MARIZ D. CASTRO

Ronaliz DJ. Ganancias

Block 39 Lot 4 Phase 3 F1 Salay-salay Street
Dagat-dagatan, Caloocan City, Philippines 1400
Mobile no.: +63.926.577.1737
E-mail: ronalizganancias09@gmail.com

OBJECTIVE

To learn the essentials of being a pharmacist and improve my knowledge

and understanding on patient care, further enhance my values toward any
responsibility that I will undertake, develop my talents, broaden my capabilities
and skills to awaken hidden potentials for further enhancement of my qualities as
a pharmacist.

SUMMARY OF QUALIFICATIONS

 Computer literate
 Efficiently handle multiple tasks and projects simultaneously.
 Able to work with minimal supervision and as a cooperative team member.
 Readily assimilate and grasp new methods and information.
 Reliable and trustworthy

EDUCATIONAL ATTAINMENT

College : Manila Central University

Bachelor of Science in Pharmacy
2012 – Present

High School : La Consolacion College- Caloocan City

2008 – 2012

Elementary : Kaunlaran Elementary School- Caloocan City

2007– 2008
 72

TRAININGS AND ATTAINMENTS

 Manufacturing Pharmacy Internship (Minor)

Growrich Manfacturing Inc.
September 2015 – March 2016

 Hospital Pharmacy Internship (Major)

Veterans Memorial Medical Center
May – August 2015
 Hospital Pharmacy Internship (Minor)
Veterans Memorial Medical Center
July – October 2014
 Community Pharmacy Internship (Minor)
Watsons Your Personal Store
July – October 2013
 Half Scholar in Manila Central University

SEMINARS ATTENDED

 Implementation of Current Good Manufacturing Practice and AO 34,

Series 2014
Filemon D. Tanchoco Auditorium, Manila Central University
December 2015
 This is W.A.R.: Win Against Resistance Seminar
Emilio T. Yap Auditorium, UP College of Pharmacy
November 2015
 Environmental Stewardship: The Role of Filipino Pharmacists
Filemon D. Tanchoco Auditorium, Manila Central University
January 2015
 Good Customer Service through Effective Communication Skills
Centennial Gymnasium, Manila Central University
September 2013

PERSONAL DATA

Date of Birth : August 09, 1996 Citizenship : Filipino

Place of Birth: Caloocan City Civil Status : Single
Gender : Female Language Spoken: Filipino and
Weight : 45kg English
Height : 4’11
 73

I hereby certify that the above information is true and correct.

RONALIZ DJ. GANANCIAS