The Efficacy of Serpentina

(Rauvolfia serpentina) and Aloe vera

(Aloe barbadensis) ointment for
Atopic dermatitis

RESEARCH PROPOSAL
In Partial Fulfillment of Requirements In Research I
Satya Chantal M. Morales
Researcher
Mrs. Rosita Pascua Romero
Project Adviser
(June 2022)
 The Efficacy of Serpentina (Rauvolfia

serpentine) and Aloe vera (Aloe barbadensis)

ointment for Atopic dermatitis

An Investigatory Project for S. Y. 2021-2022

Life Science Category

SATYA CHANTAL M. MORALES

Researcher

ROSITA PASCUA ROMERO

Research Adviser
Researcher Individual: Satya Chantal M. Morales Category: Life Science

School: Alaminos City National High School Level: School Level

School Address: San Jose Drive Poblacion, Alaminos City Pangasinan Title

Project: The Efficacy of Serpentina (Rauvolfia serpentina) and Aloe vera (Aloe barbadensis)

ointment for Atopic dermatitis Research Adviser: Mrs. Rosita Pascua Romero

A. The Problem

Skin, it’s the most bare and uncovered part of our body, due to this, skin is bare in

various microorganisms and bacteria, and because of that skin, conditions and infections are

normal, inescapable, and boundless. An example of a very usual skin conditions are

dermatitis, and one of the common and popular type is Atopic dermatitis.

Atopic dermatitis or most commonly known as Eczema is a condition that makes your

skin bothersome, rough, dry, and to redden. This dermatitis is a very common skin condition.

This condition usually appears on the stage of infancy to adulthood, but eczema can occur in

any people with any age. Eczema damages the skin’s boundary work, and because of this,

your skin becomes more delicate and more inclined to infections and dryness.

The problem is many people around the world suffers from this chronic condition..

Furthermore eczema have no permanent cure or treatment.

However, there are treatments for it that can ease itching, irritation, and hassle of

eczema for the mean time.

 One of the main causes of eczema are bacterias. Staphlococcus aureus (Staph. aureus),

it’s the bacteria that’s most usually charged with the secondary infection of eczema.

Serpentina (Rauvolfia serpetina) and Aloe vera (Aloe barbadensis) are rich in our

surroundings, Furthermore, these herbals are known to have an highly antibacterial effect

against Staphylococcus aureus. Moreover, Aloe vera has a soothing properties and is known

for its cooling effect, this would help relieve and soothe the symptoms of eczema. And that’s

the reason why the researcher chose to construct a study to determine the combined effect of

Serpentina leaves extract and Aloe vera gel in treatment of eczema.

Rational of the Study

Skin is the most exposed and uncovered part of our body, for the reason that this body

part covers whole of our frame. Furthermore, skin is used to microorganisms and bacteria,

and because of that, skin is prone to skin conditions and infections. Dermatitis are skin

conditions and infections that are familiar to the skin. Atopic dermatitis – eczema, is an

example of a popular type of dermatitis

Eczema is the most usual skin condition and a common dermatitis among all types of

dermatitis. Eczema predominantly affects young children. It appears during the time of

infancy, childhood, or even adulthood, even so, eczema can occur in people in any age.

This skin condition gives the person, skin issues – irritation, redness, bumpy, rough and

itch on the simulated skin. Because eczema destroys the skin barrier which causes your skin

to become more delicate and more inclined to infections and dryness.

 Several people around the world suffers from the symptoms of eczema, and it is known

to have no permanent treatment although there are certain medicines and treatment that may

help prevent future breakouts, flare up, ease irritation and other causes of eczema.

The issue is, some medicines and treatments for eczema like ointments and others have

chemicals that may cause more irritation, infection and dryness to the skin. Take note that

Eczema damages the skin barrier which affects your skin and make it more sensitive and

more vulnerable for irritations and dryness.

One of the main causes of Eczema are bacteria, and the most usually charged with the

secondary infection of atopic dermatitis is Staphylococcus aureus (Staph. aureus).

That’s why the researcher found two plants that have highly antibacterial effect for

Staph. aureus and are abundant to their community which are Serpentina (Rauvolfia

serpetina) and Aloe vera (Aloe barbadensis), this two plants pushes the researcher to conduct

a study to produce a natural and more effective ointment against eczema and to determine the

efficacy of the combined plants against Atopic dermatitis.

Furthermore, the findings of this study may aid the search for a more effective and

natural product for treating Atopic dermatitis – eczema.

B. Statement of the Problem

This study entitled “The Efficacy of Serpentina (Rauvolfia serpentina) and Aloe

vera (Aloe barbadensis) ointment for Atopic dermatitis” will plan to initiate a new

ointment for the treatment of the skin condition.

Specifically, it will seek to answer this following questions:

1. Is it possible to use Serpentina leaves and Aloe vera gel in the remedial of eczema

symptoms and flare up?

2. Is there a significant difference in the remedial of eczema symptoms and flare up

before and after the application of treatment concentration of Serpentina leaves and

Aloe vera gel?

3. Is there a significant difference among the treatment concentration of Serpentina

leaves and Aloe vera gel in terms of effectiveness in the remedial of eczema

symptoms and flare up?

C. Statement of the Hypothesis

This study will be posited by the following hypotheses:

Alternative Hypothesis

1. It is possible to use Serpentina leaves and Aloe vera gel in the remedial of eczema

symptoms and flare up.

 2. There is a significant difference in the remedial of eczema symptoms and flare up

before and after the application of treatment concentration of Serpentina leaves and

Aloe vera gel.

3. There is a significant difference among the treatment concentration of Serpentina

leaves and Aloe vera gel in terms of effectiveness in the remedial of eczema

symptoms and flare up.

D. Methodology

1) Research Design

The research design that will be used is Complete Randomized Design. The

research will use five (5) treatments (T0, T1, T2, T3, T4, T5) in three (3) replicates.

2) Materials/Instruments

In conducting the study, the researcher will need to prepare different instruments

and materials and will be defined operationally.

For the safety of the researchers, they will use personal protective equipment –

which includes safety gear, gloves, laboratory gown, facemask – will be used. Safety

gear will be used in order to protect the researcher while conducting the experiment.

Gloves will be needed for covering and protecting the researcher’s hand while doing

the experiment. Laboratory gown, this will be used for covering the researcher’s body.

Face mask will be also used to avoid inhalation of the chemicals and harmful

organisms that’ll be used in the experiment by covering the mouth and nose of the
researcher. Safety goggles will be used for protecting the researcher’s eyes and vision

from the chemicals that may harm the eyes While doing the experiment, the researcher

will be guided and will have close supervision of their research adviser and other

qualified and professional scientists.

For the raw materials, 400 grams of Serpentina leaves and 400 grams of Aloe

vera gel will be used in the study.

To prepare the materials needed for the experimentation, different laboratory

apparatuses and equipments will be prepared. The researcher will use basin to hold

the serpentina leaves and aloe vera while it is being washed and cleaned. To hold the

serpentine leaves and aloe vera (separate) with ethanol placed for extraction and

phytochemical testing, Erlenmeyer flask will be used. The graduated cylinder will be

used to measure the volume of ethanol used for extraction and phytochemical testing.

To measure the mass of the serpentine leaves and aloe vera gel, triple beam balance

will be needed.

The bacteria S. aureus will be cultured and collected at a qualified laboratory.

Different utensils will be used in order to conduct the experiment. Chopping

board will be used to support the knife when the leaves and aloe vera will be cut into

smaller pieces. Knife will be used to cut the serpentina leaves and aloe vera. Strainer

will be used in raising the wet leaves and aloe vera and lastly, a tray for holding the

serpentina leaves and aloe vera gel when cleaning and draining them.

3) Procedure

a. Safety Precautions
 Safety precautions will be monitored during the collection of materials and

experimentation. Safety glove, face masks, laboratory gown, and safety goggles

will be used. Additionally, the researcher will be guided by their research adviser

during the whole duration of the experiment.

b. Collection/Gathering of Materials

1000 grams of serpentina leaves and 1000 grams of aloe vera will be

gathered from the researcher’s community’s marketplace at Suki and Nepo Mart

at Alaminos City. Coconut oil and distilled water will be also bought at the CSI

Supermarket at Alaminos City.

Laboratory apparatuses will be provided by the Alaminos Laboratory and

VMUF college of Pharmacy Laboratory, example of these are weighing scales,

triple beam balance, beaker, test tubes, etc.

c. Preparation of Serpentina Leaves Extract and Aloe vera gel

The gathered leaves and aloe vera will be cleaned through the tap water. The

rotten and unwanted parts will be remove and will be properly disposed. While

the good and healthy leaves and aloe vera will be drained and set aside. After the

cleaning, the leaves will be cut into small strips while the aloe vera gel will be

extracted from the plant itself and will be cut into small cube pieces, it will be

weighed using triple balance beam balance. Also, the coconut oil will be set aside

for future usage.

 One hundred (100) grams of serpentina leaves and one hundred (100) grams

of aloe vera gel will be placed in to separate Erlenmeyer flask. One hundred fifty

(150) ml of ethanol will be added in each flask in which the leaves will be soaked

for twenty-four (24) hours. The extracts will be separated using the filter paper

and set aside for phytochemical screening and laboratory testing.

d. Phytochemical Testing

The serpentina leaves and aloe vera gel will be brought to the College of

Pharmacy Laboratory of Virgen Milagrosa University Foundation at San Carlos

City, Pangasinan for phytochemical screening to determine the presence of

saponin, flavonoids, alkaloids, and other phytochemicals.

The following procedures will be formed by an authorized medical

technologist of VMUF for the phytochemical screening.

1. Screening for Alkanoids

Seventy (70) ml of the ethanol extract will be evaporated to dryness on a

steam bath. The residue will be dissolved in 70 ml to 15 ml of HCL, aided by

warming under steam bath for 1 to 2 min. It will be cooled, filtered, and adjusted

to a volume of only 70 ml by washing the residue of the filter paper with

sufficient quantity of 1% HCL. A few drops of grains of the powdered sodium

chloride will be added to the filtrate; it was shaken, and filtered. One (1) ml of

the filtrate will be placed into each small test tube, to the 1st test tube, 3 drops of

modified Mayer’s reagent were added; to the next 3 drops of winger’s reagent

(iodine and potassium iodine TS), then 3 drops of valser’s reagent and to the last
test tube , three drops of bouchardat’s reagent. The positive indication of

screening for alkaloids will be the production of precipitate.

2. Screeing of Flavanoids

The deflated residue (Form C) will be dissolved into 30 ml of 50% ethanol

and was filtered. One (1) to 2 ml of filtrate will be placed in 3 separate test tubes.

3A. Bate-Smith-Metcalf Test

Test tube #1 will be treated with 0.5 ml of concentrated HCL wand warmed

in water bath for about5 min and observed for color change within an hour. The

positive indication of Bate-Smith-Metcalf Test will be color changing to red-

violet.

3B. Willstatter-“Cyanidin” Test

Test tube #2 will be treated with 0.5 ml of HCL and 3-4 pieces of

magnesium turings were added on it. Any color changes within 10 minutes will be

observed. The positive indication for Will-“Cyaniding” Test will be changing to

green, red, etc.

3. Screening for Steroid (Cardio active) Glycosides

4A. Presence of unsaturated sterols (Lieberman-Burchard test) result of section D

was used.

4B. Presence of unsaturated lactones since the following three tests involved color

reaction it was necessary to run concurrent test with control sample.

 a. Kedde’s Reaction

Five (5) ml of Kedde’s reagent (2gm of 3,5-dinitro-benzonic acid in 100

ml of ethanol) will be added to 5 ml of ethanol extract in an evaporating dish,

and the solution will be mixed well with a glass stirring rod. Two (2) ml of 1N

sodium hydroxide will be added to mixture and observed for color reaction. The

positive indication of Kedde’s Reaction will be a purple ring color.

4C. Keller Killiani test (2-deoxysugars)

About 10 ml of ethanol extract will be placed in evaporating dish and

dried on steam bath. 3 ml of ferric chloride reagent (mix 0.3 ml of 10% ferric

chloride solution with 50 ml of glacial acetic acid) will be added, stirred to mix

well, and then transferred to a small test tube held at a 45 degree angle, 1 ml of

concentrated sulfuric acid will be added by allowing it to run down inside the

wall of the test tube. Avoid shaking or agitation of the test tube. The positive

indication will be a purple ring.

1. Screening for Saponins

Ten (10) ml of distilled water will be added into two separate test tubes, test

tube #1 containing 2 ml of ethanol extract. Both tubes will be shaken vigorously for

30 seconds. It will be observed over a period of 30 minutes. The positive indication

for Saponins will be the formation of honeycomb.

2. Screening for Tannins and Phenolic compounds

About 100 ml of the plant extracts will be taken and evaporated to incipient

dryness over a steam bath. It will be cooled to room temperature; the residue will be

extracted with 25 ml of hot distilled water. The cooled temperature will be

 centrifuged for several minutes and the upper half from each test tube used will be

decanted 3-4 of 10% sodium chloride solution will be added to salt out undesirable

constituent through precipitation. Precipitates will be filtered off and the filtrate will

be divided into 3.

6A. Gelatin Test

The drops of 1% gelation solution will be added to the test tube #1 and

will be observed if there will be any formation of precipitate.

6B. Gelatin Black Test

Three (3) drops of gelatin salt reagent (1% gelatin solution, 10% sodium

chloride) will be added to the test tube #2 and will be observed if there will be any

formation of precipitate. The positive indication of Gelatin Block test will be

blue-black or Green-black color.

3. Screening for Anthraquinones

7A. Borntrager’s Test

Five (5) ml of the plant extract will be taken and evaporated to dryness

over a steam bath. About 5-10 ml of petroleum ether will be added to defeat the

residue and the 50 ml of distilled water will be added to the defeated residue,

mixed well and then filtered into small separator funnel. About 10 ml of benzene

will be added, mixed well and allowed 2 phases to separate. The aqueous layer

(bottom) will be drained out and the benzene phase (upper layer) as transferred to

a test tube. 5ml of ammonia T.S will be added and observed for color change in

the benzene layer. The positive indication of Borntrager’s Test will be cherry red

or pink solution.
 7B. Modified Borntrager’s Test

Three-tenths (0.3) ml of leaves powder will be heated with 10 ml of 0.5 N

of potassium hydroxide and 1 ml of dilute hydrogen peroxide for 10 min. It will

be allowed to cool and the solution will be filtered. The acidified solution will be

transferred to a small separator funnel and partitioned with 10 ml of benzene. The

benzene phase will be filtered and 5 ml will be transferred to a test tube

containing 2.5 ml of ammonia T.S. It will be observed of any color change. The

positive indication of Modified Borntrager’s Test will be cherry red or pink

solution.

4. Screening for Anthrones

2g of Anthrone is dissolved in 1 liter of concentrated H2SO4. The freshly

prepared reagent should be used for the test. Procedure and observations-Add 0.5-1

ml of the test solution to about 2ml of anthrone reagent and mix thoroughly. Observe

whether the color changes to bluish green.

a. Plant Identification

The collected plant specimens will be brought to the Bureau of Plant Industry (BPI),

Malate, Manila. The identification of the plant specimens will be achieved by the positive

comparison of morphological characteristics with authenticated, in – house plant

reference material and an authoritative technical reference description.

b. Preparation of Serpentina Leaves and Aloe vera Extracts Treatment Concentration

Five hundred (500) grams of serpentina leaves and 500 grams of aloe vera will be

extracted for the preparation of the treatments. The plant extracts will be obtained by

maceration method. The raw materials will be soaked in ethanol for three days. The plant

extract will be obtained by separating the raw material using filter paper. Then the

mixture will be kept on water bath at 60 oC until ¼ of the extract will be evaporated. The

obtained extracts will be put into an Erlenmeyer flask and will be properly labeled.

The following concentration of treatments will be used in the study:

Table 1. Different Treatment Concentration of Serpentina Leaves and Aloe vera gel

Extracts

Amount of Serpentina Amount of Aloe vera gel

TREATMENTS
Leaves Extract Extract
T0 Negative (Untreated / Water)
T1 75% (7.5 ml) 25% (2.5 ml)

T2 50 % (5.0 ml) 50% (5.0 ml)

T3 25% (2.5 ml) 75% (7.5 ml)

T4 Positive (Penicillin)

c. Collect1ion and Preparation of the Culture Media

The test organism, Staphylococcus aureus, will be gathered and tested at College of

Pharmacy Laboratory Virgen Milagrosa University Foundation.

d. Pre-Treatment Evaluation

The antibacterial test will be carried out using Agar Disc – Diffusion Technique.

Brain Heart Infusion agar will be used for the bacteria. The 48 hour old – broth culture of

the isolate (S. aureus) will be inoculated using the spread plate technique.

Circular paper discs (6 mm in diameter) will be punctured our from a Whatman No. 1

filter paper. The said discs will be sterilized using 70% alcohol and oven dried.

e. Application of Treatments

In applying the treatments, the sterilized discs will be added to the aqueous extracts of

serpentina leaves and aloe vera separately and will be allowed to absorb the extracts. The

discs bearing the extracts will be placed on the inoculated plates containing the bacterial

isolates. Then, the plates will be incubated for 48 hours. All five treatments will be

undergoing the same process except when applying to the sterilized disks. For T0,

distilled water will be applied in the sterilized discs. And commercial antibiotic will be

applied in the sterilized discs for T4.

f. Post-Treatment Evaluation

After 48 hours, the incubated plates will be observed for the growth and inhibition of

the bacterial isolates. This will be done to observe the presence of a clear zone around the

Serpentina leaves and Aloe vera gel extracts bearing discs. The clear zone will be the
 indication of the inhibition of the organism and sensitivity to the plant extract of the

pathogen.

The record of the extent of pathogen inhibition will be obtained by measuring the

diameter of the inhibition zone in each case using a ruler. The results will be based on the

Standard Zone of Inhibition.

Table 2. Standard Zone of Inhibition

Zone of Inhibition (mm) Percentage Equivalent

12 mm and below 70% Resistant

12.1 – 14 mm 75% Intermediate
14.1 – 16 mm 80% Susceptible
16.1 – 18 mm 85% Susceptible
18.1 – 24 mm 90% Susceptible
24.1 mm and above 95% Susceptible

g. Disposal of Experimental subjects and treatments

Immediately after the experimentation, all the cultured materials will be

decontaminated in the autoclave for thirty (30) minutes at 120o C.

h. Statistical Analysis

The data recorded will be subjected to data analysis in order to arrive at a conclusion. T –

test will be used to determine the significant difference before and after the application of

treatments. And Analysis of Variance (ANOVA) will also be used to determine the

statistical difference and if there is a significant difference among the treatments.

k. Level of Significance

The significant difference among the treatments will be tested at alpha = 0.05 level

of significance. If the F-tabulated value is greater than the critical value, then the null

hypotheses will be accepted. F- tabulated value is less than the critical value, and then the

null hypotheses will not be accepted.

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