Republic Of The Philippines

Department of Education
Region IV-A
City Schools Division Of Antipolo
District II-E
Antipolo City National Science And Technology High School
Sitio Cabading, Brgy. San Jose, Antipolo City
Tel No. (02) 736-04-52

Bacteriostatic of Pseudomonas
Aeruginosa and Klebsiella
Pneumoniae Screening of
Snakeroot (Rauwolfia
Serpentine) Leaves

Elizabeth DC. Abonio

(X-Ptolemy)

Jan Camille B. Lerios

(X-Ptolemy)

Jeedaiah Zxyldwynn S. Rabe

(X-Leibniz)
[approvalsheet]
Acknowledgements

We would like to acknowledge the efforts of our adviser in this research Mr.
Christian Malayan as he supported us- the researchers through this study, as well as our
former research teacher Mr. Christian Viernes for guiding us from the beginning of
conducting this study until the completion of this paper from discerning and analyzing
the whole study.

We thank Mr. James Letolio Jr. for assisting us with the correct format,
grammatical formation and comments that greatly improved the paper.

We would also like to thank the Department of Science and Technology who
provided us insight and expertise with the results that we gathered that they greatly
assisted in the research, although they may not agree with all of the interpretations
and/or conclusions of this paper.

We would also like to acknowledge the great financial support that Mayor Jun
Ynares III provided, that we used economically in conducting the experimentations in
this research.

We are also grateful for our ever-supportive parents namely Mr. & Mrs. Abonio,
Mr. & Mrs. Lerios and Mr. & Mrs. Rabe for providing us with the other financial
support that was needed as well the never-ending physical, mental and spiritual support
that they’ve given while guiding and boosting us in accomplishing this study.

Last but not the least, the Almighty God who never left us while we were doing
and completing this research study, providing us with the enough wisdom and
knowledge and lend us a long patience just to finish and defend this research study.
Abstract
Chapter 1
1 INTRODUCTION

1.1 Background of the study Page|1

1.2 Significance of the study Page|2
1.3 Statement of the problem Page|3
1.4 Research Questions Page|3
1.5 Scope and Limitations Page|4

Chapter 2
2 REVIEW OF RELATED LITERATURE Page|5-10

Chapter 3
3 METHODOLOGY

3.1 Materials and Procedures Page|1

3.2 Design Page|1
3.3 Conceptual Framework Page|1

CHAPTER 4
4 RESULTS AND DISCUSSIONS

4.1 Pytochemical Screening Page|

4.2 MIC Page|

CHAPTER 5
5 CONCLUSION AND RECOMMENDATIONS

5.1 CONCLUSION Page|

5.2 RECOMMENDATIONS Page|

CHAPTER 6
6 REFERENCES Page|
1: INTRODUCTION

- The rationale of the research being undertaken and the discussion of the general
premise or concept.

Rauwolfia serpentina, the Indian snakeroot or devil pepper, is a species of flower

in the milkweed family Apocynaceae. It is native to the Indian subcontinent and
East Asia. Rauvolfia is a perennial undershrub widely distributed in India in the
sub-Himalayan regions up to 1,000 metres. Rauwolfia serpentina contains dozens
of alkaloids of the indole alkaloid family, including ajmaline, ajmalicine, reserpine,
and serpentine, among others. R. serpentina may cause adverse effects by
interacting with various prescription drugs or via interference with mechanisms of
mental depression or peptic ulcer. The reserpine in R. serpentina is associated with
diverse adverse effects, including vomiting, diarrhea, dizziness, and headache,
anxiety, or hypersensitivity reactions.

1.1 Background of the study

Philippines is said to be a developing country that mainly shelters almost 107

million Filipinos with that, a lot of people are experiencing infections caused by
bacteria it maybe a communicable disease or not. According to the statistical data
that the World Health Organization issued in the year 2016 infectious diseases are
a major cause of death, disability, social and economic turmoil for millions of
people around the world. Respiratory infections like lower respiratory infections,
pneumonia, flu, diarrheal diseases, tuberculosis, etc. kill children and adults not
only in the Philippines but all over the world. Apparently, some microbial infections
are acquired from sick patients with active infections; contaminated water;
unhealthy environment and the food they eat.

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The common bacteria that are the main cause of this infections are Pseudomonas
Aeruginosa and Klebsiella Pneumoniae. Pseudomonas Aeruginosa is tha bacteria that
causes urinary tract infections, respiratory system infections, dermatitis, soft tissue
infections, bacteremia, bone and joint infections, gastrointestinal infections and a
variety of systemic infections, particularly in patients with severe burns and in cancer
and AIDS patients who are immunosuppressed. While Klebsiella Pneumoniae
infections refer to several different types of healthcare-associated infections that are all
caused by the Klebsiella bacteria, including pneumonia; bloodstream infections; wound
or surgical site infections; and meningitis.

However, these infections caused by Pseudomonas Aeruginosa and Klebsiella

Pneumoniae may alleviate through the use of extracted Serpentina. The purpose of this
is not only to test its effect but to have a safe alternative when you can't afford some
medicines to buy.

1.2 Significance of the study

This research study aims to help with the future pharmaceutical experiments against
malignant external otitis, endophthalmitis, endocarditis, meningitis, pneumonia,
and septicemia since those stated diseases were caused by the spreading of the
bacteria.

People that was and will be affected by the bacteria can use this study as basis for
treating the disease/s with safer medicine alternatives when they can’t afford buying
the actual medicine recommended.

If this study will be proven effected then the country will be able to treat diseases
caused by Pseudomonas Aeruginosa and Klebsiella Pneumoniae using safer and
alternative medicine that will be more economically cheaper since it can be found
anywhere, as well as receive benefits from the product since not a lot of Filipinos
can afford buying traditional medicine which cost a lot more.

The researchers’ wants to help the medical field by providing data for further studies
about R. Serpentina. Improving the chemical substance of the medicine used for the
treatment of diseases caused by Pseudomonas Aeruginosa and Klebsiella

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 Pneumoniae that will not only be beneficial for hospitals, but also the people that
will be affected with such virus.

In addition to that, this study will serve as a foundation for future researchers who
will be conducting this type of study and be able to relate to these findings that will
help them complete their research paper.

1.3 Statement of the Problem

Many human illnesses are caused by infection with either bacteria or viruses. Global
health issues have no borders. Communicable diseases are responsible for almost
half of all deaths in countries. Most deaths occur due to infectious processes. It
maybe diarrheal diseases, acute respiratory infections, malaria and measles among
children and AIDS and tuberculosis among adults. Anti-infective medications and
vaccines are available for some but not all diseases or populations. People become
displaced with an increased susceptibility due to lack of clean water, shelter, access
to health services, access to food and major overcrowding. The spread of disease
becomes difficult to control; in natural disasters or conflicts there are different
routes for diseases to cause outbreaks; contaminated water, overcrowding and air-
borne diseases.

1.4 Research Questions

This study aims to seek answers for the following questions:

1. What are the phytochemicals present in Rauvolfia Serpentine Leaves?

2. What is the effect of extracted Rauvolfia Serpentina leaves as applied to:

2.1 Pseudomonas Aeruginosa

2.2 Klebsiella Pneumoniae

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1.5 Scope and Delimitations

The focus of this study is to know the phytochemicals present in Rauwolfia

Serpentina leaves and to test the effectiveness of Rauwolfia Serpentina leaves
against Pseudomonas Aeruginosa and Klebsiella Pneumoniae with the use of MIC.
Most procedures and the study will be done here in Antipolo and in some
laboratories for experimentation.

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2: REVIEW OF RELATED LITERATURE AND STUDIES

- This chapter presents the review of related literature and studies which the
researchers have perused to shed light on the topic under study.

Rauwolfia serpentina

Rauvolfia serpentina L. Benth. Ex Kurz. is an evergreen, woody, glabrous and

perennial shrub with maximum height upto 60 cm. The plant possess tuberous root
with pale brown cork and elliptic to lanceolate or obovate leaves in whorls of three.
The plant belongs to the family Apocynaceae and occurs in habitats of tropical and
subtropical regions. The family includes 50 species, distributed worldwide in the
region of the Himalayas, Indian peninsula, Burma, Indonesia and Sri Lanka and is
indigenous to India, Bangladesh and other regions of Asia. The plant is commonly
known as Sarpagandha, Chandrabagha, Snake root plant, Chotachand, Chandrika
and Harkaya etc. The roots, leaves and juice are of medicinal importance and have
attracted the attention of practitioners of indigenous system of medicine, as it
contain a large number of secondary metabolites (Ncontaining indole alkaloids)
localized mainly in the roots and rhizomes. It has been used in India as a part of the
Ayurvedic medical system for the treatment of various ailments. In Ayurvedic
medicines, the roots of R. serpentina is used as a remedy for curing hypertension,
insomnia, mental agitation, gastrointestinal disorders, excitement, epilepsy,
traumas, anxiety, excitement, schizophrenia, sedative insomnia and insanity. In
Siddha medicine, R. serpentina roots are used for curing hypertension-associated
headache, dizziness, amenorrhea, oligomenorrhea and dysmenorrhea like
abnormalities. According to Rajendran and Agarwal (2007) fruits and seeds have
also been used for its medicinal or ethno botanical purposes by the ethnic tribes of
Virudhungar district Tamil Nadu, India.

Scientists have been working on the phytochemical analysis of the plant due to its
medicinal importance. It has been used as anthelmintic and anti-hypertensive drugs.
It is used as an antidote against snake bite and bites of other poisonous insects. In
diarrhoea, dysentery, cholera, fever, opacity of the cornea and central epilepsy and
ecbolic R. serpentina also played an important role. The plant is known to cure

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various circulatory disorders due to the presence of alkaloids. The root juices or
extract is used to treat liver and abdomen pain,various gastrointestinal disorders and
to expel intestinal worms from the childrens. Mao et al. (2009) have reported the
plant as a function of the ethnobotanical wealth of north east India. The plant also
shows the use by local people of Eastern Ghats, Uttar Pradesh, Karnataka and
Bangladesh against snake bite. The roots and leaf buds are crushed with milk, made
into a paste and used externally on the affected areas. The other diseases such as
pneumonia, malaria, body aches, eczema, burns, menstrual disorders, scabies, skin
cancers, asthma, respiratory problems, eye inflammation, spleen diseases and fever
can also be cured using R. serpentina. The present review work emphasizes on the
potential of R. serpentina as antifungal, anti-inflammatory, antioxidant,
antiproliferative, anticancerous, antidiuretic, antifibrillar, antiarrhythmic,
anticholinergic, antidysentry, antidiarrhoeal antihypotensive, anticontractile,
antidiuretic, sympathomimetic, and tranquillizing agent.

The alkaloids obtained from the root extract acts directly on central nervous system
and thereby reduces blood pressure as compared to other blood-pressure lowering
agents. R. serpentina root is reported to contain 0.7 – 3.0 % of total alkaloids and
about 0.1% of the active principle reserpine which is an indole alkaloid, present in
the root. Hence, root biomass production of this plant could be of economic
importance. On the basis of the structure there are three types of alkaloids namely,
weak basic indole alkaloids, alkaloids of intermediate basicity and strong
anhydronium bases. The various alkaloids identified in Rauvolfia include ajmaline,
ajmalimine, ajmalicine, deserpidine, indobine, indobinine, reserpine, reserpiline,
rescinnamine, rescinnamidine, serpentine, serpentinine and yohimbine etc.
Amongst all, resperine is the principle alkaloid which shows large number of
clinical applications. Along with resperine, yohimbine, serpentine, deserpidine,
ajmalicine and ajmaline are used to treat hypertensionand breast cancer.

Medicinal history of Rauwolfia serpentina

The dried root of Rauvolfia serpentina commonly known as serpentina root, in

Sanskrit as sarpagandha and in Hindi chandrabhaga is one of the most important
drugs used in modem medicine. Once it was a commonly accruing local plants in

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the plains of Darjeeling District but due to ruthless collection from the natural
habitat it has faced endangered condition. This plant is commercial source of
reserpine, an indole alkaloid, which is one of the most important therapeutically
useful drug.

The importance of Rauvolfia serpentina in modern medicine was recognised; in

1952 when Muller succeeded in isolating pharmacodynamic prineiple reserpine in
the roots of the plant which revolutionized the therapeutic use of the drug as
antihypertension and sedative. More than 20 different indole alkaloids have been
isolated so far from this plant and most of the alkaloids are confined to the roots of
the plant. The ;total alkaloid content in the root range from 1.7 to 3.0 percent which
are-mostly concentrated in the bark (about 90%). Alkaloids are also present in
leaves, stem and seeds but not in significant amount as compared to roots. Siddiqui
and Siddiqui (1931, 1935) isolated reserpine and other indole alkaloids at the very
beginning when the importance of Rauvolfia serpentine was known. They were the
pioneer workers in this line of investigation, but they followed cumbrous method.
They collected different solvent parts following the method of fractional separation.
Each fraction contained a mixture of alkaloids and the modern method of
chromatography was not used to separate pure alkaloids very easily. Besides most
of the methodologies so far recorded in connection with the quantitative estimation
of reserpine have been observed to involve costly instruments, which are generally
not available in most of the laboratory. More over all these involved cumbrous
process without the consideration of purification of reserpine. Thus an attempt has
been made to work out an easy and rapid colorimetric method for quantitative
determination of reserpine involving the newly developed methodology for
purification of the natural product.

Pseudomonas Aeruginosa

Pseudomonas aeruginosa (P. aeruginosa) is a common Gram-negative, rod-shaped

bacterium belonging to the bacterial family Pseudomonadaceae. It lives primarily
in water, soil and vegetation. In 1882 Carle Gessard, a chemist and bacteriologist
from Paris, France, discovered P. aeruginosa through an experiment that identified

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this microbe by its water-soluble pigments that turned a blue-green when exposed
to ultra-violet light.

Like other members of the genus, Pseudomonas aeruginosa is a free-living

bacterium, commonly found in soil and water. However, it occurs regularly on the
surfaces of plants and occasionally on the surfaces of animals. Members of the
genus are well known to plant microbiologists because they are one of the few
groups of bacteria that are true pathogens of plants. In fact, Pseudomonas
aeruginosa is occasionally a pathogen of plants. However, Pseudomonas aeruginosa
has become increasingly recognized as an emerging opportunistic pathogen of
clinical relevance. Several different epidemiological studies track its occurrence as
a nosocomial pathogen and indicate that antibiotic resistance is increasing in clinical
isolates.

Pseudomonas aeruginosa is an opportunistic pathogen, meaning that it exploits

some break in the host defenses to initiate an infection. In fact, it is the epitome of
an opportunistic pathogen of humans. The bacterium almost never infects
uncompromised tissues, yet there is hardly any tissue that it cannot infect if the
tissue defenses are compromised in some manner. It causes urinary tract infections,
respiratory system infections, dermatitis, soft tissue infections, bacteremia, bone
and joint infections, gastrointestinal infections and a variety of systemic infections,
particularly in patients with severe burns and in cancer and AIDS patients who are
immunosuppressed. Pseudomonas aeruginosa infection is a serious problem in
patients hospitalized with cancer, cystic fibrosis, and burns. The case fatality rate in
these patients is near 50 percent.

Pseudomonas aeruginosa is measuring 0.5 to 0.8 µm by 1.5 to 3.0 µm. Almost all
strains are motile by means of a single polar flagellum. The bacterium is ubiquitous
in soil and water, and on surfaces in contact with soil or water. Its metabolism is
respiratory and never fermentative, but it will grow in the absence of O2 if NO3 is
available as a respiratory electron acceptor. The typical Pseudomonas bacterium in
nature might be found in a biofilm, attached to some surface or substrate, or in a
planktonic form, as a unicellular organism, actively swimming by means of its
flagellum. Pseudomonas is one of the most vigorous, fast-swimming bacteria seen
in hay infusions and pond water samples.

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Klebsiella pneumoniae

In 1882, Carl Friedlander first described Klebsiella pneumoniae as an encapsulated

bacillus after isolating the bacterium from the lungs of those who had died from
pneumonia. Originally named Friedlander's bacillus, it was not until 1886 when the
bacterium garnered the name Klebsiella. Klebsiella pneumoniae is a gram-negative,
encapsulated, non-motile bacterium that is found in the environment and has been
associated with pneumonia in the alcoholic and diabetic patient population. The
bacterium typically colonizes human mucosal surfaces of the oropharynx and
gastrointestinal (GI) tract. Once the bacterium enters the body, it can display high
degrees of virulence and antibiotic resistance. Today, K. pneumoniae pneumonia is
considered the most common cause of hospital-acquired pneumonia in the United
States and the organism accounts for 3% to 8% of all nosocomial bacterial
infections.

Klebsiella pneumoniae is part of the Enterobacteriaceae family and is described as

a gram-negative, encapsulate and non-motile bacterium. Virulence of the bacterium
is provided by a wide array of factors that can lead to infection and antibiotic
resistance. The polysaccharide capsule of the organism is the most important
virulence factor and allows the bacteria to evade opsonophagocytosis and serum
killing by the host organism. To date, 77 different capsular types have been studied,
and those Klebsiella species without a capsule tend to be less virulent. A second
virulence factor is lipopolysaccharides that coat the outer surface of a gram-negative
bacteria. The sensing of lipopolysaccharides release an inflammatory cascade in the
host organism and has been shown to be a major culprit of the sequela in sepsis and
septic shock. Another virulence factor, fimbriae, allows the organism to attach itself
to host cells. Siderophores are another virulence factor that is needed by the
organism to cause infection in hosts. Siderophores acquire iron from the host to
allow propagation of the infecting organism.

Klebsiella pneumoniae is one of a handful of bacteria that is now experiencing a

high rate of antibiotic resistance secondary to alterations in the core genome of the
organism. Alexander Fleming first discovered resistance to beta-lactam antibiotics
in 1929 in gram-negative organisms. Since that time, K. pneumoniae has been well
studied and has been shown to produce a beta-lactamase that causes hydrolysis of

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the beta-lactam ring in antibiotics. Extended beta-lactamase (ESBL) K. pneumoniae
was seen in Europe in 1983 and the United States in 1989. ESBLs can hydrolyze
oxyimino cephalosporins rending third-generation cephalosporins ineffective
against treatment. Due to this resistance, carbapenems became a treatment option
for ESBL. However, of the 9000 infections reported to the Centers for Disease
Control and Prevention (CDC) due to carbapenem-resistant Enterobacteriaceae in
2013, approximately 80% were due to K. pneumoniae. Carbapenem resistance has
been linked to an up-regulation in efflux pumps, an alteration of the outer
membrane, and increases production in ESBL enzymes in the organism.

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3: METHODOLOGY

- This chapter presents the review of related literature and studies which the
researchers have perused to shed light on the topic under study.

Page | 11