Kamayo Phytomedicines: Morphology, Phytochemistry & Bioactivity

Morphological, Phytochemical, and Antibacterial Screenings of Kamayo

Phytomedicines: Towards an Ethnopharmacobotanical Remedy
for Staphylococcus Infections

Hanzel B. Caringal, Jogie L. Elcanto, Jerisna Marie Y. Alvarez

Tagbina National High School

ABSTRACT

This study aims to source out novel drugs against human pathogens. It aimed to
investigate the bioactive compound of Kamayo phytomedicines through Phytochemical
Screening and to test its bioactivity as antibiotic against Staphylococcus aureus
BIOTECH 1582 and Methicillin-Resistant Staphylococcus aureus (MRSA). Crude
extracts were prepared by soaking the homogenized plant stems in 95% ethanol. The
samples were then filtered and the filtrate was concentrated at 10,000 ppm using rotary
evaporation. The crude extract was then applied to S. aureus using filter paper disc
diffusion assay. Four (4) set-ups were made (Lunas Bagon stem extract, Himag Bagon
stem extract, and Vancomycin and distilled water for comparison). The set-ups were
made in triplicates. Zones of Inhibition (mm) were measured and computed in terms of
microbial index and compared with the standard antibacterial drug, Vancomycin discs.
Phytochemical test was also done to ascertain the presence of bioactive compound.
Phytochemical analysis revealed that Lunas Bagon contained alkaloids and
saponins while Himag Bagon contained flavonoids, saponins and tannins. Data analysis
revealed that the Phytomedicinal extracts were able to inhibit the growth of
Staphylococcus aureus and MRSA. Compared with Lunas Bagon, Himag Bagon extracts
showed more potency at inhibiting the growth of the Superbug. However, Vancomycin,
the commercial drug, still exhibited bigger zone of inhibition than the plant extracts. This
can be accounted for by the fact that the phytomedicinal extracts are still crude and thus
contain several impurities while the positive control is in its pure state (1 mg/ml).
The project reports the potential of Kamayo Phytomedicines, particularly Himag
Bagon, as antibiotic source against drug-resistant Staphylococcus infections.
Furthermore, it confirms the folkloric beliefs among Kamayo in Surigao del Sur that their
Phytomedicines --- Lunas Bagon and Himag Bagon --- possess antibacterial properties.

Keywords:
Ethnopharmacobotany, Staphylococcus aureus, MRSA, Superbug, Antibiotic Resistance

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 Kamayo Phytomedicines: Morphology, Phytochemistry & Bioactivity

INTRODUCTION

1.1 Background of the Study

The world is in a state of global antibiotic crisis. Antibiotic resistance is

jeopardizing feasibility of medical practices. Multiple antibiotic classes have lost potency

to major bacterial strains, among them are gram-positive species (Rossolini et al, 2014).

Methicillin-Resistant Staphylococcus aureus (MRSA), a drug-resistant gram-positive

bacteria, is now responsible for more deaths than AIDS in the United States; it also

represents more infections than meningitis, bacterial pneumonia and flesh-eating strep

put together (Gutierrez, 2008). Patients who are infected with Staph infections (SA) have

a substantially increased death rate. The World Health Organization (2005) cited that

patients infected with Staphylococcus aureus would likely die (by 29 percent) within 30

days, this death rate is 49 percent higher than that among patients with other bacterial

infections.

In response to this important medical challenge, the search for new antibiotics

must be intensified and new bacterial targets must be sought and used for screening

(Barbosa et al, 2002). With the continuing widespread of the disease and the

overwhelmingly developing resistance of bacteria to antibiotics, it is apparent that a novel

drug must be sought out.

A way out of reducing antibiotic resistance and adverse effects on host is the

employment of antibiotic resistance inhibitors of plant origin (Nostro et al, 2000). Gruyal,

Roasario and Palmes (2014) cited that medicinal plants and herbs have been used for

many centuries as a source of people’s drugs for the treatment and prevention of diseases,

disorders and the promotion of good health and still provide the first line of primary

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 Kamayo Phytomedicines: Morphology, Phytochemistry & Bioactivity

health-care even in the present age to major segments of the population worldwide. In

fact, today, around 40% of the drugs in pharmacies globally are natural products (Lahlou,

2013). It is therefore obvious that plants play an important role in medicine even today.

In recent years, work on ethnobotanical knowledge worldwide has increased

especially in some parts of Europe, Asia, and Africa. India, a country with a rich culture

and traditional knowledge, had contributed a major share of the world’s ethnobotanical

work (Pieroni et al, 2003). In the Philippines, ethnobotanical documents are relatively

few, with some focusing on well known indigenous groups including the

Pinatubo Negritoes and their use of plant resources; the Tasadays in Mindanao, who have

been the subject of various studies; the Itawes of Cagayan and the Ibaloi of Benguet

province and their utilization of forest resources (Balangcod & Balangcod, 2009). In

Surigao del Sur, the Kamayos (group of people with distinct dialect and cultural roots)

often marvel at the effectiveness of plants health wise. For instance, Lunas Bagon and

Himag Bagon, the local plants growing on forest thickets, are frequently collected to treat

human ailments such as diabetes, skin diseases, snake bites, high fever, among others.

These plants are often referred to as miracle plant in their native pharmacopeia.

To contribute solution to the worsening antibiotic crisis, the researchers sought

the experiential wisdom of Kamayos. In this paper, Kamayo Phytomedicines --- Lunas

Bagon and Himag Bagon --- are studied in the hope of sourcing out novel compounds to

arrest the growing cases of death caused by Staph infections. Scientific names of the

plants were not established by the researchers however, thus the plant description,

phytochemistry and bioactivity are investigated.

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 Kamayo Phytomedicines: Morphology, Phytochemistry & Bioactivity

Generally, the objective of this study is to describe the morphological

characteristics of Kamayo Phytomedicines, to identify their phytochemical components

and to screen their bioactivity against Staphylococcus aureus and Methicillin-Resistant

Staphylococcus aureus (MRSA).

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 Kamayo Phytomedicines: Morphology, Phytochemistry & Bioactivity

1.2 Objectives of the Study

The main objective of this research is to study the morphology, phytochemicals,

and bioactivity of Kamayo Phytomedicines (Lunas Bagon and Himag Bagon) against

Staphylococcus aureus BIOTECH 1582 and Methicillin-Resistant Staphylococcus aureus

(MRSA).

Specifically, the study aims to:

1. Describe the morphological characteristics of Lunas Bagon and Himag Bagon;

2. Determine the phytochemical characteristics of Lunas Bagon and Himag Bagon;

3. Determine if Lunas Bagon and Himag Bagon extracts inhibit the growth of

Staphylococcus aureus and MRSA; and

4. Determine if there is a significant difference in the zones of inhibition (ZOI) of

different extracts against Staphylococcus aureus and MRSA.

1. 3 Research Problem

The main problem of the study is to determine the morphological, phytochemical

and biological characteristics of Lunas Bagon and Himag Bagon.

Specifically, this study aimed to answer the following questions:

1. What are the morphological characteristics of Lunas Bagon and Himag Bagon?

2. What are the phytochemical characteristics of Lunas Bagon and Himag Bagon?

3. Can Lunas Bagon and Himag Bagon extracts inhibit the growth of

Staphylococcus aureus and MRSA?

4. Is there a significant difference in the ZOI of different extracts against

Staphylococcus aureus and MRSA?

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 Kamayo Phytomedicines: Morphology, Phytochemistry & Bioactivity

1.4 Statement of the Null Hypothesis

Based on the foregoing research problems, the following null hypotheses were

formulated:

1. Lunas Bagon and Himag Bagon extracts cannot inhibit the growth of

Staphylococcus aureus and MRSA; and

2. There is no significant difference in the zone of inhibition (ZOI) of different

extracts against Staphylococcus aureus and MRSA.

1.5 Conceptual Framework

Figure 1.1 Schematic Diagram of the study Morphological, Phytochemical and

Antibacterial Screenings of Kamayo Phytomedicines: Towards an
Ethnopharmacobotanical Remedy for Staph Infection
The study is based on the concept that Lunas Bagon and Himag Bagon (plants

used by Kamayo for ethnopharmacobotanical uses) exhibit activity against

microorganisms. This can be accounted for the presence of a wide array of secondary

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 Kamayo Phytomedicines: Morphology, Phytochemistry & Bioactivity

metabolites in its system. This however is yet to be proven, thus, this study was

conceptualized. In this study, the extracts from the stem of Lunas Bagon and Himag

Bagon together with the positive control (Vancomycin) and the negative control (distilled

water) were tested against Staphylococcus aureus and MRSA. Then the zones of

inhibition were measured using digital calliper. Moreover, the morphological

characteristics of the plant which include the kind of stem, venation, phyllotaxy, and root

system were identified. Phytochemical screening was also conducted to determine its

phytochemical components.

1.5 Scope and Delimitation of the Study

The study “Morphological, Phytochemical and Antibacterial Screenings of

Kamayo Phytomedicines: Towards an Ethnopharmacobotanical Remedy for

Staphylococcus Infections” is focused on the description of the Morphological

Characteristics, Phytochemicals, and Bioactivity of Kamayo Phytomedicine (Lunas

Bagon and Himag Bagon) extracts. For bioactivity, the extracts were tested against

Staphylococcus aureus and MRSA, a bacterium that commonly causes skin infections.

Lunas Bagon and Himag Bagon were collected from Tambis, Barobo, Surigao del

Sur (8°33’36’’N, 126°1’56’’E). The crude extraction of the bioactive compounds and the

bioassay against S. aureus were conducted at the University of the Immaculate

Conception-Science Resource Center (UIC-SRC) in Davao City. The procedure used for

the conduct of the antimicrobial screening was based from the book titled “A Guidebook

to Plant Screening: Phytochemical and Biological”. The phytochemical screening was

done at UIC-SRC.

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1.6 Definition of Terms

Bioactive Substance: Substance having an effect on, or causing a reaction in, the living

animal or plant tissue exposed to it. Here, the extract is bioactive if it can inhibit the

growth of the test organisms.

Bioactivity: Specific effect on, or a reaction in, living animal or plant tissue upon

exposure to a substance. In this paper, it means the effect of the extract against

Staphylococcus aureus and MRSA.

Phytochemicals: Also called phytonutrient, any of various bioactive chemical

compounds in plants, such as antioxidants. Here, it refers to the chemical components of

the plant sample.

Ethnopharmacobotanical Remedy: developing a cure for human illnesses by

conducting scientific experiments using plants used by natives in folkloric medicine

Kamayo: The minority group speaking the Kamayo dialect in Barobo, Surigao del Sur

Kamayo Phytomedicines: Folkloric plant-based medicines used by the Kamayo to treat

various human illnesses.

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1.7 Significance of the Study

Since ancient times, people use the gifts of nature found in the surrounding

environment to treat different illnesses. Medicinal plants are of great significance, and the

utilization of various plants in folk medicine has a very long history. There are about

35,000 – 70,000 plants used in folk and scientific medicine worldwide (Hamilton, 2004).

As of 2004, at least 200,000 phytochemicals have been characterized, but this is still

thought to represent only a small percentage of phytochemicals that exist in nature

(Santos, et al., 2004). This further indicates the importance of drugs of herbal origin for

folk and modern medicine.

Due to increased interest in medicinal plants from different countries, the issue of

preservation of the medically important plants is important. Habitat destruction and

environmental pollution are factors that strongly affect medicinal plants in the wild. The

issue on environmental protection however is subject to national issues relating to general

concept of environmental preservation. For conservation to proceed, it is important to

identify the plant species that are the most threatened due to over collection in the wild.

These species must receive the highest prioritization for preservation. To this end, it is

important to identify the plants with folkloric uses and test it for possible medicinal

value. Identifying the plant and its phytochemicals as well as bioactivities is important for

proper indexing and referencing.

On the other hand, the search for antibiotic drugs for mutant bacteria is one of the

major problems in the field of medicine. The prevalence of the people getting sick from

disease causing bacteria has increased year by year due to continuous poor sanitation,

pollution and bacterial drug resistance. Taking vancomycin is the only cure to fight

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 Kamayo Phytomedicines: Morphology, Phytochemistry & Bioactivity

mutant bacteria like MRSA, but recent studies suggests that even to these drugs,

“superbugs” are developing resistance.

Many antibiotics are available in the market today. Nevertheless, bacteria are

getting resistant to most of these drugs. Due to over-usage of antibiotics, the staph

bacterium for example has become resistant to the commonly used broad-spectrum

antibiotics that had previously been used to treat staph infections; and since 2005 more

than 94,000 MRSA cases have been reported in the United States, and an estimated

19,000 MRSA deaths have occurred (Scatter, 2009). Even E. Coli and Salmonella strains

are becoming resistant to broad spectrum antibiotic. Hence, there is a need to source out

novel compounds which might arrest the growing cases of death caused by bacterial

infections.

As a part of the program to search for novel antimicrobial compounds from

Philippine plants used by ethnic groups, this project is conceptualized to use the crude

extract from the stem of Kamayo Phytomedicines (Lunas Bagon and Himag Bagon)

against Staphylococcus aureus and MRSA. Furthermore, it aims to determine the

morphology and phytochemistry of the phytomedicinal extracts.

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 Kamayo Phytomedicines: Morphology, Phytochemistry & Bioactivity

1.8 Literature Review

The synthesis of large numbers of antibiotics over the past three decades has

caused complacency about the threat of bacterial resistance. Bacteria have become

resistant to antimicrobial agents as a result of chromosomal changes or the exchange of

genetic material via plasmids and transposons. Streptococcus pneumoniae, Streptococcus

pyogenes, and staphylococci, organisms that cause respiratory and cutaneous infections,

are now resistant to virtually all of the older antibiotics (Neu, 1992).

The Journal of the American Medical Association reported that antibiotic-

resistant Staphylococcus aureus (SA) is now responsible for more deaths in the United

States than AIDS. Researchers found out that 31.8 out of 100,000 U.S. residents are

being infected by SA each year, leading to 94,360 infections and 18,650 deaths across the

country. This is in comparison to 12,500 deaths from AIDS in 2005, and represents more

infections than meningitis, bacterial pneumonia and flesh-eating strep put together

(Gutierrez, 2008).

Just as the United States, SA infections have become prevalent in Asian nations.

Heterointermediately resistant to vancomycin (hVISA) strains of Methicillin-resistant

Staphylococcus aureus (MRSA) were found to thrive in India, South Korea, Japan, the

Philippines, Singapore, Thailand, and Vietnam (Song et al, 2004). In 2011, Song et al

further reported that MRSA infections in the community have been increasing in Asian

countries. MRSA clones have spread between the community and hospitals as well as

between countries.

A way out of reducing antibiotic resistance and adverse effects on host is the

employment of antibiotic resistance inhibitors of plant origin (Nostro et al, 2000). Gruyal,

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 Kamayo Phytomedicines: Morphology, Phytochemistry & Bioactivity

Roasario and Palmes (2014) cited that medicinal plants and herbs have been used for

many centuries as a source of people’s drugs for the treatment and prevention of diseases,

disorders and the promotion of good health and still provide the first line of primary

health-care even in the present age to major segments of the population worldwide. In

fact, today, around 40% of the drugs in pharmacies globally are natural products (Lahlou,

2013). It is therefore obvious that plants play an important role in medicine even today.

The following readings and articles were utilized to discuss the

ethnopharmacobotanical concepts and its possible contribution to the search for plant-

based novel drugs against antibiotic resistant bacterial strains.

Ethnopharmacobotany and the Kamayos

Schultes (1962) in Uddin (2016) cited that ethnobotany is the study of

relationships between people of a primitive society and their plant environment.

Ethnopharmacobotany, on the other hand, is the scientific study of plant-based medicines

used by different ethnic or cultural groups (Random House Inc., 2016). Even before the

creation of the term “Ethnopharmacobotany”, cultural groups have long been using plants

to treat various ailments. Balick and Cox (1996) cited that there exist an ancient

relationship between plants and people; ethnobotanical systems and herbal medicines as

therapeutic agents are of a paramount importance in addressing health problems of

traditional communities.

In recent years, work on ethnobotanical knowledge worldwide has increased

especially in some parts of Europe, Asia, and Africa. India, a country with a rich culture

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 Kamayo Phytomedicines: Morphology, Phytochemistry & Bioactivity

and traditional knowledge, had contributed a major share of the world’s ethnobotanical

work (Pieroni et al, 2003).

In the Philippines, ethnobotanical documents are relatively few, with some

focusing on well known indigenous groups including the Pinatubo Negritoes and their

use of plant resources; the Tasadays in Mindanao, who have been the subject of various

studies; the Itawes of Cagayan and the Ibaloi of Benguet province and their utilization of

forest resources (Balangcod & Balangcod, 2009).

In Surigao del Sur, the study of Blasco, de Guzman and Alejandro (2014)

confirmed that plants are still valued for their medicinal purposes by the natives of the

province, given the fact that they have difficult access to traditional modern health

facilities such as government clinics and hospitals. Gruyal, del Rosario, and Palmes

(2014) added that most of the ailments treated through ethnomedicines in Surigao del Sur

are cough, diarrhea, stomach ache, and kidney infection and that leaves are the most used

part of the plants. Both of the mentioned studies are done in the Northern part of Surigao

del Sur, and both are limited to the documentation of the folkloric uses of the plants;

hence, there is still a need for laboratory screenings of the said plants. In fact, Blasco, de

Guzman and Alejandro (2014) recommended that pharmacological and phytochemical

screenings of the plant species must be done.

In this paper, plant-based ethnomedicines (phytomedicines) of the Kamayos in the

Southern part of Surigao del Sur is explored. Beyond the folkloric uses of the plants,

phytochemical and antibacterial properties of Kamayo phytomedicines is studied.

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 Kamayo Phytomedicines: Morphology, Phytochemistry & Bioactivity

The Kamayo are minority groups concentrated in Southern Surigao del Sur ---

Lingig, Bislig, Hinatuan, Barobo, Lianga, Marihatag, and San Agustin. A scattered

population is also found in Cateel and Baganga in Davao Oriental. The dialect spoken by

Kamayo is related linguistically to the Tausug and Butuanon, and belongs to the Meso

and central Philippine family of languages. However, the people speak dialects that vary

from town to town. The disparities of the dialects are due to the interchange of

communication between the Kamayo settlers and the native Manobo (National

Commission for the Culture and the Arts, 2016).

The Kamayo, just as the Surigaonon of the North, often marvel at the

effectiveness of plants health wise. Among all phytomedicines they use, they are most

especially fond of Lunas Bagon and Himag Bagon. These local plants grow on forest

thickets, and are frequently collected to treat human ailments such as diabetes, skin

diseases, snake bites, high fever, and acute infections. These plants are often referred to

as miracle plant in their native pharmacopeia.

To contribute solution to the worsening antibiotic crisis, the researchers sought

the experiential wisdom of the Kamayo. In this paper, Kamayo Phytomedicines --- Lunas

Bagon and Himag Bagon --- are studied in the hope of sourcing out novel compounds to

arrest the growing cases of death caused by Staphylococcus infections. Scientific names

of the plants were not established by the researchers however, thus the plant description,

phytochemistry and bioactivity are investigated.

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 Kamayo Phytomedicines: Morphology, Phytochemistry & Bioactivity

The Specimen (Lunas Bagon)

Lunas Bagon in its Natural Habitat

Lunas Bagon is a vine used by the Kamayo in Surigao del Sur to treat various

diseases. It is believed to cure skin diseases, snake bites, high fever, and several various

illnesses. Lunas Bagon is also known as Kanaog by the natives in reference to the vine

plant as compared to the Lunas Bundok which is also referred to as Lunas but is a tree

instead of vine.

Lunas Bagon is a woody vine, with thick, rope-like stems adapted to twining

through forest thicket. The stems are rounded with thick cork-like bark, grayish-brown,

branched and up to 2-10 meters in height. The leaves tend to be oblanceolate. They are

arranged in alternate position in the stem, have smooth margins, and vary from bright

green to grey-green. Inflorescences (in the form of spikes) are terminal, opposite the

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 Kamayo Phytomedicines: Morphology, Phytochemistry & Bioactivity

leaves, or located in the axils. Flowers of the plant are not usually seen but they are said

to be bisexual, with no perianth, and is subtended by a peltate bract.

The Specimen (Himag Bagon)

Himag Bagon

Himag Bagon is a rambling climber. Leaves are pinnate and 30 to 50 centimeters long.

Leaves have netted venation. This plant is abundant in thickets along streams, in

secondary forests at low and medium altitudes. Kamayo herbalists claimed that the plant

can be used to cure wound and skin diseases; they also believed that this plant is

anticoagulant. Its juice is edible too and is said to regulate the blood sugar level.

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Staphylococcus aureus: the Test Organism

Staphylococcus bacteria are extremely common in the environment; is usually not

a problem to healthy individuals. Historically, staph infections begin only after the

individual has suffered a skin break or open wound. Recently, due to institutional overuse

of antibiotics, strains of Staphylococcus aureus have developed drug resistance (DEP,

2006). If an individual has a weak immune response to the bacteria, SA infection can

result even with no apparent open wound. The symptoms can range from skin boils to

necrotizing fascilitis, popularly known as flesh-eating disease. S. aureus most commonly

colonizes the anterior nares (the nostrils) although the respiratory tract, open wounds,

intravenous catheters and urinary tract are also potential sites of infection.

Patients who are infected with SA have a substantially increased death rate. WHO

(2005) cited that a patient infected with SA is five times more likely to die than other

patients. There is a death rate of 29 percent within 30 days among patients infected with

Staphylococcus aureus. The death rate among SA patients is 49 percent higher than that

among patients with other bacterial infections.

Methicillin Resistant Staphylococcus aureus (MRSA) causes as many as 20% of

S. aureus infections in populations that use intravenous drugs. These out-of-hospital

strains of MRSA, now designated as community-acquired, methicillin-resistant S. aureus,

or CA-MRSA, are not only difficult to treat but are especially virulent. CA-MRSA

apparently did not evolve de-novo in the community, but represents a hybrid between

MRSA which escaped from the hospital environment and the once easily treatable

community organisms. Most of the hybrid strains also acquired a virulence factor which

makes their infections invade more aggressively, resulting in deep tissue infections

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 Kamayo Phytomedicines: Morphology, Phytochemistry & Bioactivity

following minor scrapes and cuts, and many cases of fatal pneumonia as well (Jonsson et

al, 2003).

Methicillin-resistant Staphylococcus aureus is a specific strain of the

Staphylococcus aureus bacterium that has developed antibiotic resistance to all

penicillins, including methicillin and other narrow-spectrum B-lactamase-resistant

penicillin antibiotics. MRSA was first discovered in the UK in 1961 and is now

widespread, particularly in the hospital setting where it is commonly termed a superbug

(Rosato et al., 2003).

Although MRSA has traditionally been seen as a hospital-associated infection,

there is currently an epidemic of community-acquired MRSA in the USA. The

abbreviations CA-MRSA (community-associated MRSA) and HA-MRSA (hospital-

associated MRSA) are now commonly seen in the literature (Dyke, 2003).

Interestingly, MRSA cases chiefly emanate from institutions such as hospitals and

gymnasiums. Many professional athletes have developed MRSA infections from

exposure in stadium locker rooms. On June 22, 2006, the US Centers for Disease Control

and Prevention issued an alert regarding an outbreak in tattoo parlors, after 54 customers

developed MRSA from unlicensed artists.

Some strains of S. aureus over-express B-lactamase and appear to be resistant to

oxacillin and rarely, methicillin despite being mecA-negative. They have slightly raised

minimum inhibitory concentrations (MICs) and may thus be described as “minimally

resistant”. Other strains express modified PBPs and exhibit varying degress of B-lactam

antibiotic resistance.

Vancomycin and teicoplanin are glycopeptides antibiotics used to treat MRSA

infections. Teicoplanin is a structural congener of vancomycin that has a similar activity

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 Kamayo Phytomedicines: Morphology, Phytochemistry & Bioactivity

spectrum but a longer half-life (t1/2). Both drugs have low oral absorption thus are

administered intravenously for systemic infections, with the exception of

pseudomembranous colitis where vancomycin can be given by mouth for this GI tract

infection. Up to 53 million people are thought to carry MRSA. Scientists estimate that

around 2 billion people, some 25-30 percent of the worlds’ population, have a form of S.

aureus bacteria (WHO, 2005).

As of early 2005, the number of deaths in the United Kingdom attributed to

MRSA has been estimated by various sources to lie in the area of 3000 per year. The

Staphylococcus bacteria account for almost half of all UK hospital infections. The issue

of MRSA infections in hospitals has recently been a major political issue in the UK,

playing a significant role in the debates over health policy in the general election held in

that country in 2005 (Bumketvogt et al., 2005).

Secondary Plant Metabolites

Alkaloids

Alkaloids are one of the most diverse groups of secondary metabolites in living

organisms and have an array of structure types, biosynthetic pathways, and

pharmacological activities (Roberts and Wink, 1998). Alkaloid is generally recognized as

having strong antimicrobial, antibacterial and antifungal biological properties. Although

before, these secondary plant metabolites are found to be only abundant in plants,

nowadays, it has been known that they are also present in insects, animals, marine

invertebrates and other microorganisms (Roberts et al., 1998).

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 Kamayo Phytomedicines: Morphology, Phytochemistry & Bioactivity

Flavonoids

Dr. Buhler and Dr. Miranda (2000) noted that flavonoids are ubiquitous. This

means that it is found almost anywhere. They can be found in fruits, vegetables and

certain beverages. Aside from that, flavonoids have also become remarkable because of

its diverse beneficial biochemical and antioxidant effects. Moreover, aside from its

antioxidative-activity, it also serve for its free-radical scavenging capacity, coronary heart

disease preventive action, and anticancer activity, while some flavonoids exhibit potential

for anti-human immunodeficiency virus infections (Yao et al., 2004). Furthermore,

flavonoids also function as Vitamin C support, cell structures protector, anti-

inflammatory and antibiotic agent (Metljan, 2014).

Steroids

According to an American youth organization, steroids are popular for its physical

enhancing effects. In fact, these are often abused by some people for this purpose.

Accordingly, these chemicals are taken more than 10 to 100 times than what is prescribed

by the physician (Santos et al., 2004). Consequently, the abusive use of these drugs are

also governed by serious side effects such as male pattern baldness, permanently

deepened voice, breast shrinking, and irregularities in the menstrual cycle (Ibid).

Tannins

Tannin is a large group having a great structural diversity and is usually divided

into two basic groups: the hydrolysable type and the condensed type (Dharmananda,

2003). These are greatly known in the business industry because of its use in tanning the

animal hide into leather. However, according to Ashok et al (2012), apart from tanning,

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 Kamayo Phytomedicines: Morphology, Phytochemistry & Bioactivity

tannins are also used in dyeing, photography, refining beer and wine as well as astringent

in medicines. Significantly, tannins form a vital element of tea. Moreover, these

substances are also called as anti-nutrient because it lower the absorption level of some

material into the body. That is why, substances that contains them such as coffee and tea

are not advised to be taken in regularly and in large amounts (Ibid).

Saponins

Like the other secondary metabolites, saponins are also found as a diverse group

which is widely distributed in the plant kingdom. It is characterized by their structure

containing a triterpene or steroid aglycone and one or more sugar chains (Ustundag et al.,

2007). These substances has not only found its way to pharmaceuticals but in the

cosmetics as well. Due to its ability to create foam, ancient people used its roots as soap.

Moreover, saponins are said to be the substance that give some plants their bitter taste,

thus reducing the feed intake of non-ruminant animals. Furthermore, some plants are also

causing food poisoning to grazing animals due to toxicity of their saponin content. But on

the lighter side, there are types of these substance like those which are found in oats and

spinach can increase and accelerate the absorption of calcium and silicon in the body,

thus assisting digestion (Cornell University, 2014).

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METHODOLOGY

2.1 Research Design

An Experimental Group-Two Control Groups Design was employed. Lunas

Bagon and Himag Bagon extracts were used against Staphylococcus aureus and MRSA

in vitro as shown in the table below.

Staphylococcus aureus MRSA

Treatments
R1 R2 R3 R4 R1 R2 R3 R4
Lunas Bagon Stem Extract x1 x2 x3 x4 x1 x2 x3 x4
Himag Bagon Stem Extract x1 x2 x3 x4 x1 x2 x3 x4
+Vancomycin x1 x2 x3 x4 x1 x2 x3 x4
-Distilled Water x1 x2 x3 x4 x1 x2 x3 x4
Legend:

Replicates: Test Microorganisms:

R1=replicate 1 Staphylococcus aureus BIOTECH 1582
R2=replicate 2 Methicillin-Resistant Staphylococcus aureus BIOTECH 10378
R3=replicate 3

x=Zone of Inhibition Samples: Lunas Bagon stem and Himag Bagon stem

The study used Staphylococcus aureus and MRSA as test organisms in the

antimicrobial screening for novel drugs from Kamayo Phytomedicines (Lunas Bagon and

Himag Bagon). Four treatments were made: Lunas Bagon Stem Extract, Himag Bagon

Stem Extract, positive antibacterial control (Vancomycin) and negative control (distilled

water). Each set-up was done in triplicates.

Furthermore, it screened the phytochemical component of the plant.

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 Kamayo Phytomedicines: Morphology, Phytochemistry & Bioactivity

2.2 Procedure

A. Collection of Samples

Prior to the collection of plant samples in Tambis, Barobo, Surigao del Sur

(8°33’36’’N, 126°1’56’’E), the researchers sought permission for sampling from the

Community Environment and Natural Resources (CENRO) – Lianga, Surigao del Sur

(See Appendix).

Kamayo herbalist from the locality was tapped to help the researchers in gathering

the sample. Five kilograms (5 kg) each of Lunas Bagon and Himag Bagon stem was

collected. The leaves were then detached from the stem and each sample was cleaned

with tap water and air-dried. The air-dried stems were cut into small pieces for crude

extraction and antibacterial screening.

B. Morphological Characterization of Plant Samples

The plant morphology was investigated to identify the plants. Technical

assistance was also sought from an Ecosystems Specialist in DENR-Timber Resources

and Research Center in Bislig City. The following morphological characteristics were

observed for reference: Leaf Shape, Leaf Color, Leaf Venation (vein patterns in the

leaves), phylotaxi (arrangement of the leaves in the stem), and other morphological

characteristics.

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 Kamayo Phytomedicines: Morphology, Phytochemistry & Bioactivity

C. Request for MRSA culture

The culture of Methicillin-Resistant Staphylococcus aureus was purchased from

the Philippine National Collection of Microorganisms (PNCM), University of the

Philippines-Los Baños BIOTECH Department.

D. Preparation of Plant Crude Extract

Five hundred grams of the air-dried samples were soaked in 750 mL 95% Ethanol

for 48 hours. The soaked samples were filtered using Whatman #41 filter paper and the

filtrate was subjected to rotary evaporator. The extract was then concentrated to

1 mg/mL (10,000 ppm) in distilled water for evaluations.

E. Preliminary Screening for Antibacterial Activity

E. 1 Preparation of Culture Media

One hundred fifty millilitre (150 mL) Mueller-Hinton agar (MHA, Himedia) was

prepared. It was sterilized in an autoclave at 15 psi for 15 minutes; then cooled at 45°C

and poured in the Petri dish (10 mL per dish). This serves as the base agar for the

bioassay. A Top Agar was also prepared the same way but with only 10% Agar. Top

Agar was used to inoculate bacteria.

E.2 Antimicrobial Assay

The antibacterial assay was done using filter paper disc diffusion assay. For the

preliminary test, Staphylococcus aureus was used as test organism.

E.2.1 Reviving Test Organism

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 Kamayo Phytomedicines: Morphology, Phytochemistry & Bioactivity

A loopful of S. aureus pure culture from the stock was transferred to the 8 mL

sterile nutrient broth (NB). The test tube was incubated at 37 degree Celsius for 24 hours.

A 24-hour culture was used for the assay.

E.2.2 Inoculation of Test Organism for Bioassay

Ten microliters (μL) of S. aureus from the Broth Culture wad inoculated into each

of the flask containing Top Agar (MHA, 10% agar). The test organism was aseptically

swabbed into the solidified nutrient agar by streaking the swab over the entire surface of

the agar plate three times. The plates were rotated at 60 degrees after each application to

ensure an even distribution of the inoculum on the surface of the medium. The swabbed

plates were left to stand for 5 minutes.

E.2.3 Paper Disc Diffusion Method

Filter Paper Disc Diffusion Technique was used for the bioassay. Using flamed forceps,

the paper disc was picked out and immersed into the animal extract. The paper discs were

implanted to the Petri-dishes by pressing them down gently on the seeded agar plate. The

Petri dishes were incubated for 24 hours. Each treatment was done in triplicates.

E.2.4 Reading of the Zones of Inhibition.

After the incubation period, the zones of inhibition (ZOI) around the filter paper

discs were measured in millimetre (mm) using digital vernier caliper.

F. Phytochemical Screening

The Phytochemical analysis was done using the standard procedure. Tests on

Alkaloids (using Mayer’s and Wagner’s reagents), steroids (Keller-Kiliami Test and

Leiberman-Burchard Test), flavonoids (Bate-Smith and Metcalf Test), and Tanning

(Gelatin Test). Phytochemical testing was done at UIC-SRC in Davao City.

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 Kamayo Phytomedicines: Morphology, Phytochemistry & Bioactivity

G. Decontamination and Disposal of Microbes

All materials and laboratory apparatuses that come in contact with the test

organisms were autoclaved at 121°C for 30-40 minutes. After which, the decontaminated

objects were left to cool; then, they were washed thoroughly with soap and water. The

nutrient media were discarded in the designated waste disposal area. The working zone

was disinfected with chlorox.

H. Data Analysis

The data were recorded and analysed using SPSS v. 16.

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 Kamayo Phytomedicines: Morphology, Phytochemistry & Bioactivity

RESULTS AND DISCUSSION

The data gathered in the experimentation are hereby presented, analysed, and

statistically interpreted. Graphical presentations of data are also employed.

Morphological Characteristics of Kamayo Phytomedicines

Figure 3.1 Photograph showing the Morphological Characteristics of Lunas Bagon

Lunas Bagon has woody stem which reaches from 2-10 meters in length. It has

adventitious root system which allows it to cling on trees and shrubs in the forest thicket.

Arranged in alternate pattern, with deep green color foliage, the leaf has simple margin,

hairy texture and has netted venation. Morphologically, it resembles Betel Piper but it has

oblanceolate leaves as compared with much common Betel Piper which has Obovate

shape. Based on initial morphological characteristics, Lunas Bagon is identified as Piper

sp. This initial finding is not confirmed though, as the National Museum needed to see its

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 Kamayo Phytomedicines: Morphology, Phytochemistry & Bioactivity

flowers and seeds for its proper taxonomic classification, but its seeds and flowers are

very rarely seen.

Figure 3.2 Photograph showing the Morphological Characteristics of Himag Bagon

Himag Bagon is a rambling climber. Leaves are pinnate and 30 to 50 centimeters

long. Leaves have netted venation. This plant is abundant in thickets along streams, in

secondary forests at low and medium altitudes. Kamayo herbalists claimed that the plant

can be used to cure wound and skin diseases; they also believed that this plant is

anticoagulant. Its juice is edible too and is said to regulate the blood sugar level. Based on

its morphological characteristics, particularly its abundant white secretions, the plant

likely belongs to Euphorbiaceae Family. This initial taxonomic finding is not confirmed

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 Kamayo Phytomedicines: Morphology, Phytochemistry & Bioactivity

though, as the DENR and the National Museum needed to see its flowers and seeds, but

its seeds and flowers are very rarely seen.

Phytochemical Analysis of Lunas Bagon and Himag Bagon

*copy of the table results for Phytochemical Analysis of Lunas Bagon and Himag Bagon

As observed in the phytochemical screening, Lunas Bagon extracts have

alkaloids and saponins; Himag Bagon has flavonoids, saponins, and tannins.

Alkaloids are bioactive against bacterial and fungal pathogens while flavonoids

and hydrolysable tannins function as antioxidant (Yao et al., 2004). Saponins give

substances ability to create foam, it can also increase and accelerate the absorption of

calcium and silicon in the body, thus assisting digestion (Cornell University, 2014).

Tannins, on the other hand, are used as astringent in medicine (Ashok et al., 2012). The

presence of these secondary metabolites suggests the plants’ usefulness as medicine.

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 Kamayo Phytomedicines: Morphology, Phytochemistry & Bioactivity

Microbial Assay against Staphylococcus aureus and MRSA

Lunas Bagon Himag Bagon Vancomycin Distilled Water

Ave. ZOI (mm) of S. aureus 12.44 13.56 32.00 6.00
Ave. ZOI (mm) of MRSA 6.00 14.22 23.67 6.00

Figure 3. Average zone of inhibition (mm) of S. aureus and MRSA

The graph above shows that Himag Bagon stem extract has higher average zone

of inhibition for S. aureus (13.56 mm) and MRSA (14.22 mm) as compared with the ZOI

of Lunas Bagon stem extracts (12.44 mm for S. aureus and 6.00 mm for MRSA). This

may be attributed to the presence of more phytochemicals in Himag Bagon than in Lunas

Bagon as indicated by the Phytochemical Analysis result.

When compared with the positive control, the result suggests that the plant

extracts has smaller average ZOI as compared with vancomycin. This is expected

because the plant crude extracts could also contain other impurities while vancomycin is

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 Kamayo Phytomedicines: Morphology, Phytochemistry & Bioactivity

in its pure state (at 1mg/mL). Compared with the negative control, the extracts showed

more antibacterial potency especially Himag Bagon .

To test if the perceived difference in the ZOI is significant, ANOVA was

performed as shown in the following table.

ANOVA and Post Hoc Test for ZOI of S. aureus

ANOVA for ZOI of Staphylococcus aureus

Average ZOI Statistical Test
Treatment
(mm) (α = 0.05)
Lunas Bagon 6.00a
Himag Bagon 13.33a 0.000
+Vancomycin 32.00b
- Distilled Water 6.00 c
Note: Means with the same letter superscript within a column are
not statistically different from each other

Table 3.1 ANOVA of the Zones of Inhibitions

Based on the table above, the p-value (0.000) is less than the level of significance

(α = 0.05). The result suggests that there is a significant difference in the ZOI among

different treatments (Lunas Bagon extracts, Himag Bagon extracts, Vancomycin, and

Distilled Water). Therefore, the null hypothesis stating that there is no significant

difference in the ZOI is rejected. This means that the different treatments have different

bioactivity against the test pathogen (Staphylococcus aureus). To test which of the

extracts is more potent against S. aureus, Post Hoc analysis was conducted (as indicated

by the letter superscripts of the means of ZOI in the table above). The table shows that

Lunas Bagon and Himag Bagon have similar potency at inhibiting bacterial growth and

has higher ZOI than the negative control (distilled water). The table also shows that

vancomycin, the positive control, has the highest capacity of inhibiting bacterial growth.

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 Kamayo Phytomedicines: Morphology, Phytochemistry & Bioactivity

This, again, is expected because the crude extract may also contain other impurities while

vancomycin is in its pure state (at 1mg/mL).

Nevertheless, the extracts still exhibited bioactivity against the test organism.

ANOVA and Post Hoc Test for ZOI of MRSA

ANOVA for ZOI of Methicillin-Resistant S. aureus

Average ZOI Statistical Test
Treatment
(mm) (α = 0.05)
Lunas Bagon 6.00a
Himag Bagon 14.22b 0.000
+Vancomycin 23.67c
- Distilled Water 6.00a
Note: Means with the same letter superscript within a column are
not statistically different from each other

Analysis of variance revealed that there is a significant difference in the ZOI of

different treatments against Methicillin-Resitant Staphylococcus aureus (p-value is

0.000). To test which of the extracts differ in antibacterial potency, Post Hoc Test was

run. It was found out that Lunas Bagon has similar ZOI with distilled water, hence is not

usable for fighting against antibiotic-resistant Staph infections. Nevertheless, Himag

Bagon showed more potency against MRSA; this suggests that it can be a novel source of

bioactive compound for superbug infections. However, vancomycin, the positive control,

still exhibited higher ZOI than Himag Bagon. This, again, is expected because Himag

Bagon is still crude while vancomycin is in its pure state.

The result showed potential for Himag Bagon as source of novel compound for

fighting against Staph infections caused by drug-resistant strain of S. aureus.

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 Kamayo Phytomedicines: Morphology, Phytochemistry & Bioactivity

CONCLUSIONS

Based on the results of the experiment performed and from statistical analysis, the

following conclusions were drawn:

1. Secondary metabolites are present in Kamayo phytomedicines. Lunas Bagon has

alkaloids and saponins while Himag Bagon has flavonoids, saponins, and tannins.

2. For Staphylococcus aureus, Lunas Bagon and Himag Bagon extracts showed

similar potency at inhibiting the bacterial growth. The negative control have the

least ZOI while vancomycin had the highest zone of inhibition.

3. For Methicillin-Resistant S. aureus, Himag Bagon showed more potency at

inhibiting the growth of the drug-resistant bacteria when it is compared with

Lunas Bagon and distilled water. Vancomycin still exhibited the highest ZOI for

MRSA.

Conclusively, this paper reports the potential of Kamayo Phytomedicines,

particularly Himag Bagon, as antibiotic source against drug-resistant Staph infections.

Furthermore, it confirms the folkloric beliefs among Kamayo in Surigao del Sur that their

Phytomedicines --- Lunas Bagon and Himag Bagon --- possess antibacterial properties.

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 Kamayo Phytomedicines: Morphology, Phytochemistry & Bioactivity

RECOMMENDATIONS:

The following recommendations were formulated for future references:

1. Conduct Minimum Inhibitory Concentration (MIC) Test for Lunas Bagon and

Himag Bagon.

2. Isolate and identify the bioactive compounds of Lunas Bagon and Himag Bagon

3. Conduct Cytotoxicity and Genotoxicity assay

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 Kamayo Phytomedicines: Morphology, Phytochemistry & Bioactivity

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7

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PICTORIAL

Lunas Bagon Himag Bagon

Homogenizing of the Samples Soaking of the Samples in 95% Ethanol

Weighing of the Samples Soaking of the Samples in 95% Ethanol

Soaked Samples

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Kamayo Phytomedicines: Morphology, Phytochemistry & Bioactivity

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 Kamayo Phytomedicines: Morphology, Phytochemistry & Bioactivity

Vancomycin Discs

Plant Crude Ethanolic Extracts

Blank Discs immersed in Ethanolic Extracts

40