THE ANTIBACTERIAL ACTIVITY OF CYMODOCEA SERRULATA LEAF EXTRACTS AGAINST

ESCHERICHIA COLI

By

Katrina Mae S. Nachor

Kyrene Gail Rogando

A Thesis Presented to the

Faculty of the Department of

Natural Sciences College of

Science and Engineering

Ateneo de Naga University

In Partial Fulfillment of requirements for

the degree of

Bachelor of Science in

Biology

2019-2020
 INTRODUCTION

1.1Background of the Study

Much of the world’s population uses herbal medicine globally as their primary

health care choice (Winston 2012). Herbal medicines are naturally occurring plant

derived substances that are used to treat illnesses within local or regional healing

practices. These products are complex mixtures of organic chemicals that may come

from any raw processed part of a plant. Herbal medicine has been practiced for

thousand of years it continues to be of use in modern, western world. World Health

Organization (WHO) recently estimated that 80% of people worldwide would rely on

herbal medicines for some part of their primary health care (“Herbal Medicine.”

Miami Acupuncture, 2000 miamiacupuncture.com/services/herbal-medicine.)

Herbal medicine also called botanical medicine, uses the plants seeds, roots,

leaves, bark or flowers for medicinal purposes. The biological effects of these plants

have beneficial effects. The plant is either sold raw or as extracts, where its macerated

with water, alcohol or other solvent to extract some of the chemicals (Herb List |

Herbactive Health Clinic, www.herbactive.co.uk/daily-nutritional-herbal-powder-

herblist.php)

A variety of herbal medicines and chemicals are also prepared from seagrasses.

Despite their name, seagrasses are actually not grass at all as they do flower like land

plants and seagrass produce oxygen (EPA, Environmental Protection Agency) The depth

at which seagrass are found limited by water clarity, which determines the amount of

light reaching the plant. Light is required for the plants to make food through
photosynthesis (Florida Keys National Marine Sanctuary). Seagrass is also one of the

most productive ecosystems in the shallow marine environment that have a vital part

of life at the sea.

Existence of seagrass in the coastal areas provides substantial contributions to

nutrient cycling of coastal waters and marine environment in general. Seagrass

meadows have high biological productivity, compared to other ecosystems (Azkab

2006). They also function as primary producers, recycling nutrients, sediment traps and

barriers to erosion (Tomasick et al., 1997 & Nobi et al., 2012) Seagrass are the classic

ecosystems and high productive habitat because it is a habitat for many types of biota

such as macro algae, sponges, coral various types of sessile invertebrates that an

essential source of food and income for coastal inhabitants (Duffy 2006). Seagrass

grow both vertically and horizontally. Their blades reach upwards and their roots down

and sideways. Seagrass species come in many different shapes and sizes (Florida’s

Marine Environment, pg. 260)

Seagrasses range from species with long flat blades that look like ribbons to fern or

paddle-shaped leaves, cylindrical or spaghetti base or branching shoots. The 72 species

of seagrasses are commonly divided into for main groups: Zosteraceae,

Hydrocharitaceae, Posidoniaceae and Cymodoceae (Seagrass Ecology by M. Hemming

and C.M. Duarte)

Cymodocea serrulata occurs in the lower intertidal zone, in sheltered localities on

coarse coral sand, sand mud, or mud with coral rubble substrates. The plant grows
together with Thalassiahemprichii, Enhalusacorroides, Halophila ovalis and

Syringodiumisoetifolium. Cymodocea serrulata is abstract in places where there is

freshwater influence. In Albay, Southern Luzon, Philippines the plant was collected from

boulders near the month of stream, growing together with Halodule univernis.

Cymodocea serrulata is common in tropical and subtropical regions. C. serrulata is a

long ribbon-like leaves with blunt, rounded tips that have serrations (these are

sometimes very tiny). The leaf scars around the upright stem are not continuous. It has

thick rhizomes (underground stems). The leaf sheaths around the leaf are flattened.

Sometimes seen with reddish bands. (Ria Tan 2008)

There is a report on the Antibacterial, antioxidant and anti-inflammatory of

Cymodocea serrulata. Through this study the researcher expect a significant anti-

bacterial effect on the bacteria used. The antibacterial and inhibition activities of the

Cymodocea serrulata can contribute for a new approach of the herbal leaves for

treatment for antibacterial and a normal substitute for synthetic aids.

1.3. Objectives of the Study

 To determine the antibacterial activity of Cymodocea serrulata leaf extracts against

Escherichia coli
 To determine the zones of inhibition (ZOI) of E. coli at different concentrations of the
leaf extracts of Cymodocea serrulata namely: (25%, 50%, 75% and 100% of
concentrations)
 To determine the significant difference among the four concentrations namely: (25%,
50%, 75% & 100%)
 To compare between the most higher concentration from commercial medicine
 MIC (Bactericidal or Bacterious Tatic) or nareretired ang growth of bacteria
1.4. Research Hypothesis

 Extracts of Cymodocea serrulata is effective in inhibiting growth of S. aureus

 Cymodocea serrulata as a treatment for bacterial inhibiting bacteria and has

a high potential to serve a s treatment for open wounds

1.5. Significance of the Study

Many people will benefit in this research findings and one of them would be those

people who can’t afford branded medicine and rely on traditional treatments. Some area

within the country is left behind in terms of health care mostly because some of these are

remote areas and are very hard to reach. Moreover, some of these people can’t afford

expensive medicine in the treatment of wounds, and thus is susceptible to risk of

infections. It will help the people financially because herbal plants tend to be inexpensive

compared to drugs. On the other hand, people who prefer organic medicine will benefit

from this because not only is the medicine cheap and affordable, but it is also organic thus

has lower risk of side effects from the use of the treatment. In addition, the proposed study

can be used as reference in treating open wounds using Cymodocea serrulata as an

alternative. Future researchers can also use the study as a basis for further researches on

herbal plants as topical treatment for open wounds. On the greater whole, this study will be

helpful to the pharmaceutical and industrial manufacturing companies. This will offer more

job opportunities for the people that could help them in the longer run.
1.6. Scope and Delimitation of the Study

This study focused mainly on the determination of the antibacterial property of

Cymodocea serrulata leaf extracts. Collection and preparation of materials is done for

preliminary test for Cymodocea serrulata is conducted to prove its presence. Qualitative,

chemical, physical analysis, antibacterial screening with the use of some solvents to test or

against some bacteria is use to determine the presence and activity of the Cymodocea

serrulata extract.

LITERATURE

REVIEW

2.1. Herbal Medicine

Herbal medicine has a long history in the treatment of several kinds of disease (Holm et

al., 1998). Their use for the treatment of disease has been practised by man for many

years and is still being widely practised even today (Kokwaro, 1993). For many years,

people have developed a store of empirical information concerning the therapeutic values

of local plants before orthodox medical practice appeared. Through periods of trial, error,

and success, these herbalists and their apprentices have accumulated a large body of

knowledge about medicinal plants. According to Iwu et al. (1999) the first generation of

plant drugs were usually simple botanicals employed in more or less their crude form.

Several effective medicines used in their natural state were selected as therapeutic agents

based on empirical study of their application by traditional societies from different parts of
the world. Following the industrial revolution, a second generation of plant drugs emerged

based on scientific processing of the plant extracts to isolate ″their active constituents″.

Plant materials remain an important component in combating serious diseases in the

world; for the therapeutic approach to several pathologies. Interest in medicinal plants has

been overwhelming in the recent times especially as an important source of

medication/health care. Currently, the global market for medicinal plants has been

estimated to be around US $62 billion and the demand is growing rapidly (Indian Council

of Medical Research, 2003). It is globally recognized that medicinal plants play a

significant role in providing health benefits to human beings. The World Health

Organization (2000) has estimated that 80 % of the inhabitants of the world rely mainly on

traditional medicines for their primary health care needs, and it may be presumed that a

major part of traditional healing involves the use of plant extracts or their active principles.

2.1.1. Herbal Medicinal Plant

It is observed that many newly synthesized drugs originate from natural plant products

(Vuorelaa et al., 2004). All parts of the medicinal plants are useful for pharmaceutical

purpose. Various parts of the plants are collected by local and folk communities all over

the world for their use but these are generally collected in low quantities. However, some

plant parts are also collected in huge quantities to supply them to the market which is

used as a raw material in various herbal industries (Uniyal et al., 2006). A number of

chemical components are present in medicinal plants which can be utilized for the

treatment of infectious as well as chronic diseases. These unique therapeutical agents are

screened repeatedly by clinical microbiologist (Periyasamy et al., 2010). These chemically

active agents are secondary metabolites which show antimicrobial activity and are usually

a combination of different constituents (Lai and Roy, 2004).

2.1.2 Evidence of Herbs for Treatment

There is evidence of herbs having been used in the treatment of diseases and for

revitalizing body systems in Indian, the Egyptian, the Chinese, the Greek and the Roman

civilizations. Plants have a vast potential for their use as curative medicine. In India,

medicinal plants are widely used by all sections of people both directly as folk medicines in

different indigenous systems of medicine like Siddha, Ayurveda and Unani and indirectly in

the pharmaceutical preparations (Srinivasan et al., 2001). India has about 4.5 million plant

species and among them, several thousands have been claimed to possess medicinal

properties against human diseases. Although traditional medicinal healers have used

medicinal plants for treatment of ailments for hundreds of years, there has always been a

lingering question in scientific circles about their therapeutic efficacy. As a consequence,

the pharmacological activity of many medicinal plants has been studied, even though the

vast majority of medicinal plants remain to be studied for their phytochemical components

and pharmacological effects.

2.1.3. Health care problems

The world finds itself in the midst of a multiplicity of problems particularly in the area

of health care. The situation in this sector is alarming because of the emergence of new

diseases. Consequently, the necessity of evolving new herbal remedies is on the

ascendancy. In the present scenario where 80 per cent of the world population has no

access to the benefits of western medicines due to financial constraints, it is quite

necessary to emphasize the relevance of traditional remedies which constitute a major

part of the health care system in the developing countries and are also entering the

therapeutics in the developed countries. New efforts in the above area have been initiated

globally and the newly emerging scientific discipline of ethnopharmacology forms part of it.

The use and search for drugs derived from plants have accelerated in recent years. A

truncated history of the contribution of plants to medicine was given by Philipson (2001).

While 25 to 50 per cent of current pharmaceuticals are derived from plants, none are used

as antimicrobial. Traditional healers have long been using plants to prevent or cure

infectious diseases; western medicine also is moving in this direction. Plants are

storehouses of a wide variety of secondary metabolites, such as tannins, terpenoids,

alkaloids which have demonstrated their antimicrobial properties in vitro. Many

pharmaceutical products are of plant, bacterial or fungal origin, although synthetic organic

compounds are subsequently used and have proved their value in a broad range of human

infections.

2.2. Global Seagrass Distribution

Seagrass are specialized marine flowering plants that have adapted to the near shore

environment of most of the world‟s continents (Short et al., 2001), except Antarctica (den

Hartog, 1970). According to den Hartog (1970), the great age of seagrasses is reflected in

the geographical distribution of recent genera. Based on his well-known monograph (1970)

and a few taxonomic works that have been carried out after that, currently 60 species of

seagrass in 12 genera are recognized. Of the 12 seagrass genera recognized at present,

seven are tropical (Larkum et al. 1989). The seven tropical representatives are Halodule,

Cymodocea, Syringodium, Thalassodendron, Enhalus, Thalassia and Halophila, and the five
temperate representatives are Zostera, Phyllospadix, Heterozostera, Posidonia and

Amphibolis. It is interesting to note that the seven genera regarded as tropical seagrasses

not homogeneously distributed in the tropics. They are found concentrated in two large

areas, one comprising the Indo-west Pacific and the other, the Caribbean and the Pacific

Coast of Central America (Phang, 2000). Fortes (1989) considered the Indo-west Pacific as

the center of generic richness and diversity of seagrasses as well as of mangroves and

coral reefs. All seven seagrasses considered as tropical genera can be found in the Indo-

west Pacific while the Caribbean and the Pacific Coast of Central America shows the

presence of four genera (Halodule, Syringodium, Thalassia and Halophila).

2.2.1. Seagrass Diversity and Distribution in Malaysia

On the basis of seagrass diversity, Malaysia belongs to the Indo-West Pacific region.

The first recorded collection of a seagrass in Malaya was made by Beccari in 1866, who

collected Cymodocea serrulata from Johor, Peninsular Malaysia (den Hartog 1970). Started

with den Hartog (1970) report of seven species (Cymodocea rotundata Erhrenb. and Hemp.

ex. Aschers., C. serrulata (R. Br.), Enhalus acoroides L. f. Royle, Thalassia hemprichii

Erhrenb., Halophila ovalis (R. Br.) Hook, H. beccarii Aschers., and H. spinulosa (R. Br.)

Aschers) from Johor, Negri Sembilan, Pulau Pangkor, Pulau Langkawi and Sarawak,

Phang‟s updates (2000) bring the tally of seagrass species in Malaysian waters to 15

species. All seven Genera of tropical seagrass are found in Malaysian waters. In Peninsular

Malaysia, seagrasses are more common along the west coast where the coastal habitats

consist mainly mangrove, sandy-muddy beaches or mudflats, which provide substrate that

are more suitable for seagrass growth. Much of the west coast of Peninsular Malaysia is of

the sandy-muddy type due to the heavily silted water brought in by the rivers, and its
sheltered condition due to its close proximity to Sumatra (Phang, 1989). Records of

seagrasses on the east coast of Peninsular Malaysia are mainly from the southern islands

of Pulau Tinggi, Pulau Besar and Pulau Sibu in Johor (Japar, 1994), and further up north,

around Pulau Redang (Phang, 2000). In Malaysia, research on seagrasses has

concentrated primarily on documenting the species present and their distribution (see

Japar Sidik et al., 1995; 1999; 2006). Phang (1989; 2000) has carried out studies on the

taxonomy and distribution of seagrass resources. The 15 seagrass species recorded at

present for Malaysia and for various states including Sabah and Sarawak. The most

widespread species are Halodule Literature Review 14 uninervis and Halophila ovalis,

whose ranges span both the west and east coast of Peninsular Malaysia, as well as East

Malaysia. Halophila ovata and Ruppia maritima have been recorded in Sabah and Penang,

respectively (Phang 2000).

2.2.2. Factors Determining the Distribution of Seagrasses

Factors controlling seagrass distribution and condition are of increasing interest to the

scientific community due to their ecological and economic value (Duarte, 1999). An

extensive body of literature exists on seagrasses and factors influencing their growth,

depth limit, and distribution. A number of general parameters have been set as critical to

whether seagrass will occur along any stretch of coastline. These include physical

parameters that regulate the physiological activity of seagrass (temperature, salinity,

waves, currents, depth, substrate and day length), natural phenomena that limit the

photosynthetic activity of the plants (light, nutrients, epiphytes and diseases) and

anthropogenic inputs that inhibit the access to available plant resources (nutrient and

sediment loading). Abal and Dennison (1996) concluded that the distribution and growth of
seagrasses is regulated by a variety of water quality factors such as temperature, salinity,

nutrient availability, substratum characteristics, turbidity and submarine irradiance. Other

factors that can determine the suitability of a site for seagrasses should be added to the

list. However, different species of seagrasses have different habitat requirements.

Livingston et al. (1998) determine a hierarchy of habitat requirements for three species of

seagrass (Halodule wrightii, S. filiforme and T. testudinum). Salinity, temperature and

depth restraints are important habitat variables that control seagrass growth; when such

variables are not limiting, light, sediment and nutrient characteristics become important in

the determination of the distribution of seagrass in coastal areas.

2.3. Importance of Seagrasses

It is said that seagrasses are a vital part of the marine ecosystem. Seagrasses are

submerged flowering plants found in shallow marine waters, such as bays and lagoons and

along the continental shelf in the Gulf of Mexico. A vital part of the marine ecosystem due

to their productivity level, seagrasses provide food, habitat, and nursery areas for

numerous vertebrate and invertebrate species. The vast biodiversity and sensitivity to

changes in water quality inherent in seagrass communities makes seagrasses an

important species to help determine the overall health of coastal ecosystems (“Welcome

to Saltwater Marine Plants.” Saltwater Marine Plants, saltwatermarineplants.com/.)

2.3.1. Ecological Significance of Seagrasses

Studies to determine the ecological significance of seagrasses in Owen Anchorage,

Western Australia, have been undertaken to allow government to assess the effects of

dredging proposals that result in the removal of seagrasses. Ecological significance was

broadly defined to include physical, chemical, biological and cultural attributes. The study
area (Owen Anchorage) is characterized by a mosaic of bare sand and patchy assemblages

of a mixture of seagrass species. These seagrass meadows are quite unlike the more

prominent monospecific meadows in more sheltered waters. Previously, seagrass research

in Western Australia had focused almost exclusively on these monospecific meadows. To

assess the effects of short-, medium- and long-term dredging on the ecological significance

of the study area, a large study was implemented, with tasks based on the attributes used

in the definition. These included detailed spatial and temporal investigations of the primary

producers (seagrasses and algae), the secondary consumers (invertebrates and fish), and

their interactions. Two techniques were used to assess the ecological significance of the

study area. The first involved a matrix of biological characteristics that calculated

proportional losses of seagrass meadows relative to the areas left after dredging.

Stochastic processes were introduced using software, with values based on extensive and

intensive field measurements. Linkage with an interactive geographic information system

database was developed to better represent seagrass dynamics. The second involved

defined beneficial uses (i.e. the way society uses or values an area) of the study area.

Preliminary results specific to the individual tasks and more general modelling results are

presented to show the value of this multidisciplinary approach in addressing the ecological

significance of seagrasses

2.3.2. Stabilizing the sea bottom

Ocean bottom areas that are devoid of seagrass are vulnerable to intense wave

action from currents and storms. The extensive root system in seagrasses, which

extends both vertically and horizontally, helps stabilize the sea bottom in a manner
 similar to the way land grasses prevent soil erosion. With no seagrasses to diminish

the force of the currents along the bottom, Florida's beaches, businesses, and homes

can be subject to greater damage from storms. Saltwater Marine Plants (“Welcome

to Saltwater Marine Plants.” Saltwater Marine Plants, saltwatermarineplants.com/.)

2.3.3. Ecosystem support

Seagrasses provide food, shelter, and essential nursery areas to commercial and

recreational fishery species and to countless invertebrates living in seagrass communities.

Some fish, such as seahorses and lizardfish, can be found in seagrasses throughout the

year, while other fish remain in seagrass beds during certain life stages. Saltwater Marine

Plants (“Welcome to Saltwater Marine Plants.” Saltwater Marine Plants,

saltwatermarineplants.com/.)

2.3.4 Maintaining water quality

Seagrasses help trap fine sediments and particles that are suspended in the water

column, which increases water clarity. When a sea floor area lacks seagrass communities,

the sediments are more frequently stirred by wind and waves, decreasing water clarity,

affecting marine animal behavior, and generally decreasing the recreational quality of

coastal areas. Seagrasses also work to filter nutrients that come from land-based

industrial discharge and storm water runoff before these nutrients are washed out to sea

and to other sensitive habitats such as coral reefs. Saltwater Marine Plants. (“Welcome to

Saltwater Marine Plants.” Saltwater Marine Plants, saltwatermarineplants.com/.)

 2.4 Cymodocea serrulata

Cymodocea serrulata occurs in the lower intertidal zone, in sheltered localities on

coarse coral sand, sand mud, or mud with coral rubble substrates. The plant grows

together with Thalassiahemprichii, Enhalusacorroides, Halophila ovalis and

Syringodiumisoetifolium. Cymodocea serrulata is abstract in places where there is

freshwater influence. In Albay, Southern Luzon, Philippines the plant was collected from

boulders near the month of stream, growing together with Halodule univernis (Den

Hartog’s 1970). Cymodocea serrulata is common in tropical and subtropical regions

Cymodocea serrulata is a long ribbon-like leaves with blunt, rounded tips that have

serrations (these are sometimes very tiny). The leaf scars around the upright stem are

not continuous. It has thick rhizomes (underground stems). The leaf sheaths around the

leaf are flattened. Sometimes seen with reddish bands. (Ria Tan 2008)

2.4.1. Chemical Constituents of Cymodocea serrulata

Chemical investigation of the dichloromethane extract of Cymodocea serrulata has

led to the isolation of bis(2-ethylhexyl) phthalate (1), chlorophyll a (2), and a mixture of β-

sitosterol (3) and stigmasterol (4).  The structure of 1 was elucidated by extensive 1D and

2D NMR spectroscopy.    The structures of 2-4 were identified by comparison of their NMR

data with literature data. Chemical structures ofbis(2-ethylhexyl) phthalate (1), chlorophyll

a (2), and a mixture of β-sitosterol (3) andstigmasterol (4) from C. Serrulata (Ragasa,

Consolacion and Perez, Judy and Shen, chien-chang, 2016, Chemical Constituents of

Cymodocea serrulata R. Brown)

2.4.2. Antibacterial activity of Cymodocea serrulata root extract against chosen poultry

pathogens

The study of marine organisms for their bioactive potential, being an important part

of marine ecosystem has picked up the rhythm in recent years with the growth recognition

of their importance in human life as well as animal. The in vitro antibacterial activity of

column chromatographic fractions of root extract of Cymodocea serrulata L. were

determined for antibacterial activity against 4 poultry pathogens. The results suggest that

6 fractions from acetone extract and one fraction from hexane extract exhibited broad

spectrum of antibacterial activity. It is concluded that the bioactive compounds from the

root extract of Cymodocea serrulata can be effectively used as an alternative poultry

medicine to replace the conventional antibiotics of having adverse side effects (Ravikumar,
Suresh and Syed Ali, Mscas and Anandh, P and Ajmalkhan, M and Dhinakaraj, M, 2011,

pages 98-100, Antibacterial activity of Cymodocea serrulata root extract against chosen

poultry pathogens)