EFFICACY OF HIBISCUS ROSE-SINENSIS COROLLA EXTRACTION AS AN

ANTIBACTERIAL AGENT AGAINST GRAM NEGATIVE AND GRAM

POSITIVE BACTERIA.

MUHAMMAD HAZIQ SYAFIQ BIN ROSDI

MANAGEMENT AND SCIENCE UNIVERSITY

2017
EFFICACY OF HIBISCUS ROSE-SINENSIS COROLLA EXTRACTION AS AN
ANTIBACTERIAL AGENT AGAINST GRAM NEGATIVE AND GRAM
POSITIVE BACTERIA.

MUHAMMAD HAZIQ SYAFIQ BIN ROSDI

THESIS SUBMITTED FOR REQUIREMENT FOR THE DEGREE OF MEDICAL
SCIENCE (HONS) IN THE INTERNATIONAL MEDICAL SCHOOL
MANAGEMENT AND SCIENCE UNIVERSITY

SEPTEMBER 2016
 1

Approval Sheet

This thesis submitted to the senate of Management and Science University has been
accepted as a fulfilment of the requirement for the Degree of Medical Sciences
(Hons). The members of the supervisory Committee are as follows:

Signature:
Supervisor: Dr Mustafa Fadhil Mohamed
Date: September 2016

Signature:
Dean: Dato Dr lailanor Ibrahim
Date: September 2016

DECLARATION
 2

I hereby declare the thesis entitled EFFICACY OF HIBISCUS ROSE SINENSIS

COROLLA EXTRACTION AS AN ANTIBACTERIAL AGENT, submitted to
International Medical School, Management and Science University for the
requirement of Degree in Medical Science is a faithful record of bonafide and original
research work carried out by me under the guidance and supervision of Dr Mustafa
Fadhil Mohammed, senior lecturer, International Medical School , Management and
Science University.

ACKNOWLEDGMENTS

I wish to express my sincere thanks and gratitude to my guider Dr Mustafa Fadhil

Mohammed, senior lecturer, International Medical School, Management and Science
University for his constant inspiration, encouragement and guidance throughout my
project. I consider myself fortunate enough that he has given a decisive turn and boost
to my career.

I take this opportunity to express my indebtness to my lecturers for their enthusiastic

help, valuable suggestions and constant encouragement throughout my work. I would
also like to express my whole hearted gratitude to for their good wishes, inspiration
and unstinted support throughout my project.
 3

I deeply acknowledge the constant support, encouragement, and invaluable guidance

at every step of my project by, Senior Lab Technician, Encik Yusuf bin Ibrahim and
Laboratory Technician. I am obliged and thankful to him for providing me the
opportunity to gain knowledge and understanding of working skills of the aspects of
my work from him.

I take this opportunity to thank my friends Sharmla, Sobyanya, Prahashini and

Davanraj for their throughout co-operation. Last but not the least I take this
opportunity to thank my father Mr. Rosdi bin Shariff and my mother Mrs. Sofiah Binti
Sabir for weathering my minor crises of confidence, for never doubting. Thank you
for everything Mama and Papa. I love you both.

ABSTRACT

H. Rose-Sinensis belongs to the family Malvaceae. Traditionally the flowers can be

used as home remedies for cough, act as a laxative and is a good source of vitamin C.
Resistance towards antibiotics having become widespread among bacteria and fungi,
new class of antibacterial substances are urgently required. There are several studies
which reveal the presence of such compounds with antibacterial properties in the
flower. The petals have some protective mechanism against microbial attack in most
of the plants. Therefore, the H. Rose-Sinensis flower petals were screened for their
antibacterial activity. Fresh flowers of H. Rose-Sinensis were collected from the
Sungai Buloh Gardening Centres (Delima Tani SDN BHD), Selangor, Malaysia and
rinsed with distilled water and shade dried and then homogenized into fine powder
and stored in air tight bottles. Cold and hot extraction was used to get the supernatant.
For the antibacterial assay, the following human bacterial pathogens were studied such
as S. aureus, B. subtillis , E. coli, and Salmonella . Antibacterial activity of the
aqueous of H. Rose-Sinensis, were tested against the different test microorganisms are
shown in the table no 1. The results clearly showed that hot extractions of flower
inhibited bacillus subtilis, Staphylococcus aureus with (9.84), (14.82) mm,
 4

respectively. Cold extraction showed an antibacterial activity against Staphylococcus

aureus, Salmonella sp. at (8.11), (7.86) mm. The hot extraction showed a very low
inhibition effects against E.coli, Salmonella at (7.66), (8.86) mm respectively. The
cold extraction showed very low inhibition effects against b.subtilis (5.00) mm and
E.coli (5.00) mm.
S.aureus showed maximum antibacterial activity and so this plant can be used to
discover bioactive natural products that may serve as leads for the development of
new pharmaceuticals that address hither to unmet therapeutic needs. Such screening of
various natural organic compounds and identifying active agents is the need of the
hour, because successful prediction of lead molecule and drug like properties at the
onset of drug discovery will pay off later in drug development.

Abstrak

H. Rose-Sinensis tergolong dalam keluarga Malvaceae. Secara tradisinya bunga boleh

digunakan sebagai ubat rumah untuk batuk, bertindak sebagai julap dan merupakan
sumber yang baik vitamin C. rintangan terhadap antibiotik telah menjadi meluas di
kalangan bakteria dan kulat, kelas baru bahan-bahan anti-bakteria diperlukan segera.
Terdapat beberapa kajian yang mendedahkan kehadiran sebatian itu dengan ciri-ciri
antibakteria dalam bunga. Kelopak mempunyai beberapa mekanisme perlindungan
terhadap serangan mikrob dalam kebanyakan tumbuh-tumbuhan. Oleh itu, kelopak
bunga H. Rose-Sinensis telah disaring untuk aktiviti anti-bakteria mereka. Bunga
segar H. Rose-Sinensis telah dikumpulkan dari Sungai Buloh Gardening Pusat
(Delima Tani SDN BHD), Selangor, Malaysia dan dibilas dengan air suling dan
bayangan kering dan kemudian homogenized menjadi serbuk halus dan disimpan
dalam botol kedap udara. perahan sejuk dan panas telah digunakan untuk
mendapatkan Supernatan. Untuk cerakin anti-bakteria, berikut patogen bakteria
manusia telah dikaji seperti S. aureus, B. subtillis, E. coli, dan Salmonella. aktiviti
antibakteria akueus H. Rose-sinensis telah diuji terhadap mikroorganisma ujian yang
berbeza ditunjukkan dalam jadual tidak 1. Keputusan jelas menunjukkan bahawa
pengekstrakan panas bunga menghalang subtilis bacillus, Staphylococcus aureus
dengan (9.84), (14.82) mm, masing-masing. perahan sejuk menunjukkan aktiviti
antibakteria terhadap Staphylococcus aureus, Salmonella sp. di (8.11), (7.86) mm.
 5

Pengekstrakan panas menunjukkan kesan perencatan yang sangat rendah terhadap

E.coli, Salmonella di (7.66), (8.86) mm masing-masing. Pengekstrakan sejuk
menunjukkan kesan perencatan yang sangat rendah terhadap b.subtilis (5.00) mm dan
E.coli (5.00) mm

CONTENTS

Page
APPROVAL i
DECLARATION ii

AKNOWLEDGEMENTS iii
ABSTRACT iv
ABSTRAK v
CONTENTS vi
LIST OF TABLES viii
LIST OF FIGURES ix
LIST OF ABBREVIATIONS xi

CHAPTER I INTRODUCTION 1
1.1 Introduction to hibiscus Rose-sinensis 2
1.2 Objectives 3
1.2.1 General objectives 3
1.2.2 Specific objectives 3
1.3 Hypothesis 4
1.3.1 Null Hypothesis 4
1.3.2 Alternative Hypothesis 4

CHAPTER II LITERATURE REVIEW

2.1 Epidemiology of Hibiscus Rose-sinensis. 5
2.2 Chemical Constituents of Hibiscus Rose-Sinensis 6
 6

CHAPTER III METHODOLOGY

3.1 Plant collection and identification 15

3.2 Extraction of aqueous component 16

3.3 Test microorganism for antibacterial assay 19

3.4 Culture preparation for antibacterial assay 19

3.5 Anti-bacterial assay 20

3.6 Data analysis 21

CHAPTER IV RESULTS 22

CHAPTER V DISCUSSION 25

CHAPTER IV CONCLUSION 29

REFERENCES 30

LIST OF TABLES

Number of tables Page

 7

Table 1

Antibacterial activity of cold and hot aqueous extract of 22

H. rosa-sinensis in agar and disc diffusion method
(inhibition zone diameter (mm)).

LIST OF FIGURES

Number of figures Page

Figure 1.1 Fresh flower of H. Rosa-Sinensis washed with distilled water

15

and before being dry under the hot sun.

Figure 1.2 Flower of H. Rosa-Sinensis after being washed with distilled 16

water and dry under the hot sun.

Figure 1.3 Dried powder was soaked in 150ml of cold distilled water and 17
kept for 24hours.
Figure 1.4 Cold extract was filter through filter paper 17

Figure 1.5 Cold extract was put into water bath and left for 4 hours 17
 8

Figure 1.6 Extract after was boiled for 30min

Figure 1.7 Extract being filter through filter paper and Figure 1.8 (right) 18
extract was put in a beaker and water bath for 2 hours.
Figure 1.8 4 bacteria strain is suspended in saline in a test tube 19

Figure 1.9 Disc that contain hot and cold extraction of H. Rosa-Sinensis
20

was put after inoculate the plate with bacteria strain.

Figure 1.10 Well of 5mm is cut after inoculation process using a sterile 21

cord borer.

Figure 2.1 Hot and cold extraction of corolla H. Rosa-Sinensis in agar 23

well diffusion method act on two gram-positive bacteria

( S. aureus and B.subtilis) and two gram-negative bacteria

( Salmonella and E.coli)

Figure 2.2 Hot and cold extraction of corolla H. Rosa-Sinensis in disc 24

diffusion method act on two gram-positive bacteria ( S. aureus
and B.subtilis) and two gram-negative bacteria
( Salmonella and E.coli)

Figure 3.1 Gram-positive bacteria that is bacillus subtillis and 35

staphylococcus aureus zone of inhibition after disc

contain hot (left) and cold (right) extraction of

corolla flower was put.

Figure 3.2 Gram-negative bacteria that is E.coli and Salmonella zone 35

of inhibition after disc contain hot (left) and cold (right)

extraction of corolla flower was put.

Figure 3.3 Gram-negative bacteria that is E.coli and Salmonella zone 36

of inhibition after well 5mm contain hot (left) and cold (right)

extraction of corolla flower was put.

 9

Figure 3.4 Gram-positive bacillus subtillis and staphylococcus aureus 36

zone of inhibition after well of 5mm contain hot (left)

and cold (right) extraction of corolla flower was put.

Figure 3.5 Location of nursery garden in sungai buloh , Selangor , Malaysia. 37

Figure 3.6 Gantt Chart 37

Figure 3.7 Instrument and tools 38

LIST OF ABBREVIATIONS

H. rose sinensis Hibiscus Rose-Sinensis

s.aureus Staphylococcus Aureus
b.subtillis Bacillus Subtillis
e.coli Escherichia Coli
nacl Sodium Chloride
 10

CHAPTER 1

INTRODUCTION

The term natural products ranges from an amazingly expansive and various
range of chemical compounds derived and segregated from plants, animals and
microorganism. The enthusiasm for natural products can be followed back up to
numerous times of their helpfulness to people, and till present time it is still
exceptionally helpful. Compounds and extracts got from the Mother Natures have
discovered the usefulness in pharmaceutical. Example is allopathic, homeopathy and,
agriculture, beauty and healthcare products in old and current societies around the
world. In this manner, the visionary to access natural products, understanding their
value and derivation applications has been a major thrust in the field of natural
products research. As a rule we don't know truly what particular biological role these
products play, aside from that they represent a numerous of chemical compounds that
can interesting and gainful to us. A huge number of characteristic items have been
portrayed, however we are not even an inch close to document all the species, there
are more likely many more of compounds waiting to be discovered.

Nature has been a sources of medicative agents for thousands of years and a
variety of modern medicine are isolated from natural sources, several supported their
use in traditional medicines or phytomedicines. Over the years, World Health
Organization (WHO) advocated ancient medicines as safe remedies for aliments of
 2

each microorganism and non-microorganism origins. Over fifty percent of all modern
clinical medicine are of natural product origin and natural merchandise play a very
important role in drug development programs within the pharmaceutical business.
Some antibiotics became virtually archaic thanks to drug resistant and consequently
new medicine should be wanted, for which herbal treatment is one possible way to
treat diseases caused by multi drug resistant bacteria.

It is established that flora or plants, through lacking the typical immune

response, have an in-built system for production have an adverse effect towards biotic
and abiotic stress condition. Since flora have coevolved with numerous pathogen, they
understandably have also developed the chemical substance against the parasitic
organisms. Therefore, it is reasonable to expect a variety of plant compounds with
specific as well as general antimicrobial germicide activity and antibacterial potential.

1.1 Hibiscus Rose-Sinensis

The plant Hibiscus rosa-sinensis is a perennial shrub with tap root. The leaves
ate 3.5-12 cm. In length and 2-5.5 cm wide. Leaves are simple ovate or ovate-
lanceolate. Leaves are entire at the base and coarsely toothed at the apex. Taste is
mucilaginous. Flowers are pedicillate, Actinomorphic, pentamerous and complete.
Corolla consists of 5 petals, red in colour and about 3 inches in diameter, generally
available in many areas within its hardiness range.The plants Hibiscus rosa-sinensis
(H. rosa- sinensis) belongs to the family category Malvaceae.

Traditionally the flowers can be used as anti-asthmatic agents. Many chemical

constituents such as cyanidin, quercetin, hentriacontane, calcium atomic number 20
oxalate, thiamine, riboflavin, thiamin , riboflavin , niacin and ascorbic acids have been
isolated from this plant. Resistance towards reveling antibiotics having become
widespread among bacteria and fungi, new class of antimicrobial category of
disinfectant substances are urgently required. There are several studies which reveal
the presence of such compounds with antimicrobial properties disinfectant contain in
numerous plant components. The petals have some protecting mechanism against
microorganism attack in most of the plants. The H. rosa-sinensis flower petals of an
 3

oversized variety of plant species growing within the locality of our surroundings
were screened for its medicinal activity.

The present study has been designed to determine the role of flower in H.
rosa-sinensis extract in the in-vitro antibacterial activity against human pathogens,
Gram positive bacteria [Staphylococcus aureus (S. aureus), Bacillus subtillis (B.
subtillis)] and Gram negative bacteria [Escherichia coli (E. coli) and Salmonella]

1.2OBJECTIVE

1.2.1 General objective:

To study the efficacy of hibiscus rosa-sinensis corolla extraction as an

antibacterial agent against human pathogens.

1.2.2 Specific Objective:

1) To study the effect of hibiscus rosa-sinensis corolla extraction component as

Antibacterial agent against gram negative and gram positive bacteria.

2) To study the different type of extraction method useful as an antibacterial

agent.
 4

1.3 HYPOTHESIS

1.3.1 NULL HYPOTHESIS:

1) There is no effect of hibiscus rose-sinensis corolla extraction component as an

antibacterial agent against gram negative and gram positive bacteria
2) There is no different type of extraction method useful as an antibacterial agent

1.3.2 ALTERNATIVE HYPOTHESIS:

1) There is the effect of hibiscus rose-sinensis corolla extraction component as an

antibacterial agent against gram negative and gram positive bacteria
2) There is different type of extraction method useful as an antibacterial agent
 5

CHAPTER 2

LITERATURE REVIEW

2.1 Epidemiology of Hibiscus Rose-sinensis.

The plant Hibiscus rose-sinensis is a perennial shrub with tap root. The
leaves ate 3.5-12 cm. In length and 2-5.5 cm wide. Leaves are simple ovate or ovate-
lanceolate. Leaves are entire at the base and coarsely toothed at the apex. Taste is
mucilaginous. Flowers are pedicillate, Actinomorphic, pentamerous and complete.
Corolla consists of 5 petals, red in colour and about 3 inches in diameter, generally
available in many areas within its hardiness range.

Plants contain secondary metabolites, which are organic compounds that

are not directly involved in the normal growth, development, or reproductions of
organisms but often play an important role in plant defenses (Harbone and Baxter,
1993). Examples include alkaloids, glycosides, terpenoids, phenols, tannins,
flavonoids and saponins (Edema and Alaga, 2012). Furthermore, there is growing
interest in the chemical composition of plants towards discovery of more effective
bio-therapeutic agents (Roja and Rao, 2002). The primary benefit of using plant-
derived medicines is that they are readily affordable and accessible (Grunwald, 1995).

The flowers are considered as demulcent, emollient, refrigerant,

aphrodisiac and emmenagogue. Paste of the flowers are applied to swellings and boils.
A decoction of the flowers is given in bronchial catarrh. Ghee fried flowers are
beneficial in menorrhagia. Crushed flowers yield a dark purplish dye which was
 6

formally employed for blackening shoes. In China, the dye is used for colouring hair,
eyebrows, foods and liquors (kirtikar and Basu, 1993; Wealth of India. 1997).

In addition to its traditional use Hibiscus rosa sinensis has anti-

inflammatory, demulcent, aphrodisiac, emmenagogue, refrigerant, anodyne, laxative,
emollient and various researchers had described the use of the flower to treat heart
disorders [J. Anjaria, et. al., 2002]. Sachdewa and Khemani established the
antidiabetic activity of HRS in diabetic rats and the effect was analogous with
glibenclamide. It has been also shown to be valuable in fever and bronchial catarrh. It
is known to have various activities like antitumor, antidiarrheal, antiestrogenic
antispermatogenic, androgenic, antiphologistic [S. D. Kholkute and K. N. Udupa
1976], antiimplantation [D. R. K. Murthy et. al., 1997], wound Healing [N. Vasudeva
and S. K. Sharma 2008],and anticonvulsant [B. Shivananda Nayak et. al., 2007].

2.2 Chemical Constituents of Hibiscus Rose-Sinensis

It mainly consists of cyaniding, anthocyanins, quercetin, hydrocitric acid,

kaempferol, hydrocitric acid, flavonoids and so forth [H. Yamasaki et. al., 1976].
These chemical constituents were reported in plant. Example is cyanidin, quercetin,
flavonoids, thiamine, riboflavin, niacin and ascorbic acid [Nair R et. al., 2005]

2.2.1 FLAVONOID

Flavonoids are polyphenolic compounds that are ubiquitous in nature and

are categorized, according to their chemical structure into flavones, anthocyanidins,
isoflavones, catechins, flavonols, chalcones and flavanones. More than 4,000
flavonoids have been recognised, many of which occur in vegetables, fruits and
beverages like tea, coffee and fruit drinks. The flavonoids have provoked considerable
interest recently because of their potential valuable effects on human health-they have
been testified to have been shown to have several biological properties including anti-
inflammatory, hepatoprotective anti-thrambotic and antiviral activities many of which
may be associated, partially at least, to their antioxidant and free-radical-scavenging
ability.[Robak, J et. al., 1988]. The antiradical property of flavonoids is directed
 7

mostly toward HO and 02 as well as peroxyl and alkoxyl radicals [Husain, S. R et. al.,
1987]. Furthermore, as these compounds present a strong affinity for iron ions their
antiperoxidative activity could also be ascribed to a concomitant capability of
chelating iron. [Morel, I et. al., 1993; Afanasav, I.B. et al, 1989]. One of the
undeniable functions of flavonoids and related polyphenols is their role in defending
plants against microbial attack. This not only comprises their presence in plants as
constitutive mediators but also their accumulation as phytoalexins in response to
microbial attack [Grayer et. al., 1994; Harborne, 1999]. Because of their extensive
ability to prevent spore germination of plant pathogens, they have been suggested also
for use against fungal pathogens.

There is an ever growing interest in plant flavonoids for treating human

diseases and particularly for monitoring the immunodeficiency virus which is the
contributing agent of AIDS. The majority of flavonoids documented as constitutive
antifungal agents in plants are flavanones, isofavonoids or flavans. The recognition
that a flavone glycoside, namely luteolin 7-(200-sulphatoglucoside), is an antifungal
component of the marine angiosperm Thalassia testudinum is remarkable [Jensen et.
al., 1998]. Skaltsa et. al., (1994) claim that acylated flavone glycosides existing in the
leaf hairs of Quercus ilex affords the plant useful protection against the damage of UV
radiation. The main experiment here was to calculate the photosynthetic proficiency of
de-haired leaves. Indeed, there is a substantial reduction in photosystem II
photochemical efficacy in treated leaves. Numerous recent papers report the consistent
presence of antibacterial activity among favonoids. Thus, the retrochalcone
licochalcone C (4, 40-dihydroxy-20-methoxy-30-prenyl) is active against
Staphylococcus aureus with a MIC of 6.25 g/ml [Haraguchi et. al., 1998]. Also, the
compound 5, 7-dihydroxy-3, 8-dimethoxy favone has an MIC of 50 g/ ml towards
Staphylococcus epidermis [Iniesta et. al., 1990]. Again, the substance 5, 7, 20, 60-
tetrahydroxy-6-prenyl-8-lavandulyl-40-methoxyfavanone completely inhibits the
progress of S. aureus at concentrations among 1.56 and 6.25 g/ml [Iinuma et al.,
1994].
 8

The above favanone is chiefly active against antibiotic resistant strains of

S. aureus and could have some activity towards treating patients, who unintentionally
pick up this infection while in hospital. Das and Pereira, 1990 have shown that a
carbonyl group at C-4 and a double bond between C-2 and C-3 are also important
features for high antioxidant activity in flavonoids. Butein and other 3, 4-
dihydroxychalcones are more active than analogous flavones because of their ability
to achieve greater electron delocalisation [Dziedzicet et. al., 1983]. Likewise,
isoflavones are frequently more active than favones because of the stabilising effects
of the 4- carbonyl and 5-hydroxyl in the former [Dziedzic and Hudson, 1983]. In the
antioxidant action of o-dihydroxyfavonoids metal chelation is an important factor
[Shahidi et. al., 1991]. Flavonoids have been stated to possess many useful properties,
containing antiinflammatory activity, enzyme inhibition, antimicrobial activity,
oestrogenic activity [Havsteen B et al, 1983; Harborne JB et. al., 1999], anti-allergic
activity, antioxidant activity [Middleton Jr E et al, 1986], vascular activity and
cytotoxic antitumor activity [Harborne JB et. al., 1992]. For a group of compounds of
relatively homogeneous structure, the flavonoids inhibit a mystifying number and
variety of eukaryotic enzymes and have an extremely wide range of activities. In the
event of enzyme inhibition, this has been assumed to be due to the interaction of
enzymes with different parts of the flavonoid molecule such as carbohydrate, phenyl
ring, phenol and benzopyrone ring [Havsteen B et. al., 1983].

A recent area of research that is of particular interest is the deceptive

inhibitory activity of some flavonoids against human immunodeficiency virus (HIV).
In vitro studies have shown that baicalin inhibits HIV-1 infection and replication.
Inhibition of HIV-1 entry into the cells expressing CD4 and chemokine co-receptors
[Li BQ et. al., 2000], and antagonism of HIV-1 reverse transcriptase [Li BQ et. al.,
1993] by the flavone O-glycoside have been demonstrated by Li and colleagues.
Baicalein [Ono K et. al., 1989], robustaflavone and hinokiflavone [Lin YM et. al.,
1997] have also been shown to inhibit HIV-1 reverse transcriptase, as have several
catechins, but catechins inhibit other DNA polymerases the HIV-1 enzyme is therefore
thought to be of non-specific nature [Moore PS et. al., 1992]. In addition, it has been
demonstrated that several flavonoids, including demethylated gardenin A and 3, 2-
 9

dihydroxyflavone, inhibit HIV-1 proteinase [Brinkworth RI et. al., 1992]. Robinetin,

myricetin, baicalein, quercetagetin [Fesen MR. et. al., 1994] and quercetin 3-O-(2-
galloyl)-l-arabinopyranoside [Kim HJ et. al., 1998] inhibit HIV-1integrase, although
there are concerns that HIV enzyme inhibition by quercetagetin and myricetin is non-
specific [Ono K et. al., 1990]. It has also been reported that the flavonoids chrysin,
apigenin and acacetin prevent HIV-1 activation via an unusual mechanism that
possibly involves inhibition of viral transcription [Critchfield JW et. al., 1996].
Interestingly, in a study by Hu and his co-group, chrysin was reported to have the
highest therapeutic index of 21 natural and 13 synthetic flavonoids against HIV-1 [Hu
CQ, et. al., 1994]. Several research groups have investigated the relationship between
flavonoid structure and inhibitory activity against HIV-1 and its enzymes [Lin YM et.
al., 1997; Brinkworth RI et. al., 1992; Fesen MR et. al., 1994; Critchfield JW et. al.,
1996; Hu CQ, et. al., 1994]. Furthermore, at least two groups have proposed
mechanisms of action for HIV-1 enzyme inhibition [Brinkworth RI et. al., 1992; Fesen
MR et. al., 1994]. Flavonoids show interactions with cytochrome P450 [Ng T B et. al.,
1996], antileukemic properties [Hodek, P et. al., 2002] and mild vasodilators
properties useful for the treatment of heart disease [Hodek, P et. al., 2002].

2.2.2 SAPONIN

Saponins are a group of secondary metabolites found widely distributed in

the plant kingdom as plant glycosides, characterized by a skeleton resulting of the 30-
carbon precursor oxidosqualene to which glycosyl residues are attached along with it
they have sturdy foaming property. Conventionally, they are subdivided into
triterpenoid and steroid glycosides, or into triterpenoid which are found primarily in
dicotyledonous plants but also in some monocots, spirostanol, and furostanol
saponins. [Hostettmann, K. et. al., 1995]. Steroid saponins occur chiefly in
monocotyledons family such as the Lilliaceae, Agavaceae, Droscoraceae and in
certain dicotyledons, such as foxglove [Hostettmann et. al., 1995]. Oats are unusual
because they contain both triterpenoid and steroid saponins [Price et. al., 1987].
Steroidal glycoalkaloids are found principally in members of the family belonging to
Solanaceae, which includes potato and tomato. The saponins formed by oats and
tomato have been studied in detail in relation to their potential role in the defences of
 10

plants against phytopathogenic fungi [Osbourn, 1996]. They are stored in plant cells
as inactive precursors but are readily converted into biologically active antibiotics by
plant enzymes in reply to pathogenic attack. These compounds can also be regarded as
preformed , since the plant enzymes that activate them are already present in
healthy plant tissues [Osbourn, 1996]. The natural role of saponins in plants is thought
to be protection against attack by pathogens and pets [Price et. al., 1987; Morrissey et.
al., 1999]. These molecules also have substantial marketable value and are processed
as drugs and medicines, foaming agents, sweeteners, taste converters and cosmetics
[Hostettmann et. al., 1995]. Saponin containing plants are used as traditional
medicines, especially in Asia, and are intensively used in food, veterinary and medical
industries [Hostettmann K et. al., 1995]. The pesticidal activity of saponins has long
been reported [Irvine FR, 1961] Saponin-glycosides are very lethal to cold-blooded
organisms, but deceptively not to mammals (Hostettmann K et. al., 1995; Hall JB et.
al., 1991).

Plant extracts containing a high percentage of saponins are commonly

used in Africa to treat water supplies and wells contaminated with disease vectors;
after treatment, the water is safe for human drinking [Hall JB et. al., 1991]. Various
studies have shown the effect of saponins on the immune system. Saponins induce a
strong adjuvant effect to T-dependent as well as T-independent antigens & it also
induces strong cytotoxic CD8+ lymphocyte responses and potentiate the response to
mucosal antigens [Kensil C.R.1996] Saponin based adjuvants have the ability to
modulate the cell mediated immune system as well as to enhance antibody production
and have the advantage that only a low dose is needed for adjuvant activity [Oda K.,
et. al., 2000]. Saponins have been extensively used as adjuvants for several years and
have been incorporated in several veterinary vaccinations. The adjuvant act of
saponins was, however, not so noticeable in some of the non-mammalian species
tested [Cossarini M, 1985; Grayson T.H et. al., 1987]. Saponin has both stimulatory
effects on the components of specific immunity and non-specific immune reactions
such as inflammation [de Oliveira C.A.C., et. al., 2001] and monocyte proliferation
[Delms F et. al., 2001] The mechanisms of immunestimulating action of saponins
have not been evidently understood, Saponins apparently NIT ROURKELA 2012
 11

Page 21 induce production of cytokines such as interleukins and interferons that might
mediate their immune-stimulant effects [Kensil C.R., 1996]. Saponins have been
shown to interpolate into cell membranes, from side to side by interaction with
cholesterol, forming holesor pores, their specific capability to form pores in
membranes has backed to their common use in physiological research (Choi et. al.,
2001; Menin., 2001; El Izzi et. al., 1992; Plock et. al., 2001; Authi et. al., 1988).

It is currently unfamiliar if the adjuvant effect of saponins is related to

pore formation, which may permit antigens to gain access to the endogenous pathway
of antigens presentation, promoting cytotoxic T-lymphocyte (CTL) response
[Sjlander A.et. al., 2001]. It was believed that the adjuvant activity of saponins could
be associated to branched sugar chains or aldehyde groups or to an acyl residue having
the aglycone [Kensil C.R., 1996]. Latter lablab sides and soya saponins were found to
display strong adjuvant activity in spite of lacking acyl residues and owning only un-
branched sugar chains Oda et. al,. concluded that the overall conformation of
functional groups exaggerated adjuvant action of saponins. Saponins have been
known long to possess a lytic action on erythrocyte cell membranes and this property
has been used for their detection. The haemolytic action of saponins is alleged due to
be their affinity for the aglycone moiety of membrane sterols, mainly cholesterol
[Glauert et. al., 1962], with which they form undissolvable complexes [Bangham &
Horne, 1962]. The quantity of glycosides prerequisite for permeabilisation is much
lower for cholesterol-rich lipid layers than cholesterol-free membranes [Gogelein et.
al., 1984]. Saponins isolated from plants such as fenugreek [Petit et. al., 1993],
Phellodendron and Aralia cortex [Kim et. al., 1998], Pueraria thunbergiana [Lee et. al.,
2000], and Calendula officinalis [Yoshikawa et. al., 2001] have revealed to have
hypoglycaemic effects. Petit et. al., (1993) established chronically higher plasma
insulin levels, possibly initiated by stimulation of the b-cells in male Wister rats were
given 10 and 100 mg fenugreek extract/300 g body weight mixed with food while
Matsuda et. al., (1999) did not find insulin-like or insulin-releasing activity in rats
were given oleanolic acid glycosides orally. The hypoglycaemic act here was due to
suppression of transportation of glucose from the stomach to the small intestine and
the inhibition of glucose transport across the brush border of the small intestine. The
 12

saponin momordin was also found to be significantly dose-dependent to inhibit gastric

discharging [Matsuda et. al., 1999]. The inhibitory activity here was dependent on the
level of serum glucose and facilitated at least in part by the capsaicin-sensitive sensory
nerves and the central nervous system. Yoshikawa et.al., (2001) revealed that the
oleanolic acid 3-monodesmosides with hypoglycaemic activity also inhibited gastric
emptying showing a correlation between the two properties, while other saponins also
did not affect gastric emptying.

2.2.3 TANNIN

Tannins are naturally occurring plant polyphenols that have a

characteristic of binding and precipitating proteins. They can have a large influence on
the nutritive value of many foods eaten by humans and feedstuff eaten by animals.
Tannins are found commonly in fruits such as grapes, persimmon, blueberry, tea,
chocolate, legume forages, legume trees like Acacia spp., Sesbania spp., in grasses i.e;
sorghum, corn, etc. The characteristics of tannins are that they are oligomeric
compounds with numerous structure units with free phenolic groups, molecular
weight fluctuating from 500 - 20,000, soluble in water, with exception of some high
molecular weight structures they are able to bind proteins and form insoluble or
soluble tannin-protein complexes. Presently there is a cumulative interest in tannins as
bioactive component of foods as well as biological antioxidants. Tannins are an
exceptional group of water soluble phenolic metabolites of relatively high molecular
weight and having the ability to complex strongly with carbohydrates and proteins
[Chavan et. al., 2001].

In the past, tannins have been viewed as one of the anti-nutrients of plant
origin because of their capability to precipitate proteins, inhibit the digestive enzymes
and decline the absorption of vitamins and minerals [Khattab et. al., 2010]. However,
lately several health benefits have been recognized for the intake of tannins and some
epidemiological associations with the decreased frequency of chronic diseases have
been established [Serrano et. al., 2009]. Abundant studies have demonstrated
supposedly significant biological effects of tannins such as antioxidant or radical
 13

scavenging activity as well as inhibition of lipid peroxidation and lipoxygenases in

vitro [Amarowicz et. al., 2000; Gyamfi et. al., 2002], antimicrobial and antiviral
[Dolara et. al., 2005; De Bruyne et. al., 1999], antimutagenic [Dolara et. al., 2005;
Carlsen et. al., 2010], and antidiabetic properties [Matsui et. al., 2001; Anderson et.
al., 2002]. The antioxidant activity of tannins results from their free radical and
reactive oxygen species-scavenging properties, as well as the chelation of transition
metal ions that modify the oxidation process [Serrano et. al., 2009]. Antioxidants have
also been reported to provide synergistic benefits for the treatment of diabetes because
of their insulin enhancing potential [Madhujith et. al., 2004].

2.2.4 PHENOLS

Phenolic compounds are some of the most widespread molecules among

plant secondary metabolites, are known to act as natural antioxidants and
antinitrosating agents which are of great significance in plant development. They are
involved in various processes comprising rhizogenesis [Curir et. al., .1990],
vitrification [Kevers et. al., 1984], resistance to biotic and abiotic stress [Delalonde et.
al., 1996], and redox reactions in soils [Takalama et, al., 1992]. Additionally, they
serve as flower pigments, act as constitutive protection agents against invading
organisms, function as signal molecules, act as allelopathic compounds, and affect cell
and plant growth [Dakora, 1995; Dakora et. al., 1996; Ndakidemi and Dakora, 2003],
are important natural animal toxicants [Adams, 1989] and some may function as
pesticides [Vidhyasekaran, 1988; Waterman and Mole, 1989; Beier, 1990]. They are
also functional components of the rhizosphere and its soil organic matter [Haider et.
al., 1975; Martin, 1977]. They have long been recognised as allelochemicals for weed
control [Rice, 1984; Putnam and Tang, 1986] phytoestrogens in animals [Adams,
1989] and plant defence molecules [Vidhyasekaran, 1988]. In the rhizosphere, they
work as important precursors for the production of soil humic substances [Haider et.
al., 1975]. A regular intake of phenolic compounds is assumed to decrease the
incidence of certain forms of cancer, and for that reason they are normally regarded as
chemo-preventive agents. [dIschia M et. al., 2006; Craig WJ et. al., 1999] The
antioxidant properties of phenols are determined by their radical scavenging ability
 14

and consequent inhibitory action on lipid peroxidation under oxidative stress

situations, which link with their substitution pattern. [Rigobello MP et. al., 2004].

2.2.4 TERPINOID

Isoprenoids, also known as terpenoids, are the major family of natural

compounds, comprising of >40 000 different molecules. The isoprenoid biosynthetic
pathway produces both primary and secondary metabolites that are of great
significance to plant growth and persistence. Terpenoids are well-defined as secondary
metabolites using molecular structures comprising carbon backbones are made up of
isoprene (2-methylbuta- 1, 3-diene) units. Isoprene comprises five carbon atoms and
as a consequence, the number of carbon atoms in any terpenoids is a multiple of five.
The terpenoids comprises of two isoprene units, containing ten carbon atoms. Among
the primary metabolites produced by this pathway are the phytohormones, abscisic
acid (ABA), gibberellic acid (GAs) and cytokinins that is the carotenoids;
plastoquinones and chlorophylls involved in photosynthesis. The ubiquinones required
for respiration and the sterols that impact membrane structure. Many of the terpenoids
are commercially interesting because of their use as flavours and fragrances in foods
and cosmetics examples menthol, nootkatone and sclareol or because they are
important for the quality of agricultural products, such as the flavour of fruits and the
fragrance of flowers like linalool [Aharoni, A. et. al., . 2004; Pichersky, E. et. al., .
1994]. In addition, terpenoids can have medicinal properties such as anti-carcinogenic
(e.g. Taxol and perilla alcohol), antimalarial (e.g. artemisinin), anti-ulcer, hepaticidal,
antimicrobial or diuretic (e.g. glycyrrhizin) activity [Bertea, C.M. et. al., 2005;
Haudenschild, C, 1998; Lin, Z.J. et. al., 2005; McCaskill, D.1998; Rodriguez-
Concepcion, M. 2004]. The terpenoids have also been shown to be of great ecological
significance [Degenhardt, J. et. al., 2003; Pichersky, E et. al., 2002]. The steroids and
sterols in animals are biologically produced from precursors of terpenoid. Sometimes
terpenoids are added to proteins to increase their attachment to the cell membrane the
 15

process known as isoprenylation [Sacchettini JC et. al, 1997]. These compounds and
their derivatives also belong to other drugs such as validol, menovasin, turpentine,
bromkamfora and other more. Turpentine is extensively used as external drugs, and it
is the main raw material for other products on the base of terpenoids.

CHAPTER 3

METHODOLOGY

3.1 Plant collection and identification

Fresh flowers of hibiscus (H. rosa-sinensis) with no apparent physical, insect or

microbial damage were collected from the Sungai Buloh Gardening Centres ( Delima
Tani SDN BHD), Selangor, Malaysia. Mature flowers with uniform structure were
chosen and washed with doubly distilled water. Then samples of flowers were sun
dried till constant weight was calculated. Dried sample of flowers were ground to
powder by using commercial blender. The samples of flowers were stored in air tight
bottle for further periodical use.

Figure 1.1 show fresh flower of Hibiscus Rosa-sinensis washed with distilled water
and before being dry under the hot sun.
 16

Figure 1.2 show the flower of hibiscus rosa-sinensis after being washed with distilled
water and dry under the hot sun.

3.2 Extraction of aqueous component

3.2.1 Cold extraction

A total of 30 g of dried flower was soaked in 150 mL of cold water in a conical flask
for 24 h and then filtered off using sterile Whatman No. 1 filter paper into a sterile
conical flask and evaporated by using solvent distillation apparatus. The extract was
got with the help of muslin cloth and put in water bath for 4 hours. The supernatant
was obtained and stored at 4 C for further use.

Figure 1.3 shows dried powder was soaked in 150ml of cold distilled water and kept
for 24hours.
 17

Figure 1.4 shows cold extract was filter through filter paper

Figure 1.5 shows the cold extract was put into water bath and left for 4 hours
 18

3.2.2 Hot extraction

A total of 30 g of dried flower was soaked in 150 mL of hot water which was then
boiled for 30 min and kept for 24 hour undisturbed and then filtered through sterile
filter paper, evaporated by using solvent distillation apparatus. The extract was got
with the help of a muslin cloth, put in a water bath for 2 hours and the supernatant was
stored at 4 C for further use.

Figure 1.6 show the extract after was boiled for 30min
 19

Figure 1.7 (left) show extract being filter through filter paper and Figure 1.8 (right)
extract was put in a beaker and water bath for 2 hours.

3.3 Test microorganism for antibacterial assay

For the in vitro antibacterial assay the following human bacterial pathogens were
studied that is Gram-positive bacteria such as Staphylococcus aureus and Bacillus
subtilis and the Gram-negative bacteria such as Escherichia coli and Salmonella.

3.4 Culture preparation for antibacterial assay

The cultures were grown on nutrient agar at 37 C for 18 hours and the colonies were
suspended in saline (0.85% Nacl) and its turbidity was adjusted to 0.5 Mac Farland
standards (108 CFU/mL). This saline culture preparation was used to inoculate the
plates.
 20

Figure 1.8 show 4 bacteria strain is suspended in saline in a test tube.

3.5 Anti-bacterial assay

3.5.1 Disc diffusion method

In the agar disc diffusion method the test compounds. The flower aqueous extract
were introduced into a disc 0.5 mm (hi-media) and then allowed to dry. Thus the disc
was completely saturated with the test compound at concentration of 40 mg/mL .Then
these discs were placed directly on the surface of Muller Hinton agar plates, swabbed
with the test organism and the plates were incubated at 37 C for 24 hours.
 21

Figure 1.9 show disc that contain hot and cold extraction of hibiscus rosa-sinensis was
put after inoculate the plate with bacteria strain.

3.5.2 Agar well diffusion method

Muller Hinton agar plates were prepared and wells of 5 mm were cut and swabbed
with different cultures. The cut wells were then filled with 50 L of both aqueous
extracts of flowers separately and the plates were kept for incubation at 37 C for 24
hours.
 22

Figure 1.10 show a well of 5mm was cut after inoculation process using a sterile cord
borer

3.6 Data analysis

The results were analyzed by using standard inhibition zone diameter (mm).

CHAPTER 4

RESULT

Table 1
 23

Antibacterial activity of cold and hot aqueous extract of H. rosa-sinensis in agar

and disc diffusion method (inhibition zone diameter (mm)).
Test Agar Well Diffusion Disc diffusion method
Positive
organism Method
Hot extract Cold extract Hot extract Cold extract Control
Ampicilin
of of of of (10 g)
Hibiscus hibiscus rose hibiscus rose hibiscus rose
sinensis sinensis sinensis
rose
sinensis
Gram-
positive
bacteria
S. aureus 14.82 8.00 13.00 8.11 11
B. 9.84 5.76 9.10 5.00 12
Subtillis
Gram-
negative
bacteria
Salmonell 9.05 7.86 8.86 7.00 15
a sp.
E. coli 7.80 5.00 7.66 6.30 13

Antibacterial activity of the aqueous of Hibiscus rosa-sinensis, were tested against the
different test microorganisms are shown in the table no 1. The results clearly showed
that hot extractions of flower inhibited bacillus subtilis, Staphylococcus aureus with
(9.84), (14.82) mm, respectively. Cold extraction showed an antibacterial activity
against Staphylococcus aureus, Salmonella sp. at (8.11), (7.86) mm. The hot
extraction showed a very low inhibition effects against E.coli, Salmonella at (7.66),
(8.86) mm respectively. The cold extraction showed very low inhibition effects against
b.subtilis (5.00) mm and E.coli (5.00) mm.
 24

AGAR WELL DIFFUSION METHOD

16
14
12
10
8
Zone Diameter (mm)
6
4
2
0
S. aureus B. Subtillis Salmonella sp. E. coli
Test Microorganism

HOT EXTRACT COLD EXTRACT

Figure 2.1 shows hot and cold extraction of corolla Hibiscus Rose-Sinensis in agar
well diffusion method act on two gram-positive bacteria ( S. aureus and B.subtilis)
and two gram-negative bacteria ( Salmonella and E.coli)
 25

DISC DIFFUSION METHOD

Zone Diameter (mm)

S. aureus
Test Microorganism

HOT EXTRACT
COLD EXTRACT

Figure 2.2 shows hot and cold extraction of corolla Hibiscus Rose-Sinensis in disc
diffusion method act on two gram-positive bacteria ( S. aureus and B.subtilis) and
two gram-negative bacteria ( Salmonella and E.coli)

CHAPTER 5

DISCUSSION

Phenolic contents and flavonoid contents present in the plant

body mainly give the idea of its medicinal importance. These are
considered the index of the antioxidant and free radical scavenging
strength of the plants, as these components are involved in deleting,
neutralizing or scavenging free radicals (Duh et al., 1999; Pietta
2000) due to the presence of conjugated ring systems and
carboxylic groups. These functional group in phenolic and flavonoid
 26

compounds also involve strongly to inhibit lipid peroxidation (Rice-

Evans et al., 1995). In the previous study both extracts of H.rosa-
sinensis showed promising quantity of the TPC and TFC. High
amount of these two types of antioxidants emphasized on the
medicinal potential of plant.

The knowledge of medicinal property of plants has been aggregated

throughout many hundreds of years. The local inhabitant have acquired rich traditional
knowledge on the utilization of many plants or plant parts for treatment of regular
disease. Therapeutic plants give accessible and socially relevant sources of essential
health care. The cures in view of these plants regularly have minimize side effect. The
bioactive substances in plants are created as secondary metabolites, which may be
development stage particular as well as organ and tissue specific. While plant leaf,
stem and root separates have been broadly assessed for bioactive mixes, screening of
plant petal has not been broad. Secondary metabolites belongs to polyketide and non-
ribosomal peptide families constitute a major class of natural products with different
biological functions and they have an assortment of pharmaceutically critical
properties. Trial studies have demonstrated that the biosynthetic mechanism for
polyketide and non-ribosomal peptides includes multi-useful mega synthase.

The antibacterial activities of H. Rosa-Sinensis corolla extraction were completed.

The vast majority of the extraction demonstrates an antibacterial activity against the
human pathogens, E. coli, B. subtllis, S. aureus, and Streptococcus sp. Salmonella sp.
All of the corolla extractions have demonstrated the activity. Examinations were
completed of plant materials as alternative sources of antibacterial agents. It has
turned out to be more common in the course of recent years, because of an expanded
rate of development of antibiotic resistance microorganism.

In the present investigation, corolla extracts from H. rosa-sinensis were screened for
antibacterial activity against human pathogenic bacterial strains. In general, Gram
 27

negative bacteria have been found to be more resistant than Gram positive bacteria. So
my results are in contrast from the previous investigations (Ruban, & Gajalakshmi,
2012). From the previous investigations (Ruban, & Gajalakshmi, 2012), it was
reported that the cold extractions of the hibiscus Rosa-Sinensis inhibit greater zone of
inhibition than hot extraction. But in the present work, it can be stated that hot extracts
were more potent than cold extract. From Table 1, the results clearly showed that hot
extractions of flower inhibited salmonella, Streptococcus aureus with (14.55), (12.82)
mm, respectively. Cold extraction showed an antibacterial activity against E.
coli, Salmonella sp. at (6.30), (10.86) mm. The hot extraction showed a very low
inhibition effects against E.coli, Salmonella at (7.80), (5.00) mm respectively. The
cold extraction showed very low inhibition effects against b.subtilis (5.00) mm and
E.coli (5.00) mm.

For the agar well diffusion method, the antibacterial activity of hibiscus rosa-sinensis
corolla aqueous extract against S.aureus , B .subtilis ,Salmonella and E.coli is shown
in Figure 2.1. The hot extract showed considerably more activity than the cold extract.
Maximum antibacterial activity was shown against S.aureus, followed by B.subtilis
and salmonella. Minimum antibacterial activity was shown against E.coli and
B.subtilis.

For the disc diffusion method, the antibacterial activity of hibiscus rosa-sinensis
corolla aqueous extract against S.aureus , B .subtilis ,Salmonella and E.coli is shown
in Figure 2.2. The hot extract showed considerably more activity than the cold extract.
Maximum antibacterial activity was shown against S.aureus, followed by equalof
B.subtilis and salmonella. Minimum antibacterial activity was shown against
B.subtilis for the cold extractions.
 28

The restraint of bacterial development by the extractions of the corolla could be

because of the nearness of some active chemical constituents in the extracts. These
active compounds may act alone or in blend to inhibit bacterial development. The
corolla extracts containing various natural components including flavonoids, tannins,
alkaloids, triterpenoids, all of which are known to have antibacterial effects. Flower
extracts contain phenolic compounds like tannins that are great antimicrobial operator.
In this way it might be abridged that the class of natural compounds must exhibit the
antibacterial action. The metabolites have been showed to be responsible of various
therapeutic activities of medicinal plants. Flavonoids particularly are known to be
effective antimicrobial agent against a wide cluster of microorganisms. The activity is
ascribed to their capacity to complex with extra cellular and soluble proteins and with
bacterial cell wall. There are a few reports published on antibacterial activity of
different herbal extracts. It bolsters the earlier investigations that the tannins isolated
from the corolla have remarkable antibacterial activity against microorganisms and
may expect pharmacological significance. Numerous antimicrobial screening studies
utilize a generally little number of microorganisms for testing. It is possible that these
plant materials contain antibacterial activity against pathogenic microscopic
organisms other than those tried in this research. Likewise, the lack of activity might
be a direct result of degradation of active chemicals amid the drying procedure, the
extraction procedure.

The vast majority of the concentrates have demonstrated antibacterial activity against
these pathogens. E. coli are common member from the normal flora of digestive
organ. It is predominant facultative life organism in the gastrointestinal tract and
colonizes the tract within hours or few days. It is in responsible of bringing about
inflammatory bowel which is described by fast onset of watery non bloody liquid. S.
aureus is a facultative anaerobe that develops by aerobic respiration or by
fermentation which yields lactic acid. These are pathogenic to people. They cause a
 29

wild scope of superlative contamination and in addition nourishment harming and

poisonous stun disorder. Salmonella sp. incorporates an extensive number of
pathogens of people and also warm blooded creatures. These are pathogenic when
procured by oral route. Comprehensively they may bring about enteric fever,
septicaemia and enteritis. The enteric fever and septicaemia are brought about by
thousands of Salmonella. Subsequently the plant concentrates can be utilized as a
critical antibiotic to cure previously mentioned disease or disorders brought about by
the distinctive strains of microscopic organisms. The present studies conclude that
these extracts could inhibit the growth of human pathogens. The outcomes are
empowering yet precise assessment is totally essential before being situate in practice
as well as the most active extracts can be subjected to isolation of the therapeutic
antimicrobials and undergo secondary pharmacological evaluation

CHAPTER VI

CONCLUSION
 30

Based on literature review Hibiscus rosa-sinensis belongs to the family Malvaceae,

flowers and leaves of this plant traditionally used in the anti-diabetic activity and
cosmetics. Flowers which are used as popular remedies are rich source of biologically
active secondary metabolite. Activity guided fractionation could isolate and
characterize many compounds. Extracts or isolated compounds produced good
antimicrobial, antioxidant, anti-inflammatory activities both invitro and invivo.
Maximum inhibition was observed in hot flower aqueous extract against human
pathogens.The vast majority of the extraction demonstrates an antibacterial activity
against the human pathogens, E. coli, B. subtllis, S. aureus, and Streptococcus sp.
Salmonella sp. All of the corolla extractions have demonstrated the activity.
Examinations were completed of plant materials as alternative sources of antibacterial
agents. It has turned out to be more common in the course of recent years, because of
an expanded rate of development of antibiotic resistance microorganism. The result
obtained from present study reveal that hot aqueous extract of Hibiscus rosa-sinensis
exhibit significant antioxidant and anticancer activity due to the increased flavonoids
and terpenoids level by boiled the extracts. The phytochemical analysis of corolla
extract from the previous research indicate the constituents present are responsible for
pharmacological effects. From the above results it can be concluded that plant extracts
have great potential as antimicrobial compounds against microorganisms and that they
can be used in the treatment of infectious diseases caused by resistant microorganisms.
S.aureus showed maximum antibacterial activity and so this plant can be used to
discover bioactive natural products that may serve as leads for the development of
new pharmaceuticals that address hither to unmet therapeutic needs. Such screening of
various natural organic compounds and identifying active agents is the need of the
hour, because successful prediction of lead molecule and drug like properties at the
onset of drug discovery will pay off later in drug development.

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APPENDIX A
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Figure 3.1 show a gram-positive bacteria that is bacillus subtilis and staphylococcus
aureus zone of inhibition after disc contain hot (left) and cold (right) extraction of
corolla flower was put.

Figure 3.2 show gram-negative bacteria that is E.coli and Salmonella zone of
inhibition after disc contain hot (left) and cold (right) extraction of corolla flower was
put.
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Figure 3.3 show gram-negative bacteria that is E.coli and Salmonella zone of
inhibition after well 5mm contain hot (left) and cold (right) extraction of corolla
flower was put.

Figure 3.4 show gram-positive bacillus subtillis and staphylococcus aureus zone of
inhibition after well of 5mm contain hot (left) and cold (right) extraction of corolla
flower was put.

APPENDIX B Explanatory notes

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Figure 3.5 Location of nursery garden in sungai buloh , Selangor , Malaysia.

1) APPENDIX C Figure 3.6 Gantt Chart

2) APPENDIX D Figure 3.7 Instrument and tools

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