A

PROJECT REPORT

ON

ANTIMICROBIAL POTENTIAL OF CHLOROFORM EXTRACT

OFABRONIA MARITIMA ON SELECTED HUMAN MICROBIAL

PATHOGENS

SUBMITTED BY
A. YASHMA (U17AC089)

DEPARTMENT OF AGRICULTURAL BIOTECHNOLOGY

BHARATH INSTITUTE OF HIGHER EDUCATION AND

RESEARCH
(Declared under section 3 of the UGC Act, 1956)
SELAIYUR, CHENNAI-600073, TAMIL NADU, INDIA

MAY 2021

i
 A

PROJECT REPORT

ON

ANTIMICROBIAL POTENTIAL OF CHLOROFORM EXTRACT

OFABRONIA MARITIMA ON SELECTED HUMAN MICROBIAL

PATHOGENS

Submitted by

A. YASHMA (U17AC089)
In partial fulfillment for the requirement of the degree
Of
Bachelor of Technology
in
Biotechnology specialization in Agricultural Biotechnology
Under the guidance of
Dr.S. SELVAKUMAR

BHARATH INSTITUTE OF HIGHER EDUCATION AND

RESEARCH
(Declared under section 3 of the UGC Act, 1956)
SELAIYUR, CHENNAI-600073, TAMIL NADU, INDIA

ii
 BHARATH INSTITUTE OF HIGHER EDUCATION AND
RESEARCH
(Declared under Section 3 of UGC Act, 1956)

DEPARTMENT OF AGRICULTURAL BIOTECHNOLOGY

This is to certify that “ANTIMICROBIAL POTENTIAL OF

CHLOROFORM EXTRACT OFABRONIA MARITIMA ON SELECTED

HUMAN MICROBIAL PATHOGENS” is a bonafide work done by A.

YASHMA (U17AC089) for the major project in Agricultural Biotechnology

during the academic year 2020-2021.

Internal project guide H.O.D

(Dr.S.SELVAKUMAR) (Dr. L. JEYANTHI REBECCA)

Submitted for the viva voce held on……………………………. at BIHER

Internal examiner External examiner

iii
 DECLARATION

I hereby declare that the project report entitled “ANTIMICROBIAL POTENTIAL OF

CHLOROFORM EXTRACT OFABRONIA MARITIMA ON SELECTED HUMAN
MICROBIAL PATHOGENS” submitted to BIHER, Chennai in partial fulfillment UG for
the award of degree of Bachelor of Technology in Biotechnology specialization in
Agricultural Biotechnology is the record of the original work carried out by me under the
guidance of Dr. S. SELVAKUMAR.

I further declare that the result of the work have not been submitted to any other university or
institute for the award of any degree or diploma.

Place: Chennai Signature of student

Date:

iv
 ACKNOWLEDGEMENT

First and foremost, I thank the Lord Almighty for pouring out his strength and wisdom to carry

out this project work successfully, and also to my parents at the second place, for encouraging

me and helping me in all ways possible for the project to succeed.

I would like to express my sincere gratitude to our respected PresidentDr.J. Sundeep Anand

and Managing Director, Dr.Swetha Sundeep Anand for their support and encouragement in

technological upgrades and novel projects.

I take great pleasure in expressing our sincere thanks to our Vice Chancellor Dr. K. Vijaya

Bhaskar Raju for encouraging us in this project. I would also like to thank our Pro Vice

Chancellor (Academics), Dr. M. Sundararajan for his constant support.

I thank our Dean-Engineering, Dr. J. Hammed Hussain for providing sufficient facilities for

the completion of this project. I would like to give our hearty thanks to my Department Head,

Dr. L. Jeyanthi Rebecca for giving inspiration for this project and her unmatched guidance.

My sincere thanks to my project guide Dr. S. SELVAKUMAR for his patient and

overwhelming guidance in completing the project and guiding in publication of the research

findings.

Last but not the least, I wish to thank all those who helped me to complete this project, directly

or indirectly by giving various suggestions and extending their helping hands.

v
 CONTENTS
S.NO CHAPTERS PAGE NO

1. ABSTRACT

2. INTRODUCTION 8

3. REVIEW OF LITERATURE 17

4. OBJECTIVE 18

5. MATERIALS AND METHODS 22

6. RESULTS AND DISCUSSION 26

7. CONCLUSION 27

8. REFERENCES 31

vi
ANTIMICROBIAL POTENTIAL OF CHLOROFORM EXTRACT OFABRONIA

MARITIMA ON SELECTED HUMAN MICROBIAL PATHOGENS

ABSTRACT

This study was carried out with an objective to investigate the antibacterial and antifungal

potentials of leaves of Abronia maritima. The aim of the study is to assess the antimicrobial

activity and to determine the zone of inhibition of extracts on some bacterial and fungal strains.

In the present study, the microbial activity of chloroform extract of leaves of Abronia

maritima was evaluated for potential antimicrobial activity against medically important

bacterial and fungal strains. The phytochemical analyses of the plants were carried out. The

microbial activity of the plant extract was due to the presence of various secondary metabolites.

Hence, these plants can be used to discover bioactive natural products that may serve as leads

in the development of new pharmaceuticals research activities. Hence, it is of interest to

investigate the anti-microbial effects of chloroform extract of Abronia maritima are of our

primary aim. Our results indicate that the chloroform extract of the plant possess antimicrobial

properties.

Key words: Abronia maritima; Resistance; Microbial chemotherapy; Antibiotics; Clinical

pathogens.

vii
INTRODUCTION

Medicinal plants, since time immemorial, have been used in virtually all cultures as a source

of medicine (Ang-Lee et al., 2001; Goldman, 2001). In India and China, herbal medicines are

still widely used, and developed countries have rediscovered many of these traditional

medicines as cheap sources of complex bioactive compounds (Phillipson, 1994). Modern

pharmaceuticals are typically oral dosage forms containing single synthetic chemicals that have

potent clinical activity. However, natural products from higher plants continue to be used in

pharmaceutical preparations either pure or as extracts (Gogtay et al., 2002). Despite the

availability of different approaches for the discovery of therapeutic agents, natural products

still remain as one of the best reservoirs of new structural types (Hostettmann, 1999). To this

effect, in the constant effort to improve the efficacy and ethics of modern medicinal practice,

researchers are increasingly turning their attention to folk medicine as a source of new drugs

(Wayne, 1998; Hoareau&Dasilva, 1999).In Mauritius, the local population has a long-standing

tradition in the use of herbal medicine. Many indigenous and endemic plant species of

Mauritius have been used in folk medicine to treat various ailments of man (Baumer, 1979;

Sussman, 1980; Wong Ting Fook, 1980; Gurib-Fakim et al., 1993, 1996a,b). Available reports

tend to show that indigenous folk-medicinal plant preservation and study is vital because such

plants are fully adapted to local environments and to conditions compared to introduced species

(Qin & Xu, 1998). Pharmacologically active compounds and phytochemicals isolated from

such endemic and indigenous plants used in folk medicine have been the center of interest

during the past few decades (Farnsworth, 1994; Benzi&Ceci, 1997). In addition, it is also of

importance to preserve and maintain knowledge about medicinal uses of plant resources of

Mauritius, which is characterized by a rich flora of exotic, endemic, and indigenous plant

species. Currently, several kinds of extracts from various exotic, endemic, and indigenous

plants are sold as decoctions or “tisanes” in several markets across Mauritius to treat minor

1
ailments (Gurib-Fakim et al., 1993, 1996a)This native herbal folk medicinal practice forms an

essential part of the heritage of the local pharmacopoeia of Mauritius. Nonetheless, only a few

medicinal plants have been validated for their medicinal virtues. Moreover, the biological

activity of such medicinal plants against certain human pathogenic bacteria and fungi is poorly

investigated, and a literature search indicated limited information about the antimicrobial

activity of Mauritian endemic plants (Gurib-Fakim et al., 1993, 1996a,b). Currently, the

emergence of resistant pathogens to many of the commonly used antibiotics has provided an

impetus for further attempts to search for new antimicrobial agents to combat infections and

overcome the problems of resistance to currently available antimicrobial agents (Balandrin et

al., 1985; Xu & Lee, 2001). Plant-based antimicrobials and antibacterial represent a vast

untapped source for medicines and hence have enormous therapeutic potential (Phillipson,

1994). They are effective in the treatment of infections while mitigating many of the side effects

associated with synthetic antimicrobial and antibacterial (Mathews et al., 1999;

Bagghi, 2000)Therefore, the current in vitro. antimicrobial study was aimed at validating the

effects of two endemic medicinal plants of Mauritius, Antidesma madagascariense

and Erythroxylum macrocarpum. O. E. Schulz (Erythroxylaceae), for their antimicrobial

activity against eight clinical strains of bacteria and two strains of fungi. In addition, we also

evaluated their phytochemical profiles to correlate possible biological activities of these plants

(Anwar Hussein Subratty et al.2005).

Since their discovery, antibiotics have completely transformed humanity's approach to

infectious diseases and have substantially reduced the threat posed by infectious diseases.

However, the emergence of drug-resistant microorganisms is swiftly reversing the advances of

the previous 50 years of research. These drug-resistant microorganisms have complicated the

treatment of infectious diseases in immunocompromised AIDS and cancer patients

2
(Davis, 1994). In this context, there is an urgent need to find new antimicrobial agents against

resistant microorganisms from different sources, especially of plant origin. Scientists from

divergent fields are now investigating plants with a new view of their antimicrobial usefulness

and as an alternative source to existing drugs. Approximately 119 pure chemical substances

extracted from higher plants are used in medicine throughout the world (Farnsworth

&Soejarto, 1985). The World Health Organization estimates that 80% of the people in

developing countries of the world rely on traditional medicine for their primary health care

needs, and about 85% of traditional medicine involves the use of plant extracts. This means

that about 3.5 million to 4 million people in the world rely on plants as sources of drugs

(Farnsworth &Soejarto, 1985; Kamboj, 2000). Among 45,000 plant species in India, 3000

plants are officially documented with medicinal potential, whereas traditional practitioners use

more than 6000 plants. India is the largest producer of medicinal herbs and is appropriately

called the botanical garden of the world (Ahmedullah, 1999). In recent years, antimicrobial

properties of medicinal plants are being increasingly reported from India (Perumal Samy, 2000;

Ahmad & Beg, 2001; Srinivasan, 2001).In the current investigation, methanol extracts of 22

plants belonging to 12 different families, used by herbalists for primary health care, were

screened for their potential antibacterial activity against opportunistic bacteria, which can cause

serious infections in hospitals and the community.(Yogesh mahida et al .2006).

Tea is a major plantation crop of India and is cultivated extensively throughout north east India.

Fungal pathogens present a significant threat to tea leaves that requires use of chemical

fungicides. However, the use of chemical fungicides for tea plants has increasingly become

unpopular due to an all-round awareness of its polluting effects, leading to tighter health and

environmental regulations. This in turn has created a need for alternative sourcing of fungicidal

agents that can be developed for treatment of fungal tea-leaf diseases. The traditional practice

of using plant preparations to combat fungal infections has gained attention, and currently the

3
focus is on detection of new antifungal components from plants that have no negative effect on

the environment or on animal and human systems. Several authors have reported antifungal

activity of plant extracts against pathogens of rice, tomato, wheat, pea, and other important

crops (Rana et al., 1999; Sindhan et al., 1999; Hu et al., 2001). However, such reports involving

tea pathogens are very few (Chakraborty et al., 1991).This paper presents a study on the in

vitro. effect of aqueous and ethanol extracts of 30 plant species against four major foliar fungal

pathogens of tea. This is an approach to identify antifungal elements in natural sources that

may be used for controlling tea-leaf diseases. (saha et al.2005).

Medicinal plants are part and parcel of human society to combat diseases, from the dawn of

civilization . They usually contain many biologically active ingredients and are used primarily

for treating mild or chronic ailments. According to World Health Organization (WHO), about

80% of the world population relies chiefly on the plant based traditional medicine especially

for their primary healthcare needs. Herbal medicines are in great demand in the developed as

well as developing countries for primary healthcare because of their wide biological and

medicinal activities, higher safety margins and lesser costs Infectious diseases are a major

cause of morbidity and mortality worldwide. The number of multi-drug resistant microbial

strains and the appearance of strains with reduced susceptibility to antibiotics are continuously

increasing. This increase has been attributed to indiscriminate use of broad-spectrum

antibiotics, immunosuppressive agents, intravenous catheters, organ transplantation and

ongoing epidemics of human immunodeficiency virus (HIV) infections This situation provided

the impetus to the search for new antimicrobial substances from various sources like medicinal

plants. Synthetic drugs are not only expensive and inadequate for the treatment of diseases but

are also often with adulterations and side effects. Therefore, there is a need to search for new

infection-fighting strategies to control microbial infections Plant medicines are used on a

worldwide scale to prevent and treat infectious diseases Plants are rich in a wide variety of

4
secondary metabolites such as tannins, alkaloids, terpenoids and flavonoids having been

found in vitro since they have antimicrobial properties and may serve as an alternative,

effective, cheap and safe antimicrobial for the treatment of microbial infections Plant based

antimicrobial compounds have great therapeutic potential as they have lesser side effects as

compared with synthetic drugs and also little chance of development of resistance. Therefore

an attempt has been made to study the antibacterial activity of ten medicinally important plants

viz. Artocarpus heterophyllus, Berberis aristata, Chromolaena odorata, Embeliaribes,

Jasminum angustifolia, Mahonia leschenaultii, Pluchea lanceolata, Plumbago indica,

Terminalia chebula, Vitex negundo.(Nitha et al.2021).

Diseases are the major causes of death in the developing countries and accounts to 50% of it.

The extensive use of the antibiotics to control these diseases has led to the emergence of

multidrug resistance (Westh et al., 2004). Bacterial resistance to antibiotics increases mortality

likelihood of hospitalization and length of stay in the hospital (Winstanley, 1997). The use of

plants as source of remedies for the treatment of many diseases dates back to history and people

of many continents have this old tradition. The advent of science into the search for antibiotics

largely depends on some of these plants as raw materials. Plant based antimicrobials represent

a vast untapped source. The use of plant extract for medicinal treatment has become popular

when people realized that the effective life span of antibiotic is limited and over prescription

and misuse of traditional antibiotics are causing microbial resistance (Alam et al., 2009). At

present, nearly 30% or more of the modern pharmacological drugs are derived directly or

indirectly from plants and their extracts dominate in homeopathic or ayurvedic medicines

(Murugesan et al., 2011; Jabeen et al., 2007; Banso, 2009; Ahamunthunisa and Hopper, 2010).

Medicinal plants are finding their way into pharmaceuticals, cosmetics, neutraceuticals. Plants

have given Western Pharmacopoeia about 7000 different pharmacologically important

compounds and a number of top selling drugs of modern times eg. quinine, taxol,

5
campothecineetc. (Tshibangu et al. 2002). The objective of this research is to evaluate the

potentiality of some common medicinal plant extracts against standard microorganisms which

would lead to theDrug fill solution was prepared by accurately weighing required quantities of

ibuprofen and diphenhydramine discovery of some active secondary metabolites (Jayalakshmi

et al.2011).

Medicinal plants are assuming widespread use in the primary health care of individuals and

communities. Plant-derived chemicals are a wide group of chemical compounds that have been

found naturally in plants. The extensive existence of these compounds has demonstrated

beneficial advantages in terms of antioxidant, antibacterial, and antifungal activities. Although

synthetic antimicrobial agents have been already approved in many countries, yet the usage of

natural compounds that are derived from microbial, animals, or plants attracts the attention of

many researchers. These compounds have exhibited promising results in overcoming the

emergence of antibiotic resistance in bacterial pathogens. The plants hold great promise as a

source of novel antimicrobial agents. They are readily available, cheap and also, almost; do not

have any side effects. Plant derivative compounds including phytochemicals have even been

employed to treat various infectious diseases and have shown interesting antimicrobial activity

against several human pathogens. Abutilon indicum (Indian Abutilon, Indian Mallow; is a

small shrub in the Malvaceae family, native to tropic and subtropical regions and sometimes

cultivated as an ornamental. This plant is often used as a medicinal plant and is considered

invasive on certain tropical islands. It is extensively grown in Bangladesh., India, Pakistan,

Srilanka.2 In traditional medicine, A. indicum is used as a demulcent, aphrodisiac, laxative,

diuretic, pulmonary, and sedative (leaves). The bark is astringent and diuretic; laxative,

expectorant, and demulcent (seeds); laxative and tonic, anti-inflammatory and anthelmintic

(plant); analgesic (fixed oil); diuretic and for leprosy (roots). The previous studies revealed the

presence of chemical constituents namely luteolin, chrysoeriol, apigenin 7-O-beta

6
rhamnopyranosy, quercetin, triacontanoic acid, ursenol, methylstigmasterol, glucopyronoside

etc3 . This present study aimed to evaluate the antimicrobial activity of Abutilon indicum, a

medicinal plant used in Ayurveda and traditional medicinal system for treatment of

manifestations caused by microorganisms (Shital Wakale and Megha Kandepalli.2021).

Soil microbiota represents one of the primitive evolutionary origins of antibiotic resistance and

is a reservoir of resistance genes available for exchange with clinical pathogens. A gram of soil

may contain up to 5000 or more different species of bacteria. Multidrug resistant strains of

pathogenic bacteria such as Escherichia coli and Klebsiella pneuminiae are prominent in

hospital areas and are increasing being isolated from community acquired infections .

Pseudomonas aeruginosa can be found in nature from sources as diverse as water, soil and

plants. P. aeruginosa is more widely known as an opportunistic pathogen for humans and

animals than as soil bacteria and can produce severe infections in immune-compromised hosts

.So, soil from the hospital nearby areas was thought to be the suitable source for the isolation

of pathogenic bacteria. Plants are rich in wide variety of secondary metabolites known as

phytochemicals such as tannins, flavonoids, steroids, terpenoids, napthoquinone, inulin,

alkaloids, water soluble phenols and phlobtannins. These phytochemicals have wide

applications from the ancient times in the treatment of infectious diseases and have fewer side

effects and reduced toxicity. Previous studies were carried out on the antimicrobial activity of

methanolic extracts of Acacia nilotica, Sida cordifolia, Tinospora cordifolia, Withaniasomnifer

and Ziziphus mauritiana and they showed significant antimicrobial activity]. A study reported

that Cinnamomum zeylanicum bark can be used to design reliable and safer herbal drug and

could be used in the pharmaceutical preparations for the treatment of infectious and malignant

diseases.So, the agar diffusion study and phytochemical screening of the extracts of

Glycyrrhiza glabra roots, Ocimum sanctum leaves and Azadirachta indica leaves can help in

the understanding of the antimicrobial activity of the extracts. In the present study,

7
biochemical, confirmatory, antibiotic sensitivity and some additional tests was done to identify

the isolated bacteria from soil. Based on the above considerations, agar well diffusion study,

phytochemical tests were carried out to examine the antimicrobial activity of the medicinal

plants extract against the isolated bacterial species. The aim of the research is to search for the

effective medicinal plants with significant antimicrobial activity (Jinku Bora et al.2016).

8
REVIEW OF LITERATURE

The antimicrobial potential of seventy-seven extracts from twenty-four plants was screened

against eight bacteria and four pathogenic fungi, using microbroth dilution assay. Lowest

concentration of the extract, which inhibits any visual microbial growth after treatment with p-

iodonitrotetrazolium violet, was considered to be minimum inhibitory concentration (MIC).

Water extracts of Acacia nilotica, Justicia zelanica, Lantana camara and Saraca

asoca exhibited good activity against all the bacteria tested and the MIC was recorded in range

of 9.375–37.5 µg/ml and 75.0–300.0 µg/ml against the bacterial and fungal pathogens,

respectively. The other extracts of Phyllanthus urinaria, Thevetianerifolia, Jatropha

gossypifolia Saraca asoca, Tamarindus indica, Aegle marmelos, Acacia nilotica,

Chlorophytum borivilianum, Mangifera indica, Woodfordiafruticosa and Phyllanthus

emblica showed antimicrobial activity in a range of 75–1200 µg/ml.( Altern Med. 2007 )

Fifty medicinal plants belonging to 26 families were studied for their antimicrobial activity.

Among 50 plants tested, 72% showed antimicrobial activity. About 22 plant extracts from 15

families exhibited activity against both Gram-positive and Gram-negative bacteria. Fourteen

plants belonging to 11 families did not show activity against any of the bacteria tested. Only

nine plant extracts showed antifungal activity. The bulb extracts of A. cepa and A.

sativum exhibited activity against both filamentous and non-filamentous fungus. Eight plant

extracts belonging to seven families exhibited both antibacterial and antifungal activity

(Srinivasan et.al.2001)

Antimicrobial activities of the crude ethanolic extracts of five plants were screened against

multidrug resistant (MDR) strains of Escherichia coli, Klebsiella pneumoniae and Candida

albicans. ATCC strains of Streptococcus mutans, Staphylococcus aureus, Enterococcus

faecalis, Streptococcus bovis, Pseudimonas aeruginosa, Salmonella typhimurium, Escherichia

9
coli, Klebsiella pneumoniae and Candida albicans were also tested. The strains that showed

resistance against the maximum number of antibiotics tested were selected for an antibacterial

assay. The MDR strains were sensitive to the antimicrobial activity of Acacia nilotica,

Syzygiumaromaticum and Cinnamum zeylanicum, whereas they exhibited strong resistance to

the extracts of Terminalia arjuna and Eucalyptus globulus. Community-acquired infections

showed higher sensitivity than the nosocomial infections against these extracts. The most

potent antimicrobial plant was A. nilotica (MIC range 9.75-313µg/ml), whereas othercrude

plant extracts studied in this report were found to exhibit higher MIC values than A. nilotica

against community acquired as well as nosocomial infection. This study concludes that A.

nilotica, C. zeylanicum and S. aromaticum can be used against multidrug resistant microbes

causing nosocomial and community acquired infections.( Rosina Khan et al. 2009)

Ethnopharmacological relevance: An emerging of antibiotic resistance strains of bacteria

brings most serious public health problems. It is therefore, important to look for more effective,

safer and less toxic alternative options of treatment. Development of new antibacterial agents

from plant extract is among the proposed solutions to overcome this problem.: To study the

antibacterial activity evaluation of Leucas aspera (Willd.) Link. (L. aspera), Solanum incanum

L. (S. incanum), and Hydnorajohanis A.B. (H. johanis) against Escherichia coli (E. coli),

Salmonella typhimurium (S. typhimurium), Staphylococcus aureus (S. aureus) and

Enterococcus feacalis (E. feacalis) in comparison with GEN (Gentamicin), CIPRO

(Ciprofloxacin), PCN (Penicillin), Ampicillin (AMP) and TCN (Tetracycline). Materials and

Methods: Following plant material collection and extraction; disc diffusion method was used

for antibacterial activity test.The plants showed a promising broad spectrum of activity against

Gram-negative (Escherichia coli and Salmonella typhimurium) and Gram-positive

(Staphylococcus aureus and Enterococcus feacals) test bacteria with growth inhibition zone

values ranging from 7.40±0.6-16.70±0.4 mm. Compared with standard antibiotics; most active

10
crude extracts were showed comparative antimicrobial effect as do penicillin, ampicillin and

tetracycline. Acetone extract of H. johani on E. coli (16.70±0.4) and ethyl acetate extract of S.

incanum on S. typhimurium (16.0±2.0) were demonstrated promising activity than the activity

demonstrated by PCN, AMP and TCN. Ethanol extract of L. aspera on S. aureus (16.40±1.1)

were also displayed better activity than the activity of AMP. Conclusions: Over all, the present

investigation proves the scientific basis for the traditional use of L. aspera, S. incanum and H.

johanis as antibacterial agent for the treatment of infections caused by E. coli, S. typhimurium,

S. aureus and E. faecalis (DemisseDakone and Gizachew Zeleke.2018)

Several species of fungi belonging to 12 genera viz. Aspergillus flavus, Aspergillus niger,

Aspergillus fumigatus, Mucor mucedo, Penicillium chrysogenum, Fusarium oxysporum,

Rhizopus stolonifer, Cephalosporium acromonium, Rhizopus leguminicola, Alternaria

alternata, Colletotrichum dematium, Macrophomina phaseolina, Phoma sp., Sclerotium rolfsii

and Curvularialunata were isolated from seeds of soybean cultivars. Among these fungi

Aspergillus flavus, Fusarium oxysporum, and Alternaria alternata were found to dominant.

Seed borne fungi of soybean can be controlled by using leaf extract of medicinal plant and

biocontrol agent. The seeds were treated with leaf extracts of plants like, Azadirachta indica

A.Juss., Acacia nilotica (L.) Del., Datura stramonium L., Polyalthia longifolia (Sooner.)The.,

Allium sativum L. and Annona squamosa. An attempt has made to know the efficacy of leaf

extract by food poisoning technique. Among these plants Polyalthia longifolia The Allium

sativum L. and Azadirachta indica A. Juss. Were more effective than other plants. All these

plant extracts showed inhibitory effect on linear growth of dominant fungi.( Rathod and

Pawar.2012).

Herbs play a significant role in pharmaceutical industries as life saving drugs. This calls for the

discovery of new antimicrobial compounds with diverse chemical structures and novel

mechanisms of action. The present study focuses on imparting antimicrobial finish to the denim

11
fabric using natural herbalextract Threeherbs, Ricinus communis, Senna

auriculata and Euphorbia hirta were screened for their antimicrobial efficiency by finishing

the methanolic extracts of these herbs on the denim fabric by qualitative test method EN ISO

20645. The combinatorial antimicrobial efficiency (1:3:2) of these extracts were studied and

confirmed quantitatively by performing AATCC 100 test method. The antifungal activity was

tested according to AATCC 30 test method The combinatorial ratio (1:3:2) showed 98%

bacterial reduction against Escherichia coli and 99% against Staphylococcus aureus. Same

ratio also showed 100% mycelial reduction against Aspergillus niger. The combinatorial

antibacterial activities of the selected three herbs showed excellent results compared to the

individual finishing .(sumithra et al 2003).

The antimicrobial activity of crude ethanolic extracts of 10 medicinal plants used in traditional

Chinese medicine was tested against five species of microorganisms: Bacillus cereus,

Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans. Of

the 10 plants tested, 5 showed antimicrobial activity against one or more species of

microorganisms. The most active antimicrobial plants were Chelidonium majus, Sanguisorba

officinalis, and Tussilago farfar (Janovoska et al.2003).

The methanol, ethanol and aqueous extracts of seven medicinal plants were evaluated for

activity against medically important bacteria such as Staphylococcus sp., Escherichia coli,

Klebsiella sp., Pseudomonas sp. The invitro antimicrobial activity was performed by agar well

diffusion method and disc diffusion method. antimicrobial activity was performed by agar well

diffusion and disc diffusion method. The ethanolic and aqueous extracts showed minimum

antimicrobial activity when compared to methanolic extracts. The methanolic extract

Phyllanthus niruri (stone breaker) showed the maximum activity against Staphylococcus sp.

The use of plant extracts with known antimicrobial properties, can be of great significance in

therapeutic treatments. (Selvamohan et al.2012).

12
To evaluate the antimicrobial activity of medicinal plants against human pathogenic bacteria

and perform Minimum Inhibitory Concentration (MIC) of plant extracts. Methods: Rhizome

of Curcuma longa, dried buds of Synzygium aromaticum, seeds of Zanthoxylum armatum and

leaves of Elaeocarpus ganitrus, Psidium guajava, Azadirachta indica, and Artemisia vulgaris

were collected from hilly regions of Nepal. The plant parts were air-dried at room temperature

and grinded to powder form. The ethanolic extracts of medicinal plants were prepared by

percolation process using separating funnel and tested against human pathogenic bacteria by

disc diffusion method. Then, Minimum Inhibitory Concentration (MIC) of the plant extracts

were determined. Results: All plants extracts exhibited antibacterial properties against bacteria

under study. However, extract from S. aromaticum (Clove), P. guajava (Guava) and E. ganitrus

(Rudraksh) leaves showed most promising result against Staphylococcus aureus with zone of

inhibition of 14mm, 16mm and 16 mm respectively. Likewise, S. aromaticum (Clove), C. longa

(Turmeric) and P. guajava (Guava) showed good antibacterial activity against Escherichia coli

with zone of inhibition of 11mm, 11mm and 10mm respectively. A. vulgaris (Titepati) and A.

indica (Neem leaves) showed promising activity against Pseudomonas aeruginosa with zone

of inhibition of 11mm. Z. armatum (Timur) showed good result against E. coli with zone of

inhibition 10mm. MIC values of ethanolic extracts of S. aromaticum and E. ganitrus were found

to be at the range of 12.5-25mg/ml. Conclusion: This study has helped to understand the use

of these plants as traditional medicine as an economic and safe alternative to treat infectious

diseases. Key words: Plant extracts, antimicrobial activity, zone of inhibition, Minimum

(Sakha et al. 2018).

Medicinal plants are traditionally used for the treatment of human infections. The present study

was undertaken to investigate Bergenia ciliata, Jasminum officinale, and Santalum album for

their potential activity against human bacterial pathogens.B. ciliata, J. officinale, and S.

album extracts were prepared in cold and hot water. The activity of plant extracts and selected

13
antibiotics was evaluated against five bacterial pathogens including Staphylococcus

aureus, Bacillus subtilis, Proteus vulgaris, Pseudomonas aeruginosa, and Escherichia

coli using agar well diffusion method.Among the three medicinal plants, B. ciliata extracts

displayed potential activity against bacterial pathogens. Cold water extract of Bergenia

ciliate showed the highest activity against B. subtilis, which is comparable with a zone of

inhibition exhibited by ceftriaxone and erythromycin. J. officinale and S. album extracts

demonstrated variable antibacterial activity. Further studies are needed to explore the novel

antibacterial bioactive molecules .(Umanali khan et al .2013).

An attempt was made to analyse the antimicrobial potential of 2 medicinally important plants

viz., Lagerstroemia indica and Annona reticulata leaf extracts against human bacterial

pathogens viz., Klebsiella pneumoniae, Staphylococcus auerus, Salmonella typhi, Proteus

vulgaris and Pseudomonas aeruginosa. Antimicrobial study was carried out by disc diffusion

method against the pathogens by using the methanol and aqueous extracts. The result of the

present study showed the presence of wide spectrum of antibacterial activities against all the

above bacterial pathogens studied. The maximum zone of inhibition observed in methanol

extract compared to aqueous extract for each bacterium was as follows. S. typhi (12 mm), K.

Pneumoniae(13 mm), P. vulgaris (20 mm), P. aeruginosa (12 mm) and S. aureus (12mm). The

present study demonstrates that the selected medicinal plants are potentially good sources of

antibacterial against the pathogens viz., K. Pneumoniae, S. auerus, S. typhi, P. vulgaris and P.

aeruginosa. The phytochemical screening demonstrated the presence of different types of

compounds like terpenoids, tannins, deoxy sugars, saponins, phenolic compouns, and

flavonoids which may contribute for the antimicrobial action of the above medicinal plant

(Chandra .2013).

In the present study, soil samples were collected from hospital nearby areas and some bacteria

were isolated and purified by using spread plate and streak plate methods. The six strains named

14
A, B, C, D, E and F were cultured. Cultural, morphological and biochemical characterization

was done to identify theses bacterial isolates. Eighteen Indian medicinal plants were selected

to examine the antimicrobial activity against the cultured species using disk diffusion method.

Methanolic extraction as well as water extract of the plant leaves and roots was prepared and

phytochemical screening of these plants was performed for constituents such as flavonoids,

saponins, glycosides, tannins, Phyto steroid, phenols, alkaloids, etc. Among all the plants Citrus

Limon showed maximum antimicrobial activity (by inhibition zone method) against the

isolated microbes. However, the isolated microbes were susceptible to the methanolic extracts

of the above-mentioned plants. In future, these plants can be further subjected to isolation of

the therapeutic antimicrobials for health purposes and food applications (Mukesh et al.2018).

From the 6 Indian medicinal plants belongs to 3 botanical families were selected on the basis

of their use in the treatment of infectious diseases. Hexane and ethanol extracts of the plants

were studied for their antimicrobial activity against various human pathogenic organism

against Gram -positive bacteria S. aureus, S. epidermitis, B. subtilis, B. cereus, M. luteus, and

Gram -negative E. coli, P. aeruginosa and fungi C. albicans, A. niger was studied by disc

diffusion method. The extracts of S. fruticosa, T.asiatica, T. ciliata showed marked

antimicrobial activity. It is significant to note that though the plant extracts showed activity

against P. aeruginosa they had no activity against the other Gram - negative bacteria E. coli.

Concentration tested was 5 μl of the plant extracts (250 μg /disc) in each sterile filter paper disc

(diameter, 6 mm).(Malairajana et al .2012).

Gnaphalium polycaulon (L.) Pers. (Asteraceae) plant, locally known as Nerabu chedi,

collected from Nilgiri District, Tamil Nadu was subjected to antimicrobial screening and

minimum inhibitory concentration of methanolic extracts of leaf, stem, and flower. The

selected plant used in traditional Indian medicine was examined for antimicrobial activity and

15
minimum inhibitory concentration against human pathogenic bacteria and fungus using the

agar well diffusion method. The antilog of the corresponding value of concentration was taken

as the minimum inhibitory concentration value. Statistical Analysis: All the values of the

results of the assay were expressed as means of triplicates, mean ± standard deviation. The

antimicrobial activity of methanolic leaf extracts of G. polycaulon showed a high level of

antimicrobial activity against the studied bacterial and fungal pathogens. Based on the results

obtained, the medicinal value of this plant could be attributed to the presence of secondary

metabolites in the traditional herbal medicines. Therefore, this antimicrobial activity shows a

source for traditional use of the plant as a local health remedy to the indigenous communities

of Tamil Nadu. Further studies on knowledge of the medicinal plant used medicinally by

indigenous people could lead to further research and new drug discovery for the treatment of

different diseases .(Kaminidevi, et al .2015). The aim of the study was focused on determining

the phytochemicals, antibacterial, antiadherence, antifungal and antioxidant activities

of Glycyrrhiza glabra, Matricaria chamomilla and Eclipta alba and also their mechanism of

action towards human oral pathogens. Qualitative analysis and quantitative estimation of

phenols and flavonoids were performed in methanolic extracts. Antibacterial, anti adherence,

antifungal assays were performed by plate assays. Antioxidant assays were done by ABTS and

DPPH methods. SEM, TEM and flow cytometry analysis were executed to find out the

mechanism of action of plant extract. Â The total phenol contents were 0.85, 1.24, 0.64 GAE/g

and the total flavonoid contents were 356, 231.34 and 88 µg QE/mg for G. glabra, M.

chamomilla and E. alba respectively.Matricaria chamomilla possesses highest antioxidant

activity (DPPH and ABTS assays) among the all extracts tested. E. alba showed a highest zone

of inhibition against S. aureus (21.6 mm) whereas G. glabra and M. chamomilla revealed the

better result of 21 mm and 19.8 mm respectively against S. mutans.Glycyrrhiza glabra showed

antifungal activity against Candida parapsilosis whereas Pseudomonas aeruginosa, Candida

16
krusei, Candida tropicalis and Candida albicans showed resistance towards all the extracts

tested. The MIC, MBC and antiadherence tests were also performed. Sorbitol assay confirmed

that G. glabra has no impact on the fungal cell wall. To confirm the mode of action SEM, TEM

and flow cytometric analysis were performed which showed the cell elongation and damage in

cytoplasmic membrane resulting in oozing of cellular constituents.This work concluded that

all the plant extracts showed potent activities among the various tests. Oral care product can be

developed if the active constituents responsible for the activities were analysed. (Lavanya et

al. 2016).

17
OBJECTIVES

❖ To determine the Minimum inhibitory concentration of the control drug Penicillin

❖ To determine the antimicrobial efficacy of Chloroform extract of Abronia maritima on

clinically important microorganisms by disc diffusion method.

18
MATERIALS AND METHODS

Collection of medicinal plants

The Indian medicinal plant Abronia maritima were collected from the medicinal garden,

Chennai, India. The parts of the plants were authenticated by the botanist

Plant Materials

The Chloroform extract of leaves of the Indian medicinal plant Abronia maritima were used

for this study.

Preparation of medicinal extracts

500 grams of plant powder was taken in round bottom flask for sample extraction using solvent

namely chloroform. The extraction was conducted by 250 ml of the solvent mixture for a period

of 24 hours. At the end of the extraction the respective solvent were concentrated under reduced

pressure and keep it in water bath (at 50°c). Now the extracted experimental solutions were

stored in refrigerator.

Antibiotics

Broad spectrum antibiotics Penicillin was used as control drugs.

Bacterial Strains

The strains of microorganisms (Bacillus cereus (ATCC13061) Shigella flexneri (ATCC 12022)

and Pseudomonas aeruginosa (ATCC 6643) were used. Pure plant cultures were ordered

Antimicrobial Activity determination

Antimicrobial activity was measured using the standard method of diffusion disc plates on agar

and the MIC was calculated using dilution method.

19
Determination of Antimicrobial Activity

Antimicrobial activity was measured using the standard method of diffusion disc plates on agar

and the MIC was calculated using dilution method (Kirby- Bauer method).

Dilution Methods

Dilution susceptibility testing methods were used to determine the minimal concentration of

antimicrobial to inhibit or kill the microorganisms. This was achieved by dilution of

antimicrobial in either agar or broth media. Antimicrobials are generally tested in log2 serial

dilutions (two fold).

Broth Dilution Method

The Broth Dilution Method is a simple procedure for testing a small number of isolates, even

single isolate.

Preparation of microorganisms for experiment

The pure cultures of organisms were sub cultured in nutrient broth. They were inoculated,

separately, into nutrient broth and kept at 37°C for 24 hours. Then, they were kept at 4°C until

use.

Growth Method

At least three to five well-isolated colonies, of the same morphological type, were selected

from an agar plate culture of a particular microorganism. The top of each colony was touched

with a loop, and the growth was transferred into a tube, containing 4 to 5 ml of nutrient broth

medium. The broth culture was incubated at 35°C for 8 hours. After the incubation period broth

culture became turbid.

20
Disc Diffusion Method

Mueller-Hinton Agar Medium Mueller-Hinton Agar is considered to be the best for routine

susceptibility testing of non- fastidious bacteria for the following reasons. It shows acceptable

batch-to-batch reproducibility for susceptibility testing. Medium is transparent, so that the

inhibition zone can be visualized clearly. It gives satisfactory growth of most non fastidious

pathogens. A large body of data and experience has been collected concerning susceptibility

tests performed with this medium.

Preparation of Mueller-Hinton Agar

Mueller-Hinton Agar was prepared from a commercially available dehydrated base according

to the manufacturer’s instructions. Immediately after autoclaving, it was allowed to cool in a

450 to 50°C water bath. The freshly prepared and cooled medium was poured into glass or

plastic, flat-bottomed Petri dishes on a level, horizontal surface to give a uniform depth of

approximately 4 mm. This corresponds to 60 to 70 ml of medium for plates with diameters of

150 mm and 25 to 30 ml for plates with a diameter of 100 mm. The agar medium was allowed

to cool to room temperature and unless the plate is used the same day, stored in a refrigerator.

Plates were used within seven days after preparation unless adequate precautions, such as

wrapping in plastic, have been taken to minimize drying of the agar. A representative sample

of each batch of plates was examined for sterility by incubating at 300 to 35°C for 24 hrs or

longer.

Preparation of antibiotic stock solutions

Powders of the two antibiotics (Penicillin) were brought from authorized medical shop. They

were accurately weighted and dissolved in sterile distilled water in appropriate dilutions to

21
yield the required concentrations. The stocks were kept in aliquots of 5 ml volumes and frozen

at -20°C.

Preparation of plant extracts solutions for the experiment

The dried plant extracts were weighed and dissolved in sterile distilled water to prepare

appropriate dilution to get required concentrations (1.0mg/ ml, 1.5mg/ ml, 2.0 mg/ ml, and 2.5

mg/ ml). They are kept under refrigeration.

Preparation of dried filter paper discs

Whattman filter paper (No.1) was used to prepare discs approximately 6 mm in diameter, which

are placed in a Petri dish and sterilized in a hot air oven. After the sterilization, the discs were

poured into the different concentration of broad spectrum antibiotics and into the prepared plant

extract solutions and again kept under refrigeration for 24 hrs.

22
RESULTS AND DISCUSION

Plant based molecules are getting the vital importance in the field of antimicrobial treatment

and most of the pharma companies are directly or indirectly depends on plant-based materials

(Selvakumar, S, 2015) he extracts of plant parts or components provide the new hope for drug

development and discoveries Harborne.1973. Therefore, we would like to investigate the

antimicrobial properties of chloroform extract of Abronia maritima were undertaken.

Figure 1: Effects of Penicillin and Plant extract on Bacillus cereus

30

25

20

Concentration
15
Penicillin
Plant extract
10

0
1 2 3 4 5 6

Reading of Minimum Inhibition Concentration

Minimum inhibition concentration was expressed as the lowest dilution which inhibited growth

judged by lack of turbidity in the tube, because very faint turbidity might be given by the

inoculums itself. The inoculate tube was kept in the refrigerator overnight and was used as the

standard for the determination of complete inhibition. The plant extract were found to be

effective against the three selected bacterial species.

23
Reading Zone of Inhibition and Interpreting Results.

After 16 to 18 hrs of incubation each plate was examined. Once the resulting zones of inhibition

came uniformly circular and in a confluent lawn of growth, the diameters of the zone of

complete inhibition (as judged by the unaided eye) are measured, including the diameter of the

disc. Zones are measured to the nearest mm using a ruler, which was held on the back of the

inverted Petri plate. Clear inhibition zones indicated the presence of antimicrobial activity

(Kone et al 2004). The use of medicinal plants in the world and especially in India, contribute

significantly to primary health care. The antimicrobial medicinal plants are well documented.

The results of different studies provide evidence that some medicinal plants might indeed be

potential sources of new antibacterial agent even against some antibiotic resistant strains .In

(Shelef ,2011). the present study reveals the chloroform extract from Abronia maritima possess

antimicrobial activities against Bacillus cereus, Shigella flexneri and Pseudomonas

aeruginosa.

Figure 2: The antibacterial effects of antibiotics and plant extract on Shigella flexneri

30

25

20
Concentration
15
Penicillin
Plant extract
10

0
1 2 3 4 5 6

24
Figure 1, 2 and 3 represents the antimicrobial effects of the control drug penicillin and

chloroform extract of Abronia maritima were analysed. The extract compared favourably with

the standard antibiotics Penicillin. The plant extract exhibited broad spectrum antibiotic

activities. The results indicated that the extract of A.maritima has stronger activity than that of

standard antibiotics. Since ancient times, herbs and /or their essential oils have been known for

their varying degrees of antimicrobial activities Oyaizu.1986. More recently, medicinal plant

extracts were developed and proposed for use in food as natural antimicrobials. Antimicrobials

are powerful but controversial tools. Food animals are often exposed to antimicrobial

compounds to treat or prevent infectious diseases and/or to promote growth. The early history

of supplementing animals feeds with

Figure 3: Antibacterial Activity of Penicillin and Plant extract on Pseudomonas

aeroginosa

30

25

20

Concentration
15
Penicillin
Plant extract
10

0
1 2 3 4 5 6

antimicrobials parallels the isolation, identification and characterization of Vitamin B12 in

1948. Further research in this area showed that several feed ingredients, including dried

25
mycelia of certain fungi, were more potent as growth promoters in the diet of chicks than was

vitamin B12 alone. The reducing capacity of Mucuna pruriens was observed by the

transformation of Fe3+ to Fe2+ ions (Hatano et al.1983).

Thus this plant can serve as a potential indicator of its antioxidant capacity. Phenols are very

important plant constituents and have scavenging ability due to their hydroxyl groups. The

phenolic compounds may contribute directly to the antioxidant action. It was suggested that

polyphenolic compounds have inhibitory effects on mutagenesis and carcinogenesis in

humans, when ingested up to 1 g daily from a diet rich in fruits and vegetables. Except for

Schigellaflexineri, Bacillus anthracis and Pseudomonas aeruginosa, Enterococci may be

resistant to Ampicilline and Penicillin because of production of low affinity Penicillin binding

protein (PBPs) or less commonly because of the production of β Lactamase. The disc diffusion

test can accurately detect isolates with altered Penicillin binding proteins but it will not reliably

detect β-lactamase producing strains. The rare β lactamase producing stains are detected best

by using a direct, nitrocefin based β-lactamase test (CaiYun et al 2007.) Certain Penicillin,

Ampicilline resistance Enterococci may possess high level of resistant. The disc diffusion test

can not differentiate those with normal resistance from this high level resistance.

26
CONCLUSION

In conclusion, the chloroform exhibits antimicrobial activities and results were compared to to

broad spectrum antibiotic like penicillin. Our results indicate that the plant extract has the

significant potential against Pseudomonas aeroginosa and Shigella flexneri. On the basis of

the results obtained in the present study, we conclude that the chloroform extract of Abronia

maritima has significant antimicrobial activity. Further studies are on to isolate the active

principles responsible for their antimicrobial activities.

27
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