Abinaya

“ASSESSING THE ANTIMICROBIAL EFFICACY OF Prosopis juliflora AGAINST
ORAL MICROORGANISMS: AN INVITRO STUDY”
A dissertation submitted
In partial fulfillment of the requirements
For the degree of
MASTER OF DENTAL SURGERY
BRANCH VII
PUBLIC HEALTH DENTISTRY
THE TAMILNADU Dr.M.G.R. MEDICAL UNIVERSITY

CHENNAI – 600032
2017 - 2020
Scanned by TapScanner
URKUND MAIN
URKUND
Urkund Analysis Result

Analysed Document: MAIN DISSERTATION ABINAYA.pdf
(D61033195) Submitted: 12/14/2019 3:39:00 AM
Submitted By: abivardhini@gmail.com
Significance: 4%
Sources included in the report:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3298580/
https://ri.conicet.gov.ar/bitstream/handle/11336/49781/CONICET_Digital_Nro.10bd9fd6-
cf32-4c05-8e8c-5b2f85359cc3_A.pdf?sequence=2&isAllowed=y
https://www.opensciencepublications.com/fulltextarticles/JPSR-2349-2805-6-184.html
Instances where selected sources appear:

12
1
ACKNOWLEDGEMENT
I thank God Almighty by whose grace this work became possible.
I would like to thank all those people who helped me directly or
indirectly to complete my Main Dissertation. I am deeply inherited to those who devoted
their precious time in giving me support and guidance at every point of time. I am
overwhelmed with humbleness and gratefulness to acknowledge my depth to all those who
have helped me in completing this Main Dissertation.
My sincere thanks to Prof K.R.Arumugam and Vice chairman
Babu Thandapani for giving me an opportunity to gain knowledge through this
institution.
My acknowledgement would never be complete without mentioning my
Principal, Dr.K.S.Premkumar sir, Professor and Head, Department of Orthodontics and
Dentofacial Orthopaedics who has guided me like a father and supported me like a brother.
I have seen a strict side of sir’s when I do mistakes but at the same time he never forgets
to appreciate any good work. I owe him a lot, he is one of the reasons for I am what I am
today and words cannot express my gratitude, “I am proud to be his student”
From an Undergraduate to Postgraduate the transition though was hard at first I extend my
heartfelt gratitude to my mentors Dr.Umesh.K, Dr.Muthukaruppaiah.R, Dr.Sangeeta
Chavan and Dr.Palanivel Pandian.R
I extend my deepest gratitude to Dr.Umesh.K sir, Professor and Head, Department of
Public health dentistry for the constant support and guidance. He is A MAN OF
SIMPLICITY. Analyzing and Adapting, not just in public health dentistry but in general is
ACKNOWLEDGEMENT
an art and sir is a Picasso in it. I am in the process of learning these lessons for life to
humanity and it is my privilege to have him as my head.
I am immensely thankful to Dr.Muthukaruppaiah.R Sir, Reader and my guide for
inspiring me in all the works of mine. Like a Magnets attraction to Iron, Like an Alginates
attraction to water, he has a thirst for newer knowledge. I wanted to learn newer things
only after looking at sir. If I come out with a different ideology and concept it is because
of sir.
My sincere thanks to Dr.Sangeeta Chavan ma’am, Reader, Department of Public health
dentistry for all the guidance she had given me in all the ventures of mine. She is a
perfectionist and a creativist. Anyone can do a good work but ma’am turns it to a perfect
one. In all works of mine I look after a touch of ma’am’s magic in it and when it happens
my confidence increases.
My sincere thanks to Dr.Palanivel Pandian.R sir for being supportive since the beginning
of not just this work but in every work of mine. Whenever I look at sir I wonder how a
person can be so humble. Sir is an energetic, spiritual and an easily approachable person
who is friendly in nature.
At this moment I would like to express my gratitude to all my seniors who had all thought
me what a Post-Graduation life is. Their support and guidance is yet another reason for me
to keep striving for better, a special mention about Dr.Prathap for helping me during the
initial days of this work and Dr.Shobana for her inputs. I also thank my friend and my
senior Dr.DheepthaSri and my fellow Co-PGs Dr. Lavanya Rahavi and Dr. Shrimathi,
my juniors & Brothers Dr.Ravi Sankar, Dr.Sasidharan & Dr.Selvamani for all the help.
ACKNOWLEDGEMENT
Last but not the least I would like to acknowledge my family members for the support and
positive energy which they had given me and to my daughter ESHA ADITI for being so
cooperative even though she has sacrificed most of her time with her mother, you are my
life,
Love you dear!

ABSTRACT
INTRODUCTION: Oral health is integral to the general well-being and
relates to the quality of life. There exists great achievements in the oral health of
populations worldwide despite which problems persists in many communities
around the world, predominantly among underprivileged groups in both developed
and developing countries. There is a rising need for safer alternatives due to the
evolving multiple drug resistance. Herbal drugs could be one such as it plays an
important role in health care. Therefore this study was conducted to investigate the
antimicrobial activity of the Prosopis juliflora leaves against Streptococcus mutans,
Lactobacillus, Aggregatibacter actinomycetemcomitans and Candida albicans.
MATERIALS AND METHODS: An In-vitro, Experimental study was
designed to assess the antimicrobial efficacy of Prosopis juliflora against oral
microorganisms. Plant extract preparation was done using Maceration technique
and in-vitro antibacterial assay was conducted to determine the zone of inhibition
and minimum inhibitory concentration using Disc diffusion method and Broth
dilution method.
RESULTS: Prosopis juliflora showed inhibitory action compared to and
superior to that positive control with a minimum inhibitory concentration of
0.005% for Streptococcus mutans, 0.005% for Lactobacillus, 0.01% for
Aggregatibacter actinomycetemcomitans and 0.005% for Candida albicans.
CONCLUSION: The present study concludes that, Prosopis juliflora leaves
from Madurai, TamilNadu is an appropriate source for antibacterial and antifungal
components as it showed notable antibacterial and antifungal activity. This plant

ABSTRACT
can be used in various forms that meets the therapeutic needs. Therefore, the
present study may provide a scientific basis for the development of novel, safer and
clinically effective medicines for both systemic and topical usage.
Keywords: Antimicrobial agent, Medicinal plant, Oral Health, Oral
Microorganisms, Prosopis juliflora

LIST OF ABBREVIATIONS
S.NO ABBREVIATION DESCRIPTION
1 WHO WORLD HEALTH ORGANISATION
2. ICFRE INDIAN COUNCIL OF FORESTRY
RESEARCH & EDUCATION
3. MDR MULTI DRUG RESISTANT
4. MRSA METHICILLIN RESISTANT
Staphylococcus aureus
5. TAE TOTAL ALKALOID EXTRACTED
6. MIC MINIMUM INHIBITORY
CONCENTRATION
7. PPM PARTS PER MILLION
8. UV ULTRA VIOLET
LIST OF TABLES
TABLE. TITLE PAGE
NO NO.
1 ZONE OF INHIBITION OF Prosopis juliflora 52
AGAINST Streptococcus mutans
2. ZONE OF INHIBITION OF Prosopis juliflora 53
AGAINST Lactobacillus
AGAINST Aggregatibacter actinomycetemcomitans
AGAINST Candida albicans
5. COMPARISON OF ANTIBACTERIAL 56
ACTIVITY OF Prosopis juliflora,
CHLORHEXIDINE, POVIDONE IODINE AND
ETHANOL AGAINST Streptococcus mutans.
ETHANOL AGAINST Lactobacillus.

LIST OF TABLES
ETHANOL AGAINST Aggregatibacter
actinomycetemcomitans
8. COMPARISON OF ANTIFUNGAL ACTIVITY 62
OF Prosopis juliflora, CLOTRIMAZOLE,
NYSTATIN AND ETHANOL AGAINST Candida
albicans
9. DETERMINATION OF MINIMUM INHIBITORY 64
CONCENTRATION AGAINST VARIOUS
MICROORGANISMS.
LIST OF GRAPHS
GRAPH TITLE PAGE
NO NO.
1 COMPARISON OF ANTIBACTERIAL 56
ACTIVITY OF TEST AND CONTROL
AGENTS AGAINST Streptococcus mutans
AGENTS AGAINST Lactobacillus
AGENTS AGAINST Aggregatibacter
4. COMPARISON OF ANTIFUNGAL 62
AGENTS AGAINST Candida albicans
5. MINIMUM INHIBITORY 65
CONCENTRATION AGAINST VARIOUS
MICROORGANISMS
LIST OF FIGURES
FIGURE. TITLE PAGE
NO NO
1 Prosopis juliflora SHRUB 37
2 COLLECTED AND DRIED LEAVES 38
3 POWDERED LEAVES 38
4 POWDERED LEAVES DISSOLVED IN 39

ETHANOL
5 MAGNETIC STIRROR FOR CONSTANT 39

STIRRING
6A FILTRATION OF THE SUPERNATANT 40
6B FILTRATION OF SUPERNATANT 40
7 PRODUCT OBTAINED FROM MACERATION 41
8 ROTARY FLASH EVAPORATOR 41
9. GLASS CONTAINER FOR EXTRACT 42

COLLECTION
10. VACCINE CARRIER FOR TRANSPORTATION 42

OF THE EXTRACT TO THE LABORATORY
11. VARIOUS CONCENTRATIONS OF THE 43

EXTRACT PREPARED
12. ANTIBACTERIAL ACTIVITY OF TEST AND 44

CONTROL AGENTS AGAINST Streptococcus
mutans AT BASELINE AND AFTER 24 HOURS

CONTROL AGENTS AGAINST Lactobacillus AT
BASELINE AND AFTER 24 HOURS
LIST OF FIGURES

CONTROL AGENTS AGAINST Aggregatibacter
actinomycetemcomitans AT BASELINE AND
AFTER 24 HOURS.
15. ANTIFUNGAL ACTIVITY OF TEST AND 47

CONTROL AGENTS AGAINST Candida albicans
AT BASELINE AND AFTER 24-48 HOURS
16. MIC DETERMINATION OF Prosopis juliflora 48

AGAINST Streptococcus mutans

AGAINST Lactobacillus

AGAINST Aggregatibacter actinomycetemcomitans

20. MIC DETERMINATION OF CONTROL AND 50

BLANK SOLUTIONS AGAINST BACTERIAE
21. MIC DETERMINATION OF CONTROL AND 50

BLANK SOLUTIONS AGAINST FUNGI
CONTENTS
SL.NO TITLE PAGE

N0
1 INTRODUCTION 1
2 AIM AND OBJECTIVES 5
3 REVIEW OF LITERATURE 6
4 MATERIALS AND METHODS 23
5 PHOTOGRAPHS 37
6 RESULTS 51
7 DISCUSSION 67
8 CONCLUSION & 73
RECOMMENDATIONS
10 REFERENCES -
11 ANNEXURES -
LIST OF ANNEXURES
S.NO ANNEXURES
I ETHICAL COMMITTEE APPROVAL LETTER
II PERMISSION LETTER FROM PHARMACY COLLEGE
III PLANT IDENTIFICATION CERTIFICATE
IV PERMISSION FROM LABORATORY
V LABORATORY REPORT FRONT PAGE
VI PERMISSION FROM HEAD OF THE INSTITUTION

INTRODUCTION
Oral health is integral to the general well-being and relates to the quality
of life. Oral and general health is interrelated and it is proven by evidence, the
strong association between various oral diseases and non-communicable chronic
diseases is primarily a result of the common risk factors. Furthermore, many
general disease conditions have oral manifestations that increase the risk of oral
disease which, in turn, is a risk factor for a number of general health conditions. 1
There exists great achievements in the oral health of populations
worldwide despite which problems still persist in many communities around the
world, predominantly among underprivileged groups in both developed and
developing countries. The major oral diseases, Dental caries and periodontal
diseases have historically been considered the most important ones.1 Caries process
is largely influenced by Streptococcus mutans and Lactobacillus2, periodontitis by
various organisms such as Porphyromonas gingivalis, Prevotella intermedia,
Bacteroides forsythus, Campylobacter rectus, and Actinobacillus
actinomycetemcomitans, as well as the Treponemes3. Oral manifestations like
candida infections caused by Candida albicans occur frequently apart from dental
caries and periodontal infection.1,4
Oral diseases are major public health problems as its impact on an
individual level and at community level, owing to pain and suffering, impairment
of function and reduced quality of life, is considerable. Furthermore, traditional
treatment of oral disease is extremely costly being the fourth most expensive
disease to treat in most industrialized countries. It is said that prevention is better
than cure which also applies to oral diseases as prevention of oral diseases is easier.
1
INTRODUCTION
The WHO Global Strategy for the prevention and control of non-communicable
diseases is a new approach in managing the prevention and control of oral diseases.1
Antibiotics have revolutionized medicine in many aspects and
countless lives have been saved since their discovery. Undesirably, the use of these
chemical drugs has been accompanied by the rapid appearance of resistant strains.
The successful use of any therapeutic agent is compromised by the potential
development of tolerance or resistance to that compound from the time it is first
employed, it is true for agents used in the treatment of bacterial, fungal, parasitic,
and viral infections.5
There is a rising need for alternatives which are safer due to the
evolving multiple drug resistance. Herbal drugs could be one such as it plays an
important role in health care. Their use in developing countries is especially
because they are economical, locally available and considered being safe.
Medicinal plants acts as authentic medicines because of the bioactive medicinal
compounds present in them.6 A medicinal plant is any plant which, in one or more
of its organs, contains substances that can be used for therapeutic purpose or which
are precursors for the synthesis of useful drugs.7 Man has been using plants to treat
common infectious diseases, and are still in use. Scientific interest in medicinal
plant has prospered in recent times as a result of improved efficiency of new plant
derived drugs and growing concerns about the side effects of modern medicine. The
continuing emergence of drug resistant organisms and the increasing evolutionary
adaptations by pathogenic organisms to commonly used antimicrobials have
reduced the efficacy of antimicrobial agents currently in use. Therefore, the search
2
INTRODUCTION
for new drugs from plants continues to be a major source of commercially
consumed drugs.8
Prosopis juliflora is commonly known as mesquite and in tamil it is
Seemai karuvelam, in hindi it is Angaraji babul, in Telugu, Mulla tumma and in
Kannada, Ballaari jaali.9 It belongs to the family Fabaceae. It is a fast growing,
thorny deciduous, drought-resistant plant having wide crown and deep-roots. It is
an invader species in India that competes with the native species. It grows in all
kinds of soil conditions, including wastelands. The herb is well-known in the
folkloric system of medicine because of its ethnobotanical importance. The high-
potential activity of these extracts compared to selective antibiotics could lead to
new antimicrobial agents which would fight against the drug-resistant pathogens.
Recent studies showed that the plant can be used in various therapeutic applications
because of its non-toxic effects ensuring its quality and safety and can be used in
the formulation of several pharmacologically active compounds.10
The plant has been described to treat oral ailments like toothache.
The leaves were used against blood and venereal diseases.10 Water extracts of
leaves and bark are traditionally used to cure mouth and throat infections,
bronchitis and ulcers; internal diseases including parasites and urinary diseases;
skin parasitic infections as well as dermatitis. As early as 1993, The Indian Council
of Forestry Research and Education (ICFRE) reported that in Asia, medicinal uses
of native Prosopis species included their flowers for the prevention
of miscarriage and bark extracts for the treatment of
bronchitis, leucoderma, tremors, asthma, rheumatism, leprosy, and dysentery.
3
INTRODUCTION
Leaf smoke is traditionally used to cure eye infections and extracts are
recommended for use against snakebite and scorpion sting.11
Advanced scientific technologies demonstrated several medicinal
properties of these species, such as antioxidant hepato-protective, hemolytic,
anticancer, antibacterial, antifungal, antidiabetic, and anti-inflammatory activities.
Alkaloids, flavonoids, terpenes, and phenolic compounds are the most important
bioactive substances of the species. Terpenes have properties which includes
antibacterial, antifungal, antihelminthic, antimalarial, and molluscicidal activities.
Phenolic compounds show anti-inflammatory, antitumor, anti-HIV, anti-infective,
vasodilatory, antiulcerogenic analgesic, and immunostimulant activities.
Flavonoids are of recent interest due to the discovery of their pharmacological
activities. Alkaloids are applied as analgesics and antimalarial agents, it also
demonstrates a broad spectrum of antifungal activities. Flavonol
glycosides and hydroxycinnamic acids from the pollens of Prosopis
juliflora provide antioxidant properties. The plant Prosopis juliflora is found in
abundant and would be a good cost effective alternative apart from its numerous
properties. 11
Owing to the properties of Prosopis juliflora and considering the
need for one agent effective against a range of organisms which is also cost
effective, an in-vitro study was carried out in the untraveled path to investigate the
antimicrobial activity of the Prosopis juliflora leaves against Streptococcus mutans,
Lactobacillus, Aggregatibacter actinomycetemcomitans and Candida albicans.
4
AIM & OBJECTIVES
AIM
To assess the antimicrobial efficacy of different concentrations of Prosopis
juliflora leaf extract against common oral pathogens.
OBJECTIVES
 To assess the antimicrobial efficacy of ethanolic leaf extract of Prosopis
juliflora against Streptococcus mutans
juliflora against Lactobacillus
juliflora against Aggregatibacter actinomycetemcomitans
juliflora against Candida albicans
 To assess the antimicrobial efficacy of Chlorhexidine 0.2% & Povidone
iodine 10% against Streptococcus mutans, Lactobacillus,
Aggregatibacter actinomycetemcomitans.
 To assess the antimicrobial efficacy of Clotrimazole 1% & Nystatin
100000 units against Candida albicans
 To compare the antimicrobial efficacy of Prosopis juliflora with the
positive and negative controls.
5
REVIEW OF LITERATURE
Bragg LH et al in 1978 conducted a study on Flavonoid Patterns in the Prosopis
juliflora Complex. The results showed that five species in the Prosopis juliflora
complex, as well as two varieties and putative hybrid, were found to have similar
flavonoid patterns. A total of 12 major compounds were identified and 21
flavonoids were detected. Also there was no flavonoid correlations with ecotypes.12
Ahmed A et al in 1985 conducted a study on antibacterial activity of juliflorine
isolated from Prosopis juliflora. Juliflorine is an alkaloid which has shown to
possess antibacterial activity. The activity was tested invitro against six gram
positive and ten gram negative bacteria. The activity was compared with standard
antibiotics penicillin, streptomycin, erythromycin, sulphamethoxazole, ampicillin
and tetracycline. Juliflorine was found to be effective against Streptococcus
pyogenes, Staphylococcus aureus, Corynebacterium diphtheriae, C.hofmanni and
Bacillus subtilis among the gram positive organisms. Ampicillin and Juliflorine
were the only agents found effective against Streptococcus faecalis, as it was found
resistant to all other antibiotics. Juliflorine did not show any inhibitory action
against Salmonella, Shigella, Proteus, Pseudomonas, Klebsiella and E.coli.13
Ahmad VU et al in 1989 conducted a study on alkaloids from Prosopis juliflora.
Comprehensive investigations were carried out. From studying the fresh leaves it
was reported that two new alkaloids juliprosinene and juliflorinine were isolated.
Preliminary screening of Juliprosinene showed antibacterial activity against
6
Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Shigella
sonnei.14
Hebbar SS et al in 2003 conducted a survey on Ethnomedicine of Dharwad district
about plants used in oral health care. The survey covered Dharwad district of
Karnataka in southern India. From the survey it was revealed that 35 plants
belonging to 26 families were being used to treat different types of oral ailments
like toothache, plaque and caries, pyorrhea and aphthae. From which sixteen plants
were new claims for the treatment of oral ailments which were not previously
reported in the ethnomedicinal literature of India. Those include Basella alba,
Blepharis repens, Capparis sepiaria, Oxalis corniculata and Ricinus communis for
the treatment of aphthae; Azima tetracantha, Caesalpinia coriaria, Cleome
gynandra, Gossypium herbacium, Leucas aspera, Merremia chryseides,
Pergularia daemia, Prosopis juliflora and Solanum nigrum for the treatment of
tooth ache and Cassia hirsuta and Cassia tora for the management of plaque and
caries.15
Gibbons S in 2005 in a review on plants as a source of bacterial resistance
modulators and anti-infective agents has mentioned that the spread of multidrug-
resistant (MDR) strains of bacteria imposes the need for the discovery of new
classes of antibacterial and compounds that inhibit these resistance mechanisms.
The review has mentioned that there are no single chemical entity plant-derived
antibacterial used clinically, and this chemically diverse group justifies
7
consideration as a source for two major reasons. First, plants have remarkable
ability to produce cytotoxic agents and second there is an ecological rationale that
antimicrobial natural products should be present or synthesized in plants. Strains of
methicillin-resistant Staphylococcus aureus (MRSA), is a major clinical problem
at present. The review of literature on plant derived bacterial resistance modifying
agents and antibacterial suggest that there are many potential new classes of
antibacterial agents which should undergo further cytotoxicity, microbial
specificity and preclinical studies. 16
Silva AD et al in 2007 conducted a study on Prosopis juliflora as intoxication with
the plant has been reported, and is characterized by neuromuscular alterations and
gliosis. The study was done to check for such an activity and the results showed
that TAE and fractionated alkaloids from P. juliflora act directly on glial cells,
inducing activation and/or cytotoxicity, stimulating no production, and may have
an impact on neuronal damages observed on intoxicated animals. 17
Sathiya M and Muthuchelian K in 2008 conducted an invitro study on
investigation of phytochemical profile and antibacterial potential of ethanolic leaf
extract of Prosopis juliflora. In vitro antibacterial studies on the ethanolic leaf
extracts were carried out on ten medically important bacterial strains. The results
revealed that the extract had good inhibitory activity against all the tested pathogens
compared with standard antibiotics like streptomycin and penicillin.8
8
Hari Prasad O et al in 2011, conducted a study to compare the antibacterial
activity of Prosopis juliflora with three commercially available mouth rinses on
oral and periodontal pathogens. MIC was determined for antibacterial activity and
results were obtained. Results showed that the activity of Prosopis juliflora was an
effective antibacterial agent against all tested organisms and it was highest in
comparison with other commercial mouth rinses.18
Abdallah EM in 2011 reported a review titled Plants: An alternative source for
antimicrobials. The review has included that though there is no doubt that
antibiotics are a miracle drugs they stand against various infectious diseases for
decades and has saved millions of lives and is saving. Yet, the recent failure of
antibiotics due to the dramatic emergence of multidrug resistant pathogens and the
rapid spread of the new infections, urge the health organizations and
pharmaceutical industries all over the world to change their strategy and to stop
going on the slow growing production of more synthetic antibiotics against the fast
growing antibiotics-resistant microorganisms. There are considerable alternative
sources of natural antimicrobials from plants with different mode of actions and
some of them are employed in traditional medicine for centuries. The review
presented the potency of plants as alternative source for antimicrobials. 19
Sathiya M, Muthuchelian K et al in 2011 conducted an Invitro study on Anti-
tumor potential of total alkaloid extract of Prosopis juliflora leaves against Molt-4
cells. The total alkaloid extract from the plant leaves was obtained using acid/base
9
modified extraction method. The anti-tumor potential of the extract based on
cytotoxicity monitoring after 24, 48 and 72 hours of exposure of the MOLT-4 cells
was evaluated using MTT (3-(4,5- dimethythiazol-2yl)2,5-diphenyl tetrazolium
bromide). Cytokinesis block micronucleus assay was used to assess the genotoxic
potential of the extract. The cytotoxic and genotoxic potential of the extract were
compared with mitogen stimulated T-lymphocyte cultures derived from peripheral
blood of healthy volunteers was done simultaneously. The test showed that, the
extract exhibited higher toxicity towards the cancer cells than the normal cells at
all the time points and at all the tested concentrations. The IC 50 values of the extract
at 24, 48 and 72 hours were found to be 90.5, 42.5 and 20.0 μg/ml/1× 106 cells
against cancer cells. The genotoxic assay showed that even at the highest exposure
concentration tested it was very low when compared with that of the micronuclei
obtained. Therefore the results demonstrate that, P. juliflora leaf alkaloids are
preferentially cytotoxic to human T-cell leukemia (Molt-4) cells in a dose and time
dependent manner with the absence of genotoxicity.20
Singh S et al in 2011 conducted a study to assess the antibacterial properties of
Alkaloid rich fractions obtained from various parts of Prosopis juliflora. The
antibacterial property were assessed using disc diffusion method on several Gram-
negative and Gram-positive bacterial strains like E.coli, Staphylococcus aureus,
Bacillus cereus, Psuedomonas putida, Klebsiella, Salmonella, Acinetobacter and
Alcaligen. Strong antibacterial effect was shown by leaf, pod and flower extract,
with MIC value ranging between 25μg/ml-100μg/ml. The extracts of the leaves
10
showed highest activity among all the plant parts. The most susceptible bacteria
was klebsiella, whereas the least susceptible were Acinetobacter and Alcaligen. The
antibiotics used for comparison were the standard antibiotics, ampicillin,
tetracycline, chloramphenicol, oflaxacin, refampin, streptomycin and sulfa drug. It
showed a comparable zone of inhibition. The growth of Acinetobacter and
Alcaligen were not inhibited by antibiotics but it showed inhibition by the alkaloidal
extracts. Likewise a known ampicillin resistant E.coli strain was found to be
inhibited by the plant extracts. The extracts were analysed by DART-MS to check
for the alkaloids and it showed the presence of piperidine alkaloids.21
Napar AA et al in 2012 conducted a study to assess the antimicrobial and
antioxidant activities of Mimosaceae pants. The study was done using the
methanolic leaf extracts of three plant species of family Mimosaceae viz., Acacia
modesta, Prosopis cineraria and Prosopis juliflora were used to evaluate their
antibacterial, antifungal and antioxidant activity. The method used for the
preparation of plant extracts was Simple maceration method and the extracts were
tested against four strains of bacteria (Bacillus subtilis, Escherichia coli, Vibrio
cholera and Enterobacter aerogenes) and two strains of fungi (Aspergillus niger
and Aspergillus fumigatus). It was found that at 15 mg/ml extract concentration the
maximum inhibitory zones observed in P. juliflora was 25 mm and it showed 7.69%
inhibition against A. niger. The Antioxidant activities of these medicinal plants also
showed significant results with maximum radical scavenging activity observed in
P. cineraria and P. juliflora. 22
11
Prabha DS et al in 2012 conducted a study to assess the acute and sub-acute oral
toxicity of ethanolic extracts of Prosopis juliflora on Rattus norvegicus. The study
was aimed to ensure the quality and safety of a traditional medicinal plant, Prosopis
juliflora. Therefore acute and subacute oral toxicity of the ethanolic extract of
Prosopis juliflora on Wistar rats was investigated. P. juliflora extract was
administered orally at doses ranging from 50-500 mg kg-1 for acute toxicity and
the animals were observed for any toxic symptoms for 72 hrs. The results did not
demonstrate any toxic symptoms below a dose level of 200 mg kg-1. The subacute
toxicity study was done with the ethanolic extracts of P. juliflora. The extracts were
tested at a dose of 200 mg kg-1 orally once daily for 30 days. On 31st day the
animals were sacrificed, the blood & serum samples, collected and analyzed for
various biochemical parameters. The results of the subacute toxicity study in
experimental animals did not show any changes in hematological, biochemical,
renal and liver function parameters when compared to control animals. It was
concluded from the results that the ethanolic extracts of P. juliflora were non-toxic
and are suitable for additional long term in vivo studies of pharmacological
effects.23
Ahmed SM, et al in 2012 conducted a study titled acute systemic toxicity of four
Mimosaceous plants leaves in mice as the medicinal plants can cause some serious
damaging effects on vital organs of the body. Toxicity studies provide their safe
use in both humans and animals. The present study was conducted using methanolic
extracts of four Mimosaceous plants including Prosopis juliflora. It was concluded
12
that at 100mg/kg body weight intraperitoneally the Mimosaceous plants is
relatively safe. 6
Naji.T in 2012, conducted a study on antibacterial effects of Prosopis juliflora in
Iran. The study was done to assess the invitro antibacterial activity of Prosopis
juliflora against six gram positive and negative bacteria. The methods used for the
same were paper disk diffusion and well methods. For this purpose methanolic
extract at different concentrations (0.05, 0.1, 0.2, 0.3, 0.4 mg/ml) was obtained. The
extract was tested against Staphylococus aureus, Micrococcus luteus, Bacillus
cereus, Shigella sonee, Pseudomonas aeruginosa and Escherichia coli were tested.
Comparison were made with Amoxicillin and Methicin. Results showed that the
organism with maximum resistant against plant extract is Pseudomonas aureginosa
and the least resistant is Micrococcus luteus. 24
Choudhar P K , Nagori B P in 2013 conducted a study to phytochemically screen
and evaluate the anti-inflammatory potency of the ethanolic extract of leaves of
Prosopis juliflora at various concentrations of 100, 200, and 400 mg/kg against
carrageenan induced paw edema in rats. Presence of flavonoids, saponins,
carbohydrates, cardiac glycosides, tannins, and alkaloids were revealed in the
preliminary phytochemical screening of ethanolic extract of the leaves of Prosopis
juliflora. The oral median lethal dose was found to be 3807.9 mg/kg in mice and >
5000 mg/kg in rats. The result showed that the extract significantly attenuated the
paw edema with the highest activity observed at dose of 400 mg/kg, supporting the
13
folkloric claim of the use of Prosopis juliflora in the management of
inflammations.25
Ibrahim M et al in 2013 conducted phytochemical analysis of Prosopis juliflora
for which air dried leaves of Prosopis juliflora commonly known as Vilayati Kikar
was analyzed for its chemical composition. The results revealed the quantitative
analysis of natural constituents. It was found that flavonoids, alkaloids, saponins,
phenols and tannins in a range of (16 ± 0.39), (3.6 ± 0.06), (2.2 ± 0.23), (0.66 ±
0.11) and (0.33 ± 0.07) % were present in the leaves. The crude fiber and ash
content were also analyzed. It was found that (17.5 ± 0.14) and (9.5 ± 0.08) % was
the content of crude and ash respectively. The pectic substances were calculated as
(4.9 ± 0.18) %. 26
Thakur R et al in 2014, conducted an in-vitro antibacterial activity of Prosopis
juliflora which was evaluated against ten bacterial type cultures by agar diffusion
assay. The crude extracts were prepared by two methods separately with three
different solvents. Varying degrees of growth inhibition was shown by all the
fractions against tested organisms. The highest antibacterial activity was observed
in aqueous fractions as compared to solvent fractions 10.
Garbi MI et al in 2014 conducted a study to assess the antigiardial, amoebicidal
and cytotoxic activity of the plant Prosopis juliflora leave extracts. The study was
conducted using extract obtained from petroleum ether and methanolic extracts in
14
vitro. It was then tested using four concentrations: (1000 ppm, 500 ppm, 250 ppm
and 125 ppm). The highest activity against Giardia lamblia was obtained from
petroleum ether extract exhibiting 78.91% mortality within 72 h at a concentration
of 500 ppm followed by the methanolic extract exhibiting 77.48% mortality within
72 h at a concentration of 1000 ppm. Conversely the lowest antigiardial activity
was recorded by petroleum ether extract 38.55 % mortality at 1000 ppm
concentration in 24 hours. The highest activity against Entamoba histolytica was
obtained from methanolic extract exhibiting 71.97% mortality within 72 h at a
concentration of 1000 ppm. In contrast the lowest antiamoebic activity was
recorded by petroleum ether extract 31.88% mortality at 125 ppm concentration
within 24 hours. The cytotoxicity of methanol and petroleum ether extract had
varying degrees of toxicity.27
Prabha D S et al in 2014 presented a review on pharmacological potentials of
phenolic compounds from Prosopis compounds. It was stated that the plant
possesses several medicinal properties due to the presence of different chemical
compounds such as alkaloids, flavonoids, terpenoids, saponins and phenolic
compounds. The compounds are distributed in different parts of the body such as
woody parts as well as leaves and pollen. It was also mentioned that Prosopis
juliflora contains a diverse group of secondary metabolites with unique and
multifactorial properties. 28
15
Vedak S, Raut SV in 2014 conducted a study on antibacterial compounds from
Methanolic Extract of Bark of Prosopis juliflora (Vilayati babhul). The study was
aimed to evaluate the antibacterial potential of the Prosopis juliflora bark (Vilayati
bhabul) in an attempt to identify the potential natural sources for synthesis of new
drug to avoid the growing antibacterial resistance. Soxhlet extraction method was
used to extract the peals of bark using methanol. Antibacterial activity on various
human pathogens was determined and antibacterial susceptibility test of crude
extract was performed which showed promising activity against tested organisms.
Phytochemical compounds such as glycosides, tannins, saponins and alkaloids
were present in the crude extract. The crude extract which was subjected to activity
guided fractionation with different polarity solvents, showed varying levels of
bactericidal activity with fraction D and fraction F shows maximum activity. MIC
of fraction D was 3.38mg/ml and 1.69mg/ml for Staphylococcus aureus and
Escherichia coli respectively whereas MIC of fraction F for the two organisms was
observed to be 2.39mg/ml and 4.77mg/ml respectively. 29
Lakshmibai R et al in 2015 conducted a study on phytochemical analysis and
antioxidant activities of Prosopis juliflora Leaves. The preliminary phytochemical
analysis revealed the presence of flavonoids, steroids, phenolic compounds,
glycosides, alkaloids, carbohydrates and proteins. The antioxidant studies were
done by DPPH scavenging method with the ethanolic extracts of Prosopis juliflora.
At different concentrations, ethanolic extracts of Prosopis juliflora leaves exhibited
significant antioxidant activity and the overall experimental result suggests that the
16
presence of flavonoids, phenolic compounds, alkaloids and other secondary
metabolites are responsible for it. Therefore the study concluded that, the plant play
a vital role in maintenance of the human health and wellbeing. 30
Tajbakhsh S et al in 2015 conducted a study in Iran to investigate the in-vitro
antibacterial activity of the P. juliflora seed pods. The antibacterial activity of P.
juliflora seed pods extract against Staphylococcus aureus, Staphylococcus
epidermidis, Escherichia coli and Pseudomonas aeruginosa was assessed. The
minimum inhibitory concentration (MIC) of the extract was determined as 0.312
mg/ml and 0.078 mg/ml for S. aureus and S. epidermidis, respectively and 1.25
mg/ml for both E.coli and P.aeruginosa. 31
Alsaadi JH , Al-Maliki AD in 2015 conducted a study on oil extract obtained from
P. juliflora pods and qualitative preliminary tests showed the extract contained
terpenoids. The oil compound was isolated and then purified & identified by thin
layer chromatography, gas chromatography-mass, nuclear magnetic resonance and
infra-red spectroscopy. Alloxan induced fasted diabetic rabbits were used to check
for hypoglycaemic effect of the compound. The dose of terpenoidic a given at
different time intervals of 2,4,6 and 24 h is 0.3 gm/kg and it showed reduced blood
glucose significantly with values 321.42, 294.60, 251.40 and 172.30 mg /100 ml,
respectively. The toxicity study has showed that the 24-Methyl cycloartan
compound has no toxic effect on red blood cells; suggesting the compounds safety
and usage to treat diabetes mellitus instead of insulin. 32
17
Kapoor A et al in 2015 reported a review on antimicrobial activity of different
herbal plants extracts. The review focused on the worldwide use of traditional
plants for the treatment of infectious diseases caused by micro-organisms. It was
mentioned that the discovery of antibiotics provide a tool against the microbial
infections but due to multiple drug resistance and adverse effects on host including
hypersensitivity, immune suppression and allergic reactions shown by antibiotics
creates a need of natural medicines which is safe and has better therapeutic effect.
Hence medicinal plants from natural origin containing secondary metabolites such
as alkaloids, steroids, tannins, and phenolic compounds are capable of exerting
antimicrobial activity by various mechanisms. These active constituents has shown
its therapeutic effect via destruction of cell membrane of microbe, membrane
proteins, enzymes located on cell membrane of microbes and through other
mechanisms. It was concluded that different extracts of medicinal plants with
minimum inhibitory concentration against microbial infections meet the need of
safe and efficient therapeutic agent against infectious diseases. 33
Preeti K et al in 2015 reported a review on pharmacological and therapeutic
application of Prosopis juliflora. It has been described that the plant contains
antibacterial, antifungal, anticancer, antioxidant, antimicrobial activity. The extract
of leaves have shown very high antimicrobial activity. The review also stated that
90% of fuel in many parts is provided by the plant. Literature suggests that it has
been used as a folk remedy for catarrh, cold, diarrhea, dysentery, excrescences, flu,
hoarseness, inflammation, measles, sore throat and for wounds healing. Decoction
18
prepared from leaf and seed extracts is used as disinfectant and also to treat scury.
Syrup prepared from ground pods of Prosopis julifora is given to children showing
weight deficiency or retardation in motor development. The syrup is also believed
to increase lactation. Tea made from Prosopis juliflora is used as it was thought to
be good for digestive disturbances and skin lesions. The various properties includes
soothing, astringent, antiseptic, antibacterial and antifungal properties. Patulitrin, a
flavanoid isolated from its flowers and fruits showed significant activity against
lung carcinoma in vivo. The richest source of plant metabolite are leaves and pods
followed by flower, root and stem. Phytochemical analysis of the extracts revealed
the presence of various components in most parts of the plant. It includes tannins,
phenolics, flavonoids, alkaloids, terpenes and steroids. Alkaloids are
pharmaceutically significant. They used as analgesic, antispasmodic, antimalarial,
antiarrythmic. It is also used in the treatment of coughs and pain, in the treatment
of gout, and as pupil dilation. 34
Odhiambo RS et al in 2015 conducted a study to assess the antibacterial activity
of ethanolic extract of root (REE) and leaves (LEE) of P. juliflora against clinical
isolates of Escherichia coli and Pseudomonas aeruginosa using paper disc
diffusion method. The results of study showed that all the extracts had inhibitory
effect on the growth of all the isolates. Chloramphenicol, erythromycin and
minocycline were effective against all the bacterial strains tested. All the bacterial
strains exhibited susceptibility to erythromycin and minocycline. Penicillin,
methicillin and ampicillin were the least effective antibiotics. Both leaves and root
19
possess saponins, tannins and alkaloids. Results from the study strongly validate
use of P. juliflora in the management of bacterial infections. 7
Persia FA et al in 2016 reported on title overview of Genus Prosopis toxicity
reports and its beneficial biomedical properties. The report included that the
secondary plant metabolites were regarded as promising sources of plant-protecting
substances. The genus Prosopis (Fabaceae) includes 44 species. It is considered
among the world’s most damaging invasive species. The genus has been found in
129 countries globally. Prosopis physiology suggests a wide range of adaptability,
shows the capability to growth in several adverse conditions, accumulates heavy
metals and synthetizes chemical defence. Some Prosopis species around the world
were described as important source of ethno pharmacological treatments for several
illnesses. It was concluded that by its selective toxic effects, plant derivatives can
be used as important source of new and successful bioactive molecules. 35
Tewari P in 2016 conducted a study on acute oral toxicity evaluation of Prosopis
Juliflora in Swiss Albino Mice in order to establish consumption safety of its
mesocarp. Acute oral toxicity study in swiss albino mice was conducted for 14 days.
The general behavior of all the animals in all the groups was categorized as active
before and after exposure to mesocarp. Prosopis juliflora mesocarp fed to swiss
albino mice recorded no acute oral toxicity evidence as the animals remained active
throughout the period of study, no pre terminal deaths were observed. There were
no abnormalities in live phase, physical activities and neurological activity
20
throughout the study period. There was no significant difference in body weight of
animal which received 3.2gm/kg and 6.4gm/kg of test material. All the animals
were active throughout the study period, there were no gross necropsy changes. The
test material has proved to be safe for human consumption and its processed food
products can enormously impact the food market. 36
Patnaik P et al in 2017 reported on title, Prosopis: Blessing and Bane. The report
highlighted the dual role of P. juliflora. It details the facets that make it a blessing
in some contexts and a bane in other contexts. The paper concluded with a summary
of assessments that balances the costs of destroying P. juliflora stands or utilizing
them, with the possible benefits of either. 37
Badri AM et al in 2017 conducted an invitro study to assess the anti-bacterial
activity of Prosopis juliflora leaves. The study was carried out to investigate the in
vitro antimicrobial activity of Prosopis juliflora methanolic leaf extract against
clinical isolates of seven gram negative bacteria and three gram positive bacteria
performed by cup-plate agar diffusion method against. The seven gram negative
bacteria includes Escherichia coli, Escherichia coli ESBL, Shigella flexneri,
Salmonella typhi, Proteus mirabilis, Pseudomonas aeruginosa, and Klebsiella
pneumonia. The three Gram positive bacteria includes Enterococcus faecalis,
Listeria monocytogenes and Bacillus cereus. The methanolic extract exhibited
inhibitory effects against most of the tested microorganisms. The largest inhibition
zone were obtained from methanolic extract against the Gram negative P.
21
aeruginosa, and Gram positive L. monocytogenes in comparison with Gentamicin
10 mg. The study concluded that methanolic extract of Prosopis juliflora was found
to be effective against different species of bacteria. 38
Henciya S et al in 2017 conducted a study on biochemical potentials of Prosopis
species. The study reported the use of Prosopis for medicinal purposes. Various
parts of the plants such as leaves and pods are used for anticancer, antidiabetic, anti-
inflammatory, and antimicrobial purposes. Phytochemical analysis of the plant
reveals the presence of flavonoids, tannins, alkaloids, quinones, or phenolic
compounds that demonstrate potentials in various biofunctions, such as analgesic,
anthelmintic, antibiotic, antiemetic, microbial antioxidant, antimalarial,
antiprotozoal, antipustule, and antiulcer activities; enhancement of Hþ, Kþ,
ATPases; oral disinfection; and probiotic and nutritional effects; as well as in other
biopharmaceutical applications, such as binding abilities for tablet production.
Juliflorine, a compound present in the plant provides a cure in Alzheimer disease
by inhibiting acetylcholine esterase at cholinergic brain synapses. Prosopis species
also possess antimosquito larvicidal activity, chemical synthesis by associated
fungal or bacterial symbionts, cyanobacterial degradation products, “mesquite”
honey and pollens with high antioxidant activity, etc. The review discloses the
origins, distribution, folk uses, chemical components, biological functions, and
applications of different representatives of Prosopis species.11
22
MATERIALS AND METHODS
STUDY DESIGN:
The present research is an Invitro, Experimental study, designed to assess the
antimicrobial efficacy of Prosopis juliflora against oral microorganisms.
ETHICAL CLEARANCE:
The nature and purpose of the study was proposed to the Institutional ethical
committee of Ultra’s Best Dental science college (IRB/IEC Reference No:2018-
STU-Br VII-MAA-12) and ethical clearance was obtained.
OBTAINING PERMISSION:
The aim and objectives of the study was explained in detail and permission was
obtained from
1) The Principal, College of Pharmacy, Madurai Medical College,
Madurai, Tamilnadu for preparation of plant extract in the Department of
Pharmacognosy.
2) The Head, Harman institute, Tanjore, Tamilnadu for
Microbiological analysis.
23
PLANT MATERIAL COLLECTION:
CRITERIAE FOR COLLECTION OF LEAVES
INCLUSION CRITERIA:
Healthy Prosopis juliflora leaves
EXCLUSION CRITERIA:
Yellowing of leaves,
Spotting,
Browning
Drying
Prosopis juliflora leaves based on the inclusion and exclusion criteria were
collected from trees found in and around American college, Madurai, Tamil Nadu.
IDENTIFICATION OF THE PLANT SPECIMEN:
The collected plant materials were botanically authenticated in the Department of
Botany, American College, Madurai, Tamil Nadu. Botanical identification of the
plant specimen was based on the leaves type, leaves arrangement, leaves shape,
leaves blade edges, leaves vein patterning, leaves petiole, presence of stipule and
the key physical traits.
Based on the key elements botanical identification was done and taxonomical
identification certificate was obtained.
24
The taxonomy is as follows
KINGDOM : PLANTAE
ORDER : FABALES
FAMILY : FABACEAE
GENUS : PROSOPIS
SPECIES : JULIFLORA
BINOMIAL NAME: Prosopis juliflora
PREPARATION OF PLANT EXTRACT:
Plant extract preparation was done in the Department of Pharmacognosy, College
of pharmacy, Madurai medical college, Madurai, TamilNadu.
Ethanolic extract of the plant was obtained using MACERATION TECHNIQUE.
ETHANOLIC EXTRACT 8,18:
The collected leaves were washed in tap water, shade dried for 15 days and made
into a fine powder of 40 mesh size using the laboratory mill. 100g of the powder
was immersed in a beaker containing 1000 ml of Ethanol. Extraction was carried
out by cold maceration technique at room temperature, under constant stirring,
using 100% (w/v) of aqueous ethanol at solid to solvent ratio of 1:10 (w/v), in
closed bottles. The next day, after 20 hours of extraction process, the suspension
was centrifuged. The supernatant was removed and stored at 4o C. The extraction
25
process was repeated. The supernatants were combined and filtered through a
Whatman GF/F glass microfibre filter (0.7 lm). The solvent was evaporated in a
vacuum at 40o C in a rotary flash evaporator under reduced pressure.
TRANSPORTATION FOR MICROBIOLOGICAL ASSAY:
The obtained extract was stored in a glass container, carried to the deep freezer in
a portable icebox (vaccine carrier) for storage, maintaining a temperature of
0 o to -4o C and transported to Harman Institute, Tanjore, Tamil Nadu within 3.5
hours for microbiological assay where Zone of inhibition and Minimum inhibitory
concentrations of the plant extract at various concentrations against Streptococcus
mutans, Lactobacillus, Aggregatibacter actinimycetemcomitans and Candida
albicans were determined.
DETERMINATION OF ANTI-MICROBIAL ACTIVITY 39
The antimicrobial activity was performed by disc diffusion method. The
required media and medium for each organisms were prepared seperately,
microorganisms were purchased (Streptococcus mutans, Lactobacillus and
Candida albicans from Chandigarh, India) (Aggregatibactor
actinomycetemcomitans from Rontgen Laboratory, Thanjavur, India) and cultured,
sample solutions were prepared and microbiological assay was done.
26
PREPARATION OF MEDIA
Nutrient Agar (NA-Himedia) Media was prepared for Bacteria comprising of
the following composition:
Composition of Media
Animal’s tissue : 5.00 g
Sodium chloride : 5.00g
Beef extract : 1.50g
Yeast extract : 1.50g
Agar : 15.0g
PREPARATION OF MEDIUM:
In order to prepare the medium for bacteria, 28 grams of nutrient agar was
suspended in 1000 ml of distilled water. It was heated till boil and the medium was
obtained by dissolving the agar completely. It was then sterilized by autoclaving at
15 lbs pressure (121˚c) for 15 minutes. The obtained product was mixed well and
poured into sterile petri plates of size 100 x 15mm.
POTATO DEXTROSE AGAR (PDA-HIMEDIA) MEDIA was prepared for
FUNGI
Composition of Media.
Potatoes infusion : 200.00g
Dextrose : 20.00g
Agar : 15.00g
27
PREPARATION OF MEDIUM:
In order to prepare medium for fungi, 39.0 grams of potato dextrose agar
was suspended in 1000 ml of distilled water. It was then heated to boil and the
medium was dissolved completely. Once the medium is dissolved it was sterilized
by autoclaving at 15 lbs pressure (121˚c) for 15 minutes. The product was mixed
well before dispensing. As a pH of 3.5 is required, the medium was acidified using
10 % sterile tartaric acid. The amount of acid required for 100 ml of sterile cooled
medium is approximately 1 ml, heating the medium after addition of acid was
avoided.
MICROORGANISMS
The microbial strains employed in the biological assays were:
BACTERIAE
1) Streptococcus mutans (MTCC 497),
2) Lactobacillus (MTCC 4979), (MTCC 441), was obtained from Microbial type
culture collection (MTCC) at the institute of Microbial Technology (IMTECH),
Chandigarh, India.
3) Aggregatibactor actinomycetemcomitans was collected from Rontgen
Laboratory, Thanjavur, TamilNadu.
28
FUNGI
Candida albicans (MTCC 183) was obtained from microbial type culture collection
(MTCC) at the institute of Microbial Technology (IMTECH), Chandigarh, India.
The microorganisms were purchased and incubated at 37o C. Fresh cultures of test
organisms were prepared.
PREPARATION OF 24 HOURS PURE CULTURE
An inoculation loop full of each of the microorganisms was suspended in
about 10ml of physiological saline in a Roux bottle. Each of these was streaked on
to the appropriate culture slants and was incubated at 37ºC for 24 hours except for
fungi which was incubated at 25ºC for 48 hours. After completion of incubation
period, when growth was observed the tubes were kept into 2-8˚C until use.
PREPARATION OF SAMPLE SOLUTIONS FOR THE EXPERIMENT
The sample solutions were prepared in 0.0001, 0.0005, 0.001, 0.002, 0.005,
0.01, 0.0125, 0.025, 0.05, 0.1 % concentrations by dissolving the plant extract in
ethanolic solvent. They were kept under refrigerated condition unless used for the
experiment.
29
PREPARATION OF DRIED FILTER PAPER DISCS
Whattman filter paper (No:1) was used to prepare discs of approximately 6
mm in diameter, which were placed in hot air for sterilization After sterilization,
the discs were loaded with 0.0001, 0.0005, 0.001, 0.002, 0.005, 0.01, 0.0125, 0.025,
0.05, 0.1% of ethanolic extract and again kept under refrigeration for 24 hrs.
Standard solutions Chlorhexidine (0.2%) (20µl), Povidone iodine (10%) (20µl)
were used for all the three bacteriae as positive control and Clotrimazole (1%)
(20µl), Nystatin (10000 unit) (20µl) was used for fungi to compare the test solution.
They were kept under refrigerated condition unless they were used for the
experiment. Since the extract is an ethanolic extract a negative control of ethanol
was used for all the organisms.
Chlorhexidine is a positively charged molecule which binds to the
negatively charged sites on the cell wall. At low concentration, it destabilizes the
cell wall and attacks the cytoplasmic membrane. Damage of cytoplasmic’s delicate
semipermeable membrane allows for leakage of components leading to cell death.
At high concentrations, chlorhexidine causes the cytoplasm to congeal or solidy. 40
Povidone iodine delivers free iodine to target cell membrane thereby
disrupting the microbial metabolic pathway, as well as destabilization of structural
cellular components of cell membranes, causing irreversible damage. 41
Clotrimazole is a broad spectrum anti-fungal agent that inhibits the growth
of pathogenic yeasts by altering the permeability of cell membrane. 42
Nystatin acts by depressing the glucose uptake by an alteration of cell
permeability. Endogenous respiration and glucose intermediate metabolism are
30
accelerated and diverted from constructive assimilation. Energy is locked up in
polymetaphosphate accumulation leading to depleted supply and thereby inhibiting
growth and cell multiplication causing cell atrophy. 43
ANTIMICROBIAL ASSAY
Antibiogram was done by disc diffusion method using plant extracts. Petri
plates were prepared by pouring 30 ml of Nutrient agar /Potato Dextrose medium
for bacteria/fungi. The test organism was inoculated on solidified agar plate with
the help of cotton swab and streaked. Then the plate is rotated by 60o and the
procedure is repeated twice for even distribution. It is then allowed to dry for 5
minutes. Briefly, inoculums of bacteria strains were spread on Nutrient agar plates
for bacteria and fungi strains were spread on potato dextrose agar for fungus strains.
Using sterile forceps, the sterile filter papers (6 mm diameter) containing the crude
extracts were laid down on the surface of inoculated agar plate which is
immediately pressed down lightly to ensure complete contact. Care was taken to
not move disc. Maximum of seven discs were positioned in such a way that the
minimum center to center distance was 24mm and no closer than 10 to 15 mm from
the edge of the petri dish. The plates were incubated at 37 ºC for 24 hours for the
bacteriae and at room temperature (30±1) for 24-48 hours for fungi strains.
MEASUREMENT OF ZONE OF INHIBITION 44
The antimicrobial potential of test compounds were determined on the basis
of mean diameter of zone of inhibition around the disc in millimeters.
31
Zone of Inhibition:
Zone of inhibition is an area of media where bacteria are unable to grow, due to the
presence of a drug that impedes their growth. Fuzzy zones are avoided and it is
rounded off to the nearest mm.
It is recorded by measuring the distance between the center of the disc to the
periphery of the inhibitory zone using a millimeter scale or a vernier caliper.
The zones of inhibition of the tested microorganisms by the samples were measured
using a millimeter scale. Antimicrobial activity was assigned by measuring the
inhibition zone formed around the disc.
The experiment was repeated in triplicate for each organism and the antibacterial
and antifungal activity was expressed in terms of mean of diameter of zone of
growth of inhibition in mm.
DETERMINATION OF MINIMUM INHIBITORY CONCENTRATION
(MIC)
MINIMUM INHIBITORY CONCENTRATION: It is the lowest drug
concentration of an antimicrobial drug that will inhibit the visible growth of
microorganism after overnight incubation. Minimum inhibitory concentration can
be determined by culturing microorganisms in a liquid media like broth dilution
methods or on plates of solid growth medium. 44
32
PREPARATION OF NUTRIENT MEDIA
For bacteria, 28.0 grams of nutrient media was weighed initially. 1000 mL
of distilled water was then taken in a conical flask and to it, the weighed nutrient
media was added. The conical flask was stirred thoroughly.
For fungi, 39.0 grams of PDA was suspended in 1000 ml of distilled water.
It was heated to boil, allowed to dissolve and then sterilized by autoclaving at 15
lbs pressure (121˚c) for 15 minutes, followed by mixing it well. Before dispensing
it, the pH was adjusted to 3.5; if required 10 % of sterile tartaric acid was added for
acidification. The amount of acid required for 100 ml of sterile cooled medium is
approximately 1 ml, heating the medium after addition of acid was avoided.
Preparations of sample solutions:
Sample = 0.0001, 0.0005, 0.001, 0.002, 0.005, 0.01, 0.0125, 0.025,
0.05, 0.1 %
Inoculation of bacterial culture:
5mL of the prepared and sterilized nutrient media was poured into test tubes.
To this, 1 mL of bacterial culture (Streptococcus mutans, Lactobacillus Sp.,
Aactinomycetemcomitans Sp and Candida albicans were inoculated (inoculums
suspension contained 1 x 106 cells/ml).
33
Procedure
5ml of nutrient media was mixed with 1 ml of bacterial culture and 1mL of
different concentrations of samples as 0.0001, 0.0005, 0.001, 0.002, 0.005, 0.01,
0.0125, 0.025, 0.05, 0.1 % were added to five different test tubes for which sterile
test tubes were used. The tubes were closed with cotton plugs. The contents of each
tube were mixed on a shaker at 250 rpm for 2 minutes. After carrying out the above
procedure, the test tubes containing the inoculums and the sample were placed in
the incubator overnight at 35°C. After incubation, the test tubes were taken out of
the incubator. The control and blank were considered as with and without bacterial
culture. The lowest concentration of MIC tubes with no visible bacterial growth
(turbidity) was recorded by subjecting it to UV Spectrophotometry at 540 nm.
Spectrophotometer measures light absorption in the UV as well as visible region.
The value obtained is recorded as Optical Density (OD) values. It is a tool for
qualitative and quantitative measurements.
STATISTICAL ANALYSIS
The data collected were recorded in a Master Chart. Data analysis was
done with the help of computer using Statistical Package for Social Sciences. (SPSS
Inc., Chicago IL, version 22.0 for Windows).
Using this software Means and Standard deviations were calculated for
quantifiable variables. ANOVA was used to test the significance of difference and
‘p’ values were calculated. A ‘p’ value of less than 0.05 denotes significant
relationship.
34
METHODOLOGY FLOWCHART
INVITRO STUDY
ETHICAL COMMITTEE - APPROVAL
COLLECTION OF PLANT SPECIMEN
IDENTIFICATION OF PLANT SPECIMEN
PREPARATION OF PLANT EXTRACT
OBTAINING DIFFERENT CONCENTRATIONS
DETERMINATION OF ANTIMICROBIAL ACTIVITY
ZONE OF INHIBITION MINIMUM INHIBITORY

CONCENTRATION
V V
DISC DIFFUSION METHOD BROTH DILUTION METHOD
35
Ethanolic extract of
Prosopis juliflora
Assessment of zone of inhibition
Different concentrations in %
0.0001, 0.0005, 0.001, 0.002, 0.005, 0.01, 0.0125, 0.025, 0.05, 0.1
Test Organisms Positive Control Negative Control
Streptococcus mutans Chlorhexidine
Lactobacillus Povidone iodine
Ethanol
Aggregatibacter
Clotrimazole
Candida albicans
36
Nystatin
PHOTOGRAPHS
Figure 1, Prosopis juliflora shrub
37
PHOTOGRAPHS
Figure 2, Collected and dried leaves of Prosopis juliflora
Figure 3, Powdered Leaves of Prosopis juliflora
38
PHOTOGRAPHS
Figure 4, Powdered leaves dissolved in Ethanol (Maceration Technique)
Figure 5, Magnetic stirrer for constant stirring
39
PHOTOGRAPHS
Figure 6 A, Filtration of the supernatant
Figure 6 B, Filtration of the supernatant
40
PHOTOGRAPHS
Figure 7, Product obtained from Maceration
Figure 8, Rotary flash evaporator
41
PHOTOGRAPHS
Figure 9, Glass container for extract collection
Figure 10, Vaccine carrier for transportation of extract to the laboratory
42
PHOTOGRAPHS
Figure 11, Various concentrations of extract prepared
43
PHOTOGRAPHS
Figure 12, Antibacterial activity of test and control agents against Streptococcus
mutans at baseline and after 24 hours.
Before (0 hours)
After 24 hours
44
PHOTOGRAPHS
Figure 13, Antibacterial activity of test and control agents against Lactobacillus
at baseline and after 24 hours.
Before (0 hours)
After 24 hours
45
PHOTOGRAPHS
Figure 14, Antibacterial activity of test and control agents against

Aggregatibacter actinomycetemcomitans at baseline and after 24 hours.
Before (0 hours)
After 24 hours
46
PHOTOGRAPHS
Figure 15, Antifungal activity of test and control agents against Candida
albicans at baseline and after 24-48 hours.
Before (0 hours)
After (48hours)
47
PHOTOGRAPHS
Figure 16, MIC Determination of Prosopis juliflora against Streptococcus mutans
Before (0 hours) After 24 hours
Figure 17, MIC Determination of Prosopis juliflora against Lactobacillus.
48
PHOTOGRAPHS
Figure 18, MIC Determination of Prosopis juliflora against Aggregatibacter

Actinomycetemcomitans
Figure 19, MIC Determination of Prosopis juliflora against Candida albicans
49
PHOTOGRAPHS
Figure 20, MIC determination Control and Blank solutions against Bacteriae
Figure 21, MIC determination Control and Blank solutions against Fungi
50
RESULTS
The present study was an attempt to assess the antimicrobial efficacy of
Prosopis juliflora against Streptococcus mutans, Lactobacillus, Aggregatibacter
actinomycetemcomitans and Candida albicans in vitro.
Data collected were entered in an Excel sheet and the obtained results
were tabulated.
51
RESULTS
TABLE 1: ANTIBACTERIAL ACTIVITY OF Prosopis juliflora AGAINST
Streptococcus mutans
Prosopis juliflora concentration % Zone of inhibition (in mm)

0.0001 Nil
0.0005 Nil
0.001 Nil
0.002 Nil
0.005 4.20 ± 0.29
0.01 5.15 ± 0.36
0.0125 5.70 ± 0.39
0.025 6.85 ± 0.47
0.05 7.35 ± 0.51
0.1 10.35 ± 0.72
Table 1 shows antibacterial activity of Prosopis juliflora against Streptococcus
mutans at various concentrations.
At 0.0001%, 0.0005%, 0.001% and 0.002% concentrations, Prosopis juliflora was
not found to be effective against Streptococcus mutans. At 0.005%, P.juliflora was
found effective against Streptococcus mutans with a mean zone of inhibition of
4.20 ± 0.29 mm. As the concentrations of the plant extract was increased to 0.01%,
0.0125%, 0.025%, 0.05% and 0.1% the mean zone of inhibition against
Streptococcus mutans was also increased to 5.15 ± 0.36 mm, 5.70 ± 0.39 mm, 6.85
± 0.47 mm, 7.35 ± 0.51 mm and 10.35 ± 0.72 mm respectively. The maximum
antibacterial effect was recorded at 0.1%.

52
RESULTS
Lactobacillus
Prosopis juliflora concentration Zone of inhibition (in mm)

(%)
0.0001 3.05 ± 0.21
0.0005 5.25 ± 0.36
0.001 6.30 ± 0.44
0.002 6.90 ± 0.48
0.005 7.45 ± 0.52
0.01 8.15 ± 0.57
0.0125 8.75 ± 0.61
0.025 9.45 ± 0.66
0.05 9.80 ± 0.68
0.1 10.60 ± 0.74
Table 2 shows antibacterial activity of Prosopis juliflora against Lactobacillus at
various concentrations.
Lactobacillus was found sensitive even at a minimum concentration of 0.0001% of
Prosopis juliflora exhibiting a mean zone of inhibition of 3.05 ± 0.21 mm. The
mean zone of inhibition of lactobacillus were found to be 5.25 ± 0.36 mm at
0.0005%, 6.30 ± 0.44 mm at 0.001%, 6.30 ± 0.44 mm at 0.002%, 7.45 ± 0.52 mm
at 0.005%, 8.15 ± 0.57 mm at 0.01%, 8.75 ± 0.61 mm at 0.0125%, 9.45 ± 0.66 mm
at 0.025%, 9.80 ± 0.68 mm at 0.05% and 10.60 ± 0.74 mm at 0.1% respectively.
The maximum antibacterial activity was recorded at 0.1% and least at 0.0001%.
53
RESULTS
Aggregatibacter actinomycetemcomitans
Prosopis juliflora concentration (%) Zone of inhibition (in mm)

0.0001 Nil
0.0005 Nil
0.001 Nil
0.002 3.10 ± 0.21
0.005 3.55 ± 0.24
0.01 4.20 ± 0.29
0.0125 6.15 ± 0.43
0.025 7.00 ± 0.49
0.05 9.05 ± 0.63
0.1 10.45 ± 0.73
Table 3 shows antibacterial activity of Prosopis juliflora against Aggregatibacter
actinomycetemcomitans at various concentrations.
Prosopis juliflora did not show any effect on Aggregatibacter
actinomycetemcomitans at 0.0001%, 0.0005% and 0.001% concentrations. At
0.002% concentration the antibacterial activity began with the mean zone of
inhibition of 3.10 ± 0.21 mm. The antibacterial activity was found to increase with
an increase in concentrations. At 0.005% the mean zone of inhibition recorded is
3.55 ± 0.24 mm, at 0.01% it is 4.20 ± 0.29 mm, at 0.0125% it is 6.15 ± 0.43 mm,
at 0.025% it is 7.00 ± 0.49 , at 0.05% it is 9.05 ± 0.63 and at 0.1% it showed the
maximum inhibition of 10.45 ± 0.73 mm.
54
RESULTS
TABLE 4: ANTIFUNGAL ACTIVITY OF Prosopis juliflora AGAINST
Candida albicans
Prosopis juliflora concentration (%) Zone of inhibition (in mm)
0.0001 Nil
0.0005 Nil
0.001 1.10 ± 0.07
0.002 3.20 ± 0.22
0.005 4.55 ± 0.31
0.01 5.30 ± 0.37
0.0125 5.85 ± 0.40
0.025 6.25 ± 0.43
0.05 8.15 ± 0.57
0.1 10.10 ± 0.70
Table 4 shows antifungal activity of Prosopis juliflora against Candida albicans at
various concentrations.
Candida albicans did not show sensitivity to Prosopis juliflora at 0.0001% and
0.0005% concentrations. At 0.001% the antifungal activity was observed with an
increase in the activity as the concentration increases. The mean zone of inhibition
at 0.001% is 1.10 ± 0.07 mm followed by 3.20 ± 0.22 mm 4.55 ± 0.31 mm, 5.30 ±
0.37 mm, 5.85 ± 0.40 mm, 6.25 ± 0.43 mm, 8.15 ± 0.57mm at 0.0005%, 0.001%,
0.002%, 0.005%, 0.01%, 0.0125%, 0.025%, 0.05%, 0.1% respectively, with a
maximum antifungal activity of 10.10 ± 0.70 mm at 0.1%.
55
RESULTS
TABLE 5: COMPARISON OF ANTIBACTERIAL ACTIVITY OF
Prosopis juliflora, CHLORHEXIDINE, POVIDONE IODINE AND
ETHANOL AGAINST Streptococcus mutans
TEST A B C D Sig
ORGANISM (P.juliflora (Chlorhexidine (Povidone (Ethanol
0.1%) 0.2%) iodine 10%) 100%)
(mm) (mm) (mm)
(mm)
Streptococcus 10.35 ± 0.72 7.90 ± 0.55 4.85 ± 0.33 0 0.000
mutans
p value <0.05 is statistically significant
GRAPH 1: COMPARISON OF ANTIBACTERIAL ACTIVITY OF TEST
AND CONTROL AGENTS AGAINST Streptococcus mutans
A- P.juliflora,
10.35
B- Chlorhexidine,
C- Povidone iodine,
7.9
Zone of inhibition in mm
D - Ethanol
4.85
A B C D
Agents
56
RESULTS
Table 5 and Graph 1, shows the comparison of test and control agents against
Streptococcus mutans where Agent A is 0.1% leaf extract, Agent B is 0.2%
Chlorhexidine, Agent C is 10% Povidone Iodine and Agent D is 100%. p value of
less than 0.05% is considered for statistical significance.
Prosopis juliflora shows the maximum inhibitory action against Streptococcus
mutans with the mean zone of inhibition of 10.35 ± 0.72 mm followed by
Chlorhexidine with a mean zone of inhibition of 7.90 ± 0.55 mm, Povidone Iodine
with 4.85 ± 0.33 mm and Ethanol with no inhibition. This result signifies that the
antibacterial activity against Streptococcus mutans is the highest among
Prosopis juliflora followed by chlorhexidine & least by Povidone iodine and with
no effect noted with ethanol, with a p value less than 0.05 which was highly
statistically significant.
57
RESULTS
ETHANOL AGAINST Lactobacillus
TEST A B C D Sig
0.1%) 0.2%) iodine 10%) 100%)
(mm) (mm) (mm) (mm)
Lactobacillus 10.60 ± 0.74 10.35 ± 0.72 9.60 ± 0.67 0 0.000
p value <0.05 is statistically significant
AND CONTROL AGENTS AGAINST Lactobacillus
A- P.juliflora,
10.6 10.35
9.6 B- Chlorhexidine,
C- Povidone iodine,
D - Ethanol
A B C D
Agents
58
RESULTS
Table 6 and Graph 2 shows the comparison of test and control agents against
Lactobacillus where Agent A is 0.1% Prosopis juliflora, Agent B is 0.2%
Chlorhexidine, Agent C is 10% Povidone Iodine and Agent D is 100%. p value of
less than 0.05% is considered for statistical significance.
Prosopis juliflora shows the maximum inhibitory action against Lactobacillus with
the mean zone of inhibition of 10.60 ± 0.74 mm followed by Chlorhexidine with a
mean zone of inhibition of 10.35 ± 0.72 mm, Povidone Iodine with 9.60 ± 0.67 mm
and Ethanol with no inhibition. This result signifies that the antibacterial activity
against Lactobacillus is the highest with Prosopis juliflora followed by
chlorhexidine where the antibacterial effect is similar to that of P.juliflora, the least
by Povidone iodine and with no effect noted with ethanol, with a p value less than
0.05 which was highly statistically significant.
59
RESULTS
ETHANOL AGAINST Aggregatibacter actinomycetemcomitans
TEST A B C D Sig
0.1%) 0.2%) iodine 100%)
(mm) (mm) 10%)
(mm) (mm)
Aggregatibacter 10.45 ± 0.73 9.60 ± 0.67 6.10 ± 0.42 0 0.000
actinomycetem-
comitans
p value of <0.05 is statistically significant
AND CONTROL AGENTS AGAINST Aggregatibacter
A- P.juliflora,
10.45
B- Chlorhexidine,
9.6
C- Povidone iodine,
D - Ethanol
6.1
A B C D
B
Agents
60
RESULTS
Table 7 and Graph 3 shows comparison of Agent A, B, C and D against
Aggregatibacter actinomycetemcomitans where Agent A is 0.1% Prosopis
juliflora, Agent B is 0.2% Chlorhexidine, Agent C is 10% Povidone Iodine and
Agent D is 100%. p value of less than 0.05% is considered for statistical
significance.
The mean value obtained for Agent A is 10.45 ± 0.73 mm, whereas for agent B is
9.6 ± 0.67 mm, Agent C is 6.10 ± 0.42 mm and Agent D, 0. The result signifies that
the antibacterial activity is highest among Prosopis juliflora followed by
Chlorhexidine agent and Povidone Iodine, Ethanol does not show antibacterial
activity, with a p value less than 0.05 which was highly statistically significant.
61
RESULTS
Prosopis juliflora, CLOTRIMAZOLE, NYSTATIN AND ETHANOL
TEST A B C D Sig
ORGANISM (P.juliflora (Clotrima- (Nystatin (Ethanol
0.1%) zole 1%) 10000 IU) 100%)
Candida 10.10 ± 0.70 6.75 ± 0.47 8.90 ± 0.62 1.05 ± 0.07 0.000
albicans
p value of < 0.05 is statistically significant
GRAPH 4: COMPARISON OF ANTIFUNGAL ACTIVITY OF TEST AND
CONTROL AGENTS AGAINST Candida albicans
10.1 A- P.juliflora,
8.9
B- Clotrimazole,
6.75 C- Nystatin,
D - Ethanol
1.05
A B C D
Agents
62
RESULTS
Table 8, Graph 4 shows antifungal activity of 0.1% Prosopis juliflora, Clotrimazole
1% , Nystatin 10000 IU and Ethanol 100% against Candida albicans where Agent
A is Prosopis juliflora, B is Clotrimazole, C is Nystatin and D is Ethanol. p Value
of less than 0.05 is considered for statistical significance.
The mean value obtained for Prospis juliflora is 10.10 ± 0.70 whereas for Nystatin
is 8.90 ± 0.62, Clotrimazole is 6.75 ± 0.47 and Ethanol is 1.05 ± 0.07 signifying
that the antifungal activity is the highest in Prosopis juliflora followed by Nystatin,
Clotrimazole and Ethanol, with a p value less than 0.05 which was highly
statistically significant.
63
RESULTS
TABLE 9: DETERMINATION OF MINIMIUM INHIBITORY
CONCENTRATION AGAINST VARIOUS MICROORGANISMS
Sample Microorganism strains

Concentration Streptococcus Lactobacillus Aggregatibacter Candida
(%) mutans actinomycetem- albicans
comitans
0.005 0.286 0.778 1.068 0.411
0.01 0.482 1.325 1.056 0.431
0.0125 1.057 1.501 1.526 0.568
0.025 0.884 1.800 1.170 0.630
0.05 2.115 2.491 1.544 1.350
0.1 1.620 2.501 1.686 1.362
Control 0.805 0.805 0.805 0.539
Blank 0.000 0.000 0.000 0.000
MIC (%) 0.005 0.005 0.01 0.005
64
RESULTS
GRAPH 5: DETERMINATION OF MINIMIUM INHIBITORY
CONCENTRATION OF Prosopis juliflora AGAINST VARIOUS
MICROORGANISMS
0.005 D
0.01 C
0.005 B
MICROORGANISM
0.005 A
MIC (MINIMUM INHIBITORY CONCENTRATION)
Table 9, Graph 5 shows the minimum inhibitory concentration of the leaf extract
against Streptococcus mutans (A), Lactobacillus (B), Aggregatibacter
actinomycetemcomitans (C) and Candida albicans (D) which is a result of
quantitative analysis of antibacterial and antifungal activity observed under UV-
Visible light Spectrophotometry.
Broth dilution with various concentrations were done and minimum inhibitory
concentration for each organism was determined. Blank test tubes were placed with
no microorganism showing a count of 0.000 bacteria and fungi. Controls showed
0.805 bacterial count for all the three bacteriae and 0.539 count of fungi. MIC in
the ranges viz., (0.005%, 0.01%, 0.0125%, 0.025%, 0.05%, 0.1%) of all
65
RESULTS
Prosopis juloflora extract showed significant antibacterial and antifungal activity
against all tested microorganisms.
The present result demonstrates that Prosopis juliflora extract exhibited high
antibacterial and antifungal activity. At concentrations around 0.005% for
Streptococcus mutans, Lactobacillus, Candida albicans and 0.01%
Aggregatibacter actinomycetemcomitans displayed a relative broader antimicrobial
spectrum at a lower concentration. These results strongly suggest that Prosopis
juliflora could be potent microbial agent.
66
DISCUSSION
In the present study plant leaves are used for assessing the antimicrobial effect,
as medicinal plants are expected to be the best alternative source of new antimicrobials.
Antimicrobial resistance in the current era is a major issue and it is stated that it can be
overcome by changing the strategy and returning to natural ingredients obtained from
plants. 19
There are various medicinal plants such as Azadirachta indica, Acacia nilotica,
Syzygium aromaticum, Allium sativum, Cinnmomum verum, Curcuma longa, Ocimum
tenuiflorum etc., having benefits both on general and oral health which are already proven
scientifically. In the present study Prosopis juliflora plant is used for assessing the
antimicrobial effect as literature suggests the usage of Prosopis juliflora traditionally for
its medicinal values. Ethnobotanical surveys reveal the use of P.juliflora plant in traditional
practice for toothache, asthma, bronchitis, conjunctivitis, skin diseases, blood and venereal
diseases as it possesses pharmacological properties such as antibacterial, antifungal,
hemolytic, anti-inflammatory, wound healing activities, anti-tumor, antiprotozoal,
antioxidant, antipustule, antiulcer, antiemetic, antidiabetic and and in alzheimer
therapy.10,11,15,18
The strength of the study is that ten different concentrations of the plant extract
is tested against four organisms and minimal inhibitory concentration for each organism is
determined. Ethanolic leaf extract was obtained and tested for antibacterial assay. This is
the first study to test the plant extract against Streptococcus mutans, Lactobacillus and
Candida albicans adding to the uniqueness of the study.
Various other studies have assessed the antimicrobial efficacy using aqueous
extract, crude extract or methanolic extract. Properties of a good solvent includes low
67
DISCUSSION
toxicity, ease of evaporation at low heat, rapid physiologic absorption of extract,
preservative action and inability to cause the extract to dissociate. The choice of solvent is
influenced by what is intended with the extract. The most commonly used solvents for
investigations of antimicrobial activity in plants are methanol, ethanol and water. Most
antimicrobial active components that have been identified are not water soluble and thus
organic solvent extracts have been found to be more potent. Ethanolic leaf extract provides
antibacterial activity against various pathogenic bacteria comparable to antibiotics such as
streptomycin and penicillin, therefore in this present study ethanolic extract is used. 11,45
The positive control agent used for the bacteriae are Chlorhexidine 2% and
Povidone Iodine 10%, for the fungi, Clotrimazole 1% and Nystatin 10000 IU as these are
the gold standard agents against these pathogens.
The plant is also found to be effective against various bacteriae such as
Corynebacterium diphtheria var. mitis, Corynebacterium hofmanni, Bacillus subtilis,
Staphylococcus aureus, Streptococcus pyogenes, Streptoccus faecalis, Pseudomonas
auruginosa, Escherichia coli and against fungi Cryptococci meningitis, Cryptococcus
neoformans, Cryptococcus gattii.7,11 In the present study only bacteriae Streptococcus
mutans, Lactobacillus, Aggregatibacter actinomyctemcomitans and fungi, Candida
albicans were tested as these organisms are the common pathogens of oral and periodontal
infections and also because the plant is said to possess antibacterial and antifungal
activity.7,8,18
The study involved botanical identification of the plant specimen to make sure
no other plant species were included. Antibacterial activity was assessed using Disc
diffusion method as it is one of the classical microbiological techniques which is
68
DISCUSSION
convenient to use with an ability to test wide range of organisms and antimicrobial agents,
having a good correlation between in-vitro and in-vivo data. Minimal inhibitory
concentrations were assessed using Broth dilution method as it is a standardized method
which is easy to perform showing rapid results and also economical. 46,47
In this study leaf extract of the plant is used for analysis as the leaves contains
alkaloids, Glycosides, Flavanoids and Terpenoids which possesses antimicrobial actions.
The mechanism of action of natural compounds are related to disintegration of cytoplasmic
membrane, destabilization of the proton motive force (PMF), electron flow, active
transport and coagulation of the cell content. Chemical compounds from essential oils also
act on cytoplasmic membrane proteins. ATPases, enzymes are known to be located at the
cytoplasmic membrane of bacteria and surrounded by lipid molecules. These enzymes are
the site of action of cyclic hydrocarbons. In addition, lipid hydrocarbons may distort the
lipid-protein interaction, and the direct interaction of lipophilic compounds with
hydrophobic parts of the protein is also possible. Another possible mechanism is the
stimulation the growth of pseudo-mycelia, evidencing that they may act on enzymes
involved in the synthesis of bacterium structural components. 33
The zone of inhibition and Minimum inhibitory concentration obtained
using the plant extract at various concentrations against Streptococcus mutans,
Lactobacillus and Candida albicans limits exact comparison with other studies as this is
the first study tested against these specific pathogens. The zone of inhibition of
Aggregatibacter actinomycetemcomitans in the present study is 10.45 ± 0.73mm whereas
in a previous study conducted by Hari Prasad O et al in 2011 it was 12.30±0.30mm. The
reason for the difference may be due to the crude extract which was used in the previous
69
DISCUSSION
study and ethanolic extract used in the present study at a lesser concentration of 0.1%. For
all other organisms comparison is done with native herbs. Aloevera, Amla, Garlic, Ginger,
Neem and Tulsi showed significant zone of inhibition against Streptococcus mutans.
Though these showed larger inhibitory zones P.juliflora showed comparable results at a
very low concentration which would have increased with an increase in concentration.48
On comparing the mean zone of inhibition of Prosopis juliflora with Neem, Tulsi, Triphala
at a concentration of 25% against Lactobacillus, Prosopis juliflora showed better results
with zone of inhibition of 10.60 ± 0.74mm.49 Prosopis juliflora showed significant
antifungal activity against Candida albicans similar to that of A.tenuifolius whereas
B.aegyptiaca, C.diurnum did not show antifungal activity.50
This antimicrobial activity of P.juliflora may be due to the presence of
Quercetin which is responsible for its analgesic, antiallergenic, antibacterial, antidiabetic
and anti-inflammatory potential and Apigenin for antiallergenic, antibacterial,
antidermatitic, anti-inflammatory and antiviral activity.18
The current study assessed minimal inhibitory concentration using broth
dilution method like in other studies, where the extract was added to several test tubes. In
addition, control tubes were prepared. The test and the control tubes were incubated at 37 oC
for 24 hours. Finally the lowest concentration of the extract that inhibited the bacterial
growth was measured as minimum inhibitory concentration.31
Chlorhexidine, a positive control used for the bacteriae in the present study
is one of the best agents effective against Streptococcus mutans. It is also stated to be a
better agent Lactobacilli but there are contrasting results stating that the inhibitory effect
on Lactobacilli could not be proven which is shortcoming. Agents containing
70
DISCUSSION
Chlorhexidine have also shown to be effective against Aggregatibacter
actinomycetemcomitans. The antifungal activity of Chlorhexidine is proven invitro but
invivo studies have shown it to be ineffective, which is not in favor of usage of it against
Candida albicans. (51 – 57)
Povidone iodine on the other hand is considered to have the broadest
spectrum of antimicrobial action showing good effect against gram positive bacteria, gram
negative bacteria, fungi and several viruses but the main drawback of Povidone Iodine is
that it exacts only an immediate antibacterial effect and unlike Chlorhexidine it lacks
prolonged action. 41,58
Clotrimazole a positive control used for fungi in the present study has a very
good antifungal action but it is ineffective against gram negative bacteria, though it has
effect on gram positive bacteria. Nystatin has antifungal action but it is completely
ineffective against bacteria.59
Literature search suggests no single agent to be effective against all the four
microorganisms tested, as two are gram positive bacteriae, one is gram negative and one is
a fungi. The results of the present study is a landmark in investigating one agent effective
against all the four organisms with promising results. 59
In the present study the minimum inhibitory concentrations observed for
Streptococcus mutans, Lactobacillus and Candida albicans is 0.005% whereas for
Aggregatibacter actinomycetemcomitans is 0.010%. These concentrations obtained are
very much lesser than the safety dose of the extract, as 200mg/kg of plant extract is
considered as non-toxic dosage. The results of the present study shows, that the plant has
antimicrobial effect and the results of various other studies from literature suggesting that
71
DISCUSSION
ethanolic extracts of Prosopis juliflora were non-toxic, it is evident that the plant is suitable
for long term in vivo studies of pharmacological effects.23 The plant is found in abundance
which could be a cost effective alternative as well.
Differences in the results may be due to some factors such as dissimilarities
in plant species, solvents and geographical regions. Since the present study is compared
with other innate herbs, nature & mechanism of action of the herbs could attribute to the
difference. The study holds its limitation of testing against fewer pathogens and using only
one extraction type for obtaining plant extract. But this does not influence the specific
outcome achieved.
72
CONCLUSIONS & RECOMMENDATIONS
The present study concludes that, Prosopis juliflora leaves from Madurai,
TamilNadu is an appropriate source for antibacterial and antifungal components as
it showed notable antibacterial and antifungal activity. The plant extract was
effective even at lesser concentrations and the inhibitory activities were found to
be dose dependent. Hence it is suitable for further in-vivo investigations.
There exists a major global healthcare problem which threatens the mankind with
emergence of multidrug resistance pathogens. The battle against these organisms is
never ending, yet we can beat them by changing our strategy. Returning back to
nature is the need of the hour, as active ingredients from the plants are the ones
which has survived microbes since millions of years.
This plant can be used in various forms that meets the therapeutic needs. Therefore,
the present study may provide a scientific basis for the development of novel, safer
and clinically effective medicines for both systemic and topical usage.
In the next studies, further works will be needed to investigate their antibacterial,
antifungal and antiviral activity against wide range of microorganisms. Further
purification of the extract may increase its activity proving to be more effective.
More research need to be destined to expand the number of scientifically studied
species, as well as to describe the cellular and molecular mechanisms related to the
described properties and to support the evidence for usage.
73
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ANNEXURES
Annexure I – Ethical committee approval letter

ANNEXURES
Annexure II– Permission letter from Pharmacy College

ANNEXURES
Annexure III – Plant Identification Certificate

ANNEXURES
Annexure IV – Permission from laboratory

ANNEXURES
Annexure V – Laboratory report front page

ANNEXURES
Annexure VI – Permission from Head of the Institution

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“ASSESSING THE ANTIMICROBIAL EFFICACY OF Prosopis juliflora AGAINST

ORAL MICROORGANISMS: AN INVITRO STUDY”

In partial fulfillment of the requirements

For the degree of

MASTER OF DENTAL SURGERY

PUBLIC HEALTH DENTISTRY

THE TAMILNADU Dr.M.G.R. MEDICAL UNIVERSITY

Urkund Analysis Result

Sources included in the report:

Instances where selected sources appear:

I thank God Almighty by whose grace this work became possible.

I would like to thank all those people who helped me directly or

indirectly to complete my Main Dissertation. I am deeply inherited to those who devoted

have helped me in completing this Main Dissertation.

My sincere thanks to Prof K.R.Arumugam and Vice chairman

Babu Thandapani for giving me an opportunity to gain knowledge through this

My acknowledgement would never be complete without mentioning my

Principal, Dr.K.S.Premkumar sir, Professor and Head, Department of Orthodontics and

today and words cannot express my gratitude, “I am proud to be his student”

heartfelt gratitude to my mentors Dr.Umesh.K, Dr.Muthukaruppaiah.R, Dr.Sangeeta

Chavan and Dr.Palanivel Pandian.R

I extend my deepest gratitude to Dr.Umesh.K sir, Professor and Head, Department of

humanity and it is my privilege to have him as my head.

I am immensely thankful to Dr.Muthukaruppaiah.R Sir, Reader and my guide for

My sincere thanks to Dr.Sangeeta Chavan ma’am, Reader, Department of Public health

who is friendly in nature.

Love you dear!

INTRODUCTION: Oral health is integral to the general well-being and

populations worldwide despite which problems persists in many communities

around the world, predominantly among underprivileged groups in both developed

antimicrobial activity of the Prosopis juliflora leaves against Streptococcus mutans,

Lactobacillus, Aggregatibacter actinomycetemcomitans and Candida albicans.

MATERIALS AND METHODS: An In-vitro, Experimental study was

designed to assess the antimicrobial efficacy of Prosopis juliflora against oral

microorganisms. Plant extract preparation was done using Maceration technique

RESULTS: Prosopis juliflora showed inhibitory action compared to and

superior to that positive control with a minimum inhibitory concentration of

0.005% for Streptococcus mutans, 0.005% for Lactobacillus, 0.01% for

Aggregatibacter actinomycetemcomitans and 0.005% for Candida albicans.

CONCLUSION: The present study concludes that, Prosopis juliflora leaves

from Madurai, TamilNadu is an appropriate source for antibacterial and antifungal

components as it showed notable antibacterial and antifungal activity. This plant

clinically effective medicines for both systemic and topical usage.

Keywords: Antimicrobial agent, Medicinal plant, Oral Health, Oral

Microorganisms, Prosopis juliflora

S.NO ABBREVIATION DESCRIPTION

1 WHO WORLD HEALTH ORGANISATION

2. ICFRE INDIAN COUNCIL OF FORESTRY

RESEARCH & EDUCATION

3. MDR MULTI DRUG RESISTANT

4. MRSA METHICILLIN RESISTANT

5. TAE TOTAL ALKALOID EXTRACTED

6. MIC MINIMUM INHIBITORY

7. PPM PARTS PER MILLION

TABLE. TITLE PAGE

1 ZONE OF INHIBITION OF Prosopis juliflora 52

AGAINST Streptococcus mutans

2. ZONE OF INHIBITION OF Prosopis juliflora 53

3. ZONE OF INHIBITION OF Prosopis juliflora 54

AGAINST Aggregatibacter actinomycetemcomitans

4. ZONE OF INHIBITION OF Prosopis juliflora 55

AGAINST Candida albicans

ACTIVITY OF Prosopis juliflora,