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TRANSCRIPT
I
“COMPARATIVE EVALUATION OF THE
ANTIMICROBIAL EFFICACY OF POMEGRANATE
AND JASMINE LEAF EXTRACTS ON
STREPTOCOCCUS MUTANS: AN INVITRO STUDY”
By
Dr. ADUSUMILLI HAMSINI
Dissertation submitted to the
RAJIV GANDHI UNVERSITY OF HEALTH SCIENCES, BANGALORE
In partial fulfillment of the requirements for the degree of
MASTER OF DENTAL SURGERY In
PEDODONTICS AND PREVENTIVE DENTISTRY
Under the guidance of
Dr. NAVEEN KUMAR. R M.D.S
Professor and Head
DEPARTMENT OF PEDODONTICS AND PREVENTIVE DENTISTRY
NAVODAYA DENTAL COLLEGE & HOSPITAL
RAICHUR
2015-2018
II
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
KARNATAKA, BANGALORE
DECLARATION BY THE CANDIDATE
I hereby declare that this dissertation/thesis entitled
“Comparative Evaluation Of The Antimicrobial Efficacy
Of Pomegranate And Jasmine Leaf Extracts On
Streptococcus Mutans: An Invitro Study” is a bonafide and
genuine work carried out by me under the guidance of Dr. NAVEEN
KUMAR. R, Professor and Head, Department of Pedodontics and
Preventive dentistry, Navodaya Dental College & hospital, Raichur.
III
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
KARNATAKA, BANGALORE
CERTIFICATE BY THE GUIDE
This is to certify that the dissertation entitled “Comparative
Evaluation Of The Antimicrobial Efficacy Of Pomegranate
And Jasmine Leaf Extracts On Streptococcus Mutans: An
Invitro Study” is a bonafide and genuine research work done under
me by Dr. ADUSUMILLI HAMSINI in partial fulfillment of the
requirements for the degree of MASTER OF DENTAL
SURGERY (MDS) in Pedodontics and Preventive dentistry.
IV
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
KARNATAKA, BANGALORE
ENDORSEMENT BY HOD,PRINCIPAL/HEAD OF THE
INSTITUTION
This is to certify that the dissertation entitled “Comparative
Evaluation Of The Antimicrobial Efficacy Of Pomegranate
And Jasmine Leaf Extracts On Streptococcus Mutans: An
Invitro Study” is a bonafide research work done by Dr. ADUSUMILLI
HAMSINI under the guidance of Dr. NAVEEN KUMAR. R, Professor
and Head, Department of Pedodontics and Preventive Dentistry,
Navodaya Dental College & Hospital, Raichur .
VIII
LIST OF ABBREVATIONS USED
A.flavus Aspergillus flavus
A.fumigates Aspergillus fumigates
A.niger Aspergillus niger
Aa Aggregatibacter actinomycetemcomitans
ADV Adenovirus
ANS Autonomic Nervous system
B.subtilis Bacillus subtilis
BHI agar Brain Heart Infusion agar
BHI broth Brain Heart Infusion broth
BHT Butylated hyroxytoluene
CHX Chlorhexidine
CNS Central Nervous system
COX Cyclooxygenase
CT Condensed Tannins
DMEAG Dimethylellagic acid
DPPH 2, 2‐diphenyl‐1‐picrylhydrazyl hydrate
EA Ellagic Acid
ET Ellagitannins
HAE Hydroalcoholic extract
HPLCMS High Performance Liquid Chromatography with Mass Spectrometry
HSV Herpes Simplex Virus
HT Hydrolysable Tannins
IX
J.sambac Jasminum sambac
JLE Jasmine leaf extract
MBC Minimum Bactericidal Concentration
MIC Minimum Inhibitory Concentration
MRSA Methicillin Resistant Staphylococcus aureus
MSB Mitis Salivarius Bacitracin Agar
NCCLS National Committee for Clinical Laboratory Standards
NRCP National Research Centre on Pomegranate
P.mirabilis Proteius mirabilis
PE Pomegranate extract
Pg Porphyromonas gingivals
Pi Prevotella intermedia
PJ Pomegranate Juice
S.aureus Staphylococcus aureus
S.mutans Streptococcus mutans
S.pyogens Streptococcus pyogens
S.sobrinus Streptococcus sobrinus
S.typhi Salmonella typhi
X
LIST OF TABLES
Sl. No. Tables Pages
1 General nutritional value of pomegranate fruit 11
2 Components of pomegranate aril juice. 12
3 Classification of Jasminum species. 20
4 Engler and Prantl classification of Jasmine based on its leaves.
20
5 Gamble’s classification of jasmine. 21
6 Composition of MSB agar 32
7 Composition of BHI agar 33
8 Composition of BHI broth 34
9 MIC and MBC of pomegranate and jasmine leaf extract against S.mutans
41
10 Descriptive statistics of the mean zone of inhibition (mm) produced by pomegranate group against Streptococcus mutans.
42
11 Descriptive statistics of the mean zone of inhibition (mm) produced by Jasmine leaf extract against Streptococcus mutans.
43
12 Comparison of 0.2% chlorhexidine and ethanol with pomegranate groups against streptococcus mutans (unpaired t- Test).
44
13 Comparison of 0.2% chlorhexidine and ethanol with jasmine leaf extract against streptococcus mutans (unpaired t- Test).
46
14 Intra group comparison of pomegranate extract groups, 0.2% chlorhexidine and 99.9% ethanol groups tested against Streptococcus mutans (Posthoc- Anova Test).
48
15 Intra group comparison of jasmine leaf extract groups, 0.2% chlorhexidine and 99.9% ethanol groups tested against Streptococcus mutans (Posthoc- Anova Test).
49
16 Inter group comparison of concentrations in pomegranate and jasmine group
50
XI
LIST OF FIGURES
Sl. No Figures Pages
1 Pomegranate flower and fruit 88
2 Parts of pomegranate fruit 88
3 Pomegranate tree 88
4 Principal anthocyanins present in pomegranate juice 89
5 Principal phenolic acids present in the pomegranate juice
90
6 Principal ETs present in pomegranate – Punicalin 90
7 Principal ETs present in pomegranate- Pedunculagin 90
8 Principal ETs present in pomegranate- Punicalagin 90
9 Jasminum sambac plant description 91
10 Jasminum sambac plant 92
11 Jasminum sambac plant 92
12 Sambacoside A 92
13 Quercitin 92
14 Isoquercitin 93
15 Rutin 93
16 Kaempferol 93
17 Luteolin 93
18 Jasminol 93
19 Palmitic acid 93
20 Stearic acid 94
21 Ursolic acid 94
22 Inositol 94
XII
Sl. No Figures Pages
23 Fridelin 94
24 Lupeol 94
25 Betulin 94
26 α- amyrin 95
27 Pomegranate arils 95
28 Centrifugation of the pomegranate extract 95
29 Pomegranate extract in hot air oven 95
30 Final gelatinous extract 95
31 Fresh jasmine leaves 96
32 Dried leaves powder 96
33 Soxhlet extraction 96
34 Final desiccated jasmine leaf extract 96
35 Armamentarium used 97
36 Collected saliva samples 97
37 Prepared media (MSB agar, BHI agar, BHI broth) 97
38 0.5mm pin point streptococci colonies 97
39 Microscopic confirmation of streptococcus mutans. Short bluish round chains.
97
40 0.5 Mcfarland standardization 98
41 2-fold dilutions of pomegranate and jasmine leaf extract
98
42 MIC and MBC of pomegranate extract 98
43 MIC and MBC of jasmine leaf extract 98
44 Zones of inhibition exhibited by pomegranate (C1-C3)
98
45 Zones of inhibition exhibited by jasmine leaf extract (D1-D3)
98
1
Structured Abstract
Title:
Comparative evaluation of the antimicrobial efficacy of pomegranate and jasmine leaf
extracts on Streptococcus mutans: an invitro study
Objective:
To evaluate and compare the antimicrobial efficacy of pomegranate and jasmine leaf
extract on oral Streptococcus mutans.
Background:
Dental caries is a multifactorial, highly prevalent oral disease in humans.1 The
initiation and progression of dental caries is implicated by the bacteria Streptococcus
mutans.2 Prescribing an antibacterial agent in children plays a significant role in the
prevention of dental caries.3 Though chlorhexidine (0.2%) is considered as a gold
standard, it unfolds numerous side effects.4 Medicinal plant extracts have lesser side
effects5 and could be safely incorporated in tooth wipes for infants.6 Pomegranate has
antioxidant, antimicrobial, anti-inflammatory and anticarcinogenic potencies.7 Jasmine
leaves have many medicinal properties and antimicrobial properties. A recent study
reports about the beneficial effects of Jasmine leaf extract.8
2
Methodology:
2ml of unstimulated whole saliva sample from 5 children aged from 4-6 years
with severe early childhood caries was collected9,10, pooled and cultured to obtain
microbial isolates of Streptococcus mutans.11 Pomegranate pulp extract was prepared
from market procured pomegranates by hand using a mortar and pestle.6 Jasmine leaf
extract was custom prepared by shade drying the leaves and later subjecting it to
soxhlet extraction.8
The MIC, MBC & Agar well-diffusion method was performed as per National
Committee for Clinical Laboratory Standards (NCCL).12
Results:
MIC and MBC values of the pomegranate extract against Streptococcus mutans was
1:64, 1:32 and for jasmine leaf extract was 1:16, 1:8 respectively. Both extracts showed dose
dependent antibacterial action against both Streptococcus mutans.
Interpretation & Conclusion:
This study demonstrated that both pomegranate and jasmine leaf extract
possessed antimicrobial efficacy against Streptococcus mutans.
Keywords:
MIC ; MBC ; Pomegranate extract ; Jasmine leaf extract ; Streptococcus mutans
3
COMPARATIVE EVALUATION OF THE ANTIMICROBIAL
EFFICACY OF POMEGRANATE AND JASMINE LEAF
EXTRACTS ON STREPTOCOCCUS MUTANS: AN INVITRO
STUDY
Introduction
Dental caries is a multifactorial, highly prevalent oral disease in humans.1 It
results from the interaction between host, diet and microflora on the tooth surface over
a period of time, resulting in localized de-mineralization of hard tissues.13-15 However,
micro-flora is considered to be the most important factor mediating the caries initiation
as well as progression. The most implicated bacteria for initiation of dental caries is
Streptococcus mutans.14 Since, caries process is the result of shift in the equilibrium
favoring de-mineralization process,16 controlling the early colonization of bacteria in
the oral cavity of children aids in reducing the caries prevalence and caries risk.
Therefore, prescribing an antibacterial agent as an adjunct to other oral hygiene
practices in children plays a significant role in the prevention of dental caries.3
Though chlorhexidine (0.2%) is considered as a gold standard, its regular use is
not advised in children as it unfolds numerous side effects like taste alteration, peeling
of tongue and mucosa, staining of teeth.4
So, the need to develop biocompatible herbal formulations arises especially in
children. Herbal products have been used since ancient times in folk medicine,
involving both eastern and western medicinal traditions. Numerous medicinal plants
and their extracts are in the limelight owing to their better, safe, economical and lesser
side effects compared to the, chemical ones.5 The natural phytochemicals isolated from
medicinal plants used in traditional medicine have been considered useful alternatives
4
to synthetic drugs. These herbal extracts could be safely incorporated in tooth wipes for
infants.6
Punica granatum, commonly known as pomegranate is found to be beneficial
fruit in maintenance of oral health.17 This fruit is considered as a ‘pharmacy onto itself’
due to its various therapeutic properties.7 Pomegranates contain polyphenols like
tannins, anthocyanins, flavonoids which have a wide antioxidant and antimicrobial
activity.17
Jasminum sambac Linn. (Family-Oleaceae) commonly known as Motia or lily
jasmine is a scandent or sub-erect shrub cultivated nearly throughout the tropical and
subtropical parts of the world.18 Traditionally, the leaves of this plant are used in fever
or cough, indolent ulcer, abdominal distension, diarrhoea, lowering the blood glucose
level, regulating menstrual flow, to clean kidney waste, inflamed and blood shot
eyes.18,19 Pharmacological activities of the plant reported so far are antidiabetic20,
antitumor21, antimicrobial22, antioxidant23, anti-acne24, A.N.S stimulating effect.25
The antimicrobial efficacy of pomegranate and jasmine leaf extract on oral
microflora has scanty evidence in the literature. The purpose of this invitro study was
to evaluate and compare the antimicrobial efficacy of pomegranate and jasmine leaf
extracts on Streptococcus mutans.
5
Aim & Objectives
Aim:
To compare the antimicrobial efficacy of Herbal extracts of Pomegranate and Jasmine
leaves on oral Streptococcus mutans
Objectives:
To evaluate the antimicrobial efficacy of pomegranate extract on oral
Streptococcus mutans.
To evaluate the antimicrobial efficacy of jasmine leaf extract on oral
Streptococcus mutans.
To compare the antimicrobial efficacy of pomegranate and jasmine leaf extracts
on oral Streptococcus mutans.
6
Review of Literature
Chlorhexidine (CHX), a cationic bis-biguanide biocide with low mammalian
toxicity and broad-spectrum antibacterial activity, was first described in 1954.26 The
primary mechanism of action of this biocide is membrane disruption, causing
concentration dependent growth inhibition and cell death. With respect to dental
hygiene applications, the cationic nature of CHX enables it to bind to tooth surfaces
and oral mucosa, reducing pellicle formation and increasing substantively through
controlled release of the agent.27
According to an in-vitro study done by Solmaz G the MIC and MBC values for
Chlorhexidine gluconate were found to be 1.5mg/L and 3mg/L respectively against
Streptococcus mutans. On performing disc diffusion assay the diameter of zone of
inhibition was determined at a range of 15.8±3.4mm.28
The efficacy of CHX in reducing oral bacteria viability strongly inhibiting
plaque regrowth and preventing gingivitis has been demonstrated in many studies.29,30
However chlorhexidine gluconate has been shown to be inactivated by food and saliva,
causes taste disturbances and mucosal irritation as well as staining of teeth and tongue.31
Since chlorhexidine is widely used, practitioners should be aware of the low
potential of the rinse to induce allergic reactions including anaphylactic shock.32
Ohtoshi et al. reported more than 30 cases of anaphylactic shock after the topical
application of chlorhexidine.33 In these cases a specific IgE antibody against
chlorhexidine was shown to be a mediator of the reaction.34
An alarming increase in bacterial strains resistant to a number of antimicrobial
agents in addition to the adverse effects associated with these synthetic agents, demands
that a renewed effort be made to seek antibacterial agents that are more compatible and
potent.
7
Traditional healers have long used plants to prevent or cure infectious
conditions. Many of these plants have been investigated scientifically for antimicrobial
activity and a large number of plant products have been shown to inhibit growth of
pathogenic bacteria.
In a study by Waghmare PF et al to compare the efficacy of turmeric and
chlorhexidine mouthwash in prevention of plaque and gingivitis, significant reduction
in total microbial count was observed in both the groups. However a significant
reduction in the mean plaque index was noted in CHX group in comparison with the
turmeric group. Chlorhexidine was found to have superior antiplaque property.35
In an another in-vitro study done to ascertain the effects of Triphala mouthwash
in comparison with 0.2% chlorhexidine gluconate on dental plaque, gingival health and
microbial count. No statistical difference was noted between the two groups on dental
plaque reduction and gingival health. However triphala showed a better inhibitory
effect than chlorhexidine on microbial counts of lactobacillus acidophilus.36
Recent studies compared the antimicrobial action of an herbal mouth rinse (The
Natural Dentist Healthy Gums Daily Oral Rinse): an essential oil oral rinse (Listerine
Cool Mint) and an established 0.12% chlorhexidine gluconate oral rinse (Peridex),
against predominant oral bacteria. ‘The Natural Dentist’ contained extracts of Aloe
barbadensis, Echinacea angustifolia, Echinacea purpurea, Hydrastis, Canadensis,
Calendula officinalis and Citrus paradisi.37
Although ‘The Natural dentist’ was found to be less potent than the Peridex, it
inhibited the growth of 40 bacterial species tested. When compared to Listerine, ‘The
Natural Dentist’ exhibited significantly lower minimum inhibitory concentrations
(MIC’s) for Actinomyces species, periodontal pathogens Eubacterium nodatum,
Tannerella forsythia and Prevotella species, as well as the cariogenic S.mutans.37
8
Venka A et al conducted a study related to the antibacterial effect of neem
mouthwash against salivary levels of streptococcus mutans and Lactobacillus
acidophilus tested over a period of 2 months. Its effect in reversing incipient carious
lesions was also assessed. He found that Streptococcus mutans was inhibited by neem
mouthwashes, with or without chlorhexidine; Lactobacillus growth was inhibited by
chlorhexidine alone. This data appeared tp prove ‘neem’ effect in inhibiting S. mutans
and reversing incipient carious lesions.38
In an in-vivo study by Prathiba A et al to compare the effect of Manuka honey
0.2% Chlorhexidine mouthwash and xylitol chewing gum on dental plaque levels.
Results revealed that the plaque inhibition by manuka honey was similar to that of 0.2%
chlorhaxidine mouthwash.39
The antibacterial properties of essential oils are well-known and activity against
oral cavity pathogens has been well documented.40 Essential oils are also capable of
enhancing the activity of chlorhexidine when used in combination. Essential oils of
cinnamon and manuka were able to significantly reduce the amount of chlorhexidine
required to inhibit the growth of oral pathogens.41 This enhanced activity was also seen
against bacterial cultures grown as biofilms. A 4-fold reductions of the amount of
chlorhexidine was found to be required to inhibit biofilm bacteria when used in
combination with cinnamon, manuka and leptospermummorrisonii oils.41
POMEGRANATE:
Historical background:
Pomegranate (Punica granatum) is native to the Mediterranean region. It is a
native fruit of Iran, one of its biggest producers and exporters. Before its medicinal
9
properties were defined, the pomegranate was revered by many of the world’s main
religions.
In Greek mythology- the pomegranate represented life, regeneration, and marriage.
In Zoroastrian ceremonial and domestic adherences- the pomegranate confined
invincibility.
In Christianity- pomegranate seeds are stated to be 613 in number, represent each of
the Bible’s 613 commandments. It also signifies sanctity, fertility, and abundance. An
icon of renaissance and life eternal in Christian art, the pomegranate is often found in
devotional statues and paintings of the Virgin and Child.
In Buddhism- pomegranate is considered to be one of the three sacred fruits, along with
citrus and peach, symbolizing the essence of favorable influences. Pomegranate is
widely represented in ceramic art in China. Additionally, pomegranate denotes fertility,
abundance, posterity, and numerous and virtuous offsprings, with a sanctified future.
In Islam- a legend holds that each pomegranate contains one seed that has come down
from paradise. The pomegranate plays a distinct role as a fertility symbol in marriages
among the Bedouins of the Middle East.
In Hinduism- the pomegranate (Sanskrit: Beejpur, literally; replete with seeds) implies
prosperity and fertility and is related with both Bhoomidevi (the earth goddess) and
Lord Ganesha (who is also called Bijapuraphalasakta, or the one fond of the many-
seeded fruit).
All parts of the plant (roots, bark, flowers, fruits, and leaves) are used for
remedial purposes in Ayurveda. The Ayurvedic system of medicine regards
pomegranate as a ‘pharmacy unto itself’. It is used as an anti-parasitic agent and a blood
tonic. It heals aphthae, diarrhea, and ulcers. Pomegranate also functions as a remedy
for diabetes in the Unani system of medicine practiced in the Middle East and India.
10
The name pomegranate originates from the genus ‘Punica’, which was the
Roman name for Carthage, where the best pomegranates were known to grow.
Pomegranate is known by the French as grenade, the Spanish as Granada, which
literally translates to seeded (‘granatus’) apple (‘pomum’).42,43
Botanical Background:
The pomegranate tree typically grows 12−16 feet and has many spiny branches.
It can be extremely longlived, as evidenced by trees at Versailles, France, known to be
over 200 years old. The leaves are glossy and lance shaped. The bark of the tree turns
gray as the tree ages. The flowers are large, red, white, or variegated, and have a tubular
calyx that eventually becomes the fruit [Figure 1-3]. The ripe pomegranate fruit can be
up to five inches wide with a deep red, leathery skin. It is grenade-shaped, and crowned
by the pointed calyx. The fruit contains many seeds (arils) separated by white,
membranous pericarp, and each is surrounded by small amounts of tart.8
Phytochemistry:
The pomegranate fruit has valuable compounds in different parts of the fruit.
These can be divided into several anatomical origins: peel, seeds, and arils. About 50%
of the total fruit weight corresponds to the peel, which is an important source of
bioactive compounds such as phenolics, flavonoids, ellagitannins(ET), and
proanthocyanidin compounds44, minerals, mainly potassium, nitrogen, calcium,
potassium, magnesium and sodium45 and complex polysaccharides.46
11
Table 1: General nutritional value of pomegranate fruit (NRCP 2008).47
Parameters Values
Moisture(%) 81.17
Total ash (%) 0.46
Protein (%) 1.21
Fat (%) 0.24
Crude fibre (%) 1.4
Carbohydrates(%) 15.52
Calorific Value (K cals /100g) 69.08
Acidity (%) 0.38
Minerals (mg/100g)
Iron 0.3
Zinc 0.19
Calcium 2.71
Magnesium 7.78
Copper 0.28
Manganese 0.3
Phosphorus 28.23
Vitamins (mg/100g)
Thiamine 0.06
Niacin 0.25
Ascorbic acid 22.42
Total carotenoids(μg/100g) 27
12
COMPOSITION OF POMEGRANATE ARIL JUICE:
The edible part of the pomegranate fruit (50%) consists of 40% arils and 10%
seeds.48 Table 2 shows the composition of pomegranate aril juice. Arils contain 85%
water, 10% total sugars, mainly fructose and glucose49, 1.5% pectin, organic acid such
as ascorbic acid, citric acid and mallic acid50, fatty acids51, amino acids52 and
bioactive compounds such as phenolics and flavonoids, principally anthocyanins.53
Table 2: Components of pomegranate aril juice.
Components
Water 85%
Sugars 10% (Fructose, Glucose, sucrose)
Pectin 1.50%
Organic acids Conjugated linoleic acid, linoleic acid, punicic acid,
eleostearic acid
Amino acids Proline, valin, methionine
Bioactive compounds Phenolics, flavonoids
Polyphenolic
compounds 0.2- 1% Anthocyanins, hydolysable tannins
CHEMISTRY OF PHENOLIC COMPOUNDS:
One of the main compounds responsible for most of the functional properties
pomegranate fruit, are phenolic compounds in any of their forms.54 Natural polyphenols
can range from simple molecules (phenolic acids, phenylpropanoids, flavonoids) to
13
highly polymerized compounds (lignins, melanins, tannins), with flavonoids
representing the most common and widely distributed subgroup.55 Chemically,
phenolic acids can be defined as substances that possess an aromatic ring bound to one
or more hydrogenated substituents, including their functional derivatives.56,57
Flavonoids:
Flavonoids are low-molecular-weight compounds consisting of 15 carbon
atoms, arranged in a C6-C3-C6 configuration. Essentially, the structure consists of 2
aromatic rings joined by a 3-carbon bridge, usually in the form of a heterocyclic ring.58
Anthocyanins:
Anthocyanins are the largest and most important group of flavonoids present in
pomegranate arils, which are used to obtain the juice. These pigments give the fruit and
juice its red color.59 There are a great variety of anthocyanins present in pomegranate
juice, principally cyanidin-3-O-glucoside; cyanidin-3,5-di-O-glucoside; delphinidin-3-
O-glucoside; delphinidin-3,5-di-Oglucoside; pelargonidin-3-O-glucoside; and
pelargonidin-3,5-di- O-glucoside (figure 4).60,61 The main differences between them are
the number of hydroxylated groups, the nature and the number of bonded sugars to their
structure, the aliphatic or aromatic carboxylates bonded to the sugar in the molecule,
and the position of those bonds.62
Phenolic Acids:
The phenolic acids present in pomegranate juice (Figure 5) can be divided into 2
groups:
(1) Hydroxycinnamic acids, principally p-coumaric acid, chlorogenic acid and caffeic
acid.63
14
(2) Hydroxybenzoic acids, mainly ellagic acid (EA) and gallic acid.64
Tannins:
Polyphenols components play a major role in their color, flavor, texture as well as
antioxidant65 and antibacterial activities.66 Phenolic compounds can denature
enzymes67 but they can also bind to substrates such as minerals, vitamins and
carbohydrates making them unavailable for microorganisms.68,69 Furthermore, phenols
can be absorbed to the cell wall, resulting in a disruption of the membrane structure and
function.70 Tannins are water-soluble polyphenolic polymers of relatively high
molecular weight and have capacity to form complexes mainly with proteins, to a lesser
extent with carbohydrates due to the presence of a large number of phenolic hydroxyl
groups. Tannins are usually divided into two major groups:71,72
1. Hydrolysable tannins (HTs)/ Ellagitannins (ETs) and
2. Condensed tannins (CTs).
1. Hydrolyzable tannins are gallic or ellagic acid esters of sugars (Figure). When
they are consumed by human, they can be degraded into gallic acid and be
absorbed in the digestive tract.73
2. Condensed tannins are polyphenols of higher molecular weight and consist
mainly of oligomers or polymers of catechin (flavan-3-ols).When CTs get
depolymerized, they produce mainly cyanidin or delphinidin, and therefore
have been further classified as procyanidins or prodelphinidins. Only a low
degree of absorption of CTs by the digestive tract has been reported. One of
their most important chemical properties is the ability to form soluble and
insoluble complexes with macromolecules, such as protein, fiber and starch.73
15
FUNCTIONAL COMPONENTS OF POMEGRANATE:
The pomegranate fruit could be considered a functional food because it has
valuable compounds in different parts of the fruit that display functional and medicinal
effects.74 Pomegranate juice, which is rich in tannins, possesses anti-atherosclerotic,
antihypertensive, anti-aging, and potent anti-oxidative characteristics.75 Hence, it
provides cardioprotective benefits.76 Pomegranate juice may have cancer-
chemopreventive as well as cancer-chemotherapeutic effects against prostate cancer, in
humans.77
PHARMACOKINETICS:
Ellagitannins (ETs) from pomegranate juice (PJ) are recorded to have several
biological properties; yet their absorption and metabolism in humans are poorly
understood.
Trials performed by Seeram NP et al. 2006, on 18 healthy volunteers, who were
administered 180 mL of PJ concentrate, showed that Ellagic acid (EA) was present in
the plasma of all subjects, with a maximum concentration of 0.06 ± 0.01 μmol / L, in
the area under the concentration time curve of 0.17 ± 0.02 (μmol.h) x L(-1), time of
maximum concentration of 0.98 ± 0.06 hours, and elimination half-life of 0.71 ± 0.08
hours. EA metabolites, including dimethylellagic acid glucuronide (DMEAG) and
hydroxy-6H-benzopyran-6-one derivatives (urolithins), were also detected in the
plasma and urine, in conjugated and free forms. DMEAG was found in the urine
obtained from 15 of 18 subjects on day 0, but was not detected on day -1 or +1,
demonstrating its potential as a biomarker of intake. Urolithins, formed by intestinal
bacteria, may contribute to the biological effects of PJ, as they may persist in the plasma
and tissues and account for some of the health benefits noted after chronic PJ
consumption.78
16
Another study investigated the absorption of a standardized extract from
pomegranate in healthy human volunteers after the acute consumption of 800 mg of
extract. Results indicate that ellagic acid (EA) from the extract is bioavailable, with an
observed C(max) of 33 ng / mL at t(max) of one hour. The plasma metabolites urolithin
A, urolithin B, hydroxylurolithin A, urolithin A-glucuronide, and dimethyl ellagic acid-
glucuronide were identified by HPLCMS.79
PHARMACODYNAMICS:
Pomegranate’s wide-ranging therapeutic benefits are attributable to several
mechanisms, most research has been focused on its antioxidant, anticarcinogenic, anti-
inflammatory properties, and antimicrobial properties.
Antioxidant property:
Animal studies have demonstrated the free-radical scavenging properties of
pomegranate juice. It has also been seen to decrease macrophage oxidative stress and
lipid peroxidation. Human trials have shown that pomegranate juice increases the
plasma antioxidant capacity; whereas in vitro assays have demonstrated that
pomegranate juice has two to three times the antioxidant capacity compared to red wine
and green tea.80,81,82 Even pomegranate peel extract enhances the free-radical
scavenging activity of hepatic enzymes catalase, superoxide dismutase, and
peroxidase.83 There is a significant decrease in the plasma carbonyl content (a
biomarker for oxidant / antioxidant barrier impairment in various inflammatory
diseases) compared to apple juice.82
Anti-carcinogenic property:
Pomegranate cold-pressed oil, supercritical CO(2) extracted seed oil, fermented
juice polyphenols, and pericarp polyphenols inhibit prostate cancer cell invasiveness
and proliferation causes cell cycle disruption, induces apoptosis, and inhibits tumor
17
growth, as per the in vitro assays utilizing three prostate cancer cell lines (DU-145,
LNCAP, and PC-3).84,85 Studies in mice implanted with the prostate cancer PC-3 cell
line demonstrated that pomegranate fruit extract inhibits cell growth and induces
apoptosis via modulation of the proteins regulating apoptosis.77,86 Recent research also
indicates that pomegranate seed oil and fermented juice polyphenols inhibit
angiogenesis via the down regulation of vascular endothelial growth factor in MCF-7
breast cancer and human umbilical vein endothelial cell lines.87
Anti-inflammatory property:
Both cyclooxygenase (COX) and lipoxygenase enzymes were inhibited in vitro
by the pomegranate seed oil extract.88
Anti-microbial property:
Both animal as well as human studies have demonstrated the antimicrobial
activity of hydroalcoholic extract (HAE) and gel from pomegranate fruits against
Staphylococcus, aureus, Streptococcus pyogenes, Diplococcus pneumoniae,
Escherichia coli, and Candida albicans.89,90
There are various schools of taught regarding the mechanism of action in which
pomegranate act for the anticariogenic property First, the ellagitannin, punicalagin causes
disturbance of polyglycans synthesis, and thus acts on the adherence mechanism of
these organisms to the dental surface. Second, the tannin crosses over the cell wall of
the microorganisms and binds to its surface leading to the precipitation of proteins and
suppress enzyme like glycosyl transferase, the key enzyme for breakdown of
carbohydrates. Third, the phenolic compounds bind to the substrate such as minerals
and carbohydrate making them unavailable for the microorganisms to survive resulting
in cell wall disruption.91
18
Lalvani V et al stated that pomegranate aril extract as a mouth rinse had a
significantly higher inhibitory effect against Streptococcus in both dental caries patients
and healthy individuals. The percentage of reduction was higher in experimental
(52.2%) than control (33.8%).91
Prashanth et al. also confirmed that methanolic extracts of pomegranate rind to
be active against all microorganisms tested in their study. An in vitro study showed that
Streptococcus sanguis, a bacterial strain present in the supra gingival plaque was
sensitive to different concentration of pomegranate extract, which demonstrated
inhibitory action similar to that of chlorohexidine.92
Subramanaim et al. conducted an in vitro study and stated that hydroalcoholic
extract of pomegranate aril showed significantly higher inhibitory effect on
Streptococcus mutans at all concentration.6
Kote et al. stated that pomegranate rinse is effective against dental plaque
micro-organisms; there was a significant reduction in Streptococcus mutans colonies
(23%).17
A recent study proved that pomegranate mouthwash had antibacterial efficacy
against Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis
(Pg), Prevotella intermedia (Pi), which are the most important periodontal pathogens.93
Punica granatum has shown antimicrobial activity against Eikenella corrodens, which
is a secondary colonizer in the biofilm formation on the tooth surface significantly more
than chlorhexidine.94 Rinsing with 30 mL of pomegranate juice was effective in
reducing colony forming units of dental plaque forming organisms by 32%.17
Pomegranate mouthwash used two times daily for fifteen days resulted in more efficient
reduction of gingival and bleeding on probing scores when compared to
chlorhexidine.95 Pomegranate gel when used as an adjunct with mechanical
19
debridement was efficient in treating gingivitis with an improvement in the clinical and
microbiological parameters.96 Significant improvement was observed in plaque,
gingival and bleeding indices among 92 patients who were instructed to use toothpaste,
which contained pomegranate along with various other herbs.97 Pomegranate thereby
could be an excellent adjunct to the conventional periodontal therapy as an anti-plaque
agent due to its antibacterial properties.98
Other uses of pomegranate in dentistry
A 10% topical pomegranate gel was efficient in reducing recurrent aphthous
stomatitis pain and time for complete healing of ulcers. This was attributed to its
anti-inflammatory, antioxidant and antimicrobial properties of pomegranate.99
An 80% pomegranate peel extract lozenge was able to decrease gag reflex in
soft palate up to 88.5% and in tonsils up to 92.5%. This effect could be due to the
presence of tannins which have anesthetic effects.100
A gel based Punica granatum bark extract was effective in treating denture
stomatitis as effectively as miconazole.101
JASMINE:
Jasmine is a genus of shrubs and vines in the olive family Oleaceae with about
200 species throughout the world, out of which around 40 species are reported to be
growing in India.
Large number of species are however, centered to the region comprising of The
Himalayas, China and Malaysia. Other countries where jasmine is cultivated are
France, Italy, Morocco, Algeria, North Africa, Spain and Egypt.
20
Classification of Jasminum species:
Walpers (1852) classified Jasminum into two sections based on leaf lets and
further classfied the sections into sub sections based on calyx characteristics as stated
in table 3.
Table 3: Classification of Jasminum species
Section I- Unifoliata Section II- Trifoliata
a. Calyx- Lobules subulate and elongate a.Calyx- Shortly dentate
b.Calyx- Sub-truncate b.Calyx- Lancate
One of the earliest accounts of descriptive studies in 43 jasmine species was
accomplished by Hooker (1882) indicating two main groups of Jasminum as indicated
below
Group I - with simple leaves, calyx pubescent /glabrous, subulate / short
Group II - with compound leaves either of trifoliate or imparipinnate
Engler and Prantl (1897) cited about 160 species of jasmine in the tropical and
subtropical regions of Asia, Africa, Australia and over 40 in India. They considered that
the simple leaf was only the transformed terminal leaflet of the imparipinnately
compound leaf. They grouped jasmine into four sections (table 4):
Table 4: Engler and Prantl classification of Jasmine based on its leaves
Section I- Unifoliata Section II- Trifoliata
Section III- Alternifolia Section IV- Pinnatifolia
21
In India, Gamble (1936) published descriptions on 20 species occurring in
presidency of Madras and classified them based on Hooker’s classification (leaf
character) as shown in table 5.
Table 5: Gamble’s classification of Jasmine
Leaf characteristics Species
Group-I: Simple leaf type
Calyx pubescent, subulate and long Jasminum sambac and Jasminum
pubescens
Calyx pubescent, linear and short Jasminum arborescens
Under calyx glabrous or nearly so, linear
and long
Jasminum rigidum
Group-II: Compound Leaf type
Leaves trifoliate, lower leaflets wanting
and calyx lobes small Jasminum auriculatum
Leaves trifoliate, lateral leaves little
smaller than terminal and calyx lobes
minute
Jasminum flexile and Jasminum
calophyllum
Leaves imparipinnate and opposite Jasminum grandiflorum
22
Jasminum sambac:
Jasminum sambac is one of the species of jasmine which is native to South-
western, Southern, and South-eastren Asia, India, Philippines, Myanmar and
SriLanka.18,102 Its various parts such as the sleaf, stem, bark, flower and root are very
useful and important in pharmaceutical industries and have been reported to possess
medicinal value.103
Synonyms:102
Nyctanthes sambac L.(basionym)
Nyctanthes undulate L.
Taxonomic classification:102,104
Kingdom: Plantae
Subkingdom: Tracheobionta- Vascular plants
Superdivision: Spermatophyta- Seed plants
Division: Magnoliophyta- Flowering plants
Class: Magnoliopsida- Dicotyledons
Sub class: Asteridea
Order: Scrophulariales
Family: Oleaceae- Olive family
Genus: Jasminum- jasmine
Species: sambac (L.) Aiton- Arabian jasmine
23
Classical names:18,105-107
Sanskrit: Mallika, Ananga, Ashtapadi
Hindi: Bel, Mogra, Mugra, Motia
English: Arabian jasmine, Tuscan jasmine
Urdu: Kaliyan, Azad, Raibel, Sosan
Telugu: Mallepuvvu, Gundemalle
Tamil: Mallikaipu, Anangamu, Iruvachi
Kannada: Dundumallige
Marathi: Mogra
Malayalam: Mulla
Ayurvedic properties:108
Rasa: Tikta, Kashaya
Guna: Laghu, Rooksha
Veerya: Seeta
Botanical description:18,109,110
J.sambac is an evergreen plant which grows from one to three meters in height. The
young branches are pubescent. Leaves are opposite, membraneous, 3.8-11.5 by 2.2-6.3
cm, variable in shape, broadly ovate or elliptic, acute, obtuse or acuminate; base
rounded or subcordate, rarely acute. Flowers are white, very fragrant, solitary, usually
3-flowered. The flowers open at night and close in the morning, they fade to pink as
they age, and bloom throughout the summer and continuously in warm climates.
24
PHYTOCONSTITUENTS:
Whole plant:
The whole plant contains glycosides mainly irridoid glycosides, Saponins,
Flavonoids and Terpenoids. J.sambac contains maximum amount of terpenoids
compared to other jasmine species. 111-114
Leaves:
Leaves contain major phytoconstituents as alkaloids, glycosides, saponins,
flavonoids and terpenoids. Mainly the Iridoid glycosides are present.115 These include
sambacin, Jasminin, Sambacoside A (figure 12), Sambacoside E & Sambacoside F,
Sambacolingoside. Flavonoids include quercetin (figure 13), isoquercetin (figure 14),
rutin (figure 15), kempferol (figure 16) and luteolin (figure 17)116,117, Secoiridoid
glucoside- sambacolignoside along with oleoside 11-methyl ester.118,119 Oligomeric
irridoids like molihuasides A is a dimeric irridoid glycoside and Molihuasides C-E is a
trimeric irridoid glycoside.120 Jasminol (figure 18) is characterised as lup-20-en-28beta-
ol; C20-C30 hydrocarbons, palmitic (figure 19), stearic (figure 20), linolenic, linoleic,
malvalic acid, betulinic, ursolic (figure 21) and oleanolic acid, D- mannitol, inositol
(figure 22), xylitol and sorbitol, friedelin (figure 23), lupeol (figure 24), betulin (figure
25), alpha-amyrin (figure 26).121
PHARMACODYNAMICS:
Anti-diabetic Activity: Ethyl acetate and aqueous extracts of leaves of J.sambac were
found to be anti-diabetic in alloxan induced diabetic rats at a dose of 300mg/kg, p.o. for
21 days. The aqueous extract was found to be more effective than ethyl acetate
extract.116
25
Anti-oxidant Activity: Methanolic extract of J.sambac leaves and essential oil showed
antioxidant activity. The extract was tested by DPPH free radical (2, 2-diphenyl-1-
picrylhydrazylhydrate) and β-carotene-linoleic acid assays; the oxidation was
effectively inhibited by extract in β-carotene-linoleic acid assays; the oxidation was
effectively inhibited by extract in β-carotene-linoleic acid system where as butylated
hydroxytoluene (BHT) was used as positive control.23
Analgesic activity: Ethanolic extract of leaves of J.sambac possessed analgesic activity
against acetic acid-induced writhing in mice at the oral dose of 250 and 500 mg/kg
comparable to diclofenac sodium (25 mg/kg).122
Anti-lipidemic Activity: The methanolic extract of flowers showed anti-lipid per
oxidative effect which was similar to butyrate hydroxyltoluene (BHT), vitamin C,
vitamin E and rutin. Results of this study suggest that the methanolic extract of
J.sambac can be used as therapeutic agents to treat various diseases caused by free
radicals and other chemical agents.123
Effects on Nervous system: Jasmine oil showed ANS stimulating effects as provide
relief in depression and uplifting of human mood. It was proved by aromatherapist by
massaging the oil and recording human autonomic parameters like blood pressure,
pulse rate, blood oxygen saturation, breathing rate, and skin temperatureas the
indicators of arousal level of ANS. Jasmine tea odour was also shown to have sedative
effects on both autonomic nerve activity and mood states when investigated in 24
healthy volunteers. J.sambac also showed CNS depressant activity in mice.25,124,125
26
Anti-viral Activity: The water extract of J.sambac flowers were found to be anti-viral
against herpes simplex viruses(HSV; including HSV-1 and HSV-2) and adenoviruses
(ADV; including ADV-11) using XTT- based colorimetric assay. Results showed that
the extract exhibited anti-HSV and anti-ADV activities at different magnitudes of
potency.126
Anti- inflammatory Activity: Ethanolic and aqueous extracts of J.sambac leaves
possessed anti-inflammatory activity against carageenan induced rat paw edema in
albino rats at a dose of 300mg/kg. The aqueous extract was found to be more effective
than ethanolic extract.127
Cardiac effects: J.sambac showed angiotensin converting enzyme inhibitor activity
and also exhibit hypotensive potential.125,128
Anti-acne activity: Ethanolic extract of J.sambac flowers were found to be effective
against acne vulgarise in agar well diffusion and broth dilution methods. The extract
was more prominent in disc diffusion method by inhibiting the growth of
Propionibacterium.24
Antimicrobial activity: Antimicrobial activity using ethanol extract of Jasminum
sambac (L.) Ait was tested against an array of Gram +ve, (Staphylococcus aureus,
methicillin resistant Staphylococcus aureus (MRSA), Bacillus subtilis and Bacillus
cereus) Gram –ve bacteria (Escherichia coli, Klebsiella pneumoniae, Salmonella
typhimurium, Pseudomonas aeruginosa and Chromobacterium violaceum) filamentous
fungi Aspergillus niger, Aspergillus fumigatus, Candida albicans and Candida
27
glabrata and yeasts. In addition, their antipathogenic potential was checked by
examining the antiquorum sensing activity of such extracts using Chromobacterium
violaceum assays. Ethanol extracts of the callus of J. sambac exhibited antibacterial
activity against both Gram +ve S. aureus and Gram -ve S. typhi and P. mirabilis.
Jasminum sambac (flowers and leaves) extracts were very active (>15 mm inhibition
zone) against Gram +ve methicillin resistant S. aureus, B. subtilis, as well as against
Gram -ve E. coli, S. typhimurium and K. pneumoniae and fungi, including the
filamentous A. niger, A. fumigates, and the yeasts Candida albicans and Candida
glabrata. 22,129-133
Antifungal activity using methanol extract of Jasminum grandiflorum,
Jasminum sambac (L.) Ait was evaluated using disc diffusion method for the inhibition
of fungal growth and spore formation of Alternaria sp. Alternaria sp, Aspergillus Niger,
A. flavus, A. fumigatus and Curvularia species are the most prevalent fungi causing nail
infection in human beings. Methanol extract of Jasminum grandiflorum and Jasminum
sambac proved to be active against, Alternaria sp.130
Antibacterial activity using ethanol extract of J. sambac (L.) Ait plant was
evaluated against the following 3 strains: Proteius mirabilis, Staphylococcus albus, and
Salmonella typhii and was found to be active against all the tested strains.131
Antimicrobial activity using ethanol extract of Jasminum sambac Ait.
(Oleaceae) leaves, flowers, fruits and stem bark was evaluated against nine bacteria and
four fungi using Agar diffusion assay and Minimum Inhibitory Concentration (MIC)
determinations. Study shows that flowers and leaves extracts of Jasminum sambac
exhibited almost good activity (10-15mm inhibition zone) against Gram +ve bacteria
including the Methicillin resistant Staphylococcus aureus (MRSA) and Bacillus subtilis
28
while a moderate activity was recorded against Gram -ve bacteria including Escherichia
coli and Klebsiella pneumonia.129
In a study conducted by Sanjay K, antimicrobial efficacy of Jasminum sambac
leaf extracts was evaluated against six bacteria (Staphylococcus aureus, Streptococcus
mutans, S. pyogenes, S. sobrinus, S. sanguinis and Lactobacillus acidophilus) and one
fungi (Candida albicans) causing dental infections. Results showed that methanol
extract was more efficient in comparison to other extracts. The zone of inhibition
ranged between 12.3±0.57-17.3±0.57 mm examined at 200 mg/mL, respectively.
Minimum inhibitory concentration were recorded for methanol extract at 3.12-25
mg/mL. The results concluded the traditional uses of J. sambac in treatment of dental
diseases.134
29
Methodology
Source of Data:
The study was conducted in the Department of Pedodontics and Preventive
Dentistry, Navodaya Dental College & Hospital, Raichur, Karnataka, in collaboration
with the Department of Microbiology, Navodaya Medical College & Hospital, Raichur,
Karnataka. A total of 5 children fulfilling the below mentioned criteria were included
in the study and informed consent was obtained from the parents prior to the
commencement of the study, 2ml of unstimulated saliva was collected from the children
using a disposable sterile container for 3-5 minutes.10
Inclusion Criteria:
1. Children with Early childhood caries9 aged between 4 to 6 years.
2. Children with no systemic illness.
Exclusion Criteria:
1. Children who were unable to expectorate
2. Children who do not brush their teeth regularly
3. Children with a history of taking antibiotics and fluoride supplements 1 month
prior to the sample collection.
30
Armamentarium:
1. Mouth mask 11.Crystal violet 21.Weighing machine 31.Distilled water
2. Gloves 12. Gram’s iodine 22.Inoculation loop
(4mm internal diameter)
32. Sterile cork
borer
3. Disposable
sterile container 13. Glass slides 23.Sterile swabs 33.Autoclave
4. Petri plates 14.Cover slips 24. Mitis Salivarius
Bacitracin (MSB) agar 34.Hot air oven
5. Test tubes 15.Microscope 25. Brain Heart
Infusion (BHI) broth 35. Incubator
6. Measuring jar 16.Centrifuge 26. Brain Heart
Infusion (BHI) agar 36.Micropipette
7. Crystal violet 17.Filter papers 27. Pomegranate seeds 37. Disposable
micropipette tips
8. Gram’s iodine 18.Mortar and
pestle 28.Jasmine leaves
38. Vernier
caliper
9.Glass slides 19.Conical flasks 29. Ethanol absolute 39.Marker pen
10. Cover slips 20. Water bath 30. 0.2%
Chlorhexidine
40. Bunsen
burner
41. Test tube
holding rack
31
Study Design:
Preparation of extract:
A. Preparation of Pomegranate extract: Fresh ripe pomegranate fruits were
obtained from the local market and thoroughly washed. The peel was removed
and the arils of the fruit were separated from the membrane. The arils were then
crushed by hand with the help of a clean mortar and pestle (Figure 27). The aril
extract was separated from the seeds by filtering with a filter paper into a conical
flask. 40ml of this filtered extract was taken and 160ml of absolute ethanol was
added to it. This extract was subjected to centrifugation at 3000 rpm for 10
minutes (Figure 28).6 The supernatant thus obtained was collected and kept in a
dry oven at 60⁰centigrade until a final gelatinous extract was obtained (Figure
29,30).10 This extract was stored at 4⁰c until further use.135
B. Preparation of Jasmine extract: Leaves were separated from the plant (Figure
31), thoroughly cleaned with water, shade dried and powdered mechanically
(Figure 32). 50g of thus obtained powder was subjected to soxhlet extraction
(Figure 33) with ethanol for 48 hours. The extract was filtered and dried in a
desiccator until concentrated extract was obtained (Figure 34). This extract was
stored at 4⁰c until further use.7
Collection of Saliva Sample:
Unstimulated whole saliva samples were collected from five children with
severe early childhood caries aged between 4 to 6 years (Figure 36).9 2ml of
unstimulated whole saliva sample was collected in a sterile container by instructing the
children to drool for 3-5 minutes. Saliva collection was done in the morning between
32
10 am to 11 am in order to eliminate any bias in the concentration of saliva due to
circadian rhythm.10 The saliva sample was collected on the day experiment was
conducted and the collected 5 samples were pooled. The samples were used for
isolating Streptococcus mutans. The samples were streaked within one hour of sample
collection.10
Media Preparation:
A. Preparation of MSB agar: This agar was used for the isolation of
streptococcus mutans from the saliva sample. The composition of this agar is
mentioned in following table 6.
Table 6: Composition of MSB agar
Ingredients gms/litre
Casein enzymic hydrolysate 15
Peptic digest of animal tissue 5
Dextrose 1
Sucrose 50
Dipotassium phosphate 4
Trypan violet 0.075
Crystal violet 0.0008
Agar 15
Final pH (at 25⁰C) 7.0±0.2
33
90.07 grams of media is weighed and suspended in 1000ml of distilled water in a
conical flask. The beak of the flask is packed with a cotton plug and the media is boiled
or placed in a water bath until the media is completely dissolved in distilled water. The
media is sterilized by autoclaving at 15 lbs pressure and 121⁰C temperature for 15
minutes. The media is allowed to cool to 45⁰ - 50⁰C and 1% sterile potassium telurite is
added and stirred. The media is poured into sterile petri dishes and allowed to harden.
Once the media gets hardened, the petri dishes are kept in incubator at 37⁰C for 24 hours
for sterility check (Figure 37).136
B. Preparation of BHI agar:
The composition of BHI agar is mentioned in the following table 7.
Table 7: Composition of BHI agar
Ingredients gms/litre
Calf brain, infusion form 200
Beef heart, infusion form 250
Proteose peptone 10
Dextrose 2
Sodium chloride 5
Disodium phosphate 2.5
Agar 15
Final pH (at 25⁰C) 7.4±0.2
52grams of media is weighed and suspended in 1000ml of distilled water in a conical
flask. The beak of the flask is packed with a cotton plug and the media is boiled or
placed in a water bath until the media is completely dissolved in distilled water. The
34
media is sterilized by autoclaving at 15 lbs pressure and 121⁰C temperature for 15
minutes. The media is allowed to cool to 45⁰ - 50⁰C and poured into sterile petri dishes
and allowed to harden. Once the media gets hardened, the petri dishes are kept in
incubator at 37⁰C for 24 hours for sterility check (Figure 37).137
C. Preparation of BHI broth:
The following table 8 describes the composition of BHI broth.
Table 8: Composition of BHI broth
Ingredients Gms/litre
Calf brain, infusion form 200
Beef heart, infusion form 250
Proteose peptone 10
Dextrose 2
Sodium chloride 5
Disodium phosphate 2.5
Final pH (at 25⁰C) 7.4±0.2
9.25grams of media is weighed and suspended in 250ml of distilled water in a conical
flask. The beak of the flask is packed with a cotton plug and the media is boiled or
placed in a water bath until the media is completely dissolved in distilled water. The
media is sterilized by autoclaving at 15 lbs pressure and 121⁰C temperature for 15
minutes. The media is allowed to cool and poured into sterile test tubes (Figure 37).138
Isolation of Streptococcus Mutans:
Streptococcus mutans was isolated from the whole saliva sample by streaking
it on MSB selective agar media.6 This selective media supports the growth of
35
streptococcus mutans and inhibits the growth of other microorganisms. 10μl saliva
sample was streaked onto each MSB agar plate using a 4mm internal diameter
inoculation loop and the plates are incubated at 37⁰C for 48 hours in an incubator.10
Following incubation the growth of Streptococcus mutans was identified by its
morphological features as 0.5mm blue pinpoint colonies (figure 38) and confirmed by
gram staining (Figure 39).91
Preparation of Bacterial Inoculum:
The bacterial inoculums suspension was prepared by picking 4 – 5 colonies of
Streptococcus mutans from the agar plate with the help of an inoculation loop and
transferring them to the test tubes containing Brain Heart Infusion (BHI) broth.
Standardization of the bacterial suspension was done according to 0.5 McFarland
standards (Figure 40) which represent 1.5 × 108 cfu/ml.
Micro Broth Dilution:
Micro broth dilution test is performed to determine the Minimum inhibitory
concentration (MIC) and Minimum bactericidal concentration (MBC) of an
antibacterial agent to be tested. Broth dilution denotes the performance of the broth
dilution test in-vitro in serial test tube dilutions. In this test, microorganisms are tested
for their ability to produce visible growth in broth containing dilutions of the anti-
bacterial agent to be tested.
The lowest concentration of an antimicrobial agent that, under defined in-vitro
condition, prevents the appearance of visible growth of a microorganisms within a
definite period of time is known as MIC. At this dilution the antibiotic is considered to
36
be bacteriostatic.139 The MIC is a guide for the clinician to the susceptibility of the
organism to the antimicrobial agent and aids treatment decisions.
The minimum bactericidal concentration (MBC) is the lowest concentration of
an antibacterial agent required to kill a particular bacterium. The MBC determines the
lowest concentration at which an antimicrobial agent will behave lethal and encourage
no regrowth. The MBC is determined using a series of steps, undertaken after a
Minimum Inhibitory Concentration (MIC) test has been completed.
Minimum bactericidal concentration (MBC) was determined incubating the
three least concentrations of pomegranate and jasmine leaf extracts bacteriostatic effect
in broth for further 24 hours and the least dilution that showed no regrowth of the
microorganism was recorded as MBC. At this dilution the extract is considered to be
bactericidal.140
Minimum inhibitory concentration (MIC) and Minimum bactericidal
concentration (MBC) are recorded for pomegranate and jasmine leaves extract using
broth dilution method according to standards of NCCLS (National Committee for
Clinical Laboratory Standards) criteria.12
A series of 10 test tubes were taken for each extract and numbered 1 to 10. 1-8
test tubes were filled with 0.5ml of ethanol as diluent. Two fold serial dilution of the
test tubes acquiring eight concentrations (1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256
i.e, 50%, 25%, 12.5%, 6.25%, 3.12%, 1.5%, 0.75%, 0.37%) was carried out by adding
0.5ml of extract to the first test tube with the help of micropipette, it was mixed well
and from the first test tube 0.5 ml of the extract was added to the second, 0.5ml from
second to third and so on till the eight test tube. The ninth test tube served as a positive
control which received bacterial inoculums without the pomegranate or jasmine leaf
extract showing bacterial growth. The tenth test tube received only ethanol without
37
inoculums and served as negative control with no bacterial growth showing sterility of
the diluents (Figure 41).
0.5ml of bacterial inoculum was then added to the 1-8 test tubes making further
dilutions as (1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512 i.e, 50%, 25%, 12.5%,
6.25%, 3.12%, 1.5%, 0.75%, 0.37%, 0.18%) and incubated at 37⁰C for 24 hours and
MIC was determined. Bacterial growth was determined by presence of definite turbidity
in the test tube inoculated. MIC was recorded as the lowest concentration of
pomegranate and jasmine leaf extract dilution tested at which no bacterial growth was
seen.
MBC was further determined by selecting three values from the MIC test that
showed no bacterial growth and sub cultured further on Brain heart infusion agar for
further 24 hours to determine if the inhibition is reversible or permanent. MBC was
determined as the lowest concentration at which no visible bacterial growth was seen
on the agar medium.
MIC and MBC values provide us with the baseline range of pomegranate and
jasmine leaf extract concentrations that can be used in agar disc diffusion test for
comparing its antibacterial effect with 0.2% chlorhexidine gluconate.
In this study, the MIC & MBC was determined for the pomegranate (Figure 42)
and jasmine leaf extracts (Figure 43) but not for chlorhexidine gluconate since the latter
is traditionally used in a concentration of 0.2% in the mouth rinses.141
Agar Well Diffusion Test:
The agar well diffusion method prescribed by NCCLS (2000)142 was employed
to analyse the anti-microbial efficacy. 3 sets of 15 petri plates of BHI Agar were
prepared for analyzing the anti- microbial efficacy of pomegranate and jasmine leaf
38
extract separately against streptococcus mutans. Streptococcus mutans was uniformly
seeded onto the BHI agar plates by streaking with sterile swab dipped in the bacterial
suspension adjusted to 0.5 McFarland’s standard and incubating for 24 hours at 37⁰c in
incubator.10 Using a sterile cork borer 5 wells of 5mm diameter were cut on the agar
plates. The pomegranate extract at concentrations of 6.25%, 3.12% & 1.5% were placed
in three wells, ethanol in 4th well and 0.2% chlorhexidine were placed in 5th well. The
jasmine leaf extract was placed at concentrations of 25%, 12.5% & 6.25% were placed
in three wells separately, followed by ethanol in the 4th well and 0.2% chlorhexidine in
the 5th well.The plates were further incubated for 24 hours at 37⁰C in the incubator. The
zone of inhibition of each plate was measured using a vernier caliper. The mean of 3
sets for each reading was calculated (Figure 44, 45).10
Statistical Analysis:
Data was collected by using a structured proforma. The data was entered in MS
excel sheet and statistically analyzed using Mean, Standard deviation, One- way
variance ANOVA test in SPSS software 19.0 version IBM USA.
Quantitative data was expressed in terms of Mean and Standard deviation
Comparison of mean and SD between all groups was done by using One way ANOVA
test.
A p-value of <0.05 was considered as statistically significant whereas a p value <0.001
was considered as highly significant.
Mean (x):
X ꓿ . ∑
Where ‘x’ is the observation and ‘n’ is the number of observations.
39
:
σ = ∑ ∑
Where ‘ai’ represents observations and ‘n’ represents the number of observations.
One-way ANOVA test:
SS = ∑ ∑
Where ‘n’ is sample size
40
Sample size of Estimation
Reference article- Effect of Pomegranate and Aloe Vera extract on Streptococcus
Mutans- An Invitro study6
M1 Mean test intervention 15.33
M2 Mean control intervention 11.00
S1 Standard deviation of M1 0.58
S2 Standard deviation of M2 1
S Pooled SD 0.81744
AH One sided=1, Two sided =2 1
1-α Set level of confidence. 0.95
1-β Set level of power of test. 0.8
Z1 Z value associated with alpha ** 1.64485
Z2 Z value associated with beta 0.84162
n Minimum sample size 15
By using above formula and putting the values in it, minimum sample size came to 15
in each group.
41
Results
In the present study the Minimum Inhibitory Concentration (MIC) and
Minimum Bactericidal Concentration (MBC) of pomegranate and jasmine leaf extract
was determined using 2-fold dilutions of the pomegranate and jasmine leaf extract.
The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal
Concentration (MBC) of pomegranate and jasmine leaf extract were determined using
the 2- fold dilutions of pomegranate and jasmine leaf extract acquiring eight
concentrations (25%, 12.5%, 6.25%, 3.12%, 1.5%, 0.7%, 0.3%, 0.1%). Table 9 shows
the MIC and MBC concentrations of pomegranate and jasmine leaf extract. The MIC
and MBC of pomegranate extract was determined at 1:64 dilution which means 1.5%
concentration and 1:32 dilution which means 3.12% concentration against
Streptococcus mutans respectively. The MIC and MBC of jasmine leaf extract was
determined at 1:16 dilution that is 6.25% concentration and 1:8 dilution that is 25%
concentration against Streptococcus mutans respectively.
Table 9: MIC and MBC of pomegranate and jasmine leaf extract against S.mutans
Extract MIC MBC
Pomegranate extract 1:64 (1.5%) 1:32 (3.12%)
Jasmine leaf extract 1:16 (6.25%) 1:8 (12.5%)
The antimicrobial efficacy of the pomegranate and jasmine leaf extracts was
determined by the zones of inhibition obtained in the agar well diffusion test. Each set
of the concentration for pomegranate and jasmine leaf extract was repeated 3 times to
overcome any inadvertent technical errors. The antimicrobial efficacy of pomegranate
extract was tested by using agar well diffusion test at 6.25% (Group C1), 3.12% (Group
C2), and 1.5% (Group C3), concentrations. 0.2% chlorhexidine gluconate (Group A)
42
was used as positive control and ethanol (Group B) was used as negative control. The
mean diameter of inhibition zones produced by these groups against Streptococcus
mutans was statistically analyzed in the following table 10.
Table 10: Descriptive statistics of the mean zone of inhibition (mm) produced by
pomegranate group against Streptococcus mutans.
N Mean Std.
Deviation
Std.
Error
Range Minimum Maximum
Group
A
15 18.00 .00 .00 0 18 18
Group
B
15 5.00 .00 .00 0 5 5
Group
C1
15 14.27 .90 .23 3.0 13.0 16.0
Group
C2
15 10.97 .67 .17 2.0 10.0 12.0
Group
C3
15 7.50 .57 .15 2.0 6.5 8.5
Group A- 0.2% CHX, Group B- Ethanol, Group C1-6.25% PE, Group C2- 3.12%
PE, Group C3- 1.5%PE.
Table 10 shows the descriptive statistics of the mean zones of inhibition of 0.2%
chlorhexidine (group A), different pomegranate concentrations (group C1, C2 and C3)
and 99.9% ethanol (group B) tested against Streptococcus mutans. The pomegranate
extract showed mean inhibition zones of 14.27 ± 0.9 for 6.25% concentration (Group
C1), 10.97 ± 0.67 for 3.12% concentration (Group C2), 7.5 ± 0.57 mm for 1.5%
43
concentration (Group C3) respectively. The positive control 0.2% chlorhexidine
(Group A) showed inhibition zone of 18 mm. 99.9% ethanol (group B) being the
negative control group showed no antibacterial action with inhibition zone 5mm, as the
diffusion well size created was 5 mm, it means it has no zone of inhibition.
The antimicrobial efficacy of jasmine leaf extract was tested at 25% (Group
D1), 12.5% (Group D2), and 6.25% (Group D3) concentrations respectively. 0.2%
chlorhexidine gluconate (Group A) was used as positive control and ethanol (Group B)
was used as negative control. The mean diameter of inhibition zones produced by these
groups against Streptococcus mutans was statistically analyzed in the following table
11.
Table 11: Descriptive statistics of the mean zone of inhibition (mm) produced by
Jasmine leaf extract against Streptococcus mutans.
N Mean Std.
Deviation
Std.
Error Range Minimum Maximum
Group A 15 18.00 .00 .00 0 18 18
Group B 15 5.00 .00 .00 0 5 5
Group
D1 15 15.47 .99 .26 3.0 14.0 17.0
Group
D2 15 10.77 .92 .24 3.0 9.0 12.0
Group
D3 15 8.02 .55 .14 2.00 7.00 9.00
Group A- 0.25 CHX, Group B- Ethanol, Group D1- 25%JLE, Group D2-
12.5%JLE, Group D3- 6.25%JLE.
44
Table 11 shows the descriptive statistics of the mean zones of inhibition of 0.2%
chlorhexidine (group A), different jasmine leaf extract concentrations (group D1, D2
and D3) and 99.9% ethanol (group B) tested against Streptococcus mutans. Jasmine
leaf extract showed mean inhibition zones of 15.47±0.99mm for 25% concentration
(Group D1), 10.77 ± 0.92mm for 12.5% concentration (Group D2), 8.02 ± 0.55 mm for
6.25% concentration (Group D3) respectively. The positive control 0.2% chlorhexidine
(Group A) showed inhibition zone of 18mm. 99.9% ethanol (group B) being the
negative control group showed no antibacterial action with inhibition zone 5mm, as the
diffusion well size created was 5 mm, it means it has no zone of inhibition.
The mean zone of inhibition of control groups 0.2% chlorhexidine (group A), 99.9%
ethanol (group B) and the test groups of pomegranate (groups C1 - C3) and jasmine
leaf extracts (groups D1 – D3) against Streptococcus mutans were compared using
unpaired-t-test (table 12 and 13) respectively, for the statistical significance.
Table 12: Comparison of 0.2% chlorhexidine and ethanol with pomegranate
groups against streptococcus mutans (unpaired t- Test).
Sl.No Group N Mean SD t p Inference
1 A 15 18.00 .00 16.000 .0001 Highly
significant C1 15 14.27 .90 (<0.001)
2 A 15 18.00 .00 40.824 .0001 Highly
significant C2 15 10.97 .67 (<0.001)
3 A 15 18.00 .00 71.729 .0001 Highly
significant C3 15 7.50 .67 (<0.001)
4 B 15 5.00 .00 -39.714 .0001 Highly
significant C1 15 14.27 .90 (<0.001)
5 B 15 5.00 .00 -34.632 .0001 Highly
significant C2 15 10.97 .67 (<0.001)
6 B 15 5.00 .00 -17.078 .0001 Highly
significant C3 15 7.50 .57 (<0.001)
45
Group A- 0.2% CHX, Group B- Ethanol, Group C1-6.25% PE, Group C2-3.12%
PE, Group C3-1.5%PE.
Table 12 shows that the comparison of 0.2% chlorhexidine and ethanol with
pomegranate groups tested against Streptococcus mutans. On comparison of group A
(0.2% chlorhexidine) and C1 (6.25% pomegranate extract), group A showed a mean
inhibitory zone with standard deviation of 18mm and group C1 showed a mean
inhibitory zone with standard deviation 14.27 ± 0.9 mm with a test value (t) of 16.000
and p-value of 0.0001 which is less than 0.001. On comparison of group A (0.2%
chlorhexidine) and C2 (3.12% pomegranate extract), group A showed a mean inhibitory
zone with standard deviation of 18mm and group C2 showed a mean inhibitory zone
with standard deviation 10.97 ± 0.67 mm with a test value (t) of 40.824 and p-value of
0.0001 which is less than 0.001. When group A (0.2% chlorhexidine) and C3 (1.5%
pomegranate extract) was compared, group A showed a mean inhibitory zone with
standard deviation of 18mm and group C3 showed a mean inhibitory zone with standard
deviation 7.50 ± 0.67 mm with a test value (t) of 71.729 and p-value of 0.0001 which
is less than 0.001. All the three groups of pomegranate (C1, C2 and C3) were less potent
than 0.2% chlorhexidine (group A) and the difference was found to be statistically
highly significant (p<0.001).
On comparison of group B (99.9% ethanol) and C1 (6.25% pomegranate
extract), group B showed a mean inhibitory zone with standard deviation of 5mm and
group C1 showed a mean inhibitory zone with standard deviation 14.27 ± 0.9 mm with
a test value (t) of -39.714 and p-value of 0.0001 which is less than 0.001. On comparison
of group B (99.9% ethanol) and C2 (3.12% pomegranate extract), group B showed a
mean inhibitory zone with standard deviation of 5mm and group C2 showed a mean
inhibitory zone with standard deviation 10.97 ± 0.67 mm with a test value (t) of -34.632
46
and p-value of 0.0001 which is less than 0.001. When group B (99.9% ethanol) and C3
(1.5% pomegranate extract) was compared, group B showed a mean inhibitory zone
with standard deviation of 5mm and group C3 showed a mean inhibitory zone with
standard deviation 7.50 ± 0.67 mm with a test value (t) of -17.078 and p-value of 0.0001
which is less than 0.001. The three groups (C1, C2, and C3) of pomegranate showed
higher antimicrobial efficacy than group B (99.9% ethanol) and the difference is found
to be statistically highly significant (p<0.001).
Table 13: Comparison of 0.2% chlorhexidine and ethanol with jasmine leaf
extract against streptococcus mutans (unpaired t- Test).
S.No Group N Mean SD t p Inference
1 A 15 18.00 .00 9.90 .0001 Highly
significant D1 15 15.467 .99 (<0.001)
2 A 15 18.00 .00 30.34 .0001 Highly
significant D2 15 10.767 .92 (<0.001)
3 A 15 18.00 .00 70.29 .0001 Highly
significant D3 15 8.02 .55 (<0.001)
4 B 15 5.00 .00 -40.929 .0001 Highly
significant D1 15 15.47 .99 (<0.001)
5 B 15 5.00 .00 -24.191 .0001 Highly
significant D2 15 10.77 .92 (<0.001)
6 B 15 5.00 .00 -21.280 .0001 Highly
significant D3 15 8.02 .55 (<0.001)
Group A- 0.2%CHX, Group B-Ethanol, Group D1-25% JLE, Group D2-12.5%
JLE,GroupD3-6.25%JLE.
47
Table 13 shows that the comparison of 0.2% chlorhexidine and ethanol with
jasmine leaf extract groups tested against Streptococcus mutans. On comparison of
group A (0.2% chlorhexidine) and D1 (25% jasmine leaf extract), group A showed a
mean inhibitory zone with standard deviation of 18mm and group D1 showed a mean
inhibitory zone with standard deviation 15.467 ± 0.99 mm with a test value (t) of 9.90
and p-value of 0.0001 which is less than 0.001. On comparison of group A (0.2%
chlorhexidine) and D2 (12.5% jasmine leaf extract), group A showed a mean inhibitory
zone with standard deviation of 18mm and group D2 showed a mean inhibitory zone
with standard deviation 10.767 ± 0.92 mm with a test value (t) of 30.34 and p-value of
0.0001 which is less than 0.001. When group A (0.2% chlorhexidine) and D3 (6.25%
jasmine leaf extract) was compared, group A showed a mean inhibitory zone with
standard deviation of 18mm and group D3 showed a mean inhibitory zone with standard
deviation 8.02 ± 0.55 mm with a test value (t) of 70.29 and p-value of 0.0001 which is
less than 0.001. All the three groups of jasmine leaf extract (D1, D2 and D3) were less
potent than 0.2% chlorhexidine (group A) and the difference was found to be
statistically highly significant (p<0.001).
On comparison of group B (99.9% ethanol) and D1 (25% jasmine leaf extract),
group B showed a mean inhibitory zone with standard deviation of 5mm and group D1
showed a mean inhibitory zone with standard deviation 15.467 ± 0.99 mm with a test
value (t) of -40.929 and p-value of 0.0001 which is less than 0.001. On comparison of
group B (99.9% ethanol) and D2 (12.5% jasmine leaf extract), group B showed a mean
inhibitory zone with standard deviation of 5mm and group D2 showed a mean
inhibitory zone with standard deviation 10.767 ± 0.92 mm with a test value (t) of -
24.191 and p-value of 0.0001 which is less than 0.001. When group B (99.9% ethanol)
and D3 (6.25% jasmine leaf extract) was compared, group B showed a mean inhibitory
48
zone with standard deviation of 5mm and group D3 showed a mean inhibitory zone
with standard deviation 8.02 ± 0.55 mm with a test value (t) of -21.280 and p-value of
0.0001 which is less than 0.001. All the three groups (D1, D2, and D3) of jasmine leaf
extract showed higher antimicrobial efficacy than 99.9% ethanol (group B) and the
difference between them was statistically highly significant (p<0.001).
The intra group comparison between all 5 groups of pomegranate extract
(groups A, B, C1, C2 and C3) against streptococcus mutans was done using Posthoc-
Anova test to determine the antimicrobial efficacy of the pomegranate extract is shown
in table 14.
Table 14: Intra group comparison of pomegranate extract groups, 0.2%
chlorhexidine and 99.9% ethanol groups tested against Streptococcus mutans
(Posthoc- Anova Test).
Group N Mean SD F P Inference
Group A 15 18.00 .00
1276.75
0.0001
(<0.001)
Highly
significant
Group B 15 5.00 .00
Group C1 15 14.27 .90
Group C2 15 10.97 .67
Group C3 15 7.50 .57
Total 75 11.15 4.71
A- 0.2% CHX, B- 99.9% ethanol, C1- 6.25% PE, C2-3.12% PE, C3-1.5% PE.
Table 14 shows the comparison of mean inhibition zones produced by different
concentrations of pomegranate extract, 0.2% chlorhexidine, and 99.9% ethanol against
streptococcus mutans. The mean zone of inhibition and standard deviation of 0.2%
chlorhexidine (group A) was 18mm, ethanol (group B) was 5mm, 6.25% concentration
49
(group C1) of pomegranate extract was 14.27±0.9 mm, 3.12% concentration (group
C2) of pomegranate extract group was 10.97±0.67 and 1.5% concentration (group C3)
of pomegranate extract was 7.50±0.57. When the mean of all groups for their
antibacterial properties was compared, by applying Post-hoc ANOVA TEST, the
difference (F) value was 1276.75and p-value was 0.0001 which was found to be
statistically highly significant (p<0.001).
The intragroup comparison between all 5 groups of jasmine leaf extract
(groups A, B, D1, D2 and D3) against streptococcus mutans was done using Posthoc-
Anova test to determine the antimicrobial efficacy of the jasmine leaf extract in
comparison to 0.2% chlorhexidine and 99.9% ethanol against Streptococcus mutans as
shown in table 15.
Table 15: Intra group comparison of jasmine leaf extract groups, 0.2%
chlorhexidine and 99.9% ethanol groups tested against Streptococcus mutans
(Posthoc- Anova Test).
Group N Mean SD F p Inference
Group A 15 18.00 .00
990.85
0.0001 (<0.001)
Highly
significant
Group B 15 5.00 .00
Group D1 15 15.47 .99
Group D2 15 10.77 .92
Group D3 15 8.02 .55
Total 75 11.45 4.82
A-0.2% CHX, B-Ethanol, D1-25% JLE, D2-12.5% JLE, D3-6.25% JLE.
50
Table 15 shows the comparison of mean inhibition zones produced by different
concentrations of jasmine leaf extract, 0.2% chlorhexidine, and ethanol against
streptococcus mutans. The mean zone of inhibition and standard deviation of group A
was 18mm, group B was 5mm, group D1 was 15.47±0.99 mm, group D2 was
10.77±0.92 and group D3 was 8.02±0.55. When the mean of all groups for their
antibacterial properties was compared, by applying Post-hoc ANOVA TEST, the
difference (F) value was 990.85 and p-value was 0.0001 which was found to be
statistically highly significant (p<0.001).
The inter group comparison was done between pomegranate (group C1, C2 and
C3) and jasmine leaf extract (group D1, D2 and D3) to evaluate their antimicrobial
efficacy statistically in table 16.
Table 16: Inter group comparison of concentrations in pomegranate and jasmine
group
Groups N Mean Std. Deviation t P Inference
Group C1 15 14.27 .90
-3.466
0.002
(<0.05)
Significant Group D1 15 15.47 .99
Group C2 15 10.97 .67
.680
0.5
(>0.05)
Not
Significant Group D2 15 10.77 .92
Group C3 15 7.50 .57
-1.116
0.016
(<0.05)
Significant Group D3 15 8.02 .54
Group C1- 6.25% PE, Group D1- 25% JLE, Group C2- 3.12% PE, Group D2-
12.5% JLE, Group C3- 1.5% PE, Group D3- 6.25% JLE.
51
Table 16 shows the inter group comparison of pomegranate groups (group C1-
C3) and jasmine leaf extract groups (group D1-D3). When group C1 (6.25%
pomegranate extract) and group D1 (25% jasmine leaf extract) was compared the t
value obtained was -3.466 and p value was 0.002. As the p value obtained was less than
0.05 the difference between the groups C1 and D1 was statistically significant. This
meant that group D1 (25% jasmine leaf extract) was more potent than group C1 (6.25%
concentration). When group C2 (3.12% pomegranate extract) and group D2 (12.5%
jasmine leaf extract) was compared the t value obtained was 0.680 and p value was 0.5.
As the p value obtained was more than 0.05 the difference between the groups C2 and
D2 was statistically not significant. This meant that group D2 (12.5% jasmine leaf
extract) was less potent than group C2 (3.12% concentration), but the difference was
not statistically significant. When group C3 (1.5% pomegranate extract) and group D3
(6.25% jasmine leaf extract) was compared the t value obtained was -1.116 and p value
was 0.016. As the p value obtained was less than 0.05 the difference between the groups
C3 and D3 was statistically significant. This meant that group D3 (6.25% jasmine leaf
extract) was more potent than group C3 (1.5% concentration).
52
Discussion
Dental caries is caused by an interaction between acidogenic bacteria, a
carbohydrate substrate which is frequently sucrose, and host susceptibility.143 The
acidogenic and acid-tolerant bacterial species Streptococcus mutans is recognized to be
the primary pathogen in early childhood caries.144-147 Various studies have revealed that
Streptococcus mutans average from 20-40% of the cultivable flora in biofilms removed
from carious lesions.148,149
Four closely related species known as Mutans streptococci identified as the
primary hosts in humans are Streptococcus mutans, Streptococcus sobrinus,
Streptococcus cricetus, and Streptococcus rattus.150 It is not always practicable to
identify and distinguish at species level, as a result based on its greater prevalence, most
of the isolates will in fact be Streptococcus mutans.151 Hence in the present study we
took into consideration strains of Mutans Streptococci bacterium which is commonly
involved in caries process in order to determine antimicrobial efficacy of pomegranate
and jasmine leaf extract in comparison with gold standard 0.2% Chlorhexidine
gluconate.
India, a developing nation, has shown an inclined trend of caries in children
over a relatively short period of time. In 1940, the prevalence of dental caries in school
children in India was 55.5% and it rose to 68% in the 1960s and climbed to 89% in
subsequent years.152-154 Sohi RK (2012) reported that the prevalence of dental caries in
children of 5 years age was 48.3%.155 Kashetty MV (2016) also reported that the
prevalence of dental caries in the primary teeth of 3–6–year-old preschool children as
62.14%.156 Hence in the present study we have involved children between the age group
of 4 to 6 years to check levels of Streptococcus mutans which is the primary causative
organisms for dental caries.
53
Since dental caries is an irreversible disease which can be treated by means of
restoration that is expensive and laborious, thus prevention at the initial step is more
essential. One of the most satisfactory possibilities for the control of dental caries
involves the topical applications of fluoride or chemotherapeutic agents which reduces
microorganisms from teeth.157 The widespread use of commercially available
antimicrobials has led to the advent of multi drug resistant pathogens which ultimately
lead a threat to global public health.158
The use of plants for treating diseases is as old as the human civilization. There
are many plants which have been in use as traditional medicine, so they are called as
medicinal plants. The trial of plants for curing diseases is the need of hour, as it is
already proven that adverse effects are associated with antibiotics.158,159
Pomegranate is an ancient, magical, and distinctive fruit, of punicaceae family.
According to Lansky, it is an extraordinary fruit with ample medicinal power contained
within it. The biochemical constituents that make it beneficial against microorganisms
are ellagic acid, ellagatannins, punica acid, flavonoids, anthocyanins, estrogenic
flavonols, flavonols and flavones.7
Jasminum sambac Linn. (Family-Oleaceae) commonly known as Motia or lily
jasmine is a scandent or sub-erect. Traditionally leaves of this plant are used in fever or
cough, indolent ulcer, abdominal distension, diarrhoea, lowering the blood glucose
level, regulating menstrual flow, to clean kidney waste, inflamed and blood shot eyes.
The major phytochemicals like alkaloids, iridiod glycosides, saponoids, flavonoids and
terpenoids make s the extract efficient against microorganisms.115
In our study, 2-fold dilutions of pomegranate and jasmine leaf extracts at eight
concentrations were used, MIC and MBC of pomegranate extract ware determined at
1:64 (1.5%) and 1:32 (3.12%) dilutions respectively for the Streptococcus mutans and
54
MIC and MBC of jasmine leaf extract were determined at 1:16 (6.25%) and 1:8 (12.5%)
dilutions respectively for the Streptococcus mutans as shown in table 9.
In our study the MIC of the pomegranate extract against Streptococcus mutans
was found to be 1:64 (1.5%). This result was in contrast to the findings of Vasconcelos
LCS et al.,(2006)92, who found the MIC of pomegranate gel extract against
streptococcus mutans at 1:16 (6.25%) dilution. Our result was not in accordance to
Alsaimary IE (2008)157, who conducted an invitro study testing the antimicrobial
efficacy of pomegranate juice against Streptococcus mutans and reported the MIC of
pomegranate juice between 100-200μcg/ml (0.01-0.02%). Our finding was also
contradictory to the findings of Bhat SS and co-workers (2014)160, who found MIC
value of pomegranate aril extract against Streptococcus mutans to be at 600mg/ml
(60%).
In our study MBC of pomegranate extract against Streptococcus mutans was
found to be at 1:32 (3.12%) dilution. Our finding was not in accordance to the findings
of Millo G et al.,(2017)161, who studied the antimicrobial effect of pomegranate gel on
cariogenic bacteria and found the MBC value of pomegranate gel against Streptococcus
mutans at 250mg/ml (25%) concentration. These differences between the MIC and
MBC values of pomegranate extract against Streptococcus mutans in our study with
previous researchers can be attributed to the variation in the methodology of preparation
of the extract, diluents used in extract preparation, the geographical, environmental and
cultivation variations of the pomegranate fruit.162
In our study the MIC of Jasmine leaf extract against Streptococcus mutans was
found at 1:16 (6.25%) dilution. This result was in contrast to the findings of Sanjay K
and co-workers (2015)134 who conducted an invitro study to screen the antimicrobial
efficacy of jasmine leaf extract against dental pathogens and reported that the MIC of
55
the methanol extract of jasmine leaf extract against Streptococcus mutans at 1:32
(12.5%) dilution.
MBC of jasmine leaf extract against Streptococcus mutans was found to be at
1:8 (12.5%) dilution. In other studies MBC was not evaluated against Streptococcus
mutans. So, in the found literature the information on MBC values of jasmine leaf
extract against Streptococcus mutans was minimal.133,134
These difference in the MIC values of jasmine leaf extract against Streptococcus
mutans in our study to previous researchers may be attributed to the variation in the
methodology and the diluents used in the preparation of the extract.
In this study, the MIC and MBC was determined for the pomegranate and
jasmine leaf extracts but not for Chlorhexidine gluconate since the latter is traditionally
used in the mouth rinses at a concentration of 0.2% Chlorhexidine.141
These MIC and MBC findings provided us the bacteriostatic and bactericidal
concentration of pomegranate and jasmine leaf extracts. It also provided us with the
baseline range of pomegranate and jasmine leaf extracts concentration that can be used
for well diffusion assay for comparing their antimicrobial effect with 0.2%
Chlorhexidine gluconate.
Agar well diffusion test was conducted to evaluate the antimicrobial efficacy of
pomegranate and jasmine leaf extracts in comparison to 0.2% chlorhexidine against
Streptococcus mutans.
The antimicrobial efficacy of the pomegranate extract can be attributed to the
synergistic action of its components. Various researchers have explained the
mechanism of action of pomegranate against cariogenic bacteria. However few
researchers expressed that the ellagitannin, punicalagin may cause disturbance of
polyglycans synthesis, and thus acts on the adherence mechanism of these organisms
56
to the dental surface. Other researchers stated that, the tannins may cross over the cell
wall of the microorganisms and bind to the surface leading to the precipitation of
proteins and suppress enzyme like glycosyl transferase, which is the key enzyme for
breakdown of carbohydrates. Few other researchers stated that, the phenolic
compounds may bind to the substrates such as minerals and carbohydrate making them
unavailable for the microorganisms to survive resulting in cell wall disruption.91
The phytochemical screening of J. sambac extract had shown that plant contains
major phytoconstituents including alkaloids, flavonoids, steroids, reducing sugars,
saponins and tannins. Doughari, (2012) stated that phytochemicals are responsible for
various properties i.e., antioxidant activity, hormonal action, enzymatic activity,
interference with DNA replication, antimicrobial activity etc. of the J.sambac leafs. J.
sambac leaf extracts possess a broad spectrum of antimicrobial activity.134
In our study the inhibitory zone exhibited by 0.2% chlorhexidine against
Streptococcus mutans was 18mm. This result was in close accordance to the findings
of Solmaz G, who conducted an invitro study and determined the diameter of zone of
inhibition of 0.2% chlorhexidine at a range of 15.8 ± 3.4 mm.28
In our study the zone of inhibition exhibited by the pomegranate extract against
Streptococcus mutans at different concentrations was 14.27 ± 0.97 mm at 6.25%
concentration (group C1), 10.97 ± 0.67 at 3.12% concentration (Group C2) and 7.5 ±
0.57 at 1.5% concentration (Group C3). When these groups (C1, C2 & C3) were
compared with 0.2% chlorhexidine (group A) in table 12, the difference was found to
be statistically highly significant.
In our study, the zone of inhibition exhibited by the pomegranate extract against
Streptococcus mutans at different concentrations was 14.27 ± 0.97 mm at 6.25%
concentration (group C1), 10.97 ± 0.67 at 3.12% concentration (Group C2) and 7.5 ±
57
0.57 at 1.5% concentration (Group C3). These results were in contrast to the findings
of Alsaimary IE (2008)157, who reported a zone of inhibition of 16 mm of 0.01-0.02%
(100-200μg/ml) pomegranate juice against Streptococcus mutans in the invitro study
conducted by him.
In another study Abdollahzadeh et al.; (2011)163 evaluated the antibacterial and
antifungal effects of three different concentrations of methanolic extract of
pomegranate, and found that concentrations of extract at 8 mg/ml (0.8%), 12 mg/ml
(1.2%) showed effective inhibitory zone of 9.5 ± 0.57mm against Streptococcus
mutans. This result was not in accordance to the results obtained in our study.
Devi A and co workers (2011)158 conducted an in vitro study to evaluate the anti
microbial efficacy of pomegranate at a concentration of 500mg/ml (50%) using four
different solvents distilled water, methanol, petroleum ether and ethanol and reported a
zone of inhibition of 25mm, 24mm, 23mm and 22mm respectively against
streptococcus mutans. The results of this study were in contrary to our results.
A zone of inhibition of 15.33 ± 0.58 at 100% concentration was found by
Subramaniam P et al., (2012)6 in an invitro study conducted to test the effect of
pomegranate and aloe vera extract on Streptococcus mutans. This result was in contrast
to our findings.
These differences in inhibitory zone values of pomegranate extract against
Streptococcus mutans in our study to the previous researches, can be attributed to the
variation in the methodology of preparation of the extract, diluents used in extract
preparation, the geographical, environmental and cultivation variations of the
pomegranate fruit.162
In our study the zone of inhibition exhibited by the jasmine leaf extract against
Streptococcus mutans at different concentrations was 15.47 ± 0.99 mm at 25%
58
concentration (group D1), 10.77 ± 0.92 at 12.5% concentration (Group D2) and 8.02 ±
0.55 at 6.25% concentration (Group D3). When these groups (D1, D2 & D3) were
compared with 0.2% chlorhexidine (group A) in table 13, the difference was found to
be statistically highly significant.
In our study the zone of inhibition exhibited by the jasmine leaf extract against
Streptococcus mutans at different concentrations was 15.47 ± 0.99 mm at 25%
concentration (group D1), 10.77 ± 0.92 mm at 12.5% concentration (Group D2) and
8.02 ± 0.55 at 6.25% concentration (group D3).
This result was in close accordance to the results of the study conducted by
Sanjay K and co workers (2015)134, who evaluated the antimicrobial efficacy of jasmine
leafs using methanol, petroleum ether, acetone and water. They found that methanol
extract of jasmine leafs showed the maximum antimicrobial activity against tested
strains in comparison to other extracts followed by petroleum ether, acetone and water
extract. The best activity of methanolic extract of jasmine leafs was noted against
Streptococcus mutans (14.3 ± 0.57 mm).
In another study, Prasad et al (2015)164 evaluated the antimicrobial efficacy of
jasmine leafs against Streptococcus mutans and found that ethanolic extract of jasmine
leaf exhibited a zone of inhibition of 15mm against streptococcus mutans. This result
was in close accordance to our study.
These differences in inhibitory zone values of jasmine leaf extract against
Streptococcus mutans in our study to the previous researches can be attributed to the
variation in the methodology of preparation of the extract, diluents used in extract
preparation, the geographical, environmental and cultivation variations of the jasmine
plant.
59
In our study, 99.9% Ethanol (Group B) showed 5mm of inhibitory zone which
means that it exhibited no antibacterial action thus suggesting no involvement in the
antibacterial action of pomegranate extract. 5mm is the diameter of the well in the agar
plate. When the pomegranate groups (C1, C2 & C3) were compared with 99.9% ethanol
(group B) in table 12, the difference between the groups was found to be statistically
highly significant. When the jasmine leaf extract groups (D1, D2 & D3) were compared
with 99.9% ethanol (group B) in table 13, the difference between the groups was found
to be statistically highly significant.165
In our study when intra group comparison between groups A – C3 was done
(table 14), we found average zone of inhibition of 0.2% Chlorhexidine gluconate and
99.9% ethanol against Streptococcus mutans (18 ± 0 mm & 5 ± 0 mm) seemed higher
in comparison to the pomegranate extract at 6.25% concentration (14.27 ± 0.97 mm),
3.12% (10.97 ± 0.67) and 1.5% (7.5 ± 0.57) which was also statistically highly
significant (p<0.001). These results suggest that the pomegranate extract exhibited
antimicrobial efficacy against Streptococcus mutans greater than the negative control
ethanol and lesser than the positive control 0.2% chlorhexidine. These results also
suggest us that a dose dependant effect was seen with decrease in diameter of inhibition
zones as the concentration of the pomegranate was further diluted. Hence a significant
difference existed between the stronger to weaker concentrations of pomegranate
extract against Streptococcus mutans. (Group C1> Group C2> Group C3). The
pomegranate gel had a significant effect of antimicrobial efficacy against growth of
Streptococcus mutans. However, the edibility and palatability of pomegranate extract
makes it superior for usage in children and as 0.2 % chlorhexidine needs to be used
only after 6 years with parents supervision.166, 167
60
In our study when intra group comparison between groups A – D3 was done
(table 15), we found average zone of inhibition of 0.2% Chlorhexidine gluconate
against Streptococcus mutans (18 ± 0 mm) seemed higher in comparison to the jasmine
leaf extract at 25% concentration (15.47 ± 0.99 mm), 12.5% (10.77 ± 0.92) and 6.25%
(8.02 ± 0.55) which was also statistically highly significant (p<0.001). These results
suggest that the jasmine leaf extract exhibited antimicrobial efficacy against
Streptococcus mutans greater than the negative control ethanol and lesser than the
positive control 0.2% chlorhexidine. The jasmine leaf extract has a significant effect of
antimicrobial efficacy against Streptococcus mutans. These results also suggest us that
a dose dependant effect was seen with decrease in diameter of inhibition zones as the
concentration of the jasmine leaf extract was further diluted. Hence a significant
difference existed between the stronger to weaker concentrations of jasmine leaf extract
against Streptococcus mutans. (Group D1> Group D2> Group D3). The jasmine leaf
extract had a significant effect of antimicrobial efficacy against growth of
Streptococcus mutans. However, the edibility of jasmine leaf extract165 makes it
superior for usage in children and as 0.2 % chlorhexidine needs to be used only after 6
years with parents supervision.166, 167
In our study when inter group comparison between groups of pomegranate and
jasmine leaf extracts was done, and the difference was found to be highly significant.
By using 1:16 concentration, zone of inhibition by the pomegranate was 14.27±0.9 and
that of jasmine was 15.47± 0.99. When we compared the mean values, it was found to
be significant (p<0.05). At 1:32 concentration, zone of inhibition by the pomegranate
was 10.97±0.67 and that of jasmine was 10.77± 0.92. These mean values when
compared, found to be non-significant (p>0.05). In the same way at 1:64 concentration,
zone of inhibition by the pomegranate was 7.5±0.57 and that of jasmine was 8.02± 0.54.
61
These mean values, were found to be significant (p<0.05). These findings suggest us
that all the groups being tested do not possess same potency of antimicrobial action
against Streptococcus mutans.
62
Summary & Conclusion
It appears that this study demonstrated the antimicrobial activity of a traditional
herb ‘Pomegranate’ and ‘Jasmine’ on Streptococcus mutans which is the main
cariogenic pathogen that causes tooth decay. Our study demonstrated that ethanolic
extract of pomegranate juice and jasmine leaf extract have shown inhibitory and lethal
effects against Streptococcus mutans.
The antibacterial efficacy of 6.25% pomegranate extract and 25% Jasmine leaf
extract was found to be nearly closer to 0.2% CHX against S. mutans.
Therefore, from our study results it could be concluded that pomegranate juice
and jasmine leaf extract could be used as a key ingredient of an antibacterial mouth
rinse, as an additive antimicrobial agent incorporated into tooth paste or added into
infant tooth wipes to prevent and control dental caries. As it is a natural compound, it
is less likely that products incorporating pomegranate juice and jasmine leaf extract will
produce undesirable side effects compared to artificial, synthetic products.
As pomegranate is a nutritive fruit and jasmine leaf is traditionally used in
treating oral lesions, they can be used as safe and alternative to the synthetic
mouthwashes. For clinical application, more molecular studies need to be elucidated to
understand the antibacterial mechanism of other active components of pomegranate
juice and jasmine leaf extract and its compatibility with the oral tissues.
63
Summary
It appears that this study demonstrated the antimicrobial activity of a traditional
herb ‘Pomegranate’ and ‘Jasmine’ on Streptococcus mutans which is the main
cariogenic pathogen that causes tooth decay. Our study demonstrated that ethanolic
extract of pomegranate juice and jasmine leaf extract have shown inhibitory and lethal
effects against Streptococcus mutans.
The antibacterial efficacy of 6.25% pomegranate extract and 25% Jasmine leaf
extract was found to be nearly closer to 0.2% CHX against S. mutans.
Therefore, from our study results it could be concluded that pomegranate juice
and jasmine leaf extract could be used as a key ingredient of an antibacterial mouth
rinse, as an additive antimicrobial agent incorporated into tooth paste or added into
infant tooth wipes to prevent and control dental caries. As it is a natural compound, it
is less likely that products incorporating pomegranate juice and jasmine leaf extract will
produce undesirable side effects compared to artificial, synthetic products.
As pomegranate is a nutritive fruit and jasmine leaf is traditionally used in
treating oral lesions, they can be used as safe and alternative to the synthetic
mouthwashes. For clinical application, more molecular studies need to be elucidated to
understand the antibacterial mechanism of other active components of pomegranate
juice and jasmine leaf extract and its compatibility with the oral tissues.
64
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Format of Consent Form
NAVODAYA DENTAL COLLEGE AND HOSPITAL, RAICHUR
DEPT. OF PEDODONTICS & PREVENTIVE DENTISTRY
CONSENT LETTER
I ………………………………………………………… the undersigned hereby give
my full consent for the saliva sample collection, as a part of the study “Comparative
evaluation of the antimicrobial efficacy of Pomegranate and Jasmine leaf extracts
on Streptococcus mutans: An Invitro study” being conducted by Dr. Adusumilli
Hamsini, Post graduate student, Navodaya Dental college and Hospital, Raichur, under
the guidance of Dr. Naveen Kumar. R, MDS, Professor & Head, Dept. of Pedodontics,
Navodaya Dental College & Hospital, Raichur.
I have read the foregoing information, or it has been read to me. I have had the
opportunity to ask questions about it and any questions I have been asked have been
answered to my satisfaction. I consent voluntarily to be a participant in this study.
Signature
Name of the Participant:
Name of the investigator:
Signature of the investigator:
Navodaya Education Trust's
NAVODAYA DENTAL COLLEGE (Affiliated to Rajiv Gandhi University of Health Sciences, Karnataka)
(Recognised by Dental Council oflndia, New Delhi) Post Box No. 26, Navodaya Nagar, RAICHUR - 584 103. (Kamataka)
Ph: 08532-223361, 223448, Fax: 08532-223266, E-mail: [email protected]
CHAIRMAN Dr. VaniShree M.,
SECRETARY Dr. Santosh Hunsgi.,
ADVOCATE Mr. Sateesh V.,
SOCIAL WORKER Mr. Narasimah Reddy.,
MEMBERS
Dr. Arun Kumar A.
Dr. Chitra Chakravarthy.
Dr. Girish Galagali.
Dr. Ameet J Kurthukoti.
Dr. Krishna Prasad.
Dr. Prashant B Patil.
Dr. Suga Reddy.
Dr. Jeevanand Deshmukh.
INSTITUTIONAL ETHICAL COMMITTEE (lEC)
ETHICAL CLEARANCE CERTIFICATE
The Institutional Ethical Committee of Navodaya Dental College &
Hospital, Raichur met on 07/10/2015 at Navodaya Dental College, Raichur
to discuss & scrutinize the Synopsis/Research Projects of Post Graduate Students/
Under Graduate/ Faculty members of this College from Ethical clearance point of
view. After scrutiny, the following original/ corrected and revised version of
Synopsis of Dissertation/ Research Project/ Study protocol has been accorded Ethics
Clearance:
Title: "Comparative Evaluation of the Antimicrobial Efficacy of
Pomegranate and Jasmine Leaf Extracts on Streptococcus
mu tans: An In vitro Study".
Name of the P.G./Y:-G./F�alty member DR. ADUSUMILLI HAMSINI
Date: 13.10.2015
Place: Raichur
J�� �rl�
Signature of Chairman Institutional Ethical Committee
PRINCf PAt ··,, Navadaya Dental College,
RAtCHUR.
? A L tt�� tA� � ... -a \'.>\
gnature of Secretary Institutional Ethical Committee
84
85
Proforma format
Participant No.:
Name:
Age:
Sex:
Diagnosis:
Sample collected:
Method of Sample collection:
Master Sheet
Romegranate e�tract MEAN ZONE OF INHIBITION
Samples Chlorhexidine Distilled water 1:16 (6.25%) 11:32(3.12%) U64(l.5%) l 18 5 14 11.5 7
2 18 5 15 12 7.5
3 18 5 16 12 6.5
4 18 5 14.5 11 7
5 18 5 15 10 7.5 6 18 5 13 10.5 8
7 18 5 13.5 11 8.5
8 18 5 14 11.5 7.5
9 18 5 13 11 8
10 18 5 13.5 10 7
11 18 5 15.5 11 8
12 18 5 15 11.5 7
13 18 5 14 10.5 8
14 18 5 13.5 10 7
15 18 5 14.5 11 8
86
Mas1!er Sheet
Jlasmine extract! MEAN ZONE OF INHIBI'TIION
Samples Chlbrhexidine Distilled water 1:4\25%) 1:8(12.5%) 1:16(6.25%) 1 18 5 16 11 8 2 18 5 15 12 8.5 3 18 5 14 11 7.5 4 18 5 14.5 10!5 7.58 5 18 5 15.5 rn 8 6 18 5 16 11.5 77.5 7 18 5 17 12 8 8 18 5 16 11 8 9 18 5 16.5 rn 9 10 18 5 17 12 7 11 18 5 14.5 9.5 8 12 18 5 15 rn 8.5 13 18 5 14 9; 9
14 18 5 15 11 7.5 15 18 5 16 11 8
Q ta) lD ,�e>l'.:t--
87
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