FORMULATION AND EVALUATION OF HERBAL

MOUTHWASH CONTAINING EXTRACTS OF IPOMOEA

MARGINATA (DESR.) VERDC. AS AN ANTIBACTERIAL
AGENT TO CONTROL GINGIVITIS – AN IN-VITRO
STUDY

Synopsis of the thesis to be submitted for the award of the

Doctor of Philosophy
Under the Faculty of Dentistry
Submitted to

SRI BALAJI VIDYAPEETH

(Deemed to be University u/s 3 of UGC Act 1956) Accredited by NAAC with ‘A++’
Grade Pondicherry – 607402
By
Prof. Vandana. S, MDS
UIN: 1905001008

Under the guidance of

Dr. Saravana Kumar R
Professor& Head, Department of Periodontics
Indira Gandhi Institute of Dental Sciences, SBV
Month and Year of submission: June 2024

i
 TABLE OF CONTENTS

S.No Contents Page No

1 Introduction 1-2

2 Review of Literature 4-5

3 Materials and Methodology 6-12

4 Results and Discussion 13-34

5 Conclusion 35

6 References 36-39

ii
 1. INTRODUCTION

Oral health is a significant determinant of general health, well-being, and

overall quality of life. Gingivitis represents a widely prevalent chronic inflammatory

condition affecting the oral cavity. Gingivitis is the most common cause for oral

malodour and poor oral health. Dental plaque is a structurally and functionally

organized biofilm that forms on the surface of teeth within few minutes after tooth

brushing and is considered as the primary etiologic factor for gingivitis. 1 Dental

plaque may be supragingival or subgingival plaque with considerable variation in

their microbial composition. Streptococcus mutans is the predominant Gram-positive

microorganism in supragingival plaque and the red complex pathogens namely

Porphyromonas gingivalis, Tannierella forsythia and Treponema denticola appear

later during biofilm development.2 These bacteria trigger an inflammatory response

and evoke a immune reaction leading to inflammation of the gingiva. Gingivitis

frequently advances to periodontitis, which involves inflammation of the soft tissue

and bone. Over time, persistent periodontitis has the potential to result in the loss of

teeth.

There exists a hypothesis suggesting that low grade inflammation (LGI) may

serve as a causal risk factor for chronic ailments, including diabetes mellitus, obesity,

and cardiovascular diseases.3 Therefore, it may be argued that biofilm bacteria serve

as a covert contributing component in the emergence and advancement of chronic

systemic disorders. The prevention of periodontal problems is a public health concern

because it has been shown that these conditions contribute to the development of

various systemic diseases.

Plaque control can be of two types, namely mechanical and chemical.

Mouthwash or mouth rinse using chlorhexidine is a common form of chemical plaque

1
control. Chlorhexidine based antimicrobial agents have demonstrated moderate

efficacy in eradicating dental plaque however, achieving consistent and targeted

removal of these pathogens while preserving the natural microbial balance in the oral

cavity remains a formidable task.4 Bacterial resistance, change in oral microbiome and

alteration in taste are some of the inherent side effects of chlorhexidine. 5

Need for the study

One approach to address bacterial resistance is the utilization of herbal or

natural products that include intricate chemical compositions thereby impeding the

development of resistance in bacteria. The growing desire for novel antimicrobials has

prompted numerous studies to focus on exploring the antimicrobial properties of

phytochemicals derived from various plant species.

Plants have been used in treating ailments since time immemorial. ‘Morning

glory’ is a commonly used plant in folk medicine that belongs to genus Ipomea,

family Convolvulacea. The most common biologically active constituents from plants

of genus Ipomoea are ergoline alkaloids, indolizidine alkaloids, nortropane alkaloids,

phenolic compounds, coumarins, norisoprenoids, diterpene, isocoumarin and

benzenoids, flavonoids and antocianosides, glycolipids, and triterpenes compounds.

The remedial effects of plant materials are mainly due to the presence of substances

called secondary metabolites of plants (SMoPs). 6,7

Harnessing secondary plant metabolites would be a cost effective and

innovative strategy to develop novel antimicrobials to combat the threat of antibiotic

resistance.

With this background the present study aimed to explore the antimicrobial

property and formulate a mouth wash from leaves of Ipomea marginata.

2
 AIM AND OBJECTIVES

Aim

To formulate and evaluate an antibacterial herbal mouth wash containing

extracts of I.marginata

Objectives

a) To extract and carry out phytochemical screening from leaves of

I.marginata

b) To formulate a mouth wash from the leaf extract of I.marginata

c) To identify the minimal inhibitory concentration of bio-active

compound on organisms causing gingivitis (S.mutans , P.gingivalis ,

T.denticola and T. forsythia)

3
 2. REVIEW OF LITERATURE

Genus Ipomea is well known for its medicinal properties in different parts of

the world. They are used to treat various conditions such as diabetes, hypertension,

constipation, fatigue and arthritis. Some of the species showed antimicrobial,

spasmolytic, hypotensive, anti-inflammatory activity and anticancer activity.

Sukitha et al., 2022 in their study aimed to study enzymatic antioxidants

(superoxide dismutase, catalase, polyphenol oxidase), nonenzymatic antioxidants

(saponin, flavonoids, ascorbic acid and tannin), and anti-oxidant ability assays (total

anti-oxidant, hydroxyl radical scavenging ability, reducing power activity, inhibition

of lipid peroxide formation, DPPH scavenging activity and metal chelating assay) in I.

marginata leaves and whole plant revealed that I.marginata possessed significant

enzymatic anti-oxidants activities like superoxide dismutase, catalase, polyphenol

oxidase in whole plant of I. marginata. plant. From the results of the study, it was

confirmed that the whole plant of I. marginata showed strong antioxidant ability. Thus

I. marginata whole plant can be used as a potential source of natural anti-oxidants.8

Haritha Potluri et al., 2021 in a study evaluated the antinociceptive activity of

the methanolic extracts of I.marginata in rodents and observed that the methanolic

extracts of I.marginata demonstrated significant antinociceptive activity and

significant dose-dependent antinociceptive task at all doses. Isolated compound

Ipalbidine demonstrated a significant docking score compared to standard diclofenac

sodium. They concluded that I.marginata can be a potent resource of antinociceptive

activity.9

4
 Santosh Kumar et al., 2019 in their narrative review has mentioned the plant I.

marginata has been used from time immemorial as an antidote to snake poisoning and

treatment of skin infections.10

Sukitha et al.,2016 conducted a study to evaluate the phytochemical screening

and antibacterial activity of the whole plant and the leaves of I. marginata.

Antibacterial activity was evaluated against S. aureus, Shigella sonnei, Enterobacter

faecalis, Salmonella typhimurium, Micrococcus luteus, Vibrio cholerae, Klebsiella

pneumoniae, Streptococcus pyogenes, E. coli, and Bacillus subtilis. The observations

of their study indicated methanol, acetone, benzene, water, and ethanol extracts of I.

marginata exhibited significant antibacterial activity and further research can prove

the antibacterial formulations can be produced from the plant. 11

Research studies pertaining to I.marginata were limited hence relevant

literature from plants of the same species are also cited.

Berenice Corona-Castañeda et L., 2013 in their study observed chloroform

soluble extracts of flowers of Ipomea mucoides exerted a potentiation effect on

clinically useful antibiotics against methicillin resistant Staphylococcus aureus

(MRSA) by increasing their antibiotic susceptibility up to fourfold at concentration

25µg.12

Adsull et al., 2012 in their study showed crude acetone extract of leaves of

Ipomea carnea exhibited antibacterial activity against two strains, Ptroteus vulgaris

and Salmonella typhimurium and ethanolic extract of leaves of I.carnea exhibited

antibacterial activity against Pseudomonous auroginosa.13

5
 3. MATERIALS AND METHODOLOGY

a. Collection of Plant material and authentication

With the guidance of local traditional healers, the plant was identified and the

leaves were collected from Auroville forest (12.0052° N, 79.8069° E) Pondicherry,

South India. The plant was authenticated by a Botanist, Professor Ayappan from

French Institute of Pondicherry, Pondicherry, India. Voucher specimen was deposited

in the Herbarium of French Institute of Pondicherry (Voucher specimen code: V.S.

001).

b. Extraction

The extraction was carried out using polar solvents of different polarity such

as N hexane, ethanol and chloroform.

Ethanolic Extraction

The leaves of the plant were carefully cleaned, dried in the shade and ground

into a powder using a warren blender. The extraction process employed a Soxhlet

extraction equipment (Biocoction Manufacturing Pvt ltd, India). Within the

apparatus's upper chamber, a porous thimble held 50 g of powdered leaf material. The

lower boiling flask was filled with 200 ml of ethanol as a solvent. A heating mantle

managed by a thermostat was used to raise the temperature of the flask to above 78 0

C. After heating the solvent to reflux, it was extracted. Following collection, the

solvent extract was concentrated independently under low pressure. Following total

evaporation, the residue's weight was recorded and saved. Complete extraction was

ensured until a colourless liquid was collected at the top.

6
Chloroform Extraction

Fresh leaves of I.marginata were collected and dried in air at room

temperature. The dried leaves were powdered. Powdered leaves were macerated with

chloroform for 48 hours. Then the solution is filtered through Buchner funnel and

collected. The chloroform is collected in a separate round bottom flask. The residual

powdered leaves were then treated for extraction process with chloroform by Soxhlet

extraction technique. The chloroform extract was then collected and mixed with the

chloroform collected in the round bottom flask. The combined extracts were then

concentrated to one tenth volume. The concentrated solution was then dried on a

water batch. The residue is then subjected for the further evaluation.

N-Hexane Extraction

Fresh leaves of I marginata were collected and dried in air at room

temperature. The dried leaves were powdered. Powdered leaves were macerated with

n-hexane for 48 hours. Then the solution is filtered through Buchner funnel and

collected the Hexane in a separate round bottom flask. The residual powdered leaves

were then treated for extraction process with n hexane by Soxhlet extraction

technique. The Hexane extract was then collected and mixed with solution collected

in the round bottom flask. The combined extracts were then concentrated to one tenth

volume. The concentrated solution was then dried on a water batch. The residue is

then subjected for further evaluation.

c. Phytochemical screening

All the three extracts were subjected to preliminary phytochemical screening

and tested for alkaloids, anthroquinones, flavonoids, phenols, steroils, tannins,

terpenoids, cardiac glycosides, saponins, phlobatannin, reducing sugars,

carbohydrates and protein/amino acids.14

7
d. Gas chromatography-Mass spectrometry (GC-MS)

GC-MS analysis of leaves of I.marginata was performed using a Perkin–

Elmer GC Clarus 500 system comprising an AOC-20i auto-sampler and a Gas

Chromatograph interfaced to a Mass Spectrometer (GC-MS) equipped with a Elite-

5MS (5% diphenyl/95% dimethyl poly siloxane) fused with a capillary column (30 ×

0.25 μm × 0.25 μm df). For GC-MS detection, an electron ionization system was

operated in electron impact mode with ionization energy of 70 eV. Helium gas

(99.999%) was used as a carrier gas at a constant flow rate of 1 mL /min, and an

injection volume of 2 μL was employed (a split ratio of 10:1). The injector

temperature was maintained at 250 °C, the ion-source temperature was 200 °C, the

oven temperature was programmed from 110 °C (isothermal for 2 min), with an

increase of 10 °C·min-1 to 200 °C, then 5 °C·min-1 to 280 °C, ending with a 9 min

isothermal at 280 °C. Mass spectrum was taken at 70 eV; a scan interval of 0.5 sec

and fragments from 45 to 450 Da. The solvent delay was 0 to 2 min, and the total GC-

MS running time was 36 min. The relative percentage amount of each component was

calculated by comparing its average peak area to the total areas. The mass-detector

used in this analysis was Turbo-Mass Gold-Perkin-Elmer, and the software adopted to

handle mass spectra and chromatograms was a Turbo-Mass version-5.2.15

Identification of Phytoconstituents

Interpretation on mass-spectrum GC-MS was conducted using the database of

National Institute Standard and Technology (NIST) having more than 62,000 patterns.

The spectrum of the unknown components was compared with the spectrum of known

components stored in the NIST library. The name, molecular weight, and structure of

the components of the test materials were ascertained.

8
Test Pathogens

The microbial strain used in the study were S.mutans (ATCC 700610),

P.gingivalis (ATCC 33277), T.denticola (ATCC 35405) and T.forsythia (ATCC 43037)

standard strains that were provided from HiMedia laboratories Pvt Ltd, Mumbai,

India

Antibacterial Effect of Extract by Disc Diffusion Method

Twenty-four-hour old test pathogens were inoculated on Mueller-Hinton agar

(HiMedia, laboratories Pvt Ltd, Mumbai, India) with blood at 0.5 McFarland optical

density concentrations using sterile swab and allowed to dry for 15 min. Extract was

prepared at 1mg/ml concentration and from this 25, 50, 75 and 100 µL was loaded on

sterile discs. All the discs were placed over the agar surface inoculated with the test

pathogen and incubated at 37± 2° C for 24-48 hour under anaerobic condition.

Ethanol served as the negative control and the standard chlorohexidine was loaded on

sterile disc to a concentration of 100 µG and served as the positive control. The zone

of inhibition obtained after incubation was measured and recorded. The experiment

was performed thrice in triplicates.

Minimum Inhibitory Concentration By Alamar Blue Oxidation Method

The minimum inhibitory concentration (MIC) of the extracts was evaluated

using the broth dilution technique, with concentrations ranging from 100 to 0.78

µg/mL. MH broth was mixed with extract to make a final concentration of 200 µg/ml.

Serial two-fold dilutions were prepared to make final concentrations ranging from 200

– 0.78 µg/mL. In a similar manner, chlorhexidine was also prepared to a concentration

of. In each dilution, a volume of 10µL of bacterial solution containing 1×106 colony-

forming units per millilitre (CFU/mL) was introduced. The dilutions were then

subjected to incubation for a duration of 24 hours at a temperature of 37°C, while

9
maintaining anaerobic conditions. A viability test was used to evaluate the growth of

the bacterial isolates in the test tubes after incubation. Fifty microliters of resazurin

were added to the tubes, which were then incubated for a duration of 15 minutes. The

process of dye reduction was documented. The maximum dilution at which the dye

does not undergo reduction was determined as MIC.

Time-Kill Kinetics Assay

100 mL of mouth wash made at the MIC level was combined with 1 mL of an

inoculum size of less than 300 CFU/mL, and it was then incubated at 37°C. At 0, 6,

12, 18, and 24 hours, 1.0 mL aliquots of the medium were obtained, aseptically plated

onto 20 mL of nutritional agar, and then incubated for 24 hours at 37°C. The colony

forming unit (CFU) was recorded. Three separate experiments were conducted in

triplicate to complete the protocol, and the log CFU/mL was plotted against time on a

graph.16

TLC direct bioautography

To enable identification of biologically active constituents in the plant mixture

direct bioautography was carried out. The inoculum was prepared by growing an

overnight culture of the test bacteria and the next day the turbidity was matched to

McFarland 0.5 standard. The inoculum was spread onto MH agar plate and the plates

were swabbed with the inoculum of representative bacterial strains in the direct

bioautography procedure. Sterile lens paper was used to cover the seeded MH agar

plate, aseptically and the dried TLC plates with corresponding spots were layered on

top of it. The TLC plate was placed face down with the silica-coated side evenly in

contact with the lens paper, and it was incubated for 24 h at 35 ± 2°C. The TLC plate

readings were compared and the zone of inhibition was observed. The fractions were

gathered, placed on sterile discs, and utilized to confirm the active fraction. 17

10
Gas chromatography-Mass spectrometry (GC-MS) of Active fraction

The active fraction obtained from direct TLC directed bioautography was

subjected to GC-MS to identify the volatile constituents. Phenol, 3,5-bis(1,1-

dimethylethyl)- and triethyl citrate were two compounds eluted in the active fraction.

Fourier Tansform Infrared Spectroscopy (FTIR)

Fourier transform infrared (FTIR) spectroscopy is an instrument able to detect

the functional groups of extract. Pellets for infrared analysis were obtained by

grinding 1 mg of dried extract with 100 mg of dry potassium bromide, and the

mixture was pressed into a 16 mm diameter mould. The FTIR spectra were recorded

on a Paragon 1000, Perkin-Elmer spectrometer (4,000-400 cm-1, 2 cm-1 of spectral

resolution, and 32 scans).

Molecular docking analysis

The bioactive compounds isolated by TLC directed bioassay was proceeded

for the molecular docking studies to determine the mode of action and binding

efficacy involved in the molecular level to predict possible active sites.

Target protein identification and preparation

The three-dimensional structure of the target virulence proteins (Mirolysin,

Glucansucrase, dipeptidyl peptidase III, Cystalysin) were retrieved from the Research

Collaborator for Structural Bioinformatics (RCSB) Protein data bank (www.rcsb.org)

respectively. A chain of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA)

reductase (HMGR) was pre-processed separately by deleting other chains (B, C and

D), ligand, as well as the crystallographically observed water molecules (water

without hydrogen bonds). Using Pymol software, water molecules and ligands already

11
present in the proteins were removed; hydrogen atoms were added and saved in

Protein Data Bank (PDB) file format.

Prediction of active site

Prediction of the active site is important in structure-based drug design. Co-

ordinates of binding sites of the proteins were identified using the software UCSF

chimera Docking Molecular docking calculations were carried out with the aid of the

software AutoDock version 4.2 and binding energy of the protein—ligand adducts

were obtained.

Formulation of mouthwash from ethanolic extracts of I.marginata

50g of dried plant powder extracted with ethanol using Soxhlet and

concentrated under vacuum dryer. About 100 mg of dried sample dissolved in 100 ml

water and filtered. 20g of sodium saccharin was added in 100 ml water as sweetener.

50 mL of glycerol was mixed in 100 water to keep the moisture along with 50 g

sorbitol. All the solutions were mixed and the final total volume is made up to 10000

mL with distilled water. All the ingredients were added under magnetic stirrer

(100rpm) at room temperature in a biosafety cabinet and stored in amber bottle.

Accelerated stability studies

Based on the ICH guidelines accelerated stability studies was carried out with

parameters such as colour, odour, foam, taste, phase separation, Ph, turbidity,

viscosity, sedimentation, precipitation, homogeneity, specific gravity, density,

phenolic content and surface tension.

Cytotoxicity of cells

To 1 ml of normal saline 200ul of RBC suspension was added and used as

reaction base. Different volume of mouth wash (25, 50, 75 and 100 µL) were added to

12
RBC suspension and incubated for 4h. 1 % SDS used as positive control. Normal

saline used as negative control. After 4 h tubes were centrifuged and the OD of

aqueous phase was taken at 560 nm. The percentage of haemolysis was calculated

then by the formula OD of test- negative control / negative control x 100. 18

Heavy metal detection

Heavy metal detection in the sample was done by Inductively Coupled Plasma

Optical Emission Spectrometry (ICP-OES) method of analysis.19

IV. Results and Discussion

Genus ipomea has long been used for medicinal purposes chiefly because of

the secondary metabolites present in the leaves.

After hot soxhlet extraction of the leaves of I marginata the preliminary

phytochemical study revealed that ethanolic extract contained alkaloids, flavonoids,

phenols, sterols, tannins, terpenoids, cardiac glycosides, phlobatannin, reducing

sugars, carbohydrates and protein/amino acids. [Table 1].

Table 1: Phytochemical constituents of ethanolic extract of leaves of Ipomea marginata

S.No Phytochemical Chloroform Ethanol N Hexane

1 Carbohydrate Positive Positive Positive
2 Glycoside cardiac Negative Positive Positive
3 Glycoside Negative Positive Positive
4 Alkaloid Positive Positive Positive
5 Flavonoid Positive Positive Negative
6 Terpenoid Positive Positive Negative
7 Phenols and Tannins Negative Positive Negative
8 Saponins Negative Negative Negative
9 Sterols Negative Positive Positive
10 Phlobotanins Positive Negative Positive
11 Anthraquinones Positive Negative Positive

13
 Presence of phytochemicals in leaves are the most important reason for

manifestation of their biological activities. After the extraction with three different

solvents based on their polarity it was seen on initial phytochemical screening

ethanolic extract exhibited presence of alkaloids, flavonoids, phenols and tannins

which have proven to be the cause for significant antibacterial property.

GC-MS Analysis

The steam distillation of ethanolic extract of leaves of I marginata was

subjected to GC-MS analysis and the results are summarized in Table 2

Table 2: Volatile components identified in the ethanolic extract of I marginata by

gas chromatography coupled to mass spectroscopy

S.NO Compound Name Abundance Retention

(%) Index

1. 2,3-Butanediol, 1,4-dimethoxy- 38.54 1094

2. Ethyl 4-oxo-2-phenylpentanoate 42.72 1629

3. Neophytadiene 22.31 1840.6

4. n-Hexadecanoic acid 75.94 1968.2

5. Hexadecanoic acid, ethylester 66.43 1993.5

6. Phytol 49.35 2113.9

7. 3-Tridecen-1-yne, (Z)- 12.2 1319

8. 9,12,15-Octadecatrienoic acid, (Z,Z,Z)- 9.7 2154

9. Squalene 26.28 2835.8

14
Figure 1: Chromatogram of volatile compounds eluted from ethanolic extract of
leaves of I.marginata

Antibacterial activity

Zone of Inhibition

With regard to S.mutans the mean zone of inhibition (ZOI) was found to be

more effective for herbal extract (18±0) compared to chlorhexidine mouth wash

(12±1) (Figure 2a). This difference was also found to be statistically significant (p=

0.00024). With respect to P. gingivalis also the mean zone of inhibition (ZOI) was

found to be more effective for herbal extract (18.3±0.57) compared to chlorhexidine

mouth wash (28.3±0.577) (Figure 2b) and (p=0.00001) with respect to T.denticola the

mean zone of inhibition (ZOI) was found to be more effective for herbal extract

(10.3±0.57) compared to chlorhexidine mouth wash (0) (Figure 2c)with (p=0.00001)

and with respect to T.forsythia the mean zone of inhibition (ZOI) was found to be

statistically significant for herbal extract (14±1.73) compared to chlorhexidine mouth

wash(18±1) (Figure 2d) with (p=0.0128) (Table 3).

15
 Table 3: Zone of Inhibition (in mm) of Herbal Extract and Chlorhexidine at
Different Concentrations

Herbal
Test 50 Chlorohexidine t
10 µG 25 µG Extract p value
Pathogens µG (100 µG) statistic
100 µG

S.mutans 12.33±0.57 13.3±0.57 16±1 18±0 12±1 10.392 0.00024

P.gingivalis 0 0 0 18.3±0.57 28.3±0.577 21.213 0.00001

T.denticola 5±1 5±1 5±1 10.3±0.57 0±0 31 0.00001

T.forsythia 5±1 5±1 5±1 14±1.73 18±1 3.464 0.0128

Figure 2: Zone of Inhibition of herbal extract versus 0.2% Chlorhexidine

16
Minimum Inhibitory Concentration (MIC)

The minimum inhibitory concentration (MIC) for the herbal extract for

S.mutans, P.gingivalis, T.denticola and T.forsythia was 6.25µg/mL, 100µg/mL,

100µg/mL and 50µg/mL respectively and that standard chlorhexidine was 25µg/mL,

12.5µg/mL, 200µg/mL and 100µg/mL respectively (Table 4). The MIC values

determine the levels of susceptibility or resistance of a specific bacterial strain to a

particular drug and drugs with lower MIC scores were more effective antibacterial

agents. In the present study the herbal extract was found to be more effective against

S.mutans, T.denticola and T.forsythia compared to chlorhexidine mouth wash.

However, it was found chlorhexidine mouth wash was more effective against

P.gingivalis compared to the herbal extract (Table 4).

Table 4: MIC of Herbal Extract and Chlorhexidine

Test Pathogens MIC of Herbal MIC of CHX(µg/ml)

Extract(µg/ml)

S.mutans 6.25 25

P.gingivalis 100 12.5

T.denticola 100 200

T.forsythia 50 100

Time kill kinetics

The results obtained for the time-kill study of test pathogens tested at MIC

concentration is displayed in the growth curve in the form of log of colony forming

unit (Figure 3). The reduction in colony forming units (CFU) over a period of 24

hours showed 97.5% reduction of S.mutans,99.65% reduction of P.gingivalis, 99.67%

reduction of T.denticola and 99.8% reduction of T.forsythia (Table 5).

17
 Table 5: Reduction in CFU of test pathogens (%) at different time intervals

Test Pathogens Percentage reduction (%)

6 hours 12hours 18hours 24hours

S.mutans 36 64 79 97.5

P.gingivalis 31 47 77.8 99.65

T.denticola 13 40 71 99.67

T.forsythia 51 68.8 97.2 99.8

Figure 3: Time Kill Curve of Test pathogens (PG:P gingivalis;TD:T denticola;TF:

T forsythia; SM: S mutans)

Time kill kinetics assay in the present study showed the herbal extract

demonstrated bactericidal effect in a time dependent manner and the viability of

pathogens was abolished within 24 hours in the time-killing test for the 5 log cycle

reductions were detected. The findings of our study align with previous research

which also observed a significant bactericidal impact when the concentration of the

antimicrobial agent was doubled (2 × MIC) and the duration of contact was extended.

Different bacteria and plant extracts exhibited varying degrees of time-dependent

microbial inhibition, according to the results of the time killing experiment. Response

18
to microbial infection by plant secondary metabolites can be viewed as one of the

contributing factors for antimicrobial properties exhibited by plants.

TLC Direct bioautography

Bioassay results revealed S. mutans and T. denticola were resistant to

Fractions 1 and 2; however, Fraction 3 demonstrated sensitivity against both the

organisms. It was observed the zone of inhibition for S.mutans and T. denticola was

16 mm. 0.2% CHX was used as positive control and the zones of inhibition for

positive control for S. mutans was 16 mm (p=0.013) and for T. denticola was 17 mm

(p=0.001) respectively.(Table 6) (Figure 5)

Direct contact bioautography demonstrated 3 fractions; namely, Fraction 1,

Fraction 2 and Fraction 3 corresponding to Rf values 0.71, 0.79, 0.85 at 254 nm

(Table 7) (Figure 4)

19
 Table 6: Zone of inhibition (mm) of three fractions and chlorhexidine by direct
bioautography

Fraction 3 Ethanolic Chlorhexidine

Fraction Fraction extract p
Pathogens (mm) (Mean ± SD)
1 2 (100µg/ml) value
(Mean±SD) (100µg/ml)

S.mutans 0 0 16 15 15 15.6 16 0.013

T.denticola 0 0 16 16 17 16.3 17 0.001

Table 7: Rf values of the fraction at 365 nm and 254 nm

Fraction Absorption at 365 nm Absorption at 254 nm

1 0.71 0.71

2 - 0.79

3 - 0.85

Figure 4: Chromatography at 365 nm and 254 nm

20
 Figure 5: Zone of Inhibition of fraction 1(f1),fraction 2(f2),fraction 3(f3) and
positive control chlorhexidine (PC)

GC-MS of active fraction

The active fraction obtained after TLC direct autobiography was subjected to

GC-MS. Two peaks were observed in the chromatogram first peak corresponded to

phenol, 3,5-bis (1,1- dimethylethyl) and second peak triethyl citrate. (Table 8) (Figure

6)

Figure 6: Chromatogram of Eluted Compounds from Active Fraction

21
 Table 8: Volatile compounds eluted by GC-MS of the active fraction

Molecular Molecular
S.no RT Compound name
wieght formula

1 15.910 Phenol, 3,5-bis (1,1- 206.3239 C14H22O

dimethylethyl

2 19.351 Triethyl citrate 276.2830 C12H20O7

Fourier Tranform Infrared Spectroscopy (FTIR)

FTIR was done to identify the functional group. Two bands appear at 2923 and

2852 cm-1, which is due to carboxylic acid stretching frequency. A sharp band

appeared at 1630 cm-1 can be attributed to the ν (-C=C) stretching due to alkene

group of the compound. Vibration at 1384 cm-1 denotes C-H bending corresponding

to aldehyde. C=C bending of alkene group was recorded at 799 cm-1. The above

observations denote the presence of functional OH group in the extract suggesting a

phenolic compound as evidenced by the GC-MS report wherein phenol, 3,5-bis (1,1-

dimethylethyl) was eluted. The FTIR spectrum was matched to NIST library and

confirmed the IR spectrum of phenol, 3,5-bis (1,1- dimethylethyl)(Table 9)(Figure 7)

Table 9: Vibration of different functional groups

O-H C-O
C-C C-H C=C
O-H stretching stretching
Group stretching bending bending
stretching Carboxylic Secondary
Alkene aldehyde alkene
acid alcohol

Vibration 3440 2923 1630 1384 1099 799

cm-2

22
 Figure 7: FTIR of different functional groups

In silico molecular docking analysis

Mirolysin is a secretory protease of T.forsythia docked with isolated

compound and compared with standard. The interaction of Phenol, 3,5-bis(1,1-

dimethylethyl)- showed docking score -5.7. ASN amino acid showed hydrophobic

interaction with ligand C6 and C13 atom. The Oxygen atom of O1 interacted with

proline residue and formed hydrogen bond. Triethyl citrate showed hydrophobic and

hydrogen bond formation between O1- TYR with affinity of -5.3cal/mol. Likewise

C14 and C15 atom of Chlorhexidine interacted with ASN and the docking score was -

7.4 kcal indicates that it has superior specificity. In addition to that standard showed

formation of ionic interaction, pi-pi stacking and hydrophobic interaction with ASN of

Mirolysin.

Glucansucrase secreted by S.mutans creating adherent environment for

structural bacteria colony forming dental biofilm. The target is docked with isolated

compound and compared with standard. The interaction of Phenol, 3,5-bis(1,1-

dimethylethyl)- showed docking score -6.2 kcal/mol with one hydrogen bond

23
hydrophobic interactions. The carbon atom of Phenol 3,5-bis(1,1-dimethylethyl)-

interacted with ASN and SER and formed hydrophobic interaction. Docking of

Triethyl citrate showed binding affinity -6.2 kcal/mol by forming hydrophobic

interaction and no hydrogen bonds were detected. The docking score of Chlorhexidine

was -8.3 kcal/mol. It formed hydrogen bond with ASN, PRO and GLN. Further it

showed ionic interaction, hydrophobic contact with PRO (N5,N7) and ASP (C4,10,

21).

Dipeptidyl peptidase III which is secreted by P.gingivalis docked with isolated

compound and compared with standard. The interaction of Phenol, 3, 5-bis(1,1-

dimethylethyl)- showed docking score -6.0 kcal/mol along with hydrophobic and pi-pi

stacking against ASN. The oxygen atom of Phenol 3, 5-bis (1,1-dimethylethyl)-

interacted with ARG and TYR and formed hydrogen bonding. Interaction of Triethyl

citrate with dipeptidyl peptidase III shows O1, O7 and O6 atoms interacted with TYR,

ASP and GLN and hydrogen bond having affinity of-5.4 kcal/mol. The docking score

of Chlorohexidine was -8.3 kcal/mol. It formed hydrogen bond with ALA (N8) and

PHE (N8, N4).

Cystalysin1C7O secreted by T denticola docked with isolated compound and

compared with standard. The interaction of Phenol, 3,5-bis(1,1-dimethylethyl)-

showed docking score -5.9 kcal/mol indicates moderate binding efficiency. The atom

of Phenol 3,5-bis(1,1-dimethylethyl)- interacted with VAL and LEU formed 2

hydrogen bond, pi-pi stacking with LEU and hydrophobic interaction with LYS. The

docking score of Triethyl citrate was -4.8 with hydrogen bond towards SER and LYS

whereas Chlorhexidine was -7.4 kcal/mo indicates that it may have superior

24
specificity than the isolated compound. It formed Hydrogen bond with TYR and

hydrophobic interaction with VAL.

A similar in silico study was conducted to assess the antinociceptive activity of

the methanolic extract of I.marginata by autodock 4.0 and date warrior software

applications, results of the study revealed Ipalbidine compound isolated from

methanolic extract of I.marginata demonstrated a very good docking score of -8.26

compared to standard diclofenac which showed a docking score of -7.03 guaranteeing

a good binding compatibility among the ligand and receptor. 9

Table 10: Docking score of ligand Phenol, 3,5-bis(1,1-dimethylethyl),triethyl

citrate and chlorhexidine with protein Microlysin

Table 11: Docking score of ligand Phenol, 3,5-bis(1,1-dimethylethyl), triethyl

citrate and chlorhexidine with protein Glucansucrase

25
Table 12: Docking score of ligand Phenol, 3,5-bis(1,1-dimethylethyl)-,triethyl
ethyl citrate and chlorhexidine with protein dipeptidyl peptidase III

Table 13: Docking score of ligand Phenol, 3,5-bis(1,1-dimethylethyl)- , triethyl

ethyl citrate And chlorhexidine with cystalysin.

26
Figure 8: Three dimensional interaction of Microlysin with Phenol, 3,5-bis(1,1-
dimethylethyl

Figure 9: Three dimensional interaction of Glucansucrase with Phenol, 3,5-

bis(1,1-dimethylethyl

Figure 10: Interaction of Dipeptidyl peptidase III with Phenol, 3,5-bis(1,1-

dimethylethyl)

27
Figure 11: Interaction of Cystalysin1C7O with Phenol, 3,5-bis(1,1-dimethylethyl)

From the above observations it can be concluded that docking score of ligands

Phenol, 3,5-bis(1,1-dimethylethyl) and triethyl citrate with proteins Microlysin,

Glucansucrase, dipeptidyl peptidase and Cystalysin exhibited docking scores

comparable to that of standard chlorhexidine.

Absorption Distribution Metabolism and Excretion

Brain Or IntestinaL EstimateD Egg (BOILED Egg) Pharmacological

Absorption Property for prediction of GI absorption and prediction of blood brain

barrier penetration plays an important role.

Brain Or IntestinaL EstimateD permeation method (BOILED‐Egg) is proposed for

two compounds to predict the passive gastrointestinal absorption and brain access.

Triethyl citrate Yellow region (yolk) is the molecule predicted to be passively

permeated through the blood-brain barrier (BBB), while Phenol, 3,5-bis(1,1-

dimethylethyl)- in the white region is the molecule predicted to be passively absorbed

by the gastrointestinal tract. (Figure 12)

28
Figure 12: BOILED Egg Model depicting gastrointestinal absorption of molecule
1(Phenol, 3,5-bis(1,1-dimethylethyl) and molecule 2 (triethyl citrate)

Formulation of Herbal mouth wash

The formulation of mouth wash was carried out using different concentrations

of the active ingredient (ethanolic extract of I marginata) keeping other variables

constant. The concentration of the extract was initially 10µg and later increased to

25µg,50µg and 100µg. (Table 14).

At 100µg concentration of ethanolic extract of I marginata statistically

significant zone of inhibition was observed for S.mutans, P.gingivalis, T.forsythia,

T.denticola. Hence a concentration of 100µg/mL of ethanolic extract of I.marginata

was considered for formulation of mouth wash.

29
Table 14: Formulations of mouth wash with different concentrations of ethanolic
extract of I.marginata

S.No Ingredient F1 F2 F3 F4

1 Ethanolic extract 10µg 25µg 50µg 100µg

of I.marginata

2 Glycerol 50 50 50 50

3 Saccharin 20 20 20 20

4 Sorbitol 50 50 50 50

5 Distilled Water 1000 ml 1000 ml 1000 ml 1000 ml

Table 15: Final composition of mouth wash

Composition Water Final

Concentration

Herbal Extract Lyophilised 0.1 g 100 100µg/mL

Glycerol 50g 100 0.005 g/mL

Saccharin 20 100 0.02 g/mL

Sorbitol 50 g 100 0.005 g/mL

Water 600

Total volume 1000 mL

0.1g of lyophilised herbal extract was used for the formulation of the mouth

wash .50g of glycerol was added as a humectant,20g saccharin was used as sweetener.

To preserve the moisture and sweetness sorbitol was added. 600ml of water was

added to make up the volume to 1000ml.

30
Accelerated Stability Studies

The formulation was subjected to accelerated stability studies to determine the

shelf life according to the ICH (International Council for Harmonisation of Technical

Requirements of Pharmaceuticals for Human Use) guidelines for the following

parameters.20

31
There were no observable physical or chemical changes seen on observing the
sample for period of 6 months

Cytotoxicity of cells

In vitro determination of hemolysis is important to determine the cytotoxicity

of newer drugs. RBC haemolysis assay was done to assess the cytotoxicity of the

formulation.21

The Scientific Committee for Consumer Safety (SCCS) established the

hemolysis test as a necessary test for human consumption product approval. 22

According to the American Society for Testing and Materials (ASTM), less than 5%

hemolysis is considered null.23 Above this limit and up to 10% is assumed as low, and

beyond 10% is perceived as marked hemolysis. Hemolysis represents the rupture or

alteration of the integrity of the red blood cell membrane, causing the release of

hemoglobin.24

Table18: Hemolysis of different concentrations of mouthwash and control

Sample OD % of Hemolysis

Control 0.008 0

25 0.008 0

50 0.008 0

75 0.0081 1.25

100 0.0081 1.25

1% SDS 0.65 80.25

Heavy metal detection

Plant based formulations are becoming increasingly common for the treatment

of several conditions. The World Health Organization recommends that the finished

products be checked for their heavy metal concentrations.

32
 Table 19: Maximum permissible levels and test result of heavy metals

S.No Test Parameter Maximum permissible Test result

levels mg/L
mg/L

1 Cadmium 0.3 <0.1

2 Lead 10 <0.1

3 Arsenic 3 <0.1

4 Mercury 1 <0.1

The presence of heavy metals namely cadmium, lead, arsenic and mercury in

the formulation was less than 0.1mg/L. The observed values were well within the

prescribed values mentioned in the Ayurvedic Pharmacopoeia. 25

Antibacterial Activity of freshly prepared mouth wash

Initial antibacterial activity tests performed with ethanolic extracts of

I.marginata demonstrated significant antibacterial activity against S.mutans,

P.gingivalis, T.denticola and T.forsythia, thereby indicating that an effective

antibacterial mouth wash can be formulated. Also, the presence of phenolic compound

phenol 3,5 -bis (1,1-dimethyl ethyl) may have contributed significantly to the

antibacterial property in accordance to previous studies that have shown phenolic

compounds are important secondary metabolites that contribute to its antimicrobial,

and antioxidant property.26

33
 Table 20: MIC of mouth wash formulation

Test Pathogen MIC of freshly MIC of mouth pvalue

prepared mouth wash after 6
wash months

S.mutans 7.25 15 ≤0.001

P.gingivalis 100 100 1.000

T.denticola 100 100 1.000

T.forsythia 75 75 1.000

There was no statistically significant difference in MIC value of freshly

prepared mouth wash and MIC of mouth wash after a period of 6 months. This

observation suggests that there is no significant deterioration in the antibacterial

property of the mouth wash against S.mutans, T.denticola, T.forsythia and

P.gingivalis.

34
 CONCLUSION

Based on the findings of this investigation, it can be deduced that the ethanolic

extract of I.marginata exhibited demonstrable antibacterial activity against specific

oral microorganisms that are known to be critical in the development of plaque, the

primary etiologic factor for periodontitis and gingivitis. Mouthwashes made of natural

ingredients, such as herbs and organic oils, are free of alcohol and chemical

preservatives and offer special medicinal benefits.

35
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