Preparation of Nanoemulsion of cinnamon essential oil for yeast infection i,e.Candida albicans.

1. University Institute of Pharmacy
CSJM UNIVERSITY, KANPUR
A Thesis Presentation on
PREPARATION OF ESSENTIAL OIL’S NANOEMULSION AND ITS
ANTIMICROBIAL PROPERTY
Presented By:
Ruchi Pal
Roll No.- 9561008
M. Pharm (Pharmaceutics)
Session- 2019-21
Guide
Dr. Nisha Sharma
(Associate Professor & Director)
University Institute of Pharmacy,
C.S.J.M. University, Kanpur.
Co-guide
Dr. B.N Singh
(Senior Scientist),
Pharmacology Division,
CSIR-NBRI,
Lucknow.
1

2. CONTENTS
Introduction
Aim & Objective
Material & Methods
Results
Conclusion
References
2

3. INTRODUCTION
• Essential oil
• The growing resistance of microorganisms to chemicals and conventional drugs has prompted scientists to search for new
sources of broad-spectrum biocides. Since ancient times, plants and their derivatives, such as essential oils (EO), have been
used in folk medicine.
• EO is a liquid, volatile, clear and colored, soluble in lipids and organic solvents of lower density than water. They can be
present in any organ of the plant, including buds, flowers, leaves, seeds, branches, stems, flowers, fruits, roots, wood or bark,
but are usually stored in secretory cells by the plant., cavity, duct, gland. trichomes or epidermal cells.
 Limitations of essential oils
• Highly volatile
• Water insolubility
• Thermally unstable
• Degradation on exposure to heat
• They get easily oxidized.
3

4. • Cinnamon oil is an essential oil obtained from the bark, leaves and twigs of the genus
Cinnamomum of the family Lauraceae. Cinnamon contains manganese, iron, dietary
fiber, and calcium.
• The active components of cinnamon oil responsible for the antifungal and antibacterial
activities are cinnamaldehyde, cinnamyl cinnamate and benzyl cinnamate.
• The mechanisms involved in the antifungal effects of cinnamon oil are cytoplasmic
granulation, cytoplasmic membrane rupture, and inactivation of intracellular and
extracellular enzymes.
• Cinnamon is widely used as a food spice, but due to its antibacterial and
pharmacological properties, it can also be used in processing, medicine and agriculture.
• Cinnamon and its compounds effectively hinder bacterial and fungal growth, and the
phytotoxic effects of cinnamon make it a possible herbicide.
CINNAMON OIL
4

5.  C. verum
 C. cassia
 C. zeylanicum
 C. burmanni
 C. camphora
 C. tamala
 C. osmophloeum
 C. altissimum
 C. loureiroi
 C. wilsonii
 C. dubium
 C. rivolorum
 C. sinharajense
 C. citriodorum
• Decreases inflammation
• Reduces blood sugar
• Decreases bad cholesterol
• Fights infections
• High antioxidant content
• Stimulates the immune system
• Stimulates libido
• Fights parasites
Various cinnamon species Some Health benefits of cinnamon oil
5

6. • Nanoemulsion are a colloidal dispersion system in the submicron size range (10-1000 nm).
• It is composed of oil, water, and surfactant & co-surfactant in appropriate ratios.
• Nanoemulsion (as a drug delivery system) can enhance the therapeutic value of the essential oil and
reduce adverse effects & toxic reactions.
• It is a non-toxic and non-irritant vehicle for skin & mucous membrane delivery.
• They can combine both lipophilic drugs and hydrophilic drugs.
• It can be prepared by both high energy (high-pressure homogenization) and low energy (phase
inversion emulsification) methods .
Nanoemulsions:
Oil in water
Nanoemulsions
Water in oil
Nanoemulsion
Double Nanoemulsions
NANOEMULSION
6

7. The nanoemulsion are classified into three major forms on the basis of their compositions.
a) Water in oil (w/o)
b) Oil in water (o/w)
c) Double emulsion w/o/w type
(a) Water in oil (w/o)
• Water in oil (w/o) type in which water droplets are dispersed in the continuous oil phase.
• Water in oil emulsion is formulated for hydrophilic drugs and are non-common nanoemulsion formulations
than o/w type emulsion for transdermal route.
• In water in oil type the drug is in water phase not in oil. Since the drug used for this type of formulation are
hydrophilic hence, selection of surfactant is based on hydrophile lipophile balance (HLB) value, to bring
stability and tension reduction between the oil and water phase.
Types of Nanoemulsion
7

8. (b) Oil in water (o/w)
• Oil in water (o/w) type in which oil droplets are dispersed in the continuous aqueousphase.
• In most cases, drugs are poorly soluble in water and are thus produced by pharmaceutical industries to
formulate it as such making it bioavailable to produce therapeutic effect.
• Nanotechnology has gained higher interest in this regard because of its ability to solubilize drug, improved
bioavailability and increase capacity of drug loading.
(c) Double emulsion w/o/w type
• Double emulsion is a colloidal system in which there is a primary emulsion phase of water in oil dispersed in an
aqueous phase using hydrophilic surfactant, and referred as water-in-oil-in-water double emulsion.
• Double emulsions are thermodynamically unstable because of huge interfacial area that leads to Ostwald
ripening.
• o/w/o type emulsion use surfactants twice one as primary surfactant and secondary surfactant, both to maintain
the interfacial tension of primary emulsion and secondary emulsion.
8

9.  High stability.
 Increase the rate of absorption.
 Reduced variability in absorption.
 A lesser amount of organic solvent is required during preparation.
 Helps to solubilize the lipophilic drug.
 Provides aqueous dosage form for water-insoluble drugs.
 Delivery of lipophilic drugs after solubilization.
 Enhanced delivery systems to enhance effectiveness while reducing the total dose and side effects.
 Increases bioavailability of the active drug.
 Less amount of energy is required.
 Improve the efficiency of a drug.
Why we prepare Nanoemulsion?
9

10. • Yeast like fungus (produce pseudohyphae)
• Normally a part of human anatomical flora (Candida)
• Capable of causing infection in presence of opportunities such as low immunity.
• Candida albicans is an opportunistic human pathogen that causes disease mainly in
immunocompromised patients. The activity of hydrolytic enzymes is essential for the virulence of
C. albicans, as well as the ability of these cells to convert from yeast to growing mycelium.
• C. albicans readily forms biofilms on various biological and abiotic surfaces, and these biofilms
can cause local and systemic infections. The biofilms of C. albicans are more resistant to free
antifungal agents than free yeast and are less affected by host immune responses. The transition
from yeast cells to mycelium cells is required for biofilm formation and is considered an important
virulence factor.
• The biofilm protects encapsulated C. albicans cells from elimination by the action of shear or the
killing action of antimicrobial agents and the host immune response.
Candida albicans-
10

11.  Mayer L. François et al., (2013) reported that Candida Albicans Polymorphs is a member of normal bacteria. Several factors and
activities have been identified that contribute to the pathogenicity of this fungus. In this review, update our current understanding of
the pathogenic mechanism of this important human pathogen. [29]
 Rojas Cortés Francisco Diego et al., (2014) reported that the various studies reviewed in this work have confirmed the traditional
use of cloves as a food preservative and medicinal plant, highlighting the importance of this plant for different applications. Clove
represents a very interesting plant species with great potential as a food preservative and as a rich source of antioxidant
compounds.[30]
 Sugumar Saranya et al., (2015) reported that the hydrophobic character of EO leads to a decrease in the solubilization efficiency of
foods, thereby creating a negative impact on the quality of foods and their antimicrobial efficacy. Focusing on this, we attempted to
prepare NE nicely using orange oil, Tween 80 (organic phase) and water (water phase) by sonication technique.[31]
LITERATURE REVIEW
11

12.  Mandras Narcisa et al., (2016) reported that the objective of this study was to extend the study to evaluate the activity of similar oils on clinical
strains of Candida albicans, C. glabrata and C. Tropicalis, as well as the effects of related components. The selection of essential oils was based
both on ethnic use and on the proven antibacterial and/or antifungal activity of some of these oils. Fluconazole and voriconazole were used as
reference drugs. [32]
 de Almeida Dantas de Fátima Leopoldina et al., (2016) reported that in this study aimed to evaluate the anti-fungal and antibiotic efficacy of
two essential vegetable oils of Cymbopogon winterianus (lemongrass) and Cinnamon cassia (cinnamon). It is concluded that lemongrass and
cinnamon essential oils are capable of daily cleaning of dentures.[33]
 Donsì Francesco et al., (2016)reported that this review focuses on the facilitating contribution of nanoemulsion to the use of EOs as natural
preservatives in foods, (a) specifically addressing the formulation and production of stable EO nanoemulsion , (b) critically reported antimicrobial
activity data analysis, both in vitro and in product, to infer the effect of the delivery system on the mechanism of action of EOs, as well as ( c)
discuss regulatory issues related to their use in the food system.[34]
 Campos De V. E. B. et.al., (2017) reported that the aim of this study was to prepare NEs containing ketoconazole (KTZ), a poorly water-soluble
drug, to characterize them and evaluate their antifungal activity against Candida albicans. The solubility of KTZ has been studied in various
essential oils. Essential oils of clove and sweet fennel were selected as the oil phase and Pluronic F127® and Cremophor RH40® as non-ionic
surfactants joined the aqueous phase.[35]
12

13. AIM- To prepare nanoemulsion of essential oils and its antimicrobial property.
OBJECTIVES
To extract essential oil from
selected medicinal plant
Preparation of nanoemulsion
of essential oil
Assessment of anticandidal
activity of essential oil
AIM & OBJECTIVE
13

14. Material
All the chemicals, oil, and other compounds were provided by the Department of Pharmacology, C.S.I.R-NBRI, Lucknow.
S. No. Chemicals Suppliers
1 API-Essential oil CSIR-NBRI, Lucknow
2 Tween 20 Thomas Baker
3 Tween 60 Himedia Laboratories Pvt.
Ltd.
4 Tween 80 Thomas Baker
5 Ethanol Absolute Ethanol
6 Isopropyl alcohol Shilex Chemicals Pvt Ltd.
7 Span 20 CDH
8 PDA Himedia
9 PDB Himedia
10 Polyethylene Glycol 400 SDFCL
11 Crystal violet solution Alfa Chem laboratories
Table 3.1 List of chemicals used along with their suppliers
S. No. Equipment Suppliers
1 Autoclave Unilabs
2 Beakers and other glass-
wares
Borosil
3 Homogenizer IKA (T25 digital ULTRA TURRAX)
4 Magnetic stirrer Precisa
5 Micropipette Finn Pipette
6 Micro centrifuge tube Appendrope
7 Centrifuge NEYA 16R
8 Vortex Spinix
9 Weighing Balance Precisa XB 220A
10 pH meter FE20-1 Meteller Toledo
11 Viscometer Brookfield (DV LV- pro model)
12 Particle size analyzer Malvern Zetasizer
Table 3.2 List of equipment along with their suppliers
MATERIAL & METHODS
14

15.  Pre-liminary studies
 Organoleptic characterization (Color and odour)
 pH determination
 Determination of density
 Determination of viscosity of oil
 Extraction of Essential Oil (EO)
 Gas Chromatography Mass spectrometry
 Selection of excipients
 Screening of Surfactants
 Screening of Co-surfactants
 Preparation of Nanoemulsion
 Methods
15

16.  Physicochemical evaluation of nanoemulsion
 Determination of particle size
 pH Determination
 Stability Studies
 Visual inspection
 Determination of viscosity
 Refractive index
 Culture and Culture conditions
 Disc diffusion method
 Alamar blue assay for cell viability
 Assessment of antibiofilm activity in C. albicans
 Spectrophotometric method
 Light microscopy
 Assessment of Antimicrobial activity
16

17. Selection of excipients
• The selection of excipients was determined by the screening procedure where the surfactant and co-surfactant
were screened with their solubility properties.
Figure 3.2 Screening Surfactant and Co-surfactant
17

18. Methodology- (High pressure Homogenization)
Figure 3.3 Preparation of Nanoemulsion
Firstly, prepared a coarse of emulsion by mixing
oil and surfactant (tween 20) with the help of
magnetic stirrer.
In separate beaker water & co-surfactant
(Ethanol) were mixed on homogenizer.
Then homogenized the aqueous phase at
15000rpm for 20 min.
During homogenization the mixture of oil &
surfactant was added drop by drop.
A small droplet size nanoemulsion was prepared.
Preparation of Nanoemulsion
18

19. Physicochemical Evaluation of Nanoemulsion
• Determination of particle size
• pH Determination
• Stability Studies
• Visual inspection
• Determination of viscosity
• Refractive index
19

20. Assessment of Antimicrobial activity
• Culture and Culture conditions
• Fluconazole resistant isolates of Candida albicans, used in this study were obtained by sub culturing pathogenic yeast and
checked for purity in pharmacology department CSIR-NBRI, Lucknow. Pure isolates were used in the study. These strains
were maintained on Sabouraud's Dextrose medium prior to the experiments in presence of 20ug/mL Fluconazole (Forcan,
Cipla) Cultures were propagated for two days in Sabouraud's medium at 37°C on an orbital shaker at 200 rpm. Cells were
harvested by centrifugation and suspended in sterile saline before use.
• Disc diffusion method
• Different concentration of oil and emulsion will be subjected to the targeted microorganisms to obtain the optimum
inhibitory concentration of oil. Agar disc-diffusion testing developed in 1940, it is the official method used in
many clinical microbiology laboratories for routine antimicrobial susceptibility testing. Nowadays, many accepted
and approved standards are published by the Clinical and Laboratory Standards Institute (CLSI) for bacteria and
yeasts testing. In this well-known procedure, agar plates are inoculated with a standardized inoculum of the test
microorganism. Then, filter paper discs (about 6 mm in diameter), containing the test compound at a desired
concentration, are placed on the agar surface. The Petri dishes are incubated under suitable conditions. Generally,
antimicrobial agent diffuses into the agar and inhibits germination and growth of the test microorganism and then
the diameters of inhibition growth zones are measured
20

21. Alamar blue assay for cell viability: -
The Alamar blue assay will be performed essentially according to the method of kit manufacturer and optical densities will be
measured at 570 nm keeping 600 nm as a reference wavelength. The percentage growth inhibition will be calculated using following
formula,
% Cell inhibition = 100 −
(A𝑡−
𝐴𝑏
)
(A𝑐−
𝐴𝑏)
× 100
Where, At = absorbance value of test compound,
Ab = Absorbance value of blank and
Ac = Absorbance value of control.
The effects of extracts will be expressed by IC50 values (the drug concentration reducing the absorbance of treated cells by 50% with
respect to untreated cells).
21

22. Assessment of antibiofilm activity in C. albicans
Spectrophotometric method
The effect of CNM EO on biofilm formation was determined by the spectrophotometric method. The overnight grown culture was
diluted to an optical density of 0.2 & 500µl of it was transferred to a 12-well plate.
The CNM EO were added at different conc. The plate was incubated at 24 h at 37ºC. After incubation, the planktonic cells in the
LB medium were removed by discarding the culture medium and gentle washing with PBS. The biofilm adhering on the wells was
stained with 500 µL of 0.2% crystal violet (CV) for 15 min. The excess stain was then eliminated by washing with PBS. The dye
bound to the cell surface was dissolved in ethanol and transferred to 96-well plate. The absorbance was taken at 590 nm.
Light microscopy
The prevention of biofilm formation was also examined through light microscopy. The biofilm was prepared in a 6-well plate on
glass coverslips, as mentioned above. The CV-stained biofilm was examined under the light microscope at 400× magnification.
22

23. Result & Discussion
Preliminary studies
S.no. Parameter Observation
1 Appearance Pale Yellow Clear Liquid
2 Odour Characteristic odour
3 pH 7.2
4 Density 0.920 g/mL
5 Specific Gravity 0.938 g/mL
6 Refractive index 1.4205 n D
23

24. Gas Chromatography Mass spectrometry
Active component of Cinnamon Essential Oil
S. No. Compound Content%
1 α-pinene 0.21
2 α-terpinene 0.42
4 Cinnamaldehyde 59.47
5 Eugenol 7.8
7 Cinnamyl acetate 27.91
8 Caryophyllene oxide 1.02
9 Total (%) 96.83
24

25. Screening of Surfactant/ optimization of formulation
The screening of surfactant was determined by visual observation. There was different surfactant identified such as tween-80
and amongst them tween 20 showed better solubility (Table 4.3).
Table 4.3 Screening of Surfactant
S.no. Surfactant
(Tween 20)
Oil
10%
Cosurfactant
(Ethanol)
Observation Stability
1 5% 10% 10% Opaque Unstable
2 5% 5% 10% Opaques,
Semitransparent
Unstable
3 5% 2.5% 10% Transparent Stable till
one month
4 5% 1% 10% Transparent Stable
during
study
tenure
25

26. 4.4.1 Determination of Particle size of Nanoemulsion
The particle size of the NE was determined by photon correlation spectroscopy using a Malvern Zetasizer and it was found to be
21.06 nm diameters.
4.4.2 Determination of pH
The pH of NE has important role for compatibility of formulation with skin. Our formulation has pH value of 7.2 i.e., compatible to
pH of our skin. Skin pH range (4.7 - 7.5).
4.4.3 Determination of viscosity
The viscosity of CNM EO NE was determined to be 1.32 cps at the torque of 20% determined using the Brookfield viscometer.
4.4.4 Stability of the prepared nanoemulsion
The physical stability of the NE was observed for 90 days period and it was found to be microscopically homogenous, measurement
with no phase separation, and did not destabilize even after infinite dilution and addition of alkalizer.
Physicochemical evaluation parameter
26

27. Table 4.4 Physical stability of the developed nanoemulsion
S.no. Parameters Days
0 30 60 90
CNM- 3 Visual
appearance
Transparency Good Maintained Maintained separation
Separation None None None None
Fungal growth None None None None
CNM-4 Visual
appearance
Transparency Good Maintained Maintained Maintained
Separation None None None None
Fungal growth None None None None
27

28. Assessment of Antimicrobial activity
Disc diffusion method
Table 4.5. 1. Zone in diameter inhibition for CNM EO
Conc. Of
Emulsion
CNM EO
5% 32 mm
1% 25 mm
(a) 5% CNM EO (b) 1% CNM EO (c)Control (d)NE (e) Fluconazole
(100µg/ml)
Figure 4.5.1 (a) zone of inhibition is 32mm by using 5% EO. (b) zone of inhibition is 25mm by using 1% EO. (c) control
(d) zone of inhibition is 25mm by using 1% NE. (e) zone of inhibition is 10mm by using fluconazole.
28

29. Determination of growth inhibition through Alamar blue assay: -
Control 0.01% conc. of CNMEO NE is used
Figure 4.5.2.1 In this fig. first three column is control and other is
having conc. of 0.01% CNMEO NE which shows less growth and
more killing of bacteria.
-20
0
20
40
60
80
100
Control 0.010% 0.025% 0.050% 0.075% 0.100% 0.200% 0.500%
%
Growth
reduction
Figure 4.5.2.1 CNMEO NE shows growth reduction of bacteria
at different concentration.
29

30. Assessment of Anti-biofilm activity
Spectrophotometric analysis
0
10
20
30
40
50
60
70
80
90
100
Control 0.025% 0.050%
% Biofilm reduction
Figure 4.5.3.1 This graph shows 90% reduction of biofilm at
0.050% and 40% reduction at 0.025%.
The biofilm formation ability of C. albicans was
determined in the presence and absence of CNMEO
NE through crystal violet staining. The CNMEO
NE inhibited the biofilm formation up to 90% at
concentration of 0.050%. CNMEO NE also inhibit
biofilm formation up to 40% at concentration of
0.025%.
30

31. Light Microscopic analysis
The crystal violet staining was performed to visualize the biofilm under a microscope. As
the conc. of CNM EO increased, the complexity of the biofilm architecture was lost.
We have got thick mat of candidal cells while in the treatment of 0.05%, cells were
dispersed and no clamp formation has been detected.
(a) Control (0.025%) (b) NE (0.025%) (c) NE (0.05%)
Figure 4.5.3.2 (a) Control with conc. of 0.025%, (b) Treated with (0.025%) of NE,
(c) Treated with (0.05%) of NE.
31

32. CONCLUSION
 To conclude this study, cinnamon oil was tested for antifungal activity and findings shows better activity of cinnamon oil over
fungal strains of disc diffusion method at 5% conc. of emulsion in which 32mm zone of inhibition is found and 1% conc. of
emulsion in which 25mm zone of inhibition.
 In assessment of antibiofilm activity of Light microscopy we have got thick mat of candidal cells while in the treatment of 0.05%,
cells were dispersed and no clamp formation has been detected and in spectrophotometric method shows 90% reduction of biofilm
at 0.050%. In determination of growth inhibition at conc. of 0.01% shows less growth and more killing of bacteria’s that’s why
color is not oxidized.
 Therefore, we proceed our work with cinnamon essential oil as dual ingredient having more active effect at less concentration of oil
against fungi as well as oily phase excipient for our nanoemulsion formulation. For preparing an optimum nanoemulsion
formulation we incorporated cinnamon oil into emulsion system using water as an aqueous phase and surfactant to overcome
interfacial tension and maintain stability of colloidal system.
 Emulsion or nanoemulsion can increase the surface area of drug exposure with minimum utilization of essential oil for such
pathogenic diseases. This study demonstrates the viability of essential oil base nanoemulsion for pathogenic diseases.
32

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35. THANKYOU
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