TYPE Original Research

PUBLISHED 26 September 2022

DOI 10.3389/fnut.2022.944856

Woodfordia fruticosa extract

OPEN ACCESS nanoemulsion: Influence of
processing treatment on droplet
EDITED BY
Kandi Sridhar,
Agrocampus Ouest, France

REVIEWED BY
Zaixiang Lou,
size and its assessment for in
Jiangnan University, China
Nidhi Kalia,
University of Florida, United States
vitro antimicrobial and
*CORRESPONDENCE
Agnieszka Najda
anti-inflammatory activity
[email protected]
Aarti Bains
[email protected] Agnieszka Najda1*, Aarti Bains2*, Joanna Klepacka3 and
Prince Chawla
[email protected] Prince Chawla4*
1
SPECIALTY SECTION Department of Vegetable and Herbal Crops, The University of Life Science in Lublin, Lublin, Poland,
This article was submitted to 2
Department of Microbiology, Lovely Professional University, Phagwara, India, 3 Department of
Nutrition and Food Science Commodity Science and Food Analysis, Faculty of Food Science, University of Warmia and Mazury
Technology, in Olsztyn, Olsztyn, Poland, 4 Department of Food Technology and Nutrition, Lovely Professional
a section of the journal University, Phagwara, India
Frontiers in Nutrition

RECEIVED 15 May 2022

ACCEPTED 05 September 2022 Recently, plant-derived bioactive compounds have been utilized in the
PUBLISHED 26 September 2022
preparation of several functional food products; however, stability and water
CITATION
Najda A, Bains A, Klepacka J and
solubility are major constraints to these compounds. Therefore, to overcome
Chawla P (2022) Woodfordia fruticosa this problem, the synthesis of nanoemulsion (oil in water) with varying
extract nanoemulsion: Influence of concentrations of Woodfordia fruticosa flower extract (1%−10% w/v) was
processing treatment on droplet size
and its assessment for in vitro carried out and characterization of nanoemulsion was done. The average
antimicrobial and anti-inflammatory droplet size of nanoemulsion samples ranges from 149.25 to 244.33 nm. The
activity. Front. Nutr. 9:944856.
control and WFNE3 nanoemulsion showed significantly (p < 0.05) higher
doi: 10.3389/fnut.2022.944856
thermal stability when correlated with average droplet size. An insignificant
COPYRIGHT
© 2022 Najda, Bains, Klepacka and difference (p > 0.05) was observed in the average droplet size and zeta
Chawla. This is an open-access article potential WFNE3 (−30.3mV) with the increased temperature rate. At varied pH
distributed under the terms of the
ranges, WFNE3 showed significantly higher (p < 0.05) stability in comparison
Creative Commons Attribution License
(CC BY). The use, distribution or with the control nanoemulsion sample. In terms of ionic strength, WFNE3
reproduction in other forums is nanoemulsion sample showed significantly (p < 0.05) higher stability, and
permitted, provided the original
author(s) and the copyright owner(s) with an increasing concentration of salt, the colloidal system of the WFNE3
are credited and that the original sample showed significantly (p < 0.05) higher droplet size (318.91 nm).
publication in this journal is cited, in
Therefore, the antimicrobial potential of WFNE3 nanoemulsion in comparison
accordance with accepted academic
practice. No use, distribution or with extract of W. fruticosa flower extract was studied against Gram-positive
reproduction is permitted which does Staphylococcus aureus, Gram-negative bacteria Pseudomonas aeruginosa,
not comply with these terms.
and fungal strain Candida albicans, respectively. WFNE3 nanoemulsion
sample in comparison to flower extract showed a significantly higher (p
< 0.05) zone of inhibition against gram-negative bacteria as compared
to the control nanoemulsion sample upon storage for 7 days. WFNE3
nanoemulsion sample showed significant (p < 0.05) higher inhibition of protein
denaturation (57.89%−87.65%) and (55.36%−83.58%) in comparison to control
nanoemulsion sample (54.67%−80.28%) and flower extract (51.56%−79.36%),

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respectively. Due to these biological activities, the WFNE3 nanoemulsion

sample could be scaled up to the industrial level for the formulation of varied
types of functional foods.

KEYWORDS

Lythraceae, nanoemulsion (nanoE), antioxidant, anti-inflammatory, zeta potential

(ZP), gum arabic

Introduction was done using gas chromatography-mass spectroscopy.

The compounds identified during characterization were:
Woodfordia fruticosa Kurtz is a well-known plant in the γ-terpinene, dihydrocarvyl acetate, 1-decalone (cis-trans),
Lythraceae family that grows up to 1,500 m in altitude and is cis-7-decen-1-al, tetradecanoic acid, palmitic anhydride,
found in India’s tropical and subtropical regions. Traditional pentadecanoic acid, octadecanoic acid, n-hexadecanoic acid,
practitioners have used the plant for a long time, particularly and 3-decyn-1-ol, 2,6-octadien-1-ol, 3,7-dimethyl-, acetate,
in Southeast Asian countries (1). Although all parts of plants and e-(geranyl acetate), as well as caryophyllene epoxide,
have medical properties, flowers are in high demand in both cyclopropaneoctanoic acid, Cyclopropaneoctanoic acid, 2H-1-
domestic and international markets. The blossoms are reddish- benzopyran-2-one, 2H-1- benzopyran-2-one, and γ-elemene.
brown and possess pungent, acrid, cooling, and alexiteric All these compounds have potential health benefits, therefore,
characteristics, acting as a uterine sedative and anthelmintic (2). incorporation of these phytocompounds isolated from the
Sprue, dysentery, intestinal complaint, rheumatism, hematuria, flowers into functional food or as dietary supplements can
wounds, bleeding, and injuries are all treated with the dried play a vital role for both consumers and food industries. The
flowers of W. fruticosa. Due to their therapeutic characteristics, isolated compounds are least soluble in water, therefore, are
these flowers are also used in the formulation of fermented more prone to environmental oxidative stress (8). It is essential
Ayurvedic drugs known as “Aristhas and Asavas” (3). Myricetin, to increase the water solubility of these components to achieve
oenothein B, isochimacoalin-A, –terpinene, limonene, and substantial floral extract properties. Emulsification is the
oligomers such as woodfordins A, B, C, E, G, H, and I, as most effective and common way to improve the functionality
well as quercetin, are among the phenolic compounds found of phytocompounds (1). Emulsification has been shown to
in the flowers. The anti-inflammatory activities of quercetin improve the stability and bioavailability of these components
and myricetin are related to their inhibition of the two key in several in vitro and in vivo experiments (8). In comparison
enzymes, lipoxygenase and cyclooxygenase of pelargonidin, that to plant extract, nanoemulsion has a better antibacterial
play a key role in the pathophysiology of inflammation (1, 4). activity (9). Furthermore, gum arabic is a polysaccharide
–terpinene and limonene have antibacterial and antifungal complex that contains 2% polypeptide and the branches are
characteristics, and they act by entering the cell membrane mainly comprised of 1,3-linked-D-galactopyranosyl units with
and destroying cellular components (5, 6). The consumption 1,6-linked -D-galactopyranosyl side chains to which several
of these naturally occurring compounds can reduce the side arabinosyl, uronic acid, and rhamnose residues are attached.
effects such as edema, heaving, queasiness, diarrhea, coughing, In addition, a 43-amino-acid-residue peptide sequence is also
loss of appetite, and abdominal pain caused by synthetic drugs present in the native molecular structure of the gum arabic (10).
(7). Scientists and various researchers are, therefore, working The strong emulsifying characteristics of gum arabic have also
on the preparation of products from naturally occurring herbs been reported due to the existence of inter and intramolecular
to compensate for the therapeutic gap as well as reactions CH...π interactions (11, 12). The oil phase was desirable for
that are required during the secondary treatment. W. fruticose the preparation of W. fruticosa flower extract nanoemulsion,
flower due to the presence of phytocompounds has potential hence sunflower oil was selected to disperse the flower extract
health benefits and can be utilized as a remedy for the treatment and to formulate the continuous phase of the nanoemulsion.
of various biological disorders. In our previous study, Najda There are several studies available on the formulation of plant
et al. (1), Woodfordia fruticosa flower extract was prepared by extract-based nanoemulsions; however, no data are available on
modified solvent evaporated technique, and its characterization the formulation of W. fruticosa flower extract nanoemulsion.
Therefore, the current research work was performed with the
Abbreviations: DLS, dynamic light scattering; DPPH, 2, 2-diphenyl- following objectives in mind: (i) Preparation and stabilization
1-picrylhydrazyl; RRE, red rice extract; GA, Gum arabic; TBA, 2- of W. fruticosa extract nano-emulsion; (ii) Characterization of
thiobarbituric acid; BSA, bovine serum albumin. the emulsion; (iii) in vitro mineral bioaccessibility of zinc, iron,

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calcium, and ferritin contents of nanoemulsion using transwell with the high-energy process using a probe sonicator (Sonics and
assay; and (iv) Antimicrobial and anti-inflammatory activity Materials Inc. New Town, CT, USA) at 5◦ C, 5.0 s pulse rate, and
of emulsion. 25 min (formulation time). The freshly prepared nanoemulsion
samples were then kept in air-tight glass containers for further
screening (8).
Materials and methods
Materials
Selection of suitable nanoemulsion
Creaming index
Flowers of W. fruticosa were harvested from the forest of
Creaming stability of freshly prepared nanoemulsion
district Mandi located in Himachal Pradesh, India. Analytical
samples was assessed by following the method proposed by
reagent grade emulsifying and a surface-active agent such
Pengon et al. (13) for the selection of suitable nanoemulsion
as Gum arabic (spray dried) and tween-80 were procured
during 15 days of storage. In brief, glass bottles filled with 50 ml
from Sigma Aldrich Co. St. Louis, MO, USA. From Hi-
nanoemulsions were kept at accelerated temperatures (80◦ C)
Media Laboratories Pvt. Ltd., Mumbai, India, ethanol, L-
and the physical stability of the nanoemulsion samples was then
ascorbic acid, calcium chloride, sodium chloride, phosphate
examined by calculating the creaming index.
buffer, Muller Hinton Agar (MHA), and Sabouraud Dextrose
The following equation was used to determine the
Agar (SDA) were purchased. For the oil phase and dispersion
creaming index:
of flower extract, sunflower seed oil was used and it
was obtained from CDH Pvt. Ltd., Mumbai, India. For
antimicrobial studies, standard pathogenic bacterial strains such VC
Creaming index (%) = × 100
as Staphylococcus aureus (MTCC 3160), Klebsiella pneumoniae VE
(MTCC 3384), Pseudomonas aeruginosa (MTCC 2295), and
Salmonella typhmurium (MTCC 1254) and fungal strain of Here, VC is the volume of cream layer after heating; VE is
Candida albicans (MTCC 183) were obtained from IMTECH, the volume of the emulsified layer.
Chandigarh, India.
Droplet size and zeta-potential of nanoemulsion
The dynamic light scattering (DLS) technique is an effective
method for determining the droplet size of an emulsion system
Methods at the nanoscale. All of the emulsion samples (1% v/v) were
diluted in deionized water for the droplet size evaluation.
Preparation of ethanolic extract
Measurements were recorded in triplicates for each sample at
Woodfordia fruticosa flowers were picked fresh and to
25◦ C using particle size and zeta potential analyzer (Zetasizer
remove dirt and debris, flowers were rinsed with triple distilled
Nano ZS, Malvern Instruments Ltd. Malvern, WR14 1XZ, UK).
water before drying at 30◦ C in a hot air oven (SGM lab Solutions
Private Limited, India). A mechanical grinder was used to
ground the dried flowers into a uniform powder (Bajaj, mixer Influence of processing treatment on droplet
grinder, 900 watts, New Delhi, India). The powdered sample size and zeta potential of nanoemulsion
(10 g) was dispersed in 100 ml absolute ethanol (1:10 w/v ratio) Effect of thermal processing
in a 250-ml conical flask and placed in an orbital shaker for Freshly prepared nanoemulsions were exposed to a series of
72 h (Thermo Fisher Scientific Pvt. Ltd., Mumbai, India). The processing treatments that might come transversely in industrial
samples were then filtered using Whatman Filter Paper No. 1 applications. The effect of all the processing treatments on
and kept at refrigerator temperature (4–7◦ C) for a further 72 h. droplet size and zeta potential was examined.
The dried extract was then obtained and stored in an airtight
amber-colored glass vial at −20◦ C for further investigations. Effect of pH
Hydrogen ion concentration imparts a great influence on
the droplet and particle size of the food materials; hence,
Preparation of nanoemulsion nanoemulsions were transferred into 50 ml glass beakers and
Different concentrations of W. fruticosa flower extract adjusted to different pH ranges (2.0–9.0) using 0.1N NaOH and
(WFE; 0.25, 0.50, 0.75, 1, 1.5, 2, 2.5, 3, 4, and 5%) were 0.1N HCl solutions. All the samples were then subjected to
dispersed in 10 ml of sunflower (SF) oil using a magnetic stirrer droplet size and zeta potential evaluation.
(SPINOT MC 02, Tarsons, Kolkata, India) and the oil phase
(WFE + SF) was then mixed in an aqueous phase containing Effect of salt concentration
1% gum arabic and 250 µl tween-80 for 20 min. The final After the storage of the samples for 1 month at a temperature
nanoemulsion samples (WFNE0.25-WFNE5) were formulated of 30◦ C, the influence of varied salt concentrations on the

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droplet size of nanoemulsions was carried out by following distilled water). The mixture was centrifuged at 3,000 × g for
the literature (14). In brief, sodium chloride in different 15 min. After centrifugation, the packed cell thus obtained was
concentrations (0.5, 1, and 2 M) was added to confirm the washed with iso saline solution. An equal volume of HRBC
alteration in droplet size and zeta potential. suspension and extract (500 µl) mg/ml was dissolved in 0.36%
hypo saline solution (2 ml) containing phosphate buffer pH 7.4.
Oxidative stability of nanoemulsion The mixture is then incubated in biological oxygen demand
Oxidative stability of formulated nanoemulsion was (BOD) incubator for 30 min at 37◦ C and after incubation, the
examined by determining the TBA values by following the suspension was centrifuged at 3,000 ×g for 20 min. Diclofenac
literature (15). Fresh 0.025 M TBA solution was prepared and sodium and deionized water were used as the positive and
it was neutralized with 2 M NaOH and citric acid, respectively. negative controls.
TBA reagent was then added to nanoemulsion (5 ml) and Percentage membrane stabilization assay was calculated as:
was mixed and the mixture was heated instantly in a boiling
water bath for 10 min. The sample was then cooled in ice-cold Ot
Stabilization (%) = 100 − × 100
water followed by the addition of cyclohexane (10 ml) and Op
4 M ammonium sulfate (1 ml). Samples were centrifuged at
where: Ot is the optical density of the sample and Op is the
5,000 ×g and the orange-red colored solvent was obtained
optical density of the control.
and absorbance was then measured at 532 nm using a UV-
visible spectrophotometer (Shimadzu UV-2600i/UV2700i,
Tokyo, Japan).
Albumin denaturation assay
In vitro antimicrobial assay
The potential antimicrobial activity of nanoemulsion was In this assay, reaction mixture (5 ml) containing fresh egg
examined against pathogenic Gram-positive and Gram-negative albumin (200 µl), 2.8 ml buffer solution (pH 6.4), and 2 ml of
bacteria viz. Staphylococcus aureus, Pseudomonas aeruginosa, extracts were mixed and incubated at 37◦ C at 15 min for 15 min
and fungi Candida albicans using the agar well diffusion method. followed by heating up to 70◦ C for 5 min and absorbance was
Herein, 1 × 108 cells/ml bacterial inoculum and 1 × 105 measured at 660 nm. Deionized water and diclofenac sodium
cells/ml fungal inoculum were inoculated on 4% enriched were used as the negative and positive control. Percentage
Mueller Hinton agar (MHA) and Sabouraud Dextrose agar inhibition of protein denaturation was calculated as:
plates, respectively. The wells on agar plates were made using
Oa
a sterile cork borer. The extract was eluted in 8% DMSO and 50 Inhibition (%) = 100 × −1
Oc
µl of it was transferred into the agar well using a micropipette.
The MHA plates containing bacterial inoculum were incubated where: Oa test sample absorbance and Oc control
for 24 h at 37◦ C and the SDA plate containing fungal inoculum absorbance (13).
was incubated at 27◦ C for 72 h. The zone of inhibition diameter
was measured using a vernier caliper (12).
Statistical analysis
Time kill study
Time kill study was measured by methods proposed by Statistical analysis of obtained results was carried out by
Majeed et al. (16) Herein, 100 µl of the extracts for each bacterial following the method given by Kaushik et al. (17). Statistical
and fungal sample were taken after a time interval of 0, 18, 24, differences and standard deviations were calculated by one-way
and 48 h and 0, 24, 48, and 72 h, respectively. Then, samples ANOVA (analysis of variance) and Microsoft Excel, respectively.
were diluted serially and spread on plates containing MHA and The comparison between the mean calculated by difference
SDA. The plates were incubated at 37and 72◦ C, respectively. Log value and the standard mean error was determined by a
CFU/ml was then calculated after incubation. descriptive statistical tool.

In vitro anti-inflammatory activity

HRBC membrane stabilization Results and Discussion
Human red blood cell membrane stabilization was
performed by the following method proposed by Bains et al. Synthesis of nanoemulsion
(12). In brief, from a healthy volunteer not administered with
non-steroidal anti-inflammatory drugs (NSAID) for 15 days, The effect of varying concentrations of W. fruticosa extract
5 ml of blood was taken and dissolved in an equal volume of on the formulation of nanoemulsion was initially investigated
Alsever solution (20.5 g dextrose, 8 g sodium citrate, 0.55 g for droplet size and the results are depicted in Figure 1A. The
citric acid, and 4.2 g of sodium chloride dissolved in 1,000 ml average droplet size of all the nanoemulsions ranged from

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FIGURE 1
(A) Average droplet size of WFNE3 nanoemulsion; (B) Zeta potential of WFNE3 nanoemulsion. The error bar represents the standard deviation
from the mean values.

149.25 to 244.33 nm, respectively, and increased droplet size in zeta potential. According to the literature, zeta potential
was observed with increasing concentration of extract. Herein, characterizes the surface charge and that can be employed for the
among all the nanoemulsion samples, significantly higher (p < understanding of physical and thermal stability of nanoemulsion
0.05) droplet size was observed for WFNE5; however, samples due to electrostatic repulsion. Moreover, zeta potential depends
ranging from WFNE 0.25 to WFNE3 revealed an insignificant on the composition of emulsion and it is a function of the
(p > 0.05) difference. Furthermore, even at a high concentration droplet’s environment. Overall, negative charge distribution on
of flower extract, no visual sign of flocculation or creaming was formulated nanoemulsion was majorly due to the arrangement
observed. This emulsion behavior (no visual sign of flocculation) of gum arabic molecules with oil phase, surface active agent, and
was attained due to a change in the charge distribution of the flower extract through hydrophobic binding moiety. Our
exterior and interfacial viscosity of the emulsion system in the results were well supported by the findings of Khan et al. (18)
presence of gum arabic and tween-80 (8). The Zeta potential of and Yao et al. (19) who observed the electrostatic interaction of
all emulsion samples was observed and the results are depicted in emulsifier with continuous phase and surface-active agent with
Figure 1B. Herein, all the samples showed a significant difference high total charge distribution.
in zeta potential and it ranged from −34.4 to −23.5 mV.
However, it could be revealed from the results that samples
ranging from WFNE0.25 to WFNE1 showed insignificant Creaming index of nanoemulsion
differences (p > 0.05) in zeta potential values of all the emulsion To understand how extract influenced the stability of
samples, with increasing concentration nanoemulsion samples emulsions, it was necessary to understand the creaming
(WFNE1.5–WFNE5) showed significant difference (p < 0.05) stability processes responsible for destabilizing emulsions in

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FIGURE 2
Creaming index of WFNE3 nanoemulsion. The error bar represents the standard deviation from the mean values.

the first place, and the results of creaming stability of with a lower concentration of W. fruticosa were substantially
control and WFNE0.25–WFNE5 nanoemulsion samples are stable in terms of creaming index due to considerably mono-
presented in Figure 2. Herein, the control emulsion sample dispersed, small droplet sized, and strong repulsion between
showed a significantly (p < 0.05) higher creaming index extract nanodroplets, adequately coated by gum arabic (8).
(24.56%) in comparison with flower extract nanoemulsion
(10.56%−18.54%), and during the 10th day of the storage, visual
creaming was observed in the control sample. However, WFNE5 Thermal stability
showed a 10.56% to 18.54% creaming index during the 5th, 10th, The impact of food processing temperature on food
and 15th days of storage, respectively. The colloidal stability of matrixes or packaging components comprising nanoemulsion
an emulsion can be controlled by the colloidal interaction forces that reveal the stability during final consumption was examined
keeping the droplets apart and evenly dispersed. by illustrating the thermal stability of nanoemulsion [8].
Creaming is the mass transfer of emulsion droplets on The thermal stability of control and WFNE3 nanoemulsion
the surface of the emulsion, driven by the resistance of the is depicted in Figures 3A,B and during observations, both
droplets of emulsion in the continuous phase. Moreover, the WFNE3 and control samples showed significant (p < 0.05)
movement of the emulsion droplets can be driven by the density differences from each other. Herein, as compared to the
difference between the droplets and the continuous phase, control nanoemulsion (194.22 nm), the WFNE3 (146.56 nm)
the size of the droplet, and the viscosity of the continuous nanoemulsion showed significantly (p < 0.05) higher thermal
phase. Moreover, according to Derjaguin–Landau–Verwey– stability when it was correlated with average droplet size.
Overbeek theory, the creaming index of colloidal systems Moreover, with an increasing temperature rate, an insignificant
potentially depends on the balance of electrostatic repulsive (p > 0.05) difference was observed in the average droplet size
forces and van der Waals attractive forces acting on the of the nanoemulsion samples. During the thermal processing
interface. Therefore, a theoretical model of nanoemulsion can be of the selected WFNE3 nanoemulsion, no visual creaming or
anticipated, where the hydrophilic part of the emulsifier projects coalescence was observed. On the other hand, in the case of
into the aqueous phase which remains toward molecules of charge distribution of the colloidal system, with the increase in
flower extract. Cavitation during the ultra-sonication process temperature, an insignificant difference in the zeta potential of
exerted chemical distortion of the oil phase. Hence, samples WFNE3 (−30.3 mV) was observed. This could be due to the

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FIGURE 3
Effect of different ranges of (A) temperature on droplet size of WFNE3 nanoemulsion; the (B) temperature on zeta potential of WFNE3
nanoemulsion; (C) pH on droplet size of WFNE3 nanoemulsion; (D) pH on zeta potential of WFNE3 nanoemulsion; (E) ionic concentration on
droplet size of WFNE3 nanoemulsion; (F) ionic concentration on zeta potential of WFNE3 nanoemulsion. The error bar represents the standard
deviation from the mean values.

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developed steric repulsion by surface-active agent and emulsifier nanoemulsion did not affect due to increased salt concentration,
and it was not extensively adequate due to the creation of hence no visual coalescence and oiling off were observed.
thin interfacial layers. Therefore, as a result, the droplets of
the colloidal system were highly stable against aggregation. As
well, interfacial colloidal surface tension in selected WFNE3 TBA value
nanoemulsion was interrelated to the stimulus of Laplace Oxidative stability was measured for both of the
pressure, in which lower values enable the rupture of the nanoemulsion samples and the results of TBA value in
droplets, creating minor droplet sizes and higher stability against comparison with sunflower oil are depicted in Figure 4. Varied
aggregation or coalescence with increasing temperature (20). range and significant (p < 0.05) difference of oxidative stability
in terms of TBA value for sunflower oil (0.00–0.157), control
nanoemulsion, and WFNE3 nanoemulsion (0.00–0.017) was
pH stability observed during the storage. However, the control emulsion
The impact of variation in pH upon the droplet size sample and WFNE3 emulsion sample revealed in-significant
and charge distribution (zeta potential) of the control and differences (p > 0.05) with each other, whereas, a significant (p
WFNE3 nanoemulsion was examined and results are presented < 0.05) difference was observed in the TBA value of sunflower
in Figures 3C,D. Herein, at varied pH ranges, WFNE3 showed oil and nanoemulsion samples during the 3rd, 5th, and 7th
significantly higher (p < 0.05) stability in comparison with day of storage, respectively. The higher oxidative stability of
the control nanoemulsion sample. The colloidal system of the the colloidal system of WFNE3 nanoemulsion was due to
WFNE3 nanoemulsion sample at pH 5–9 showed significantly the occurrence of W. fruticosa flower extract (22) and our
lower (p < 0.05) average droplet size as compared to pH ranging results were well supported by the findings of Chawla et al.
from 2 to 4, and it was found to be highly stable coalescence and (8) who observed higher oxidative stability in terms of TBA
Ostwald ripening. However, at pH 4, a thin layer of lipid droplets value for gum arabic-stabilized Rhododendron arboreum flower
on the surface of the WFNE3 nanoemulsion with a lower droplet extract nanoemulsion.
size (290.27 nm) was observed. Despite the small droplet size,
there was calescence in the sample. The WFNE3 nanoemulsion
sample was highly unstable to phase separation at pH 2 and 3. Antimicrobial activity
This result was possibly due to the revelation of amino chains The antimicrobial potential of WFNE3 nanoemulsion in
of emulsifier molecules with an amplified rate of protonation, comparison with extract of W. fruticosa flower extract was
triggered by the demulsification (21). assessed against pathogenic gram-positive and gram-negative
bacterial and fungal strains. The results were represented in
Figures 5A,B. Herein, the flower extract and nanoemulsion
Ionic strength were stored at 37◦ C for 7 days and then evaluated for their
Different salt concentrations were induced to the colloidal antimicrobial activity. In the present study, it has been observed
system of the selected nanoemulsion and the influence on that stored WFNE3 nanoemulsion samples in comparison
droplet size and zeta potential was evaluated (Figures 3E,F). to flower extract showed a significantly higher (p < 0.05)
Herein, WFNE 3 nanoemulsion sample showed significantly (p zone of inhibition against gram-negative bacteria. However, in
< 0.05) higher stability in terms of ionic strength in comparison comparison to positive control in terms of zone of inhibition,
with the colloidal system of the control nanoemulsion sample. there observed a non-significant difference. Furthermore, the
However, with an increasing concentration of salt, the colloidal flower extract showed significantly (p < 0.05) less antimicrobial
system of the WFNE3 sample showed a significantly (p < activity against gram-negative bacteria P. aeruginosa during the
0.05) higher droplet size (318.91 nm). This result was obtained 7th day of storage, and the zone of inhibition decreased from
due to structural chemistry and polyanionic charge distribution 227.76±0.1 mm to 17.34±0.05 mm; however, on the 5th and
on the surface of gum arabic which resulted in coulombic 7th day of storage, non-significant difference (p < 0.05) in
repulsion; however, negatively charged carboxylic moiety led to antimicrobial activity was observed. During storage in the case
electrostatic interaction between positive and negative charges of WFE nanoemulsion, no decrease was observed in the zone
of sodium ion which increased in droplet size. Furthermore, the of inhibition. In comparison to flower extract, WFNE3 is highly
zeta potential of the colloidal system of WFNE3 nanoemulsion susceptible to gram-positive bacteria and fungus and showed a
was greatly influenced by the electrostatic interaction and a significantly higher (p < 0.05) zone of inhibition against them.
significant decrease (p < 0.05) in the overall charge distribution Also, during the 7th day of storage, WFNE3 nanoemulsion
of the nanoemulsion sample was observed. The presence of showed high antimicrobial susceptibility as compared to the
positive charge on amino acid groups of gum arabic is bound control nanoemulsion sample and have a non-significant (p <
with negatively charged chloride ions (17). Apart from the 0.05) difference in the zone of inhibition. There was a significant
increase in droplet size of nanoemulsion, the colloidal stability of decrease (p < 0.05) in the zone of inhibition for both bacterial

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FIGURE 4
Effect of storage on TBA value of WFNE3 nanoemulsion. The error bar represents the standard deviation from the mean values.

FIGURE 5
Effect of storage on the antimicrobial efficacy of WFNE3 nanoemulsion against (A) Pseudomonas aeruginosa and Staphylococcus aureus; and
(B) Candida albicans. The error bar represents the standard deviation from the mean values.

and fungal strains shown by flower extract during storage, the cell wall is composed of lipopolysaccharides that cause the
hence it was concluded from the results that the antimicrobial difference in hydrophobic properties and induce mutation in
susceptibility of flower extract decreases when stored at 37◦ C porins and other factors that result in resistance against several
for 7 days while WFNE3 showed promising results. Here, the extracts, nanoemulsion, and synthetic antibiotics. The WFNE3
WFNE3 extract was least susceptible to gram-negative bacteria nanoemulsion showed remarkable antifungal activity against C.
P. aeruginosa in comparison to S. aureus and Candida albicans. albicans with a zone of inhibition of 25.62 mm. The bioactive
In the case of gram-positive bacteria S. aureus, the cell wall compounds present in the sample diffuse resulting in the
is composed of a thick hydrophobic cell wall that permits disruption of the cell membrane of fungal strain. Another reason
the passage of bioactive components to pass through the cell for remarkable antimicrobial activity is that nanoemulsion due
membrane (23), while in the case of gram-negative bacteria, to their nano size results in an increase in the contact surface

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FIGURE 6
Time kill kinetics of WFNE3 nanoemulsion against (A) S. aureus and P. aeruginosa, (B) C. albicans, (C) Anti-inflammatory activity of WFNE3
nanoemulsion with HRBC membrane stabilization, and (D) Anti-inflammatory activity of WFNE3 nanoemulsion using BSA denaturation assay.

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Najda et al. 10.3389/fnut.2022.944856

of the sample with microorganisms, thereby resulting in an nanoemulsion sample (54.67%−80.28%) and flower extract
improvement of accessibility of bioactive compounds and their (51.56%−79.36%), respectively. However, in comparison to
ability to penetrate inside the cell membrane (24). WFNE3 nanoemulsion, control nanoemulsion and flower
extract standard Diclofenac sodium salt showed significantly
higher (p < 0.05) percentage protein denaturation inhibition
Time kill study and HRBC membrane stabilization. HRBC membrane is similar
to the lysosomal membrane; therefore, the assay is considered
Time kill study of the WFNE3 nanoemulsion sample was for limiting inflammation response (28, 29). In addition to this,
performed and results are presented in Figures 6A,B. In the the anti-inflammatory response in the case of inflammatory
present study with the increase in time interlude, nanoemulsion disease is well correlated with the denaturation of proteins.
WFNE3 showed less significance (p < 0.05; 7.82 and 7.76 log Tissue injuries result in the denaturation of proteins that
CFU/ml) in comparison to control nanoemulsion (7.90 and 7.82 are constituted by cells and intracellular components. The
log CFU/ml) and flower extract (7.98 and 8.02 log CFU/ml) emulsification of flower extract improves the functionality of
for both bacterial strains. The inhibitory effect of the WFNE3 flower extract which is supposed to inhibit the synthesis or
nanoemulsion sample on S. aureus is due to the bioactive release of inflammatory mediators as well as stabilization of cell
compounds present in it. These bioactive compounds suppose membrane (5). Also, the effective anti-inflammatory activity
to inhibit the synthesis of protein and biochemical pathways of flower extract results due to the synergistic effect of both
and result in the destruction of cell membrane structure. The the sunflower oil and droplet size in a continuous phase of
gram-negative bacterial strain P. aeruginosa showed a higher the emulsion.
log CFU/ml value, this is due to the reason that in gram-
negative bacteria, the outer cell membrane consists of a layer
of a phospholipid bilayer and lipopolysaccharides that are Conclusion
embedded with porin proteins and β-barrel channels. This
structure prevents the penetration of antibiotics and other active The less bioactivity and release of undesirable metabolites
components to enter the cells (25). Another foremost reason for from plant extract results due to environmental stress. To
the resistance of gram-negative bacteria is the development of overcome this problem, the formulation of gum arabic-
efflux pumps that expel the components toxic to the cell and the stabilized W. fruticosa nanoemulsion was formulated to enhance
production of enzymes that act either by breaking or causing the bioactivity and functionality of phenolic compounds,
alteration in the antibiotic structure and bioactive components which showed a strong health-promoting effect. Among
by using mutational changes or genes acquisition (26). Similarly, the samples with various additives of W. fruticosa extract,
in the case of C. albicans, nanoemulsion showed significantly emulsions with 3% addition showed the best properties
lower (p < 0.05) 7.72 log CFU/ml values as compared to during the stability test, antimicrobial, anti-inflammatory,
control nanoemulsion (7.80 log CFU/ml) and flower extract and antioxidant activities. It was observed that the WFNE3
(7.98 CFU/ml). The reduction in log CFU/ml value is due to the nanoemulsion sample also showed higher stability to creaming
reason that yeast lacks surface-active hydrophobin proteins and or flocculation over a wide range of temperature, pH,
has hydrophilic nature; therefore, the hydrophobic antifungal and salt concentrations. Also, during storage, the stability
components can readily be accumulated at the conidia of fungal of sunflower oil and flower extract toward oxidation is
strains (1, 27). improved by nanoemulsion. In comparison to flower extract,
the nanoemulsion sample showed effective antimicrobial
activity against standard bacterial and fungal strains. The
Anti-inflammatory activity WFNE3 also showed significantly (p < 0.05) enhanced anti-
Albumin denaturation assay and HRBC membrane inflammatory activity and antioxidant activity. Therefore, in
stabilization assay were used for the anti-inflammatory the present study, it is determined that gum arabic-stabilized
assay and the results are depicted in Figure 6C. In the nanoemulsion showed remarkable antimicrobial, antioxidant,
present study, a significant (p < 0.05) difference was and anti-inflammatory activities and hence can be used as an
observed in the anti-inflammatory activity of flower effective food preservative.
extract, WFNE3, and the control nanoemulsion sample.
WFNE3 nanoemulsion, control nanoemulsion sample, as
well as flower extract showed inhibition of protein and Data availability statement
stabilization of HRBC membrane in a dose-dependent
manner (20–100 µg/ml). WFNE3 nanoemulsion sample The original contributions presented in the study are
showed significant (p < 0.05) higher inhibition of protein included in the article/supplementary material, further inquiries
denaturation (55.36%−83.58%) in comparison to control can be directed to the corresponding authors.

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Author contributions Conflict of interest

AN, PC, and AB: conceptualization, formal analysis, The authors declare that the research was conducted in
investigation, and writing original draft preparation. AN the absence of any commercial or financial relationships
and PC: methodology. PC and AB: software validation. that could be construed as a potential conflict
AB: resources. JK and PC: data curation, visualization, and of interest.
supervision. AN, JK, and PC: review and editing. All authors The handling editor KS declared a past co-authorship with
contributed to the article and approved the submitted version. the author PC.

Funding
Publisher’s note
The support by the Department of Food Technology
and Nutrition, Lovely Professional University is gratefully All claims expressed in this article are solely those of the
acknowledged. The research project was financially supported authors and do not necessarily represent those of their affiliated
by the Minister of Education and Science under the program organizations, or those of the publisher, the editors and the
entitled Regional Initiative of Excellence for the years 2019– reviewers. Any product that may be evaluated in this article, or
2023, Project No. 010/RID/2018/19, and the amount of funding claim that may be made by its manufacturer, is not guaranteed
was 12.000.000 PLN. or endorsed by the publisher.

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