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Accepted Manuscript

Encapsulation of orange and lavender essential oils in chitosan


nanospherical particles and its application in leather for aroma
enrichment

Punitha Velmurugan , Vasudevon Ganeshan ,


Nishad Fathima Nishter , Raghava Rao Jonnalagadda

PII: S2468-0230(17)30093-7
DOI: 10.1016/j.surfin.2017.08.009
Reference: SURFIN 133

To appear in: Surfaces and Interfaces

Received date: 12 June 2017


Revised date: 17 August 2017
Accepted date: 22 August 2017

Please cite this article as: Punitha Velmurugan , Vasudevon Ganeshan , Nishad Fathima Nishter ,
Raghava Rao Jonnalagadda , Encapsulation of orange and lavender essential oils in chitosan
nanospherical particles and its application in leather for aroma enrichment, Surfaces and Interfaces
(2017), doi: 10.1016/j.surfin.2017.08.009

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service
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ACCEPTED MANUSCRIPT

Highlights

 Essential oils were encapsulated using chitosan by emulsion polymerization technique


 Oil encapsulates were characterized by particle size, morphology, FTIR & TGA
 Aroma encapsulates incorporated in leather after neutralization process
 Aroma oils release in leather is persist after three wash with dry cleaning solvent &
water

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Encapsulation of orange and lavender essential oils in chitosan nanospherical particles


and its application in leather for aroma enrichment

Punitha Velmurugan1, Vasudevon Ganeshan1, Nishad Fathima Nishter2, Raghava Rao


Jonnalagadda*2

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Centre of Advanced Study in Crystallography and Biophysics, University of Madras,
Guindy Campus, Chennai 600 025, India.

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2
Inorganic and Physical Chemistry Laboratory, Council of Scientific and Industrial Research

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- Central Leather Research Institute, Adyar, Chennai 600 020, India.

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__________________________________________________________________
*To whom correspondence should be addressed.
Tel.: +91 44 24411630; Fax: +91 44 24911589; E-mail: jrrao@clri.res.in

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ABSTRACT
Incorporating “smartness” into leathers leads to enormous value addition. One such smartness
is development of scented leather. Lavender and orange oils have been microencapsulated
and infused into the leather. Viscosity, opacity, and transmittance have been measured in
order to optimize oil:surfactant ratio. Emulsion polymerization technique has been adopted
for encapsulation of scent oils using chitosan as wall material. Percentage encapsulation
efficiency, oil load and content have been studied for the encapsulated products. Lavender oil
encapsulates hold better oil content and loading than orange oil encapsulates. Further,

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encapsulates have been characterized using particle size, surface morphology, FT-IR and

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TGA and results were corroborating. The encapsulated product withstands high temperature

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up to 500ºC and also it also shows antimicrobial activity. Encapsulated scent oils have been
incorporated into leather and subjected to perception, strength, washability and diffusion
coefficient studies. Thus, successful value addition to leather through incorporating of

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pleasant smell has been made possible resulting in “smart scented” leathers.
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Keywords: scented leather; smart leather; lavender oil; orange oil; nanospheres;
encapsulation
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1. INTRODUCTION

Developing leather products with additional functionalities such as cool or thermo-sensitive


leathers1and conductive leathers2 can lead to new dimensions in value addition to leather.
Leather garments and upholstery have great demand in western countries, and in order to
capture the market; two main areas like innovation and value addition need to be focused.
One such approach is embedding scent in leather thereby leather garments, upholstery get
value addition. Scent oils are predominantly used so far in the field of medicine3, cosmetics4,

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liquid detergents and fabric conditioners5,6.

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Humans have applied scents and fragrance to skins since ancient times. Perfume

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compositions are an important part of many industries ranging from luxury goods sectors,
food service industries to manufactures of various chemicals. The purpose of using perfume
or fragrance composition in these industries is to affect the customers through their sense of

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smell and entice them to purchase the perfumed products. As such, there is significant interest
in producing a perfume formulation that people would find aesthetically pleasing. Scent oils
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such as orange oil (Citrus sinensis) and lavender oil (Lavandula angustifolia) belong to the
family of essential oils and have profound application in various areas. Hence, it would be of
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greater benefit if the scent is incorporated into leather matrix as such. Also leather garments,
which cannot be washed more often, can be made out of scented leathers, which would also
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mask the bad odour.


Attempts towards scent infusion in leather are in scarce. The scent oils applied to
leather as microcapsules employing hydrophobic core materials, polyisocyanates7,
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polyamines, poly urea8 contains mixture of scent oil, essential oils, fragrances as water
soluble9and lipid soluble substances10 etc., some are susceptible to degradation in the
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presence of surfactants causing rupturing and tend to have aggressive action on capsules. In
finishing stage, scent infusion in leather is applied as top coats and in case of shoes, patches
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were fixed, which contain deodorizer for sustained release11. Since scent oils are volatile in
nature, sustained release cannot be achieved when it is applied during the finishing stage of
leather. The volatility of the scent oils bottlenecks their application in the leather field
although it has potential application. Our research group recently achieved the infusion of
encapsulated lemon grass oil in leather12. In the present study, the scent oils such as lavender
and orange oil have been infused in the leather by encapsulating these scent oils using the
chitosan as a shell material. Biocompatibility, antimicrobial activity of chitosan as well as the
re-tanning ability in leather processing13, makes chitosan a suitable wall material to

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encapsulate scent oils. These encapsulated scent oils have been characterized by tensiometer,
DLS, TGA, FT-IR, optical microscopy, and SEM. Further, these scent oils infused leathers
have been studied for physical and organoleptic properties, morphology, washability test,
perception evaluation and porosity.

2. EXPERIMENTAL SECTION

The chemicals chitosan, Triton X-100 (Non-ionic surfactant), potassium persulphate and

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acrylic acid were procured from Sigma. Leather chemicals were purchased from Stahl, BASF,

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Colourtex and Clarient India. The scent oils (Orange and lavender oil) were purchased from

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Aromax Trading Corporation Chennai.

2.1 Optimization of the Scent Oil: Surfactant Ratio

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Optimization of the product has been carried out by varying the surfactant to oil ratio.
Throughout the process, the oil (1 mL) has been kept constant and the surfactant
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concentration has been altered from 0.2 to 1.0 mL. Transmittance opacity and viscosity for all
the ratios has been carried out.
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2.2 Encapsulation of Scent Oils


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The scent oils (orange and lavender oil) were emulsified using Triton X-100, under stirring
until a white solution was obtained. The emulsion stability was studied using transmittance
measurement. Chitosan was dispersed in acrylic acid in the ratio of 1:2, which was added to
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the emulsions under stirring condition. To this mixture, potassium per sulfate was added to
initiate the polymerization process and the total reaction was carried out at 50 ◦C for 8 hours.
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The intactness and effectiveness of encapsulated product has been maintained by


lyophilization of the product and maintained at 4 ◦C. All the experiments were carried out in
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triplicates and data were reported in terms of mean ±standard deviation.

2.3 Characterization of Encapsulates

2.3.1 Microencapsulation efficiency, oil content and oil loading efficiency


Lyophilized encapsulated scent oils were crushed and dissolved in 1% (v/v) Triton X-100.
The lyophilized samples were agitated and vortexed until the oil from the shell ooze out into

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the solution. The oil content and encapsulation efficiency was then calculated using the
equation below 12.
( )

( )

( )

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where W is the weight of the nanospheres; W1 is the weight of the scent oil (orange and

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lavender oil) encapsulated in a known amount of nanospheres; W2 is the total amount of scent
oil introduced in the same amount of nanospheres; W3 is the total amount of polymer used

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including the cross linker.

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2.3.2 Analysis of the particle size and morphology
The encapsulated scent oils were subjected to dynamic light scattering measurement studies
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in-order to determine the stability of the encapsulated product and hydrodynamic size. The
samples were dissolved in milliQ water and the measurement was performed at 25 ˚C using
Zetasizer Malvern instruments U.K. Optical microscopy study has been carried out for the
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lavender and orange oil encapsulated products and emulsions. The encapsulated products and
emulsions were smeared over the glass slide and kept for drying. The optical images of the
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products have been obtained with the aid of camera fitted to the microscope. The scanning
electron microscope study was carried out for the encapsulated products to study the surface
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morphology and size using Quanta series 200 SEM. The film formation of the encapsulated
product on the glass plates have been done under controlled drying and with the aid of
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adhesive, the glass plate was fixed on the stubs and coated with thin layer of gold by gold
sputtering. SEM images were captured for the encapsulated lavender and orange oils at 12
KV.
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2.3.3 Surface tension measurement


Surface tension was performed for the scent oils, emulsified scent oils and scent oil loaded
microspheres using GBX 3S tensiometer (France) with 0.1mN/m DU Nuoy probe. The
instrument was standardized using milliQ water prior to the experiment. The entire
experiment has been carried out at 25 ºC.

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2.3.4 Oxidation index measurement


A known volume of scent oil was vortexed with hexane. Oxidation index of the mixture was
determined spectrometrically at 234 nm and as a result of oxidation the concentration of
hydro peroxides formed, which was quantified with the aid of extinction coefficient of 2900
mol-1 cm-1.14

2.3.5 Fourier transform - infrared spectral analysis


The FT-IR spectral analysis for the chitosan, acrylic acid, blank microspheres and scent oil

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loaded microspheres was carried out using JASCO FT-IR instrument. The samples were

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pelletized using potassium bromide and scanned in the frequency range of 4000-600 cm-1.

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2.3.6 Thermo gravimetric analysis (TGA)
Thermo gravimetric analyses of the chitosan, blank microspheres and scent oil loaded

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microspheres were studied using thermo gravimetric analyzer TA-TGA-Q 50 TA instruments,
USA. A known amount of above said samples were weighed and heated from 25 to 800 ˚C
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with the heating rate of 10 ˚C/min under the constant nitrogen flow of 300 mL/min.
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2.4 Application of the Scent Oil Loaded Microspheres into the Leather
In order to evaluate the efficiency of these scent oil loaded nanospheres infusion in leather,
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wet blue sheep skins were used as a raw material. Wet blue sheep skins were shaved to a
thickness of 1 mm and neutralized in the drum using 100 % (w/v) water, 0.5 % (w/v) sodium
formate and 1 % (w/v) sodium bicarbonate (added in intervals in order to adjust the pH). pH
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of the wet blue was raised from 4 to 5.5-6.0 then neutralization water was drained. Further
the wet blue was washed with water prior to the next leather processing. To analysis the
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ability of scent infusion in leather, these neutralized wet blue leathers were cut through
backbone and made into two pieces, in which one part has been used for scent infusion and
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other one was kept as control. The difference between the control and scent infused leathers
was the addition of scent oil loaded nanospheres in the latter. Rest of the post tanning process
such as retaining, fatliquoring, dyeing and fixing and other mechanical operations were kept
unchanged in the manufacturing of both control and scent infused garment leather. The
detailed process recipes have been tabulated in Table 1.

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2.5 Characterization of Scent Infused Leather

2.5.1 Physical testing, organoleptic properties and perception evaluation


The control and scent oils infused leather have been subjected to physical testing using
INSTRON series IX as per the IUP protocol15-17. The softness was measured as per IUP
protocol18 using a MSA ST 300 digital leather softness tester supplied by MSA Engineering
systems. Organoleptic properties such as fullness, softness, grain tightness, smoothness and
color uniformity and perception evaluation were evaluated by experienced tanners (hand and

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visual examination). And ratings were given for each property on a scale of 0-10. In ratings,

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higher value indicates that the leather possesses transcendence property.

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2.5.2 Release characteristics of the scent infused leathers: Swatch test and wash-ability
test in aqueous and dry-cleaning solvent

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In order to understand the release of fragrance from leather matrix two methods were adopted
such as swatch test and washability test. For the swatch test, lavender and orange oil infused
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leathers were cut into small specimens with the dimensions of 10 cm × 10 cm. Further the
specimens were conditioned at 27 ± 2 ºC and 65 ± 2 % relative humidity (R.H.) over a period
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of 90 days. On each day of a predefined series of time intervals (24 h), samples were taken
and tested for release of fragrance with the positive and negative scale for the presence and
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absence of the fragrance, respectively. The assessment will be carried out by an experienced
tanner by smelling the specimens after scratching an ‟X‟ with the plastic stick19-20. For the
washability test, control and scent infused leather were weighed and soaked in the beaker
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containing 10 mL of water and perchloroethylene (dry cleaning solvent). The samples were
soaked for 5 minutes for each wash. The retentivity of the scent oils has been analyzed after
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drying by the expertise panel.


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2.5.3 Water diffusion coefficient measurement


Water diffusion coefficient for the experimental and control leathers were measured. The
leather samples of uniform length and thickness were taken and their dried weight been
measured accurately. Then the samples were immersed in the beaker containing water and
periodically the wet weights of the samples have been measured. During the wet weight
measurement, the surface water has been removed. The water uptake and diffusion coefficient
has been measured using the formula reported earlier12.

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2.5.4 Porosity measurements


The pore size and its distribution of the control and scent infused leathers were measured by
using PMI Capillary flow porometer. Leathers from different areas were cut into pieces with
20 mm diameter and its thickness was measured. For porosity measures, crust leathers in dry
condition were subjected to flow of (non-reacting gas) nitrogen gas then the same leathers
were subjected for wetting with Calwick liquid (surface tension=15.9 dynes.cm-1) and again
leathers were subjected to the flow of above mentioned gas. For all the leathers, changes in
the flow rate in dry and wet condition were measured as a function of pressure, and the pore

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size and its distribution was calculated by the following equation using software.

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4𝛾 𝑐𝑜𝑠𝜃
𝐷
𝑝

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where, D is diameter of pore; γ is surface tension of Calwick liquid; θ is contact angle of the
Calwick liquid; p is differential pressure.

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2.5.5 Antimicrobial activity of the encapsulated scent oils
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The antibacterial and antifungal efficacy of the scent oil loaded microspheres were
determined by testing against bacteria such as, Bacillus cereus and Bacillus subtilis and fungi
such as Rhizoctonia solani, Macrophomina pahaseolina and Aspergillus fumigatus,
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respectively. The bacterial culture was inoculated on the nutrient agar plate for antibacterial
analysis, whereas for antifungal analysis, fungus agar plug was placed on the potato dextrose
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agar. The wells were created using sterile cork borer. Different concentration of the scent oil
loaded nanospheres were loaded into the wells and plates, which were incubated at 37 ºC for
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24 h (bacteria) and 72 h (fungi), respectively. Zone of inhibition in mm was measured.


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3. RESULTS AND DISCUSSION


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3.1 Optimization of the scent oil: surfactant ratio


Triton X-100 has been selected as an emulsifier, since it is not afflicted by salinity of water as
well as doesn't change the oil property. It is most used and suitable surfactant to form oil in
water emulsion in industry21. In order to form stable emulsion, non-ionic surfactant (Triton
X-100) concentration has been varied from 0.2 to 1, because concentration can affect the
emulsion stability, droplet size and distribution and viscosity of the emulsion. The opacity,
density, relative viscosity, and transmittance of the emulsion with varied concentration of
surfactant has been collected and collated in Table 2. Opacity is a measure of the cloudy

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appearance rises by the formation emulsion by the scent oil and Triton X-100, due to the
scattering effect of the larger fraction of light by the dispersed oil droplets. Opacity value
decreases with an increase in surfactant concentration, which indicates that smaller size of oil
droplets are formed at the higher surfactant ratio. From the table it can be observed that,
increase of Triton X-100 increases the transmittance, density and viscosity of the liquid
phase. This may be due to the enhancement in the distribution of scent oil droplets in water
phase with smaller droplet size as well as this avoids the scent oil droplets from coalescence.
Similar observation has been reported by several researchers on the addition of emulsifiers in

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preparing oil/water emulsions22-24.

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3.2 Encapsulation of scent oils and characterization of encapsulates
Based on the opacity, viscosity, density and transmittance results, scent oil: surfactant ratio
has been optimized as 1:1 and the same has been used for the preparation of both the scent oil

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encapsulates. Encapsulation of these scent oils such as orange and lavender has been carried
out using chitosan and acrylic acid as wall material by emulsion polymerization technique,
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which has been established by our research group for lemongrass oil encapsulation12. Several
factors, which affects the encapsulation effciency of essential oils such as wall and core
materials, emulsifier characteristics24. The encapsulation efficiency (%), oil content (%) and
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oil load (%) values of the scent oil loaded nanospheres are shown in Table 3. Slight variation
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has been observed for orange and lavender oil nanospheres on oil loading (%), oil content
(%), encapsulation efficiency (%). Encapsulation efficiency (%) and oil content (%) has been
decreased in the formation of orange oil loaded nanospheres than the lavender oil loaded
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nanospheres. On the contrary, orange oil loaded nanospheres possess high oil load (%) than
the lavender oil loaded nanospheres. This may be due to the higher loss/volatility of orange
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oil than lavender oil during the encapsulation process 19, 25.
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3.2.1 Particle Size and Morphological Analysis


DLS results revealed that, orange and lavender oil encapsulated nanospheres exhibited a
bimodal and trimodal size distribution with the Z-average of 213.6 and 273.8 nm with the
polydispersity of 0.69 and 1, respectively (Table 3 and Figure 1). Figure 1a reveals a
bimodal size distribution of orange oil loaded nanospheres in which the first peak has been at
14.1 nm diameter, representing 6.8% of the nanospheres and second peak at 327 nm diameter,
corresponding to 93.2% of the nanospheres. In the case of lavender oil loaded nano and
nanospheres, trimodal distribution has been observed (Figure 1b), in which first, second and

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third peak has been at 45.2, 163.5 and 1080.6 nm diameter, representing 7.2, 16 and 76.8% of
nano and nanospheres, respectively. Collagen is a leather making protein and it is also the
dominating material in the skin, which approximately holds one third of the skin. Unique
properties of leather such as, breathability and viscoelasticity arises due to hierarchical
structure of collagen at the nano and microscale. Breathability property of leather is due to
presence of different types of pores, pore size distribution in different parts, and pore
diameters along with pore path as well as it is a determining factor which will influence the
leather manufacturing through controlling the penetration of leather chemicals. Several

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researchers have investigated the changes in pore size and its distribution of skin matrix

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during leather processing using various techniques such as, thermoporometry, nitrogen

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adsorption, capillary flow porometry26-31. It has been reported that, chrome tanned wet blue
sheep skin possess average pore radii in the range of 3-20 nm with high pore volume (by
thermoporometry analysis)32, whereas BET nitrogen adsorption method reveals that (small

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through-pores and blind pores), surface area (6.73 m2/g), pore volume (0.0612 cm3/g) with an
average pore diameter (385.8 nm)33. Capillary flow porometry explains about the throat pore
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diameter, which is the main factor deciding about the diffusion of chemicals based on its size
and it is reported that, the average largest throat pore diameter is 1313 ±170 nm with the
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range of 1126-1566 nm, average mean flow throat pore diameter is 426 ± 125 nm with the
range of 269- 617 nm and the average smallest throat pore diameter 161±59 nm with the
range of 108-263 nm33. Based on the above observation on pore size and its distribution, the
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prepared orange oil and lavender oil encapsulated nanospheres (Z-average) have a compatible
size for the well diffusion in the range of average mean flow throat pore present in the skin
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matrix. Recently reported encapsulated lemongrassoil has ability to penetrate up to average


smallest throat pore due to its Z-average particle diameter of 117 nm, since it is
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comparatively smaller than the encapsulated orange and lavender oil12.


Surface morphology of the encapsulated scent oils has been examined using optical
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and scanning electron microscopy, which has been shown in Figure 2 and Figure 3,
respectively. It has been observed that, both the scent oil droplets dispersed in uniform
manner and showed a distinct spherical shape surrounded by the wall material, which can be
spotted by the contrast nature of oil and wall material. On the other hand, scanning electron
micrographs of encapsulated lavender oil (Figure. 3a) showed surface imperfections with
different shapes and size with crowding of nanospheres. This may corroborate with the
trimodal distribution of DLS measurements. This type of large size nanospheres with surface
imperfections may favour high shielding effect against oil evaporation and oxidation34.

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Micrographs of the encapsulated orange oil (Figure. 3b) showed a spherical shape of
encapsulates, but at a lower magnification (500x) agglomerated spheres with distinct
imperfections is observed whereas at higher magnification (1000x) separate spheres in close
proximity between two spheres is seen.

3.2.2 Surface Tension and Interfacial Energy


Surface tension of the encapsulated scent oils, emulsions and oils were measured and are
collated in Table 3. The value of the surface tension mainly depends on the nature of the

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scent oil and surfactant used to lower the surface tension values. Form the results it is evident

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that, the surface tension of the encapsulated product and emulsion is lower than that of the

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scent oil. In order to reduce the surface tension between two immiscible liquids such as scent
oil and water, the emulsion has been formed using non-ionic surfactant namely, Triton X-100.
An emulsion is formed by reducing the interfacial energy between the scent oils and water

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thereby assisting in the emulsion formation as well as reducing the ability of coalescence of
water or scent oil by the formation of smaller drops of the dispersed phase within the
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continuous phase. Hence, from the results it is determined that, the oil has been encapsulated
as the surface tension of the product is reduced when compared to surface tension of the oil.
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Lavender oil and orange oil has interfacial energy of 2.58, and 6.16 mN/m, respectively. In
this study, the interfacial energy determines the nature of interaction between the oil and the
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wall material. Lavender oil has low interfacial energy indicates the presence of higher
nonpolar groups, which may increase the hydrophobic interaction with chitosan.
Comparatively, orange oil has high interfacial energy indicates that the presence of higher
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polar groups than the lavender oil, which may enhance the interaction with the wall material
electrostatically in addition with hydrophobic interaction. The stability of the encapsulated
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product relies on the dynamic interaction with the wall material12.


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3.2.3 Oxidation index measurements


Oxidation index measurements were carried out to quantify the oxidation behaviour of scent
oil and encapsulated products by the formation of hydro peroxides. This helps to study the
efficacy of wall material to engulfs the scent oils in order to prevent the oxidation process
such that quantum amount of the oil will be retained. During the formation of hydro
peroxides from oils, the formation of conjugated dienes occurs due to shift/rearrangement in
their double bond positions such as isomerization and conjugation14. The concentration of the
lavender and orange oil is susceptible for the hydro peroxide or diene formation, which have

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been observed to be 10.57 and 9.8 μM/L and in products it has been found to be 2.68 and
2.33 μM/L, respectively (Table 3). The reduction in the formation of hydro peroxides or
dienes in the encapsulated product has been observed, which may be due to the protective
effect of wall material against the oxidation process of scent oils.

3.2.4 FT-IR Studies


The FT-IR spectra for the optimized ratio of the formulated product, empty nanospheres,
chitosan, lavender oil and orange oil have been studied and are shown in Figure 4. The

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chitosan FT-IR spectrum corroborates the presence of amide I and amide III bands at 1662

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and 1371 cm-1, respectively. Other functional groups present in the chitosan have been

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observed, it is majorly by the presence of carbohydrates peaks35. The blank nanosphere
(chitosan-acrylic acid) FT-IR spectra reveals that interaction between the carboxylate group
of acrylic acid and the positive charged amino group in chitosan resulted in amidation, as the

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characteristic band has been observed at 3522 and 3442 cm-1 due to N-H stretching.
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The FT-IR spectrum of the lavender oil reveals that, the characteristic absorption peak such as
1729 cm-1, which represents the presence of linalool, linalyl acetate. These are the two major
constituents present in the lavender oil. Two distinct peaks at 2950 and 1463 cm-1 have been
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indicates the presence of alkane group to the C-H stretching and bending vibrations,
respectively36. The chemical composition of orange oil consists of pinene, sabinae, myrcene,
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limonene, linalool, and citronellal37. FT-IR spectrum of orange oil showed major peaks at
2948, 1462, 1356, 1247, 1185, and 945 cm-1, which corroborates the presence of functional
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groups such as alkane group due to the methyl C-H asymmetrical stretch as the peak
observed at 2948 cm-1. Aromatic ring stretch has been observed (C=C) around 1461 and 945
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cm-1 (C-H out of plane bend). The spectra of lavender and orange oil loaded nanospheres
reveal that, there is no loss of the major constituents present in these oils by the encapsulation
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process. FT-IR spectra of the orange oil encapsulated product reveals that, shift in peak from
1662 to 1608 cm-1 has been observed due to NH bending vibrations. This may be due to the
interaction between the NH group of chitosan and the scent oils. From this it is evident that,
scent oils are encompassed by the wall material through physical interaction such as van der
Walls or electrostatic interaction and no chemical interactions are involved as there no
deviations in the chemical constituents of the scent oils is seen25.

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3.2.5 TGA Analysis


Thermogravimetric analysis for the blank nanospheres and the scent oil loaded nanospheres
was carried out and is shown in Figure 5. The three stage decomposition model has been
observed in the case of blank nanocapsules. Initial degradation in the range of 78-250 ºC
indicates the loss of moisture, formed as a result of amidation. The amidation between the
acrylic acid and chitosan has been confirmed in the FT-IR analysis. The degradation of
acrylic acid and chitosan has been observed at 250- 380 ºC and major degradation of chitosan
observed at 512 ºC. Degradation profile with different temperature ranges of blank

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nanospheres elucidates the complex mechanism. Mechanism unravels that dehydration,

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decarboxylation of carboxylic acid group of acrylic acid and chain scission in the polymer

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chitosan aids in the degradation of the wall material38. Thermograms of lavender oil and
orange oil loaded nanospheres reveal that the loss of moisture (initial degradation) is at 248
ºC. Loss of residual oil and its constituents has been observed at 457 ºC. The degradation of

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core material and the complete evaporation of the oil has been recorded above 550 ºC. Hence,
the thermogravimetic analysis reveals that the wall material plays a predominant role in
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retaining the oil at high temperatures (above 100 ºC) and it can be applicable in thermal
wears.
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3.3 Application of Scent Oil Loaded Nanospheres into Leather


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Recently, our research group has optimized the percentage offer and stage of incorporation of
lemongrass oil loaded nanospheres into leather for the better infusion and exhaustion12. The
process step for addition was optimized as after neutralization process in fresh water, which
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means before the retanning, dyeing and fatliquoring wetblue as well as percentage offer was
optimized as 5% oil content offer on shaved weight12. These optimized results lead to better
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exhaustion of scent oil loaded nanospheres, this may be due to lot of available pores in the
wetblue for diffusion. Hence, 5% of the lavender and orange loaded nanospheres have been
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incorporated into leather after neutralization process in the fresh water. The efficacy of the
scent infusion in leather has been analysed.

3.3.1 Perception evaluation, physical strength and organoleptic properties


Perception evaluation of the scent infused leather has been carried out. Experienced tanners
evaluated the experimental leathers and the intensity of the scent is measured as degree of
intensity on the scale of 0-5, which has been tabulated in the Table S1. Lavender oil has very
intense perception value compared with orange oil infused leather. Both the scent oils are

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showing good perception of fragrance, which may be due to well diffusion these scent oils
into the pore present in the leather matrix.

The scent oil infused leather and control leather were analysed for the strength properties.
The strength characteristics of the leathers are tabulated in the Table 4. The strength
properties of the scent infused leathers are comparable to that of control leather. The
organoleptic properties of the experimental leathers were evaluated by the experienced
tanners. Organoleptic properties such as softness, fullness, grain smoothness were found to be

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better in orange oil infused leather than the lavender oil infused leather. The colour

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uniformity was found to be unchanged for both the leather. Grain tightness of the lavender oil

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infused leather has been found to be higher than the orange and control leather.

3.3.2 Release characteristics of the scent infused leathers: Swatch test and wash-ability
test with water and dry cleaning solvent
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Release of fragrance from the leather has been evaluated by the tanner through the aroma
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release using swatch test, and the procedure has been adopted from the cotton fabrics19-20. For
the first 60 days, the aroma has been smelt from the lavender and orange oil infused leather
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by hanging the leather specimens in the open air without any scratch on the leather. After 60
days, for a longer period, fragrance has been smelt upon scratching „X‟ on the leather matrix.
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Over all, lavender oil infused leather releases aroma for longer period than the orange oil
infused leather. Release kinetics of fragrance oil from the leather matrix in the solvent
medium has been not addressed, because no need of washing is required for leather.
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The experimental leathers were subjected to wash ability test with water and dry cleaning
solvent. The perception of fragrance after the washing was evaluated. And the results are
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tabulated in Table 5. The lavender oil infused leather perceived fragrance after three washing;
in contrast the intensity perceived by the orange oil infused leather was low. Solvent washed
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scent infused leathers shows that, the lavender oil infused leather perceived high intensity
after the first wash and intensity decreased in subsequent washing. Orange oil infused leather
perceived high intensity in first washing and less intensity was perceived in second and third
washing. From the wash-ability studies, intensity of the lavender oil perceived while washed
with water, in case of solvent wash orange oil treated leather perceived the fragrance.

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3.3.3 Water diffusion coefficient and porosity


In the porous medium the effective diffusion coefficient differs from the real diffusion
coefficient. Since, leather is porous matrix, let us assume that, if the space of the diffusion
decreases, the pores are blocked by the encapsulated products. The water uptake and
diffusion coefficient of the lavender and orange oil encapsulated product are presented in
Table S1. The pore size distributions of the scent infused leathers are uniform in nature,
which has been determined by the porosity measurement. The absolute D value of the
lavender oil determined is found to be higher than the orange oil encapsulated product. This

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results in the blockage of pores by the lavender encapsulated product as compared to orange

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oil encapsulated product. It is evident from the particle size studies that, the size of the

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formulated lavender encapsulated product is higher value compared to the orange oil, which
resulted in lower diffusion coefficient value of lavender encapsulated leather than the orange
and control leathers. The digital image of lavender and orange oil infused leathers has been
shown in Figure 6.
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3.3.4 Antimicrobial activity
Encapsulated scent oil possesses the antimicrobial activity as they inhibited the bacteria and
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fungi, which has been tabulated in Table 6. Chitosan exhibits excellent antimicrobial
activity39. The mechanism behind the antimicrobial activity is quite tricky as the chitosan and
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the encapsulated oil possess the antimicrobial activity. Chitosan mechanism of inhibition is
quite notable as the charge of the chitosan is cationic which is due to presence of the NH3+
which interacts with anionic charged bacterial cell wall and results in the disruption of the
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cell wall leading to the release of the cellular materials13. In the case of fungi, the charge
interaction between the chitosan and cell wall of fungi establishes a thick layer around the
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cell wall. As a consequence, the ion channel gets blocked, which ultimately leads to lysis of
fungi40. The acidic environment in which the chitosan is dissolved exhibits greater
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antimicrobial activity. The essential oils used in this study also possess antimicrobial efficacy.
The essential oils majorly consist of terpenoids, phenylpropanoids, benzenoids and amino
acid derivatives41. Hence, the synergistic effect of chitosan and essential oils augments the
antimicrobial efficacy of the formulated product. Since the leather is generally prone for the
fungal attack, minimization of the microbial attack on the leather can be achieved by
incorporating the encapsulated product in the leather.

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4. CONCLUSIONS

Scent oils like orange and lavender oil has been emulsified using different concentration by
varying the ratio of oil: surfactant. Based on the studies 1:1 ratio has been used for the
preparation of encapsulation. The oil load and oil content for the encapsulated scent oils has
been analysed, which reveals that lavender oil takes an edge over the orange oil in
encapsulation efficiency and oil content. Particle size for the encapsulated oils were
analysed, which reveals that orange oil encapsulates (213.6 nm) shows bi-modal

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distribution and lavender oil encapsulates (273.8 nm) shows tri-modal distribution.

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Morphological analysis of lavender and orange oil encapsulates shows the uniform
distribution of scent oils and the encapsulation has been ensured by contrast nature of oil and

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wall material as spherical oil droplet is surrounded by wall material. FTIR analysis of
lavender and orange oil encapsulated nanospheres infers that physical interaction such as

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van der Waals or electrostatic interaction has been observed and chemical constituents of
scent oils has been unaffected during the encapsulation process. Thermogravimetric analysis
of the orange and lavender oil encapsulated nanospheres reveal that complete degradation of
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encapsulates occurs above 550 ºC and hence they can be used in thermal wears. Application
of the encapsulated scent oils has been carried out and incorporated into the leather after
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neutralization process. 5% of the scent oil encapsulates (product) has been offered and
results revealed that better exhaustion of the product as diverse pores are available in the wet
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blue for better diffusion. The scent oil incorporated leather has been tested for its physical,
organoleptic, wash-ability and water diffusion coefficient, which infers that slight variations
were found between orange and lavender oil incorporated leathers. An antimicrobial study
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has been carried out against B. cereus, B. subtilis, Aspergillus fumigatus, Macrophomina
phaseolina. Lavender oil encapsulated nanosphere possess antimicrobial activity against
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bacteria and fungi whereas orange oil has less efficacy when compared to lavender oil. From
the above characterization studies it is evident that, slight variations are found between
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orange and lavender oil but the efficacy of both the oils when applied in leather is similar in
performance though some minor changes were observed in organoleptic properties. Even
though the development of apt method for the reinfusion of scent oil on the leather will be a
futuristic approach in this study. Essential oil encapsulates in cotton fabrics serves the
mosquito repellent property20,42-43 and this similar behaviour is also expected to perform by
the scent infused leathers.

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ACKNOWLEDGEMENTS
The authors thank CSIR for funding under the Supra institutional Project–S&T Revolution in
Leather with a Green Touch (CSC0201). Punitha Velmurugan and Vasudevon Ganeshan
thank the DST-INSPIRE (IFA13-ENG-54) for the Faculty Award. Authors thank Dr. Geetha
Baskar and Dr. Aruna Dhathathreyan for the valuable inputs.

Notes. The authors declare no competing financial interest.

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REFERENCES

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1. Sangeetha, S.; Nishad Fathima, N.; Sreeram, K.J.; Raghava Rao, J.; Nair, B.U.

CR
Stimuli responsive leathers using smart retanning agents. J. Am. Leather. Chem.
Assoc. 2012, 107(6):196-204.
2. Wegene, J.M.; Thanikaivelan, P. Conducting Leathers for Smart Product Applications.

3.
US
Ind. Eng. Chem. Res. 2014, 53, 18209−18215.
Motomura, N.; Sakuraai, A.; Yotsuya, Y. A Psychophysiological Study of Lavender
AN
Odorant Memoirs of Osaka Kyoiku University, Ser, DI. 1999, 47, 2, 281-287.
4. Martins, I.M.; Barreiro, M.F.; Coelho, M.; Rodrigues, A.E. Microencapsulation of
M

essential oils with biodegradable polymeric carriers for cosmetic applications. Chem.
Engg.J. 2014, 245, 191–200.
ED

5. Tsaur, L. S.; Lin, S. Fragrance microcapsules for fabric conditioning, US patent


US5246603 A, 1993.
6. Popplewell, L. M., K. D. Lee, et al. Encapsulated fragrance chemicals, US patent
PT

US7196049 B2, 2007.


7. Markus, A. and C. Linder. Encapsulated essential oils, US patent US20060165746 A1
CE

2006.
8. Jacquemond M.; Jeckelmann, N.; Ouali, L.; Haefliger, O.P. perfume-containing
AC

polyurea microcapsules with undetectable levels of free isocyanates. J. Appl. Polym.


Sci. 2009, 114(5):3074 – 3080.
9. Braunlich, F. Water-soluble essential oils and perfumes. US patent US1580952
A1926.
10. Bara, I. Fragrancing composition of Pickering emulsion type, US patent
US20140329735 A, 2013.
11. Laughlin, T. J.; Dever, G. R. Foot and shoe deodorizer, US patent US5732485 A,
1994.

18
ACCEPTED MANUSCRIPT

12. Punitha, V.; Fathima, N.N.; Geetha, B.; Aruna, D.; Rao, J. R. Development of smart
leathers: incorporating scent through infusion of encapsulated lemon grass oil. RSC
Adv. 2015, 5, 59903-59911.
13. Xiao Fei Liu.; Yun Lin Guan.; Dong Zhi Yang.; Zhi Li.; Kang De Yao. Antibacterial
Action of Chitosan and Carboxy methylated. J. Appl. Polym. Sci.2001,79,1324–1335.
14. Shahidi, F.; Wanasundara, U. N.; Akoh, C. C.; Min, D. B.; eds., Food Lipids:
Chemistry, Nutrition and Biotechnology, Marcel Dekker, Inc: New York, 2002, 465–
487.

T
15. IUP 2, Sampling, J. Soc. Leather Tech. Ch.2000, 84, 303.

IP
16. IUP 6, Measurement of tensile strength and percentage elongation, J. Soc. Leather

CR
Tech. Ch.2000, 84, 317-321.
17. IUP 8, Measurement of tear load double edge tear, J. Soc. Leather Tech. Ch. 2000, 84,
327-39.
18.
US
IUP 36, Measurement of leather softness, J. Soc. Leather Tech. Ch. 2000, 84, 347-
349.
AN
19. Li, S., Boyter, H., Qian, L. UV curing for encapsulated aroma finish on cotton. J. Text.
Inst. 2005, 96, 407–11.
M

20. Specos, M. M. M., Garcíac, J. J., Tornesello, J., Marino, P., Della Vecchia, M., Defain
Tesoriero, M. V., Hermid, L. G. Microencapsulated citronella oil for mosquito
ED

repellent finishing of cotton textiles. Transactions of the Royal Society of Tropical


Medicine and Hygiene, 2010, 104, 653–658.
21. Liyana Nadirah, M.S; Abdurahman H.N; and Rizauddin, D. Rheological studies of
PT

petroleum fluid and oil in water emulsions. International journal of engineering


sciences and research technology 2014, 3, 129-134.
CE

22. Weirong, J. I.; Lempe, D. A. Calculation of viscosities of liquid mixtures using


Eyringstheoryin combination with cubic equation of state. Chinese. J. Chem. Eng.
AC

2006, 14(6), 770-779.


23. Ashrafizadeh, S.N.; Kamran, M. Emulsification of heavy crude oil in water for
pipeline transportation. J. Pet. Sci. Eng. 2010, 71(3–4), 205-211.
24. Zuobing Xiao.; Ting Tian.; Jing Hu.; Mingxi Wang.; Rujun Zhou. Preparation and
characterization of chitosan nanoparticles as the delivery system for tuberose.
fragrance Flavour Fragr. J. 2014, 29, 22–34.
25. Zettlemoyer, A. C.; Schweitzer, E. D.; Walker, W. C. Internal surface area of hide. J.
Am. Leather. Chem. Assoc. 1946, 41, 253.

19
ACCEPTED MANUSCRIPT

26. Li, Y., Ai, L., Yokoyama, W., Shoemaker, C. F., Wei, D., Ma, J., Zhong, F. Properties
of Chitosan-Microencapsulated Orange Oil Prepared by Spray-Drying and Its
Stability to Detergents. J. Agric. Food Chem. 2013, 61, 3311−3319.
27. Kanagy, J. R. Macro-pores in leather as determined with a mercury porosimeter J.
Am. Leather Chem. Assoc. 1963, 58, 524.
28. Fathima, N. N.; Dhathathreyan, A.; Ramasami, T. Mercury intrusion porosimetry
Nitrogen Adsorption and scanning Electron Microscopy analysis of pores in skin
Biomacromolecules.2002, 3, 899.

T
29. Fathima, N. N.; Dhathathreyan, A.; Ramasami, T. Influence of crosslinking agents in

IP
the pore structure of skin, Colloid Surf. B.2007, 57, 118.

CR
30. Fathima, N. N.; Pradeep Kumar, M.; Rao, J. R.; Nair, B. U. A DSC investigation on
the changes in pore structure of skin during leather processing. Thermo chim. Acta
2010, 501, 98.
31.
US
Gil, R. R.; Ruiz, B.; Lozano, M. S.; Fuente, E. The role of crosslinking treatment on
the pore structure and water transmission of biocollagenic materials. J. Appl. Polym.
AN
Sci. 2013, 130, 1812.
32. Dagnew, N.; Punitha, V.; Sreeram, K. J.; Rao, J. R.; Nair, B. U.; An Assessment of
M

Differences between Butt and Belly Regions of Indian Sheep Skin. J. Am. Leather.
Chem. Assoc. 2015, 110, 165-176.
ED

33. Murali S., Rao, J.R., Fathima, N.N. Analysis of variations porosity of metal
crosslinked collagen matrix. J. Appl. Polym. Sci. 2014, 131, 40835.
34. Chang, Y. I.; Scire, J.; Jacobs, B. Effect of Particle Size and Microstructure
PT

Properties on Encapsulated Orange Oil. ACS Symposium Series 10, 2009, 370, 87–
102.
CE

35. Shanmugapriya, A.; Srividhya. A.; Ramya, R.; Sudha, P.N. Graft copolymerization of
Chitosan with acrylic acid used in waste water treatment. International Journal of
AC

Environmental Sciences. 2011, 1, 7.


36. John, A. Manthey. Fourier Transform Infrared Spectroscopic Analysis of the
Polymethoxylated Flavone Content of Orange Oil Residues. J. Agric. Food Chem.
2006, 54, 3215-3218.
37. John, C. Interpretation of Infrared Spectra, a Practical Approach In Encyclopedia of
Analytical Chemistry 2000, 10815–10837.
38. Trong-Ming Don,; Chia-Fong King,; Wen-Yen Chiu. Synthesis and properties of
chitosan modified poly (vinyl acetate). J. Appl. Polym. Sci.2002, 86, 3057-3063.

20
ACCEPTED MANUSCRIPT

39. Sudarshan, N. R.; Hoover, D. G.; Knorr, D. Antibacterial action of chitosan. Food
Biotechnol, 1992, 6, 257.
40. Mohamed, E.; Badawy, I. Structure Antimicrobial Activity Relationship of Quaternary
N-Alkyl Chitosan Derivatives against Some Plant Pathogens. Journal of applied
polymer science. 2010, 117,960–969.
41. Dudareva, N.; Negre, F.; Nagegowda, D. A.; Orlova, I. Plant volatiles: Recent
advances and future perspectives. Crit Rev Plant Sci. 2006, 25, 417–40.
42. Anitha, R., Ramachandran, T., Rajendran R., Mahalakshmi, M. Microencapsulation of

T
lemon grass oil for mosquito repellent finishes in polyester textiles. Elixir Bio. Phys.

IP
2011, 40, 5196-5200.

CR
43. Yuvasri. V., Anchana devi, C., Leela, K. A comparative study on microencapsulated
essential oils for mosquito repellent finished cotton fabrics, Journal of Medical
Pharmaceutical and Allied Sciences, 2016, 161-176.

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Table 1. Post Tanning Process recipe for lavender and orange oil infused leathers
Process/chemicals % of offer Time in minutes Remarks
Washing
Water 100 20 Drained
Neutralization
Water 150
Sodium formate 1.5 20
Sodium bicarbonate 1.0 3X5+60
Checked for pH 5.5-6.0 and drained
Washing (twice)
Water 200 15 Drained
Addition of microencapsulated oils (such as lavender and orange oil encapsulated nanopheres)

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5% of oil content + 10% water

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Retanning, dyeing, fatliquoring
Water 100
Verminol liquor ASN 2 10

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Tanicor PW 6
Basyntan FB6 3 60
Wattle-GS powder 1
Dye (Violet 5R 0.5% for 2 60 Check penetration
Lavender oil infused
leather and Orange dye
2% for orange oil infused
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leather)
Lipodermliquor FB2 2
Lipodermliquor SA 1
Fixing
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Formic acid 2 3X5+40 Check exhaustion and


drained
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Table 2. Opacity, viscosity, and transmittance measurements of different ratios of


oil:surfactant
Oil: Orange Lavender
T-X- Κ Ρ ηrel T Κ Ρ ηrel T
100 (a.u.) (g/mL) (%) (a.u.) (g/mL) (%)
1:0.2 0.30 0.60 - 67.81 0.29 0.72 - 86.12
1:0.4 0.16 0.62 - 96.1 0.08 0.73 - 87.39
1:0.6 0.11 0.67 - 96.16 0.05 0.75 1.69 87.8

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1:0.8 0.01 0.70 1.80 96.62 0.01 0.75 1.86 89.15

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1:1.0 0.004 0.72 2.15 97.43 0.002 0.889 3.02 92.1

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Table 3. Characterization of the encapsulated product and oil


Parameter Lavender Orange
Oil Product Oil Product
Oil load (%) - 196.19±1.76 - 183.27±2.48
Oil content (%) - 9.80±2.19 - 9.01±1.84
Encapsulation efficiency (%) - 34.34±0.98 - 40.23±1.18
Surface tension (mN/m) 29.2±0.75 28±0.98 30.6 ±0.34 28.3±0.35
(29± 51 (28.6±0.44

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Emulsion) Emulsion)

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Particle size (nm) - 273.8±10.2 - 213.6±9.10

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Oxidation index (μM/L) 10.57 2.68 9.8 2.33
Density (g/cm3) 0.953 1.448 0.86 0.835
Interfacial energy (mN/m) 2.58 - 6.16 -

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Table 4. Strength and organoleptic characteristics of scent infused leather


Characteristic study Control Lavender Orange
Tensile strength (kg.cm-2) 264.72± 2.5 222.50±3 207.10±5
Tear strength (Kg.mm-1) 121.40 ±3 93.29±3 97.71 3±3
Softness 9 7 8
Fullness 7 7.5 8.5
Grain smoothness 9 7.5 8
Colour uniformity 8 7.5 7.5

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Grain tightness 8 9 8

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Table 5. Characterization of scent infused leather with respect to wash


Sample Water Solvent
Washing 1 Washing 2 Washing 3 Washing 1 Washing 2 Washing 3
Control Nil Nil Nil Nil Nil Nil
Lavender Perceived Perceived Perceived High Perceived Less intensity
intensity Perceived
Perceived
Orange Less intensity Less Less High Less Less intensity

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Perceived intensity intensity intensity intensity Perceived

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Perceived Perceived Perceived Perceived

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Table 6. Antimicrobial properties of scent infused leathers


Zone of inhibition (mm)
B.cereus B.subtilis Aspergillus fumigatus Macrophomina
phaseolina
Conc. 25 50 75 100 25 50 75 100 25 50 75 100 25 50 75 100
(mg/mL)
Orange 4.5 5 10 12.5 9 11 13 16 2 4.5 6.8 9.2 - - - -

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Lavender 5 6.5 8 9.5 13 14 12 17.5 3.8 5.3 7.2 8.9 2.5 3.8 4.6 6.2

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Figure Captions
Figure 1. Particle size profile of encapsulated (a) orange oil and (b) lavender oil

Figure 2. Optical microscopy images of (a) orange (b) lavender oil encapsulated
products
Figure 3. Scanning electron micrographs of (a) Lavender and (b) Orange oil
encapsulated leather surface at different magnifications
Figure 4. FT-IR spectra of the chitosan, acrylic acid, blank nanosphere, lavender oil,

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orange oil and their encapsulated products, respectively.

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Figure 5. TGA thermograms of lavender and orange oil encapsulated product.
Figure 6. Digital images of scent oil infused leathers. (a) Orange oil (b) lavender oil

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infused leathers.

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Figure 1.

(a) (b)

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Figure 2.

(a) (b)

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Figure 3.
(a)

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(b)

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Figure 4.

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Figure 5.

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Figure 6.

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(a) (b)

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Graphical abstract

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