Chemical Mapping of Essential Oils, Flavonoids

and Carotenoids in Citrus Peels by Raman

Microscopy
Ying Yang, Xiaohe Wang, Chengying Zhao , Guifang Tian, Hua Zhang, Hang Xiao, Lili He, and Jinkai Zheng

Abstract: Citrus peels, by-products in large quantity, are rich in various functional and beneficial components which have
wide applications. Chemical analysis of these components in citrus peels is an important step to determine the usefulness
of the by-products for further applications. In this study, we explored Raman microscopy for rapid, nondestructive, and in
situ chemical mapping of multiple main functional components from citrus peels. The relative amount and distribution in
different locations (flavedo, albedo, and longitudinal section) of 3 main functional components (essential oils, carotenoids,
and flavonoids) in citrus peels were systematically investigated. The distribution profiles of these components were
heterogeneous on the peels and varied between different species of citrus peels. Essential oil was found mainly existed
Food Chemistry

in the oil glands, while carotenoids were in the complementary location. Some flavonoids were observed in the oil
glands. This study showed the capability of Raman microscopy for rapid and nondestructive analysis of multiple bio-
components without extraction from plants. The information obtained from this study would assist the better production
and application of the functional and beneficial components from citrus by products in an effective and sustainable manner.

Keywords: chemical mapping, citrus peels, functional components, Raman microscopy

Practical Application: This study indicated the capability of Raman microscopy for rapid and nondestructive analysis of
multiple bioactive components in plant tissues. The information obtained from the study would be valuable for developing
effective and sustainable strategy of utilization of citrus peels for further applications.

Introduction
Citrus is one of the most cultivated and popular fruits around
the world. In recent years, the annual production of citrus is ap-
proximately up to 102 million tons, widely grown in tropical area,
subtropical area, and many other areas (Mehl and others 2014).
Thirty percent of them are used to produce citrus juice, with
large amount of peels as by-products which are waste and cause
environmental problems. However, the citrus peels are especially
rich in a variety of functional and beneficial components. In vari-
ous citrus peels, including the common species like Citrus sinensis,
C. reticulate, C. clementina Hort ex Tan., and C. bergamia, the main
functional components are essential oils, flavonoids, carotenes and
so on (Ranganna and others 1983; Ruberto 2002; Chen and oth-
ers 2011). They have been widely applied in many fields. Extensive
researches have shown that the main component of essential oil in
different citrus is limonene, including (Ruberto 2002; Geraci and
others 2016). Limonene, an important functional component, ex-
hibits multiple biological activities, such as antimicrobial (Geraci
and others 2016), antioxidant (Phi and others 2015), larvicidal
(Campolo and others 2016), and antiacetylcholinesterase (Aazza
and others 2016). They are widely applied in pharmacology (Hosni
JFDS-2017-1059 Submitted 7/2/2017, Accepted 9/16/2017. Authors Yang,
Zhao, Tian, and Zheng are with Inst. of Food Science and Technology, Chinese Acad.
of Agricultural Sciences, Beijing 100193, P. R. China. Author Wang is with Inst.
of Food and Processing, Liaoning Acad. of Agricultural Sciences, Shenyang 110161,
China. Authors Zhang, Xiao, He, and Zheng are with Dept. of Food Science, Univ.
of Massachusetts, Amherst, Mass. 01003, U.S.A. Direct inquiries to authors He and
Zheng (E-mail: lilihe@foodsci.umass.edu, zhengjinkai@caas.cn).
Authors Yang and Wang contributed equally to this work.
and others 2010), food (Geraci and others 2016), and aromather-
apy (Hoenen and others 2016). There are also various flavonoids
in high levels in citrus peels and they have gained great interest
due to their excellent functions and bioactivities, including an-
ticancer (Zheng and others 2013), anti-lipogenic (Nakajima and
others 2016), antioxidant activity (Ademosun and Oboh 2014),
and anti-inflammation (Inafuku-Teramoto and others 2011). An-
other important component in citrus peel is carotenes, mainly
beta-carotene, often used as nutraceutical to inhibit various dis-
eases, such as cataracts, cardiovascular diseases, multiple sclerosis
and cancer, due to the excellent single oxygen and free radi-
cal scavenging property (Thakur and Azmi 2013). Therefore, the
chemical analysis of these bioactive components in citrus peels is
important for further application of the by-products.
Several methods have been used for components analysis of
citrus peels, including HPLC-UV, LC-MS, GC-MS, and UPLC-
PDA-ESI/MS (Inafuku-Teramoto and others 2011; Molina-Calle
and others 2015; Guccione and others 2016). These mainstream
methods for components analysis have many advantages, such as
high sensitivity, accuracy, and good repeatability. However, there
are also several disadvantages such as complex and time consuming
sample preparation which involves solvent extraction and establish-
ment of analysis conditions and manipulation procedures. More
importantly, these methods are not capable of providing valuable
information related with distribution profiles of multicomponents
in the peels. Understanding the distribution profiles would as-
sist the better production and application of the functional and
beneficial components from citrus by-products in an effective and
sustainable manner. The objective of this study is to determine
the biochemical profiles of multiple bio-components in the cit-
rus peels (flavedo, albedo, and longitudinal section) of 3 kinds

C 2017 Institute of Food Technologists

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2840 Journal of Food Science r Vol. 82, Nr. 12, 2017 doi: 10.1111/1750-3841.13952
Further reproduction without permission is prohibited
Chemical mapping of essential oils . . .
Figure 1–Three kinds of citrus fruits (A), scan samples (B) and chemical structures of their major functional components (C). Raman spectra of different
location rich in essential oil, carotenoids, and flavonoids (D). The characteristic Raman peaks were labeled in red, blue and black for essential oil,
carotenoids and flavonoids, respectively.
of citrus fruits, including California oranges, Seedless California
clementines, and Honey Florida tangerines using Raman mapping
technique.
Raman spectroscopy is based on Raman scattering effect in
which information about molecular vibration, rotation, and other
low-frequency modes can be obtained nondestructively as fin-
gerprint (Zhang and others 2017). This nondestructive detection
along with another large advantage of less interference from water
molecules, makes Raman a useful tool with various application,
such as detection of protein chromophore (Quinones-Ruiz and
others 2017), identification of breast cancer (Depciuch and others
2016), detection of water quality (Depciuch and others 2016),
and so on. Coupled with a confocal microscope and mapping
technique, Raman microscopy can be used for semi-automatic
data collection where hundreds of spectra can be collected auto-
matically at every pixel of the defined area, and then integrated to
generate artificial color images based on the intensity of a designate
peak (Liu and others 2016a). It has been a valuable tool for broad
applications in pharmaceutics (Gordon and McGoverin 2011),
materials (Perevedentsev and others 2016), and food sciences (Liu
and others 2016b). In our previous studies, Raman microscopy
was used for imaging and characterization of cancer cell responses
to polymethoxyflavones (Zhang and others 2016), and real-time
and in situ monitoring of pesticide penetration in leaves (Yang and
others 2016). For in situ bio-component analysis in plant tissues,
FT-Raman microscopy was used to discriminate of carotenoid and
Food Chemistry
flavonoid content in petals of pansy cultivars, which demonstrated
the application of components analysis in flower (Gamsjaeger and
others 2011). More recently, Raman microscopy was used for in
situ detection and identification of hesperidin crystals, as well as the
location in citrus peels with the assistance of fluorescence (Inoue
and others 2015).
To the best of our knowledge, this is the first time that Ra-
man microscopy was successfully used to simultaneously determine
the relative amount and distribution profiles of multiple bioactive
components in different locations (flavedo, albedo, and longitudi-
nal section) of citrus peels and compared between 3 different kinds
of citrus. The information obtained here is valuable for developing
effective and sustainable strategy of utilization of citrus peels for
further applications.
Materials and Methods
Materials
Three kinds of citrus fruits, California oranges (Citrus sinensis)
from Sunkist growers, Inc. (Sherman Oaks, Calif., U.S.A.), Seed-
less California clementines (Citrus clementina Hort. Ex Tan.) from
Cuties cooperative (Bakersfield, Calif., U.S.A.) and Honey Florida
tangerines (Citrus reticulata) from Noble World Wide Florida Cit-
rus Sales (Winter Haven, Fla., U.S.A.), were used in this study.
The citrus fruits chosen in this study were all fresh, hydrating, and
with bright color and regular shape (Figure 1A).
Vol. 82, Nr. 12, 2017 r Journal of Food Science 2841
 Chemical mapping of essential oils . . .
Table 1–The characteristic peak shifts in Raman spectra of major functional components.
Components Wavenumber (cm−1 )
Essential oil (Gudi and others 2014) 1678
1646
1609
1435
758
Carotenoid (Quilitzsch and others 2005) 1509 to 1535
1156
1007
Flavonoid (Inoue and others 2015) 1550 to 1700
1225 to 1272
764 to 767
Sample preparation
Three kinds of citrus fruits (orange, clementine, and tangerine)
Food Chemistry
were soaked in brine solution for 10 min, rubbed with hands, and
washed with water for 3 times to remove fungicides, wax and resin
on the citrus peels. Then they were cut into 6 pieces longitudi-
nally and peeled off. The thicknesses of the 3 citrus peels were
3.97, 2.29, and 1.44 mm for oranges, clementines, and tangerines,
respectively. 1.5 × 1.5 cm2 pieces for flavedo side and albedo side
were further cut and pasted via double-faced tape on glass slides for
Raman detection to make sure the scan surface flat (Figure 1B), re-
spectively. Furthermore, in order to obtain better understanding of
the components distribution in citrus peels, pieces in 0.3 × 1.5 cm
were also obtained and one of the longitudinal sections was pasted
on the slide, and used for Raman detection.
Instrument
In this study, a DXR Raman microscope Thermo Fisher
Scientific (Madison, Wisconsin, U.S.A.) with spectral resolution
of 5 cm−1 , facilitated with a 780 nm excitation and 24 mW
laser power through a 60× water immersed objective (Olympus,
Japan), was used. The measured condition for each sample was as
follows: 50 μm slit width, 400 lines/mm, spot size 1.0 μm and
integration time 1 s. Each spectrum was collected in the 400 to
3300 cm−1 through the Thermo Fisher Scientific OMNICTM
Software (Madison, Wisconsin, U.S.A.).
Raman mapping
The components distribution of citrus peels in different posi-
tions was evaluated, including flavedo side, albedo side, and lon-
gitudinal section. For flavedo and albedo side, 2 kinds of maps,
area maps and line depth profiles, were collected. For area maps,
the scanning area was about 150 × 300 μm in width and length.
For line depth profiles, the scanning area was about 200 × 90 μm
in width and depth. The internal components distribution of the
peels was investigated through surface scanning of the longitudinal
section with the scanning area about 900 μm × 300 μm. Raman
mapping was performed by collecting 150 spectra with (or less
than) a 10-μm-step size over the surface or depth in sequence.
The scanning area in 1 video frame is limited which cannot meet
the requirement when a large sample area is needed. Mosaic, made
up of multiple video images, is an efficient method in this situa-
tion. In briefly, mosaic in Raman is a method to split each video
frame into a large image, which can provides much more informa-
tion about the material investigated and makes the analysis results
more accurate. Thus, in order to obtain more information from
a relative large area, the method of Mosaic was also used in this
study. The total scanning area was about 4500 μm × 7500 μm
2842 Journal of Food Science r Vol. 82, Nr. 12, 2017
Assignments
C═C bond stretching of the ring
C═C bond stretching of the vinyl group
C–C vibrations
C═C bending
C–H vibrations
polyene chain (–C═C– stretching vibrations)
polyene chain (–C–C– stretching vibrations)
in-plane rocking of CH3
benzenic ring vibrations (C═O and C2 –C3 stretching vibrations)
para-disubstituted benzene ring vibration
daromatic C–H out-of-plane deformation vibration
with a 50 μm × 50 μm step size over the surface in constant. All
experiments were repeated at least 5 times, independently.
Data analysis
The distribution and relative amounts of 3 main functional com-
ponents (essential oils, carotenoids, and flavonoids, Figure 1C)
were analyzed in different citrus peels. The characteristic peak
shifts in Raman spectra of different components were shown in
Table 1. The images were integrated based on the Raman spectra
at specific peak shifts using the Atlμs function in the OMINICS
software (Thermo Fisher Scientific). All the spectra were put in the
same common scale to compare, no other application, including
subtraction, was done. The relative quantity for each component
was determined through the color on the image.
One-way ANOSIM (Analysis of Similarity) was used here to
compare spectral data between groups (Clarke and Gorley 2006).
The comparison was based on 9999 permutations, the Euclidean
distance as the dissimilarity metric and the first 2 principle com-
ponent scores (PC1 and PC2) by principal component analysis
(PCA). PCA is a mathematical procedure to convert a set of possi-
bly correlated variables into a set of principal components, which
are values of linearly uncorrelated variables. PC score reflected
the percentage of the data analyzed of the real characteristics be-
tween different samples (Zhang and others 2016). In this study,
twenty points were chosen randomly from the carotenoids rich
locations in the flavedo map of each citrus species. The spectra at
the points from the same citrus species were saved as 1 group and
transported into TQ Analyst software (Thermo Fisher Scientific),
through which PCA were performed with discriminant analysis
without any other preprocessing procedure. A 0.01% significant
level was used. In ANOSIM, the difference between 2 groups
was characterized by R value. R-value is a statistic parameter to
reflect the difference between different groups. It is calculated as
following, R = (rB – rW )/(N (N-1) / 4), where rB , rW , and N
are mean ranks between groups (rB ) and within groups (rW ) and
number of detection, respectively. The range of R-value is (–1, 1).
An R-value >0.75, 0.5 to 0.75, and <0.25 indicates clearly differ-
ent, difference with some overlapping and almost no differences,
respectively (Pearson and others 2014). R-values below 0 suggest
more similarity between groups than within groups and indicate
a problem in experiment design.
Results and Discussion
Identification of characteristic Raman peak of functional
components
In order to interpret a Raman spectrum obtained from a mul-
ticomponent material, such as citrus peel, characteristic peaks
Chemical mapping of essential oils . . .

of each component need to be identified first. As shown in
Figure 1D, the major marker bands at 1528, 1156, and 1010 cm−1
confirmed the presence of carotenoids, assigned to polyene
─C═C─, ─C─C─ stretching vibrations and in-plane rocking of
CH3 , respectively (Quilitzsch and others 2005). All the 3 bands
have no overlap with other components and can be used as marker
bands for carotenes and 1156 cm−1 was chosen for carotenes iden-
tification in this study. The observation of peaks at 1679, 1646, tial oil. The locations of essential oil were aligned with the places
1434, and 761 cm−1 suggested essential oil in the flavedo layer,
assigned to C═C bond stretching of the ring and vinyl group,
C═C bending and C─H vibrations, respectively, according to
previous literature data (Gudi and others 2014). Characteristics
peaks at 1628, 1605, 1378, and 1275 cm−1 indicated the exis-
tence of flavonoids, assigned to benzenic ring vibrations (C═O
and C2 ─C3 stretching) and para-disubstituted benzene ring vi-
bration, respectively (Inoue and others 2015). However, bands
1646 cm−1 of essential oil overlaps with bands 1628 and 1605 cm−1
(761, 1156, and 1275 cm−1 for essential oils, carotenoids and
flavonoids, respectively). As shown in Figure 2, the relative content
for each component present in the flavedo layer was determined
based on the colors which represent the intensity of the char-
acteristic peak. The red color represents the highest level of the
components and the blue color indicates the region with lowest
content. As seen in Figure 2B, the flavedo layer was rich in essen-
of oil glands in the optical images, which were about 0.2 mm in
diameter (Figure 2A). Carotenoids were the most abundant func-
tional component with high level and wide distribution in Raman
mapping (Figure 2C). The result was aligned with the fact that the
orange color of ripened citrus peel was mainly determined by
carotenoids, mainly beta-carotene. Figure 2D also showed that,
there were little flavonoids in the outside of flavedo. Comparing
the distribution and intensity of the mappings allowed drawing the

Food Chemistry
conclusion on the relative content of the 3 components in citrus
of flavonoids. Thus, taking the intensity of each band into account,
flavedo, thats carotenoids, essential oils and flavonoids from high to
761 and 1275 cm−1 were used as marker bands for identification low. By comparing these images, we found carotenoids existed in
of essential oils and flavonoids, respectively. Raman mapping basedthe place outside of oil gland. Some of flavonoids existed exactly in
on above-mentioned was then carried out for the analysis of the oil gland with relative high concentration, while the others were
relative intensity and distribution of 3 functional components, es-observed in the places with carotenoids. Figure 1C also confirm
sential oils, flavonoids, and carotenoids in citrus peels. this conclusion, in which some marker bands of flavonoids can be
seen in both spectra of location rich in essential oil and carotenoids
Analysis of multi-component in the flavedo layer obtained from mappings. The calculation of flavonoids in oil gland
In the flavedo layer, all the 3 functional components were found might due to the high expression of the corresponding genes in
in the Raman image according to the characteristic peak shifts epithelial cells (Voo and others 2012). The result also indicated

Figure 2–Mosaic Raman mapping of flavedo of clementine peel under microscope (A), and the wavenumber of 761 cm−1 (B), 1156 cm−1 (C), and
1275 cm−1 (D). The color according to the individual band intensities at 761, 1156, and 1275 cm−1 indicated the relative content and distribution of
essential oil, carotenoids and flavonoids, respectively. The positions in red circles were the locations of oil glands.

Vol. 82, Nr. 12, 2017 r Journal of Food Science 2843

 Chemical mapping of essential oils . . .

that flavonoids, a group of lipophilic compounds, might exist in information about the internal region of the flavedo and albedo
(surface and internal region) were obtained through line scanning
2 physical formats of being solution in essential oil or crystal in
citrus peel. Essential oil could be used as a good natural solventof flavedo and albedo layer (Figure 3). As it can be seen, essen-
for flavonoids to deal with poor solubility in accordance with tial oil and carotenoids mainly existed in the flavedo layer, and
our previous study, in which different citrus essential oils were the relative content was varied according to the vertical depth.
successfully used to improve citrus peels derived flavonoids (YangFlavonoids existed both in the flavedo and albedo layers, and the
and others 2017). later was more abundant than the former. To better interpret the
internal part of a citrus peel, Raman mapping of longitudinal sec-
Analysis of component distribution in different positions of tion of the citrus peel was performed, and the images that was
a citrus peel integrated from the wavenumber of 761, 1156, and 1275 cm−1 ,
The distribution features of the functional components in dif- which were presented of essential oil, carotenoids and flavonoids,
ferent position were also investigated by Raman mapping. The respectively (Figure 4). The flavedo layer (right part) and albedo
Food Chemistry

Figure 3–Raman mapping of flavedo (A) and albedo (B) of clementine peel via line depth scanning under the wavenumber of 761, 1156, and 1275
cm−1 .

Figure 4–Mosaic Raman mapping of longitudinal section of clementine peel under microscope (A), and the wavenumber of 761 cm−1 (B), 1156 cm−1
(C), and 1275 cm−1 (D).

2844 Journal of Food Science r Vol. 82, Nr. 12, 2017

Chemical mapping of essential oils . . .

Food Chemistry
Figure 5–Raman mapping of albedo of 3 different citrus peels (orange, clementine, and tangerine) detected at the wavenumber of 761 cm−1 (A), 1156
cm−1 (B), and 1275 cm−1 (C).

Figure 6–Raman spectra (A) of the location rich in carotenoids of 3 different citrus peels (orange, clementine, and tangerine), and their PCA plot (B).

layer (left part) can be easily recognized under the microscope
(Figure 4A). The distribution profiles of essential oil, carotenoids
and flavonoids in the longitudinal section were quite similar to
those in the flavedo layer. Essential oil was mostly existed in the
oil gland, while carotenoids with the mutually complementary dis-
tribution. Flavonoids appeared both in and out of the oil glands,
depending on its physical structure (solution or crystal). Further-
more, the relative content of essential oil and carotenoids in the
albedo layer were much lower than those in the flavedo layer,
indicating that they mainly existed in the flavedo layer.
Comparison of relative amount of each component
in different citrus peels
The relative content and distribution of the 3 functional compo-
nents in 3 different types of citrus peels were also investigated. As
shown in Figure 5, essential oil was most abundant in the tangerine
peel. While carotenoids was most abundant in the clementine peel,
followed by orange and tangerine peel. The content sequence was
in agreement with the color of each kind of citrus peels (shown in
Figure 1A), which was consisted with the fact that the orange color
of ripened citrus peel was mainly determined by carotenoids. In

Vol. 82, Nr. 12, 2017 r Journal of Food Science 2845

 Chemical mapping of essential oils . . .
terms of flavonoids, the level was highest in clementine, followed
by orange and tangerine. It was easy to come into the conclusion
that different citrus peels can be distinguished from each other
efficiently by Raman mapping. Raman spectra of the carotenoids
were further used for the discrimination of 3 different citrus peels
(orange, clementine, and tangerine). The Raman spectra peak
intensity as well as pattern significantly varied between different
citrus peels (Figure 6A). The PCA plot also confirmed this con-
clusion (Figure 6B) in which the clusters of the 3 citrus peels set
apart significantly. PC1 and PC2 explained 95.3% and 2.57% of
the total data variance, respectively. In addition, from ANOSIM
result, R-value between each citrus peel is almost 1.0 which was
>0.75, and the P-value < 0.01%, indicating obvious difference
between them. The result showed the potential of using Raman
mapping as a tool to discriminate between different citrus species
as a base for identifying possible adulteration or mislabeling.
Food Chemistry
Conclusions
In conclusion, we have successfully applied Raman microscopy
for rapid and nondestructive chemical mapping of 3 impor-
tant bioactive components, that is essential oil, carotenoids, and
flavonoids in citrus peels. The distribution profiles of different lo-
cations (flavedo, albedo, and longitudinal section) of peels of 3 dif-
ferent citrus were systematically investigated. This study indicated
the capability of Raman microscopy for rapid and nondestructive
analysis of multiple bioactive components in plant tissues. The in-
formation obtained from the study would assist the development
of an effective and sustainable strategy to better utilize the citrus
by-products.
Acknowledgments
The authors would like to acknowledge the financial support of
the Program of Introducing International Advanced Agricultural
Science and Technology of the Ministry of Agriculture of P. R.
China (948 Program, No. 2016-X31) and the National Natural
Science Foundation of China (No. 31401581).
Author Contributions
Ying Yang and Xiaohe Wang conducted the main experiment,
data collection and analysis. Chengying Zhao participated in the
experiment and writing of the manuscript. Guifang Tian and Hua
Zhang participated in data collection and analysis. Hang Xiao took
part in experiment design and the writing progress. Lili He and
Jinkai Zheng designed the experiment, and participated in the
data analysis and the manuscript writing.
Conflicts of interest
The authors declare no competing financial interests.
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