Abstract
The current study investigated the scabicidal potential of Egyptian
mandarin peel oil (Citrus reticulata Blanco, F. Rutaceae) against
sarcoptic mange-in-rabbits. Analysis of the oil's GC–MS identified a
total of 20 compounds, accounting for 98.91% of all compounds found.
Mandarin peel oil topical application improved all signs of infection,
causing a scabicidal effect three days later, whereas in vitro
application caused complete mite mortality one day later. In comparison
to ivermectin, histopathological analysis showed that the epidermis'
inflammatory-infiltration/hyperkeratosis-had disappeared. In addition
to TIMP-1, the results of the mRNA gene expression analysis showed
upregulation of I-CAM-1-and-KGF and downregulation of ILs-1, 6, 10,
VEGF, MMP-9, and MCP-1. The scabies network was constructed and
subjected to a comprehensive bioinformatic evaluation. TNF-, IL-1B, and
IL-6, the top three hub protein-coding genes, have been identified as
key therapeutic targets for scabies. From molecular docking data,
compounds 15 and 16 acquired sufficient affinity towards the three
screened proteins, particularly both possessing higher affinity towards
the IL-6 receptor. Interestingly, it achieved a higher binding energy
score than the ligand of the docked protein rather than displaying
proper binding interactions like those of the ligand. Meanwhile,
geraniol (15) showed the highest affinity towards the GST protein,
suggesting its contribution to the acaricidal effect of the extract.
The subsequent, MD simulations revealed that geraniol can achieve
stable binding inside the binding site of both GST and IL-6. Our
findings collectively revealed the scabicidal ability of mandarin peel
extract for the first time, paving the way for an efficient,
economical, and environmentally friendly herbal alternative for
treating rabbits with Sarcoptes mange.
Subject terms: Biochemistry, Drug discovery, Microbiology
Introduction
Sarcoptic mange (Sarcoptes scabiei) is a serious infectious disease
that invades humans and animals all over the world^[48]1. The mites are
highly adapted to contact with their host as contagious, burrowing, and
obligate parasites. Sarcoptic mange Grower pig production is negatively
impacted by adult female mites; because they mate on the skin's
surface, burrow into the skin, lay eggs, and cause irritations that can
lead to bleeding, reduced feeding and development, chronic stress, and
decreased welfare^[49]2,[50]3. The clinical picture represents chronic
hyperkeratotic, which is characterized by the presence of aural crusts
and many mites on the animal^[51]4. Similar to people, rabbits are
susceptible to Sarcoptes infection, or mange, which reduces production
and causes economic losses for rabbits, especially in the absence of
effective treatment^[52]5. Therapy options include the systemic
treatment of macrocyclic lactones, local administration of amitraz or
pyrethroids, or both^[53]6,[54]7. Despite their long history of
effectiveness in treating mange, their extensive use has led to a
decline in effectiveness because of the emergence of drug resistance.
Thus, it is crucial to create novel scabicides that are both efficient
and secure in order to treat and control mammalian scabies^[55]6.
In rabbits, goats, and pigs, several essential oils derived from Citrus
limon, Lavandula angustifolia, Citrus aurantium amara, Pelargonium
asperum, Melaleuca alternifolia, Syzygium aromaticum, Eucalyptus
radiata, Leptospermum scoparium, Juniperus oxycedrus, Cryptomeria
japonica, and Cymbopogon martini, were put to the test in real time
against S. scabiei^[56]8–[57]12. Essential oils are typically favoured
over chemical acaricides since they are less harmful to animals and
have a shorter environmental persistence. Also, the complex chemistry
of essential oils is known to considerably impede the emergence of drug
resistance against these chemicals^[58]13. Yet, because essential oils
consist of a complex mixture of components, it might be challenging to
attribute an essential oil's acaricidal properties to a specific
ingredient or combination of compounds^[59]14. Skin irritation is yet
another potential drawback that has been reported in humans^[60]15.
Some of the most coveted Citrus fruits for fresh consumption are
mandarins, C. reticulata^[61]16. The more frequent name for them is
"mandarin," but they are also occasionally called "tangerines." The
Mandarin species includes a number of cultivars and hybrids^[62]16.
Popularly grown varieties include C. unshiu Marcovitch (also known as
Unshiu mikan in Japanese), C. nobilis Loureiro (also known as king
mandarins), C. deliciosa Tenore (also known as Mediterranean
mandarins), and C. reticulata Blanco (common mandarins)^[63]16,[64]17.
Mandarins are one of the main Citrus fruits grown in many countries
such as China, Brazil, USA, India, Mexico, Spain, etc. The fruits have
a great commercial worth for their essential oils and other fragrant
compounds, even though they are primarily used to make pastries^[65]18.
A lot of beverages, candies, cookies, and desserts use Citrus
flavours^[66]19, while the peels of C. reticulata are used to flavour
alcohol^[67]19. Citrus reticulata EO shown an anti-proliferative
activity against rat pulmonary fibrosis produced by bleomycin (BLM) and
protective properties against human embryonic lung fibroblasts (HELFs).
The method is believed to involve correcting the imbalance between
oxidation and antioxidation, lowering collagen deposition and fibrosis,
and down-regulating lung tissue expressions of connective tissue growth
factor (CTGF) and mRNA^[68]20. Due to its high d-limonene
concentration^[69]21, C. reticulata EO demonstrated a moderate level of
radical scavenging action^[70]22. Mandarin oil is well known for its
broad spectrum antibacterial and antifungal actions. It inhibits the
growth of several bacteria including Escherichia coli, Bacillus
subtilis, Pseudomonas aeruginosa, and Staphylococcus
aureus^[71]22,[72]23, as well as several fungi including Penicillium
italicum, P. chrysogenum, P. digitatum, Aspergillus niger,-A. flavus,
Alternaria alternata, Curvularia lunata, Rhizoctonia solani, Fusarium
oxysporum, and[-]Helminthosporium oryzae^[73]23–[74]26.
The GC–MS profiling of mandarin peel oil has been used in the current
study. Additionally, for the first time, through-in vitro,-in
vivo,-histopathology,-mRNA-expression, and network/in silico analysis,
the extract's scabicidal potential against-Sarcoptic-mange-in-rabbits
has been investigated, allowing for the incorporation of natural
candidates to proper and secure management of infectious diseases. The
present investigation's framework is shown in Fig. [75]1.
Figure 1.
[76]Figure 1
[77]Open in a new tab
General outflow of the study.
Material and methods
Ethical permission
Plant materials and experiments were conducted in accordance with
relevant institutional, national, and international guidelines. The
study took place according to the ethical committee's permission number
of 9/5/2022 at Deraya College. It was done in accordance with the
National Institute of Health's guidelines for the care and use of
laboratory animals and ARRIVE guidelines^[78]27.
Fruit collection
In January 2021, C. reticulata cultivated fruits were harvested from a
house garden on Atia Street in Beni-Suef, Egypt. A voucher specimen
(2021-BuPD-88) was deposited at Pharmacognosy-Department,
Faculty-of-Pharmacy, Beni-Suef-University, Egypt.
Sample preparation
Using the Clevenger apparatus, the fresh peels (0.5 kg) were
hydrodistillated for two hours at 75 °C. The oil was gathered, dried
over anhydrous sodium sulphate, and kept in airtight amber glass vials
at 4 °C for storage. On the basis of the plant material's fresh weight,
the yield (v/w%) was computed^[79]28,[80]29.
GC–MS analysis
Gas chromatography-mass spectrometry (GC/MS) was used to perform
chromatographic analysis on the oil recovered from peels^[81]28,[82]30.
The GC–MS apparatus combines a thermal mass spectrometer detector (ISQ
single quadrupole mass spectrometry) with a TRACE GC ultra-high
performance gas chromatograph (THERMO Scientific Corp., USA). A TR-5 MS
column (30 m × 0.32 mm i.d., 0.25 mm film thickness) was installed in
the GC–MS system. For the analyses, He-lium was used as the carrier
gas, and the split ratio was set at 1:10 using the following
temperature program: 60 C for 1 min, followed by 4.0 C/min to 240 C and
a 1-min hold. At 210 °C, the injector and detector were maintained.
One-liter samples of the mixes were always administered as diluted
samples (1:10 hexane, v/v). By using a spectral range of m/z 40–450 and
electron ionization (EI) at 70 eV, mass spectra were produced. Using
AMDIS software ([83]www.amdis.net), the chemical components of the
essential oil were deconvoluted and identified by their retention
indices (relative to n-alkanes C8-C22), mass spectra matching to
genuine standards, and retention times (when available). (NIST Standard
Reference Database, 78 Version 5.10) Wiley spectral library
collection^[84]28,[85]31,[86]32.
In vitro assays
In-vitro-antioxidant-activity
Hydrogen-peroxide-scavenging-activity
The reaction with a defined amount of exogenously provided hydrogen
peroxide (H[2]O[2]) was used to determine H[2]O[2] scavenging activity
that reflects the anti-oxidative capacity of the peel oil. Colorimetric
analysis was used to estimate the residual H[2]O[2]^[87]33. In brief,
20 µl of the sample was mixed with 500 µl of H[2]O[2] and incubated at
37 °C for 10 min. 500 l of the enzyme/3,
5-dichloro-2-hydroxyl-benzensulfonate solution were then added, and it
was incubated at 37 °C for 5 min. The colored product's intensity was
quantified colorimetrically at-510-nm. A positive control was ascorbic
acid. By comparing the test results to those of the control group, the
percentage-of H[2]O[2]-scavenging activity-was calculated and applying
the following formula:
[MATH: scavenging
activity=A
control-A
sampleA
control×100 :MATH]
IC[50] of each sample was calculated after performing the assay at
eight different concentrations : (1000, 750, 500, 375, 250, 187.5, 125
and 0 µg/mL) using Graph pad prism 7 software.
Superoxide radical scavenging activity
The scavenging activity of superoxide anion was measured^[88]34. In a
Tris-HCL solution (16 mM, pH 8.0) containing 90 l of NBT (0.3 mM), 90 l
of NADH (0.936 mM), 0.1 ml of peel oil (125, 250, 500, and 1000 g/mL),
and 0.8 ml of Tris-HCl buffer, superoxide anion radicals were produced
(16 mM, pH 8.0). After adding 0.1 ml of PMS solution (0.12 mM) to the
mixture, the reaction was started. The mixture was then incubated at
25 °C for 5 min, during which time the absorbance was measured at
560 nm. Ascorbic acid was used as a model substance. Using the formula
below, the percentage inhibition was calculated by comparing the test
results to those of the control:
[MATH: Superoxide scavenging
activity=A
control-A
sampleA
control×100 :MATH]
IC[50] was estimated by doing the test at four different concentrations
and using the GraphPad Prism 7 software.
Biological investigation
Collection-of-Sarcoptes-scabiei-mites
Adult-mites-were collected from rabbits that were-naturally-infected,
Deraya University, Minia, Egypt's Animal House. Scraped from the
borders of the lesions, the infected skin samples were then
shifted-to-petri plates and-incubated within a
biochemical-oxygen-demand (BOD) for an incubator for 30 min at 35 °C.
In vitro-application of peels oil on-sarcoptic-mange
A petri dish containing mites was filled with 2 ml of diluting extract
(20%), along with the plates were then incubated-in-BOD. Reaction
observations were made-at 1, 12, and 24 h after application. Petri
plates were incubated at an ambient temperature of 25 °C and with a
relative moisture of 75%, with a 5% ivermectin (1 cm3/l) group as the
positive-control-and distilled water as the-negative-control. By
stimulating the mites with a needle, the death of the mite was
confirmed; the mite was deemed dead if it showed no response.
In vivo application of peels oil
The study took place on male adult rabbits for 4 weeks (weighing
2.8–3.2 kg) that were infected. The animals' ears showed clinical
indicators of mange infection, such as hyperkeratinization,
inflammation, redness, itching, and irritability. Microscopic mite
identification in skin scrapings further corroborated this. Four groups
of five rabbits each were made up of twenty animals, as follows: Five
rabbits made up the normal group, the paraffin
oil-positive-control-group. The-ivermectin-treated-group
(5%-ivermectin). The peel-oil-group (20%-peel-oil in paraffin-oil).
Paraffin oil, which is a-mineral-oil, was reportedly chosen as a
diluent for the peel oil because it has little impact-on-mites^[89]35.
Each group were kept in a separate cage, and each group received
treatment by dipping the infected ears once daily. Steel hoppers were
used to feed all of the rabbits, and water was available at all times.
The rabbits were observed every two days to assess their clinical
recovery. The goal was to find any signs of improvement in the lesions,
such as the absence of irritation and redness, cutaneous smoothing, the
start of the development of hair from the infection, and the cessation
of scab development^[90]10. Skin scrapings from each rabbit's sick and
healed areas were taken every three days, and Throughout the course of
the therapy, they were microscopically investigated to check for
sarcoptic mites with a LEICA, DM1000 microscope with a digital camera
(LEICA, EC3, Germany)^[91]10.
Histopathological-examination
Tissue samples were collected at zero and three weeks following the
start of the course of therapy via 20% peeling oil as well as
ivermectin from healthy and infected ears. Following that, samples were
dried in ethyl alcohols of increasing strength, sterilized via xylene,
infused with paraffin that had been melted at 55–60 °C, and finally
inserted into paraffin wax. The samples were then preserved in 10%
buffered formalin. Deparaffinized, rehydrated, and stained with
hematoxylin and eosin (H & E), “3–5 m thick” tissue sections were
examined using-a-light-electron-microscope^[92]36.
RNA-isolation-and-qRT-PCR-assay
Using-a-digital-homogenizer-(Branson-Digital-Homogenizer®,-Danbury,-CT,
-USA), 100 mg of the tissues under investigation were homogenised in
1 ml of TRIzolTM RNA Extraction Reagent (Amresco, Solon, OH, USA). RNA
extraction from the biopsy sample was done in accordance with the
manufacturer's instructions. RevertAid H-minus First Strand cDNA
Synthesis Kits (#K1632, Thermo Science Fermentas, St. Leon-Ro, Germany)
were used to create cDNA from the extracted RNA for comparable amounts
of total RNA in all samples. The-qRT-PCR-was carried out
on-the-Applied-Biosystems Step One Plus system using the cDNA as a
template. The primers were created using the NCBI primer blast software
and were produced by Invitrogen. Using the GAPDH gene as a housekeeping
gene, data were analyzed using the 2CT approach^[93]31. Table [94]1
lists the primer sequences that were employed.
Table 1.
Gene primers sequences.
Gene Forward Reverse
GAPDH GTC AAG GCT GAG AAC GGG AA ACA AGA GAG TTG GCT GGG TG
VEGF CAT CAG CCA GGG AGT CTG TG GAG GGA GTG AAG GAG CAA CC
IL-1 AGC TTC TCC AGA GCC ACA AC CCT GAC TAC CTT CAC GCA CC
IL-6 GCC AAG TTC AGG AGT GAC GA AGA GCC CAT GAA ATT CCG CA
MCP-1 GAT CCC AAT GAG TCG GCT GG ACA GAA GTG CTT GAG GTG GTT
ICAM GGC GGC TCA GTG TCT CAT T TTC GTT CCC AGA GCG AGT G
IL-10 AAC AAG AGC AAG GCA GTG GA CTA GCC GAG TTG CCA TCC TG
KGF ACA ATG TGG CCA AAA ATG GCT AGG AGA TTT TTC CCC TGG CG
MMP-9 GCA GAG GAG TAC CTG TTC CG ATT ATC CAG CTC CCC CGT CT
TIMP-1 CCT TCT GCA ACT CCG ACC TT GTA CCC GCA GAC ACT TTC CA
[95]Open in a new tab
In silico studies
Construction of protein–protein interaction (PPI) network
Using Cytoscape 3.9.1 software ([96]https://www.cytoscape.org/)^[97]37
and by lunching STRING disease query tool incorporated in it which
retrieves network for the top human proteins associated with the
queried disease from a weekly updated web source of diseases database
([98]https://string-db.org/)^[99]38 Scabies was chosen as the search
term, and "Human sapiens" was chosen as the species type. The
confidence score was set to 0.4, and the default settings for the
remaining parameters were used to create the PPI network^[100]39.
Hub gene expression analysis
The plugin for cytoHubba the hub genes are identified using ranking
techniques such as degree, edge percolated component (EPC), maximum
neighbourhood component (MNC), the density of maximum neighborhood
component (DMNC), and maximal clique centrality (MCC), as well as
bottleneck, eccentricity, closeness, radiality, betweenness, stress,
and clustering coefficient. Cytoscape is regarded as a useful
exploration interface for the most significant nodes in PPI
networks^[101]40,[102]41.
Gene ontology and enrichment analysis
We employed a freely accessible bioinformatics web tool in the current
investigation (ShinyGO v0.76.3). Using the many bioinformatics
databases accessible, it is possible to perform both gene ontology
enrichment analysis and pathway enrichment analysis. ShinyGO was used
to perform the gene ontology and enrichment analysis on the 16 genes to
determine the cellular elements, molecular functions, and biological
processes that were impacted by this set of genes. ShinyGO retrieves
comprehensive descriptions of biological signal transduction pathways
from numerous databases^[103]42.
Molecular docking study
The methodologies of molecular docking intend to predict the best
binding orientation of a ligand to a receptor. It proposes several
suitable poses of the ligand within the active or docking site of a
receptor molecule.in this study, twenty compounds that were identified
underwent an in silico study by using screening for three different
important-protein-targets that are heavily involved in the scabies
infection process, as well as screening for potential targets at the
mite itself as an acaricidal effect, in an attempt to get deep inside
the mechanistic anti-scabietic effect of orange oil. The chosen targets
include IL-1, which is highly effective in stimulating T cells with
regulatory functions, and IL-6, which is involved in the formation of
Th17 lymphocytes and the release of IL-17^[104]43. These cy-tokines
have been identified as one of the primary molecules responsible for
allergic Th2-type inflammation in the immunological response to
scabies, along with TNF-, which is significant in alternative
macrophage activation^[105]44. GSH, which is linked to the scabies
defense system, takes part in a variety of processes crucial to the
preservation of cells from oxygen and free radical oxidative
damage^[106]45, Its distinctive anti-oxidant action makes it a
potential target for the oil's acaricidal impact^[107]46. In our
docking investigation, we validated the ligand and visualized the many
docked poses using the computer programme MOE 2019.010. TNF- complexed
with its ligand (PDB ID code: 2AZ5) is the last one, and GST is the
other protein target of the mite delta class. The first protein target
is (IL-1), depicted by the protein's PDB ID code of 6Y8M in
co-crystallization with IL-6, as reflected by PDB ID code 1ALU, and its
inhibiting ligand SX2 (a-bromo-amido-pyridine-derivative)^[108]47
represented by proteins (PDB ID code: 3EIN), the selected targets were
acquired via the web from the Database of Proteins
([109]http://www.rcsb.org/pdb).
Molecular dynamic simulation
The MD simulations were carried out using NAMD 3.0.0.
software^[110]48,[111]49. The Charmm-36 force field is implemented in
this piece of software. The protein structure was examined for missing
hydrogens, the protonation states of the amino acid residues were set
(pH = 7.4), and the co-crystallized water molecules were removed using
the QwikMD toolkit of the VMD software. The entire assembly was then
packed into a 20 solvent buffer containing 0.15 M Na + and Cl- ions in
an orthorhombic box of TIP3P water. After 5 ns of equilibration, the
systems were subjected to an energy minimization protocol. Force Field
Toolkit (ffTK), a plugin for the VMD software, was used to determine
ligand properties and topologies. After the parameters and topology
files were prepared, they were imported into VMD so that the
protein–ligand complexes could be read accurately, and the simulations
could be run.
Statistical analysis
The data were tabulated using the statistical
programme-GraphPad-Prism-version-9 (GraphPad,-La-Jolla,-CA,-USA). To
evaluate statistical differences between the groups, the ANOVA test was
performed, followed by the-Bonferroni-post-hoc-test-for
multiple-comparisons. The threshold for statistical significance is a
p-value of 0.05 or less.
Results
GC–MS profiling of mandarin peels oil
Egyptian C. reticulata peels gave 2.6% v/w volatile oil fresh weight,
being colourless with a characteristic odor, lighter than water, clear,
transparent, and not viscous at room temperature as well as at 4 °C.
GC–MS analysis was used to identify a total of 20 compounds, accounting
for 98.91% of all compounds found (Table [112]2, Figs. [113]2, [114]3).
The identified compounds 1–20 belonged to different chemical classes,
including monoterpene, phenylpropene, fatty alcohol, and sesquiterpene
(Table [115]2, Fig. [116]3). where monoterpenes represented 92.16% of
the total identified compounds, followed by phenylpropene (3.01%),
fatty alcohol (2.36%), and sesquiterpene (1.38%) (Table [117]2).
Fourteen monoterpenes compounds (92.16%) were identified; ranging from
cyclic hydrocarbon (D-limonene 4, γ-terpinene 6, 73.32%) which
represented the major oil fraction, to oxygenated cyclic hydrocarbon
((-)-isomenthone 10, terpinen-4-ol 11, (-)-carvone 14, 3.32%), and
oxygenated acyclic hydrocarbon (linalool 8, citronellol 13, geraniol
15, 8.78%), acyclic hydrocarbon (α-myrcene 3, α-ocimene 5, 2.84%),
bicyclic hydrocarbon (α-pinene 1, sabinene 2, 3.45%), to oxygenated
bicyclic hydrocarbon (camphor 9, 0.45%) (Table [118]2, Fig. [119]3).
Also, phenylpropene class (3.01%) contained 2.70 and 0.31% of estragole
12, and anethole 17, respectively. The detected fatty alcohol class
contained only 1-octanol 7 and 1-decanol 16, representing 2.36% (Table
[120]2, Fig. [121]3). On the other hand, three sesquiterpene compounds
(1.38%) were identified, varying from a bicyclic hydrocarbon
(caryophyllene 19, ( +)-valencene 20, 1.00%), to a tricyclic
hydrocarbon (α-copaene 18, 0.38%) (Table [122]2, Fig. [123]3).
Table 2.
Citrus reticulata oil composition using GC/MS analysis isolated from
peels.
Nu Identified Compound MF Area % RT RI
1 α-Pinene C[10]H[16] 1.81 4.72 947
2 Sabinene C[10]H[16] 1.64 5.60 957
3 α-Myrcene C[10]H[16] 2.66 6.07 955
4 D-Limonene C[10]H[16] 71.72* 7.50 933
5 α-Ocimene C[10]H[16] 0.18 8.00 929
6 γ-Terpinene C[10]H[16] 1.60 8.16 935
7 1-Octanol C[8]H[18]O 1.71 8.54 943
8 Linalool C[10]H[18]O 5.39 9.33 941
9 Camphor C[10]H[16]O 0.45 10.21 912
10 (−)-Isomenthone C[10]H[18]O 1.00 10.49 936
11 Terpinen-4-ol C[10]H[18]O 1.34 11.20 857
12 Estragole C[10]H[12]O 2.70 11.80 943
13 Citronellol C[10]H[20]O 2.71 11.97 934
14 (−)-Carvone C[10]H[14]O 0.98 13.05 927
15 Geraniol C[10]H[18]O 0.68 13.45 937
16 1-Decanol C[10]H[22]O 0.65 13.91 948
17 Anethole C[10]H[12]O 0.31 14.21 910
18 α-Copaene C[15]H[24] 0.38 16.39 926
19 Caryophyllene C[15]H[24] 0.39 17.52 944
20 (+)-Valencene C[15]H[24] 0.61 20.15 941
Total 98.91%
[124]Open in a new tab
RI Retention index relative to n-alkanes, RT Retention time (min), MF
Molecular formula.
*Major compound, % Percentage.
Figure 2.
[125]Figure 2
[126]Open in a new tab
GC/MS spectrum for Citrus reticulata peels oil.
Figure 3.
[127]Figure 3
[128]Open in a new tab
Structures of identified compounds, using GC/MS analysis, from Citrus
reticulata oil isolated from peels.
According to the literature, the chemical composition of essential oils
varies depending on the age of the plant, harvesting time, geographical
location, and environmental conditions^[129]50. The Indian C.
reticulata peels volatile oil differently from the Egyptian, having 80
compounds, where monoterpene (63.80%), represents mainly limonene
(50.42%), myrecene (3.03%), and α-terpineol (1.19%), while
sesquiterpene (12.98%) represents mainly α-copaene (1.49%), β-copaene
(1.30%), and α-humulene (1.23%). The Indian C. reticulata oil is
characterised by its high content of fatty acids (8.73%) and aldehyde
content (7.08%), mainly n-hexadecanoic acid (5.65%) α-sinensal
(3.14%)^[130]51. The essential oil isolated from fully matured, ripened
Indian fruit peels of C. reticulata, on the other hand, contained 37
different components (99%). The primary ingredients included limonene
(46.7%), geranial (19.0%), neral (14.5%), geranyl acetate (3.9%),
geraniol (3.5%), -caryophyllene (2.6%), nerol (2.3%), neryl acetate
(1.1%), and others^[131]26.
The essential oil constituents reported in C. reticulata grown in
Burundi contained 58 constituents^[132]52. The most prevalent chemical
category was monoterpene hydrocarbons (94.7%). Limonene accounted for
84.8% of the total composition, with -terpinene (5.4%), myrcene (2.2%),
and -pinene (1.1%) following. Germacrene D and valencene were the
primary components of the sesquiterpene hydrocarbons, which made up
only 0.2% of the total composition. Compounds containing oxygen from
different chemical groups made up 2.3%^[133]52. The two main chemical
groupings were terpene alcohols (0.7%) and aliphatic aldehydes (0.7%).
Linalool (0.7%), octanal (0.5%), and decanal (0.2%) made up the bulk of
the mixture. In concentrations of 0.1%, octyl acetate, α-sinensal,
decanol, and perillaldehyde were present. Thymol, α-sinensal, methyl
thymol, as well as the acetate esters bornyl, ɣ-terpinyl, geranyl,
citronellyl, and decyl acetates, were all found at concentrations of
less than 0.05%^[134]52.
The essential oil constituents of C. reticulata cultivated in Algeria
were reported to contain 24 constituents. Monoterpene hydrocarbons
accounted for the most abundant chemical group (89.56%). The main
components were limonene (67.04%), -terpinene (15.50%), and -pinene
(2.75%). Sesquiterpene hydrocarbons accounted for a minor quantity
(3.26%), where l-caryophyllene was the main constituent^[135]53.
The literature review on essential oil components in C. reticulata
cultivated in different regions corroborates some commonalities.
Consequently, limonene, a hydrocarbon monoterpene, is invariably the
most common ingredient in essential oils made from Citrus peels, making
up typically between 60 and 70 percent of the oil. However, limonene
can show lower levels, as in fully matured, ripened Indian fruit peels
of C. reticulata, in which it can decrease to 46%^[136]26. Also
prevalent are the following substances: monoterpenes, which typically
account for less than 15%, γ-terpinene, myrcene, and α-pinene, which
can reach an abundance of 6.0%, 3.6%, and 1.5%, respectively.
Non-terpenoid or terpenoid compounds (aldehydes, ketones, esters, fatty
acids, and phenyl) are reported to be present (1–10%) or absent
according to the cultivated region, but there are no commonalities
among studies reporting these compounds to have an impact on the
essential oil activity or not. Sesquiterpene hydrocarbons are the most
varied group of all known chemicals, and this is true for the majority
of species. The most prevalent groupings also frequently include
oxygenated monoterpene alcohols and monoterpene hydrocarbons.
The antioxidant potential of mandarin peels oil
This study looked into the antioxidant activity of mandarin peel oil
as-a-scavenger-potential-against-H[2]O[2]. The outcomes showed that
mandarin peel oil had H[2]O[2] scavenging capacity at a concentration
of 1000 µg/mL increased in an exceedingly dose-dependent manner,
compared with a standard-ascorbic-acid (IC[50] = 139.2 µg/mL). This
means that the higher the concentration of the oil, the more
effectively it scavenges the H[2]O[2] radicals (Fig. [137]4A).
Figure 4.
[138]Figure 4
[139]Open in a new tab
The H[2]O[2] scavenging activity of both the mandarin peel oil and the
standard increased in a concentration-dependent manner (Fig. 4A).
Interestingly, at a concentration of 1000 μg/mL, mandarin peel oil
exhibited the highest superoxide removal action, with an IC50 value of
176.2 μg/mL (Fig. 4B). This indicates that the oil was more effective
at scavenging the superoxide radicals than the standard, ascorbic acid.
The SOD activity of both the mandarin peel oil and the standard also
increased in a concentration-dependent manner (Fig. [140]4B).
Interestingly, at a concentration of 1000 µg/mL, mandarin peel oil
exhibited the highest superoxide removal action, with an IC[50] value
of 176.2 µg/mL (Fig. [141]4B). This indicates that the oil was more
effective at scavenging the superoxide radicals than the standard,
ascorbic acid.
Overall, these findings indicate that mandarin peel oil is a potent
antioxidant with a high ability to scavenge H[2]O[2] and superoxide.
Evaluation of the in vitro scabicidal potential of mandarin peels oil
According to in vitro data, the mandarin peel oil (20%) achieved a
remarkable acaricidal impact. The mites displayed a slow movement that
began at one-hour post-application-(PA) and terminated at 24 PA via 99
percent death rates, as determined by microscopic analysis.
Evaluation of the in vivo efficacy of mandarin peels oil on infected rabbits
Sarcoptic mange, some chronic lesions, and scabs were visible on and
inside the ears of rabbits infected with Sarcoptes scabiei. These
animals suffered from itching, congestion, scratching, and anorexia,
while those treated with mandarin peel oil (20% peel oil in paraffin
oil) showed a gradual improvement in clinical symptoms from the fourth
day PA through the experiment's conclusion (three weeks-PA). The lack
of irritation, bleeding, scale formation, restlessness, and the
appearance of smooth skin and new hair growth were signs of the
recovery^[142]54. The ivermectin-treated animals, on the other hand,
gradually improved but did not completely eradicate the condition from
the seventh day PA till the investigation's conclusion (Fig. [143]5).
Figure 5.
[144]Figure 5
[145]Open in a new tab
Inspection of mange-infected rabbits under a microscope, (A) control
group (paraffin oil), (B) mandarin peels oil group (20% peels oil in
paraffin oil), (C) ivermectin group (5% ivermectin).
On the fifth day PA, each the peels oil along with ivermectin groups of
infected animals' skin scrapings contained dead mites. By the time the
animals were checked once more, on day 10, the dead mites had totally
disappeared.
Histopathological investigation
The normal skin's epidermis and dermis were clearly visible in the
histological analyses of the normal group. The stratum corneum and
stratum granulosum made up the epidermis, and the reticular layer, hair
follicles, sebaceous glands, and sweat glands are visible in the dermis
(Fig. [146]6A). Skin samples from the control group, on the other hand,
displayed a changed histology, which is usual for this parasite
infection^[147]55. Skin erosion could be seen as a result of the
stratified squamous epithelial sloughing, hyperkeratosis, akanthosis,
and folded, seemingly injured skin. Moreover, the epidermis,
inflammatory cellular infiltration, and hypergranulating dermis all
displayed necrotic debris mixed with various stages of mites
(Fig. [148]6B).
Figure 6.
[149]Figure 6
[150]Open in a new tab
Microscopical-examination-of-skin from-different-groups of-animals, (A)
normal-architecture-of the-skin: e; epidermis, d; dermis, h.f.;
hair-follicles, (B)
control-group-showing-skin-damage-with-hyperkeratosis (red arrows),
mites-remnants-embedded-in the-skin (blue arrows),
hypergranulation-of-dermis (green-arrows),
severe-akanthosis-with-cellular-infiltration (black-arrows), (C)
mandarin-peels-oil group showing-restoration of-normal-architecture,
with-mild-infiltration (red-arrow), healthy-sebaceous-glands
(yellow-arrow) and hair-follicles (black-arrows), (D) ivermectin-group
showing-moderate damage-with-hyperkeratosis (red-arrow), mature-mites
with-eggs-remnants-embedded in-the-dermis (black-arrow)
surrounded-by-cellular-infiltration (green-arrow), and
some-sebaceous-adenitis (yellow-arrows).
Also, biopsy samples from the animals given mandarin peel oil (20% peel
oil in paraffin oil) demonstrated a slight cellular infiltration, a
lack of mites, an increase in the number of hair growth follicles, and
the appearance of normal sebaceous glands are signs of progress in the
skin's surface layers (Fig. [151]6C). The skin condition was improved
in the group that had ivermectin treatment, where only a few
inflammatory cells and hyperkeratosis were seen. In the outermost layer
of skin, the mites' remains could be seen embedded. Cellular filtration
and sebaceous adenitis were present in certain regions (Fig. [152]6D).
Gene expression results
The-pro-inflammatory-cytokines-(IL-1β,-IL-6),-the-pleiotropic-cytokine-
(IL-10)-and-the-monocyte chemoattractant-protein-1-(MCP-1) were all
downregulated in the animals treated with mandarin peel oil (20% peel
oil in paraffin oil), according to the results of q-PCR. On the other
hand, 2–sevenfold increases in ICAM-1, MMP-9, VEGF, KGF, and TIMP-1
were seen (Fig. [153]7).
Figure 7.
[154]Figure 7
[155]Open in a new tab
Relative gene expression in skin tissue of different animal groups
using qRT-PCR After normalisation to GAPDH, (A) I-CAM, (B) IL-1, (C)
IL-10, (D) MCP-1, (E) TIMP-1, (F) MMP-9, (G) KGF, (H) IL-6, and (I)
VEGF. In comparison to the healthy control group, the data show an
increase in expression by a factor of two. The mean ± SD are shown as
bars. A one-way ANOVA test is used to determine whether there's a
significant difference between categories, via (a) p < 0.05 in contrast
to the normal control grouping and (b) p < 0.05 in contrast to the
marketplace drug-induced category.
Molecular docking study
The methodologies of molecular docking intend to predict the best
binding orientation of a ligand to a receptor. It proposes several
suitable poses of the ligand within the active or docking site of a
receptor molecule.
Construction of protein–protein interaction (PPI) network
The created PPI network comprised of 296 nodes and 1725 edges, which is
illustrated in Figs. [156]S1–[157]S3.
Hub gene expression analysis
The cytoHubba plugin Cytoscape is considered a useful exploring
interface for the most important nodes in the PPI networks, it used to
determine the hub genes using ranking methods ,The results shown in
(Table [158]3) demonstrated that 16 nodes repeated in more than
analysis method, regarding the occurrence, IL1B possessed the highest
score as it appeared in 10 methods from the 12 methods followed by Il6
and TNF-α with score of 9 for each, while CD4 appeared 8 times, IL10
and IL2 seven times (Figs. [159]S2 and [160]S3). In protein–protein
interaction networks, it is believed that the most connected nodes
(hubs) are the key players, being responsible for the most extensive
pathological effects^[161]56 a circular layout for the filtered nodes
revealed that TNF-α and IL6 possessed the highest node degree in the 16
nodes (Fig. [162]8A), the highest occurred protein in cytoHubba
analysis IL1B with TNF-α and IL6 were chosen for in silico molecular
modelling^[163]57,[164]58.
Table 3.
List of the protein coding genes present in at least two methods from
twelve different methods of the cytoHubba plugin Cytoscape.
No Name Occurrence
1 IL1B 10
2 IL6 9
3 TNF-α 9
4 CD4 8
5 IL10 7
6 IL2 7
7 IL4 6
8 STAT3 5
9 CD8A 4
10 CSF2 4
11 ALB 3
12 CASP3 3
13 DSG1 3
14 TSLP 3
15 CCL3L3 2
16 FLG 2
[165]Open in a new tab
Figure 8.
[166]Figure 8
[167]Open in a new tab
(A) Through a circular network design, the margins represent
interactions between proteins, and the nodes serve as the hub protein
criteria. Each protein's connectivity is represented by the dimension
of the nodes; the larger the node, the greater its connection with
other nodes in the network, (B) Functional enrichment analysis of
filtered 16 protein coding genes by ShinyGO
([168]https://www.genome.jp/kegg/, accessed on 12 September 2022);
([169]http://bioinformatics.sdstate.edu/go/, accessed on 13 September
2022, a graphical gene set enrichment tool).
Gene ontology and enrichment analysis
The Gene Ontology (GO) is considered a computational bioinformatic
model of biological systems, beginning with the molecular level
reaching to the organism level, GO aims to provide knowledge about the
functions of gene products, namely, proteins and non-coding RNA
molecules. GO is organized in three aspects. GO Molecular Functions
(MF) describe activities that occur at the molecular level, Biological
Processes (BP) represent the larger processes or ‘biological programs’
accomplished by multiple molecular activities and Cellular Components
(CC) which are the cellular structures in which a gene product performs
a function, the specific genes expressed in each cell define the
identity and functionalities of that cell. Regulation of transcription
is highly complex and leads to differential gene expression in specific
cells or under specific conditions^[170]59. The analysis of the
selected genes revealed that Positive regulation, and phosphorylation
of STAT on JAK/STAT pathway were the top biological process in the same
order while Interleukin 6 receptor complex was the top molecular
component followed by keratohyalin granule and T cell receptor complex.
For the Molecular function category screened genes were correlated with
interleukin 4 and 8 receptor binding followed by toxic substance
binding, Finally, the KEGG pathway for the selected protein coding
genes were found to be involved in inflammatory bowel disease, Malaria
and Legionellosis. (Fig. [171]8B).
Docking with PDB ID: 6Y8M
The X-ray crystallographic structure of (IL-1β) complexed with its
ligand was obtained from the Protein Data Bank
([172]http://www.rcsb.org/pdb/,code 6Y8M). The ligand was re-docked in
an active pocket at an acceptable RMSD of 1.311 and an energy score of
-5.870 kcal/mol in five interactions of hydrogen bonds along with one
ionic bond interaction to verify the results of our research. The
involved amino acid residues in the-H-bond-interactions were Thr 147,
Met 148, Gln 149, and-Arg 11 as H-acceptor, and another one
with-Met-148 as-H-donor, while the ionic interaction was encountered
with Arg 11. The-dock-score-of-the-twenty compounds against 6Y8M is
summarized in Tables [173]S1–[174]S4. According to docking outcomes,
compound 15 (geraniol) had a docking score of − 5.881 kcal/mol, which
was less favorable than the kinetic energy obtained by the
co-crystallized ligand (Table [175]S5),
with-two-hydrogen-bond-interaction one as H-donor with Asn 108 and
other as H-acceptor with Lys 109, meanwhile compound 16 (1-decanol)
achieved approximately similar energy score of − 5.625 kcal/mol when
compared with the co-crystalized ligand showing three hydrogen bond
interactions as H- acceptor through the hydroxyl group of 1-decanol and
the amino acid residues Gln 149,Thr 147 and Arg 11 which resemble the
interactions of the-co-crystalized-ligand (Fig. [176]9A,B).
Figure 9.
[177]Figure 9
[178]Figure 9
[179]Open in a new tab
(A) 2D actions and 3D docking represent compound 15 (geraniol) in the
successful pocket location of IL-1 (PDB: 6Y8M), (B) 2D actions and 3D
docking represent compound 16 (1-decanol) in the successful pocket
location of IL-1 (PDB: 6Y8M), (C) 2D actions and 3D docking present in
compound 15 in the successful pocket location of IL-6 (PDB: 1ALU), (D)
2D relationships and 3D docking present in compound 16 in the
successful pocket location of IL-6 (PDB: 1ALU), (E) 2D relationships
and 3D docking present in compound 17 in the successful pocket location
of IL-6 (PDB: 1ALU), (E) 2D relationships and 3D docking represent
compound 16 in TNF- successful pocket location (PDB: 2AZ5), and (F) 2D
relationships and 3D docking present compound 15 in GST successful
pocket location (PDB: 2AZ5) (PDB: 3EIN).
Docking with PDB ID:1ALU
The X-ray crystallographic structure of IL-6 complexed with its ligand
was made available by the Protein Data Bank
([180]http://www.rcsb.org/pdb/, code 1ALU) (tartaric acid). The
co-crystallized ligand (l-( +)-tartaric acid) posture was predicted by
the docking method with an RMSD-of-1.758-and-an-energy-score-of
− 4.191 kcal/mol. In the style of contacts shown in Table [181]S6,
H-acceptor-interactions with Arg-182 and-Arg-179 were present, as were
ionic interactions and one-hydrogen-bond-with-Gln-acting
as-the-H-donor. It's interesting to note that many the 20
phytochemicals' docking results showed strong affinity for the
receptor, with scores similar to the co-crystallized ligand (Table
[182]S2). It is worth mentioning that both compounds 15 and 16
exhibited better affinity towards the binding site of IL-6 than the
ligand, as they showed ΔG of − 4.372 and − 4.401 kcal/mol
respectively. The hydrogen bond interactions appeared as three hydrogen
bond acceptors with amino acid residues Arg 179 and Arg 182 in both,
which match the interaction pattern of the co-crystallized ligand,
moreover, compound 8 also achieved a good energy score of
− 4.151 kcal/mol when compared with the ligand score of
− 4.191 kcal/mol. (Fig. [183]9C,D).
Docking with PDB ID: 2AZ5
The Protein Data Bank ([184]http://www.rcsb.org/pdb/, code 2AZ5)
provided the X-ray crystallographic structure of (TNF-) complexed with
its ligand. It was demonstrated that the co-crystallized ligand was
associated with 16 residues of amino acids and attached inside a small
pocket, with seven of those residues coming from-chain-A and the
remaining nine-from-chain-B, including-six-tyrosine-residues, from each
subunit of the TNF- dimer. This inhibitor works by attaching to the
cytokine's active trimer form, stimulating its dissociation into the
inactive dimer form, and stabilizing it^[185]60. The ligand was
re-docked in the active pocket to verify our research. During
interactions with receptors, the ligand established
hydrogen-bonds-with-Gln-61 as an H-donor-and-with-Tyr-119 as a pi-H
interaction. The co-crystallized ligand pose was predicted by the
docking method with the least RMSD and an energy score of
− 6.923 kcal/mol. Figure [186]9E, Table [187]S7. Table [188]S3
summarizes the dock scores of the 20 compounds against 2AZ5. Due to its
hydroxyl moiety and the amino acid sequence Gln 61, compound 16 was the
only one to obtain a dock score of − 5.129 kcal/mol with a single
hydrogen bond interaction as an H-donor (Fig. [189]9).
Docking with PDB ID:3EIN.
The-Protein-Data-Bank-([190]http://www.rcsb.org/pdb/,code 3EIN)
has-the-X-ray-crystallographic-structure-of-Drosophila melanogaster's
delta-class-GST. When glutathione was redocked, it revealed four
hydrogen bond interactions, two of which included Arg-67-and-Ser-66 as
H-acceptors and the-other-two-involving Glu 65-and Ile 53-as H-donors.
The ligand's energy score was − 5.945 kcal/mol in addition to the two
ionic interactions with Arg 67 and Glu 65 (Table [191]S8). based on the
investigated compounds' docking results reported in Table [192]S4,
compound 15 (geraniol), which displayed a G of − 5.861 kcal/mol,
exhibits strong similarities to glutathione in terms of energy score.
In a similar manner to the co-crystallized ligand, geraniol interacted
with the binding site of the GST receptor by forming two hydrogen
bonds, one with the amino acid residue Ser-66 as an H-acceptor-and
the-other with-Glu 65 as an-H-donor (Fig. [193]9F).
Molecular dynamics simulation
To validate the docking outcomes, the best-scoring docking pose of
geraniol with both GST and IL-6 were subjected to 50 ns-long MD
simulation. As shown in Fig. [194]10, geraniol modelled structure
achieved stable binding inside the binding site of each protein with
RMSD profiles (~ 2.5 Å) comparable with that of the co-crystallized
ligands (~ 1.7 Å). These results suggest geraniol as a probable
inhibitor of both GST and IL-6.
Figure 10.
[195]Figure 10
[196]Open in a new tab
RMSDs of geraniol inside both GST and IL-6 in comparison with the
co-crystalized ligand of each protein [(A) and (B) respectively] over
the course of 50 ns-long MD simulation.
In silico druglikeness of compounds 15 and 16.
Various physicochemical properties of a given drug may have a
significant impact on its bioactivity, as they are closely related to
interactions between the drug and its potentially suspected target.
Recently, in silico approaches introduced a powerful tool for drug
discovery to assess the proposed pharmacokinetics (ADME) of compounds,
which play a vital role in their pharmacological activities, especially
at the early stages of screening for lead compounds. Consequently, the
measurement of these parameters is of great value in the selection of
an efficient drug candidate. Lipinski and Veber’s rules are successful
tools to perform such screening, as Lipinski’s rule of five states that
a compound has drug-like activity if at least three of the following
criteria are achieved: a molecular mass less than 500 Da, a maximum of
five hydrogen donors, a maximum of 10 hydrogen bond acceptors, and a
partition coefficient between octanol and water (LogP (o/w)) smaller
than 5. According to Veber’s rule, a compound is orally active if it
has 10 or fewer rotatable bonds and a polar surface area (PSA) greater
than 140 Å. For predicting drug-like properties, we used Reaxys. The
screening of compounds 15 and 16 revealed that all of them complied
with Lipinski and Veber’s rules (Fig. [197]11).
Figure 11.
[198]Figure 11
[199]Open in a new tab
In silico druglikeness (Lipinski and Veber rules) of compounds 15 and
16.
Discussion
Essential oils (EOs) are generally well tolerated, as evidenced by
their widespread use in food, hair, and skin preparations^[200]61. In
comparison to conventional drugs, EOs are less likely to cause
resistance due to their multiple active components^[201]61. EOs may
have antibacterial, anti-inflammatory, and antipruritic properties in
addition to their scabicide properties^[202]8,[203]14. All these
adjuvant properties are especially appealing for the treatment of
scabies.
As a result of mites burrowing deeply into the skin, scabies
pathogenesis is complicated and involves a number of mechanisms,
including parasite persistence, which has an impact on both the
structure and function of skin^[204]62. All these elements work
together to make treatment ineffective, especially given that most
synthetic medications kill mites rather than altering the immune system
or promoting tissue repair. In light of this, plant-derived
phytochemicals can operate as safe substitutes for synthetic options
for the eradication of infectious diseases due to their broad
therapeutic potential and negligible adverse effects^[205]63. Citrus
fruits have reportedly been found to have immunostimulatory,
anti-inflammatory, antimicrobial, and antioxidant
properties^[206]64–[207]66. It had considerable antibacterial efficacy
against S. aureus and Candida-skin-infections,
including-oral-and-vaginal-candidiasis^[208]65,[209]67–[210]70.
Fascinating studies have shown that Citrus oil may change the way that
inflammatory responses are expressed, suppressing pro-inflammatory
cytokines and enhancing skin's defensive barriers^[211]71. No
investigation has yet been conducted, to the best of the information we
have, on the acaricidal-potential-of mandarin peel oil against
Sarcoptes scabiei.
Therefore, the current study examined the GC/MS makeup of mandarin peel
oil and assessed the oil's ability to kill Scabies-mites-in-both in
vitro-and in vivo testing. There were no symptoms of skin irritability,
inflammation, or unease during or after the application of mandarin
peel oil. Our findings showed that orange peel oil could have a
substantial acaricidal effect on Sarcoptes scabiei mites 24 h after
application. The animals' skin began to exhibit healthy symptoms after
the mites died, including the cessation of
inflammation-and-hyperkeratosis, the emergence of new-skin-layers,
and-the beginning of new hair growth. This occurred at the same time as
reports of the effective treatment of rabbit mange^[212]72. This full
recovery was seen after 3 weeks, whereas the ivermectin group's healing
continued until the completion of the experiment (4 weeks) without
leading to full recovery. The histopathological findings revealed that
the dermis and epidermis of the treated animals improved, inflammation
cells decreased, and mite remains were not present in the skin layers.
The death of mites, as well as the absence of inflammation, pruritis,
skin damage, and scale formation, are the primary causes of the
improvement^[213]55. In contrast, the skin layers of the ivermectin
group saw gradual alterations throughout therapy, and at the end of the
investigation, some inflammatory cells as well as traces of deceased
mites were still visible. This could be explained by the potent
anti-mite effects of ivermectin as well as the common itching and
allergic reactions brought on by topical deltamethrin use, which
extends inflammation and causes additional delays the emergence of good
indicators^[214]73.
Epidermal-keratinocytes-as the-first line-of defense against hazardous
external invaders must be used to obtain understanding of
the-modulative-effects-of mandarin peel oil on the-pathophysiology-of
scabies. To recognize various infections and launch immune responses,
keratinocytes generate recognition receptors on their surfaces. These
receptors allow them to secrete cytokines, chemokines, and
anti-microbial peptides that help attract inflammatory cells^[215]74.
Any imbalance in the activity of keratinocytes, which is crucial for
the control of skin immunological homeostasis, might lead to illness.
Our results show that when exposed to live digging scabies mites or
their waste products (including saliva or eggs as well), a significant
amount of genes in the skin fibroblasts and keratinocytes change their
expression, which further activates other cell types^[216]75. Hence, in
response to scabies, many other skins cell categories, such as lymphoid
cells, endothelial cells, or LCs, and dendritic cells, have complex
interactions (cross talk), which results in the development of
inflammatory and oxidative stress states. This could increase reactive
oxygen species like H[2]O[2], which leads to lipid peroxidation and
negatively affects the skin's structure and permeability. In our
investigation, faster clinical and parasitological recovery confirmed
the potential antioxidant action of mandarin peel oil by restoring the
altered oxidant/antioxidant balance in treated animals to normal.
Antioxidants are believed to hasten wound healing by reducing oxidative
stress on the wound. They are essential in preventing harm from being
done to biological elements like DNA, proteins, lipids, and bodily
tissue when reactive species are present^[217]31. Because the elevated
levels of ROS at the site of injury are the main promoters of collagen
disintegration, the breakdown of the extracellular matrix (ECM), a
decrease in vascular development and re-epithelialization, and a rise
in cytokines that are pro-inflammatory, all of which extend
inflammation, an extract with ROS scavenging potential could be a key
component of the healing protocol^[218]31.
The anticipated mechanism of action of mandarin peel oil on
scabies-infected rabbits was shown schematically in Fig. [219]12.
Infiltrating mites, based on reports, activate the skin's
keratinocytes, and exhibit a capacity to suppress the immune system's
response by lowering the expression of genes of i-CAM-1, an
intracellular adhesion molecular structure noticed on endothelial
surface cells. This decreases the blood-supply and-immune cells to the
penetration site and lessens the protective abilities of both
lymphocytes and neutrophils. On the other hand, an infection increases
MCP-1, a chemokine that stimulates immune cells and causes
inflammation^[220]76. Clinical signs are not noticed for 4–6 weeks
after an individual has been diagnosed with scabies mites. This is
since the regulatory T cells (type 1) are induced to produce IL-10, a
cytokine with anti-inflammatory properties that is required by humans
to prevent inflammatory and autoimmune illnesses^[221]77. Furthermore,
mite products that sensitize keratinocytes are likely to raise the
production of VEGF, which is additionally induced by the mites to raise
angiogenesis. The mites raise the flow of blood in the region in order
to get the nourishment they require from the food being consumed, which
worsens inflammation^[222]77. A delayed re-epithelialization of the
wound is the result of decreased KGF receptor signaling, which also
lowers the rate of proliferation of epidermal keratinocytes along the
wound edge. Matrix metalloproteinase (MMP-9) is one of a set of
hydrolase enzymes that are expressed in many severe conditions,
including wounds, osteoarthritis, ischemia, and viral disorders. The
inflammation also significantly increases MMP-9 levels^[223]78. Nearly
all parasite infections use MMP-9 to remodel tissue, which often slows
down the production of ECM molecules including collagen II and
aggrecan^[224]79. TIMP-1 (tissue inhibitor of met-alloproteinase)
tightly regulates the biological activities of MMPs, and proteolysis
results from an imbalance in the MMPs/TIMPs ratio^[225]80. Reversing
the activation of this network of interrelated genes may therefore be a
useful treatment approach to slow the spread of scabies. When CSE was
applied topically, the expression of IL-1, 6, 10, VEGF, MMP-9, and
MCP-1 significantly decreased, whereas the expression of i-CAM-1, KGF,
and TIMP-1 significantly increased.
Figure 12.
[226]Figure 12
[227]Open in a new tab
The suggested mechanism for the effect of mandarin peel oil on
scabies-infected rabbits.
The outcome was an improvement in host immunity against invading mites,
a decrease in pro-inflammatory cytokines and an increase in
anti-inflammatory ones, which could reverse the unfavorable symptoms
and lead to improved re-epithelialization, rapid recovery, and a
decrease in inflammation.
Conclusion
With instances of treatment failure and the emergence of resistance,
controlling scabies effectively using the available acaricidal
medicines has proven to be extremely difficult. With a biocidal
performance comparable to that of traditional synthetic treatments,
this study demonstrated the mandarin peel oil's acaricidal efficacy
against mange mites in rabbits. The work examined the composition of
the oil and revealed the presence of different hydrocarbons and their
oxygenated forms, with proved biocidal activities. Additionally, the
oil has been tested against naturally infected rabbits with mange using
different techniques and proved higher efficacy and safety compared to
market agents. The mandarin peel oil presents an ideal alternative to
commercial medications used for the control of arachnids that can harm
humans and animals while being economical, safe, and environmentally
friendly. These candidates can be successfully employed to create novel
biocides for applications in agricultural improvement and livestock
protection.
Supplementary Information
[228]Supplementary Information 1.^ (11.6MB, zip)
[229]Supplementary Information 2.^ (2.4MB, docx)
Acknowledgements