Pharmacological Research - Modern Chinese Medicine 8 (2023) 100272

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Pharmacological Research - Modern Chinese Medicine

journal homepage: www.elsevier.com/locate/prmcm

“Botanical description, chemical composition, traditional uses and

pharmacology of Citrus sinensis: An updated review”
Priti Dongre a,∗, Chandrashekhar Doifode a, Shaily Choudhary b, Neeraj Sharma b
a
Shri Sachhidanand Shikshan Sanstha’s Taywade College of Pharmacy Koradi, Nagpur, Maharashtra 441111, India
b
Patel College of Pharmacy, Madhyanchal Professional University, Ratibad, Bhopal, Madhya Pradesh 462044, India

a r t i c l e i n f o a b s t r a c t

Keywords: Introduction: Citrus sinensis (L.) Osbeck is one of the extensively studied plants in the Rutaceae family. It is a
Citrus sinensis popular and commonly used ingredient in Traditional Chinese Medicine with preparations derived from peels,
Phenolic compounds young fruit, mature fruit, flower, and other tissues. The plant is known for numerous activities such as antioxidant,
Toxicity
antibacterial, antidiabetic, antifungal, antiosteoporosis, hypocholesterolemic, antiobesity, etc. due to the presence
Mechanism of action
of phenolic acids, flavonoids, and essential oil components.
Pharmacological activity
Material and Method: The online databases (Scopus, Google Scholar, PubMed, Science Direct, and Web of Sci-
ence) search engine was searched using keywords: phytochemical compositions, traditional uses, antibacterial,
antiproliferative, hypocholesterolemic, insecticidal, cardiovascular, neurodegenerative diseases and toxicity with
sweet orange and Citrus sinensis. The available information was comprehensively studied and compiled.
Result: Scientific studies suggested that C. sinensis is a popular medicinal plant used by local and traditional com-
munities to get its nutritional and medicinal benefits. Crude extract, fractions, and isolated compounds showed
significant pharmacological potentials that can improve health when added to the diet.
Conclusion: Oranges are an incredibly valuable source of bioactive compounds that could have good potential
for incorporation into human food and hold the promise for the management of mild and deadly health condi-
tions. Further investigation may pave the way for the development of value-added products for current disease
management.

Introduction tant phytochemicals such as flavonoids, vitamins, and phenolic acids

confirming that food and medicine came from the same source [8].
The genus Citrus L., which belongs to the Rutaceae family consists About 48 million tonnes of C. sinensis (Orange) were produced
of different forms such as trees, shrubs, and herbs in the world [1,2]. worldwide in January 2021–2022 with China, Brazil, the European
The citrus genus is the most cultivated and traded variety in the world Union, Mexico, and United States being the top producers. Several na-
as a garden plant and one of the most important commercial fruit crops tions are using oranges primarily for juice extraction, which accounts
grown on all continents of the world [3–5]. Various species of Citrus for 1.7 million tonnes of orange production by January 2021–2022 [9].
are C. limon (lemon), C. medica (citron), C. aurantium (sour orange), C. Global C. sinensis production for 2022–2023 is projected to be 5% lower
paradise (grapefruit), C. reticulata (mandarin, tangerine), C. clementina at 47.5 million tonnes. This is because of decreased production in US and
(clementine) and C. sinensis (sweet orange). Amongst them, C. sinensis EU. The decreased production decreases fruit consumption and process-
(Orange) is extensively grown for its fruits, which have food and medic- ing worldwide [10].
inal properties [6]. Thirst-quenching fruit attracts all age groups with its multifold
C. sinensis probably originated from South East Asia and were cul- health benefits, mainly consumed fresh or in juice. C. sinensis fruit
tivated in China by 2500 BCE [7]. It is one of the most commonly em- is rich in vitamins, minerals, and dietary fibers which are essential
ployed ingredients in Traditional Chinese Medicine with preparations for normal growth and development of healthy life. Starting with the
derived from peels, young fruit, mature fruit, flower, and other tissues. pulp, seeds, leaves, and skin of the fruit, oranges have various advan-
All of these parts are processed to prepare food items in China and are tages. One noteworthy example is that a healthy, antibiotic-free product
popularly used in Chinese prescriptions due to the presence of impor- with no hazardous residues for humans can be produced by employ-

∗
Corresponding author.
E-mail address: [email protected] (P. Dongre).

https://doi.org/10.1016/j.prmcm.2023.100272
Received 4 March 2023; Received in revised form 26 May 2023; Accepted 3 June 2023
Available online 10 June 2023
2667-1425/© 2023 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)
P. Dongre, C. Doifode, S. Choudhary et al. Pharmacological Research - Modern Chinese Medicine 8 (2023) 100272

ing the proper amounts of orange waste and by-products as a supple- Traditional/ ethnomedicinal uses
ment in the poultry diet [11]. C. sinensis essential oil in clinical studies
showed reduced anxiety levels in patients of dental treatment as well C. sinensis is consumed all over the world as an excellent source
as in healthy volunteers submitted to anxiogenic situations [12]. Re- of vitamin C, which is a powerful natural antioxidant that builds the
cent studies indicate that C. sinensis supplementation may be useful as body’s immune system. It has been used traditionally to treat various ail-
a prophylactic agent against SARS-CoV-2 infection and complementary ments like constipation, cramps, colic, diarrhea, bronchitis, tuberculosis,
treatment for COVID-infected patients [13]. These few examples had cough, cold, obesity, menstrual disorder, angina, hypertension, anxiety,
proved the worth of the nutritional benefits of C. sinensis in everyday depression and stress, asthma, vomiting, fever, hiccoughs and for indi-
life. gestion [6,14]. In Chinese Traditional medicine, the orange has a long
Further, the current review highlighted primarily the pharmacologi- history as a cooling agent for coughs, colds, and respiratory disorders.
cal actions of C. sinensis, along with its botanical description, traditional It is used as a traditional symbol of good luck in China [18].
usage, phytochemicals present, applications, and toxicological studies.
Applications
Methodology
C. sinensis is rich in many valuable phytoconstituents and are widely
used for their beneficial effects.
Online databases (Scopus, Google Scholar, Pubmed, Science Direct,
Orange essential oils have many applications in different sectors. In
and Web of Science) were used with keywords such as phytochemical
the food industry, oil is used as an antifungal agent to avoid crop and
compositions, traditional uses, production, antibacterial, antiprolifera-
food deterioration during the transportation stage. In the cosmetic and
tive, hypocholesterolemic, insecticidal, cardiovascular, covid, neurode-
cleaning sectors, oils are commonly applied due to their aromatic prop-
generative diseases and toxicity with sweet orange and C. sinensis in the
erties. In the Pharmaceutical industry, oils find a place for their preven-
search up to February 2023. The available information was comprehen-
tive properties against some diseases like cancer, diabetes, antiviral, and
sively studied and compiled. This review also consolidates the evidence-
inflammations. In the chemical industry, the orange peel can be used to
based information on C. sinensis - collected from online databases (Sco-
produce methane, after the essential oil is extracted [19].
pus, Google Scholar, PubMed, science direct, and Web of Science up to
Essential oil is widely used in the preparation of toilet soaps, per-
February 2023).
fumes, cosmetics, and other home care products, personal hygiene prod-
ucts, or as a green solvent [20]. It is one of the popular oils in aroma
Botanical description therapy including massage, inhalations, and baths since ancient times.
In the textile, plastics, and paint industries, oil is used as a masking
Scientific classification of Citrus sinensis Kingdom: Plantae Division: agent in order to cover up undesirable odors. Oil is extensively used in
Magnoliophyta Class: Magnoliopsida Subclass: Rosidae Order: Sapin- pharmaceutical formulations for its flavoring properties. Recently, food
dales Family: Rutaceae Genus: Citrus [14] and packaging industries are using the oil for its natural antimicrobial
C. sinensis tree grows in tropical, semitropical, and warm temperate properties as consumer concerns increase and to ensure safe foodstuff
regions, becoming the world’s most widely cultivated citrus fruit tree. [21].
The tree is native to Asia and is now widespread throughout the Pacific Phenolic compounds: Several scientists proved that regular con-
and warm areas of the world. It is an evergreen flowering tree typically sumption of phenolic compounds present in orange reduces the risk
growing to 7.5 m, but occasionally reaching heights up to 15 m, con- of disease and is also beneficial for human health. Phenolic compound
sists of a compact crown with mostly spiny branches. Leaves are smooth, plays a noteworthy role to combat the incidence of diseases such as
dark green, 3–5 mm wide, 6.5–15 cm long alternate with toothed blades cancer, heart disease, hepatic injury, and neurodegenerative disorders.
differently shaped, oval or elliptical, connected to the stem by winged They are useful against many other health issues such as osteoarthritis
petioles. As copious oil is present in leaves radiates a strong character- and ocular disorders and act as phytoestrogen and anti-thyroidal com-
istic citrus odor. pounds. These compounds have strong antioxidant and free radical scav-
Flowers are small, waxy white, and fragrant which are axillary in enging activity and may have practical applications in the food industry
whorls of 6 (5 cm wide) with five white petals and 20 to 25 yellow by replacing the chemical antioxidant with this natural antioxidant [20].
stamens. Pectin is one of the compounds found everywhere in the peel part
Fruits are varied in their shape and size (from round to oblong). mainly in flavedo (epicarp), albedo (mesocarp), and edible portion (en-
These are mostly greenish in color after gaining their full size it shows docarp). They are mostly found in higher plants’ cell walls and are called
bright yellow to orange color instead of green. Fruits are round, 4– complex polysaccharides compounds. Pectin is produced industrially
12 cm, consisting of a leathery peel 6 mm thick, tightly adherent, pro- and is widely used as a thickening, coating, and encapsulating material
tecting the juicy inner pulp, which is divided into segments that may due to its stabilizing, gelling, and thickening characteristics in jam and
not contain seeds, depending on the cultivar. Fruits are full of flavor, confectionery products. Due to its film-forming capacity, pectin may be
fragrance, and juice [6,15,16]. used as a packaging material to improve the sensory attributes of some
The external coloured surface of the orange peel is called a flavedo, foods and for their shelf life [20].
whereas an inner white spongy structure is called an albedo. Flavedo
contains bags with essential oils (EO) mostly composed of limonene, Chemical composition
whereas the albedo has a foamy structure and features a high content
of pectin. Fruit is covered by skin-like tissue called an endocarp, which The chemical composition of C. sinensis varies with cultivars, year of
is composed of pulp and juice, where there are also membranes to de- harvest and postharvest storage conditions, planting area, fruit variety,
limit each of the segments. Peels are rich in structural polysaccharides part of the fruit, climate, and degree of maturation. Despite of this fruit
and related polymers, including cellulose [18–20% w/w based on dry is rich in many important phytoconstituents present in various parts of
solid (DS)], hemicellulose (14–16% w/w DS), pectin (20–22% w/w DS) C. sinensis viz leaves, seed, flower, juice, and peels. Different scientists
and lignin (5–7% w/w DS). It is also abundant in free sugars namely studied and reported several types of chemical compounds which in-
glucose, fructose, and sucrose (about30–36%w/w DS) as well as other clude the groups like volatile oil, flavonoids (e.g., flavanones, flavanone
components such as EO (0.3 –0.5% w/w DS) and proteins (up to 8% glycosides, and polymethoxylated flavones), carbohydrates, coumarins,
w/w DS), the peel being particularly plentiful in pectin (up to 42–43% peptides, fatty acids, steroids, alkanes, hydroxyamides, carotenoids, car-
w/w DS) [17]. bamates, vitamins and alkylamines [Table 1].

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P. Dongre, C. Doifode, S. Choudhary et al. Pharmacological Research - Modern Chinese Medicine 8 (2023) 100272

Table 1
Phytochemistry of C. sinensis.

Sr. No Class Compounds Plant part Reference

1 Volatile compounds Limonene, 𝛼-pinene,ϒ-Terpinene, 𝛼-Terpinene, Leaves, flower,peels [22–27]

𝛼-Terpineol,Neral, 𝛽-Pinene, Geranial, Myrcene, Linalool,
(-)-Carvone, Geranyl acetate, Valencene, □-Cadinene, Fernesol,
Sinensal,Vanillin,Guaiacol, Homofuraneol, Malic acid,Octanal,
Decanal, Acetaldehyde, Ethyl butanoate,Ethyl
etanoate,4-Acetyl-1-methylcyclohexene, Dimethyl
trisulphide,3-Mercapto-2-butanone,
4-Mercapto-4-methyl-2-pentanone, ϒ-Butyrolactone,
2-Phenylethnaol, Geraniol, 3- Methyl-1-pentanol,
3-(Methylthio)-propanol, р-Coumaric, Sabinene, Cis-
𝛽-Ocimene, Geranyl pyrophosphate, (-)−4S-Limonene, Linalyl
acetate, (3R)-(-)-Linalool, (3S)-(+)-Linalool, □−3-Carene, (E)-
𝛽-Ocimene, Terpinen-4-ol, Octopamine, Synephrine, Tyramine,
N-Methyltyramine, Terbutaline, 𝛼-Cubebene, 𝛽-Elemene,
𝛼-Carophyllene,1-Octanol, 𝛽-Terpineol, Nerol, E-Carveol,
𝛽-Geraniol, Ethylbutanoate, Ethyl acetate, Ethyl propanoate,
Methyl butanoate, Hexanal, 2E-hexanal, Heptanal, Udecanal
2 Flavonoids Hesperedin, hesperetin, naringin, naringenin,sakuratin, Peels [28–38]
3,5,4′-Trihydroxy-7, 3′ -dimethoxy-flavanone-glc, Narirutin
4′-glucoside, Narirutin, Isosakuranetin, Tangeretin, Nobiletin,
Chrysoeriol, Limocitrin, Limocitrol, Quercetagetin,
Isosakuranetin,
Daidzein,Genistein,Formononetin,Isoformononetin,Biochanin
A, prunetin, glycitein, Daidzin, Sissotrin,
Glytin,Ononin,Genistin
3 Steroids 𝛽 -Sitosterol, 𝛽-Sitosterol-3-O- 𝛽-d-glucopyranoside Leaves [31]
4 Hydroxylamide, alkane, Tetracosane, (E)-N-(1,3,4,5-Tetrahydroxyhexadecan-2-yl) Leaves [31]
Fatty acids dec‑4-enamide
5 Coumarins Scoparone, Limetin, Osthol, Xanthotoxin, Bergapten, Peels roots [39–42]
Isopimpinellin, Bergaptol.
6 Peptides Citrusin-I, Citrusin-II, Citrusin-III, Peels [43]
7 Carbohydrates Glucose, Sucrose, Fructose, Galactose Fruit [44]
8 Carbamates, alkylamines Carbosulfan, Carbofuran,3-Hydroxycarbofuran, Dibutylamine Fruit [45]
9 Carotenoids Zeaxanthin, Zeinoxanthin, 𝛽-Cryptoxanthin, Lutein, Fruit, [46]
Auroxanthin, 𝛼-Carotene
10 Minerals Potassium, magnesium, calcium, sodium, iron, magnese, Natural and commercial juices, [47]
magnesium, zinc seed
11 Vitamins A, D, E, K, B1, B2, B3, B5, B6 and C Fruit, Seed [48]

The primary and secondary metabolome of C. sinensis is chemi- Trolox equivalents (TE) as the reference antioxidant, it also was able
cally diverse. Numerous structural classes and thousands of unique com- to scavenge DPPH radicals with an antioxidant power corresponding to
pounds have been identified and described [49]. These studies’ findings 14.39± 0.19 μM TE and eliminated about the 87% of hydroxyl radical
undeniably showed that oranges are a good source of chemicals that generated at 16.40 μM TE. Five C-glycosyl flavones—lucenin-2, vicenin-
could be used as functional additives in human health products and po- 2, stellarin-2, lucenin-2–41 -methyl ether, and scoparin-one 3‑hydroxy-
tential medication candidates. 3-methylglutaryl glycosyl flavonol, 3‑hydroxy-3-methylglutaryl glyco-
syl quercetin, and one flavone O-glycoside— the 7-O-neoesperidoside
Pharmacological activity of chrysoeriol are responsible for the aforementioned antioxidant ef-
fects [51]. An amperometric biosensor for the quantification of the scav-
A large number of therapeutic potentials of C. sinensis have been re- enging capacity of orange juices displayed values for natural orange
ported and they might be due to the presence of a variety of phytocon- juice at 1.01 mM of IC50 /μM 10.6± 0.5 while one bought commer-
stituents. The therapeutic potentials were described comprehensively in cially gave values at 2.27, 1.56, 0.91, 2.22 mM of IC50 values 17.0 ±
the further sections. 0.8 μM, 17.2 ± 0.7 μM, 26.4 ± 0.8 μM, and 18.9 ± 0.9 μM, respectively.
Substances such as ascorbic acid (IC50 30.3± 0.9 μM), caffeic acid (no
Antioxidant activity significant results), gallic acid (no significant results), ferulic acid (no
significant results), curcumin (no significant results), catechol (no sig-
Over the years, research on antioxidants and free radicals for under- nificant results), quercetin (no significant results) were used for control
standing their role has grown exponentially. The antioxidant capacity and also for comparing. The orange juice showed the best activity [52].
of C. sinensis juice purchased from the local market was determined C. sinensis seed extract exhibited antioxidant activity using reducing
by the kinetics of hydrogen peroxide scavenging method showed k val- power and DPPH radical-scavenging assays, gallic acid (IC50 = 29.5 μM)
ues of 1.2±0.3 × 103 s−1 and 0.4± 0.1 × 103 s−1 . H2 O2 may be used was used as a control standard [53]. The flavonoid content of several
as a relative indicator and regulator of antioxidant activity due to the methanolic extract fractions of Navel orange peel (flavedo and albedo
substantial variance in kinetic constants of H2 O2 elimination in dietary of Citrus sinensis) was first analyzed phytochemically and then eval-
samples [50]. In different study, the antioxidant activity of Moro C. uated for its antioxidant activity in vitro. The chemical structures of
sinensis crude juice was determined by different methods (2,2-diphenyl- the fractionated constituents were determined by comparing their re-
1-picrylhydrazyl (DPPH), OH, 2,2′-azino-bis(3-ethylbenzothiazoline-6- tention times and the obtained UV spectral data with the available bib-
sulphonic acid (ABTS+ ) and Ferric reducing antioxidant power and the liographic information and further confirmed by detailed LC-DAD-MS
result showed that Moro juice efficiently scavenges ABTS radical cations (ESI+) analysis. The main flavonoid groups found within the fractions
reaching up to 64% of quenching corresponding to 14.30 μM using examined were polymethoxylated flavones, O-glycosylated flavones,

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P. Dongre, C. Doifode, S. Choudhary et al. Pharmacological Research - Modern Chinese Medicine 8 (2023) 100272

C-glycosylated flavones, O-glycosylated flavonols, O-glycosylated fla- tial for intracellular signaling defensive responses to pathogens
vanones and phenolic acids along with their ester derivatives. In ad- [65]. C. sinensis contains phytochemicals that may serve as a bar-
dition, the quantitative HPLC analysis confirmed that hesperidin is the rier against the build-up of ROS/RNS and eliminate them from
major flavonoid glycoside found in the orange peel. The antioxidant ac- the body. Through irreversible dehydrogenation, vitamin C di-
tivity of the orange peel methanolic extract fractions was evaluated by rectly reacts with O2 , HOO− and OH− clearing 1 O2 [66]. The an-
DPPH assay and the Co(II)/EDTA-induced luminol chemiluminescence tioxidant activity of vitamin carotenoids is also associated with
assay. Results showed that orange peel methanolic extracts possess mod- the restraining of free radical production and quenching 1 O2
erate antioxidant activity as compared with the activity observed for [66,67,68]. Reactive oxygen species can be effectively eliminated
the aglycones, diosmetin (EC50 71.79±13.58 mg and hesperetin (EC50 by flavonoids’ dual ability to serve as preventatives and chain
29.18 ± 2.80 7 mg). Quercetin (EC50 = 0.06 mg quercetin/mg DPPH) scission factors [62]. Moreover, limonoids work by reacting with
was used as a positive control and the tested aglycones exhibited a signif- free radicals to reduce their activity and prevent further chain
icantly higher hydroxyl scavenging activity than quercetin [54]. Oboh reactions, and then achieve the effect of free radical scavenging
G et al. investigated the DPPH antiradical action of C. sinensis juice. [69].
Results showed that C. sinensis juice exhibited 84.81% DPPH antiradi- (4) Chelators with transitional metals- Vitamin E in Citrus fruits may
cal effect at 100 μg/mL. The ascorbic acid (96.36%) was used as posi- chelate transitional metals, such as Fe2+- and Cu2+. It was re-
tive control showing 96.36% DPPH antiradical effect [55]. Citrus peel’s ported that vitamin E from Citrus fruits reduced ferrous iron to
bound phenolic content demonstrated DPPH scavenging at 1 mg/mL, ferric iron contents [66]. Aalikhani et al. [70] examined the iron
OH scavenging at 4 mg/mL, ability to chelate Fe2+ at 0.48 mg/mL, chelation activity of hesperidin and coumarin on the brain tissue
and prevention of Fe2+ induced lipid peroxidation in the pancreas at of iron-overloaded mice and found that hesperidin and coumarin
142.8 μg/mL [56]. Acetone-water extract from red oranges (C. sinen- strongly chelate excessive iron from the serum and deposit iron
sis, Torocco) exhibited an intracellular antioxidant activity of 85% in from the brain tissue which confirms that both the powerful op-
Caco-2 cells at a concentration of 50 mg/mL. Gallic acid and vitamin tions to chelate iron ions and to increase the activity of antioxi-
C were employed as positive control. The red-orange extract showed dant enzymes.
significant higher antioxidant activity than positive controls [57]. The (5) Ingredient synergism- The interaction or synergistic effect among
antioxidant activity of methanolic and ethanolic C. sinensis peel ex- the nutrients and/or bioactive compounds contained in citrus
tracts demonstrated significant levels of free radical scavenging activ- fruits may also contribute to their antioxidant capacity. Polyphe-
ity produced by ABTS, with values of 55.8% and 60.7%, respectively, nols had synergistic effects with vitamins C and E by their mu-
and DPPH scavenging activity based on its capacity as a hydrogen dona- tual reduction [66]. The interaction between naringenin and hes-
tor, with values of 70% and 80%, respectively. Ascorbic acid and water peridin of naval orange (C. sinensis Osbeck.) provides an exam-
(20–150 μL) were employed as a control [58,59]. The scavenging ac- ple of the synergism among phenolic compounds [71]. However,
tivity of dichloromethane extract, and diethyl ether, ethyl acetate, and more research is undoubtedly required to identify the underlying
n-butanol fractions ranged from 3 to 1.1 mg dry extract/mg DPPH. Ethyl mechanism of action. The benefits of C. sinensis as an antioxidant
acetate fraction exhibited the best antioxidant activity which is due to medicinal agent are demonstrated by the state of knowledge to-
the presence of C-glycosylated flavones, O-glycosylated flavones, poly- day. As a result, we support the use of C. sinensis as a nutraceu-
methoxylated flavones, O-glycosylated flavanones, and esters of pheno- tical ingredient.
lic acids [60]. All these findings clear that C. sinensis is a good source
of antioxidant compounds. Antibacterial activity

Mechanism of antioxidant action of C. sinensis Essential oil, crude extracts, and isolated compounds of C. sinensis
have been studied for antibacterial activity showing notable results. Us-
Numerous potential methods of antioxidant action of C. sinensis have ing the agar well-diffusion method, silver nanoparticles made from C.
been proposed in the available literature. We gathered the literature sinensis peel extract displayed a range of zones of inhibition against
data and summarised it in Fig. 1, which we based on our following dis- Escherichia coli (25 °C 12.5 mm, 60 °C 16.0 mm), Pseudomonas aerugi-
cussion. nosa (25 °C 11.7 mm, 60 °C 13.4 mm), and Staphylococcus aureus (25 °C
7.8 mm, 60 °C 9.2) [72]. According to a different study, silver nanopar-
(1) Inhibition of oxidant enzymes- C. sinensis may exhibit antioxi- ticles synthesized by mixing silver nitrate solution with C. sinensis juice
dant activity by inhibiting the oxidant enzymes such as nitric ox- for 2 h at 37 °C displayed minimum inhibitory concentration (MIC) val-
ide synthase (NOS), lipoxygenase (LOX), xanthine oxidase (XO), ues of 20 μg/mL for Bacillus subtilis and Shigella and 30 μg/mL for S.
cyclooxygenase (COX), NADPH oxidase (NOX), and myeloperox- aureus and E. coli. Antibiofilm activity of 80% to 90% was observed
idase (MPO). These enzymes are the main promotors of cellular at 25 μg/mL [73]. C. sinensis cold-pressed terpeneless (CPT) oil dis-
ROS/RNS [61]. Hesperetin is capable of reducing the cellular pro- solved in ethanol or dimethylsulphoxide (DMSO) displayed MICs for
duction of free radicals by inhibiting XO [62]. According to Lin Listeria monocytogenes at 0.3% and 0.25% v/v, and for Salmonella ty-
et al. [63] coumarins are able to decrease the cellular production phimurium at 1% v/v. Both ethanol and DMSO oil dispersion systems
of free radicals by inhibiting XO. Thus, polyphenols and other exhibited an intermediate MIC of 0.75% v/v for Lactobacillus plan-
phytochemicals may help C. sinensis antioxidant capability. tarum [74]. CPT at 0.5% v/v also showed an inhibitory zone effect
(2) Interaction with redox signaling pathways- The redox signaling at 10 L against S. aureus organisms that were sensitive to methicillin
pathway is one of the most important ways by which Citrus fruits (31.50± 3.02 mm), resistant to methicillin (65.83± 3.76 mm), and in-
exhibit their antioxidant activity. Studies have shown that both termediately resistant to methicillin and vancomycin (76.67± 4.08 mm
Hesperidin and Hesperetin counter oxidative stress by enhancing and 32.50 ± 2.74 mm, respectively). The inhibition of bacterial growth
the antioxidant cellular response by involving the Nrf2 pathway in the plates containing test oil was judged by comparing it with the
[64]. visible growth of untreated control plates [75]. When used against P.
(3) Direct reaction with ROS/RNS: Citrus bioactive compounds may aeruginosa, the essential oil containing 1,8-cineole and hydrocarbons
directly react with reactive oxygen species (ROS) and/or reactive demonstrated MIC90% ≥10% (v/v) [76]. Another study investigated that
nitrogen species (RNS), and show antioxidant activity. Higher 0.1 mL of C. sinensis oil inhibited E. coli (18 ± 2 mm), L. monocyto-
concentrations of ROS/RNS play a crucial role in the etiology of genes (27± 2 mm), B. cereus (19± 2 mm), and S. aureus (14± 3 mm)
many human diseases, whereas low levels of ROS/RNS are essen- with varying inhibition diameters. [77]. By molecular distillation and

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P. Dongre, C. Doifode, S. Choudhary et al. Pharmacological Research - Modern Chinese Medicine 8 (2023) 100272

Fig 1. A schematic representation of the potential antioxidant mechanism of Citrus sinensis. Five pathways are suggested in existing literature: (1) inhibition of
oxidant enzymes, reducing the cellular production of ROS/RNS, (2) interaction with redox signaling pathways, leading to the cellular antioxidant response, (3) direct
reaction with ROS/RNS as a ‘‘free radical scavenger”, (4) chelate with transitional metals yielding less oxidative damage, (5) interaction of the ingredient synergism,
which influence the whole antioxidant system. ∗ NOS- Nitric Oxide Synthase, LOX- Lipoxygenases COX- Cyclooxygenases, NOX-NADPH oxidase, NADPH- Nicotinamide
Adenine Dinucleotide Phosphate, MPO-myeloperoxidase Nrf-2 – Nuclear factor erythroid 2-related factor2, Nf-kB- nuclear factor kappa B, SOD-superoxide dismutase,
CAT-catalase, GPx-glutathione peroxidase, Prx-peroxiredoxin, GR-glutathione reductase, TR-thioredoxin, ROS-Reactive oxygen species, H2O2,- hydrogen peroxide,
O2− superoxide anion, OH- hydroxyl radical, 1O2- singlet oxygen, NO- Nitric oxide, ONOO_ - peroxynitrite, HOCL- Hypochlorous acid, CO2−3- Carbonate, ROO_-
Peroxy radical, ↑-Increase, ↓-Decrease, damage.

column chromatography Sweet orange oil and its primary compounds sitive strain whereas for the resistant strain was >1 μL/mL. Ethambutol
decanal (73.36%), octanal (78.12%), and linalool (90.61%) inhibited displayed a MIC value of 2 μL/mL for the sensitive strain and for the
the growth of bacteria such as E. coli (MIC 100–25 μg/mL; MBC 200– resistant strain was >32 μL/mL. Finally, rifampicin gave a MIC value of
50 μg/mL), S. aureus (MIC 100–50 μg/mL; MBC 200–100 μg/mL), Sac- 0.60 μL/mL for the sensitive strain and a MIC >2 μL/mL for the resistant
charomyces cerevisiae (MIC 100–6.25 μg/mL; MBC 200–25 μg/mL), As- strain. In this investigation, the examined extracts had lower activity
pergillus niger (MIC 50 μg/mL; MBC 200–100 μg/mL) and no activity than the standard medications [84]. C. sinensis leaf extracts in acetone
for Penicillium citrinum [78]. A blend of (1:1 v/v) of C. sinensis and C. and hexane demonstrated 27 mm inhibitory zones against Helicobacter
bergamia essential oils displayed inhibitory activity with a MIC value of pylori. As a positive control, clarithromycin (0.05 g/mL) was applied
0.25%−0.5% (v/v) and a minimum inhibitory dose (MID) of 50 mg/L and this antibiotic showed greater action than the examined extracts. It
against vancomycin-susceptible and vancomycin-resistant Enterococcus is important to recognize that active extracts may contain components
faecium and E. faecalis, respectively. The main components of the mix- with increased activity. The above-mentioned studies clearly demon-
ture were limonene (45%–73%), citral (0.7%–3%), and linalool (0.5%– strate C. sinensis’s broad antibacterial spectrum, supporting its usage as
15%) [79,80]. Terpene oil (10 μL) from C. sinensis essence was found an antibacterial agent.
to inhibit eleven Salmonella strains and serotypes in zones reaching Regarding the mechanism of action of the essential oil of C. sinen-
29.2 ± 3.7 mm (S. enteritidis, S. senftenberg, S. senftenberg, S. ten- sis, its antibacterial efficacy is directly linked to its lipophilic nature.
nessee, S. kentucky, S. heidelberg, S. montevideo, S. michigan, S. ty- It has been noted that gram-positive food-contaminating bacteria are
phimurium, and S. Stanley). The most predominant compound in the more sensitive to the EOs’ harmful effects compared to gram-negative
oil was d-limonene at a level of about 94% and myrcene (3%) was bacteria as both differ in their cell wall content. The antibacterial effect
the second-most prevalent component [81]. Strong antibacterial prop- of EOs has been reported due to disorganization of the plasma mem-
erties against L. monocytogenes, E. coli, S. enteritidis, P. mirabilis, and brane, potential interference with the electron transport system, efflux
B. cereus were exhibited by C.sinensis oil. [82]. An anti-acne formu- of DNA, RNA, and proteins, the disintegration of the proton pump, and
lation based on C. sinensis (3%), Ocimum basilicum L (5%) essential ATP depletion during biosynthesis as shown in Fig 2. [85].
oils, and acetic acid (12%) inhibited Propionibacterium acnes. This an-
tibacterial activity is due mainly to the 94.0% of limonene in C. sinen- Antiproliferative activity
sis, and limonene (2.54%), linalool (21.0%), and eugenol (7.17%) in
O. basilicum L [83]. The drug-sensitive (MIC 200 μL/mL), isoniazid- Juice of three different varieties of C. sinensis Moro, Tarocco, San-
resistant (MIC 25 μL/mL), and ethambutol-resistant (MIC 50 μL/mL) guinello were used for the preparation of standardized extract and
strains of Mycobacterium tuberculosis H37Rv were all susceptible to the studied for antiproliferative activity. Results showed the inhibition
peel hexane extract of C. sinensis. Streptomycin exhibited a MIC value of proliferation of human prostatic epithelial cell line PZ-HPV-7 at
of 0.50 μL/mL for the sensitive strain whereas a MIC >8 μL/mL for the 10−3 g/mL and lung fibroblast cell line of Chinese hamsters V79–4
resistant strain. Isoniazid showed a MIC value of 0.60 μL/mL for the sen- at 10−4 g/mL. Untreated controls just with 5 × 104 cells/well were

5
P. Dongre, C. Doifode, S. Choudhary et al. Pharmacological Research - Modern Chinese Medicine 8 (2023) 100272

Fig 2. Antibacterial Mechanism of action of essential oil of Citrus sinensis. ATP- Adenosine triphosphate, PI- Inorganic phosphate, ADP -Adenosine diphosphate,
DNA Deoxyribonucleic acid, RNA-Ribonucleic acid,↑-Increase, ↓-Decrease.

employed [86]. C. Sinensis Osb. (cv. Washington Navel and cv. San- -pentamethoxyflavone (IC50 52.72 ± 0.22 μM; AC50 94.62 ± 1.50 μM),
guinello) fruit juice at a concentration of 10% showed 90.5% antipro- 4′ ‑hydroxy-5,6,7,8,3′ -pentamethoxyflavone (IC50 47.41 ± 3.64 μM;
liferative activity against MCF-7 cells in human breast adenocarcinoma. AC50 87.10 ± 7.83 μM), 5‑hydroxy-3,6,7,8,3′,4′ -hexa-methoxyflavone
The juice of the same varieties at concentrations of 82.6% and 73% (IC50 4.16 ± 2.33 μM; AC50 5.90 ± 0.11 μM), 5‑hydroxy-3,6,7,3′,4′ -
showed 100% antiproliferative activity against the cell lines: K562 (hu- pentamethoxyflavone (IC50 2.07 ± 2.56 μM; AC50 5.87 ± 0.13 μM)
man chronic myelogenous leukemia) and HL-60 (human leukemia) [87]. [92]. Volatile oil rich in d-limonene obtained from blood oranges
Anti-proliferative and cytostatic effects of C. sinensis juice on the growth showed decreased in proliferation of colorectal cancer cells HT-29 at
of guinea corn radicle has been studied and the result showed the per- 1000 ppm [93]. Flavones and isoflavones showed inhibition of cell
centage of inhibition at concentrations of 5%, 10%, 20%, 40% and proliferation and induction of cell apoptosis on MCF-7 breast can-
60% (v/v) were 18.94%, 72.37%, 91.96%, 99.72%, and 100% respec- cer cells: 5‑hydroxy-3,6,7,8,3′,4′ -hexamethoxyflavone (IC50 2.50 μM;
tively. Juice at the concentration of 40% and 60% (v/v) showed cyto- (ECmin) 1.56 μM), 5,6,7,4′ -tetramethoxyflavone (IC50 10.5 μM; ECmin
static effects compared with the standard drug methotrexate (50 μg/mL) 3.15 μM), 3,5,6,7,8,3′,4′ -heptamethoxyflavone (IC50 > 50 μM; ECmin
which showed 77.71% of inhibition [88]. Polymethoxyflavones iso- 50 μM), 5,6,7,3′4’ -pentamethoxyflavone (IC50 > 50 μM; ECmin
lated from peels of C. sinensis showed activity on human lung can- >50 μM). Cell numbers (1 × 103 cells) were presented as control
cer cells. Nobiletin and 3,5,6,7,8,3′4’ -heptamethoxyflavone had a half [94]. The aqueous extract of C. sinensis L. (Osbeck) showed a signif-
inhibitory concentration (IC50 ) of 50 μM against H1299 cells, while icant cytotoxic effect on cells of the Yoshida ascites sarcoma [78]. 4′
5‑hydroxy-3,7,8,3′,4′-pentamethoxyflavone and 5‑hydroxy-3,6,7,8,3′,4′ -Geranyloxyferulic (0.141 ± 0.011 mg/g) obtained from C. sinensis de-
-hexamethoxy-flavone showed IC50 values of 16.5 μM against H1299 picted a potential chemopreventive effect [95].
cells. The above four flavonoids had shown the same activity to- C. sinensis has been shown to have significant promise as an an-
wards human lung cancer cells H441 and H460 [89]. Oil extracted by ticancer in a number of different investigations. Subramanian Pillai
cold pressed method from orange peels containing a mixture of non- et al. [97] provided evidence that p53-mediated G1/S arrest of DMBA-
hydroxylated polymethoxyflavones (75.1%) and hydroxylated poly- induced mammary carcinoma is elicited via p21 up-regulation. The in-
methoxyflavones (5.44%) induces apoptosis in breast cancer cells MCF-7 crease in the levels of p53 and p21 and the decrease in the levels of cy-
with a Minimal Effective Concentration (ECmin) of 9.25 μg/mL. Sim- clin D1, cyclin E, CDK2, and CDK4 upon tangeretin treatment in DMBA-
ilarly, a mixture containing only hydroxylated polymethoxyflavones induced animals indicates that the CDK inhibitor p21 WAF/CIP1 acts via
(97.2%) induces apoptosis in breast cancer cells MCF-7 with a Mini- p53 up-regulation. p21 WAF/CIP1 binds and inhibits the cyclin D1 and
mal Effective Concentration (ECmin) of 4.62 μg/mL [90]. Decreased de- E dependent kinases and regulates the G1 to S phase transition of the
velopment of tumor was studied in Apc (Min/+) mice (a mouse model cell cycle and hence cell proliferation [96] Fig. 3. In an azoxymethane-
for human familial adenomatous polyposis) when fed with 5% orange induced mouse model of colon cancer, hesperidin was found to alter the
peel extract containing 30% polymethoxyflavones (tangeretin 19.0%, anti-apoptotic scenario by modulating Bax/Bcl-2 ratio, together with en-
heptamethoxyflavone 15.24%, tetramethoxyflavone 13.6%, nobiletin hanced release of cytochrome-c and activation of caspase-3/9. Experi-
12.49%, hexamethoxyflavone 11.06 and sinensitin 9.16%) [91]. Sev- mental studies revealed that Hesperidin initiates apoptosis by inhibiting
eral compounds also obtained from peel extract presented inhibitory constitutively activated Aurora-A-mediated PI3K/Akt/GSK-3b pathway,
activities against the proliferation of cells (IC50 ) and induced apop- and mTOR (mammalian target of rapamycin) pathway coupled with the
tosis (AC50 ) of HL-60 cell lines: 3,5,6,7,8,3′4’ -heptamethoxyflavone stimulation of autophagy in colon cancer model [97] Fig 3. Numerous
(IC50 13.31 ± 1.28 μM; AC50 33.88± 0.01 μM), nobiletin (IC50 solid tumors induce vascular proliferation through the production of an-
1.50 ± 7.01 μM; AC50 > 100 μM), 3,5,6,7,3,4′ -hexamethoxyflavone giogenic factors, especially vascular endothelial growth factor (VEGF).
(IC50 20.59 ± 1.01 μM; AC50 92.10 ± 5.67 μM), 3′ ‑hydroxy-5,6,7,8,4′ Nobiletin showed an antiangiogenic activity with the LD50 value being

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P. Dongre, C. Doifode, S. Choudhary et al. Pharmacological Research - Modern Chinese Medicine 8 (2023) 100272

Fig 3. Anticancer activity of polymethoxyflavone- Tangeretin, Nobiletin and Hesperidin. ∗ CDK2- cyclin-dependent kinase inhibitors, p53 -protein 53, p21-protein 21,
ROS- Reactive oxygen species, ERK1/2- extracellular signal-regulated kinase 1/2, c-JNK-c-jun N-terminal kinase, VEGF- vascular endothelial growth factor, VEGF-R-
vascular endothelial growth factor receptor, HIF-1- Hypoxia-inducible factor-1, Bax: Bcl-2- B-cell lymphoma-2, GSK 3 𝛽-glycone synthase kinase, PD1- programmed
cell death protein 1, PDL1- programmed cell death protein ligand.

10 lg (24.9 nmol) per egg in a chick embryo chorioallantoic membrane pergillus niger (MIC≥ 1.6 mg/ml) [103]. Six separate varieties of C.
assay and inhibited angiogenic differentiation induced by VEGF and FGF sinensis yielded hydrodistilled essential oils that were effective against
by downregulation of ERK1/2 and c-JNK and activation of the caspase P. digitatum (ED50 2389.9–1004.6 ppm) and P. italicum (ED50 5407.5–
pathway [98] Fig. 3. Another study demonstrated that naringin treat- 3142.2 ppm). Using the agar dilution technique, essential oil from
ment decreased the level of secreted VEGF from MDAMB-231 breast peels prepared by cold-pressing demonstrated efficacy against Mucor
tumor cells [98]. T cells can kill the tumor cells by preventing PD-L1 hiemalis, P. expansum, and F. proliferatum with inhibition of 36.5%,
from binding to PD-1 by employing a PD-1 or PD-L1 inhibitor, such 34.9%, and 59.5% [104]. All the above studies showed that various
as tangeretin [99] Fig. 3. Above reports proved that C. sinensis a rich compound present in C. sinensis has remarkable antifungal activity. The
source of phytochemicals especially flavonoid is effective in the preven- increasing prevalence of fungal infections around the world has created
tion and treatment of cancers. Still, there is a need for more studies a need for new antifungal treatments, and in this line, C. sinensis offers
and data which would lead to the discovery of novel drugs from these a diversity of compounds with antifungal action.
phytochemicals.
Anti-Obesity activity
Antifungal activity
Recently few studies have shown the beneficial effects of C. sinen-
Crude extracts, oils, and isolated compounds of C. sinensis have sis and its bioactive compound in obesity and weight management. 4
shown remarkable antifungal activity. Hexane extract of injured peel weeks of treatment with Moro juice extract (Morosil®, 400 mg/die) re-
C. sinensis of L. Osbeck cv. Valencia or C. paradisa MacFaden cv has sulted to induce a significant reduction in body mass index (BMI). On
been prepared and the compound 3-[4‑hydroxy,3-(3-methyl-2-butenyl)- the other hand, when subjects were treated with Moro in extract form,
phenyl]−2-(E)-propenal is isolated. This isolated 7 μg of the com- BMI, body weight, waist, and hip circumference were significantly dis-
pound showed antifungal activity against Penicillium digitatum and parate from the placebo group. It was revealed that the synergistic effect
against Cladosporium cucumerinum on Si gel thin layer chromato- of bioactive compounds like flavone glycosides, ascorbic acid, antho-
graphic plates [100]. Ethanolic, aqueous, and petroleum ether extracts cyanins, and hydroxycinnamic acids present in Moro juice reduced fat
of C. sinensis L. (Osbeck) displayed activity against Candida albicans in humans [105] High-fat (HF) diet-induced obese mice were studied
[101]. Another study found that a hydrodistillation oil mixture (1:1) by using citrange (C. sinensis × Poncirus trifoliata) fruit extract. Female
of C. maxima Burm and C. sinensis L. (Osbeck) completely inhibited C57BL/6 mice were fed with a chow diet (control), an HF diet, HF diet
the mycelial growth of Trichoderma viride, Aspergillus fumigatus, Al- supplemented with 1% w/w citrange peel extract (CPE) or 1% w/w cit-
ternaria alternate, Fusarium oxysporum, and Aspergillus fumigatus at range flesh and seed extract (CFSE) for 8 weeks. It was found that both
750 ppm [102]. Using a micro broth dilution assay, polymethoxy- CPE and CFSE regulated glucose metabolic disorders in obese mice. In
lated flavones produced from C. sinensis peel extract (flavone-7-O-[6- CPE and CFSE-treated groups, the body weight, blood glucose, serum
acyl]-glucoside, tetramethyl-O-scutellarein, nobiletin, natsudaidai, tan- total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-c)
geretin, and heptamethoxyflavone) demonstrated efficacy against As- levels were significantly reduced relative to those in the HF group. Re-

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P. Dongre, C. Doifode, S. Choudhary et al. Pharmacological Research - Modern Chinese Medicine 8 (2023) 100272

lated gene expressions in the liver were examined in order to inves- Insecticidal activity
tigate the mechanisms of action of CPE and CFSE on the metabolism
of glucose and fat. Peroxisome proliferator-activated receptorᵧ (PPARᵧ) Compounds of terpineol and 1,8-cineole present in the essential oil
and its target genes were down-regulated by CPE and CFSE in liver tis- extracted from leaves of C. sinensis showed insecticidal activity against
sue, according to qPCR testing. Additionally, liver X receptors (LXR) 𝛼 larvae of Culex pipiens molestus (LC50 60 mg/L). Both compounds are
and 𝛽, which are both implicated in lipid and glucose metabolism, have effective against C. pipiens molestus bites offering complete protec-
lower expression levels after exposure to CPE and CFSE. Results showed tion during 1.6 and 2 h, respectively [113]. The essential oil extracted
that CPE and CFSE administration might reduce obesity and metabolic from peels of C. sinensis has proven activity against Musca domestica
abnormalities in HF diet-induced obese mice, most likely through in- L. (LC50 3.9 mg/dm3). Dimethyl 2,2-dichloro vinyl phosphate (LC50 of
hibiting the expression of the genes for PPAR and LXR. Naringin and 0.5 mg/dm3), a volatile organophosphate was used as a positive con-
poncirin were two of the major flavonoids found in citrus extracts; they trol. The better activity presented by positive control compared to es-
may both function as bioactive substances in this process [106]. With no sential oil [114]. (+)-limonene present in orange essential oil (LC50
harmful side effects, aqueous-methanol extracts of the flavedo, albedo, 3.9 (1.2–13) mg/dm3 (95% CI) kill M. domestica L. within 15 min or
and pulp of combined samples of two citrus fruit species (C. reticulate less. The positive control used was Deltamethrin (LC50 9.2 (2.8–29.5)
and C. sinensis) effectively reduced oxidative stress in human adipocytes mg/dm3 (95% CI)), and this worked better than an essential oil [115].
[107]. Obesity is a major health problem in all countries. Many medica- C. sinensis essential oil showed activity against larvae of M. domes-
tions have been used to manage obesity over the years. Most successful tica with lethal concentrations between 3.93 and 0.71 μL/cm2, while
weight loss medication has shown serious adverse effects and has now lethal time LT50 varied between 5.8 to 2.3 days [116]. C. sinensis es-
been withdrawn from the market. The above findings showed that the sential oil prepared from seeds and fruit peels killed the larvae and
positive effects of C. sinensis and the active components might be em- adults of Triboluim castaneum by contact action. Larval toxicity was
ployed in obesity and weight loss therapy. found at 42.48 μL, 41.58 μL, and 40.28 μL at 24, 48, and 72 h ex-
posure. The adult toxicity was 45.46 μL, 53.28 μL, and 44.55 μL at
24, 48, and 72 h exposure, respectively [117]. C. sinensis peels ex-
Antiparasitic activity tract killed a certain number of mosquitos (41 ± 8; 78 ± 5), houseflies
(22 ± 4; 72 ± 7) and cockroaches (31 ± 4; 85 ± 5) proved insecticidal
Globally, parasitic infections pose a severe threat to public health, potency during 30 and 60 min [118]. The vine mealybug, Planococ-
and C. sinensis offers an option in their treatment and management. cus ficus is a pest in grapevine-growing areas worldwide. The essential
The ethyl acetate (IC50 26.67 μg/mL), acetone (IC50 > 100 μg/mL), and oils C. sinensis plant was tested for its insecticidal activity against P.
methanol (IC50 > 100 μg/mL) hexane (IC50 42.13 μg/mL) and chloro- ficus. The LC50 and LC90 in adults were 5.4 and 16.2 mg/mL, respec-
form (IC50 88.03 μg/mL), extracts of C. sinensis peel displayed moderate tively. Whereas pre-ovipositing adult females LC50 and LC90 were 5.4
antimalarial activity against chloroquine (CQ)-sensitive (3D7) strain of and 13.5 mg/mL, respectively [119]. When tested on the adult cowpea
Plasmodium falciparum. Artemisinin (3D7 strain IC50 0.0045 μg/mL), bruchid Callosobruchus maculatus, C. sinensis oil proved poisonous af-
chloroquine (3D7 strain IC50 0.021 μg/mL), CQ diphosphate (D6 strain ter 24 h (LC50 = 269 μL/L) [120]. C. sinensis peel extracts exhibited
IC50 0.00311 μg/mL), mefloquine (D6 strain IC50 0.01608 μg/mL) and larvicidal and nematicidal effects. Chloroform and methanol extract of
quinine (3D7 strain IC50 0.02 μg/mL) were used as standard drug for this C. sinensis proved activity against the larvae of Anopheles subpictus
study. [108]. The petroleum ether and methanol extracts of C. sinensis (LC50 = 58.25 ppm and LC90 = 298.31 ppm) and larvae of Culex tri-
exhibited moderate antimalarial activity against the P. falciparum FCK taeniorhynchus (LC50 = 38.15 ppm and LC90 = 184.67 ppm) respec-
2 strain having IC50 values of 51.06 and 53.61 μg/mL, respectively. Un- tively whereas hexane extract was active against the nymph of Aphis
treated controls, consisting of parasitized red blood cells and 10 μL Ci of gossypii (LC50 = 162.89 ppm and LC90 = 595.40 ppm) [121]. Anophe-
[35S]-methionine were used [109]. Trypanosoma evansi in 3 min and les stephensi (LC50 182.24–490.84 ppm), Aedes aegypti (LC50 92.27–
Trypanosoma brucei in 5 min died after exposure to a dose of 0.4 g/mL 497.41 ppm), and Culex quinquefasciatus (LC50 244.70–530.97 ppm)
of essential oil of C. sinensis peel. A set of positive control (25 mg/mL mosquitoes all were susceptible to larvicidal and pupicidal effects from
of diminavetor), negative control (infected blood suspended in heparin the ethanol extract of C. sinensis peels. The C. sinensis ethanol ex-
and Phosphate Buffer Saline Glucose pH 7.2), and diluent control (pure tract demonstrated total protection against A. stephensi, A. aegypti,
vegetable oil 100%) were set up. Diminavetor-induced total lysis after and C. quinquefasciatus at 90 min at 350 ppm and 100% repellency at
2 min for both parasites [110]. The separation, purification, and struc- 150 min at 150 ppm. Adult mortality against A. stephensi, A. aegypti,
tural elucidation of pure chemicals from extracts and essential oil of C. and C. quinquefasciatus was observed using an ethanol extract of C.
sinensis are required in order to develop promising antiparasitic medica- sinensis (LC50 272.19 ppm; LC90 457.14 ppm), (LC50 289.62 ppm; LC90
tions, despite the fact that extracts only demonstrated weak antiparasitic 494.88 ppm) and (LC50 320.38 ppm; LC90 524.57 ppm) respectively
activity. [122]. C. sinensis oil was effective against C. quinquefasciatus larvae
(LC50 11 μg) [123]. Insecticidal action against A. aegypti, C. quinque-
fasciatus, and A. dirus was demonstrated using C. sinensis oil [124].
Hypocholesterolemic activity C. sinensis oil exhibited fumigant toxicity against Tetranychus urticae
with LC50 2.22 μL/L. In this study eugenol and phosphine were used as
Literature data show that C. sinensis helps the body to absorb low positive controls having LC50 = 0.004 μL/L air and 100% mortality at
cholesterol which is a serious heart-related issue. Adult male Wistar 2 × 10−3 g/L [125]. C. sinensis var. pear peel essential oil showed in-
rats (weighing 200–250 g) were given lyophilized C. sinensis juice at secticidal activity against Bemisia tabaci at 8.5 μL/L which caused 97%
a dose of 5 g/kg in an aqueous vehicle for 15 days. This resulted mortality and LC50 3.80 μL/L of air. Eugenol (LC50 0.20 μ/L/L of air)
in lower plasma concentrations of cholesterol (31%), LDL (44%), and was used as positive control. Eugenol showed better fumigant action
triglycerides (33%) [111]. By promoting the elimination of cholesterol than essential oil [126]. C. sinensis essential oil showed a strong toxic
(123%−126%) and bile acids (129%−133%) in feces, microsized insol- effect on the eggs of Hyalomma dromedarii, especially in earlier em-
uble fibers from C. sinensis fruits reduced blood triglyceride and total bryonic development at a concentration of 1:20 (oil: ethanol 95% v/v)
cholesterol concentrations (15.7%−17.0%) and 15.6%−17.8%, respec- [127]. The hexane extract of C. sinensis leaf possessed moderate larvici-
tively [112]. There aren’t many studies on the hypocholesterolemic ef- dal efficiency against the dengue vector. The bioassays resulted in LC50
fects of C. sinensis, but the ones that do exist are interesting and perti- and LC90 values of 446.84 and 1370.96 ppm, respectively after 24 h of
nent, indicating that more work on this subject would be very beneficial. exposure [128]. Coumarins xanthyletin and seselin, and the limonoid

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P. Dongre, C. Doifode, S. Choudhary et al. Pharmacological Research - Modern Chinese Medicine 8 (2023) 100272

limonin isolated from hexane extract of C. sinensis leaves displayed in a balanced state and subsequently in the management of osteoporosis.
vitro activity on the growth of Xylella fastidiosa at concentrations rang- However, more research in this field is required.
ing from 1.00 to 2.00 mg/mL. A blank experiment was performed with
DMSO/H2O (1:1) alone (100 μL of bacterial culture, 33.5 μL of DMSO, Relaxant, sedative, and anxiolytic activities
and 33.5 μL of H2O) in the well and was used as a positive control
[129]. Extract of C. sinensis presented strong bioactivity against Semia Aromatherapy is a holistic healing treatment that uses essential oil
phis heraclei, Aphis craccivora, Tetranychus viennensis, and T. tranca- and natural plant extracts to promote health and well-being. It helps
tus [130]. The findings of the above result suggested that it is possible to improve the health of the body, mind, and spirit. It enhances both
to utilize C. sinensis as an alternative crop protectant against the most physical and emotional health. The natural essential oil present in C.
likely pest species. C. sinensis natural compounds have a clear effect on sinensis worked as a relaxant and sedative in dental patients [137].
the growth of a variety of pests. The methanol and dichloromethane extracts prepared from C. sinensis
(L.) Osbeck flower part when studied in the exploratory cylinder model
Activity in cardiovascular system mice with ED50 (ip) values displayed a dose-dependent sedative effect
of 47.04 ± 12.03 mg/kg and 129.15 ± 21.25 mg/kg respectively [138].
Citrus varieties contain a bunch of dietary flavonoids, which lower Hesperidin present in the methanol extract of C. sinensis showed a seda-
the risk of harmful cardiovascular events. Consumption of 500 mL/day tive effect (ED50 = 11.34 ± 2.48 mg/kg). Anxiolytic activity of sweet
of C. sinensis commercial juice twice a day for four weeks in healthy orange aroma was demonstrated in forty male volunteers who were al-
volunteers showed a decrease in diastolic and systolic blood pressure located to five different groups for the inhalation of sweet orange essen-
in 5.13% (p = 0.03) and 5.91% (p = 0.003) respectively. However, the tial oil (test aroma: 2.5, 5, or 10 drops). Psychologic parameters (state
drinking of natural Citrus sinensis juice for four weeks did not show any anxiety, subjective tension, tranquilization, and sedation) and physio-
significant effects on either diastolic or systolic blood pressure [131]. logic parameters (heart rate and gastrocnemius electromyogram) were
In another study, guinea pigs of both sexes (300–500 g) treated with evaluated. Results gave scientific support to use as a tranquilizer by aro-
ethanol leaf extract showed inotropic depression on the atria (EC50 of matherapists [139]. Sweet orange aroma (C. sinensis oil) in Wistar rats
300 μg/mL). Similarly, acetone leaf extracts of C. sinensis in guinea pigs demonstrated anxiolytic-like activity. The animals were exposed to the
of both male and female sex (300–500 g) showed inotropic depression orange aroma (100, 200, or 400 μL) for 5 min within a plexiglass cham-
on the atria with EC50 of 140 μg/mL. Drugs control atropine sulfate ber and were then immediately submitted to the behavior tests. All doses
(1 μM propranolol, naloxone (10 μM), and propranolol (1.5 μM atropine of C. sinensis oil showed an anxiolytic effect. Diazepam (2 mg/kg) was
sulfate), did not change the effect of the crude extract [132]. Cardio- used as a positive control. C. sinensis almost showed the same effect
vascular diseases (CVDs) are a significant health burden with an ever- as diazepam [140]. Orange is a great natural alternative because it has
increasing prevalence and the leading causes of morbidity and mortal- the well-known medicinal potential of C. sinensis extract and its com-
ity worldwide. The use of orange could be an alternative, cost-effective ponents to offer relaxing and sedative-hypnotic effects.
treatment approach for CVDs over conventional medicines that shows
severe adverse effect. Other pharmacological activities

C. sinensis fruit peel is rich in flavonoids and clinically documented

Protective of uv activity for roles in the relief of cough and promotion of digestive health. Among
the flavonoids due to their ability to bind to the essential proteins of the
Overexposure to UV radiation causes genetic changes that may lead SARS-CoV-2 coronavirus, hesperidin is currently attracting the interest
to skin cancer. Red-orange (Bionap®, 15 and 30 μg/mL) standardized of researchers. Several computational methods independently applied
extract showed protective effects on ultraviolet B (shortwave) which by different researchers, showed that hesperidin has low binding energy
induced damage in human keratinocytes. This activity could occur to both with the coronavirus "spike" protein and with the main protease
block cellular oxidative stress-related events such as inflammation and that transforms the early proteins of the virus (pp1a and ppa1b) into
apoptosis [133]. A supplement of red orange complex® (C. sinensis va- the complex responsible for viral replication [13].
rieties Moro, Tarocco, and Sanguinello), a product of Bionap Company N B Maulydia et al. [141] performed a study to analyze the flavonoid
administered at the dose of 100 mg/daily for 15 days showed a reduc- compounds found in the orange peel to determine its potential as
tion in UV-induced skin erythema on human healthy volunteers. More- anti-Mpro through molecular docking. The compounds were initially
over, the use of the red-orange extract in subjects showed a reduction in screened for drug-like properties and then docked using Autodock Vina
melanin content from 27% to 7% [134]. These findings suggest that the in the PyRx emulator software. The docking results were visualized us-
genotoxic effects of UV radiation on cells can be protected by orange. ing the BIOVIA Discovery Visualizer 2020. The result showed that the
Thus, orange can be a good ingredient for sun protection formulation. binding free energy of hesperidin (−8.6 kcal/mol) was higher than nel-
finavir (−8.5 kcal/mol). In addition, hesperitin (−7.3 kcal/mol), saku-
Antiosteoporotic activity ranetin (−7.1 kcal/mol), isosacuranetin (−7.2 kcal/mol), and tetra-o-
methylscutallerin (−6.8 kcal/mol) exhibited lower binding free energy
Osteoporosis is a systemic disorder characterized by decreased bone value than control. The author further concluded that hesperidin has the
mass and microarchitectural deterioration of bone tissue. Due to its potential as an inhibitor of the main protease’s SARS-CoV-2. Hesperetin
important prevalence worldwide, osteoporosis is considered a serious has shown an anti-SARS-coronavirus activity through the inhibition of
public health concern associated with disability and pain. Citrus fruits 3-chymotrypsin-like cysteine protease. Naringin is used to prevent and
showed a safer and more effective treatment approach in the treatment decrease plasma cytokines levels of TNF-𝛼, IL-1𝛽, IL-10, and IFNɤ in-
of osteoporosis. Ethanolic extract of leaves and peels of C. sinensis in the patient with SARS-CoV-2. Hesperetin revealed a potential binding with
dose of 5 mg/kg when administered to rats whose one or both ovaries the ACE2 protein, which could prevent COVID-19 infection [142]. The
are removed surgically showed an increase in trabecular bone mineral treatment of COVID-19 involves the use of Traditional Chinese Medicine
content and mineral density of tibia. It also showed improved levels of like C. sinensis. To combat coronavirus in the future, we need to con-
phosphorus and calcium reducing bone loss [135]. Orange pulp feeding centrate more on natural substances like C. sinensis, a power bank of
to male rats with osteoporosis displayed improvement in characteristics vital components.
of bone structure [136]. These few studies support that Citrus sinensis Flavonoids derived from C.sinensis peel have been shown to exhibit
could be an effective herbal treatment to keep the bone metabolism in neuroprotective effects in several in vitro and in vivo investigations done

9
P. Dongre, C. Doifode, S. Choudhary et al. Pharmacological Research - Modern Chinese Medicine 8 (2023) 100272

for neurodegenerative diseases like Alzheimer’s disease and Parkinson’s abnormalities in body weight, clinical signs and symptoms and blood
disease. Nobiletin exhibited memory-improving effects in animal models parameters [13]. Thus, all these studies indicate C. sinensis to be safe
of Alzheimer’s disease. In addition, nobiletin treatment improved motor for acute dosing. However, chronic studies are required to explore the
and cognitive deficits in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine long-term adverse effects of the medicinal plant. It is essential that hu-
(MPTP)-induced Parkinson’s disease model mice. Administration of hes- man administration of C. sinensis isolated compound be investigated for
peridin for 16 weeks helped improve learning and memory function safety, tolerability, and bioavailability.
by enhancing the recognition index in the APPswe/PS1dE9 transgenic
mouse model. It corrected the A𝛽-induced mitochondrial abnormalities Conclusion and future prospective
by reducing MDA and H2 O2 levels as well as restoring depleting GSH
levels and total antioxidant capacity (T-AOC) [143]. Scientific studies revealed that C. sinensis is a popular traditional
Hesperidin inhibited learning and memory impairments resulting Chinese medicinal plant used in local and traditional medicine because
from aluminum chloride (AlCl3)—induced Alzheimer’s disease, acting of its nutritional and therapeutic advantages. Crude extract, fractions,
as an AChE inhibitor. Hesperidin attenuated amyloid precursor protein and isolated compounds exhibited various pharmacological activities
(APP) expression via NF-𝜅B dependent pathway and suppressed the lev- such as antiproliferative, antibacterial, antioxidant, insecticidal, antimi-
els of A𝛽1–40 and 𝛽- and 𝛾-secretases (which both modulate the cleav- crobial, antiparasitic, and antifungal and also help in managing obesity,
age of APP) in the hippocampus and the cortex of the brain of rats cholesterol, osteoporosis, and neurodegenerative diseases. Recent stud-
[144]. Administration of nobiletin (50 mg/kg, i.p. or 50–100 mg/kg, ies indicate that C. sinensis supplementation may be useful as a pro-
p.o.) from postoperative day 3 for 11 days to olfactory Bulbectomized phylactic agent against SARS-CoV-2 infection and as a complementary
mice significantly improved short-term memory impairment in the Y- treatment for COVID-infected patients. Chinese scientists proved the in-
maze test [145]. Yabuki et al. [145] reported that nobiletin administra- credible benefits of C. sinensis in some severe illnesses when paired
tion (50 mg/kg, i.p.) for 2 consecutive weeks, starting 1 day after the with Western therapy. Evidenced-based literature clearly demonstrates
final MPTP injection, improved motor deficits in MPTP-induced Parkin- that Oranges are a unique valuable source of bioactive compounds that
son’s disease model mice in the rotarod test, beam-walking test and im- could have good potential for incorporation into human food and hold
proved MPTP-induced cognitive deficits in the passive avoidance test the promise for the management of mild and deadly health conditions.
and novel object recognition test. Preliminary studies showed that the Moreover, further extensive research investigations are needed to ex-
essential oil of C. sinensis significantly inhibits acetylcholinesterase a plore (in vitro and in vivo) for determining the real and unseen benefits
total of 73% in group 50 mg kg−1, 83% in group 100 mg kg−1, and of extracts obtained from C. sinensis that may open the possibilities of
76% 200 mg kg−1 and significantly improves memory of the animals finding new clinically effective drugs against some life-threatening dis-
in promising preclinical studies [146]. The acute effect of flavanone- eases.
rich orange juice on neurological responses has also been assessed. In
24 healthy middle-aged adults (30–65 years), a flavonoid-rich beverage Declaration of Competing Interest
(272–220.46 mg hesperidin, 34.54 mg narirutin, and 17.14 mg other
flavonoids) improved the cognitive scores at 2 and 6 h post-consumption None.
[147]. Results from C. sinensis indicate that the isolated compound has
a sizable pharmacological impact on neurodegenerative disorders.
CRediT authorship contribution statement

Toxicological evaluation and risks

Priti Dongre: Writing – original draft, Writing – review & editing.
Chandrashekhar Doifode: Writing – original draft, Supervision. Shaily
Due to the decreasing efficacy and increasing contraindications of
Choudhary: Writing – original draft. Neeraj Sharma: Supervision.
synthetic drugs, the use of natural drugs is on the rise and as a result,
self-medication is very frequent. It is essential to ascertain the safety pro-
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