Review

Citrus aurantium Flowers: Overview of Chemistry, Functionality,

and Technological Applications
Sepidar Seyyedi-Mansour , Pauline Donn , Paula Barciela , Ana Perez-Vazquez, Rafael Nogueira-Marques,
Franklin Chamorro, Maria Carpena * and Miguel A. Prieto *

Universidade de Vigo, Nutrition and Bromatology Group, Department of Analytical Chemistry and Food Science,
Instituto de Agroecoloxía e Alimentación (IAA)—CITEXVI, 36310 Vigo, Spain; [email protected] (S.S.-M.);
[email protected] (P.D.); [email protected] (P.B.); [email protected] (A.P.-V.);
[email protected] (R.N.-M.); [email protected] (F.C.)
* Correspondence: [email protected] (M.C.); [email protected] (M.A.P.)

Abstract: Bitter orange (Citrus aurantium L.), a member of the Rutaceae family, finds global
utility in both the treatment of various ailments and its role as a rootstock for Citrus species
in agriculture. Various parts of Citrus aurantium L. have been employed in traditional
medicine due to their multifarious therapeutic potential. The blossom of this plant serves
as a rich source of bioactive compounds, notably polyphenols, alkaloids, and terpenes. Ad-
ditionally, it harbors substantial quantities of functional, nutritive, and biologically active
compounds, which manifest their presence through antioxidant, antidiabetic, anticancer,
antimicrobial, cardiovascular, and neuroprotective properties. The recovery of bioactive
compounds is significantly affected by extraction methods. Many conventional methods
have been explored for the recovering of bioactive compounds from bitter orange flowers.
However, in response to the limitations of conventional techniques, green extraction meth-
ods, characterized by their ability to significantly increase the yield and reduce the time,
energy, and solvent requirements, have also been assessed for this matrix. Therefore, the
study of the functionalities of bitter orange blossoms represents a domain with unexplored
research opportunities. Consequently, this review aims to offer a comprehensive insight
into the biological properties and medicinal applications of the active compounds found
Academic Editor: Luisella Verotta within C. aurantium.
Received: 31 December 2024
Revised: 10 February 2025 Keywords: biological properties; Citrus aurantium L.; extraction methods
Accepted: 12 February 2025
Published: 17 February 2025

Citation: Seyyedi-Mansour, S.; Donn,

P.; Barciela, P.; Perez-Vazquez, A.; 1. Introduction
Nogueira-Marques, R.; Chamorro, F.;
Carpena, M.; Prieto, M.A. Citrus
Plants have profoundly impacted the human diet throughout history, showcasing
aurantium Flowers: Overview of notable health-enhancing attributes. Given increasing health concerns and the persistent
Chemistry, Functionality, and quest for healthier lifestyles, there has been a surge in interest surrounding naturally
Technological Applications. Molecules derived substances extracted from plants. Citrus stands as one of the paramount fruit
2025, 30, 930. https://doi.org/ trees crops globally, not solely due to its substantial total production, but also owing to
10.3390/molecules30040930
its notable economic value [1]. Different parts of the citrus tree hold significance within
Copyright: © 2025 by the authors. traditional herbal medicine for addressing diverse health issues. Notably, in Asian herbal
Licensee MDPI, Basel, Switzerland. practices, the whole unripe and dried fruit is employed to facilitate digestion. In contrast,
This article is an open access article
regions like South America and Mexico use the leaves to cause a sense of tranquility for the
distributed under the terms and
conditions of the Creative Commons
mind and soul. Furthermore, the essential oils extracted from the fruits find application in
Attribution (CC BY) license creating liquors and perfumes [2].
(https://creativecommons.org/
licenses/by/4.0/).

Molecules 2025, 30, 930 https://doi.org/10.3390/molecules30040930

Molecules 2025, 30, 930 2 of 18

Within the array of Citrus species, Citrus aurantium L. stands out as a distinct variety
with versatile applications spanning functional foods, cosmetics, and the pharmaceutical
sector. C. aurantium L., commonly referred to as sour orange or bitter orange, is an evergreen
tree mainly cultivated in tropical and subtropical areas and belongs to the Rutaceae family.
C. aurantium has garnered recent attention due to its wide range of biological activities.
These include anticancer, antibacterial, antioxidant, antidiabetic, and immune-enhancing
properties, and potential applications in treating neurological disorders [3]. C. aurantium
is renowned for its fragrant white flowers, characterized by their delicate nature and ar-
rangement along the shoot axis, occurring singly or in multiple formations [4]. C. aurantium
flowers have a rich history of usage as a food flavoring agent, contributing to various
beverage types and pastries. Beyond culinary applications, these flowers have also found
a place in medicinal products due to their attributed antidepressant, anti-infectious, and
sedative properties. Moreover, their incorporation into skincare products highlights their
versatility across various domains [5]. Many biological properties have been attributed to
these flowers, including antioxidant, anticancer, anti-complement, anti-inflammatory, and
anti-tumor properties [6–8]. Specifically, compounds such as phenolic acids, flavonoids,
alkaloids, and terpenoids (essential oils) are potentially responsible for these mentioned
properties. They have garnered significant attention owing to their diverse functionalities
and inherent potential as nutraceutical agents [9].
Extraction technology plays a fundamental role in influencing the extraction efficiency
of phytochemical components and significantly affects their subsequent biological activ-
ities. Conventional extraction techniques have been used the most to extract bioactive
compounds in citrus. This technique has disadvantages, such as the use of large amounts
of solvent; in some cases, it is not environmentally friendly and causes a longer duration
of extraction. Consequently, there is an urgent need for a new and efficient extraction
approach for materials derived from citrus blossoms. In this context, green extraction
techniques have shown promise. For example, Sandhu et al. showed that the ultrasonic
method can significantly increase the biological potential of citrus extracts [10]. Also, these
techniques are considered more environmentally friendly because smaller amounts of
solvent are required, significantly reducing the extraction time. In addition, heat-sensitive
compounds are preserved depending on the chosen method and conditions. However, the
extraction conditions must be carefully evaluated for the best recovery efficiency. Prior
research has commonly employed solvents like methanol, ethanol, acetone, and ethyl
acetate, often mixed with water, to extract phenolic and flavonoid substances from plants.
The polarity of these solvents is a key consideration in phenolic and flavonoid extraction
procedures. Consequently, selecting an appropriate standardized extraction method for
these plant compounds is challenging. Due to this, there can be differences in the total
levels of phenolics and flavonoids based on the extraction solvents used [11].
The prime objective of this review was to consolidate recent research endeavors
pertaining to the bioactive compounds within C. aurantium flowers. This entailed a compre-
hensive exploration of extraction techniques conducive to isolating these compounds, as
well as a thorough examination of the biological attributes exhibited by extracts derived
from the plant. The aim was to consider the prospective utilization of these extracts as
ingredients within the domains of food, pharmaceuticals, and cosmetics. Furthermore,
this review serves as a resource for recognizing existing gaps in knowledge, potentially
facilitating further investigation into this relatively underexplored species.
 2. Literature Review Search
Molecules 2025, 30, 930 3 of 18
The methodology employed for the literature search was structured through
different research tools such as Google Scholar, Pubmed, and Science Direct, by finding
2. Literature Review Search
articles related to the topic, and using a combination of the specific key words, “Citrus
The methodology employed for the literature search was structured through different
aurantium L.”, “Bitter orange flowers”, “Biological activities”, “Extraction methods”, and
research tools such as Google Scholar, Pubmed, and Science Direct, by finding articles
“industrial applications”. The final inclusion was made in accordance with articles and
related to the topic, and using a combination of the specific key words, “Citrus aurantium
book chapters that were within the scope of the referenced keywords within the last 20
L.”, “Bitter orange flowers”, “Biological activities”, “Extraction methods”, and “industrial
years in order to applications”.
compile the The maximum amount
final inclusion wasof information,
made in accordanceand only
with appropriate
articles and book chapters
studies were utilized.
that were within the scope of the referenced keywords within the last 20 years in order to
compile the maximum amount of information, and only appropriate studies were utilized.
3. Bioactive Compounds of Citrus Flowers
3. Bioactive Compounds of Citrus Flowers
The C. aurantium flower is a rich source of bioactive compounds, which makes it an
The C. aurantium flower is a rich source of bioactive compounds, which makes it an
interesting topic to some authors, and their health-enhancing properties can be attributed
interesting topic to some authors, and their health-enhancing properties can be attributed to
to these chemicalthese
constituents (Figure 1).
chemical constituents The 1).details
(Figure of each
The details compound
of each withinthethe
compound within metabolites
metabolites of C. aurantium flowers
of C. aurantium areare
flowers elaborated
elaboratedininthe
thebelow section.
below section.

Figure 1. Chemical Figure

constituents attributed
1. Chemical to Citrus
constituents aurantium
attributed flowers
to Citrus are associated
aurantium flowers arewith health-
associated with health-
promoting properties. Createdproperties.
promoting in https://BioRender.com (accessed on 20(accessed
Created in https://BioRender.com Januaryon
2025).
20 January 2025).

3.1. Phenolic Compounds

3.1. Phenolic Compounds
As one of the vital chemical components in C. aurantium, phenolics have been rec-
As one of the vital as
ognized chemical components
having beneficial in C.foraurantium,
properties human health.phenolics have are
Polyphenols been
a group of
recognized as having beneficial
secondary properties
metabolites forwidespread
that are human health. Polyphenols
in the plant kingdom.are Theya group of to serve
are known
secondary metabolites
variousthat are widespread
protective in the
functions for plant
plants thatkingdom. They
are significant to are
theirknown to serve
adaptability and survival.
various protectiveThe antioxidant
functions foractivity
plantsof Citrus
that areflowers involves to
significant various
theircomponents,
adaptability butand
polyphenols,
which comprise
survival. The antioxidant flavonoids
activity and flowers
of Citrus phenolic acids,
involveshavevarious
received considerable
components, attention
but due to
polyphenols, whichtheir importance
comprise and extensive
flavonoids and investigation.
phenolic acids, have received considerable
The flowers of C. aurantium contain a diverse array of compounds, notably (1) flavonoids,
attention due to their importance and extensive investigation.
including rutin, apigenin, luteolin, quercetin, naringin, hesperetin, rhoifolin, eriocitrin,
The flowers hesperidin,
of C. aurantium contain a diverse array of compounds, notably (1)
and neohesperidin; and (2) phenolic acids, such as quinic acid, caffeic acid,
flavonoids, including rutin,acid,
rosmarinic apigenin,
ferulic luteolin, quercetin,
acid, pyrogallol, naringin,
syringic acid, andhesperetin,
gallic acid. rhoifolin,
Some authors have
eriocitrin, hesperidin, andthat
reported neohesperidin; and (2)
C. aurantium flowers arephenolic acids,
a rich source such as compounds,
of phenolic quinic acid,especially
caffeic acid, rosmarinic acid,which
flavonoids, ferulicare
acid, pyrogallol,
present syringic
in significant acid, and
quantities gallic acid.
[3,5,11,12]. In theSome
C. aurantium
authors have reported that C. aurantium flowers are a rich source of phenolic compounds,into three
flower, a noteworthy class of compounds are flavonoids, which can be classified
especially flavonoids, which are present in significant quantities [3,5,11,12]. In the C.
aurantium flower, a noteworthy class of compounds are flavonoids, which can be classified
into three categories: flavones, flavanones, and flavanols. Notably, flavones may also be
Molecules 2025, 30, 930 4 of 18

categories: flavones, flavanones, and flavanols. Notably, flavones may also be found in
the methoxylated form, where most or all hydroxyl groups are methylated. It has been
observed that the total amount of flavonoids (TFC) and total phenolics (TPC) present in the
C. aurantium flowers is 46 ± 2.40 mg/g and 81 ± 1.80 mg/g, respectively [13]. This indi-
cates that the sample under investigation may contain significant quantities of flavonoids,
which are natural compounds found in plants and are known for their potential health
benefits, including antioxidant and anti-inflammatory effects [14]. According to two studies
investigating methanolic C. aurantium flower extracts, the phenolic compound pyrogallol
exhibited the highest amount, measuring 541 and 526 µg/g in dry weight (DW). In con-
trast, rosmarinic acid with 75.4 µg/g DW and gallic acid with 212 µg/g DW had a lower
amount [11,12]. Moreover, it has been found that 5-hydroxy-6,7,3′ ,4′ -tetramethoxyflavone
(HTF) is present in the flowers of Citrus aurantium L. var. amara Engl (C. aurantium var.
amara) and exhibited remarkable antioxidant and anti-tumor properties [15]. Tannins, a
group of polyphenolic biomolecules, have been identified as existing within the flowers,
leaves, and peels of C. aurantium L. These compounds play a crucial role in protecting
against predation and regulating plant growth. These molecules play a crucial role in
defending the plant against predation while also serving as a regulator for its growth [16].
Taken together, these findings underscore the influence of chemical structures on the
interaction with solvents, consequently influencing the extraction yield of compounds.

3.2. Other Compounds

C. aurantium flowers are a veritable source of active metabolites, comprising various
constituents beyond phenolic compounds. Some research has reported the detection of
various bioactive compounds, including alkaloids, essential oil, polyterpenes, and sterols,
within samples of C. aurantium flower extracts [9,16]. Alkaloids with nitrogen atoms
constitute the largest category of naturally occurring compounds identified in C. aurantium.
Every single alkaloid found in bitter orange contributes to an elevation in the metabolic rate
as well as an increase in the rate of fat oxidation [2]. A total of nine alkaloid compounds were
successfully identified within the flower of Citrus aurantium L. var. amara Engl. (CAVA)
through the employment of an LC–MS assay. Among these compounds, stachydrine,
caffeine, and cathine emerged as prominent alkaloids. It has been substantiated that this
flower holds considerable potential for inducing anti-obesity effects and regulated lipid
metabolism in steatosis Hep G2 cells. Synephrine, hordenine, cytisine, N-methylcytisine,
coniine, and (R, S)-anatabine are additional compounds that were identified in this study.
Across various studies, these compounds have exhibited a range of activities, including the
inhibition of glucose production, cardiovascular benefits, and protective properties against
lung damage [17]. Citrus essential oils are generally acknowledged as safe (GRAS), owing
to their extensive range of biological activities. Besides the activities mentioned, undiluted
essential oils are traded at considerable prices within the international market, finding
significant demand in the perfumery sector and the cosmetic industry. Based on the findings
of one study, it has been observed that the essential oil extracted from C. aurantium flowers
is composed of a complex combination of terpenes, terpenoids, aliphatic and aromatic
hydrocarbons, sulfur-containing compounds, phenolics, polyhydric alcohols, and other
oxygenated phytochemicals [18]. The main component in the essential oil of C. aurantium
flowers is linalool, followed by d-limonene, linalyl acetate, and β-pinene [19–21]. However,
according to the findings of [22], it has been observed that the essential oil derived from the
flowers of C. aurantium in Tunisia primarily comprises limonene as its principal constituent.
Limonene, a prevalent terpene compound widely found in nature, constitutes a significant
proportion of various essential oil compositions. The variations observed in the composition
of essential oils can be attributed to multiple factors encompassing environmental aspects
Molecules 2025, 30, 930 5 of 18

(such as geography, climate, and soil conditions), plant parts, collection time, genetic
influences, and disparities in the extraction techniques employed. Similar findings were
reported in the study by Liang et al., where oxygenated monoterpenes were identified
as the primary constituents of the essential oil from Citrus aurantium flowers, followed
by monoterpene hydrocarbons. The major components include linalool, linalyl acetate,
and limonene. Pharmacological studies have shown that the oral and intraperitoneal
administration of C. aurantium essential oil reduces sleep latency, prolongs sleep duration,
and decreases locomotor activity in mice. Additionally, GC-MS analysis has identified
several key volatile compounds, including α-pinene, 3-carene, 1,2-oxolinalool, hotrienol,
sabinene, terpinolene, and geraniol [20].

4. Biological Activities of Citrus Flowers

4.1. Antioxidant Activity of Citrus aurantium Flowers
The antioxidant activity of a plant extract is the capacity of the extract to delay or
prevent the oxidation of free radicals and thus limit the harmful consequences of oxidation.
Nowadays, migration from synthetic to natural antioxidants is increasing due to health
reasons. Foods, pharmaceuticals, and cosmetics industries are looking for new natural
sources of ingredients with antioxidant properties for health and environmental purposes.
In the literature, the antioxidant activity of the extracts of Citrus aurantium flowers (CAF)
has been reported by many authors (Table 1). For example, in the study conducted by
Değirmenci and Erkurt, 2020, the radical scavenging activities of hydrogen peroxide (H2 O2 )
and 1,1-diphenyl-2-picrylhydrazyl (DPPH) of an ethanolic extract of CAF were evaluated.
From their findings, it was found that this extract presents antioxidant properties, as seen
from the results of the half inhibitory concentrations (IC50 ) that were 66.50 ± 2.70 and
96.07 ± 1.26 µg/mL, respectively, for H2 O2 and DPPH free radical scavenging activities.
They also determined the total phenolic content (TPC) and total flavonoids content (TFC)
of the ethanolic extract of CAF, estimated at 81.37 ± 3.2 mg gallic acid equivalent (GAE)/g
of extract (E) and 20.34 ± 2.68 mg of quercetin equivalent (QE)/g E, thus correlating
with their antioxidant activity [9]. Other authors [11] have studied the antioxidant ac-
tivity of CAF by determining the ferric reducing antioxidant power (FRAP) and DPPH
radical scavenging of three different extracts of the Citrus aurantium flower: an ethanolic
extract (EE), a methanolic extract (ME), and an infusion extract with boiled water (IE).
Their results showed that the ME presented higher DPPH free radical scavenging activity
and ferric reductive antioxidant power in comparison to the other extract; meanwhile,
it still had a smaller concentration of 300 µg/mL of BHT or α-tocopherol. The results
presented as the percentage of antioxidant power at 300 µg/mL of the ME, EE, and IE
of CAF were as follows for the DPPH radical scavenging activity: 55.32%, 52.41%, and
50.46%; and for FRAP: 51.70%, 47.60%, and 43.50%. Similarly, the evaluated TPC and TFC
of the tree extract followed the same behavior, with the ME presenting higher values (TPC:
4.83 ± 0.05 mg GAE/g DW, TFC: 4.11 ± 0.05 mg rutin equivalent (RE)/g DW) followed
by the EE (TPC: 4.55 ± 0.01 mg GAE/g DW, TFC: 3.83 ± 0.05 mg RE/g DW) and the IE
(TPC: 3.93 ± 0.58 mg GAE/g DW, TFC: 1.88 ± 0.01 mg RE/g DW). The variability of re-
sults amongst different authors could be due to the difference in extraction methods, the
type of solvent, and the origin and treatment method of the sample.
Molecules 2025, 30, 930 6 of 18

Table 1. Biological activities of Citrus aurantium L. extracts.

Solvent Activity Ref.

Antioxidant Activity
EY TPC TFC DPPH H2 O2 NO Fe2+
% mg GAE/g DW mg QE/g DW IC50 (µg/mL) IC50 (µg/mL) at 800 µg/mL at 800 µg/mL
H2 O 15.52 71.45 19.02 191.08 142.86 - -
EtOH 17.46 87.96 28.20 87.15 54.96 - -
[9]
EA 4.63 40.48 10.42 707.88 465.76 - -
Hexane 3.14 27.12 12.61 809.19 674.56 - -
EtOH - 4.55 3.83 52.41% * 47.60% * - -
H2 O - 3.93 1.88 50.46% * 43.50% * - - [11]
MeOH - 4.83 4.11 55.32% * 51.70% * - -
EtOH 12.00 78.76 12.11 723.10 902.30 30.00% 6.30% [23]
Antimicrobial Activity
L. monocy- K.
E. coli S. typhimurium S. aureus MRSA B. cereus P. aeruginosa C. albicans
togenes pneumoniae
EO (MIC, µg/mL) 75 75 <75 75 500 75 75 75 [16]
H2 O (MIC, mg/mL) 6250 6250 781 - 1562 781 - - -
H2 O (MBC, mg/mL) 12,500 12,500 1562 - 1562 1562 - - -
MeOH (MIC, mg/mL) 3124 3124 390 - 390 1562 - - -
[14]
MeOH (MBC, mg/mL) 6250 6250 390 - 781 1562 - - -
EA (MIC, mg/mL) 12,500 12,500 12,500 - 6250 6250 - - -
EA (MBC, mg/mL) 25,000 25,000 12,500 - 12,500 12,500 - - -
Anticancer Activity
MeOH IC50 µg/mL: Chang liver (>); MCF-7 (152.34); MDA-MB 231(49.74); HT-29 (96.23) [11]
HTF Inhibition rate: SMCC-7721 (88.20% at 200 µg/mL); Hela cells (71.00% at 200 µg/mL); B16 (54.20% at 50 µg/mL)
[15]
LA Inhibition rate: SMCC-7721 (50.30% at 200 µg/mL); Hela cells (42.80% at 200 µg/mL); B16 (39.60% at 50 µg/mL)
Abbreviations: EY: extraction yield; TPC: total phenolic content; TFC: total flavonoid content; DPPH: 2,2-Diphenyl-1-picrylhydrazyl radical scavenging; H2 O2 : hydrogen peroxide
scavenging activity; NO: nitric oxide scavenging activity; Fe2+ : ferric chelating ability; FRAP: ferric reducing antioxidant power; IC50: half inhibitory concentration; MIC: minimum
inhibitory concentration; MBC: minimum bactericidal concentration, EtOH: ethanol; EO: essential oil; H2 O: water; EA: ethyl acetate; MeOH: methanol; HTF: 5-hydroxy-6,7,3′ ,4′ -
tetramethoxyflavone; LA: limonexic acid. Note: results with (*) for DPPH and H2 O2 were tested at 300 µg/mL.
Molecules 2025, 30, 930 7 of 18

4.2. Anticancer Properties of Citrus aurantium Flowers

The management of cancer with compounds or extracts from natural sources is a recent
and efficient treatment method that, additionally, has been shown to exert a lesser secondary
effect. Through their antioxidant activities, phenolic compounds in general and particularly
flavonoids, identified in plant extracts like CAF, can play the role of antiproliferative agents
to avoid or slow down the apoptosis of cancer cells [24]. In this way, the anticancer potential
of CAF extracts has been studied by many authors; some of the results obtained are shown in
Table 1. For example, the effects of the methanolic extract of CAF were evaluated on Chang
liver cancer cells, breast cancer cells (MCF-7, MDA-MB 231), and colon cancer cells (HT-29).
These authors found that the cytotoxicity of the extract was more efficient on MDA-MB 231
with an IC50 of 49.74 ± 0.75 µg/mL [11]. Also, other authors have found that limonexic
acid (LA) and 5-hydroxy-6,7,3′ ,4′ -tetramethoxyflavone (HTF), two bioactive compounds
isolated from CAF, present a concentration-dependent inhibitory effect on the proliferation
of a human skin cancer model (B16) and a hepatocellular carcinoma cell line (SMCC-7721).
They show that HTF presents greater inhibitory activity on SMCC-7721 cell growth, with a
value of 88.20% for an extract concentration of 200 µg/mL. They concluded that HTF has a
higher inhibition effect on the morphological variation and proliferation of the tested cancer
cells compared to the control ‘5-Fluorouracil’ [5,15]. Additionally, polysaccharides extracted
from Citrus aurantium have been studied for their anticancer activity with plausible results,
showing their high potential in the treatment of cancer [25]. Also, limonene, a compound
present in the essential oil of CAF, has proven, through a test on animals, that it presents a
positive response against cancers of the liver, pulmonary system, breast, skin, colon, and
pancreas by slowing down the growth of their development [26]. Thus, the presented
results of the different studies conducted on this plant lead to the conclusion that it is a
very good source of anticancer compounds for further application in pharmacology as a
natural ingredient.

4.3. Antimicrobial Activity Citrus aurantium Flowers

Pathogenic microorganisms are hazards that could be very harmful to human beings,
especially above a certain dose once ingested. In nature, there are plant extracts or com-
pounds extracted from plants that can fight or inhibit the activities/proliferation of these
pathogenic microorganisms. The capacity of a plant extract to play this role is characterized
by its antimicrobial activity. Thus, the plant extracts of CAF as well as their essential oil
extracts (EO) have been studied by many authors for this purpose, as seen in Table 1. This
is the case of the study conducted by Dhekra Trabelsi and their collaborators, which ana-
lyzed the in vitro antimicrobial activity of the EOs and methanolic extracts (MEs) of CAF
against the yeast culture Candida albicans, five Gram-negative bacteria (Klebsiella pneumoniae
cefotaxime resistant, Klebsiella pneumoniae ATCC 1388, Salmonella typhimurium, Pseudomonas
aeruginosa ATCC 27853 (CIP 76110), and Escherichia coli ATCC 25922 (CIP 7624)), and three
Gram-positive ones (Methicillin-resistant Staphylococcus aureus, Staphylococcus aureus ATCC
25923 (CIP7625), and Listeria monocytogenes ATCC 19111). They found that the MEs of
CAF only inhibited the development of Staphylococcus aureus ATCC 25923 with 1.2 mm as
inhibition diameter, while the EO of CAF presented antimicrobial activity against all the
tested microorganisms with a minimal inhibition concentration (MIC) of 75 µg/mL for most
of them, except Staphylococcus aureus ATTC (<75 µg/mL) and Listeria monocytogenes ATTC
(500 µg/mL) [16]. Moreover, in the research conducted by Degirmenci and Erkurt, 2020,
three different studied extracts of CAF (water, methanol, and ethyl acetate, respectively,
WE, ME, EE) presented effective antimicrobial activity against five foodborne pathogens in
rice pudding (Escherichia coli O157:H7, Staphylococcus Typhimurium Staphylococcus aureus,
Bacillus cereus, and Listeria monocytogenes), with the ME being the best. They found that
Molecules 2025, 30, 930 8 of 18

the MIC was between 781 and 6250 mg/L for the WE, 390 and 3124 mg/L for the ME,
and 6250 and 12,500 mg/L for the EE; while the minimal bactericidal concentration (MBC)
was between 1562 and 12,500 mg/L for the WE, 390 and 6250 for the ME, and 12,500 and
25,000 mg/L for the EE [14]. These authors concluded that the ME of CAF or isolated bioac-
tive compounds of this extract are possible sources of natural ingredients for application as
natural preservatives in food and pharmaceutical industries.

4.4. Anti-Inflammatory Activity of Citrus aurantium Flowers

Inflammation is defined as the body’s immune response to infections, injury, or
metabolic stress [27]. This phenomenon can be harmful to human beings and needs
to be regulated through anti-inflammatory compounds. Thus, some flavonoid-rich plant
extracts have demonstrated anti-inflammatory activity. That is the case of the CAF extract
that was studied by Karimi and collaborators. These authors found that the ME of CAF
at different concentrations was able to inhibit induced nitric oxide (NO) production (by
IFN-γ and LPS) from the macrophage RAW 264.7 cells. They evaluated the NO production
at 14.40 µM while applying the ME at a concentration of 100 µg/mL. Also, they found that
even at the highest concentration of the extract amongst the studied ones (100 µg/mL), the
RAW 264.7 cell viability was still higher than 90%, showing the safety of the tested CAF
methanolic extract [11].

4.5. Anti-Obesity Activity of Citrus aurantium Flowers

Obesity, referring to an excessive accumulation of fat, is a metabolic disorder generally
associated with diabetes and hypertension. Some plants of traditional pharmacopeia have
been suggested as a natural treatment for obesity-associated diseases with the advantage
of presenting fewer adverse effects [28,29]. Some authors have studied the anti-obesity
activity of CAF extract through its ability to inhibit lipid accumulation. Thus, in the study
carried out by Li et al., (2021) in vitro and in vivo screening of the anti-obesity activity
of the chloroform (CHL) extracts of CAF were evaluated. These authors found that this
extract is not only able to decrease the metabolic syndrome of high-fat-diet mice, but in
addition, can inhibit the differentiation of 3T3-L1 preadipocytes, a key factor of excessive
energy storage [29,30]. These results support the reason why they have been used for many
years in traditional medicine to manage disorders related to obesity. However, the authors
suggested that additional research regarding the bioavailability and toxicity of this extract
needs to be carried out [29].

4.6. Anti-Amnesic Activity of Citrus aurantium Flowers

Alzheimer’s disease (AD), known as a progressive neurodegenerative disorder that
mainly affects the older population, is associated with a loss in memory and cogni-
tive impairments [31]. In vivo studies of the anti-amnesic potential of plant extracts on
scopolamine-induced memory impairments have been studied by Rahnama et al., (2015) in
order to evaluate capacity for memory and learning impairments to be reduced. Their re-
sults show the significant efficiency of the tested CAF extracts in passive avoidance response
tests. Also, the results of a Morris water maze test indicate that during trial sessions, this
extract can reduce escape latency. Concerning the effect of CAF extract on malondialdehyde
(MDA) serum, this extract significantly reduces MDA serum levels. Finally, these authors
concluded that there is a beneficial effect of the ME of CAF in the treatment of Alzheimer’s
disease and the possibility for application of the extracted bioactive compounds of CAF in
pharmaceutical industries [32].
Molecules 2025, 30, 930 9 of 18

5. Extraction Techniques of Bioactive Compounds of Citrus Flower

In recent decades, there has been an increasing interest in obtaining several compounds
(e.g., phenolic compounds, tannins, and flavonoids) naturally present in different matrixes
(e.g., plants and seaweed) due to their biological activity. In this way, the extraction step
is an essential feature, so that the compounds previously mentioned can be released from
the cellular medium [33]. Therefore, extraction is defined as the process in which the cell
is disrupted to liberate the compounds inside it. Extraction techniques can be classified
into two groups: conventional extraction techniques, in which maceration, percolation,
digestion, and Soxhlet extraction are included [33]; and new extraction techniques, in
which ultrasound- assisted extraction, pressurized liquid extraction, ultrasound-assisted
extraction, enzyme-assisted extraction, and supercritical fluid extraction are included [33].
In this section, the studies conducted regarding C. aurantium flowers to this date using
either conventional or new extraction techniques are compiled. Moreover, the most relevant
results are included in Table 2.

Table 2. Comparison between conventional and new extraction techniques to obtain compounds
from Citrus aurantium flowers.

Operational Conditions
Extraction Pre- Activity
T P t F Pw Form Compound Yield (%) Application Ref.
Technique S Treat.
(◦ C) (bar) (min) (Hz) (W)
Conventional Extraction Techniques
Mechano- n-hexane 1:5 Ball
RT ns 30 ns ns 6.60 ns ns
chemical (w/v) milling
Reflux n-hexane 1:8 Powder EO [34]
70 ns 240 ns ns Grinding 2.08 ns ns
extraction (w/v)
Hydro- H2 O 1:20
140 ns 360 ns ns Grinding 0.25 ns ns
distillation (w/v)
EO 0.31 Antimicrobial
activity
Limonene 40.81 against E.
Linalool 26.66 aerogenes, S.
Hydro- typhi, M.
H2 O 100 ns 360 ns ns Dried ns luteus, K. ns [35]
distillation γ-elemene 7.97
pneumoniae,
α-terpineol 4.97 E. coli, B.
α-terpynil subtilis, S.
2.07 aureus
acetate
Hydro-
H2 Od 100 ns 240 ns ns Crushed ns Linalool 25.7 ns ns
distillation
8.78 mg
TPC
EAG/g DW
Scavenging [36]
Percolation MeOH RT ns ns ns ns Dried Powder 4.86 mg ns
TFC activity
EC/g DW
0.06 mg
Tannin
EC/g DW
Tyrosinase Cosmetic:
MeOH 5.10 inhibition, skin
Solvent ns ns ns ns antioxida- whiteners
30 Grinding Powder Extract and [12]
extraction tive,
EtOH 50% 8.60 anti-wrinkle anti-wrinkle
treatments treatments
H2 O 15.52 Natural
preservative
Soxhlet MeOH 17.46 Antioxidant against food
BTB ns 360– ns ns Grinding Powder Extract [14]
extraction 480 and antimi- spoilage in
EtOAc 4.63 crobial
dairy
Hexane 3.14 desserts
Steam Anti-
H2 O 100 ns 60 ns ns Dried ns EO NS ns [37]
distillation hyperlipidemia
Soxhlet 360–
EtOH BTB ns ns ns Dried ns Extract 16.38
extraction 480 Antioxidant, Functional [9]
Steam antibacterial food
H2 O 100 ns 240 ns ns Grinding Powder EO 0.57
distillation
Hydro-
H2 O 100 ns 180 ns ns ns ns EO 0.31 ns ns [35]
distillation
Hydro-
ns 0.36
distillation
H2 O ns 180 ns ns ns ns EO ns ns [4]
Steam
100 0.04
distillation
Molecules 2025, 30, 930 10 of 18

Table 2. Cont.

Operational Conditions
Extraction Pre- Activity
T P t F Pw Form Compound Yield (%) Application Ref.
Technique S Treat.
(◦ C) (bar) (min) (Hz) (W)
New Extraction Techniques
Protection
hepatocellu- Food
MAE H2 O 25 ns 60 ns ns ns ns EO 1.19 (v/w) additives as [18]
lar I/R flavoring
damage
Anti-
UAE EtOH 80% ns ns 30 100 ns Grinding Powder EO NS ns [37]
hyperlipidemia
OAHD H2 O EO 0.05
SLME H2 O ns ns 50 ns ns ns ns EO 0.21 ns ns [4]
SFME - EO 0.17
Inhibition
lipid accu- Pharmaceutical
UAE EtOH 80% 80 ns 180 ns ns ns ns Alkaloids 5.66 [17]
industry
mulation
Abbreviations: UAE: ultrasound-assisted extraction; OAHD: ohmic assisted hydro-distillation; SLME: solventless
microwaved extraction; SFME: solvent-free microwave extraction; S: solvent; T: temperature; t: time; RT: room
temperature; ns: not specified; EO: essential oil; TPC: total phenolic compounds; TFC: total flavonoid content;
MeOH: methanol; EtOH: ethanol; EtOAc: ethyl acetate; I/R ischemia/reperfusion; DW: dry weight; EAG: gallic
acid equivalents; EC: catechin. BTB: below the boiling point.

5.1. Conventional Extraction Techniques

Conventional extraction techniques have been the most studied techniques applied in
C. aurantium flowers. Zhu et al. (2021) extracted the essential oils (EOs) of C. aurantium L.
var amara flowers by applying mechanochemical-assisted extraction. Mechanochemistry is
a process where the mechanical energy produced by applying force (e.g., shear, friction,
or impact force) in solid matrixes leads to changes produced by the substance’s internal
energy [34]. Flowers were pretreated by applying ball milling for several minutes until a
powder was achieved. Then, the extraction was set using n-hexane as an extraction solvent
for 30 min at room temperature. Since no heat is applied, the milling step is crucial to
obtain high recovery yields. Hydrodistillation and reflux extraction were also performed as
a control. For the hydrodistillation extraction, water at 140 ◦ C was used as a solvent for 6 h,
while reflux extraction was performed with n-hexane at 70 ◦ C for 4 h. The recovery yields of
the EO obtained using mechanochemical-assisted extraction, reflux, and hydrodistillation
were 6.60%, 2.08%, and 0.25%, respectively. Thus, by applying MCAE, Zhu et al. (2021)
achieved a recovery yield 26 times higher than hydrodistillation and three times higher
than reflux extraction. Moreover, MCAE was able to obtain higher yields in less time and
without applying heat, which may be an advantage for the stability of the thermolabile
compounds extracted. Hydrodistillation was also applied by Sidi et al. to extract the EO
from C. aurantium flowers [35]. Hydrodistillation is a traditional extraction methodology
usually applied to obtain the EO from plant materials [38]. It can be performed in different
ways: water distillation, water and steam distillation, and direct steam distillation [39].
Different authors have studied hydrodistillation applied in flowers from C. aurantium,
especially for EO extraction. For EO extraction, water at 100 ◦ C for 360 min was employed,
obtaining 0.31% of the extraction yield, with limonene being the main compound of the EO.
Moreover, the antimicrobial activity against E. aerogenes, S. typhi, M. luteus, K. pneumoniae,
E. coli, B. subtilis, and S. aureus was also studied, showing positive results against all these
pathogens [35]. Degirmenci and Erkurt, 2020, also obtained positive antimicrobial and
antioxidant activity results from EOs extracted from C. aurantium flowers by applying steam
distillation for 4 h and obtaining 0.57% yield recovery. In this study, Soxhlet extraction was
also applied using ethanol for 6–8 h, obtaining 16.38% of the EO and showing the suitability
of this process [9]. In another study run by J. Li et al., steam distillation was applied
in C. aurantium flowers to obtain the EO. The results show a high content in flavanones
(17.93% of the EO extracted), showing great anti-hyperlipidemia activity [37]. Continuing
with Soxhlet extraction, this is a traditional extraction technique that has been already
used in flowers of C. aurantium. It is identified as the standard methodology to which
Molecules 2025, 30, 930 11 of 18

new extraction techniques are compared. In this way, Soxhlet extraction combines both
maceration and percolation extraction techniques since the solvent reaches the boiling
point and the condensed drops pass through the solid matrix in a process that takes
almost 8 h [40]. Degirmenci and Erkurt, 2020, ran a study in which this technique was
applied in C. aurantium flowers using four different solvents (water, methanol, ethyl acetate,
and hexane) in their respective boiling points for 8 h. In this way, a comparison of the
different solvent suitabilities was made, where methanol showed the highest recovery with
17.46%, followed by water with 15.52%. Moreover, the authors studied the antioxidant and
antimicrobial activity of the extracts against different pathogens in rice pudding, showing
positive results and suggesting their potential application as shelf-life extenders in dairy
desserts [14]. Percolation is also a traditional extraction technique that consists of passing
a solvent through a solid material drop by drop. The flow ratio of the extracted solvents
is 5 mL/min when 1 kg of plant material is used [33]. Hydrodistillation and percolation
extraction were used for the phenolic, flavonoid, and tannin determination and EO recovery
from C. aurantium blossoms, respectively. Methanol was used for percolation extraction
to determine the phenolic, flavonoid, and tannin content, while water at 100 ◦ C for 4 h
was applied for EO extraction. The authors determined 26 volatile compounds in the EO,
with linalool being the most abundant (25.70%) of the total EOs extracted (which was not
specified in the work). Moreover, the total phenolic compounds, the total flavonoids, and
the tannins present in the extract were quantified, being 8.78 mg GAE/g, 4.86 mg EC/g, and
0.06 mg EC/g, respectively. In this way, the authors determined the free radical scavenging
activity of the methanolic extracts, showing an IC50 of 20 µL/g [36]. Solvent extraction is
a process where the transfer of compounds takes place by putting the solvents in contact
with different solubility or distribution coefficients [41]. This extraction technique was
applied to C. aurantium flowers with no petals. Two runs were carried out for 30 min: one
using methanol as extraction solvent, and the other using ethanol 50%, being 5.10% and
8.60% of the extraction recovery obtained, respectively. Moreover, the extracts showed a
potential tyrosinase inhibition, and antioxidative, antibacterial, and anti-wrinkle activity,
with potential application in the cosmetic industry [12].
Considering the data shown, traditional extraction techniques have been applied in C.
aurantium flowers to extract different compounds, mainly EOs. In this way, higher recovery
yields were obtained with Soxhlet extraction. However, this methodology needs long
extraction times and high quantity solvents, so alternative pathways should be considered
since the application of this EO in both the food and pharmacology industries could
be interesting.

5.2. New Extraction Techniques

New extraction techniques have been slightly studied using flowers from C. aurantium
as the matrix. In this way, a study compared both traditional and new extraction method-
ologies for EO recovery. The new methodologies were solvent-free microwave extraction
(SFME), solventless microwave extraction (SLME), and ohmic-assisted hydrodistillation
(OAHM). Water was used as the extraction solvent in both SLME and OAHM, and the
extraction time was 50 min for all the treatments. The highest extraction yield of the three
new methodologies was obtained when SLME was applied, achieving 0.21%. For SFME
and OAHM the extraction yields were 0.17 and 0.05%, respectively. Moreover, the results
of the EO showed how SLME caused the selective extraction of linalool, which was also
the main compound of all the EOs obtained. Finally, in this study, it was shown that
the extraction technique can affect the yields of the compounds extracted, with limonene
and b-pinene showing significant differences [4]. Microwave-assisted extraction was also
applied in C. aurantium flowers by Almalki, W.H. In this study, a mixture of water and
Molecules 2025, 30, 930 12 of 18

dichloromethane at 25 ◦ C were used as the extraction solvents for 25 min, achieving 1.19%
EO. Moreover, the antioxidant, anti-inflammatory, and antiapoptotic activity, as well as
the protection against hepatocellular I/R-induced damage, was studied, showing positive
results [18]. Another study, in which ultrasound-assisted extraction was applied for the EO
recovery of C. aurantium flowers, showed the anti-hyperlipidemia activity of the extract
obtained using ethanol 80% for 30 min and 100 Hz [37].
As well as with traditional extraction techniques, new extraction methodologies ap-
plied in C. aurantium flowers are conducted for EO recovery. However, the studies using
novel extraction techniques applied in this matrix are limited. Moreover, the extraction
yields obtained are lower than those obtained with Soxhlet extraction, so further research
is needed, especially considering the current global concern to apply less toxic and more
environmentally friendly procedures during industrial processes. Table 3 summarizes the
benefits and drawbacks of the different conventional and advanced extraction methods.

Table 3. Evaluation of the advantages and limitations of conventional and modern extrac-
tion techniques.

EM Advantages Limitations Ref

Conventional Methods

Mechanochemical - Increasing the extraction rate - Modification of crystal structure, physical, and [34]
- Reduction in solvent use and extraction time chemical properties

Reflux extraction - High yield shorter extraction time - Loss of heat-sensitive compounds [42]
- Less solvent use

Hydro-distillation - Increase the extraction yield - Long extraction time [34,43]

- Centrifugation and filtration not required - Loss of heat-sensitive compounds

- Shorter extraction time - Very long extraction time

Percolation - Thermolabile compound extraction - Higher solvent consumption [44]
- Shorter extraction time - Complex protocol
- Automatic continuous process - Loss of heat-sensitive compounds
Soxhlet - Increase extraction yield - Higher solvent consumption [44,45]
- No filtration or centrifugation needed - Long extraction time

Steam distillation - Safe and easy protocol - Long extraction time [46,47]
- Higher purity - High energy consumption
New Extraction Methods
- Lower solvent consumption - Loss of heat-sensitive compounds
MAE - Higher extraction yield - High energy consumption [48]
- Better selectivity, and reproducibility - Safety precautions
- Higher extraction yield
- Cost of equipment
UAE - Short extraction time [49]
- Limitation on compound extraction
- Lower solvent consumption
- Impact on functional properties
- Preservation of thermosensitive compoundsc
- Cost of equipment
OAHD - Shorter extraction time [50]
- Loss of sensitive compounds
- Higher extraction yield
- Safety precautions
- Lower solvent consumption
SLME - Loss of heat-sensitive compounds [4]
- Higher extraction yield
- High energy consumption
- Shorter time
- No solvent consumption
SFME - Loss of heat-sensitive compounds [4]
- Higher extraction yield
- High energy consumption
- Shorter time
Abbreviations: MAE: microwave-assisted extraction UAE: ultrasound-assisted extraction; OAHD: ohmic-assisted
hydro-distillation; SLME: solventless microwaved extraction; SFME: solvent-free microwave extraction.

6. Potential Applications in Food, Pharmaceutical, and

Cosmetic Industries
C. aurantium flowers have a widespread application as a flavoring agent in several
food products, such as dairy products, or in the pharmaceutical and cosmetic industries as
an ingredient in many anti-infective, antidepressant, sedative, and even skin care products,
as is shown in Figure 2 [5,9].
A popular use is the essential oil of C. aurantium, also called neroli oil, utilized in
aromatherapy for its central nervous system stimulation, blood pressure reduction, seda-
Molecules 2025, 30, 930 13 of 18

tive, analgesic, anti-inflammatory, antispasmodic, digestive, and diuretic properties [51].

These properties are based on the chemical composition of the species; in fact, among
the most prominent active compounds that compose C. aurantium, pectin and hesperidin
represent a large proportion, which has meant a significant increase of development and
application [52]. In addition, D-limonene, closely followed by several other monoterpenes
such as linalool, β-myrcene, and α-terpineol, are often predominant compounds in C. auran-
tium [53]. Its composition also excels in many other phytochemicals; for example, in a study
conducted to demonstrate the protective effects of the essential oil (EO) of C. aurantium
Molecules 2025, 30, 930 13
flowers on liver injury, the results revealed that the oil had a high concentration of terpenes,
along with their oxygenated derivatives, hydrocarbons (aliphatic and aromatic), phenolics,
alcohols, aldehydes, and other essential oxygenated phytochemicals, which caused rats to
6. Potential Applications in Food, Pharmaceutical, and
produce fewer apoptotic cells than rats exposed to ischemia accompanied by reperfusion
Cosmetic Industries
without the administration of the EO. C. aurantium EO was proven to have antioxidant and
C. aurantium
anti-inflammatory effects flowers
[18]. The have a widespread
antimicrobial application
properties of the EOas a flavoring
are agent in several
also remarkable,
products, such as dairy products, or in the pharmaceutical and
with demonstrated effects against Gram-negative and Gram-positive strains and against cosmetic industries as an
opportunistic gredient in many
pathogenic anti-infective,
bacteria, which haveantidepressant,
a crucial rolesedative, and even
in food-borne skin careor
infections products,
shown
small skin wounds [54].in Figure 2 [5,9].

Figure 2. An overview
Figure 2. of
Anthe differentofapplications
overview the Citrus aurantium
the differentofapplications flower.
of the Citrus Created
aurantium in Create
flower.
https://BioRender.com (accessed on 20 January 2025).
https://BioRender.com (accessed on 20 January 2025).

A popular use is the essential oil of C. aurantium, also called neroli oil, utilized in aro
therapy for its central nervous system stimulation, blood pressure reduction, sedative, a
gesic, anti-inflammatory, antispasmodic, digestive, and diuretic properties [51]. These p
erties are based on the chemical composition of the species; in fact, among the most promi
active compounds that compose C. aurantium, pectin and hesperidin represent a large pro
Molecules 2025, 30, 930 14 of 18

In a separate study, they were able to demonstrate the potential of C. aurantium flower
extract in its application as a natural preservative against bacteria responsible for food
spoilage to extend the shelf life of dairy desserts. In the study, the phytochemical com-
position and the in vitro antioxidant and antibacterial activity of different extracts were
ascertained. The methanolic extract had a total phenolic content (87.96 mg GAE/g) and
flavonoids (28.20 mg QE/g) that directly correlated with the antioxidant activity that
showed an IC50 value of 87.15 µg/mL for DPPH. Moreover, the extracts were effective,
showing their inhibitory action on all the tested bacterial species and highlighting a mini-
mum bactericidal concentration (MIC) of 390 µg/mL for Staphylococcus aureus. In addition,
the presence of bioactive agents such as polyphenols, flavonoids, alkaloids, and terpenes
was identified, which is key for further use in the food industry [14]. In the field of pharma-
ceutical and cosmetic application but also based on the potential for bitter orange blossom
to be used as an antibacterial, Benzaid et al. analyzed the effects of C. aurantium EO on
the oral bacterium Streptococcus mutans and evaluated its toxicity in contact with gingival
epithelial cells. The results showed a decrease in the growth of S. mutans (OD600 decreased
to 0.09 compared to the control (0.24) with 0.30 µg/mL of the EO in 24 h) and the degrada-
tion of its mature biofilms (equivalent or superior to the effect obtained with gentamicin at
3 µg/mL), in addition to a marked decrease in certain virulence genes (comC, comD, comE,
gtfB, and gtfC). This study proved that, even at low concentrations, the EO of C. aurantium
is effective in the reduction in bacterial outgrowth and the degradation of biofilms, which
constitutes an interesting approach in the design of mouth rinses [55].
The study examined the impact of Citrus aurantium L. flower extract on primary
dysmenorrhea in young women through a double-blind, randomized controlled trial.
Participants received either the extract, mefenamic acid, or a placebo for three days at the
beginning of menstruation. The extract significantly reduced pain severity, outperforming
mefenamic acid on the first and second days. It also demonstrated a favorable safety
profile with minimal side effects. The therapeutic effects are attributed to its bioactive
compounds, including flavonoids (linalool, hesperidin, and naringin), polyphenols, and
limonoids, which exhibit anti-inflammatory, antioxidant, and antispasmodic properties.
These findings suggest C. aurantium is a promising natural remedy for menstrual pain
management [43].
Applying it also to the cosmetic industry, Chen et al., (2022), tested the anti-aging and
anti-wrinkle effects of C. aurantium flowers by studying their toxic and tyrosinase inhibitory
effects on human epidermal melanoma cells (HEMn) along with their melanin content.
The 50% fermented ethanolic extract exhibited remarkable antityrosinase, antioxidant,
antibacterial, and anti-wrinkle activity (largely attributed to the presence of neohesperidin,
which can reduce the activity of the MMP-1 enzyme). They also reported that it may be
considered safe as it exerts no toxic effect on cells of the normal human skin fibroblast line
CCD-966 and that it is capable of reducing melanin formation [12].
On the other hand, Pasandideh et al., (2021) screened the antioxidant and inhibitory
effects of C. aurantium petal extract on acetylcholinesterase and amyloid nanobiofibril pro-
duction from bovine serum albumin (BSA). The phytochemical screen identified chemical
compounds including carbohydrates, phytosterol, saponins, tannins, proteins, essential oils,
terpenoids, and flavonoids. D-limonene and daphnetin were the predominant antioxidant
compounds in the extracts, with 9.53% and 5.54%, respectively. The antioxidant capacity
assessed by a DPPH assay was 8 mg/mL at an extract rate of 94.25% (2.36 mg/mL EC50 )
and the extract reduced acetylcholinesterase activity by less than 47.04% (42.80 mg/mL
IC50 ). The results are supportive of the application of C. aurantium petals in its use as a
natural product to decrease Alzheimer’s disease complications [56]. Due to the richness of
secondary metabolites in C. aurantium, it has been used to treat several other pathologies
Molecules 2025, 30, 930 15 of 18

or ailments, such as anxiety, lung and prostate cancers, gastrointestinal disorders, and
obesity [25]. For instance, the benefit of lavender and C. aurantium EO in the recovery of
anxiety and nervousness in patients has been reported in conscious ICU patients [57].
Sweet orange (Citrus sinensis) and sour orange (Citrus aurantium) are globally popular
and marketable citrus varieties. They account for 50% of world citrus production, with
73 million metric tons per year [58]. By employing both species (Citrus sinensis and C.
aurantium), Sevindik et al. determined their chemical components and antiproliferative
activities with the aim of assessing their use in the cosmetic and pharmaceutical industry
as natural perfume, as well as anticancer agents. The results pointed out compounds such
as nerolidol (28.07%), 2,6,10-dodecatriene-1-ol, (15.11%), and linalool (14.47%) in the EO
of C. aurantium, and nerolidol (22.13%), linalool (14.06%), and sabinene (10.83%) were
identified in the EO of C. sinensis. These findings support that these two EOs exhibit
a strong antiproliferative effect along with low cytotoxicity values at low and medium
concentrations. Nevertheless, they caused higher apoptotic deformation of the cellosaurus
cell line (FL), which suggests the necessity of investigating the cytotoxic effect [59].
By way of discussion, after compiling the available bibliography, it is seen that the
applications taken from the flower of the species C. auranitum are scarce, despite its potential
and abundant chemical composition in active compounds. In the preceding sections, we
have described current studies in which the capacity of this species of bitter orange has
been tested with the objective of real applications in the cosmetic, pharmaceutical, and
food industries.

7. Conclusions
The scientific evidence confirmed the benefits of medicinal plants, particularly flowers,
and underscored their potential as valuable resources for wide-ranging functions and
health advantages. However, certain species have not undergone comprehensive research
regarding their chemical composition and biological efficacy. The analysis of C. aurantium
flowers has exposed their extraordinary chemical composition. Regarding obtaining a more
thorough understanding of their scientific properties, this present review has outlined the
various bioactive substances discovered in C. aurantium, focusing on the phenolic, alkaloid,
and terpene compounds that have been documented. Also, eco-friendly and sustainable
extraction techniques have been summarized to optimize the yield and preserve the bioac-
tive compounds of C. aurantium flowers. Methods such as supercritical fluid extraction,
ultrasound-assisted extraction, and microwave-assisted extraction have demonstrated great
potential in improving the efficiency and environmental sustainability of the extraction
process. These advanced techniques not only enhance the quality of the extracted com-
pounds but also align with the increasing demand for green and sustainable practices in the
industry. To sum up, the flowers of Citrus aurantium are a valuable and flexible resource
with substantial potential in diverse industries such as the food, nutraceutical, and pharma-
ceutical industries. It is imperative to continue research and development efforts to fully
exploit their capabilities, enabling the development of innovative applications that make
use of their chemical diversity and biological functions, thereby promoting sustainability
and economic progress.

Author Contributions: Conceptualization, M.A.P., M.C. and S.S.-M.; methodology, S.S.-M., P.D.,
P.B., A.P.-V., R.N.-M. and F.C.; software, P.D., P.B. and A.P.-V.; validation, M.C. and M.A.P.; formal
analysis, S.S.-M., P.B. and A.P.-V.; investigation, S.S.-M., P.D., P.B., A.P.-V., R.N.-M. and F.C.; resources,
M.A.P.; data curation, S.S.-M., P.D., P.B. and A.P.-V.; writing—original draft preparation, S.S.-M., P.D.,
P.B., A.P.-V., R.N.-M. and F.C.; writing—review and editing, S.S.-M., M.C. and M.A.P.; visualization,
M.A.P., M.C. and S.S.-M.; supervision, M.C. and M.A.P.; project administration M.A.P.; funding
acquisition, M.A.P. All authors have read and agreed to the published version of the manuscript.
Molecules 2025, 30, 930 16 of 18

Funding: The research leading to these results was supported by Xunta de Galicia, supporting the
pre-doctoral grant of P. Barciela (ED481A-2024-230). The authors thank the EU-FORA Fellowship
Program (EUBA-EFSA-2023-ENREL-01) that supports the work of F. Chamorro.

Data Availability Statement: No new data were created or analyzed in this study. Data sharing is
not applicable.

Conflicts of Interest: The authors declare no conflict of interest.

References
1. Ballistreri, G.; Fabroni, S.; Romeo, F.V.; Timpanaro, N.; Amenta, M.; Rapisarda, P. Anthocyanins and Other Polyphenols in
Citrus Genus: Biosynthesis, Chemical Profile, and Biological Activity. In Polyphenols in Plants; Elsevier Inc.: Amsterdam, The
Netherlands, 2019; pp. 191–215.
2. Putzbach, K.; Rimmer, C.A.; Sharpless, K.E.; Sander, L.C. Determination of Bitter Orange alkaloids in dietary supplements
standard reference materials by liquid chromatography with ultraviolet absorbance and fluorescence detection. J. Chromatogr. A
2007, 1156, 304–311. [CrossRef] [PubMed]
3. Hao, K.; Hu, W.; Hou, M.; Cao, D.; Wang, Y.; Guan, Q.; Zhang, X.; Wang, A.; Yu, J.; Guo, B. Optimization of ultrasonic-assisted
extraction of total phenolics from Citrus aurantium L. Blossoms and evaluation of free radical scavenging, Anti-HMG-CoA
Reductase Activities. Molecules 2019, 24, 2368. [CrossRef]
4. Mohagheghniapour, A.; Saharkhiz, M.J.; Golmakani, M.T.; Niakousari, M. Variations in chemical compositions of essential
oil from sour orange (Citrus aurantium L.) blossoms by different isolation methods. Sustain. Chem. Pharm. 2018, 10, 118–124.
[CrossRef]
5. Maksoud, S.; Abdel-Massih, R.M.; Rajha, H.N.; Louka, N.; Chemat, F.; Barba, F.J.; Debs, E. Citrus aurantium l. Active constituents,
biological effects and extraction methods. an updated review. Molecules 2021, 26, 5832. [CrossRef]
6. Meneses, C.; Valdes-Gonzalez, M.; Garrido-Suárez, B.B.; Garrido, G. Systematic review on the anxiolytic and hypnotic effects of
flower extracts in in vivo pre-clinical studies published from 2010 to 2020. Phytother. Res. 2023, 37, 2144–2167. [CrossRef]
7. Motaghi, S.; Jonaidi, H.; Abbasnejad, M.; Usofi, M.; Khaki, M.A.; Sheibani, V. Behavioral and electrophysiological evidence for
attenuation of CNS by aqueous extract from Citrus aurantium (CaL) flowers in rat. Comp. Clin. Path 2016, 25, 769–774. [CrossRef]
8. Seyyedi-Mansour, S.; Carpena, M.; Donn, P.; Garcia-Oliveira, P.; Echave, J.; Barciela, P.; Fraga-Corral, M.; Cassani, L.; Simal-
Gandara, J.; Prieto, M.A. Profiling of Phenolic Compounds in Citrus Flowers and Their Biological Activities. Eng. Proc. 2023,
48, 37. [CrossRef]
9. Değirmenci, H.; Erkurt, H. Relationship between volatile components, antimicrobial and antioxidant properties of the essential
oil, hydrosol and extracts of Citrus aurantium L. flowers. J. Infect. Public Health 2020, 13, 58–67. [CrossRef] [PubMed]
10. Sandhu, H.K.; Sinha, P.; Emanuel, N.; Kumar, N.; Sami, R.; Khojah, E.; Al-Mushhin, A.A. Effect of ultrasound-assisted pretreatment
on extraction efficiency of essential oil and bioactive compounds from citrus waste by-products. Separations 2021, 8, 244. [CrossRef]
11. Karimi, E.; Oskoueian, E.; Hendra, R.; Oskoueian, A.; Jaafar, H.Z.E. Phenolic compounds characterization and biological activities
of Citrus aurantium bloom. Molecules 2012, 17, 1203–1218. [CrossRef] [PubMed]
12. Chen, C.Y.; Hu, C.Y.; Chen, Y.H.; Li, Y.T.; Chung, Y.C. Submerged fermentation with Lactobacillus brevis significantly improved
the physiological activities of Citrus aurantium flower extract. Heliyon 2022, 8, e10498. [CrossRef] [PubMed]
13. Hashemi, S.M.B.; Amininezhad, R.; Shirzadinezhad, E.; Farahani, M.; Yousefabad, S.H.A. The Antimicrobial and Antioxidant
Effects of Citrus aurantium L. Flowers (Bahar Narang) Extract in Traditional Yoghurt Stew During Refrigerated Storage. J. Food Saf.
2016, 36, 153–161. [CrossRef]
14. Degirmenci, H.; Erkurt, H. Chemical profile and antioxidant potency of Citrus aurantium L. flower extracts with antibacterial
effect against foodborne pathogens in rice pudding. LWT 2020, 126, 10927. [CrossRef]
15. Zhao, H.Y.; Yang, L.; Wei, J.; Huang, M.; Jiang, J.G. Bioactivity evaluations of ingredients extracted from the flowers of Citrus
aurantium L. var. amara Engl. Food Chem. 2012, 135, 2175–2181. [CrossRef] [PubMed]
16. Trabelsi, D.; Ammar, A.H.; Bouabdallah, F.; Zagrouba, F. Antioxidant and Antimicrobial Activities of Essential Oils and Methanolic
Extracts of Tunisian Citrus aurantium L. IOSR J. Environ. Sci. Toxicol. Food Technol. 2014, 8, 18–27. [CrossRef]
17. Cai, W.F.; Yan, M.M.; Wang, Z.; Jiang, M.P.; Yan, B.; Shen, C.Y. Optimization of the extract from flower of Citrus aurantium L. var.
amara Engl. and its inhibition of lipid accumulation. J. Food Biochem. 2022, 46, e14332. [CrossRef] [PubMed]
18. Almalki, W.H. Citrus aurantium flowers essential oil protects liver against ischemia/reperfusion injury. S. Afr. J. Bot. 2021, 142,
325–334. [CrossRef]
19. Dosoky, N.S.; Setzer, W.N. Biological Activities and Safety of Citrus spp. Essential Oils. Int. J. Mol. Sci. 2018, 19, 1966. [CrossRef]
[PubMed]
Molecules 2025, 30, 930 17 of 18

20. Liang, Z.; Shang, X.B.; Su, J.; Li, G.Y.; Fu, F.H.; Guo, J.J.; Shan, Y. Alternative Extraction Methods of Essential Oil from the Flowers
of Citrus aurantium L. Var Daidai Tanaka: Evaluation of Oil Quality and Sedative-Hypnotic Activity. Nat. Prod. Commun. 2021,
16, 1934578X211004061. [CrossRef]
21. Sarrou, E.; Chatzopoulou, P.; Dimassi-Theriou, K.; Therios, I. Volatile Constituents and Antioxidant Activity of Peel, Flowers and
Leaf Oils of Citrus aurantium L. Growing in Greece. Molecules 2013, 18, 10639–10647. [CrossRef] [PubMed]
22. Hsouna, A.B.; Hamdi, N.; Halima, N.B.; Abdelkafi, S. Characterization of essential oil from Citrus aurantium L. flowers: Antimi-
crobial and antioxidant activities. J. Oleo Sci. 2013, 62, 763–772. [CrossRef]
23. Nabavi, S.F.; Nabavi, S.M.; Ebrahimzadeh, M.A. Antioxidant activity of hydro-alcoholic extracts of 4 citrus species flower. Prog.
Nutr. 2016, 18, 74–80.
24. Garcia-Oliveira, P.; Otero, P.; Pereira, A.G.; Chamorro, F.; Carpena, M.; Echave, J.; Fraga-Corral, M.; Simal-Gandara, J.; Prieto, M.A.
Status and challenges of plant-anticancer compounds in cancer treatment. Pharmaceuticals 2021, 14, 157. [CrossRef] [PubMed]
25. Suntar, I.; Khan, H.; Patel, S.; Celano, R.; Rastrelli, L. An overview on Citrus aurantium L.: Its functions as food ingredient and
therapeutic agent. Oxid. Med. Cell. Longev. 2018, 2018, 7864269. [CrossRef] [PubMed]
26. Makhoul, S.; Bakkour, Y.; El-nakat, H.; Omar, F.E. The Lebanese Citrus aurantium: A Promising Future in Medicinal Phytochemistry.
J. Pharmacogn. Phytochem. 2012, 1, 63–66.
27. Antonelli, M.; Kushner, I. It’s time to redefine inflammation. FASEB J. 2017, 31, 1787–1791. [CrossRef]
28. Cox, A.J.; West, N.P.; Cripps, A.W. Obesity, inflammation, and the gut microbiota. Lancet Diabetes Endocrinol. 2015, 3, 207–215.
[CrossRef]
29. Li, X.Y.; Hao, Y.F.; Hao, Z.X.; Jiang, J.G.; Liu, Q.; Shen, Q.; Liu, L.; Yi, Y.-K.; Shen, C.-Y. Inhibitory effect of chloroform extracts from
Citrus aurantium L. var. amara Engl. on fat accumulation. Phytomedicine 2021, 90, 153634. [CrossRef]
30. Torres-Villarreal, D.; Camacho, A.; Castro, H.; Ortiz-Lopez, R.; de la Garza, A.L. Anti-obesity effects of kaempferol by inhibiting
adipogenesis and increasing lipolysis in 3T3-L1 cells. J. Physiol. Biochem. 2019, 75, 83–88. [CrossRef] [PubMed]
31. Parihar, M.S.; Hemnani, T. Alzheimer’s disease pathogenesis and therapeutic interventions. J. Clin. Neurosci. 2004, 11, 456–467.
[CrossRef]
32. Rahnama, S.; Rabiei, Z.; Alibabaei, Z.; Mokhtari, S.; Rafieian-kopaei, M.; Deris, F. Anti-amnesic activity of Citrus aurantium flowers
extract against scopolamine-induced memory impairments in rats. Neurol. Sci. 2015, 36, 553–560. [CrossRef] [PubMed]
33. Bitwell, C.; Indra, S.S.; Luke, C.; Kakoma, M.K. A review of modern and conventional extraction techniques and their applications
for extracting phytochemicals from plants. Sci. Afr. 2023, 19, e01585. [CrossRef]
34. Zhu, P.; Hao, M.; Su, F.; Xu, W.; Zhang, Q.; Su, W.; Adams, E. Mechanochemical-assisted extraction of essential oils from Citrus
aurantium L. var. amara Engl. Ind. Crops Prod. 2022, 188, 115703. [CrossRef]
35. Moutaouafiq, S.; Farah, A.; Benbrahim, K.F.; Salma, M.; Abdellah, F.; El Houssine, A.; Kawtar, B.; Dalila, B. Comparison of the
Chemical Composition and the Bioactivity of the Essential Oils of Three Medicinal and Aromatic Plants from Jacky Garden of
Morocco. Int. J. Pharmacogn. Phytochem. Res. 2016, 8, 537–545.
36. Dhifi, W.; Mnif, W.; Jelali, N.; Beyrouthy, M.E.; Salem, N.B. Citrus aurantium (Bitter Orange) blossoms essential oil and methanolic
extract: Composition and free radical scavenging activity. Acta Hortic. 2013, 997, 195–200. [CrossRef]
37. Li, J.; Luo, Y.; Zhan, L.; Gu, Y.; Zhang, W.; Wen, Q.; Feng, Y.; Tan, T. Comprehensive chemical profiling of the flowers of Citrus
aurantium L. var. amara Engl. and uncovering the active ingredients of lipid lowering. J. Pharm. Biomed. Anal. 2022, 211, 114621.
[CrossRef] [PubMed]
38. Oreopoulou, A.; Tsimogiannis, D.; Oreopoulou, V. Extraction of Polyphenols From Aromatic and Medicinal Plants: An Overview
of the Methods and the Effect of Extraction Parameters. In Polyphenols in Plants; Academic Press: London, UK, 2019; pp. 243–259.
39. Aramrueang, N.; Asavasanti, S.; Khanunthong, A. Leafy Vegetables. In Integrated Processing Technologies for Food and Agricultural
by-Products; Academic Press: Cambridge, MA, USA, 2019; pp. 245–272.
40. Malik, J.; Mandal, S.C. Extraction of herbal biomolecules. In Herbal Biomolecules in Healthcare Applications; Academic Press:
London, UK, 2022; pp. 21–46.
41. Chen, H.; Wang, L. Posttreatment Strategies for Biomass Conversion. In Technologies for Biochemical Conversion of Biomass;
Metallurgical Industry Press: London, UK, 2017; pp. 197–217.
42. Zhang, Q.W.; Lin, L.G.; Ye, W.C. Techniques for extraction and isolation of natural products: A comprehensive review. Chin. Med.
2018, 13, 20. [CrossRef] [PubMed]
43. Aboualsoltani, F.; Bastani, P.; Khodaie, L.; Fazljou, S.M.B.; Salehi-Pourmehr, H. The effect of Citrus aurantium L. flower extract on
the severity of primary dysmenorrhoea: A double-blind, randomised, controlled clinical trial. J. Herb. Med. 2024, 45, 100878.
[CrossRef]
44. More, P.R.; Jambrak, A.R.; Arya, S.S. Green, environment-friendly and sustainable techniques for extraction of food bioactive
compounds and waste valorization. Trends Food Sci. Technol. 2022, 128, 296–315. [CrossRef]
Molecules 2025, 30, 930 18 of 18

45. Fernández-Cabal, J.; Avilés-Betanzos, K.A.; Cauich-Rodríguez, J.V.; Ramírez-Sucre, M.O.; Rodríguez-Buenfil, I.M. Recent Devel-
opments in Citrus aurantium L.: An Overview of Bioactive Compounds, Extraction Techniques, and Technological Applications.
Processes 2025, 13, 120. [CrossRef]
46. Nour, A.H.; Modather, R.H.; Yunus, R.M.; Elnour, A.A.M.; Ismail, N.A. Characterization of bioactive compounds in patchouli oil
using microwave-assisted and traditional hydrodistillation methods. Ind. Crops Prod. 2024, 208, 117901. [CrossRef]
47. Yani, S.; Gao, X.; Grayling, P.; Wu, H. Steam distillation of mallee leaf: Extraction of 1,8-cineole and changes in the fuel properties
of spent biomass. Fuel 2014, 133, 341–349. [CrossRef]
48. Hamid Nour, A.; Ruth Oluwaseun, A.; Hamid Nour, A.; Suliman Omer, M.; Ahmed, N. Microwave-Assisted Extraction of
Bioactive Compounds (Review). In Microwave Heating—Electromagnetic Fields Causing Thermal and Non-Thermal Effects; IntechOpen:
London, UK, 2021; p. 13. Available online: https://www.intechopen.com/books/advanced-biometric-technologies/liveness-
detection-in-biometrics (accessed on 15 July 2024).
49. Yusoff, I.M.; Mat Taher, Z.; Rahmat, Z.; Chua, L.S. A review of ultrasound-assisted extraction for plant bioactive compounds:
Phenolics, flavonoids, thymols, saponins and proteins. Food Res. Int. 2022, 157, 111268. [CrossRef] [PubMed]
50. Gavahian, M.; Farahnaky, A.; Javidnia, K.; Majzoobi, M. Comparison of ohmic-assisted hydrodistillation with traditional
hydrodistillation for the extraction of essential oils from Thymus vulgaris L. Innov. Food Sci. Emerg. Technol. 2012, 14, 85–91.
[CrossRef]
51. Mannucci, C.; Calapai, F.; Cardia, L.; Inferrera, G.; D’Arena, G.; Di Pietro, M.; Navarra, M.; Gangemi, S.; Spagnolo, E.V.; Calapai,
G. Clinical Pharmacology of Citrus aurantium and Citrus sinensis for the Treatment of Anxiety. Evid. Based Complement Altern. Med.
2018, 2018, 3624094. [CrossRef]
52. Zhou, P.; Zheng, M.; Li, X.; Zhou, J.; Shang, Y.; Li, Z.S.; Qu, L. A consecutive extraction of pectin and hesperidin from Citrus
aurantium L.: Process optimization, extract mechanism, characterization and bio-activity analysis. Ind. Crops Prod. 2022,
182, 114849. [CrossRef]
53. Badalamenti, N.; Bruno, M.; Schicchi, R.; Geraci, A.; Leporini, M.; Gervasi, L.; Tundis, R.; Loizzo, M.R. Chemical Compositions
and Antioxidant Activities of Essential Oils, and Their Combinations, Obtained from Flavedo By-Product of Seven Cultivars of
Sicilian Citrus aurantium L. Molecules 2022, 27, 1580. [CrossRef] [PubMed]
54. Napoli, M.D.; Castagliuolo, G.; Badalamenti, N.; Maresca, V.; Basile, A.; Bruno, M.; Varcamonti, M.; Zanfardino, A. Citrus
aurantium ‘Crispifolia’ Essential Oil: A Promise for Nutraceutical Applications. Nutraceuticals 2023, 3, 153–164. [CrossRef]
55. Benzaid, C.; Belmadani, A.; Tichati, L.; Djeribi, R.; Rouabhia, M. Effect of Citrus aurantium l. Essential oil on streptococcus mutans
growth, biofilm formation and virulent genes expression. Antibiotics 2021, 10, 54. [CrossRef] [PubMed]
56. Pasandideh, S.; Arasteh, A. Evaluation of antioxidant and inhibitory properties of Citrus aurantium L. on the acetylcholinesterase
activity and the production of amyloid nano–bio fibrils. Int. J. Biol. Macromol. 2021, 182, 366–372. [PubMed]
57. Karimzadeh, Z.; Azizzadeh Forouzi, M.; Rahiminezhad, E.; Ahmadinejad, M.; Dehghan, M. The Effects of Lavender and Citrus
aurantium on Anxiety and Agitation of the Conscious Patients in Intensive Care Units: A Parallel Randomized Placebo-Controlled
Trial. BioMed Res. Int. 2021, 2021, 5565956. [CrossRef] [PubMed]
58. Aydeniz-Guneser, B. Cold pressed orange (Citrus sinensis) oil. In Cold Press Oils: Green Technology, Bioactive Compounds, Functionality,
and Applications; Academic Press: Cambridge, MA, USA, 2020; pp. 129–146.
59. Sevindik, E.; Aydin, A.; Yildirim, E.M. Chemical Compositions and Antiproliferative Effect of Citrus sinensis and Citrus aurantium
Flowers in The West Anatolian Ecological Conditions. J. Essent. Oil Bear. Plants 2018, 21, 1011–1020. [CrossRef]

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