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Article

Antibacterial and Mosquito Repellent Potential of Eight Citrus Cultivars and Their Chemical Composition

by
Mehwish Nawaz
1,
Bait Ullah
1,
Muhammad Ghazanfar Abbas
2,
Muhammad Binyameen
2,
Violeta Apšegaitė
3,
Raimondas Mozūraitis
3,4,* and
Muhammad Azeem
1,*
1
Department of Chemistry, COMSATS University Islamabad, Abbottabad Campus, Abbottabad 22060, Pakistan
2
Laboratory of Insect Chemical Ecology, Department of Entomology, Faculty of Agricultural Sciences & Technology, Bahauddin Zakariya University, Multan 60800, Pakistan
3
Laboratory of Chemical and Behavioral Ecology, Institute of Ecology, Nature Research Centre, LT-08412 Vilnius, Lithuania
4
Department of Zoology, Stockholm University, SE-10691 Stockholm, Sweden
*
Authors to whom correspondence should be addressed.
Horticulturae 2025, 11(1), 9; https://doi.org/10.3390/horticulturae11010009
Submission received: 2 December 2024 / Revised: 20 December 2024 / Accepted: 23 December 2024 / Published: 26 December 2024
(This article belongs to the Section Processed Horticultural Products)
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Abstract

:
Citrus fruit peels are a rich source of essential oils (EOs), which contain biologically active compounds; however, they are often discarded as waste, which causes pollution. The fresh peels of eight citrus cultivars growing in Pakistan were used to extract EOs through steam distillation. Gas chromatography-mass spectrometry (GC-MS) analysis of fresh peel EOs revealed that limonene was the most abundant compound, constituting 94.5%, 96.1%, 95.3%, 93.3%, 56.2%, 91.5%, 96.4%, and 96.7% of Citrus jambhiri, C. aurantium, C. sinensis var. Malta cv. Blood Malta, C. sinensis var. Malta cv. Shakri Malta, C. limon, C. pseudolimon, C. reticulata var. Mandarin cv. Feutrell’s Early, and C. reticulata var. Mandarin cv. Kinnow, respectively. The dried peel EO of C. reticulata var. Mandarin cv. Kinnow contained 95.2% limonene. C. limon peel EO exhibited the highest antibacterial activity among all citrus peel EOs with the minimum inhibitory concentration of 312 μg/mL against Staphylococcus aureus. The C. aurantium and C. sinensis var. Malta cv. Shakri Malta peel EOs exhibited the highest mosquito repellent activity against Ae. aegypti females, providing protection for 45 min when tested at a concentration of 166 µg/cm2. This study showed C. aurantium and Shaki Malta peel EOs could be used to formulate natural mosquito repellent.

1. Introduction

Most cultivated citrus are hybrids between two or more ancestral species of the genus Citrus (Sapindales: Rutaceae). They are distributed throughout the tropical, subtropical, and temperature zones with over 250 known commercial varieties [1], including orange, pomelo, grapefruit, kinnow, lemon, sweet orange, kumquat, lime, and others [2]. About 140 countries produce 70 million tons of citrus fruits annually. Pakistan is the 12th largest citrus producer, with an annual production of about 1,816,000 tons [3]. Swat, Mardan, Nowshera, Malakand, Lower Dir, Multan, Sahiwal, Sargodha, Bahawalpur, Toba Tek Singh, and Vehari are the citrus-producing districts in Pakistan [4].
Citrus peels comprise approximately 20–30% of the total weight of fruit [5]. Tons of solid citrus peel waste are produced during fruit processing, such as canning and juicing, and are often discarded as waste, contributing to significant environmental pollution with a lack of practical reuse. Even though they are not edible, citrus peels could be used as fish feed, as a raw material for conventional paper, as an activated carbon adsorbent in cosmetics [6], and in bioethanol production [7]. Citrus peels are a good source of bioactive compounds such as ascorbic acid, carotenoids, and flavonoids [8]. Moreover, citrus peels are rich in essential oils (EOs). It is estimated that 0.5–3.0 g/kg of EO can be obtained from citrus fruit peels [9]. Citrus peel oil is produced by cold press, solvent extraction, or distillation. The cold press is the most commonly used industrial method for citrus EOs extraction, producing complex mixtures of about 400 compounds, 85–99% of which are volatile constituents, including several types of sesquiterpenes, hydrocarbons, and monoterpenes [10]. The composition of the mixture of terpenes varies from species to species and includes different compounds like limonene, α-pinene, β-pinene, β-myrcene, linalool, and terpinene [11]. Limonene is a major component of citrus peel EO, as it ranges between 32 and 98% [12].
Besides Citrus species, limonene is found in different proportions in diverse types of plant essential oils. Several previous studies demonstrated various biological activities of limonene. For example, a limonene racemic mixture and individual enantiomers exhibited antibacterial activity against different bacteria [13,14,15,16]. A study described the anti-fungal activity of limonene against food-spoiling yeast [17]. In another study, both enantiomers of limonene exhibited similar mosquito larvicidal activity against Ae. albopictus; however, in repellency bioassay, (-)-limonene showed higher activity, whereas (+)-limonene exhibited comparatively lower activity against female Ae. albopictus [18]. Miller et al. reported that limonene showed potential as an anti-cancer agent to treat breast cancer [19]. Vieira et al. (2018) reviewed several studies summarizing various health-beneficial effects of limonene, such as anti-inflammatory, anti-bacterial, anti-oxidant, and anti-cancer [20]. Both enantiomers of α-pinene and (-)-β-pinene exhibited moderate repellency, whereas (+)-β-pinene showed good repellency towards Ae. albopictus female mosquito [18]. A study showed the anti-bacterial activity of α-pinene and β-pinene against different bacteria [21]. A recent study reported the biological activity of α-pinene and β-pinene for controlling cattle tick Rhipicephalus microplus [22].
Infectious ailments are significant public health issues worldwide [23,24]. Though various antibiotic agents are available to treat microbial infection, microbes have acquired resistance against many antibiotics [24,25,26]. To overcome this problem, natural products from plants could be a better alternative to antibiotics since plants are the foremost source of bioactive compounds. Plant natural products are considered safe for personal use and are effective and readily available for treating various ailments [27,28]. Several studies reported that citrus peel EOs possessed a wide range of biological activities such as anti-viral [29,30], antibacterial [14,31,32,33], anti-inflammatory [34], antioxidant [35], anticancer [36], and anti-fungal activities [33,37,38].
Mosquitoes are significant carriers of several tropical diseases, including dengue, yellow fever, malaria, etc. An evident practical and most economical way of avoiding the spread of these diseases to people is the use of repellents [39]. Mosquito repellents are preferred to prevent insect-borne diseases and are a cost-effective healthcare practice. In the market, various synthetic and natural insect repellents are available, including the most famous formulation, N, N-diethyl-3-methylbenzamide (DEET), which has proven to show excellent repellency against mosquitoes and other blood-feeding insects [40]. However, several studies showed that prolonged use of DEET could pose some adverse effects. The research demonstrated that EOs-based mosquito repellents could be the best alternative to synthetic formulations as they are considered safe and show effective repellency against several mosquito species [41,42,43].
Numerous studies from various countries have reported the chemical composition of citrus peel EOs [12,44,45], as well as their antibacterial [32,33,44,46,47] and mosquito larvicidal [48,49] activities. However, a few studies in the literature describe the mosquito-repellent activities [50,51,52,53] of citrus peel EOs. To our knowledge, no previous study has reported the mosquito-repellent activity of Citrus jambhiri, C. pseudolimon, and C. sinensis var. Malta cv. Shakri Malta, C. sinensis var. Malta cv. Blood Malta, and C. reticulata (L.) var. Mandarin cv. Feutrell’s early. Moreover, no detailed investigation has been carried out to compare the chemical composition and bioactivity of EOs extracted from the peels of various citrus cultivars, minimizing variations in sample preparation methods that affect the chemical composition and biological activities. To fill this knowledge gap, this study aimed to compare the chemical composition of EOs extracted from fruit peels of various citrus cultivars growing in Pakistan and to evaluate their mosquito-repellent activity against outdoor-biting Aedes aegypti as well as their antibacterial activity against pathogenic bacteria: Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa PAO1. Moreover, the enantiomeric composition of limonene present in different citrus EOs was also investigated.

2. Materials and Methods

2.1. Collection and Maintenance of Citrus Peels

Different cultivars of citrus fruits were collected from their respective orchards located in various districts of Pakistan (Table 1). The plant specimens were identified by comparing the diagnostic morphological characters of the plant with those presented in the Flora of Pakistan and with those available from the literature sources [54,55,56]. In addition, the names of the plants were verified using World Flora Online [57] and the Flora of Pakistan [58]. Voucher specimens were submitted to the herbarium of the Department of Environmental Sciences, COMSATS University Islamabad, Abbottabad Campus, Abbottabad, Pakistan. The fruits were thoroughly washed with tap water and wiped with a cotton cloth. Afterward, the peels were carefully removed with a sharp knife, cut into pieces of 2–3 inches, and subjected to EO extraction on the same day or stored in the freezer at −30 °C until used for EO extraction within 24 h.

2.2. Extraction of EOs

Steam distillation was used to extract EOs from the fresh peels of citrus fruits using a previously reported method [42,59]. Weighed citrus fruit peels of 1500 g were subjected to steam distillation in a stainless-steel distillation apparatus (Liaqat Engineering, Faisalabad, Pakistan). A 2 L of distilled water was added to the bottom of the stainless-steel vessel to avoid direct contact with the peels packed in a meshed container adjusted above water level. The vessel was then heated using an electric hotplate. The released steam passed through the packed peels, extracting the volatile compounds. The steam containing peel volatiles was cooled down using a water condenser connected externally to the top of the vessel. The distillate, consisting of water and peel volatiles, was collected in a 1 L glass separating funnel for 3 h. The lower water layer was disposed of, and the upper layer of EO was recovered through decantation and weighed using a digital analytical balance after removing traces of water over anhydrous MgSO4 (Daejung Chemicals, Siheung-si, South Korea). The percentage yield of extracted EO was calculated by dividing the mass of EO by the mass of fresh peels and multiplying by a hundred. From each citrus peel sample, the EO was extracted in a triplicated manner. The extracted EOs were stored in glass vials at −20 °C until used for chemical analysis and bioassays.

2.3. Chemical Analysis of EOs by GC-MS

The chemical analysis of citrus peel EO was investigated using a Hewlett-Packard 6890 N gas chromatograph (GC) and an HP 5973 mass spectrometer (MS, Agilent Technologies Inc., Santa Clara, CA, USA). The GC was fitted with a DB-5 capillary column (Agilent Technologies Inc., Santa Clara, CA, USA) having 30 m length, 0.25 mm internal diameter, and 0.25 µm stationary phase film thicknesses. The parameters of GC and MS were set as previously reported by Azeem et al. [42]. In short, the GC injector was isothermally set at 225 °C. The initial temperature of the column oven was isothermally set at 40 °C for 2 min after that, increased at the rate of 4 °C/min to 230 °C, and finally isothermally set at 230 °C for 5 min. High-purity helium (99.99%) was used as the carrier gas that flowed at a steady rate of 1 mL/min through the column. Diluted solutions of EO samples were injected in a GC injector in the splitless mode set for 30 s. The parameters for the mass spectrometer were as follows: an electron ionization energy of 70 eV was used for ionization in positive mode. MS ion source temperature was isothermally set at 180 °C. Mass spectra of the separated compounds were acquired in the 30 to 400 m/z range. The GC peaks were used to calculate the percentage composition of every component of an EO. To identify the separated compounds, their mass spectra were first compared to those in the NIST-2008 (National Institute of Standard Technology) MS library, in the NIST webbook, and to published data [60]. The retention times of n-alkanes (C9–C24) were determined to calculate the retention indexes of the isolated compounds by applying the same GC-MS parameters used for the analyses of the EOs. The computed retention indices were compared to the published data to determine the elution sequence and identify the separated substances. Lastly, the identification of EO constituents was confirmed by injecting available pure reference compounds such as α-pinene, β-pinene, β-myrcene, limonene, and linalool, etc. (Sigma-Aldrich, St. Louis, MO, USA) using the identical GC-MS parameters applied for analyses of EOs.
The enantiomeric composition of limonene in EOs was determined by a Shimadzu GC-2010 Plus gas chromatograph equipped with an AOC-20i liquid autosampler, an FID detector (Shimadzu Corporation, Kyoto, Japan), and an Rt®-bDEXsm column (30 m × 0.25 mm × 0.25 µm) (Restek Corporation, Bellefonte, PA, USA). The stationary chiral phase of the column was composed of 2,3-di-O-methyl-6-O-tert-butyldimethylsilyl-β-cyclodextrin and cyanopropylphenyl/dimethylpolysiloxane. Cyclodextrin-based GC stationary phases provide excellent separation for a wide range of chiral compounds and are the most widely used [61]. The injector and the detector temperatures were set isothermal at 250 °C and 260 °C, respectively. The oven’s initial temperature was 50 °C; afterward, it ramped by 2 °C/min to 160 °C and then increased by 10 °C/min to 210 °C. Helium was used as the carrier gas at a 1.5 mL/min flow rate. Nitrogen was used as a make-up gas at a flow rate of 30 mL/min. Standards of (S)-(-)-limonene and (R)-(+)-limonene were obtained from Fluka Chemicals, Gillingham, UK.

2.4. Antibacterial Activity

To test the antibacterial potential of extracted EOs, four human pathogenic bacterial strains, Escherichia coli ATCC 25922, Pseudomonas aeruginosa (PAO1), Bacillus subtilis ATCC 6633, and Staphylococcus aureus ATCC 6538, were obtained from the National Institute of Health, Islamabad, Pakistan. The bacterial strains were streaked on nutrient agar (NA) Petri plates and grown overnight at 37 °C. The broth dilution method was used to find the minimum inhibitory concentration (MIC) against selected bacterial strains by adopting a reported method [62]. Briefly, freshly grown bacteria colonies were suspended in 4 mL of sterilized distilled water. The suspension’s optical density was adjusted to the equivalent of 0.5 McFarland standard, which consisted of 108 colony-forming units per mL (CFU/mL). The bacterial suspension was further diluted serially in sterilized distilled water to obtain the required concentration of 104 CFU/mL. To determine the MIC of test substances, an aliquot of 10 µL of bacterial suspension was mixed in 990 µL of sterilized water to be used as a water reference to count the number of CFU originally added in any sample or control test tube. In another similar test tube, 980 µL of sterilized nutrient broth was taken, to which 10 µL of bacterial suspension and 10 µL of test substance solution were added. After overnight incubation at 37 °C, a 100 µL aliquot of the mixtures from the water reference and sample test tubes was spread evenly on separate NA Petri plates and incubated at 37 °C for 24 h, and viable CFUs were counted on each Petri plate. If the number of CFU in the test substance is less than or equal to the number of CFU in the water reference, then the concentration was considered MIC [62]. The concentrations ranging from 0.312 to 20 mg/mL were used in determining the MIC of test substances. In this experiment, ciprofloxacin was used as a positive control, whose two-fold dilutions of 2.5–40 µg/mL were employed. At least five replicates of each concentration of test or control samples were employed. The same bacterial strains were also used to find bacterial growth inhibition by adopting the reported method [62], with details presented in Supplementary Data.

2.5. Mosquito Rearing

Ae. aegypti colony was maintained under laboratory conditions as described earlier [42,63]. Briefly, Ae. aegypti eggs were added in distilled water maintained in a climate chamber set at 25 ± 2 °C and 80 ± 10% relative humidity at the photoperiod 12 h:12 h light: dark. The hatched larvae were fed with a fish diet (Osaka green fish food, Chennai, India). The larvae were observed daily, and the emerged pupae were transferred to a separate plastic container containing distilled water. The container was placed in the Plexiglas mosquito cages till the emergence of adults. Cotton soaked with 10% sucrose solution was placed in adult mosquito cages to provide food for adult mosquitoes. The mated female (4–5 days old) mosquitoes were fed with the blood of an immobilized pigeon. The polypropylene jars (200 mL) filled with distilled water and lined with wax paper were placed in each adult cage as egg-laying media. The eggs were shifted to fresh distilled water in a tray for hatching. The procedure was repeated until the number of adult mosquitoes was sufficient for mosquito repellency bioassays.

2.6. Mosquito Repellency Bioassay

The repellent activity of citrus peel EOs was investigated against adult female Ae. aegypti by using the human bait method previously described [42,50]. Briefly, 3–4 days old and blood-starved 20 female mosquitoes were released in a separate experimental cage. Before the experiment, the volunteer hands were washed with fragrance-free soap and dried in the air. The volunteer wore gloves on both hands that covered the entire hand and arm except for a circular area of 30 cm2 on the dorsal side of both hands. An aliquot of 100 μL solution of negative control (ethanol solvent) or test substance (1% or 5% w/v) solution was evenly applied to the exposed area of the hand. In this way, the concentration of pure EO on the test hand was 33.3 μg/cm2 or 166 μg/cm2. The solvent was evaporated for 3 min in the air before starting the repellency bioassay. The hand was exposed to female mosquitoes in an experimental cage for 5 min, and the number of mosquitoes’ successful landings was counted on the negative control or sample-treated hand. To check the repellent persistence, the bioassay was carried out in the same way described above, except using the same treated hand after each 15 min period and counting females’ landings for 5 min until the number of mosquito landings on control and treated hands became equal. The human subjects (3 volunteers) were informed about the test procedure, and informed consent was obtained before conducting repellency bioassays. Moreover, permission for human subjects use was obtained from the Ethical and Biosafety Committee of Bahauddin Zakariya University, Multan. The experiment was repeated five times for each test or control substance, and fresh mosquitoes were used in each replicate. The percent repellency was calculated using the formula: % Repellency = [(Mc − Mt)/Mc] × 100, where Mc is the number of mosquito landings on the negative control and Mt is the number of mosquito landings on the test substance-treated hand.

2.7. Statistical Analysis

To determine the statistical difference between CFU percent inhibition (Supplementary Data) and the repellent effect of EO samples, the data were analyzed by one-way ANOVA with a post-hoc Bonferroni test. The statistical tests were performed using the computer software SPSS 20 (IBM, Armonk, NY, USA).

3. Results

3.1. Percentage Yield of EOs from Citrus Fruit Peels

Fresh fruit peels of different citrus cultivars produced 0.05–0.38% of EO. The fresh peels of C. jambhiri and C. reticulata yielded the highest quantity of EO, whereas C. limon peel yielded the least amount of EO compared to all other citrus cultivar samples (Table 1).

3.2. Chemical Composition of EOs

Limonene was the most abundant compound in the EOs of all citrus samples, composing over 90% of the oil content except C. limon EO, which comprised 56% of this monoterpene (Table 2, Figure S1). β-Myrcene was also found in the EOs of all citrus samples, and its proportion ranged from 0.8 to 2.7% (Table 2). β-Pinene composed 20.2% of C. limon EO, and its relative abundance was significantly higher than those determined in the EOs of all other samples (Table 2, Figure 1).
The chiral analysis of limonene present in different cultivar peel EOs showed that all citrus cultivars consisted of about 99% of (R)-(+)-limonene except C. limon EO, which contained 96.91% (R)-(+)-limonene and 3.09% (S)-(-)-limonene (Table 3).

3.3. Antibacterial Activity of EOs

All citrus peel EOs showed antibacterial activity with varying degrees against tested bacterial strains. Among all citrus peel EOs, the C. limon EO was the most active, with MIC values ranging from 0.312 to 0.625 mg/mL against all bacteria except PAO1, against which this EO showed MIC 1.25 mg/mL. The EOs of C. reticulata KF and KD showed moderate activity with MIC values of 1.25 and 2.5 mg/mL against E. coli, whereas 2.5 and 1.25 mg/mL against S. aureus, respectively, both these EOs exhibited MIC of 2.5 mg/mL against the PAO1 (Table 4). EOs of C. sinensis BM and SM, C. pseudolimon, and C. reticulata FE showed the least activity against all tested pathogenic bacteria (Table 4). EO of C. limon showed the best inhibition of colony-forming units against all four bacteria species, i.e., Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa (PAO1) compared to other EOs tested (Figure S2).

3.4. Repellent Activity of EOs Against Aedes aegypti Females

Mosquito repellent activity data showed that all citrus EOs showed activity against female Ae. aegypti. The statistical data analysis revealed that these EOs imparted significantly different bioactivity against female mosquitoes at 33.3 µg/cm2 (df = 9, F = 171, p < 0.0001). Overall, C. reticulata KD exhibited the highest repellency, whereas C. jambhiri showed the least repellency among all tested EOs. After 5 min of sample application, EOs of C. aurantium, C. reticulata FE, and KD showed similar (p > 0.05) repellency. After the same time frame, the EO of C. sinensis SM peels showed 73% repellent activity that was similar (p > 0.05) to those of C. aurantium and C. reticulata FE but different (p < 0.05) from C. reticulata KD. The repellency of all EOs decreased over time, and after 30 min of exposure, only the EOs of C. aurantium, C. sinensis SM, and C. reticulata KD showed some repellency (Figure 2).
The ANOVA analysis revealed that at the higher concentration of 166 µg/cm2, the tested EOs exhibited significantly different repellency against female mosquitoes when tested after 5 min (df = 9, F = 108, p < 0.0001) and 30 min (df = 9, F = 805, p < 0.0001). Among all tested citrus peels, EOs of C. aurantium, C. sinensis SM, C. reticulata FE, and C. limon showed 100% repellency, which was comparable (p > 0.05) to that of the positive control DEET (Figure 3). After 30 min of exposure, EOs of C. aurantium, C. sinensis SM, and C. reticulata KD showed 84%, 71%, and 56% repellency, respectively, whereas the repellency of all other samples decreased below 20%. After 75 min of exposure, only EOs of C. aurantium and C. sinensis SM displayed mosquito repellency of about 10% and 5%, respectively, against Ae. aegypti females (Figure 3). The mosquito-repellent activity of C. reticulata KD was higher than that of C. reticulata KF throughout the testing period.

4. Discussion

The yield of EOs distilled from peels of citrus cultivars was in the range of 0.05–0.38% of fresh peel mass. A previous study from Pakistan reported that C. reticulata, C. paradisii, and C. sinensis fresh peels produced 0.3%, 0.20%, and 0.24% of EOs [64]. Another study from Pakistan reported a 0.29% yield of C. reticulata peel EO [50]. An Iranian study revealed that C. aurantium produced 0.7% EO [65]. A recent study from Morocco reported that C. limonum, C. reticulata, and C. paradisii peels yielded 1.02%, 0.80%, and 0.90% of EOs, respectively [66]. In the current study and previous studies from Pakistan, large pieces of whole citrus peels were used for the extraction of EO, whereas in the study from Morocco, peel zest was utilized to extract EO, determining that the results of the current study are similar to previously reported studies from Pakistan and differ from those of Morocco. The variation in the percentage yield of EOs could be explained based on the differences in extraction method and the condition of the plant sample. Besides this, the growth conditions of plants, such as soil type, climate, and altitude, also affect the yield of an extracted EO [62,67,68].
In the current study, the major compound in the EO of C. aurantium peel was limonene, constituting more than 96%. A previous study from Greece reported that fresh peels of C. aurantium contained 94.7% limonene, 2.0% β-myrcene, and 0.7% linalool [69]. Another study from Iran described 81.6% limonene and 5.7% β-myrcene in EO of C. aurantium [44]. The chemical composition of C. aurantium determined in the current study is similar to that described in Greece; however, it differs to some extent from that reported by the Iranian study. Our results showed that the EO of C. jambhiri peels comprised limonene, β-myrcene, and terpinene-4-ol. A study from Egypt reported 92.4% limonene, 1.5% β-myrcene, 0.6% sabinene, and 0.5% terpinene-4-ol in EO of C. jambhiri peel [45]. Another study from Sudan reported 84.5% limonene as well as sabinene, β-myrcene, and α-terpineol [70]. The chemical composition of C. jambhiri EO reported in the current study is similar to previously reported data. The EOs of C. sinensis BM and SM cultivars investigated in the current study contained limonene, β-myrcene, and linalool as main compounds. Previously, Tao et al. from China described that limonene (77.5%), β-myrcene (6.3%), α-farnesene (3.6%), and γ-terpinene (3.4%) were the major components in oven-dried sweet orange (C. sinensis) peels EO [71]. A recent study from Pakistan showed that limonene (95.8%), α-pinene (0.3%), and β-pinene (0.5%) were the major compounds in the EO of shade-dried C. sinensis peels [33]. Interestingly, the chemical composition of BM and SM peel EOs is very similar to each other except for the relative proportion of linalool and some other minor components. Moreover, the data of C. sinensis cultivars described in this study exhibited some similarities to a previous Pakistani study [33] while showing significant differences from that reported by Chinese [71] investigators.
The current investigation revealed that the EOs of C. reticulata cultivars were comprised of limonene, β-myrcene, and α-phellandrene. However, a study from Morocco showed that the main components of C. reticulata zest EO were 76.6% limonene, 2.3% β-myrcene, and 16.7% ρ-cymene [66]. A study from India reported that the EO of shade-dried C. reticulata peel contained 50.4% limonene, 3.0% β-myrcene, and 3.1% trans-carveol [72]. The data published from Bulgaria reported 85.2% limonene, 4.3% β-myrcene, and 1.3% α-pinene as the most abundant components of C. reticulata EO [73]. A recent study from Pakistan showed 92.7% limonene, 2.5% β-myrcene, and 1.6% sabinene, along with some minor compounds composed of C. reticulata peel EO [50]. The differences in the chemical composition of C. reticulate EOs reported in the current and previous studies carried out in Pakistan could be determined by different cultivars of this species used for the experiments as well as the growth conditions of the plants.
Besides other citrus cultivars studied, the chemical composition of C. limon EO was significantly different due to the presence of α-pinene, β-pinene, sabinene, limonene, γ-terpinene, terpinene-4-ol, α-terpineol, and citral. Several previous studies also reported similar chemical compositions. For example, a previous study from India identified limonene (29.0%), β-pinene (15.5%), γ-terpinene (8.6%), neral (4.2%), terpinen-4-ol (3.3%), and geranial (5.3%) as major constituents in EO of C. limon [74]. In 2014, Al-Jabri and Hossain [32] compared the chemical composition of Indian and Turkish lemon peel EOs. They found that the Indian lemon peel EO consisted of 53.6% limonene, 15.1% α-terpineol, 7.4% β-pinene, 4.3% α-terpinolene, and 3.6% citral, whereas in Turkish lemon peel EO there were 78.9% limonene, 5.1% β-pinene, 4.6% α-terpineol, and 0.9% citral [32]. A study from Greece reported the presence of 59.3% limonene, 13.4% β-pinene, 8.6% γ-terpinene, 3.5% β-myrcene, and 1.6% geranial in the EO of lemon [18]. The EO of C. pseudolimon is rarely studied for chemical composition and biological activities. The main compounds composing the EO of the cultivar Galgal were sabinene, β-myrcene, limonene, and β-bisabolene. The chemical composition of C. pseudolimon described in the current study is significantly different from a previous study conducted in Pakistan that reported the presence of 47.1% limonene, 10.2% eugenol, and 3.7% γ-terpinene in EO of C. pseudolimon extracted from peels collected from Sargodha district of Pakistan [75]. The difference in chemical composition could be due to the difference in cultivation area and the method of identification of compounds in both studies.
The enantiomeric composition analysis of limonene found in various citrus cultivars showed that (R)-(+)-limonene was the most abundant enantiomer in most of the citrus cultivars, constituting more than 99% except EO of C. limon, where the amount of (R)-(+)-limonene reached 97%. In literature, there are a few studies where the chemical composition of citrus EO and the enantiomeric composition of chiral compounds were described. For example, a study from Greece reported the presence of 99.2% (R)-(+)-limonene in C. limon EO [18], whereas a Norwegian study reported 99.9% (R)-(+)-limonene in commercial lemon oil and orange oil [76], which is significantly different from current reported data.
Overall, the tested EOs were more active against Gram-negative bacteria compared to Gram-positive bacteria. All citrus peel EOs showed antibacterial activity with varying degrees against different tested bacterial strains. Here, both cultivars of C. sinensis showed similar antibacterial activity against all tested bacteria. A previous study from China [71] showed that the MIC values of C. sinensis EO against B. subtilis were 9.33 µL/mL (~9.33 mg/mL), S. aureus 4.66 μL/mL, and E. coli 18.75 μL/mL. Another study from Pakistan showed that MIC values of C. sinensis EO against E. coli, S. aureus, and S. agalactia were 13.020, 10.410, and 6.510 mg/mL, respectively [33]. The MIC result of C. senensis cultivars investigated in the current study is similar to those previously reported in Pakistani and Chinese studies.
Our data demonstrated that among all tested citrus EOs, C. limon EO was the most active and showed low MIC values compared to other citrus samples. The possible reason for this difference might be the difference in the chemical composition of these EOs. In all other cultivars EOs, limonene is the most abundant compound, whereas, in the case of C. limon, there are several compounds, including limonene, β-pinene, p-cymene, and other minor compounds whose synergetic effect made C. limon EO comparably more active compared to all other EOs. A previous study from Morocco indicated that C. limon EO showed MIC values of 60 µg/mL against S. aureus and 750 µg/mL against E. coli [66]. A study from Egypt reported the MIC of C. limon EO against B. cereus (510 μg/mL), E. coli (260 μg/mL), P. aeruginosa (200 μg/mL), and S. aureus (430 μg/mL) using the microdilution method [77]. The antibacterial activity of C. limon EO, determined in the current study, differs from previously reported data. Moreover, the bioactivity of C. limon EOs presented in various studies also differs from that of other species. This might be due to the different chemical compositions of the EOs as well as the difference in susceptibility of bacterial strains tested in these studies.
In the current study, MIC values of C. aurantium peel EO were relatively lower compared to those reported in the previous study from Iran that showed 100 mg/mL MIC against S. aureus and 50 mg/mL against E. coli, S. typhi, and B. cereus [44]. Similarly, another study from Bulgaria described MIC in the range from 60 to >600 µg/mL for C. aurantium EO against S. aureus, E. coli, P. aeruginosa, and B. subtilis, which is lower than that reported in the present study against S. aureus and B. subtilis, whereas similar to that of E. coli and P. aeruginosa [73]. The difference in the bioactivity of different EO samples could be due to the difference in their chemistry as well as the varied susceptibility of microbial strains.
Among the EOs of three C. reticulata cultivars, the EO of the sample KD was the most active, whereas the EO of the FE cultivar possessed the least activity against all tested bacteria. A recent study from India reported 1250 μg/mL MIC values of C. reticulata EO against E. coli and S. aureus [78], which is similar to the bioactivity of EOs derived from the KF and KD cultivars determined in the current study. A study from Spain reported 1000 μg/mL MIC of C. reticulata EO against S. aureus and 5000 μg/mL against E. coli and P. aeruginosa [79]. The P. aeruginosa (PAO1) strain studied here is quite resistant under natural conditions due to its ability to biofilm formation [80]. Despite that, the EOs of C. reticulata inhibited the growth of the P. aeruginosa (PAO1) strain even at moderate concentrations. Though the major compound in all C. reticulata cultivars was the same, however, the synergistic effect of minor compounds could be the reason for variations in their biological activity.
The effect of EOs on different pathogenic microbes includes degradation of a cytoplasmic membrane, destruction of the cell wall of pathogenic bacteria, destruction of membrane proteins, coagulation of the cytoplasm, and enhanced permeability of the cell membrane that causes the outflow of the essential cellular ingredients, decreasing the proton motive force or pressure and the cellular ATP by reducing the energy synthesis [81,82,83]. Though studies explained diverse types of mechanisms of action of essential oils on bacterial cells, most studies showed that essential oils are capable of degrading bacterial cell walls and causing damage to the bacterial cell structure [14,15,81,84] that leads to increased permeability due to the non-separable nature of EOs from the bacterial cell wall. In several studies, limonene was reported to destroy the cell morphology and structure by affecting the cell membrane [14,15,16], whereas a study reported a similar effect of C. medica EO on bacterial cells [85].
Interestingly, EO extracted from C. aurantium was among the least active EOs against bacteria; however, it showed the highest repellency against female Ae. aegypti that lasted for an extended period of time. The good mosquito repellent activity of C. aurantium EO could be due to the bouquet of monoterpenes, including limonene, occurring at a higher proportion compared to other EOs. To our knowledge, only a few publications reported the mosquito-repellent activity of C. aurantium EO. A study from India reported 50% bite protection activity of C. aurantium EO against Ae. aegypti females after four hours of application when a 1000 µg/cm2 dose was tested [51]. A study from Thailand reported that C. aurantium EO exhibited 10 min protection time against Ae. aegypti mosquito bites when approximately 330 µg/cm2 dose was applied [86]. The repellency results of the current study are different from the Thailand study, whereas they are similar to the Adhikari et al. [51] study from India. Here at a lowest tested dose of 33.3 µg/cm2, C. aurantium EO exhibited moderate repellency after 15 min, whereas when the dose increased five times to 166 µg/cm2, the repellent longevity increased four times, indicating the importance of a higher dose for a repellent activity for an extended period.
We have studied the repellent activity of EOs derived from two C. sinensis cultivars, i.e., SM and BM. The mosquito-repellent activity of the SM cultivar was significantly higher than that of BM. Though EOs of both cultivars contained almost similar proportions of limonene, however, the relative abundance of linalool was higher in the SM cultivar. Kline et al. [87] demonstrated that linalool showed similar spatial repellency against Ae. aegypti females compared to DEET; therefore, the repellency difference between these two cultivars of C. sinensis could be explained by a four times larger amount of linalool in the EO of a more repellent SM cultivar. A previous study from Thailand reported a 30 min protection time of 100 µL of pure C. sinensis EO against Ae. aegypti; however, this EO lost its activity when it was combined with ethanol [88]. Another study from Thailand reported the mosquito-repellent activity of C. sinensis EO against Ae. aegypti and Culex quinquefasciatus at 330 µg/cm2 dose and reported mean protection times of 20.9 and 42.8 min, respectively [86]. The repellent results of the SM cultivar seem higher than the Phasomkusolsil et al. [88] report, whereas they are comparable to those of Soonwera [86]. The repellent activity of these studies points out that EO extracted from different cultivars of C. sinensis cultivated at different places showed diverse bioactivities against mosquitoes due to differences in their EO chemistry.
EO extracted from C. limon showed the least mosquito repellency among all tested EOs. The mosquito repellency of C. limon reported here is similar to a reported study from India where C. limon EO exhibited only 27% repellency when a 1000 µg/cm2 dose was tested after 4 h of application [51]. A study from Iran reported 71.1% mosquito repellency of C. limon while testing 1% EO solution against Anopheles stephensi [52]. A study from Greece showed moderate repellency of C. limon EO against Ae. albopictus when 0.2 µL/cm2 (~200 µg/cm2) dose was applied [18]. The repellent activity of C. limon studied here is in accordance with the reported data. Interestingly, the EO of C. limon was the most active against pathogenic bacteria but showed the least mosquito repellent activity compared to its counterpart EOs.
Among three samples of C. recticulata EOs, KD exhibited excellent mosquito repellency for about 60 min, whereas the other two EO samples, i.e., KF and FE, showed repellency for a shorter period of time against Ae. aegypti females. Interestingly, KF and KD were extracted from Kinnow peel collected from the same orchard, and the only difference between them was the condition of the peel, fresh or dried, which significantly changed the mosquito repellency of these two EOs. Effiom et al. [53] from Nigeria reported that C. reticulta peel extract did not show any repellency when 5–10% solution was applied, but the repellent longevity increased to 5 h when 25% solution was tested. A previous study from Pakistan reported mosquito repellent activity of fresh C. reticulata peel EO and demonstrated repellency for more than 90 min [50] at a similar dose used in the current study. The repellency of fresh Kinnow peel EO reported here is lower than previous Pakistani reported data, whereas it is higher than a Nigerian study. The difference in bioactivity could be explained based on the difference in the chemical composition of EOs reported in studies.
The mosquito-repellent activity of C. pseudolimon and C. jambhiri was similar, though the chemical composition of these two EOs was qualitatively the same, but the relative proportion of some minor components differed a little. There is no previous study reporting the mosquito repellency activity of C. pseudolimon and C. jambhiri EOs. Moreover, a previous study reported good insecticidal activity of C. jambhiri EO against stored grain beetle, Tribolium castaneum, when a 27 µL/L dose was tested in a fumigation bioassay [89].

5. Conclusions

Limonene was the most abundant compound, and (R)-(+)-limonene was the most abundant enantiomer in all EO samples. C. limon EO exhibited the highest antibacterial activity against tested bacterial strains, whereas 5% solutions of C. aurantium and Citrus sinensis var. Malta cv. Shakri Malta peel EOs exhibited 60% and 35% mosquito repellent activity against Ae. aegypti females for 45 min compared to DEET, which showed about 90% repellency for 75 min. The results showed that EOs extracted from C. aurantium and Citrus sinensis var. Malta cv. Shakri Malta peel has the potential to be used for formulating plant-based mosquito repellent.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae11010009/s1, Figure S1: Citrus peel essential oils chromatograms; Figure S2: Percent bacterial growth inhibition with respect to negative control (DMSO) of different citrus peels essential oils against (a) Bacillus subtilis; (b) Staphylococcus aureus; (c) Escherichia coli; (d) Pseudomonas aeruginosa (PAO1). References [84,90] are cited in supplementary file.

Author Contributions

Conceptualization, M.A. and R.M.; methodology, M.N., M.A., M.B., M.G.A., and B.U.; data analysis, M.N., M.A., V.A., and R.M.; writing—original draft preparation, M.N. and M.A.; writing—review and editing, M.A., M.B., and R.M.; visualization, M.A. and R.M.; supervision, M.A.; funding acquisition, M.A. and R.M.; project administration, M.A. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the International Foundation for Science (IFS), Sweden (Grant No. I-1-F-6041-1), available to M.A., and by the Lithuanian state grant through Nature Research Centre, program 2 Climate and Ecosystems, Vilnius, Lithuania, available to R.M. and V.A.

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethical and Biosafety Committee of Bahauddin Zakariya University, Multan (protocol code No. 04 lURECl2022, approval date 21 September 2022).

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Data Availability Statement

The original contributions presented in the study are included in the article; further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Horticulturae 11 00009 g001
Figure 1. The chemical structures of components constitute over 2% of essential oils of citrus peels.
Figure 1. The chemical structures of components constitute over 2% of essential oils of citrus peels.
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Figure 2. Repellent persistence of nine citrus EO samples and DEET tested at 33.3 μg/cm2 against Ae. aegypti females. RL—Citrus jambhiri, SO—Citrus aurantium; BM—Citrus sinensis var. Malta, cv. Blood Malta; SM—Citrus sinensis var. Malta, cv. Shakri Malta; DL—Citrus limon; GA—Citrus pseudolimon; FE—Citrus reticulata var. Mandarin cv. Feutrell’s early; KF—Citrus reticulata var. Mandarin cv. Kinnow (from fresh peel); KD—Citrus reticulata var. Mandarin cv. Kinnow (from dried peel). Bars having different letters depict significant differences (p < 0.05) among the repellency of test substances after different periods independently (ANOVA post-hoc Bonferroni test). Error bars denote the standard error (n = 5).
Figure 2. Repellent persistence of nine citrus EO samples and DEET tested at 33.3 μg/cm2 against Ae. aegypti females. RL—Citrus jambhiri, SO—Citrus aurantium; BM—Citrus sinensis var. Malta, cv. Blood Malta; SM—Citrus sinensis var. Malta, cv. Shakri Malta; DL—Citrus limon; GA—Citrus pseudolimon; FE—Citrus reticulata var. Mandarin cv. Feutrell’s early; KF—Citrus reticulata var. Mandarin cv. Kinnow (from fresh peel); KD—Citrus reticulata var. Mandarin cv. Kinnow (from dried peel). Bars having different letters depict significant differences (p < 0.05) among the repellency of test substances after different periods independently (ANOVA post-hoc Bonferroni test). Error bars denote the standard error (n = 5).
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Figure 3. Repellent persistence of nine citrus EO samples and DEET tested at 166 μg/cm2 against Ae. aegypti females. RL—Citrus jambhiri, SO—Citrus aurantium; BM—Citrus sinensis var. Malta, cv. Blood Malta; SM—Citrus sinensis var. Malta, cv. Shakri Malta; DL—Citrus limon; GA—Citrus pseudolimon; FE—Citrus reticulata var. Mandarin cv. Feutrell’s early; KF—Citrus reticulata var. Mandarin cv. Kinnow (from fresh peel); KD—Citrus reticulata var. Mandarin cv. Kinnow (from dried peel). Bars having different letters depict significant differences (p < 0.05) among the repellency of test substances after different time periods independently (ANOVA post-hoc Bonferroni test). Error bars denote the standard error (n = 5).
Figure 3. Repellent persistence of nine citrus EO samples and DEET tested at 166 μg/cm2 against Ae. aegypti females. RL—Citrus jambhiri, SO—Citrus aurantium; BM—Citrus sinensis var. Malta, cv. Blood Malta; SM—Citrus sinensis var. Malta, cv. Shakri Malta; DL—Citrus limon; GA—Citrus pseudolimon; FE—Citrus reticulata var. Mandarin cv. Feutrell’s early; KF—Citrus reticulata var. Mandarin cv. Kinnow (from fresh peel); KD—Citrus reticulata var. Mandarin cv. Kinnow (from dried peel). Bars having different letters depict significant differences (p < 0.05) among the repellency of test substances after different time periods independently (ANOVA post-hoc Bonferroni test). Error bars denote the standard error (n = 5).
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Table 1. Description and yield percentage of essential oils extracted from citrus cultivars.
Table 1. Description and yield percentage of essential oils extracted from citrus cultivars.
EO TypeThe Local Name of a CultivarPeels ConditionVoucher NoLatin NameLocation% Yield
RLRough lemonFreshCUHA-465Citrus jambhiri LushAbbottabad0.38 ± 0.04
SOSour orangeFreshCUHA-25Citrus aurantium (L.)Abbottabad0.19 ± 0.02
BMBlood maltaFreshCUHA-466-1Citrus sinensis Osbeck var. MaltaKhanpur0.12 ± 0.01
SMShakri maltaFreshCUHA-466-2Khanpur0.21 ± 0.01
DLDesi lemonFreshCUHA-467Citrus limon (L) OsbeckMultan0.05 ± 0.00
GAGalgalFreshCUHA-468Citrus pseudolimon WesterHaripur0.27 ± 0.02
FEFeutrell’s earlyFreshCUHA-469-1Citrus reticulata Blanco var. MandarinSargodha0.21 ± 0.01
KFKinnowFreshCUHA-469-2Sargodha0.29 ± 0.02
KDKinnowDriedCUHA-469-2Sargodha0.35 ± 0.03
Table 2. Chemical composition of citrus peel EOs.
Table 2. Chemical composition of citrus peel EOs.
Identified CompoundsRIRLSOBMSMDLGAFEKFKD
α-Pinene9270.4 10.60.50.52.70.60.40.40.7
Camphene942 0.3
Sabinene9690.50.30.20.21.22.50.20.10.2
β-Pinene9720.20.1trtr20.20.40.1 tr
β-Myrcene9882.31.81.71.80.82.72.21.52.1
α-Phellandrene10020.10.20.20.30.10.20.10.90.7
3-Carene1008 tr0.30.40.1
α-Terpinene1016 tr0.10.1 0.1tr tr
p-Cymene1021 8.3
Limonene103294.596.195.393.356.291.596.496.795.2
cis-β-Ocimene1035 0.1
trans-β-Ocimene10470.10.1 0.2 0.1 tr
γ-Terpinene1058trtrtr0.11.6 tr tr
Terpinolene10880.1tr0.30.20.70.10.1trtr
Linalool10990.40.10.52.10.20.20.1 0.1
Nonanal1104 0.1 0.1
Chrysanthenone1106 0.2
β-Citronellal1153 0.1 tr
Terpinene-4-ol11790.60.2 1.30.3tr tr
α-Terpineol11920.3 1.60.1tr tr
Decanal1201 tr 0.2
Carveol1230 0.5
α-Citral1270 0.6
δ-Elemene1342 tr0.1
Copaene1381 0.1 tr
trans-β-Caryophyllene14260.1trtr 0.40.1tr
trans-α-Bergamotene1440 0.80.3
Valencene1500 0.60.4tr 0.2
β-Bisabolene1514 1.30.4
Spathulenol 0.2
RI—retention index was determined using a DB-5 GC column; 1 value is percent; tr—traces; RL—Citrus jambhiri, SO—Citrus aurantium; BM—Citrus sinensis var. Malta, cv. Blood Malta; SM—Citrus sinensis var. Malta, cv. Shakri Malta; DL—Citrus limon; GA—Citrus pseudolimon; FE—Citrus reticulata var. Mandarin cv. Feutrell’s early; KF—Citrus reticulata var. Mandarin cv. Kinnow (from fresh peel); KD—Citrus reticulata var. Mandarin cv. Kinnow (from dried peel).
Table 3. The relative abundance of limonene enantiomers in citrus peel essential oils.
Table 3. The relative abundance of limonene enantiomers in citrus peel essential oils.
EO TypesEnantiomeric Composition %
(R)-(+)-Limonene(S)-(-)-Limonene
RL99.510.49
SO99.560.44
BM99.510.49
SM99.460.54
DL96.913.09
GA99.520.48
FE99.360.64
KF99.350.65
KD99.330.67
RL—Citrus jambhiri, SO—Citrus aurantium; BM—Citrus sinensis var. Malta, cv. Blood Malta; SM—Citrus sinensis var. Malta, cv. Shakri Malta; DL—Citrus limon; GA—Citrus pseudolimon; FE—Citrus reticulata var. Mandarin cv. Feutrell’s early; KF—Citrus reticulata var. Mandarin cv. Kinnow (from fresh peel); KD—Citrus reticulata var. Mandarin cv. Kinnow (from dried peel).
Table 4. The minimum inhibitory concentration (MIC) of different citrus peel EOs and Ciprofloxacin.
Table 4. The minimum inhibitory concentration (MIC) of different citrus peel EOs and Ciprofloxacin.
SampleMIC (mg/mL)
Gram PositiveGram Negative
B. subtilisS. aureusE. coliP. aeruginosa (PAO1)
RL1020510
SO52.5510
BM2020520
SM2020520
DL0.6250.3120.6251.25
GA2010520
FE2010520
KF102.51.252.5
KD51.252.52.5
Ciprofloxacin0.010.010.0050.02
RL—Citrus jambhiri, SO—Citrus aurantium; BM—Citrus sinensis var. Malta, cv. Blood Malta; SM—Citrus sinensis var. Malta, cv. Shakri Malta; DL—Citrus limon; GA—Citrus pseudolimon; FE—Citrus reticulata var. Mandarin cv. Feutrell’s early; KF—Citrus reticulata var. Mandarin cv. Kinnow (from fresh peel); KD—Citrus reticulata var. Mandarin cv. Kinnow (from dried peel).
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Nawaz, M.; Ullah, B.; Abbas, M.G.; Binyameen, M.; Apšegaitė, V.; Mozūraitis, R.; Azeem, M. Antibacterial and Mosquito Repellent Potential of Eight Citrus Cultivars and Their Chemical Composition. Horticulturae 2025, 11, 9. https://doi.org/10.3390/horticulturae11010009

AMA Style

Nawaz M, Ullah B, Abbas MG, Binyameen M, Apšegaitė V, Mozūraitis R, Azeem M. Antibacterial and Mosquito Repellent Potential of Eight Citrus Cultivars and Their Chemical Composition. Horticulturae. 2025; 11(1):9. https://doi.org/10.3390/horticulturae11010009

Chicago/Turabian Style

Nawaz, Mehwish, Bait Ullah, Muhammad Ghazanfar Abbas, Muhammad Binyameen, Violeta Apšegaitė, Raimondas Mozūraitis, and Muhammad Azeem. 2025. "Antibacterial and Mosquito Repellent Potential of Eight Citrus Cultivars and Their Chemical Composition" Horticulturae 11, no. 1: 9. https://doi.org/10.3390/horticulturae11010009

APA Style

Nawaz, M., Ullah, B., Abbas, M. G., Binyameen, M., Apšegaitė, V., Mozūraitis, R., & Azeem, M. (2025). Antibacterial and Mosquito Repellent Potential of Eight Citrus Cultivars and Their Chemical Composition. Horticulturae, 11(1), 9. https://doi.org/10.3390/horticulturae11010009

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