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Article

Chemical Analyses of Volatiles from Kumquat Species Grown in Greece—A Study of Antimicrobial Activity

by
Vasileios Ziogas
1,*,
Christos Ganos
2,
Konstantia Graikou
2,
Antigoni Cheilari
2 and
Ioanna Chinou
2,*
1
Institute of Olive Tree, Subtropical Plants and Viticulture, Hellenic Agricultural Organization—DIMITRA (ELGO-DIMITRA), 73134 Chania, Greece
2
Laboratory of Pharmacognosy and Chemistry of National Products, Department of Pharmacy, National and Kapodistrian University of Athens, 15771 Athens, Greece
*
Authors to whom correspondence should be addressed.
Horticulturae 2024, 10(2), 131; https://doi.org/10.3390/horticulturae10020131
Submission received: 20 December 2023 / Revised: 18 January 2024 / Accepted: 26 January 2024 / Published: 30 January 2024
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Abstract

:
The volatiles of different aerial parts of three kumquat species (Fortunella margarita Swingle–Nagami, Fortunella japonica Swingle–Marumi, and Fortunella crassifolia Swingle–Meiwa) growing in Greece were analyzed via GC-MS and evaluated for their antimicrobial properties against nine human pathogenic microorganisms. A total number of 23 compounds were identified in the peel, 38 in the leaves, and 30 in the flowers of the examined species. Limonene was the dominant metabolite in the peels of all three species, germacrene-D was present in the leaves of Nagami and Marumi kumquats, while limonene was the most abundant in the flower of Marumi and Meiwa kumquat but with significant differences in the composition of the total fracture of the essential oil, since compounds with high antimicrobial activity were only present in the flower of Meiwa kumquat. The essential oils from the leaf and peel of the three kumquat species were either inactive or showed weak antimicrobial activity, respectively, against Gram-positive and Gram-negative bacterial strains and pathogenic fungi. Only the essential oil from the flower of F. crassifolia Swingle (Meiwa) showed a stronger effect (MIC values 3.5–7.48 mg/mL) against all the assayed microorganisms. Furthermore, through multivariate statistical analysis, we studied the relationships between the samples regarding their origin (species and plant part), as well as between the chemical composition of the corresponding essential oils and their antimicrobial activity. Considering its chemical profile and antimicrobial activity, the Greek Meiwa flowers’ essential oil seemed a promising essential oil for further exploitation in the food and/or medicinal industry.

1. Introduction

The Citrus genus is one of the most significant genera of the Rutaceae family, since it includes economically important species such as oranges, mandarins, lemons, limes, and grapefruit [1]. Among the Rutaceae family, there are species whose fruit is used mainly for medicinal and food purposes, like the kumquat. The name kumquat is said to derive from the early Chinese word “chin kan”, which means “gold orange”. The kumquat is a cold-hardy, vigorous, and prolific small-sized tree that produces small round or oval-shaped fruits. Initially, kumquats were classified into the genus Citrus, but a century ago, they were reclassified into the genus Fortunella, which has six species (Fortunella japonica, Fortunella margarita, Fortunella obovata, Fortunella crassifolia, Fortunella polyandra, and Fortunella hindsii) [2]. Until recently, the genus Fortunella was renamed Citrus japonica [3]. The kumquat tree is native to Southeast China, particularly Taiwan, where it has been cultivated since the 12th century, and its cultivation has now expanded to Japan, India, the Philippines, South Asia, Argentina, Brazil, Florida, California, the Mediterranean region, Australia, and South Africa [2,4]. Among the most cultivated kumquat species are the round “Marumi” (F. japonica Swingle), the oval “Nagami” (F. margarita Swingle), and the “Meiwa” or “Jingdan” (F. crassifolia Swingle), with the latter being a natural hybrid of the “Marumi” and “Nagami” kumquats [5]. In Greece, the kumquat (Nagami) was introduced in 1924 and is cultivated systematically on the island of Corfu, with an annual production of 140 tonnes. The kumquat (Nagami) is one of the three Greek citrus species characterized as a Product of Protected Geographical Indication [6].
The unique characteristic of the kumquat fruit, compared to most Citrus, is that as a fruit, it is consumed whole and has a characteristic intense sweet start-flavor and slightly bitter end-flavor. Detrimental to the overall sensory experience, the taste and the aroma qualities of the fruit are the content and composition of the rind essential oils [7]. The kumquat fruit is mainly consumed fresh or as a processed fruit product (liqueurs, marmalades, sauces, juices), but it can be preserved in sugar syrup. Furthermore, kumquat is also used in folk medicine, and the therapeutic, antimicrobial, and antioxidant attributes of isolated metabolites from the fruit rind have drawn the attention of scientific groups [8,9].
The kumquat fruit has been characterized as a valuable source of primary and secondary metabolites of exceptional nutritional value. The discrepancy between the reported results regarding the nutritional composition and secondary metabolite footprint is attributed to several factors, such as the implemented cultivation techniques, production areas, microclimate, genetic diversity, and non-standardized extraction and analytical methods [2,10,11,12]. Essential oils represent one of the most important fractions of secondary metabolites in kumquat, and they are mostly accumulated in the oil glands of the rind (peel), flowers, and leaves. Essential oils are volatile hydrophobic liquid substances with rather low molecular weights, which provide a distinguished aroma profile [13]. When the kumquat fruit is ripe, there is an increased number of oil glands in the subepidermal tissue of the outer pericarp, and the essential oil content is near 1–2%, along with yellow pigments and aromatic oils [14,15]. The kumquat-derived essential oil is of special importance due to its rich content of unsaturated fatty acids, which contribute to reductions in cholesterol levels and the prevention of cardiovascular diseases [16]. Also, it has been reported that kumquat essential oil can reduce the proliferation of human prostate cancer cells for 24–72 h by 55–63% [17]. Kumquat essential oil is characterized by its mild nature and considered to have superior quality compared with other citrus oils [14]. Due to the latter fact, it is used extensively as an edible flavoring, a common fragrance, an antiphlogistic agent, an antivirus agent, a carminative, a deodorant, and an expectorant [9,14]. It is noted that the dominant compound of the kumquat essential oil cluster is limonene [18].
Several studies have pinpointed the antimicrobial potential of kumquat essential oils from the peel or leaves [3,5,9,19]. It has previously been highlighted that essential oils extracted from aromatic or medicinal plants are a safe alternative to chemical pesticides, since they can inhibit the growth of various postharvest pathogens [20]. Furthermore, essential oils from basil and bergamot could be a potential alternative to traditional antibiotics and contribute to animal and public health [21]. Kumquat essential oils are utilized in the food industry and Generally Recognized As Safe (GRAS) for implementation in food production as potential alternatives to chemical antimicrobials [3]. It is well-established that kumquat essential oils, in vapor or liquid form, have the potential to inhibit the development of a range of Gram-positive and Gram-negative bacteria [5,22].
In comparison to other citrus fruits (oranges, lemons, mandarins), studies dedicated to the beneficial attributes of kumquat are rather limited [23]. To the best of our knowledge, there is no report in the literature regarding the essential oil compositions of the three major kumquat species (F. japonica Swingle, F. margarita Swingle, and F. crassifolia Swingle) cultivated in Greek climatic conditions, and there is a parallel scientific gap regarding the essential oil profiles of kumquat flowers. The current work aimed to determine the essential oil footprint of the peel, flowers, and leaves for the three kumquat species cultivated under Greek microclimatic conditions and evaluate their antimicrobial potential, as well as study further the relationships between the samples regarding their origin (species and plant part) and between the chemical compositions of the corresponding essential oils and the antimicrobial activity through multivariate statistical analysis.

2. Materials and Methods

2.1. Plant Material and Sample Preparation

Mature kumquat fruits and leaves were harvested from three species, “Marumi” (F. japonica Swingle), “Nagami” (F. margarita Swingle), and the “Meiwa” (F. crassifolia Swingle), in March. Flowers from the “Marumi” and “Meiwa” kumquat species were harvested during July, at full bloom, during the first morning hours, just after sunrise (8 months before the harvest of mature fruit). All fruits, leaves, and flowers were randomly selected from five (5) 20-year-old trees grafted upon trifoliata orange rootstock (Poncirus trifoliata Raf.) located at the Arboricultural Station in Poros (South Greece) during the 2021 cultivation period. All trees were healthy and were cultivated under the same climatic conditions and agricultural practices. All fruits were of similar size and shape, with a mean diameter of 1.0 cm and a length of 3 cm. Fifty fruits and leaves (ten fruit–leaves per tree of similar developmental stage) were sampled from the exterior and the interior of the canopy from all four directions. After harvest, the fruit, leaves, and flowers were randomly divided among three groups for each replication.

2.2. Essential Oil Extraction

After washing the fruits and leaves with distilled water, the peel of the fruit (flavedo and albedo) was separated carefully by hand. Flowers were not washed due to the extreme sensitivity of the tissue. The fruits were cut into two portions and the flesh was removed. The fresh albedo layer of the fruits (120 g) was carefully peeled off and discarded. The peel of fresh fruits was cold-pressed, and the essential oil was then separated from the crude extract by centrifugation (10 min at 15,000× g rpm). Leaves (approx. 60 g) and flower tissue (30 g) were subjected to hydro-distillation using a Clevenger-type steam distillation apparatus for 3 h. The essential oil was isolated from the aqueous emulsion via centrifugation (15 min at 15,000× g rpm). The essential oil yield was recorded, and the oil was dried using anhydrous sodium sulfate. All essential oils were stored at 4 °C in amber vials until further analysis [24]. Analyses were performed within a week of the essential oil extraction.

2.3. Gas Chromatography-Mass Spectroscopy (GC-MS) Analysis

The chemical compositions of the essential oils were analyzed using GC-MS. The analysis was performed on an Agilent Technologies Gas Chromatograph 7820A connected to an Agilent Technologies 5977B mass spectrometer system (Agilent, Santa Clara, CA, USA) based on electron impact (EI) and 70 eV of ionization energy. The gas chromatograph was equipped with a split/splitless injector and a capillary column HP5MS of 30 m, internal diameter of 0.25 mm, and membrane thickness of 0.25 μm. The temperature program included an initial temperature of 60 °C for 5 min; then, an increase at a rate of 3 °C/min until the temperature reached 130 °C; then, an increase at a rate of 2 °C/min up to 180 °C; and finally, an increase at a rate of 5 °C/min with a final temperature of 240 °C, where the program was completed. The total analysis time was 65.33 min. The carrier gas was He at a flow rate of 0.7 mL/min, injection volume of 2 μL, split ratio of 1:10, and injector temperature of 280 °C [25]. The Kovats retention index (RI) values were determined based on a homologous series of n-alkanes (C8–C25). The compounds were identified via mass spectra comparison with libraries (Wiley Registry of Mass Spectral Data) and confirmed via a comparison of Kovats retention indices (KIs) with literature data [26]. The relative proportions of the essential oil constituents were expressed as percentages obtained by peak area normalization, and all relative response factors were taken as one.

2.4. Antimicrobial Activity

To investigate the antimicrobial activity, the essential oils were dissolved in dimethyl sulfoxide (DMSO) using Muller-Hinton broth or RPMI with MOPS for the successful cultivation of the fungi. The antimicrobial activity was studied against six (6) strains of human pathogenic bacteria and three (3) fungi, implementing the micro-dilution broth method, which evaluates the Minimal Inhibitory Concentration (MIC) according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST). The experiments were conducted against Gram-positive bacteria, Staphylococcus aureus (ATCC 25923) and Staphylococcus epidermis (ATCC 12228), and Gram-negative bacteria, Escherichia coli (ATCC 25922), Enterobacter cloacae (ATCC 13047), Klebsiella pneumoniae (ATCC13883), and Pseudomonas aeruginosa (ATCC 227853), as well as and three human pathogen fungi, Candida albicans (ATCC 10231), Candida tropicalis (ATCC 13801), and Candida glabrata (ATCC 28838). Before the assay, stock solutions were freshly prepared (at concentrations of 10 and 1 mg/mL). The stock solutions were serially diluted in the broth medium (100 μL of Sabouraud broth) in 96-well plates, and 1 μL of the microbial suspension (prepared in sterile distilled water) was inoculated in each well. Plates were stored and incubated at 37 °C for 24 h. After incubation, the MICs were assessed as the lowest concentrations preventing visual growth of the reference microbial strains. In addition to the used essential oils, standard antibiotics such as netilmicin, amoxicillin, and clavulanic acid (for bacteria), as well as 5-flucytosine and amphotericin B (for fungi) were tested (Sanofi Paris, France, Diagnostics Pasteur). For each experiment, a pure solvent solution was used as the blind control. All experiments, in all cases, were replicated three times and results were expressed as mean values [27].

2.5. Statistical Analysis

All experiments were performed in triplicate. Three batches of essential oils were prepared for each tissue, and every batch was analyzed three times, resulting in a total of nine measurements. All statistical analysis was performed using the SPSS software (version 26) (SPSS Inc., Chicago, IL, USA).
The correlation coefficient (Spearman r) of essential oil’s chemical content with antimicrobial activity was determined in GraphPad Prism 8 (GraphPad Software, version 8, San Diego, CA, USA). Multivariate statistical analysis was conducted using SIMCA 14.1 software (Sartorius, formerly MKS Umetrics, Umeå, Sweden). Data were Pareto-scaled for PCA and PLS data analysis, and the models’ quality was checked through R2X, R2Y, and Q2 cumulative values. Supervised PLS models were validated by applying 100 permutations.

3. Results and Discussion

3.1. Volatile Composition from Kumquat Leaves, Peel, and Flower Essential Oils

The expressed essential oils from fresh peels (pericarp) of kumquat gave a sub-yellow, semi-solid oil with a characteristic pleasant citrus odor. The yield of the extraction was estimated to be 1.5% (v/w). The received essential oils were analyzed via GC-MS to determine their profile and the dominant compounds. The results of the analysis are provided in Table 1. It has been reported that terpenes and their derivatives, alcohols, and aldehydes are the dominant metabolites in the peel essential oil of citrus [28]. A total of 23 volatile compounds were identified across the essential oils from the peel of the three studied kumquat species (Nagami, Marumi, Meiwa). From these, eleven were alcohol derivatives of terpenes, and eight were alcohols, making those two classes the most common volatile compounds identified in the kumquat peel. As can be seen in Table 1, for the Nagami kumquat (F. margarita Swingle), 14 volatile compounds were identified, constituting 99.29% of the total amount of the essential oils within the peel. Among these, nine were terpene compounds (96.47%), four were alcohol compounds (1.13%), and one was an ester (1.69%). For the Marumi kumquat (F. japonica Swingle), 12 volatile compounds were identified, accounting for 98.29% of the total amount of peel essential oil. Among these, five were terpene (95.28%), four were alcohols (1.29%), two were esters (1.55%), and one was an epoxide (0.17%). Regarding the Meiwa kumquat (F. crassifolia Swingle), 16 volatile compounds were identified, constituting 92.97% of the total fraction of the peel essential oil compounds. Among these, eight were terpenes (88.52%), five were alcohols (3.01%), two were esters (1.33%), and one was a ketone (0.11%). It is well-documented that terpenoid hydrocarbons are the most characteristic and abundant compounds in the peel of citrus fruit [5].
GC-MS analysis revealed that limonene was the most abundant terpene in the peel of all three kumquat species, with a percentage of 84.85% (Nagami), 88.92% (Marumi), and 76.62% (Meiwa). In our study, Nagami peel’s essential oil had an adequate amount of myrcene (7.79%), germacrene-D (2.08%), and geranyl acetate (1.69%), and a minor amount of linalool (0.8%), α-pinene (0.63%), bicyclogermacrene (0.46%), and sabinene (0.23%). It is noted that δ-elemene, α-phellandrene, α-eudesmol, germacrene-b, α-terpineol, and viridiflorol occurred at <0.19% in the total fraction of the peel essential oil. Also, Marumi peel’s essential oil had an adequate amount of terpene myrcene (5.34%), and a minor amount of octanol acetate (0.86%), geranyl acetate (0.69%), linalool (0.53%), α-pinene (0.49%), germacrene-D (0.42%), α-Terpinol (0.36%), and cis-para-mentha-2,8-dien-1-ol (0.23%). Terpinen-4-ol, trans-linalool oxide, and sabinene occurred at <0.17% in the total fraction of the peel essential oil. Furthermore, Meiwa kumquat peel had an adequate amount of terpene β-pinene (6.31%) and germacrene-D (2.2%) and a minor amount of α-pinene (1.55%), trans-carveol (1.49%), geranyl acetate (1.08%), δ-elemene (0.69%), trans-para-mentha-2,8-dien-1-ol (0.53%), cis-para-mentha-2,8-dien-1-ol (0.44%), sabinene (0.36%), viridiflorol (0.28%), β-elemene (0.27%), and octanol acetate (0.25%). Carvone occurred at 0.11% in the Meiwa peel essential oil.
In our study, it is noted that in all three kumquat species (Nagami, Marumi, and Meiwa), limonene was the predominant compound, ranging from 76.62 to 88.92%, followed by myrcene (5.34 to 7.79%) and germacrene-D (0.42 to 2.2%). Our data are in line with those of Liu et al. [7], who stated that in kumquat peel, limonene dominated by 67.47% to 72.98%, myrcene by 3.91 to 4.83%, and germacrene-D by 1.86 to 3.00%. Vincenzo and Poioana [29] reported that in the peel of Nagami kumquat (F. margarita Swingle), elevated levels of limonene (96%), minor amount of myrcene (1.6–1.7%), and germacrene-D (0.9–1.4%) were detected. In the present investigation, regarding the volatile profile of the Marumi kumquat (F. japonica Swingle), our data are similar to those reported by Quijano and Pino [30], who stated that limonene was the major compound (73.7%), followed by myrcene (6.3%), germacrene-D (3.4%), linalool (2.3%), α-pinene (1.7%), and geranyl acetate (1.5%). Our data also support the fact that the % of limonene in Meiwa (F. crassifolia Swingle) peel essential oil is comparatively less than that of Nagami (F. margarita Swingle) and Marumi (F. japonica Swingle) [3,5]. Previous reports also indicated that limonene was the dominant volatile compound in the peel of four types of kumquats (Jindou, Youpi, Huapi, and Suichuan) and two types of calamansi (Citrus macrocarpa) (from the Philippines and Indonesia), contributing by 90–96% to the total amount of volatiles [31]. The dominance of limonene in the peel of citrus was also reported in oranges, mandarins, lemons, and bergamot [32,33]. Bora et al. [34] reported that citrus essential oils from various species like orange, lemons, pummelo, grapefruit, lime, etc., consist of major compounds with biological activity, like α-/β-pinene, sabinene, β-myrcene, d-limonene, linalool, α-humulene, and α-terpineol. It is useful to note that essential oil from the kumquat peel is rich in limonene content, compared with other essential oils from the peel of citrus species (bergamot and lemon), a fact that pinpoints that kumquat peel is a valuable source of limonene [14].
It is also stated that even though limonene is the dominant component of the peel, some volatile components (mainly mono- and sesquiterpenes), present even in minor quantities, play a crucial role in the development of the characteristic odor and flavor of the kumquat fruit [18]. Also, in the case of kumquats, the chemistry of the terpenoids and their concentration in the matrix strongly determine the odor of the fruit [3]. The ratio of these latter volatiles also contributes to the palatability of the fruit and its overall attraction for consumption from animals/humans and is one of the reasons why the kumquat fruit is eaten with its peel [7,35]. It is noteworthy that in citrus fruit, the pungent odor is determined by the concentration of camphene (camphor odor), α-phellandrene (turpentine and spicy odor), caryophyllene (woody and spicy odor), and α-bisabolol (spicy odor), volatiles which are not present when the fruit is ripe [7]. In the current work, α-phellandrene (0.14%) was detected only in the fruit of Nagami (F. margarita Swingle), and this could be the reason why the fruit of this species exhibited a more pungent taste.
Noticeable differences were recorded among the composition of the three kumquat species (Nagami, Marumi, and Meiwa) since Nagami was the only kumquat where the presence of α-phellandrene, α-eudesmol, and bicyclo-germacrene was recorded. Also, only in the peel of the Marumi kumquat was the presence of the furanoid trans-linalool oxide recorded. Furthermore, only in the peel of Meiwa kumquat was an increased amount of β-pinene recorded along with a minor amount of the alcohols trans-para-mentha-2,8-dien-1-ol, trans-carveol, ketone carvone, and terpene β-elemene, while myrcene was absent. Similar variances were recorded in other kumquat species in the work of Goh et al. [31].
The water-distilled essential oil from the fresh kumquat leaves, for all studied species (Nagami, Marumi, Meiwa), produced a nearly solid green-yellow oil with a characteristic citrus odor. The yield of the extraction was estimated to be 0.082% (v/w). The received essential oil was analyzed via the use of GC-MS to determine the dominant compounds that constitute their profile. The results of the analysis are provided in Table 1. A total number of 38 volatile compounds were identified across the essential oils from the leaves of the three studied kumquat species (Nagami, Marumi, Meiwa). From these, twenty-one were alcohol derivatives of terpenes, sixteen were alcohols, and one was a ketone, making the first two the most common volatile compound classes identified in the leaf tissue. As can be seen in Table 1, for the Nagami kumquat (F. margarita Swingle), 14 volatile compounds were identified, which constituted 76.68% of the total amount of essential oils within the leaf. Of these, ten were terpenes compounds (41.01%), while four were alcohol compounds (35.67%). For the Marumi kumquat (F. japonica Swingle), 29 volatile compounds were identified, which accounted for 71.28% of the total amount of leaf essential oil. Of these, sixteen were terpene compounds (36.79%), twelve were alcohol compounds (34.21%), and one was a ketone (0.28%). Regarding the Meiwa kumquat (F. crassifolia Swingle), 24 volatile compounds were identified, which included 72.8% of the total fraction of the leaf essential oil compounds. Of these, fifteen were terpene compounds (37.49%) and nine were alcohol compounds (35.31%).
Several volatile compounds, mainly alcohols and alcohol terpene derivatives, were commonly identified in the leaves of all three kumquat species, but a difference in their abundance was observed. In detail, Nagami kumquat (F. margarita Swingle) leaves revealed germacrene-D as the dominant monoterpenic compound (16.4%) followed by elemol (13.17%), α-eudesmol (13.01%), germacrene-B (9.88%) and viridiflorol (8.9%), bicyclogermacrene (3.78%), δ-elemene (3.61%) and β-elemene (2.22%), caryophyllene (2%), and limonene (1.27%). γ-elemene, α-humulene, δ-cadinene, and phytol occurred at <0.75% in the total fraction of the leaf essential oil.
Marumi kumquat (F. japonica Swingle) leaves also had germacrene-D as the dominant monoterpenic compound (12.03%), followed by γ-eudesmenol (9.75%), elemol (8.65%), β-elemene (5.26%), caryophellene (4.97%), viridiflorol (4.83%), α-eudesmol (4.00%), limonine (3.62%), δ-elemene (3.42%), spathulenol (3.14%), nerolidol (2.23%), bicyclogermacrene (1.35%), and α-humulene (1.31%). Sabinene, myrcene, 2δ-carene, linalool, trans-para-mentha-2,8-dien-1-ol, cis-para-mentha-2,8-dien-1-ol, α-terpineol, trans-carveol, cis-carveol, carvone, α-ylangene, α-copaene, γ-elemene, α-guaiene, α-bulnesene, and α-cadinene occurred at <0.92% in the total fraction of the leaf essential oil.
Meiwa kumquat (F. crassifolia Swingle) leaves had alcohol elemol (9.59%) as the dominant volatile followed by germacrene-D (9.04%), viridiflorol (8.39%), cubenol (7.27%), δ-elemene (6.03%), β-elemene (5.97%), α-eudesmol (4.19%), α-muurolol (3.19%), β-bourbonene (2.97%), γ-elemene (2.84%) and α-gualene (2.19%), α-humulene (1.84%), limonene (1.75%), caryophyllene (1.3%), α-bulnesene (1.29%), α-ylangene (1.04%). β-ocimene, linalool, α-terpineol, α-cubebene, valencene, δ-cadinene, and eudesmol-7(11)-en-4-ol occurred at <0.62% in the total fraction of the leaf essential oil.
Noticeable differences were recorded among the composition of the three studied kumquat species (Nagami, Marumi, and Meiwa) since germacrene-B was only detected in the leaves of Nagami kumquat and was one of the most abundant volatiles (9.88%), while Nagami leaves also contained a very high amount of α-eudesmol (13.01%) compared with the other two kumquat leaves (4.19% for Meiwa and 4.00% for Marumi). Traces of sabinene, myrcene, carene-δ2, trans-para-mentha-2,8-dien-1-ol, cis-para-mentha-2,8-dien-1-ol, trans-carveol, cis-carveol, carvone, and α-copaene, and an adequate amount of spathulenol (3.14%) and nerolidol (2.23%) were only detected in the leaves of Marumi kumquat. Trace amounts of β-ocimene, α-cubebene, valencene, and eudesm-7(11)-en-4-ol were only detected in the leaves of Meiwa kumquat. An abundant amount of cubenol (7.27%), α-muurolol (3.19%), and β-bourbonene (2.97%) were only detected in the leaves of Meiwa kumquat and not in the other two studied kumquat species.
There is limited knowledge regarding the essential oil profile of kumquat leaves, and in general, it is non-existent and undocumented. An initial attempt was made in the work of Satyal et al. [36], where the leaf essential oil composition of Marumi kumquat (F. japonica Swingle), grown in Nepal, was examined. In their work, a total number of 42 compounds was identified, accounting for 99.6% of the total oil composition. The major compound of the leaf essential oil was linalool (35.1%), followed by eugenol (14.8%), geraniol (12.7%), and its aldehyde counterpart geranial (7.9%), with an adequate amount of nerol (5.3%) and (Z)-asarone (5.0%). Oulebsir et al. [37] reported that in sour orange (Citrus aurantium L.), 43 volatile compounds were detected in the essential oil from the leaves, with the major volatiles being linalool, linalyl acetate, and a-terpineol. The witnessed difference in the leaf essential oil profile of Marumi kumquat (F. japonica Swingle) could be attributed to the different locations, regarding their cultivation (Greece vs. Nepal), since it is well-documented that cultivation conditions and locality exert a strong influence upon the essential oil profile [14,17].
The water-distilled essential oils from the flowers of Marumi (F. japonica Swingle) and Meiwa (F. crasssifolia Swingle) kumquat yielded a sub-yellow, semi-solid oil with a characteristic citrus odor. The extraction yield was estimated to be 0.28% (w/v). The received essential oil was analyzed via GC-MSto determine the dominant compounds constituting their profile. The results of the analysis are provided in Table 1. A total of 30 volatile compounds were identified in the essential oils from the flowers of Marumi and Meiwa kumquats. Among these, sixteen were terpenes, ten were alcohols, one was an aldehyde, one was an ester, and one was an epoxide. Terpenes and alcohol were the most dominant volatile compounds in the essential oil of kumquat flower. For the Marumi kumquat (F. japonica Swingle), 16 volatile compounds were identified, constituting 85.83% of the total amount of essential oils within the flower. Of these, fourteen were terpene compounds (83.75%), while two were alcohol compounds (2.79%). In Meiwa kumquat (F. crassifolia Swingle), 30 volatile compounds were identified, accounting for 83.53% of the total amount of flower essential oil. It is noted that the essential oil profile of Meiwa kumquat flowers consisted of double the number of metabolites compared to Marumi kumquat flowers. Among these, sixteen were terpenes (52.33%), ten were alcohol compounds (25.42%), one was an epoxide (4.41%), one was a ketone (0.82%), one was an aldehyde (0.28%), and one was an ester (0.27%).
GC-MS analysis revealed that limonene was the most abundant terpene in the flowers of both species (Marumi and Meiwa). However, there was a significant difference in the percentage of this specific terpene, with Marumi kumquat flower having almost double the amount (63.67%) compared to Meiwa (27.75%). Marumi flower essential oil also contained adequate amounts of germacrene-D (4.79%), β-gurjunene (2.94%), germacrene-B (2.79%), valencene (2.65%), elemol (1.76%), α-eudesmol (1.03%), δ-elemene (1.02%), myrcene (0.99%), and β-ocimene (0.87%). Trace amounts of δ-cadinene, β-selinene, α-pinene, γ-elemene, and α-humulene occurred at <0.71% in the total fraction of the flower essential oil.
In the flowers of Meiwa kumquat, nine volatile compounds constituted 43.83% of the total amount of essential oils. Specifically, Meiwa flowers exhibited significant amounts of valencene (8.69%), β-eudesmol (6.6%), elemol (5.33%), β-gurjunene (4.03%), caryophyllene oxide (4.41%), spathulenol (4.1%), germacrene-D (4.06%), α-eudesmol (3.39%), citronellol (3.22%), caryophyllene (1.27%), β-elemene (1.12%), germacrene-B (1.02%), linalool (0.94%), and geraniol (0.94%). Trace amounts of α-pinene, myrcene, β-ocimene, trans-para-mentha-2.8-dien-1-ol, trans-carveol, geranial, δ-elemene, citronellyl acetate, γ-elemene, α-humulene, β-selinene, carvone, bicyclogermacrene, δ-cadinene, and nerolidol occurred at <0.82% in the total fraction of the flower essential oil.
It is highlighted that β-eudesmol, caryophyllene oxide, spathulenol, citronellol, along with a minor amount of linalool, trans-para-mentha-2.8-dien-1-ol, trans-carveol, carvone, geraniol, geranial, citronellyl acetate, caryophyllene, bicyclogermacrene, and nerolidol were only detected in the Meiwa kumquat flower. The minor existence of the ester citronellyl acetate provides a very characteristic kumquat odor to the flower of Meiwa, as proposed by various research groups [18,30,38]. Noticeable differences were recorded in the composition of the two kumquat species (Marumi and Meiwa). There is a significant difference regarding the dominant compound in the flower essential oil, with Marumi flowers producing almost triple the amount of limonene compared to Meiwa. Also, in Meiwa kumquat flowers, a triple amount of the terpene valencene was detected (8.69%), compared to Marumi (2.65%). These differences arise due to the different genotype profiles, as proposed by Sarrou et al. [39]. Furthermore, this work highlights the fact that limonene is the dominant essential oil compound in the kumquat flowers, similar to sour orange (Citrus aurantium L.) [40,41] and Citron (Citrus medica L.) var. sarcodactylis [42]. To the best of our knowledge, this is the first report that examines the kumquat flower essential oil composition and elucidates aspects of an unexplored research domain of this specific citrus fruit (kumquat).

3.2. Antimicrobial Activity

The essential oils from the three kumquat species (Nagami, Marumi, and Meiwa) were evaluated for their antimicrobial potential against nine (9) human pathogenic microbes (two Gram-positive (S. aureus and S. epidermidis) and four Gram-negative bacterial strains, as well as three fungi strains). As per the results presented in Table 2, the essential oils from the peel of all three kumquat species exhibited moderate activity against the Gram-positive bacteria (MIC values 11.15–13.00 mg/mL) and weak activity against the remaining Gram-negative bacteria (MIC values 13.78–18.50 mg/mL). The essential oil from the leaves of all examined kumquat species demonstrated no antibacterial activity (MIC values >20 mg/mL) against all six bacterial strains. Notably, only the essential oil from the flowers of Meiwa kumquat exerted strong antibacterial activity (MIC values 3.5–7.48 mg/mL) against all Gram-positive and Gram-negative bacterial strains. Furthermore, our results (as shown in Table 2) indicate a lack of and minor antifungal activity in the examined leaf (MIC values >20 mg/mL) and flower (MIC values 7.45–12.50 mg/mL) essential oils from all three kumquat species against all Candida fungal strains.
The strong antibacterial effect of Meiwa kumquat flower essential oil could be attributed to its low levels of limonene and elevated levels of germacrene-D, the epoxide caryophyllene oxide, and linalool—compounds with known antibacterial activity [5,43,44]. Furthermore, the fact that the essential oil from the flower of Meiwa kumquat exerts a strong antibacterial effect against Gram-positive bacteria (MIC values 3.50–3.48 mg/mL) compared to Gram-negative bacteria (MIC values 6.23–7.48 mg/mL) provides a valuable asset for potential use. This is particularly significant since most citrus species typically exhibit strong antibacterial activity against Gram-negative bacteria compared to Gram-positive bacteria, given the latter’s cell envelope that blocks antimicrobial infiltration [45]. Our results align with those of Bozinou et al. [24], who highlighted a similar protective ability of the essential oil from orange (Citrus sinensis L.) peel cv. New Hall against Gram-positive bacteria. Furthermore, the relatively low antimicrobial activity of the examined essential oils from the peel of all three kumquat species could be attributed to the dominance of limonene. It is well-established that limonene, due to its highly volatile and hydrophobic nature, has limited antimicrobial effects [45]. This aligns with the findings of Fisher and Phillips [22], which stated that limonene from the essential oil of lemon, orange, and bergamot failed to exert antimicrobial activity against Camphylobacter jejuni, E. coli O157, L. monocytagenes, B. cereus, and S. aureus. Similarly, Wang et al. [5] reported that the essential oil from the peel of Meiwa (F. crassifolia Swingle) kumquat, rich in limonene, exhibited antifungal activity but limited antibacterial effects. The latter was attributed to the fact that the antimicrobial activity of kumquat essential oil is enhanced by the presence of minor amounts of linalool, carveol, and carvone [3,5]. The observed high antimicrobial activity of the essential oil from the flowers of Meiwa (F. crassifolia Swingle) kumquat could be attributed to the synergistic effect of the diverse major and minor compounds present in the essential oil, as proposed by Değirmenci and Erkurt [45]. In the essential oil of Meiwa (F. crassifolia Swingle) kumquat flowers, limonene is the dominant component but in a relatively low percentage, while other minor compounds such as α-pinene, linalool, trans-carveol, carvone, myrcene, caryophyllene, nerolidol, spathulenol, and caryophyllene oxide exist, all with well-documented antimicrobial activity [20,46,47,48,49].
The mode of action through which essential oils exert their antimicrobial activity is still a subject of debate [14]. Papoutsis et al. [50] proposed that the antifungal activity is based on the rapture of the phospholipid bailer and the deregulation of the membrane permeability. The lipophilic nature of essential oils facilitates their translocation from the aqueous phase into the membrane structure. Ultee et al. [51] suggested that carvacrol increases membrane fluidity, causing the leakage of protons and potassium ions, disrupting membrane potential, and inhibiting ATP synthase activity. Cho et al. [52] proposed that the antibacterial activity of essential oils is exerted by altering intercellular components, interfering with cellular metabolism, and arresting bacterial mycelium spore formation and growth.

3.3. Principal Component Analysis (PCA) Regression Analysis

The correlation of the chemical content of the essential oils with microbial activity was initially tested using Spearman’s correlation coefficients. However, no solid conclusions could be drawn as p > 0.05 for all values. A two-tailed non-parametric Spearman’s analysis was chosen due to the data not passing normal distribution testing. However, Spearman’s heatmap is shown in Supplementary data (Figure S1). No rank correlation was taken into consideration, and to draw stronger conclusions, larger datasets are needed. Additionally, multivariate statistical analysis was employed to further study the relationship among the samples regarding their origin (species and plant part) and the chemical composition of the corresponding essential oils with antimicrobial activity. A PCA model with two principal components (PCs) was constructed, accounting for 89.2% of the explained variance. The score plot (Figure 1) showed clustering of essential oils according to the plant part they are distilled from and not based on the species. Peels and leaves exhibited significant discrimination in PC1. The PCA loading plot (Figure 2) revealed that limonene, myrcene, α-pinene, and trans-carveol contribute to peel sample clustering, while germacrene-B, germacrene-D, δ-elemene, and viridiflorol contribute to leaves. Furthermore, another PCA model (two PCs and 88.6% explained variance) was generated considering both chemical constituents (X variables) and antimicrobial activity (Y variables). The PCA X&Y score plot (Figure 3) showed differentiation of the flower samples, consistent with the measured MIC values. The PCA X&Y loading plot can be found in Supplementary data (Figure S2). In addition, supervised PLS regression analysis was used to facilitate the identification of compounds related to the antimicrobial activity for each microbial strain separately. The PLS coefficient plots were calculated from the corresponding PLS models with three principal components for S. aureus, S. epidermis, K. pneumoniae, E. cloacae, and E. coli and are shown in Supplementary data (Figures S3–S7). Models for P. aeruginosa, C. albicans, C. tropicalis, and C. glabrata were not considered, as they were not valid. PLS coefficient plots for S. aureus, S. epidermis, K. pneumoniae, E. cloacae, and E. coli demonstrated a clear correlation of antimicrobial activity with limonene, myrcene, and trans-carveol for all strains. Additionally, germacrene-B followed for S. aureus and S. epidermis, and a-pinene for K. pneumoniae, E. cloacae, and E. coli, respectively.

4. Conclusions

The industry has shown significant interest in the utilization of plant-origin antimicrobial substances. This study represents the first known analysis of the essential oil’s profile derived from different aerial parts (peel, leaf, flower) of kumquat species (Nagami, Marumi, and Meiwa) cultivated in Greece. The current report highlights valuable attributes associated with Nagami kumquat, the sole kumquat with Protected Geographical Indication in Greece, offering added value to this agricultural product. The essential oils studied exhibited a diverse array of specific volatiles present in each tissue and kumquat species. Limonene emerged as the dominant compound in the peel of all three species, while germacrene-D was the dominant compound in the leaves of Nagami and Marumi. In the flowers, limonene was dominant in Marumi and Meiwa, albeit with significant differences in the composition of the total fracture of the essential oil. The current study emphasized that the essential oil from the leaves and peel of the three kumquat species exhibited little to no antimicrobial activity against Gram-positive and Gram-negative bacterial strains and pathogenic fungi. Interestingly the essential oil from the flower of F. crassifolia Swingle (Meiwa) displayed the strongest antimicrobial effect, evidenced by its low MIC values against all studied microorganisms. This study offers valuable data for the potential use and commercial exploitation of essential oils from kumquat leaves and flowers (from Greece). It supports the use of kumquat as a novel natural source of antimicrobial substances for incorporation into functional foods and as a source of useful substances for the healthcare industry. Considering the chemical profile and antimicrobial activity observed in this study, the Greek Meiwa kumquat flower essential oil appears to be a promising candidate for further exploitation in the food and medicinal industries. Comprehensive data on toxicity, in vivo potential use, and detailed understanding of the mode of antimicrobial and antioxidant action are required to fully elucidate their potential applications.

Supplementary Materials

The following supporting information can be downloaded via this link: https://www.mdpi.com/article/10.3390/horticulturae10020131/s1. Figure S1: Spearman’s heatmap regarding essential oils (EOs) chemical content correlation with antimicrobial activity. No rank correlation was taken under consideration since p > 0.05 for all values. Figure S2: PCA X&Y loadings plot of EOs from the leaves, peels, and flowers of kumquat species. Variables are colored according to the chemical category of the containing compounds and antimicrobial variables (black) are also shown. Figure S3: PLS coefficient plot of PC1 for S. aureus. Data are colored based on the chemical categories of the compounds in the EOs. Figure S4: PLS coefficient plot of PC1 for S. epidermis. Data are colored based on the chemical categories of the compounds in the EOs. Figure S5: PLS coefficient plot of PC1 for K. pneumoniae. Data are colored based on the chemical categories of the compounds in the EOs. Figure S6: PLS coefficient plot of PC1 for E. cloacae. Data are colored based on the chemical categories of the compounds in the EOs. Figure S7: PLS coefficient plot of PC1 for E. coli. Data are colored based on the chemical categories of the compounds in the EOs.

Author Contributions

Conceptualization, I.C. and V.Z.; methodology, C.G., A.C. and K.G.; data curation, C.G., A.C. and K.G.; writing—original draft preparation, V.Z.; writing—review and editing, V.Z. and I.C.; supervision, I.C.; project administration, I.C. and V.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

The original contributions presented in this study are included in the article and Supplementary Materials, further inquiries can be directed to the corresponding author.

Acknowledgments

The authors thank the staff of the Arboricultural Station of Poros (Hellenic Ministry of Rural Development and Food, Greece) for their help with the sampling of the material.

Conflicts of Interest

The authors declare no conflicts of interest.

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Horticulturae 10 00131 g001
Figure 1. PCA score scatterplot of essential oils from the leaves, peels, and flowers of kumquat species. Observations are colored according to the plant part.
Figure 1. PCA score scatterplot of essential oils from the leaves, peels, and flowers of kumquat species. Observations are colored according to the plant part.
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Horticulturae 10 00131 g002
Figure 2. PCA loading plot of essential oils from the leaves, peels, and flowers of kumquat species. Variables are colored according to the chemical category of the containing compounds.
Figure 2. PCA loading plot of essential oils from the leaves, peels, and flowers of kumquat species. Variables are colored according to the chemical category of the containing compounds.
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Horticulturae 10 00131 g003
Figure 3. PCA X&Y score scatterplot of essential oils from the leaves, peels, and flowers of kumquat species. Observations are colored according to the plant part.
Figure 3. PCA X&Y score scatterplot of essential oils from the leaves, peels, and flowers of kumquat species. Observations are colored according to the plant part.
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Table 1. Volatiles of the leaves, peels, and flowers from kumquat species growing in Greece, expressed as peak area percentage.
Table 1. Volatiles of the leaves, peels, and flowers from kumquat species growing in Greece, expressed as peak area percentage.
LeafPeelFlower
No.CompoundsKIaKIbF. mF. jF. cF. mF. jF. cF. jF. c
1.α-pinene934939---0.630.491.550.440.20
2.sabinene972975-0.04-0.230.110.36--
3.β-pinene975979 ----6.31--
4.myrcene987990-0.84-7.795.34-0.990.21
5.δ-2-carene10001002-0.07------
6.α-phellandrene10021002 --0.14----
7.limonene102410291.273.621.7584.8588.9276.6263.6727.75
8.trans-β-ocimene10451050--0.12---0.870.45
9.trans-linaool oxide (furanoid)10811086 ---0.17---
10.linalool10951096-0.370.140.80.53--0.94
11.trans-para-mentha-2,8-dien-1-ol11191122-0.15---0.53-0.36
12.cis-para-mentha-2,8-dien-1-ol11331137-0.30--0.230.44--
13.terpinene-4-ol11741177----0.170.27--
14.α-terpineol11861188-0.210.270.100.36---
15.octanol acetate12101213----0.860.25--
16.trans-carveol12161216-0.50---1.49-0.28
17.citronellol12231225-------3.22
18.cis-carveol12241229-0.08------
19.carvone12391243-0.28---0.11-0.82
20.geraniol12461252-------0.94
21.geranial12631267-------0.28
22.δ-elemene133613383.613.426.030.19-0.691.470.68
23.α-cubenene13441348--0.15-----
24.citronellyl acetate13501352-------0.27
25.a-ylagene13711375-0.471.04-----
26.α-copaene13731376-0.44------
27.geranyl acetate13781381---1.690.691.08--
28.β-bourbonene13861388--2.97-----
29.β-elemene138713902.225.265.97--0.271.021.12
30.trans-caryophyllene141514192.004.971.30----1.27
31.β-gurjunene14291433------2.944.03
32.γ-elemene143114360.570.922.84---0.360.25
33.α-guaiene14341439-0.872.19-----
34.α-humulene145014540.751.311.84---0.340.49
35.germacrene-D1483148516.4012.039.042.080.422.204.794.06
36.β-selinene14871490------0.710.63
37.valencene14941496--0.34---2.658.69
38.bicyclogermacrene150015003.781.35-0.46---0.82
39.α-bulnesene15061509-0.271.29-----
40.δ-cadinene152015230.530.910.62---0.710.66
41.elemol1547154913.178.659.59---1.765.33
42.germacrene-B155915619.88--0.10-0.522.791.02
43.trans-nerolidol15601563-2.23-----0.26
44.spathulenol15751578-3.14-----4.1
45.caryophyllene oxide15821583-------4.41
46.viridiflorol159015928.904.838.390.09-0.28--
47.γ-eudesmol16281632-9.75------
48.α-muurolol16411646--3.19-----
49.cubenol16431646--7.27-----
50.β-eudesmol16481650-------6.60
51.α-eudesmol1650165313.014.004.190.14--1.033.39
52.eudesm-7 (11)-en-4-ol16981700--0.51-----
53.phytol193919430.59-1.76-----
Total 76.6871.2872.899.2998.9792.9783.5385.83
KIa: Retention index calculated, KIb: Retention index from literature, F. m.: F. margarita, F. j.: F. japonica, F. c.: F. crassifolia.
Table 2. Antimicrobial activities of essential oils are measured in terms of the MIC (mg/mL).
Table 2. Antimicrobial activities of essential oils are measured in terms of the MIC (mg/mL).
SpeciesSampleS. aureusS. epidermidisP. aeruginosaK. pneumoniaeE. cloacaeE. coliC. albicansC. tropicalisC. glabrata
Nagami
(F. margarita)		Peel12.5012.3713.7815.9016.9517.6510.508.707.45
Leaf>20>20>20>20>20>20---
Marumi
(F. japonica)		Peel13.0011.1514.3516.3017.4018.5012.5012.0011.75
Leaf>20>20>20>20>20>20---
Flower16.3215.7216.2015.8816.5517.45---
Meiwa
(F. crassifolia)		Peel12.3412.2016.7815.4518.3017.2510.029.759.00
Leaf>20>20>20>20>20>20>20>20>20
Flower3.503.847.456.237.487.10---
Netilmicin4 × 10−34 × 10−38.8 × 10−38 × 10−38 × 10−310 × 10−3
Amoxicillin2 × 10−32 × 10−32.4 × 10−32.2 × 10−32.8 × 10−32 × 10−3
Clavulanic acid0.5 × 10−30.5 × 10−31 × 10−31 × 10−31.6 × 10−31.2 × 10−3
5-flucytocine itraconazole 0.1 × 10−31 × 10−310 × 10−3
Ammphotericin B 1 × 10−30.5 × 10−30.4 × 10−3
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Ziogas, V.; Ganos, C.; Graikou, K.; Cheilari, A.; Chinou, I. Chemical Analyses of Volatiles from Kumquat Species Grown in Greece—A Study of Antimicrobial Activity. Horticulturae 2024, 10, 131. https://doi.org/10.3390/horticulturae10020131

AMA Style

Ziogas V, Ganos C, Graikou K, Cheilari A, Chinou I. Chemical Analyses of Volatiles from Kumquat Species Grown in Greece—A Study of Antimicrobial Activity. Horticulturae. 2024; 10(2):131. https://doi.org/10.3390/horticulturae10020131

Chicago/Turabian Style

Ziogas, Vasileios, Christos Ganos, Konstantia Graikou, Antigoni Cheilari, and Ioanna Chinou. 2024. "Chemical Analyses of Volatiles from Kumquat Species Grown in Greece—A Study of Antimicrobial Activity" Horticulturae 10, no. 2: 131. https://doi.org/10.3390/horticulturae10020131

APA Style

Ziogas, V., Ganos, C., Graikou, K., Cheilari, A., & Chinou, I. (2024). Chemical Analyses of Volatiles from Kumquat Species Grown in Greece—A Study of Antimicrobial Activity. Horticulturae, 10(2), 131. https://doi.org/10.3390/horticulturae10020131

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