Chemical profile of persian lime seeds ( Citrus Limettioides T.): Focus on limonoids and polyphenols

: Citrus fruit industrial processing generates tons of waste composed of peels, seeds and pulp. Incorrect disposal of these residues may harm the environment. The extraction of oil and bioactive compounds from citrus fruit seeds may be considered a sustainable alternative to the disposal of waste by the citrus agroindustry. In order to provide safe disposal of citrus waste an evaluation of its composition is necessary. Here we report the results of the application of a methodology to evaluate the composition the seeds of Citrus limettioides . In the first step, extraction with supercritical carbon dioxide was used. This work allowed the isolation and identification of four aglycone-type limonoids by High Performance Liquid Chromatography and Nuclear Magnetic Resonance, identified as limonin, nomilin, deacetylnomilin, and obacunone. In addition, six other polar limonoids and two glycosyl flavonoids were identified by HPLC-ESI/MS/ MS.


INTRODUCTION
The global production of citrus fruits was about 158.5 million tons, in 2020.Asia has the highest production of these fruits (47%), followed by Africa (43.7%),America (8.1%),Europe (0.4%) and Oceania (0.1%).China leads citrus production with 28.16% of world production.Brazil, India and Mexico each account for about 5% of global production (Suri et al. 2022).The residues from the citrus agroindustry are rich sources of vitamins, mineral salts and dietary fiber, in addition to containing biologically active substances such as flavonoids, carotenoids, low molecular weight phenolics, terpenes, and considerable amounts of lipids, sugars, polysaccharides and organic acids.Some of these compounds have therapeutic properties such as: antioxidant, anticancer, antitumor, anti-inflammatory and antiviral, in addition to being a source of raw material of industrial interest (citrus pectin).For this reason, peels, bagasse and seeds can be considered important raw materials for the recovery of these bioactive compounds (Banerjee et al. 2017, Sharma et al. 2017).Part of the waste from this agroindustry supplies the production of animal feed or fertilizers, but the high cost of drying and transporting this material makes the cost of its processing a limiting factor for their use.Incorrect disposal of these residues may harm the environment.However, their high availability aroused the interest of researchers in adding value to waste from citrus fruit processing industries (Kobori & Jorge 2005).
Citrus fruit seeds are rich in vegetable oils with high nutritional value, proteins, limonoids, phytosterols, tocopherols and polyphenols (Kobori & Jorge 2005, Waheed et al. 2009).Limonoids are highly oxygenated tetranortriterpenoids present in species of the Rutaceae and Meliaceae families.Seeds, fruits and peels of oranges (sweet and sour), lemons, limes, grapefruits, bergamots and mandarins are the main source of limonoids in Citrus genus.These compounds may be found both in the form of free aglycones as well as in the corresponding β-D-glycosides, being free aglycones mostly present in seeds and peels, while glycosides are formed during fruit maturation (Gualdani et al. 2016).Limonoids have antioxidant, anti-inflammatory, neuroprotective, antiviral, antimicrobial, antiprotozoal, antimalarial and antifungal activities, besides insecticidal activity.Figure 1 displays the structures of the main Citrus limonoids.Limonin is the major limonoid present in species of the genus Citrus (Zhang & Xu 2017, Hasegawa et al. 2000).
Citrus fruits belong to the genus Citrus of the Rutaceae family (Waheed et al. 2009).The genus Citrus is represented by several species with agroeconomic importance such as Citrus sinensis (common orange), Citrus limon (Sicilian lemon), Citrus latifolia (Tahiti acid lime), Citrus grandis (pomelo), Citrus paradisi (grapefruit), Citrus medica (cider), Citrus reticulata (mandarin, tangerine), and Citrus aurantium (bitter orange), being one of the most cultivated genera in the world (Araujo & Salibe 2002).The species Citrus limettioides Tanaka, popularly known as the Chick lime, sweet lime, navel lime or Persian lime originates from northern India and is commercially cultivated in several countries, mainly in warm and temperate tropical regions (Fronza & Hamann 2015).The fruit of this plant has a smooth peel, pale greenish-yellow in color and is very juicy (more than 50% by weight in juice) having an average of ten seeds (Lopes et al. 2013).Seeds have 20 -37% of its weight as an oil (Waheed et al. 2009).
Industrial strategies leading to more efficient extractions of bioactive substances promote the valorization of these products.In addition to avoiding degradation of thermolabile substances and the extensive use of solvents, they satisfy environmental regulations regarding waste disposal.In this context, extraction with supercritical fluid (SFE) appears as an alternative to conventional extraction methods.SFE is in line with Green Chemistry postulates and has the potential to provide high yields, reduce chemical degradation and overcome environmental constraints by reducing waste disposal (Putnik et al. 2017, Herrero et al. 2010).
In the present report, we present a separation scheme that allowed the extraction/ characterization of the chemical composition of C. limettioides seeds, specifically, its oil, limonoids and polyphenols present therein.The methodology adopted allowed the characterization of the seed oil and the identification of sixteen substances, namely eight aglycones, six glycosylated limonoids, and two flavonoids.Our results contribute to the characterization of the phytochemical profile of Citrus limettioides seeds, being the first report on the presence of glycosylated limonoids in this species.

Standards and Reagents
Solvents used in extraction, HPLC and HPLC/MS were all of high purity.The carbon dioxide (CO 2 ) solvent used was of high purity (99.9%), from Air liquide (America Corp, Augusta, GA).

Botanical material
Persian lime (Citrus limettioides) seeds were obtained from fruits purchased at CEASA commercial center, Rio de Janeiro.The seeds were dried in an oven at 40 °C and crushed in a laboratory blender (Waring Blender) before extraction.

Preparation of Extracts
The supercritical fluid extractor used was a TOP INDUSTRIE (France) equipped with an XU032 oven and HPFlow PUMP 50-1000 pump.The crushed and dried material (26.6 g) was subjected to supercritical fluid extraction after packaging in a stainless steel extractor cell whose volume is 100 mL.The extraction parameters were those optimized by Yu et al for the extraction of limonoids from C. paradisi (Yu et al. 2007).Therefore, the operational conditions used were: 483 bar pressure, 50 °C temperature, supercritical CO 2 flow rate: 40 g/ min, extraction time: 60 minutes.A viscous, yellowish oil was obtained (extraction yield: 10.4 g, 39%).
The cake resulting from the supercritical CO 2 extraction (16 g) was placed in a Soxhlet extractor and was extracted with methanol (400 mL) for 6 hours.The resulting material was concentrated under vacuum on a rotary evaporator.3.3 g (20%) of dry extract were obtained.

Isolation and identification of limonoid aglycones
The crude extract obtained by supercritical CO 2 extraction was fractionated by partitioning between hexane, methanol and water (2:1:1, v/v/v), into three 200 mL portions.The hexane fraction obtained was concentrated under vacuum to obtain an oily material with a predominant composition of triglycerides.After alkaline hydrolysis and methylation with BF 3 / methanol (EDER 1995), the oil was submitted to gas chromatography coupled to mass spectrometry (Shimadzu QP 2010 Plus) under the following conditions: DB5-MS capillary fused silica column (30 m, 0.25 mm I.D., 0.25 µm film thickness).Quantitation was made on the basis of their chromatographic peak area percentages.The initial oven temperature was set at 50 °C (1min), then raised by 50 °C/min to 170 °C, 4 °C /min to 300 °C and then held for 20 min.He (99.999%) was used as the carrier gas with a flow rate of 1.0 mL/min; the injector temperature was set to 220 ºC and a split ratio of 1:50 was used.The oil was diluted in chloroform (5 µL oil in 450 µL chloroform) and 1 µL of the dilution was injected in the chromatograph.Mass spectra were taken at 70 eV.The mass range values used were of 40-700 Da.

Nuclear Magnetic Resonance
1 H/ 13 C NMR spectra were obtained at 500/125 MHz and 400/100 MHz (Varian VNMRSYS-500), in the indicated solvents, with TMS as internal standard, using 3 mm tubes.The 2D correlation experiments were performed with the help of proprietary software.For 2D heteronuclear experiments (edited HSQC, HMBC), typical acquisition parameters included 1K or 2K x 512 data points, filled with zeros to 1K or 2K x 2K points, and processed using linear prediction in F1.All spectral data were processed using MestReNova software version 12.0.

HPLC-UV-MS/MS
The analyzes by high performance liquid chromatography with ultraviolet detector coupled to tandem mass spectrometry (HPLC-UV-MS/MS) were performed using a Dionex UltiMate 3000 system, coupled to a Fleet LCQ mass spectrometer (Thermo Fisher Scientific, Waltham, MA.A Waters Symmetry RP-18 column (150 x 4.6 mm, 5 µm) was used at a flow rate of 0.4 mL min −1 .Separation was performed at room temperature and the mobile phase used was: 0.1% aqueous trifluoroacetic acid (A) and acetonitrile (B).The gradient used was: 100% A to 100% B in 65 minutes.The mass spectrometer, equipped with an electrospray source (ESI), was operated in negative ionization mode.High purity nitrogen was used as sheath gas (35 arbitrary units) and auxiliary gas (10 arbitrary units).High purity helium was used as collision gas.The instrumental parameters used were as follows: source voltage: 5 kV, capillary voltage: 7 V, tube lens: 65 V and capillary temperature: 400 °C.Mass spectra were acquired in the range of 140-1500 Da.For the fragmentation study, a data-dependent scan was performed and the normalized collision energy of the collisioninduced dissociation (CID) cell was set to 30 eV and the isolation width of precursor ions was set to m/z 2.0.

RESULTS AND DISCUSSION
An extraction scheme for the phytochemical study of C. limettioides seeds was developed and its application enabled the extraction/ characterization of the chemical composition of fruit seeds, specifically, their oil, limonoids and polyphenols present therein.In the first phase of the scheme, application of supercritical fluid extraction with supercritical CO 2 yielded 39% of oil composed of triglycerides of palmitic (26.7%), stearic (3%), oleic (30.3%), and linoleic acids (29.9%), and other minor fatty acids, besides other compounds.Partition of the oil between water, methanol and chloroform and preparative HPLC led to the isolation and identification of four limonoids, namely: deacetylnomilin (1), nomilin (2), limonin (3) and obacunone (4).The structures of the isolated substances were determined by comparison of their spectroscopic data obtained by 1 H and 13 C NMR, with data from the literature (Manners et al. 2000, Khalil et al. 2003, Poulose et al. 2007).Rouseff and Nagy (1982), using gas chromatography, studied the content and identity of limonoids in seeds of several Citrus species, including C. limettioides, having identified the same limonoids as above, noting that limonin is the major limonoid in this and the other studied species.
In the aqueous fraction obtained from the cake resulting from the CO 2 extraction, eleven substances were characterized by HPLC-UV/ DAD/ESI-MS/MS analysis in the negative mode, as shown in Table I.The mass spectra of the fraction produced abundant deprotonated molecular ions by ESI in negative mode, with little fragmentation in the analysis under conditions of Collision Induced Dissociation (Energy of 30 eV).The compound identification criteria was based on the ultraviolet absorption spectra and their MS/MS spectra, with data obtained in MS1 (mass of the deprotonated molecular ion) and by the fragmentation pattern (in MS2).
Mass spectrometry of limonoids has been an important tool in studies on the structure of limonoids.Tian et al. (2003) studied the fragmentation of citrus limonoids after electroionization.Still in 2003, Tian and Schwartz carried out a very comprehensive study of the fragmentation of aglycones and limonoid glycosides, this time with electrospray ionization and atmospheric pressure ionization, in negative and positive modes using collision-induced dissociation and sequential mass spectrometry (Tian & Ding 2000).Avula et al. (2016) studied limonoids and flavonoids in citrus fruit seeds.
Fragmentation of limonoid glycosides is dominated by the elimination of the glucosyl moiety (Jayaprakasha et al. 2010).In the mass spectrum of obacunone 17-β-D-glucopyranoside, OG (7) and nomilin 17-β-D-glucopyranoside, NG (5) intense deprotonated ions [M-H] -at m/z 633.4 and m/z 693.4,respectively, were observed.The fragmentation of the ion at m/z 633.4 resulted in an intense ion at m/z 427.1, which corresponds to loss of a neutral fragment [CO 2 +glucose] for OG (Tian & Ding 2000).The fragment at m/z 565.1 in the NG mass spectrum was already noticed by Tian & Ding (2000) and by Raman et al. (2005), but no proposal for the fragmentation was suggested by these authors.
Limonoids are widely distributed in a wide variety of species of the genus Citrus.However, reports dealing specifically with this class of compounds are scarce.In a work carried out by Rouseff & Nagy (1982) limonoids in seeds of C. limettioides T. were determined by HPLC by comparing the retention time of limonoids with standards.The following limonoids were identified: limonin, obacunone, nomilin, deacetylnomilin and deoxylimonine.However, the presence of glycosylated limonoids was not reported.

CONCLUSION
An extraction scheme was designed and applied to the phytochemical study of C. limettioides seeds.In the first step, the seeds were extracted with supercritical CO 2 .The resulting solid residue was then submitted to liquid-liquid partitions.The application of this extraction scheme resulted in the isolation of four aglycone-type limonoids (deacetylnomilin, nomilin, limonin and obacunone) from the extract obtained with CO 2 .The subsequent liquid-liquid partition steps led to a fraction rich in polar components, whose composition was elucidated with the aid of HPLC-MS/MS.Four aglycone-type acidic limonoids (limonoic acid, deacetylnomilinic acid, isolimonoic acid or limonoic acid A-ring lactone and nomilinic acid), six glycosylated limonoids were identified: nomilin 17-β-D-glucopyranoside, limonin 17-β-D-glucopyranoside, obacunone 17-β-D-glucopyranoside, nomilinic acid 17-β-Dglucopyranoside, deacetylnomilinic acid 17-β-Dglucopyranoside and deacetylnomilinic 17-β-Dglucopyranoside, in addition to two flavonoids: vicenin-2 (apigenin 6,8-di-C-glycoside) and brutieridin.This is the first report describing the presence of glycosylated limonoids in Citrus limettioides T. seeds.The review by Gualdani et al. (2016) pointed out that the reason for the low number of studies on the biological activities of these compounds may be attributed to their difficult isolation from natural sources and to their poor pharmacokinetics.Thus, the separation/analysis scheme reported here can be considered a general scheme for the analysis and separation of metabolites in citrus seeds in general.

Figure 1 .
Figure 1.Chemical structures of the main limonoids found in Citrus limettioides.

Figure 2 .
Figure 2. Structures of compounds identified in the aqueous fraction of C. limettioides.

Table I .
Characterization of the substances present in the aqueous fraction of C. limettioides.