Exploration of the biochemical composition of Citrus L. seeds for industrial applications

SUMMARY: This study aimed to assess the biochemical profiles of Citrus L. seeds and elucidate the correlation patterns among varieties based on their biochemical compositions. Samples of Citrus L. seeds were subjected to extraction, and the resulting oils were analyzed for their biochemical levels. Principal Component Analysis (PCA) was usedto unveil the relationship patterns among Citrus L. The research findings revealed that several citrus seeds, including C. Paradise , C. limon (L.) Burm.f., Citrus reticulate , C. maxima (Burm.) Merr . , and Citrus sinensis , exhibited similarities in their compositions of flavonoid compounds, phenolic acids, and carotenoids. However, aromatic volatile compounds displayed variations among varieties without discernible patterns of similarity. The N-serotonin compound exhibited significant variations among varieties; whereas fatty acid compounds demonstrated similarities between Citrus latifolia and C. limon (L.) Burm.f. The eight samples showed no similarity in their biochemical variation patterns.


INTRODUCTION
Citrus plants boast a diverse array of varieties, each characterized by significant differences in their biochemical composition.Citrus seeds, often overlooked, contain bioactive compounds such as flavonoids, phenolic acids, carotenoids, N-serotonin, volatile compounds, and fatty acids.Despite extensive prior research on these compounds, their potential for further exploration remains untapped.
The flavonoids, phenolic acids, and carotenoids identified in citrus seeds exhibit robust antioxidant properties (Tundis et al., 2014) and hold promise as crucial components in pharmaceutical, culinary, and cosmetic applications (Maqbool et al., 2023).However, focused research on distinct citrus seed varieties and their biochemical compounds is limited, particularly in comprehending compositional variations among citrus varieties.
Serotonin, a seldom-discussed component, presents intriguing potential.Citrus amblycarpa and other citrus species' seed inner shells house numerous previously unidentified Acyl-Nω-methylserotonins and Branched-chain Acylserotonins (Kruk et al., 2022).This acyl derivative of N-methylserotonin is exceptionally rare in other plants (Servillo et al., 2015).Initial research indicates an association with antioxidant potential and other neurological functions, warranting further exploration.
Continuing exploration of volatile compounds and fatty acids in citrus seeds is also of interest.These seeds contain undiscovered volatile compounds, encompassing a variety of aromatic and organic elements (Park et al., 2021), with potential applications in pharmaceuticals (Mahmoud et al., 2014), cosmetics (Burnett et al., 2021), and the culinary domain (Kim et al., 2018).Despite their significance, the specific roles and broad uses of these volatile compounds in citrus seeds remain largely unexplored and necessitate further investigation.
Our research mapping from 2010 to 2023 revealed a progression in focus.The initial stage (2010)(2011)(2012)(2013)(2014)(2015)(2016) emphasized psychochemical properties and variations in types of citrus sinensis.The production of citrus seed oil began in 2017, and its impact on pharmaceuticals, biodiesel, and other sectors was evaluated.However, from 2019 onwards, there has been a notable lack of increased research focus on the impact of citrus seeds.In 2020, heightened interest emerged in biochemical components associated with microbiological effects.Nevertheless, psychochemical comparisons of citrus seed oils and their effects have not yet achieved prominence.This suggests a potential research gap, necessitating further exploration into other overlooked biochemicals.
Furthermore, this research offers a broader and more comprehensive understanding of the biochemical composition of citrus seeds, focusing specifically on flavonoids, phenolic acids, carotenoids, volatile compounds, fatty acids, and serotonin.The anticipated outcome of this research is to pave the way for harnessing the potential applications of these compounds across various industries.Moreover, the findings from this study serve as a catalyst for the development of innovative products based on the collection of bioactive compounds identified in citrus seeds.
The main objective of this study was to examine the biochemical profile of citrus seeds and uncover patterns of relationships among different citrus based on their biochemical compounds.The overarching goal is to unlock the untapped potential inherent in citrus seeds and promote their utilization across diverse industrial sectors.

Material
This study used citrus seeds that are abundant in Indonesia, including citrus latifolia, C. limon (L) Burm f., citrus sinensis, C. paradise, citrus amblycarpa, C. maxima (Burm.)Merr., citrus reticulata, and citrus maxima.The eight citrus seeds were obtained from waste from the beverage industry, and some were purchased from farmers on March 20, 2023.
Among the eight samples, four of them could be identified as original Indonesian varieties, namely citrus maxima, C. maxima (Burm.)Merr., citrus paradise, and citrus amblycarpa.Meanwhile, citrus reticulata comes from China, citrus latifolia from Persia, and C. Limon (L.) Burm f. from India.

Oil extraction
Oil extraction followed the method adapted from Aydeniz et al. (2014).The extraction process began with manual separation, washing, air drying, and freezing of citrus seeds at -20 °C.The seeds were then roasted in an oven at 30-35 °C for 30 minutes.Afterward, the seeds were cooled to room temperature, and the water content was measured using an Ohaus MB45 water content meter (OHAUS Instruments; Shanghai, Co., Ltd.).Cold pressing of the seeds was conducted using a laboratory-scale pressing machine.Fine particles and any remaining water in the pressed oil were separated through filtration.The centrifugation (Magal M16R, Shanghai.China) process operated at a speed of 6800 x g for 10 minutes.Oil samples were transferred into colored glass bottles and stored at −18 °C.

Biochemical analysis
The extraction of polyphenolic compounds from citrus seed oil followed the procedure outlined by García-Villalba et al. (2010).Initially, all oil samples underwent sep pak C-18 cartridge filtration, assisted by a vacuum in the manifold.To initiate the process, 3 grams of seed oil were dissolved in 3 mL of hexane (ChemStationAsia, Malaysia), applied to the cartridge, and rinsed with 5 mL of hexane to eliminate non-polar components.The residual hexane in the cartridge was removed using a nitrogen stream.The remaining phenolic fraction was subsequently extracted with 10 mL of methanol, and the resulting solution was filtered through a 0.45 μm pore size PTFE (polytetrafluoroethylene) membrane from Fisher Scientific before inclusion in the HPLC chromatogram.All samples were stored at -18 °C and analyzed within 24 hours.The flow through each cartridge was consistently managed concurrently on the manifold.
The identification process for specific phenolic acids and flavonoids adhered to the methodology outlined by Yilmaz and Karaman (2017) with minor adjustments.A C18 reversed-phase column (Zorbax Eclipse Plus, 250 mm x 4.6 mm i.d.x 5 μm) facilitated separation, and the analysis was conducted at 25 °C at a flow rate of 0.55 mL/min, and 25-μL injection volume.The chromatographic analysis involved the concurrent monitoring of phenolic extracts at 280 nm.Mobile phase A consisted of 100% acetonitrile (Merck, Darmstadt, Germany), and mobile phase B was ultra-pure water with 0.2% (v/v) sulfuric acid (Merck, Darmstadt, Germany) 95-97%, analytical grade) following the guidelines of the National Institute of Standards and Technology (NIST, 2014).Separation was performed using the following gra-dient program: 80% A / 20% B for 0-20 min, 70% A / 30% B for 20-26 min, 60% A / 40% B for 26-32 min, 50% A / 50% B for 32-38 min, 40% A / 60% B for 38-42 min, 45% A /55% B for 42-47 min, 35% A / 65% B for 47-52 minutes, 20% A / 80% B for 52-54 minutes, 10% A / 90% B for 54-56 minutes, 100% B for 56-72 minutes, 45% A /55% B for 72-76 minutes, and 80% A/ 20% B for 76-80 minutes.The identification of individual phenolic compounds depended on their retention time, and quantification was performed by measuring the peak area at 280 nm, using a standard curve prepared from the corresponding standard, according to NIST guidelines.All analyses were performed in triplicate.
In this study, TC was presented in units of mg/kg.To verify chlorophyll carotenoids as pheophytin-a, the AOCS Cc 13i-96 method (AOAC, 1997) was employed.This involved measuring absorbance at 630, 670, and 710 nm.
The determination of serotonin compounds in this study was made using HPLC and referring to Kruk et al. (2022).The mobile phase, composed of elution solvent A = 0.1% formic acid (BASF, China) in water and B (0.1% formic acid in acetonitrile), was employed for elution based on a scheme of 85-60% A and 15-40% B from 6-15 minutes, with a post-run phase of 5 minutes involving 60-20% A and 40-80% B. The detection of serotonin derivatives took place at 324 nm, using a C18 column (1.8 μm, 2.1 × 50 mm).Parameters such as injection volume (2 µL), flow rate (0.4 mL/min), and column temperature (25 °C) were standardized.The contents of CS and FS were determined by referencing standard curves developed from standard solutions.
Fatty Acid Methyl Esters (FAMEs) were synthesized following the Ce 2-66 protocol (AOAC, 1997) and subsequently quantified using a Gas Chromatograph (Shimadzu's Nexis GC-2030; Maryland, USA) equipped with an HP 88 capillary column (100 m x 0.25 mm ID x 0.2 µm film thickness).The Gas Chromatograph was operated at various temperature settings: initially set at 120 °C for 1 minute, followed by an increase to 175 °C (10 °C/min) for 10 minutes, then to 210 °C (5 °C/min) for 5 minutes, and finally to 230 °C (5 °C/min) for an additional 5 minutes.For the analysis, a 1-µL injection volume was used with an injector split ratio of 1:50 and a flow rate of 2 mL/min, with hydrogen as the carrier gas.The injector and detector temperatures were maintained at 250 and 280 °C, respectively.Fatty acid identification was made through chromatography, employing a standard mixture of FAMEs for reference.
The volatile compound identification procedure was based on Guneser and Yilmaz (2017).Volatile compounds were gathered through headspace solid-phase microextraction (SPME) with specific fibers (2 cm to 50/30 µm DVB/Carboxen/PDMS; Supelco, Bellafonte). 2 grams of the oil sample, 1 gram of NaCl, and 20 μl of the internal standard (IS) (1 μl of 2-methyl-3-heptanone dissolved in 10 ml of methanol) were combined n a 40-ml SPME bottle, and stirred for 2 minutes.This mixture was then placed in a water bath at 45 °C for 15 minutes to stabilize the volatiles into the headspace.Subsequently, a needle was inserted into the bottle, and the needle fiber was introduced into the headspace at a depth of 2 cm for 10 minutes in a water bath.The volatiles collected on the needle fibers were then injected into a GC/ MS equipped with an HP5 MS column (30-m x 0.25mm i.d.x 0.25-µm).For the identification of volatiles, databases such as the National Institute of Standards and Technology (NIST, 2014) and The Wiley Registry of Mass Spectral Data (Wiley, 2006) were consulted, along with the Retention index (Kovats).

Statistical test
The statistical approach employed was analysis of variance (ANOVA), complemented by a post-hoc Tukey's Honestly Significant Difference (HSD) test to discern variations in biochemical composition among different Citrus L seeds.The significance threshold was set at p < 0.05.The analysis was conducted in three replicates, and the results were presented as Mean ± Standard Deviation.This analytical framework facilitated the identification of noteworthy differences in the biochemical composition of citrus seeds.Additionally, we conducted a Principal Component Analysis (PCA) using Minitab 21 software.This facilitated a comprehensive exploration of both similarities and distinctions in the biochemical composition among various citrus varieties, providing a more detailed understanding of their profiles.

Flavonoids, phenolic acids, and carotenoids
The flavonoid content, specifically catechin, in citrus latifolia seeds did not exhibit significant differences (p > 0.05) compared to C. maxima (Burm.)Merr.and citrus seeds.Similar findings were observed for citrus amblycarpa seeds against citrus reticulate and the C. paradise variety against citrus maxima.Notably, the variety C. limon (L.) Burm.f.displayed significantly different seeds (p < 0.05) across the seven samples.
Concerning eriocitrin compounds, no significant differences were observed in citrus sinensis, C. paradise, citrus reticulate, or citrus maxima seeds (p > 0.05).C. maxima (Burm.)Merr.seeds, however, exhibited the highest eriocitrin levels and were significantly different from the seven samples.
The phenolic acid compounds exhibited varying levels across the eight seeds, with citrus latifolia containing both the highest and lowest levels of gallic acid (tr-ferulic acid, rosmarinic acid, and tr-2-hydrocinnamic acid), showing significant differences from the other seven samples.
Additionally, citrus latifolia seeds demonstrated the highest levels of carotenoids, including total carotenoids, β-carotene, and lutein, and the lowest levels of total chlorophyll (pheophytin a).These differences were found to be significant (p < 0.05) across the seven samples.Detailed results regarding the levels of flavonoids, phenolic acids, and carotenoids are provided in Table 1.
The categorization of citrus seed varieties based on flavonoids, phenolic acids, and carotenoids is illustrated in Figure 2.Among the seed varieties, including C. paradise, C. limon (L.) Burm.f., citrus reticulate, C. maxima (Burm.)Merr., and citrus sinensis, there was a prominent influence on component 1, indicating their similarity in terms of the tested compounds.Conversely, the citrus maxima, citrus amblycarpa, and citrus latifolia varieties displayed dissimilarities, positioned distinctly apart in different quadrants (Figure 2A).
The grouping of flavonoids, phenolic acids, and carotenoids in citrus varieties is further depicted in Figure 2B.A narrow angle, indicative of similarity, was observed for compounds such as naringin, naringenin, total chlorophyll (pheophytin a), rosmarinic acid, rutin, tr-2-hydrocinnamic acid, neohesperidin, tr-ferulic acid, and eriocitrin.These compounds demonstrated a strong correlation in component 1.
Total carotenoid compounds, lutein, β-carotene content, and hesperidin exhibited similarities in component 2, albeit with a weak correlation.In contrast, syringic acid and catechin were positioned closely, indicating a strong correlation.

Acylserotonin
All seed varieties exhibited N-Acylserotonin compounds that did not display significant differences (p > 0.05) for < C21, ai-C21, Me-C20, n-C21, n-C25, and ai-C27.The prevalence of N-Acylserotonin compounds was found in the C22 to C24 homologs for all varieties, and the distribution of values was consistent across all samples.Notably, there was a substantial difference for Me-C22, with the citrus maxima variety recording the highest level (12 ± 0.3 mg/kg oil), which was found to be significantly different (p < 0.05) when compared among the seven samples.Comprehensive results regarding the composition of N-Acylserotonin for each variety are presented in Table 2.
The grouping of citrus seed varieties based on the N-Acylserotonins compound did not reveal a strong correlation between varieties.Each variety displayed a wide angle, but C. paradise and citrus sinensis varieties exhibited similarity compared to other varieties.As illustrated in Figure 2C, the even distribution of N-Acylserotonins compounds in seed varieties did not indicate any noticeable similarity among the samples.
Furthermore, the n-C21 compound demonstrated similarity to ai-C27, n-C25, n-C23, and Me-C22.As illustrated in Figure 2D, the levels of N-Acylserotonins were evenly distributed into four quadrants, indicating similarity despite having distinct strong correlations for each N-Acylserotonin compound.
The second dominant compound was b-Myrcene, reaching its highest levels at 124.89 ± 0.4 ppm in the citrus latifolia variety, and it was significantly different (p < 0.05) from other varieties.Comprehensive results for the composition of volatile compounds are provided in Table 3 The grouping of citrus seed varieties based on aromatic volatile compounds did not reveal a strong correlation between varieties.All samples exhibited very wide angles between each other, and the distri-  bution of aromatic volatile compounds was uniform across all varieties, indicating a lack of similarity among them (see Figure 3A).A strong correlation, indicated by a narrow angle, was observed for the compounds 4-carnomenthol, butyl iso butyrate, 2,5 dimethylpyrazine, 1-octenol, and phenylethyl alcohol.Similar properties were also noted in the compound group 3-Methylbutanol, benzaldehyde, and (E)-limonene oxide in component 1 (see Figure 3B) In component 2, a narrow angle was observed for the compounds butyrolactone, isopropyl pentanoate, (z)-linalooxide, and α-thujene.A negative correlation was seen for the furfural, hexanal, and 2-furan menthol compounds.

Fatty acids
None of the varieties exhibited significant differences (p > 0.05) for the compounds lauric acid, ara-  chidic, and behenic.However, palmitic compounds showed significant differences (p < 0.05) among varieties, except for citrus latifolia and citrus maxima.Polyunsaturated fatty acids (PUFA) dominated, reaching 53.27 ± 0.8 ppm in the citrus seed variety, and were significantly different from other varieties.The second dominant group was saturated fatty acids (SAFA), which exhibited significant differences in Citrus maxima varieties.The highest levels of monounsaturated fatty acids (MUFA) were observed in the C. limon (L.) Burm.f.variety (27.8±0.4 mg/ kg oil), and it was significantly different from other varieties.The complete composition of fatty acid compounds is detailed in Table 4.
Based on the composition of fatty acid compounds, the citrus latifolia and C. limon (L.) Burm.f.varieties exhibited similarity in the component 1 area, characterized by very narrow angles.In contrast, other varieties demonstrated different conditions, showing no strong correlation with each other (see Figure 3C).
In the composition of fatty acid compounds, palmitic, linoleic, and stearic exhibited a narrow angle or strong correlation in component 1.Another group displaying a strong correlation consisted of behenic, linolenic, lauric acid, and margaric.In component 2, the myristic acid compound was very strongly correlated (narrow angle) with PUFA, while this condition did not apply to oleic or arachidic.Complete results can be observed in the biplot of the fatty acid composition test (Figure 3D).

DISCUSSION
The seed varieties of C. paradise, C. limon (L.) Burm.f., Citrus reticulate, C. maxima (Burm.)Merr., and citrus sinensis exhibited similarities in terms of flavonoid composition, phenolic acids, and carotenoids.In contrast, citrus maxima, citrus amblycarpa, and citrus latifolia varieties demonstrated no similarities.Notably, hesperidin was the predominant compound among flavonoids, with concentrations ranging from 890 to 930 ppm.Additionally, tr-Ferulic acid dominated among phenolic acid compounds.These results are in agreement with the findings of Güneşer et al. (2018).
Despite reported findings by various researchers, the utilization of flavonoids, phenolic acids, and carotenoids in citrus varieties on an industrial scale remains limited.Notably, bioflavonoid com-pounds have been shown to facilitate the apoptosis of hepatocellular carcinoma cells.Specifically, flavone glycosides such as neohesperidin, hesperidin, and naringin have demonstrated the ability to induce the death of liver cancer cells.The use of Annexin V-FITC/PI staining and flow cytometry has revealed the apoptosis of HepG2 cells in this context (Banjerdpongchai et al., 2016).
In human kidney cells (HEK 293T cells) subjected to H 2 O 2 -induced oxidative stress, flavonoids from citrus amblycarpa seeds (FLS) have demonstrated a protective effect.FLS was observed to decrease malondialdehyde levels, indicative of reduced oxidative damage, while concurrently elevating the levels of crucial antioxidant enzymes, including CAT, SOD, GSH, and GSH-Px (Yang et al., 2020).The key phenolic substances identified in Citrus amblycarpa seeds, namely 1,2-dihydroxybenzene, kaempferol, catechin, and isorhamnetin, have been recognized for their ability to safeguard against oxidative damage to cells (Yang et al., 2020).Citrus amblycarpa seed extract has been identified as a potential source of various phytochemical substances, encompassing alcaloids, flavonoids, saponins, tannins, steroids, and glycosides.The extract possesses the ability to significantly increase sleep duration while concurrently reducing sleep time (Rahman et al., 2022).
Aromatic volatile compounds were found uniformly across all varieties without exhibiting similarities.Notably, discrepancies in previous research (Güneşer et al., 2018) highlight variations in the citrus sinensis variety, where compounds such as 3-Methylbutanal and 3-Methoxy-1-butanol were absent under cold-pressing conditions.
Despite reports of the effectiveness of these compounds in cancer treatment by earlier researchers, their optimal utilization remains unexplored.Mahmoud et al. (2014) conducted research revealing that Limonene, administered at a dose of 100 mg/ kg, was more effective in reducing bilirubin, while a dose of 50 mg/kg demonstrated greater efficacy in reducing oxidative stress and mitigating liver damage.Additionally, compounds such as α-pinene, 1,8-cineole, karyophyllene, and geraniol have been identified as having anticancer properties by inhibiting cancer cell proliferation (Tunjung et al., 2020) The grouping of citrus seed varieties based on the N-Acylserotonins compound reveals dissimilarities, with an exception observed in the C. paradise and citrus sinensis varieties.Our findings diverge from those of Kruk et al. (2022), particularly regarding the absence of the N-serotonin (ai-C25) compound in citrus reticulate and citrus maxima varieties.Similarly, iso-C26 was not detected in citrus reticulate, C. maxima (Burm.)Merr., and citrus maxima varieties, and ai-C27 were absent from citrus reticulate varieties according to Kruk et al. (2022) The germination of citrus seeds has been shown to enhance antioxidant activity and increase the content of phenolic components.Despite this, a lack of correlation between the content of phenolic compounds and antioxidant activity suggests the potential presence of other antioxidants (Falcinelli et al., 2020).Addressing this, Kruk et al. (2022) highlighted that the inner skin of citrus seeds contains acylserotonin, an active antioxidant.This acyl derivative of N-methylserotonin, rarely found in plants, and citrus seeds also contains serotonin compounds with branched chains.
In the context of bone cancer therapy, the combination of gold nanoparticles (AuNPs) with citrus reticulata seed extract has proven effective in reducing gold-to-gold nanoparticles, as evidenced by FT-IR testing (Ahati et al., 2022).
The utilization of citrus sinensis seed oil, coupled with alkaline catalytic transesterification, has successfully met biodiesel quality standards (ASTM6751 and EN14214) (Ezekoye et al., 2019).Biodiesel derived from citrus seed oil exhibits a higher density than petroleum-derived biodiesel at 15 °C (Agarry et al., 2013).Citrus sp.seeds with various catalysts such as green copper oxide nanoparticles, NaOH, and CaO yield comparable results, as observed in citrus medica (Dhanasekaran et al., 2016).The antibacterial properties of citrus sinensis seed oil, containing 36% linoleic acid and 27% oleic acid, have been harnessed in the production of medical soap (Atolani et al., 2020)

CONCLUSIONS
The seeds of C. paradise, C. limon (L.) Burm.f., citrus reticulate, C. maxima (Burm.)Merr., and citrus sinensis shared similarities in their flavonoid, phenolic acid, and carotenoid profiles.However, distinct differences were observed in other varieties.The uniformity in these chemical compositions offers promising opportunities for sustainable innovation, particularly in harnessing the positive effects of flavonoids on oxidative damage and antioxidant activities in Citrus L. seeds.
Although volatile aromatic compounds exhibit variances without discernible patterns across varieties, their reported potential in cancer treatment underscores their significance.Noteworthy diversity in N-serotonin compounds exists among varieties, while certain varieties, such as citrus latifolia and C. limon (L.) Burm.f., share similarities in fatty acid compounds.
The versatile applications of Citrus L. seed oil, including biodiesel production, medical soap formulation, cancer treatment, and the development of modern chemotherapy drugs, underscore the manifold variations and potential uses that warrant further exploration in the industry.

Figure 1 .
Figure 1.Overlay visualization of citrus seed research from 2010 to 2023

Figure 3 .
Figure 3. Grouping of citrus seed varieties.based on the composition of volatile aromatic compounds: PCA score plot based on volatile aromatic compounds (A), biplot (B).Score-plot based on fatty acids (C) and biplot (D), using the mean result of 3 repetitions.

Table 1 .
Composition of flavonoid, phenolic acid, and carotenoid compounds in citrus seed varieties.

Table 2 .
Composition of N-Acylserotonin compounds in citrus seed varieties.

Table 3 .
Composition of volatile aromatic compounds in citrus seed varieties.

Table 4 .
Composition of fatty acid compounds in citrus seed varieties.