Recently, polysaccharides from diverse plant sources have been widely researched for various purposes, including medicine, food, animal feed, and cosmetics (Huang et al., 2015). Polysaccharides are natural, easily accessible, inexpensive, and non-toxic (Kaczmarek et al., 2020). Furthermore, they are biologically active with anti-inflammatory, antioxidant, anti-tumor, immuno-modulating, and antimicrobial properties (Wang et al., 2012, 2015).

Citrus fruits are abundant sources of nutrients, such as vitamins A, C, and E, and phytochemicals, including carotenoids, pectins, flavonoids, coumarins, and limonoids (Rafiq et al., 2018). Vitamin E, mainly found in the peel and seeds, protects cell membranes against lipid oxidation due to reactive oxygen species (ROS) (Rafiq et al., 2018). The juice-processing industries generate large amounts of citrus fruit residues, including peels, pulps, and seeds. Citrus fruits represent approximately 50% of the raw fruit processing industry (Anwar et al., 2008). These residues might lead to pollution, disposal, and other environmental problems due to microbial contamination (Fisher and Phillips, 2008). Therefore, several reports have explored the processing of citrus residues to recover the natural value-added components, such as fibers and bioactive components, including flavonoids and additives (Casquete et al., 2014; Sharma et al., 2017).

Citrus by-products, such as peels and seeds, can be potentially used in the food industry owing to their nutrients and bioactive compounds (Casquete et al., 2014; Sharma et al., 2017). Among them, citrus seeds have been reported to contain diverse components, such as polyphenols, phytosterols, tocopherols, and an abundance of unsaturated fatty acids, which can be valuable food additives (Anwar et al., 2008; Adeyeye and Adesina, 2015).

Furthermore, due to the increased demand for safer and more nutritious foods ingredients, researchers have been exploring natural raw materials, such as the natural value-added components from citrus residues (Anwar et al., 2008; Casquete et al., 2014; Adeyeye and Adesina, 2015). Moreover, research interest is steadily rising in uncovering novel biologically active compounds, such as the bioactive substances from citrus residues (Anwar et al., 2008; Casquete et al., 2014; Adeyeye and Adesina, 2015; Sharma et al., 2017; Rafiq et al., 2018).

Seeds are valuable food sources as they can be stored in the dried form for prolonged periods. Recently, edible seeds have been used to prepare food items, such as rice balls and rice cakes, in Japan (Kimura et al., 2017). Increased utilization of citrus fruits creates abundant waste, exacerbating environmental pollution. Due to the increasing demand for newer and safer food additives with special health benefits by the food processing industry, their production using by-products, including citrus seeds, has increased worldwide (Sharma et al., 2017).

Trifoliate orange or Poncirus trifoliate belongs to the family Rutaceae in the genus Poncirus, which is similar to the genus Citrus. The hot-water extract of these fruits has been used in traditional Oriental medicine as an anti-inflammatory and anti-allergy agent and to treat stomach disorders (Chun and Sankawa, 1989; Takase et al., 1994). Chemicals, such as limonin and imperatorin, isolated from trifoliate orange seeds, have been shown to have anticancer properties (Rahman et al., 2015). However, studies investigating the bioactive compounds in trifoliate orange seeds are relatively few. Therefore, it has been underutilized as a major residue compared with the Citrus genus.

Therefore, I isolated the crude polysaccharides from trifoliate orange seeds and investigated their immuno-potentiating role by evaluating their anti-complementary property and free radical quenching ability.

Isolation of crude polysaccharides from trifoliate orange seed extraction powder (TSEP)

Trifoliate orange seeds were obtained from the Ceil Food Company. They were processed by washing under tap water and oven drying (Sanyo Electric Co., Ltd.) for 12 h at 80°C. The dried seeds were ground into powder using an electronic grinder (Hanil Electronics Corp.). One hundred of the powder was extracted by l L of distilled water with stirring at a 40°C incubator (Sanyo Electric Co., Ltd.) for 8 h. And then, it was centrifuged at 6,000 g for 20 min, and, the supernatant was lyophilized to give the TSEP. Then, 12 g of TSEP was dissolved in 100 mL of distilled water. The sample was precipitated by adding four volumes of 95% ethanol and centrifuging at 6,000 g for 30 min. The precipitate was then re-diluted using distilled water. The sample was dialyzed using a 12 kDa molecular weight cut-off filter and then centrifuged at 6,000 g for 30 min. The supernatant was lyophilized and used for isolating the trifoliate orange seed crude polysaccharide (TSCP).

Analysis of the sugar and protein content

We determined the content of neutral sugar using the phenol-sulfuric acid method with galactose as the standard (DuBois et al., 1956). The uronic acid content was measured using the m-hydroxybiphenyl method using galacturonic acid as the standard (Blumenkrantz and Asboe-Hansen, 1973). The 2-keto-3-deoxy-D-manno-2-octulosonic acid (Kdo) content was measured using the modified thiobarbituric acid method with Kdo as the reference (Karkhanis et al., 1978). The protein content was determined using Bradford’s method using bovine serum albumin as the standard (Bradford, 1976). The monosaccharide content was analyzed by modifying the alditol acetate method by Jones and Albersheim (1972) using a gas chromatography (GC) system (6000 series, Young Lin Co.) equipped with an SP-2380 capillary column (0.2 μm×0.25 mm×30 m; Supelco) and flame ionization detector. The GC temperature program was 60°C for 1 min, 60 → 220°C (30°C/min), 220°C for 12 min, 220 → 250°C (8°C/min), and 250°C for 15 min. The molar ratio of the monosaccharides was calculated using the peak areas and response factors of the slope of the standard curve for each monosaccharide.

Total polyphenol content

The total polyphenol content was measured using the Folin-Ciocalteu method (Sulc, 1984). Briefly, 0.01 mL of the appropriately diluted sample, 0.79 mL of distilled water, and 0.05 mL of Folin-Ciocalteu reagent were mixed into the test tubes. After 1 min, 20% sodium carbonate (0.15 mL) was added, and the mixture was left standing at room temperature for 2 h. The absorbance was measured at 750 nm, and the total polyphenol concentration was calculated using a calibration curve with gallic acid as the reference.

2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity

The DPPH radical scavenging activity was measured based on the method by Cheung et al. (2003). Briefly, 0.2 mM DPPH ethanolic solution (0.8 mL) was added to 0.2 mL of the diluted sample, mixed vigorously, and left standing for 10 min in the dark. The decrease in absorbance was determined at 520 nm compared to a blank (without sample) using a spectrophotometer. The DPPH radical scavenging activity was calculated using the following equation:

DPPH radical scavenging activity (%)=(1−As/Ac)×100

where As and Ac are the absorbance values measured with and without the sample, respectively.

2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging activity

The ABTS radical scavenging activity was measured according to the method by Re et al. (1999). Briefly, the ABTS radical was formed by adding 7 mM ABTS to a 2.45 mM potassium persulfate solution and then leaving the mixture overnight at room temperature in the dark. The ABTS radical solution was then diluted using distilled water to attain an absorbance of 1.4∼1.5 at 414 nm. Then, 1 mL of properly diluted ABTS radical solution was added to 50 μL of the sample. The mixture was incubated for 1 h and its absorbance was determined at 414 nm. The ABTS radical scavenging activity was calculated using the following equation:

ABTS radical scavenging activity (%)=(1−As/Ac)×100

where As and Ac are the absorbance values with and without the sample, respectively.

Anti-complementary property

The anti-complementary property was assessed using a complement fixation test based on complement consumption and the degree of red blood cell lysis using the residual complement (Kabat and Mayer, 1964). Briefly, a 50 mL aliquot of the sample was mixed with an equal volume of normal human serum (NHS) and gelatin veronal buffered saline (GVB²+; pH 7.4), containing 500 mM Mg²⁺ and 150 mM Ca²⁺. The mixtures were then cultured at 37°C for 30 min, and the residual total hemolytic complement (TCH₅₀) was measured by IgM hemolysin-sensitized sheep erythrocytes (EA cells) at a density of 1×10⁸ cells/mL. NHS was cultured with water and GVB²+ as a negative control. Polysaccharide-K (PSK), a known immune-active polysaccharide from Coliolus versicolar, was used as a positive control for the anti-complementary property assay. The anti-complementary property of the samples was calculated as the percentage inhibition of the control TCH₅₀:

Inhibition of TCH₅₀ (%)=[TCH₅₀ (control)−TCH₅₀ (treated with sample)]/TCH₅₀ (control)

Statistical analysis

All data were analyzed using IBM SPSS version 20.0 (IBM Corp.). Statistically significant differences among the samples were determined by t-test or one-way ANOVA and Duncan’s multiple range test. The statistical significance level was set at P<0.05, P<0.01, and P<0.001.

Isolating TSCP and analyzing its sugar and protein content

Polysaccharides are natural polymers in plant cell walls, animal cells, and microorganisms. They comprise diverse monosaccharide units and their derivatives (DelattreI et al., 2011). Mainly, plants contain abundant natural polysaccharides, which structurally consist of homo or hetero monosaccharides and uronic acids linked with glycosidic bonds (Li and Wang, 2015; Zhang and Wang, 2015). Their monosaccharide content varies widely (Zhang and Li, 2018). Naturally occurring polysaccharides have different monosaccharide components, molecular weight, charge properties, and glycosidic bonds, which affect their functional characteristics and enhance their applicability (Yuan et al., 2020).

Here, I isolated TSCP from the TSEP prepared using water extraction and ethanol precipitation. The final yield of TSCP was 7% (dry weight, Table 1). Table 2 shows the sugar and protein content of the TSCP. The neutral sugar and uronic acid contents were 63.8% and 13.6%, respectively. However, I did not detect any Kdo-like material. The protein content was 22.6%. Table 2 and Fig. 1 show the monosaccharide component of the TSCP that was measured using GC. The monosaccharides with the highest content was arabinose (28.28 mol%), followed by galactose (16.76 mol%)> galacturonic acid+glucuronic acid (13.6 mol%)> glucose (12.45 mol%)> rhamnose (4.18 mol%)> mannose (0.57 mol%)> fucose (0.32 mol%).

Table 1 . The yield of the TSCP isolated from TSEP.

TSEP (g)	TSCP (g)	Yield (%)
12	0.84	7.00

TSCP, trifoliate orange seed crude polysaccharide; TSEP, trifoliate orange seed extraction powder..

Table 2 . The chemical composition of TSCP.

Variable	Value
Chemical composition (%)
Neutral sugars	63.8±8.1
Uronic acid	13.6±1.4
Kdo-like material	0
Protein	22.6±1.5
Monosaccharide component (mol%)
Arabinose	28.28±2.4
Galactose	16.76±3.8
Rhamnose	4.18±0.5
Xylose	1.24±0.2
Glucose	12.45±0.9
Mannose	0.57±0.1
Fucose	0.32±0.0
Galacturonic acid+glucuronic acid	13.60±1.4
Kdo	0

Values are presented as mean±SD..

TSCP, trifoliate orange seed crude polysaccharide; Kdo, 2-keto-3-deoxy-D-manno-2-octulosonic acid..

Figure 1. Gas chromatography chromatogram of the standards (A) and the trifoliate orange seed crude polysaccharide (B) for analyzing the monosaccharide composition.

Total polyphenol content and antioxidant activities

Recent studies have shown that natural polysaccharides, including microorganisms, plants, and animals, have potential antioxidant properties (Wang et al., 2013; Huang et al., 2017). Especially the polysaccharides of plant origin, such as those from leaves, seeds, roots, stems, fruits, and wood, have outstanding antioxidant potential (Krishnaiah et al., 2011). Several polysaccharides have been used to develop safe and effective food additives or medicines due to their potent antioxidant properties (Jiang et al., 2005; Zhang et al., 2011).

Phenolic compounds, such as polyhydroxylated substances, vary in structure, from simple to complex phenolics, and the degree of polymerization. The phenolic compounds with more complex structures and high molecular weights are often known as polyphenols (Balasundram et al., 2006). The total polyphenol content is commonly quantified using the Folin-Ciocalteu method (Singleton et al., 1999), which is also ideal for separating phenolic compounds (Khoddami et al., 2013). Therefore, I used this method to assess the total polyphenol content of TSCP (Fig. 2), which was 28.66 and 70.96 μg/mL for 1 and 10 mg/mL of TSCP, respectively (P<0.01).

Figure 2. The total polyphenol content of the trifoliate orange seed crude polysaccharide (TSCP). **Significant difference at P<0.01 based on t-test.

Over accumulation of ROS species, such as superoxide (O₂·⁻) and hydrogen peroxide (H₂O₂) radicals, cause lipid peroxidation and membrane damage (Jaspers and Kangasjärvi, 2010). Antioxidants can scavenge and stabilize unstable free radicals, thus inhibiting ROS-mediated oxidation. The chemical assays used to determine the free radical scavenging property are based on the capability of certain compounds to scavenge synthetic free radicals using diverse free radical generating systems and methods for detecting oxidation endpoints. The DPPH and ABTS radical scavenging assays utilize general spectrophotometric procedures for measuring the antioxidant properties of substances (Güllçin and Daştan, 2007).

Therefore, I determined the antioxidant activities of TSCP using ABTS radical and DPPH radical scavenging assays, as presented in Fig. 3. TSCP showed higher ABTS radical scavenging ability at 10 mg/mL (31.67%) than 1 mg/mL (8.07%; P<0.01). However, there were no significant differences in the DPPH radical scavenging activity between 1 mg/mL (11.72%) and 10 mg/mL (11.97%) of TSCP (P>0.05).

Figure 3. Antioxidant activities of the trifoliate orange seed crude polysaccharide. ABTS, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) radical scavenging activity; DPPH, 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity. P<0.01 based on ANOVA; different letters (a-c) represent significant differences among samples.

Phenolic compounds are secondary metabolites in plants that have significant biological functions, including antioxidant activities, activation of the endogenous immune systems, and protection against degenerative diseases (Balasundram et al., 2006). Phenolics are potent antioxidants owing to their capacity to scavenge free radicals, such as O₂·⁻, HO·, HOCl, and H₂O₂ radicals (Aruoma, 1994; 2003). These bioactive phenols are important in food products (Tepe et al., 2006). The antioxidant activity of the TSCP shown here is most likely due to these phenolic compounds. Studies have shown that the association between the total polyphenol content and the ABTS free radical scavenging activity is more robust than that with the DPPH system (Floegel et al., 2011). Here, I found significant differences in the ABTS radical scavenging activity of TSCP with low (30.42 μg/mL) and high total polyphenol contents (86.12 μg/mL). Whereas I did not observe any significant difference in the DPPH radical scavenging activity (P>0.05). Based on this, I suggest that TSCP is a potential natural alternative to synthetic antioxidants.

To date, the detailed mechanism underlying the antioxidant systems is poorly understood. Nevertheless, the characteristics of natural polysaccharides, such as monosaccharides content, molecular weight, water solubility, structural conformation, polarity, and intramolecular hydrogen linkages, have been linked to their antioxidant properties (Jia et al., 2013; Zhang et al., 2013). I showed that the TSCP isolated using water extraction had relatively higher monosaccharide content, with arabinose (28.28 mol%) and galactose (16.76 mol%) more abundant than other monosaccharides.

Anti-complementary property

Generally, the complement system is essential for the host’s immune system to fight against pathogens, including bacteria and viruses (Yamagishi et al., 2003). This system comprises over 30 plasma- and membrane-bound proteins and is regarded as a nonspecific host immune response, which includes macrophages and lymphocytes (Reid, 1988; Fruchterman, 1998; Gan et al., 2021). Previously, several polysaccharides have been shown to have immuno-modulating properties, such as complement activation, macrophage stimulation, anti-cancer properties, and B-cell proliferation (Khan et al., 2019; Li et al., 2019; Zhao et al., 2020; Gan et al., 2021).

We determined the anti-complementary property of TSCP, as shown in Fig. 4, which showed a significant concentration-dependent increase (P<0.001). The values were 61.36, 44.22, and 22.9% at 1,000, 500, and 100 μg/mL of TSCP, respectively. Moreover, 1,000 μg/mL of TSCP revealed a similar anti-complementary property as the positive control (60% PSK).

Figure 4. Anti-complementary activities of the trifoliate orange seed crude polysaccharide (TSCP). Polysaccharide-K (PSK), a known immnoreactive polysaccharide from Coliolus versicolar, was used as the positive control. Significant difference at P<0.001 based on ANOVA, different letters (a-c) represent significant differences among samples.

In summary, I successfully isolated TSCP, a crude polysaccharide, from trifoliate orange seeds. I found that it possesses a significant immuno-modulating effect, based on its anti-complementary property and potent antioxidative potential, as analyzed using in vitro systems. Moreover, I also examined the sugar and protein content of TSCP. However, further studies are required using in vivo models to reveal the main characteristics underlying its bioactivity, including molecular weight, and glycosidic bond patterns. The details regarding the functional conformations of its sugars also need to be further analyzed.

None.

The author declares no conflict of interest.