2021 Volume 27 Issue 6 Pages 907-914
To identify the components in Hyuganatsu orange (Citrus tamurana Hort. ex Tanaka) that improve bone metabolism function, the structure of the polysaccharide PWH fractionated from water extracted from Hyuganatsu orange juice was investigated. The findings showed that PWH consisted of arabinogalactan with a molecular mass of 68 kDa. Monosaccharide compositional analysis indicated that PWH contained galactose, arabinose, and rhamnose at a ratio of 57:27:16. Using the methods of methylation analysis, partial acid hydrolysis, and NMR, PWH was indicated to be a type II arabinogalactan with a backbone of 1,3-linked galactopyranosyl residues. Furthermore, it was found that PWH effectively inhibited the fusion of osteoclasts. The results showed that the functional component of Hyuganatsu orange that inhibits osteoclast formation contains PWH.
Hyuganatsu orange (Citrus tamurana Hort. ex Tanaka) is a citrus fruit native to Miyazaki Prefecture, Japan. It was found that Hyuganatsu orange homogenate and its water extract can increase bone density in ovariectomized rats, which are the animal model for osteoporosis (Yamaguchi et al., 2012). Hyuganatsu orange water extract was divided into five fractions (Hata et al., 2015). Among them, the fraction with the smallest molecular mass and the fraction with the largest molecular mass showed inhibitory effects on osteoclast formation. These active components were estimated to be low molecular mass hesperidin and high molecular mass polysaccharides. Further, the preventive effects of Hyuganatsu orange juice on osteoporosis in postmenopausal women were investigated, and the effect of calcium absorption in the intestine on the mechanism of action was evaluated (Nishizono et al., 2019). In a calcium absorption test using an inverted intestinal tract, a dramatic calcium absorption-promoting effect was observed in the water-soluble polymer derived from Hyuganatsu orange juice and the polysaccharide fractionated from it by ultrafiltration. The results suggested that Hyuganatsu orange juice is a functional food that can prevent the onset and/or progression of osteoporosis induced by ovarian hormone deficiency in postmenopausal women. Furthermore, the specific components of Hyuganatsu orange responsible for the improvement of bone metabolism were analyzed to realize practical applications of these research results. The active components were extracted from the water fraction of Hyuganatsu orange juice residue using an organic solvent. From an analysis of the water fraction, the active component responsible for improving bone metabolism was clarified to be a polysaccharide, although hesperidin or β-cryptoxanthin in citrus fruit had been previously reported to have a similar effect.
The polysaccharide components of plants have been reported to improve immunostimulatory action and increase the amount of intestinal flora (Classen et al., 2006; Robinson et al., 2001). Among these components, arabinogalactans (AGs) have physiological functions such as differentiation and growth of plant tissue, and discovery of new functions can be expected (Burgalassi et al., 2011; Yu et al., 2005). To date, many reports have described in detail the structure of polysaccharide components originating from plants, but there are few reports on structures that can be applied to functional expression and elucidation of the mechanism of action.
In the present study, to identify the active components of bone metabolism in Hyuganatsu orange, the structure of the polysaccharide fractionated from the water extract of Hyuganatsu orange juice residue was investigated. Furthermore, the effects of the polysaccharide on osteoclast formation were confirmed.
Polysaccharide sample preparation For the experiment, a concentrated water extract of Hyuganatsu orange juice residue, which was prepared by Miyazakiken Nokyokajyu Co., Ltd., was used. To extract the water-soluble component from the Hyuganatsu orange, water was added to frozen Hyuganatsu orange juice residue, and the mixture was stirred while heating. Then, the supernatant recovered by centrifugation was concentrated under reduced pressure. The concentrated water extract was fractionated by ultrafiltration using Biomax® Membrane (100K) (Merck KGaA, Darmstadt,Germany) and Ultracel® Membrane (30K) (Merck KGaA, Germany), and then the concentrate was freeze-dried. A sample dissolved in distilled water was inserted into a Cellufine A-500 anion-exchange column (100 × 1.5 cm, JNC Co., Tokyo, Japan) equilibrated with distilled water. The column was washed with distilled water and the polysaccharides were eluted with a linear gradient of 0–2.0 M NaCl eluent. Among the fractionated fractions, the fraction confirmed to inhibit fusion of osteoclasts was called PWH.
Partial acid hydrolysis PWH (100 mg) was hydrolyzed using a 0.1 M sulfuric acid solution (50 mL) at 100 °C for 2 h before being neutralized by adding barium sulfate. Then, the solution was collected by suction filtration, and was concentrated in vacuum. After ultrafiltration using an Ultracel® membrane (10K) (Merck KGaA), the concentrate was freeze-dried and the resulting sample was called PWH-A.
Total sugar content, uronic acid and protein analysis The total sugar content in the sample was measured using the phenol-sulfuric acid method (Dubois et al., 1956). Uronic acid in the sample was measured using the carbazole-sulfuric acid method (Bitter and Muir, 1962). Protein in the sample was measured using a BCA assay using a TaKaRa BCA Protein Assay Kit (Takara Bio Inc., Shiga, Japan).
Monosaccharide composition analysis The sample (100 mg) was hydrolyzed by incubating with 2 M sulfuric acid at 100 °C for 2 h. After neutralization with barium sulfate, the solution was collected by vacuum filtration. The collected solution was concentrated in vacuum and analyzed by high-performance liquid chromatography (HPLC).
Methylation composition analysis After 5 mg of the sample was fully dried in vacuum, it was dissolved in 2 mL DMSO and dehydrated by molecular sieves. Then, 100 mg of NaOH powder was added and the solution was ultrasonicated for 30 min in a nitrogen atmosphere. After that, 1 mL methyl iodide was added, and the resulting mixture was ultrasonicated for a further 1.5 h. Then, 4 mL of 0.88 % KCl was added, and dialysis was performed for 12 h. After the in-membrane liquid was dried in vacuum using an evaporator, the methylated polysaccharide was retreated again as described above. Finally, the methylated polysaccharide was obtained upon extraction with chloroform. The methylated polysaccharide was hydrolyzed by incubation in 2 M trifluoroacetic acid (TFA) at 110 °C for 4 h. The TFA was completely removed by repeated concentration to dryness with methanol, and the resulting residue was dissolved in 2 mL of distilled water. Then, it was reduced with 40 mg of NaBH4 at 30 °C for 1.5 h. Next, after neutralization with acetic acid and concentration to dryness, the product was acetylated at 100 °C for 1 h by adding 0.5 mL of acetic anhydride and 0.5 mL of pyridine. The partially methylated alditol acetate obtained upon extraction with chloroform was analyzed by gas chromatography-mass spectrometry (GC-MS).
HPLC analysis To determine the molecular mass and to identify monosaccharide species in the samples, HPLC analysis was conducted. To determine the molecular mass, the samples were measured by a Refractive Index Detector RID-10A (Shimadzu Co., kyoto, Japan) at a flow rate of phosphate buffer (pH 6.8) of 0.5 mL/min and a column temperature of 40 °C using a TSKgel G5000-PWXL column (Tosoh Co., Tokyo,Japan) with a 7.8 mm ID and a 300 mm length. To construct the calibration curve, various kinds of dextrans (Sigma-Aldrich Co., St. Louis, USA) with known molecular masses were used. For identification of monosaccharides, the samples were measured using the RID-10A at a flow rate of 1.0 mL/min using 75 parts of acetonitrile and 25 parts of purified water, and a column temperature of 40 °C using a Wakosil 5NH2 column (FUJIFILM Wako Pure Chemical Co., Osaka,Japan) with a 4.6 mm ID and a 150 mm length. A commercially available monosaccharide was used as a standard.
GC-MS analysis GC-MS analysis was performed using a GCMS-QP2010SE (Shimadzu Co.). As a capillary column, an InertCap RTX 5MS (GL Sciences Inc., Tokyo, Japan) was used. The temperature profile was as follows: First, the sample was kept at 180 °C for 5 min, the temperature was then raised from 180 °C to 250 °C at a rate of 2°C/min, and finally it was kept at 250 °C for 5 min.
NMR analysis A sample (∼40 mg) was dissolved in 0.4 mL of D2O. Then, 1H NMR and 13C NMR spectra were measured at 313 K using a Bruker AV-400M spectrometer (Bruker Instruments Inc., Billerica, USA). The obtained data were analyzed using proprietary Bruker software.
Enzymatic hydrolysis PWH (10 mg) was dissolved in a 100 mM sodium acetate buffer (pH 4.0), and then endo-1,5-α-L-arabinanase from Aspergillus niger (Megazyme Ltd., Bray, Ireland) was added and the solution was incubated at 40 °C for 1 d. The reaction solution was then analyzed by HPLC.
Effect on osteoclast formation Rat osteoclast precursor cells (Primary Cell, Cosmo Bio Co., LTD., Tokyo, Japan) were disseminated in a culture medium with 96-well plates (the culture medium contained 10 % fetal bovine serum, 50 ng/mL Macrophage Colony Stimulating Factor, and 15 ng/mL Receptor Activator of NF kappa B Ligand). Then, they were incubated at 37 °C for 3 d under a 5 % CO2 atmosphere. Subsequently, the culture medium was replaced with another containing the sample, and the sample was incubated at 37 °C for 2 d under 5 % CO2. After incubation, the cells were immobilized in 10 % neutral buffered formalin solution (FUJIFILM Wako Pure Chemical Co.), stained with a Tartrate-Resistant Acid Phosphatase (TRAP) staining kit (Cosmo Bio Co., LTD.). Among the TRAP-positive cells, fused osteoclasts were counted by eye under an optical microscope. The obtained data were expressed as mean ± standard error, and were analyzed by a one-way analysis of variance. p < 0.05 was considered to be statistically significant.
Monosaccharide composition analysis First, PWH was fractionated from a concentrated water extract of Hyuganatsu orange juice residue by ultrafiltration using two types of membrane. Among the fractionated fractions, those concentrates that were confirmed to inhibit the fusion of osteoclasts were further fractionated by anion-exchange chromatography. As shown in Fig. 1, three separated peaks (P1–P3) were observed, and P2 was confirmed to inhibit the fusion of osteoclasts; this compound is referred to as PWH. The HPLC analysis revealed that PWH produced a symmetrical peak (Fig. 2), and its molecular mass was 68 kDa. A BCA assay of PWH showed a negative response indicating that it was free of protein. PWH was shown by the carbazole-sulfuric acid method to contain no uronic acid, indicating that it was a neutral polysaccharide. The results of a monosaccharide composition analysis showed that PWH contained galactose, arabinose, and rhamnose at a ratio of 57:27:16.
Anion-exchange chromatography on Cellufine A-500.
HPLC chromatograms of PWH and PWH-A.
In order to simplify the structure determination of polysaccharides and identify structures useful for functional expression, partial acid hydrolysis of PWH was performed and a structural analysis of the products was carried out. PWH-A is a polysaccharide from a concentrate obtained by partially hydrolyzing PWH and fractionating it by ultrafiltration. According to the HPLC analysis, PWH-A exhibited a symmetrical peak (Fig. 2), and its molecular mass was 5 kDa. The monosaccharide composition analysis revealed that PWH-A contained galactose and rhamnose at a ratio of 96:4.
Linkage analysis To determine the glycosyl linkage types, a methylation analysis was conducted. As shown in Table 1, the intrachain residues of PWH were 1,3-linked Galp (13.3 %), 1,6-linked Galp (7.8 %), and 1,5-linked Araf (7.9 %). The nonreducing terminals of PWH were composed of Araf (19.0 %), Rhap (16.3 %), and Galp (0.5 %), indicating that PWH contained many branches. The branch point was 1,3,6-linked Galp (35.2 %). These ratios were consistent with those obtained in the monosaccharide composition analysis. PWH-A was composed of 1,3-linked Galp (57.3 %) and 1,6-linked Galp (42.7 %).
| Methylated sugars | Linkages | Molar ratio (%) | |
|---|---|---|---|
| WE | WEA | ||
| 222,3,5-Me3-Araf | T-Araf | 19.0 | |
| 2,3-Me2-Araf | 1,5-Araf | 7.9 | |
| 2,3,4,6-Me4-Galp | T-Galp | 0.5 | |
| 2,4,6-Me3-Galp | 1,3-Galp | 13.3 | 57.3 |
| 2,3,4-Me3-Galp | 1,6-Galp | 7.8 | 42.7 |
| 2,4-Me2-Galp | 1,3,6-Galp | 35.2 | |
| 2,3,4-Me3-Rhap | T-Rhap | 16.3 | |
NMR analysis The assignment of the 13C NMR spectra (Fig. 3) of PWH and PWH-A was determined mainly by comparison with reported values and by correlation with the respective 1H NMR, heteronuclear singular quantum correlation (HSQC) and heteronuclear multiple bond correlation (HMBC) spectra (Fig. 4, S1–4) (Liang et al., 2014; Nagel et al., 2016; Wang et al., 2005; Wang et al., 2015; Xu et al., 2010). The signals due to galactose, arabinose, and rhamnose were simply distinguished by comparing the spectra of PWH and PWH-A. The obtained chemical shifts are summarized in Table 2.
13C NMR spectra of PWH and PWH-A.
A(T-α-Araf), B(1,5-α-Araf), C(1,3-β-Galp), D(1,6-β-Galp), E(1,3,6-β-Galp), F(T-α-Rhap), C′(1,3-β-Galp), and D′(1,6-β-Galp) represented the different residues from PWH and PWH-A as shown in Table 2.
HMBC spectrum of PWH.
E(C6)-F(H1): cross peak between C-6 of residue E and H-1 of residue F; E(C3)-A(H1): cross peak between C-3 of residue E and H-1 of residue A.
1H NMR spectrum of PWH.
1H NMR spectrum of PWH-A.
HSQC spectrum of PWH.
HSQC spectrum of PWH-A.
| Residues | C-1 | C-2 | C-3 | C-4 | C-5 | C-6 | |
|---|---|---|---|---|---|---|---|
| A | T-α-Araf | 110.4 | 83.6 | 78.0 | 85.2 | 62.5 | |
| B | 1,5-α-Araf | 108.7 | 82.5 | 77.9 | 85.2 | 69.7 | |
| C | 1,3-β-Galp | 104.6 | 74.1 | 81.3 | 69.9 | 76.3 | 62.3 |
| D | 1,6-β-Galp | 104.6 | 72.0 | 73.9 | 69.9 | 75.0 | 70.6 |
| E | 1,3,6-β-Galp | 104.4 | 72.0 | 82.2 | 71.1 | 77.8 | 71.6 |
| F | T-α-Rhap | 101.9 | 73.2 | 70.1 | 74.5 | 71.4 | 17.8 |
| C′ | 1,3-β-Galp | 104.6 | 73.9 | 83.1 | 69.8 | 76.3 | 62.2 |
| D′ | 1,6-β-Galp | 104.6 | 71.9 | 73.8 | 69.8 | 74.9 | 70.6 |
The signals appearing at 110.4 and 108.7 ppm in the 13C NMR spectrum of PWH were attributed to C-1 in residues A and B, respectively. The low-field chemical shifts indicate that the arabinosyl residues were of the furanose form. For residue A, the signal at 110.4 ppm attributed to C-1 and that at 62.5 ppm belonging to the C-5 of arabinose suggest the existence of terminal T-α-Araf (Liang et al., 2014). Residue B was assigned as 1,5-α-Araf according to the low-field shifted signal for C-5 (69.7 ppm). The resonances at 104.6, 104.4, and 101.9 ppm were attributed to C-1 in residues C and D, C-1 in residue E, and C-1 in residue F, respectively. Comparison of the chemical shifts among residues C, D, and E showed that C-3 in residues C and E, and C-6 in residues D and E shifted to lower field. From these results, residues C, D, and E were determined to be 1,3-β-Galp, 1,6-β-Galp, and 1,3,6-β-Galp, respectively. For residue F, the signals at 101.9 ppm for C-1 and at 17.8 ppm were attributed to C-6 of rhamnose, proving the existence of terminal T-α-Rhap (Nagel et al., 2016). In the 13C NMR spectra of PWH-A, residues C′ and D′ were attributed to 1,3-β-Galp and 1,6-β-Galp, respectively. In the HMBC spectra, the 13C NMR resonance for C-3 for residue E was correlated with the 1H NMR signals for C-1 in residue A. It was also observed that the 13C NMR resonance for C-6 of residue E was correlated with the 1H NMR signals for C-1 of residue F (Fig. 4). From these results, it can be concluded that in residue E, Araf is linked to C-3, and Rhap is linked to C-6.
Enzymatic hydrolysis As a result of hydrolyzing PWH with endo-1,5-α-L-arabinanase and measuring the molecular mass by HPLC, the molecular mass hardly changed before and after the enzymatic hydrolysis (data not shown). From this result and the results of the monosaccharide composition analysis, 1,5-linked α- L-Araf was not present in the backbone chain. These results suggest that almost all Araf and Rhap are located at the periphery of PWH, and that the backbone chain is composed of Galp. This, combined with the results of the linkage analysis, suggests that about 55 % of 1,3-linked-β-Galp is substituted at C-6 by Rhap, and about 71 % of 1,6-linked-β-Galp-is substituted at C-3 by Araf.
AGs, which are widely distributed in the cell walls of plants, are polysaccharides with high β-D-Galp and α-L-Araf contents. Generally, AGs can be roughly classified into type I (AGs-I) and type II (AGs-II), depending on their structure. AGs-I have a backbone chain of 1,4-linked-β-Galp and a side chain where α- L- Araf is linked to C-3 of the Galp backbone chain (Luonteri et al., 2003; Voragen et al., 2009). AGs-II have a backbone chain of 1,3-linked-β-Galp and a side chain composed of 1,6-linked β- D-Galp, 1,5- and 1,3,5-linked, and terminal α- L- Araf attached to the C-6 of the Galp backbone chain (Caffall and Mohnen, 2009; Xu et al., 2010). Our results demonstrate that the PWH isolated from Hyuganatsu orange is AGs-II, which has a backbone chain of 1,3-linked β- D-Galp. Alternatively, since the PWH-A was linear with no side chains, PWH may be an AGs-II with a backbone chain of 1,3- and 1,6-linked β- D-Galp and side chains of terminal and 1,5-linked α- L-Araf, and terminal α- L- Rhap. AGs-II with a backbone chain of 1,3- and 1,6-linked β- D-Galp has been reported in AG from green tea (Wang et al., 2015), but there are few reports.
Effect on osteoclast formation The effect of PWH and PWH-A on osteoclast formation was investigated. Osteoclast progenitor cells express TRAP as they differentiate into osteoclasts. Therefore, osteoclast formation was evaluated by counting the number of fused cells among TRAP-positive cells. Rat osteoclast precursor cells were incubated in a culture medium in which PWH and PWH-A were added at 10 µg/mL and 100 µg/mL, respectively (Fig. S5). The number of fused cells was counted after TRAP staining (Fig. 5). It was found that the number of fused cells was clearly reduced in PWH, even at a concentration of 10 µg/mL compared with the control. In a similar experiment, staining dead cells with trypan blue showed no difference in the number of dead cells compared to the control (data not shown). From these results, it was demonstrated that PWH clearly inhibited the fusion of osteoclasts, even at a concentration of 10 µg/mL. For PWH-A, on the other hand, although a decrease in the number of fused cells was observed after increasing the concentration of additives, no significant difference was observed when compared to a control that did not contain polysaccharide. If not affected by the reduction in molecular mass, this result may indicate the ability of the side chains of PWH to inhibit the fusion of osteoclasts and may be one of the structures useful for functional expression.
Micrograph of a) cont., b) PWH 100 µg/mL, c) PWH 10 µg/mL, d) PWH-A 100 µg/mL and e) PWH-A 10 µg/mL at the end of culture before TRAP staining.
Effect of PWH and PWH-A on osteoclast formation.
Data are expressed as mean ± SE (n = 8). *p < 0.05 for control.
In our previous studies, PWH promoted the absorption of Ca from the rat inversion intestine, and human studies have shown improvements in bone resorption and bone formation markers involved in osteoclast differentiation. Since PWH is a polymer, it is considered that part of it is hydrolyzed and absorbed in the intestinal tract by organisms such as intestinal bacteria, which affect bone metabolism; however, PWH-A has an effect of suppressing the formation of osteoclasts. PWH may affect bone metabolism, primarily by promoting Ca absorption. Further research is needed on the digestion and absorption of PWH. In several studies where arabinogalactan was administered orally, it was shown to lower the concentration of cytokines such as IL-6, but it is unclear how it acts (Velikova et al., 2020). It is also possible that such cytokines may affect bone metabolism.
In this study, a homogeneous and neutral polysaccharide (PWH) was fractionated from a water extract of Hyuganatsu orange juice residue. PWH has a molecular mass of 68 kDa and contains a backbone chain of 1,3- linked galactopyranosyl residue. This suggests that PWH is an type II arabinogalactan (AG-II). Furthermore, the effect of PWH on osteoclast formation was investigated. The results clearly indicated that PWH inhibited the fusion of osteoclasts, even at a concentration of 10 µg/mL. In this study, the functional components in Hyuganatsu orange that inhibit osteoclast formation were identified. These research results are an important step toward the elucidation of the structure responsible for the functional expression and its mechanism of action.
Conflict of interest There are no conflicts of interest to declare.
