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Preparation of Lentinula edodes polysaccharidecalcium complex and its immunoactivity a

a

a

Yujiao Cui , Huidan Yan & Xuewu Zhang a

College of Light Industry and Food Sciences, South China University of Technology, Guangzhou, China Published online: 27 May 2015.

Click for updates To cite this article: Yujiao Cui, Huidan Yan & Xuewu Zhang (2015): Preparation of Lentinula edodes polysaccharide-calcium complex and its immunoactivity, Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2015.1044930 To link to this article: http://dx.doi.org/10.1080/09168451.2015.1044930

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Bioscience, Biotechnology, and Biochemistry, 2015

Preparation of Lentinula edodes polysaccharide-calcium complex and its immunoactivity Yujiao Cui, Huidan Yan and Xuewu Zhang* College of Light Industry and Food Sciences, South China University of Technology, Guangzhou, China Received November 20, 2014; accepted April 10, 2015

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http://dx.doi.org/10.1080/09168451.2015.1044930

Polysaccharide is a major bioactive component of mushrooms. In this study, for the first time, starting from a new Lentinula edodes polysaccharide L2, we prepared a novel L2–calcium complex and the process was optimized. Scanning electron microscopy and Fourier Transform infrared spectrometry were used for characterization. The immunostimulating activities of L2 and L2–calcium complex were measured by enhancing the production of two cytokines TNF-α and IL-6 in RAW264.7 cells. While L2–calcium complex significantly stimulates the secretions of TNF-α and IL-6 compared with the control, complex with calcium ion decreased the secretion of them. These facts indicate that calcium ion can modulate immune stimulating activity of Lentinula edodes polysaccharide L2. Key words:

Lentinula edodes; polysaccharide– calcium complex; preparation; calcium supplement; immuno-stimulator

Mushroom is regarded as a health food that is low in fat, but high in proteins and dietary fiber (non-starch polysaccharides). Polysaccharide is the major component of mushrooms. The biological activities of these polysaccharides have attracted more attention recently in the medical fields due to their immunomodulatory and antitumor effects.1) For example, lentinan, a cell wall beta-glucan from the fruiting bodies of Lentinula edodes, exhibits evident antitumor and immunostimulating activities by stimulating natural killer cells, T-cells, B-cells, neutrophils, and macrophage-dependent immune system responses.2,3) In previous study,4) we isolated a new heteropolysaccharide (L2) from the fruit body of Lentinula edodes, which consists of glucose (87.5%), galactose (9.6%), and arabinose (2.8%) and has an average molecular weight of 26 kDa. Most importantly, L2 does not possess a triple-helical conformation but also exhibits immunostimulating activities involving the toll-like receptor-2 in RAW264.7 cells. Calcium is an essential mineral for the human body. However, many individuals do not obtain the optimum *Corresponding author. Email: [email protected] © 2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry

amount of calcium from diets and depend on bioavailable calcium supplements. Now, various calcium supplements have been developed; organic calcium (e.g. calcium citrate, calcium lactate, and calcium gluconate) was considered to increase calcium absorption efficiency,5) compared with inorganic calcium carbonate. Calcium supplementation has been found to be beneficial for bone health in children, young adults, and menopausal women.6) The evidence from randomized clinical trials also suggested that calcium supplementation generated significant weight loss in overweight and obese individuals.7) In this study, a new organic calcium was created. Specifically, starting from the novel Lentinula edodes polysaccharide L2, we prepared a L2–calcium complex, which could be a new food additive both as an organic calcium supplement and also as an immunostimulator.

Materials and methods Extraction of Lentinula edodes polysaccharide L2. The extraction and purification of L2 was performed as described before.4) Briefly, the fruit body of Lentinula edodes was extracted with boiling water at a ratio 20:1 (w/w) for 2 h, and the supernatants were concentrated under 80 °C, separated on a centrifuge, and the precipitates were dried under vacuum. After deproteination, the solution was centrifuged and redissolved in distilled water, and the resulting solution was then freeze-dried at −40 °C to obtain crude polysaccharides. Subsequently, a DEAE-52 cellulose anion exchange column was applied to the crude polysaccharides and eluted with distilled water, 0.05 mol/L NaCl solution, 0.1 mol/L NaCl solution, and 0.2 mol/L NaCl solution, respectively. Four fractions, CL1, CL2, CL3, and CL4, were obtained by freeze-drying. Then, a Sephadex G-200 column was applied to the second fraction CL2 and eluted with 0.05 mol/L NaCl solution at a ratio of 1.5 mL/10 min. After gel filtration, dialysis, and freeze-drying, the new polysaccharide L2 was obtained. Preparation of Lentinula edodes polysaccharide L2– calcium complex. Lentinula edodes polysaccharide

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L2 (100 mg) was dissolved in deionized water (5 mL). Calcium chloride solution was added to the prepared polysaccharide solution with the ratio 4.5–5.5 (w/w), stirring under temperature 55–65 °C, and pH 8.5–9.5. A dialysis of the polysaccharide–calcium complex solution was performed for 2 days with a dialysis bag of 3,500 Da. The filtrate was freeze-dried to obtain polysaccharide–calcium complex.

Fig. 1. to L2.

Orthogonal experiment for the process optimization. Based on single-factor experiments, the effects of temperature (°C), pH value, time (h), and the ratio of calcium chloride to polysaccharide L2 on the formation of L2–calcium complex were determined. Then, the orthogonal design for the four variables was applied to optimize the preparation process of L2–calcium complex.

Effects of environmental factors on the formation of L2–calcium complex. (A) Time, (B) temperature, (C) pH, and (D) the ratio of CaCl2

Table 1.

Orthogonal experiments.

Factors

Level

A:Temperature (°) B:pH C:Time (min) D:Ratio of CaCl2 to L2

55 8.5 45 4.5

60 9.0 60 5.0

65 9.5 75 5.5

No.

A

B

C

D

Calcium content (mg/g)

1 2 3 4 5 6 7 8 9 K1 K2 K3 R

1 1 1 2 2 2 3 3 3 5.493 5.664 5.692 0.199

1 2 3 1 2 3 1 2 3 5.520 5.972 5.358 0.614

1 2 3 2 3 1 3 1 2 5.345 5.827 5.678 0.482

1 2 3 3 1 2 2 3 1 5.525 5.660 5.664 0.139

5.034 6.102 5.344 5.825 5.989 5.178 5.700 5.824 5.553 —— —— —— ——

Factor A B C D Error

Sum of square 0.070 0.364 0.607 0.037 1.08

Freedom 2 2 2 2 8

Mean of square 0.035 0.182 0.303 0.019 0.135

F 1.1596 6.025 19.044 0.611

Significance

*p < 0.05.

*

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Polysaccharide–calcium complex

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The calcium content of L2–calcium complex was analyzed as below: 50 mg of the complex was dissolved in 10 mL of acids solution (nitric acid: hydrochloric acid = 1:1). After 24 h, the solution was heated to be transparent, moved to another flask, and diluted by distilled water to the final 25 mL. Based on internal standards, the calcium content was determined by atomic emission spectrometry (Hitachi Z-2000, Japan). Each determination was performed in triplicate.

the orthogonal table L9(4 ). The results (Table 1) demonstrated that the effects of temperature (°C), pH value, time (h), and the ratio of CaCl2:L2 on the formation of L2–calcium complex could be listed as: pH > time > temperature > the ratio of CaCl2:L2. The optimal conditions were determined as: 65 °C, pH 9.0, 60 min, and the ratio of CaCl2:L2 = 5.5:1. The variance analysis indicated that the reaction time exhibited significant influence on the formation of L2–calcium complex, but other factors did not.

Characterization of L2–calcium complex. The morphology of L2–calcium complex was visualized by Phenom scanning electron microscopy (SEM) (Phenom G2 Pure, Phenom-World). The Fourier Transform infrared (FT-IR) spectra were recorded on 500–4,000 cm−1 by a Nexus 670 FT-IR spectrophotometer (Thermo Nicolet Co., USA).

Characterization of L2–calcium complex and its immunostimulating activity. The surface structures of L2 and L2–calcium complex were examined by SEM. Fig. 2 showed that L2–calcium complex displayed

Immunostimulating activity of L2–calcium complex. RAW264.7 cells were purchased from Medical College of Sun Yat-Sen University culturing with RPMI 1640 plus the 100 U/mL of penicillin, 100 μg/mL of streptomycin, and 10% fetal bovine serum. The logarithmic growth phase cells were adjusted to a concentration of 1 × 106 cells/mL. The cell solutions (100 μL) and sterilized PBS (100 μL) were added into each well. Cells were cultured at 37 °C in a 5% CO2 humidified atmosphere cultivator for 24 h. Then, the culture medium was refreshed, and cells were incubated with 100 μl of L2 or L2–calcium samples (250 μg/mL). For control, cells were incubated with 100 μl of lipopolysaccharide (50 μg/mL). The supernatants were collected after 24 h incubation. TNF-α and IL-6 were determined by Mouse IL-6 ELISA Kit (Neobiosience Technology Co., LTD) and Mouse TNF-α ELISA Kit (Neobiosience Technology Co., LTD) according to the instruction. Each determination was performed in triplicate. Statistical analysis. Data are presented as means ± SD and were analyzed for statistical differences. Student’s t-tests were used for all statistical analysis between different groups. The P-values less than 0.05 were considered to be significant.

Results and discussion Optimal preparation of L2–calcium complex. The results for single-factor experiments indicated that the calcium content in L2–calcium complex increased with reaction time (Fig. 1(a)), temperature (Fig. 1(b)), or pH (Fig. 1(c)) in the beginning; after 1 h, 60 °C, or pH = 9, the calcium content decreased slowly. While for the mass ratio of calcium chloride to polysaccharide L2 (Fig. 1(c)), the calcium content in L2–calcium complex increased with the ratio of CaCl2:L2, after the ratio = 5, basically arrived at equilibrium. Subsequently, four factors including temperature (°C), pH value, time (h), and the ratio of CaCl2:L2 were selected for orthogonal experiments according to

Fig. 2. The surface structures of L2 (A) and L2–calcium complex (B) by SEM.

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larger flakes than L2 did, although both exhibited smooth flake patterns. This could be explained by the fact that calcium bridges polysaccharides L2 molecules and increases their interactions; thus, more L2 molecules can assembly together, leading to the formation of larger flakes. Because the binding of calcium to polysaccharide is a complex process, the binding of calcium to alginates follows a three-step process.8) However, the structure of L2 is different from alginate, so the detailed binding mechanism of calcium to L2 requires further study. The structural changes of L2 and L2–calcium complex by FT-IR spectra are displayed in Fig. 3. Compared with L2, the peaks in the spectrum of L2– calcium complex become weak, although the backbones of them are similar. The characteristic peaks at 3,381 and 2,918 cm−1 in the spectra indicate the existence of O–H and C–H groups, respectively. Specially, the peak 3,381 cm−1 moves to 3,406 cm−1, suggesting the association of O–H bond. Moreover, the peaks at 1,414 cm−1 (C–H vibration) and 1,045 cm−1 (C–O vibration) are significantly weakened, and the peak 1,414 cm−1 is shifted to 1,421 cm−1, suggesting that the complexation of L2 and calcium could be primarily based on C–O bonds.

Fig. 3.

FT-IR spectra of L2 (A) and L2–calcium complex (B).

The immunostimulating activities of L2 and L2– calcium complex were measured by enhancing the production of two cytokines TNF-α and IL-6 in RAW264.7 cells. It can be seen from Fig. 4 that different concentrations of L2–calcium complex can significantly stimulate the secretions of TNF-α and IL-6, compared with the control. But, L2–calcium complex basically showed reduced responses compared to nonCa-binding polysaccharide, meaning that Ca did not increase the immune response of polysaccharide L2. Calcium ions play a pivotal role in the physiology and biochemistry of organisms and the cell, such as second messenger in signal transduction pathways, neurotransmitter in neurons, contraction of all muscle cell types/ fertilization, and enzymes cofactor.9) However, no report about immunomodulation of free calcium ions is available in literature indeed. The reduced immune response of L2–calcium complex could be due to the fact that calcium partly occupied the stimulating sites of IL-6 and TNF-α after its complexation with L2, hence influencing the production of IL-6 and TNF-α. Since TLR2 was found to be one of L2’s acting receptors,4) it is possible that TLR2 is also one receptor of L2–calcium complex; of course, further study is required to confirm this.

Polysaccharide–calcium complex

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Author contribution YC collected references and wrote the manuscript, HY performed the experiments, and XZ designed the experiment and revised the manuscript.

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References

Fig. 4. Production of two cytokines TNF-α (A) and IL-6 (B) in RAW264.7 cells stimulated by L2 and L2–calcium complex.

Conclusion For the first time, based on a new Lentinula edodes polysaccharide L2, we prepared a L2–calcium complex, which provides foundation for future research and development as an organic calcium supplement and also as an immunostimulator.

Conflict of interest All authors have no conflict of interest to declare.

[1] Guo CX, Choi MW, Cheung PCK. Mushroom and immunity. Curr. Top. in Nutraceut. Res. 2012;10:31–41. [2] Bisen PS, Baghel RK, Sanodiya BS, Thakur GS, Prasad GBKS. Lentinula edodes: a macrofungus with pharmacological activities. Curr. Med. Chem. 2010;17:2419–2430. [3] Zhang YY, Li S, Wang XH, Zhang LN, Cheung PCK. Advances in lentinan: isolation, structure, chain conformation and bioactivities. Food Hydrocolloids. 2011;25:196–206. [4] Xu XF, Yan HD, Zhang XW. Structure and immuno-stimulating activities of a new heteropolysaccharide from lentinula edodes. J. Agric. Food. Chem.. 2012;60:11560–11566. [5] Adluri RS, Zhan LJ, Bagchi M, Maulik N, Maulik G. Comparative effects of a novel plant-based calcium supplement with two common calcium salts on proliferation and mineralization in human osteoblast cells. Mol. Cell. Biochem. 2010;340: 73–80. [6] Straub DA. Calcium supplementation in clinical practice: a review of forms, doses, and indications. Nutr. Clin. Pract. 2007;22: 286–296. [7] Onakpoya IJ, Perry R, Zhang JH, Ernst E. Efficacy of calcium supplementation for management of overweight and obesity: systematic review of randomized clinical trials. Nutr. Rev. 2011;69:335–343. [8] Fang Y, Al-Assaf S, Phillips GO, Nishinari K, Funami T, et al. Multiple steps and critical behaviors of the binding of calcium to alginate. J. Phys. Chem. B. 2007;111:2456–2462. [9] Civitelli R, Ziambaras K. Calcium and phosphate homeostasis: concerted interplay of new regulators. J. Endocrinol. Invest. 2011;34:3–7.

Preparation of Lentinula edodes polysaccharide-calcium complex and its immunoactivity.

Polysaccharide is a major bioactive component of mushrooms. In this study, for the first time, starting from a new Lentinula edodes polysaccharide L2,...
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