Characterisation of the chemical composition and in vitro anti-inflammation assessment of a novel lotus (Nelumbo nucifera Gaertn) plumule polysaccharide

Food Chemistry 125 (2011) 930–935

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Characterisation of the chemical composition and in vitro anti-inflammation assessment of a novel lotus (Nelumbo nucifera Gaertn) plumule polysaccharide Chun-Huei Liao, Su-Jen Guo, Jin-Yuarn Lin ⇑ Department of Food Science and Biotechnology, National Chung Hsing University, 250 Kuokuang Road, Taichung 40227, Taiwan, ROC

a r t i c l e

i n f o

Article history: Received 19 May 2010 Received in revised form 14 September 2010 Accepted 19 September 2010

Keywords: Anti-inflammatory activity Non-obese diabetes (NOD) mice Lotus plumule polysaccharide (LPPS) Type 1 diabetes

a b s t r a c t Lotus plumule has been recognised as an anti-inflammatory agent. A novel lotus plumule polysaccharide (LPPS) was prepared, characterised and cultured with splenocytes from non-obese diabetes (NOD) and BALB/c mice (as a control) to further evaluate its anti-inflammatory effects in this study. The results showed the molecular weights of LPPS respectively distributed at 391 kDa (89.8 ± 0.5%) and 5 kDa (10.1 ± 1.5%) using HPLC analysis. The neutral sugar composition in LPPS comprised xylose (33.4 ± 10.0%), glucose (25.7 ± 1.6%), fructose (22.0 ± 15.0%), galactose (10.5 ± 3.3%), and fucose (8.1 ± 3.5%). LPPS administrations at appropriate concentrations significantly (P < 0.05) increased interleukin (IL)-10, IL-6, tumour necrosis factor (TNF)-a secretions by splenocytes from both NOD and BALB/c mice at ages of 15, 22 and 26 weeks, respectively. However, LPPS administrations markedly (P < 0.05) increased secretion ratios of anti-/pro-inflammatory (IL-10/IL-6) cytokines by splenocytes. The present study suggests that LPPS may have potential to treat type 1 diabetes via its potent anti-inflammatory activity. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Helper T lymphocytes can be broadly divided into type 1 helper T lymphocytes (Th1) and Th2 cells on the basis of the array of secreted cytokines (Anderson & Bluestone, 2005). Th1 cells produce cytokines such as interferon (IFN)-c and tumour necrosis factor (TNF)-a and promote cell-mediated immune responses. In contrast, Th2 cells produce cytokines such as interleukin (IL)-4, IL-5, and IL-10 and promote selected humoral immune responses. Both Th1 and Th2 immune responses reciprocally regulate each other through their respective cytokines to keep balance in body. However, an imbalance between Th1 and Th2 subgroups in vivo may cause differential diseases. It is found that type 1 diabetes (T1D) is an organ-specific and chronic autoimmune disease that is related to an imbalance between Th1 and Th2 subgroups in vivo (Chen, Li, & Yu, 2008). Th1-skewed immune responses in T1D patients result in chronic inflammation and specifically destroy pancreatic islet b cells (Forestier et al., 2007). Undoubtedly, Th1-skewed immune responses are associated with the disease progression in non-obese diabetes (NOD) mice (Delovitch & Singh, 1997). It is found that treatment of NOD mice with cytokines or reagents that block Th1 immune responses decreases an incidence of diabetes; on the other hand, treatment of NOD mice with cytokines or reagents that enhance Th1 immune responses increases an incidence of diabetes (Anderson & Bluestone, 2005). An appropriate ⇑ Corresponding author. Tel./fax: +886 4 22851857. E-mail address: [email protected] (J.-Y. Lin). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.09.082

regulation of Th1-skewed immune responses to Th2-inclination in patients may suppress the development of T1D at early stage, although this is still an oversimplification of the existing data. This study attempts to investigate a potential anti-T1D agent, lotus plumule polysaccharide (LPPS). Lotus seeds have been widely consumed by people in Asia. Lotus plumule is a germ of lotus seed with bitter taste and generally removed before lotus seeds are eaten. Lotus plumule has been recognised as an anti-inflammatory agent in traditional Chinese medicine. In our previous studies, we have found that supplementation with lotus (Nelumbo nucifera Gaertn) plumule significantly increased the level of anti-inflammatory cytokine IL-10, but inhibited the production of pro-inflammatory cytokine TNF-a produced by peritoneal macrophages from lipopolysaccharide (LPS)-challenged mice (Lin, Wu, Liu, & Lai, 2006). Furthermore, a 3-week supplementation of lotus plumule before LPS challenge alleviated acute systemic inflammation in vivo via decreasing the visceral organ inflammation and increasing the production of anti-inflammatory cytokine IL-10 from splenocytes (Lin, Lai, Liu, & Wu, 2007). It is suggested that lotus plumule has potent anti-inflammatory activities. We further hypothesised that lotus plumule may serve as an anti-T1D agent for its potent anti-inflammatory activity. We have found that polysaccharides exist in the hot water extract of lotus plumule. Recently, polysaccharides are recognised as biological response modifiers (BRMs) via interactions with Toll-like receptors (TLR) and scavenger receptors (SRs) of immune cells to modulate subsequent cell proliferation, cytokine expression and complement activation(Leung, Liu, Koon, & Fung, 2006). However,

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characterisation of LPPS and its effect on Th1-skewed inflammation has not been investigated yet. In recent years polysaccharides from different sources have been reported to exhibit a variety of biological activities, such as antioxidant (Yang et al., 2009), and immunostimulatory (Guo et al., 2009; Niu, Liu, Zhao, Su, & Cui, 2009). Crude polysaccharide aqueous extracts from Phaeodactylum tricornutum extract exhibit anti-inflammatory and immunostimulatory effects (Guzman, Gato, Lamela, Freire-Garabal, & Calleja, 2003). A polysaccharide isolated from the mesocarp of Orbignya phalerata fruits enhances phagocytosis and exhibits anti-inflammatory activity in vivo (da Silva & Parente, 2001). We suggested that LPPS might contribute to antiinflammatory effects against systemic or local inflammation. However, characterisation of polysaccharides from different plants is still limited. NOD mice with pathogenic Th1-skewed autoimmune diseases first show severe insulitis by 10 weeks of age (Graham et al., 2008). Diabetes onset typically occurs at ages of 12–14 weeks in female NOD mice and slightly later in male mice (Anderson & Bluestone, 2005). The incidence of diabetes typically reaches 60–80% in NOD female mice and 10–20% in NOD male mice by 30 weeks of age (Graham et al., 2008). Undoubtedly, the immune cells in NOD mice are suggested to be dramatically changed and bear differential inflammation status during the process of developing T1D (Anderson & Bluestone, 2005). The spleen, the largest lymphoid organ in body collecting antigens from the blood, mostly comprises B and T lymphocytes, possibly reflecting the systemic inflammation status. Therefore, splenocytes from NOD mice at different ages after diabetes onset (>14 weeks) were selected for bio-assay of LPPS in vitro. To characterise LPPS and its anti-inflammatory effects on T1D, LPPS were isolated, characterised and incubated with primary splenocytes from female NOD and BALB/c mice (as a control) at ages of 15, 22 and 26 weeks in this study. The present study suggests that LPPS having 391 kDa molecular weight and rich in xylose, glucose, and fructose, exhibited potent potential to treat Th1-skewed autoimmune disease, such as T1D, via its potent anti-inflammatory activity. 2. Materials and methods 2.1. Sample preparation of lotus plumule polysaccharides (LPPS) The lotus, Nelumbo nucifera Gaertn which is generally cultivated in Taiwan, was selected to conduct in this study. Briefly, fresh lotus seeds were provided by a farmer in Tainan, Taiwan. The lotus plumule was carefully collected and then air dried at 40 °C overnight. The air-dried lotus plumule was milled into powder, and then placed into plastic bottles. To isolate polysaccharides, the lotus plumule powder was added with five volumes of deionised water, thoroughly mix and heated at 100 °C water bath for 4 h to obtain hot water extracts. The hot water extracts were centrifuged at room temperature, 5000  g for 30 min. The supernatant was collected and added with three volumes of 95% ethyl alcohol at 4 °C for 12 h to precipitate polysaccharides. The mixture was centrifuged at room temperature, 5000  g for 30 min to isolate insoluble polysaccharides from the supernatant. The insoluble pellet (lotus plumule polysaccharide, LPPS) was obtained, lyophilised and stored at 30 °C until use.

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against a deionised water blank using bovine serum albumin (BSA) as a standard. Total sugar content was estimated using phenol–sulphuric acid reaction with sugars (Dubois, Gilles, Hamilton, Rebers, & Smith, 1951). The reaction mixture absorbance was measured at 488 nm against a deionised water blank using glucose as a standard. Data were expressed as gram (g) glucose equivalents (GAE)/100 g lyophilised powder. Phenolics content was determined using Folin–Ciocalteau method (Lin, Li, & Hwang, 2008). The reaction mixture absorbance was measured at 750 nm against a deionised water blank using gallic acid as a standard. Data were expressed as gram (g) gallic acid equivalents (GAE)/100 g lyophilised powder. The water content of LPPS was determined using standard gravimetric comparisons by heating at 135 °C for 2 h. All of the approximate chemical composition in LPPS was determined in triplicate, respectively. 2.1.2. Molecular weight of LPPS The molecular weight of LPPS was assayed using a high performance liquid chromatography (HPLC) method. The LPPS powder was re-dissolved in deionised water. The LPPS solution was filtered through a 0.45 lm filter and ultrasonically degassed before use. For chromatographic separation, a PolySep-GFC-P 4000 column (300  7.8 mm, Phenomenex Inc., CA, USA) that was connected to precolumn of the same type was applied. The column oven operated at 25 °C. The mobile phase (deionised water) was filtered through a 0.45 lm filter under vacuum and ultrasonically degassed before use. The refractive index (RI) detector (model 410) and pump (model 600E) were used. The flow rate of the mobile phase was 0.8 ml/min. Samples were injected using an autosampler (model 717). Aliquots of 20 ll sample solution were subjected to the HPLC analysis. The dextran standards (Sigma, Missouri, USA), 5 kDa, 50 kDa, 80 kDa, 150 kDa, and 410 kDa, were used. Using a five point standard curve (5–410 kDa versus retention time, respectively), the molecular weight of LPPS was determined in triplicate, respectively. 2.1.3. Neutral sugar composition in LPPS To analyse the neutral sugar composition in LPPS, the LPPS was first fully digested to be simple sugars. Briefly, an aliquot of 3 g of LPPS was added with 5 ml of 2 M trifluoracetic acid (Sigma, Missouri, USA). The resultant mixture was heated at 100 °C water bath for 16 h. The digested solution was evaporated under vacuum to dry. An aliquot of 10 ml deionised water was added to re-dissolve the mixture. The mixture was adjusted to pH 7.0 using 1 N NaOH (Wako, Osaka, Japan). The volume of the mixture was adjusted to a fixed amount. The sample solution was filtered through a 0.45 lm filter and ultrasonically degassed before use. The neutral sugar composition in LPPS was determined using HPLC method. The mobile phase (acetonitrile:water = 88%, v/v) was filtered through a 0.45 lm filter under vacuum and ultrasonically degassed before use. The flow rate of the mobile phase was 1 ml/min. Samples were injected using an autosampler (model 717). Aliquots of 10 ll sample solution were subjected to the HPLC analysis. The refractive index (RI) detector (Shimadzu RID-10 A) and chromatographic separation column (Phenomenex Luna 5 NH2 100 A; 250  4.6 mm, 5 lm, Phenomenex Inc., CA, USA) were used. Six sugar standards (Sigma, Missouri, USA), including fructose, fucose, galactose, glucose, mannose, and xylose, were chosen (Hardy, Townsend, & Lee, 1988). 2.2. Experimental animals

2.1.1. Approximate chemical composition of LPPS LPPS was taken for analyses of total protein, total sugar, phenolics and water. In brief, total protein concentration was determined by the method of Bicinchoninic acid (BCA) solution (Smith et al., 1985). The reaction mixture absorbance was measured at 550 nm

Female NOD/LtJ (non-obese diabetes (NOD) mice) and BALB/ cByJNarl (normal mice, as a control) mice at different ages were purchased from the Laboratory Animal Center at Medicine College of National Taiwan University and the National Laboratory Animal

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Center, National Applied Research Laboratories, National Science Council in Taipei, Taiwan, ROC, respectively. The experimental mice were maintained in the Department of Food Science and Biotechnology at National Chung Hsing University, College of Agriculture and Natural Resources in Taichung, Taiwan, ROC. The mice were provided chow diet (laboratory standard diet) and water ad libitum. All mice were housed in stainless steel cages and kept at a controlled temperature (25 ± 2 °C) and ambient humidity (50– 75%). Lights were maintained on a 12 h dark-light cycle. Mouse body weight was measured twice a week during the study period. The NOD and BALB/c mice at different ages respectively sacrificed for splenocytes isolation. The animal use protocol listed in this study was reviewed and approved by the Institutional Animal care and Use Committee (IACUC), National Chung Hsing University, Taiwan, ROC. 2.3. Preparation of primary splenocyte cultures Single splenocytes were isolated from experimental mice as described previously (Lin et al., 2007). Briefly, the splenocytes were prepared by aseptically removing spleens from NOD and BALB/c mice at different ages, respectively. Viable cell numbers were assessed by a trypan blue exclusion method using a hemocytometer. Isolated splenocytes from each animal were adjusted to 1  107 cells/ml TCM medium for use. 2.3.1. Possible cytotoxic effects of LPPS administration on primary splenocytes To evaluate possible cytotoxicity of LPPS, effects of LPPS administered at different concentrations on splenocytes viability were determined. Each cell population (5  106 cells/ml) from BALB/c mice at age of 10 weeks was respectively treated with LPPS at the indicated concentrations of 0, 39, 78, 156, 312, 625, 1250, and 2500 lg/ml, as well as lipopolysaccharide (LPS, as a positive control, Sigma–Aldrich Co., Missouri, USA) at 2.5 lg/ml in 96 well microtiter plates. The plates were incubated at 37 °C in a humidified incubator with 5% CO2 and 95% air for 72 h. Viability of splenocytes was determined using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma–Aldrich Co., Missouri, USA). The absorbance was measured at 550 nm on a plate reader (ELISA reader, ASYS Hitech, GmbH, Austria). The cell viability was directly expressed as the absorbance average (Lin et al., 2007). 2.3.2. Effects of addition of Polymyxin B (PMB) on LPPS- or LPSstimulated splenocyte cultures Polymyxin B (PMB) neutralises endotoxin LPS effects by high affinity to lipid A of LPS. To confirm whether LPPS was contaminated with LPS, PMB was respectively added to LPPS- or LPS-stimulated splenocyte cultures. Briefly, PMB (Sigma, Denmark) was aseptically added with LPPS or LPS (a B-cell mitogen, Sigma–Aldrich Co., Missouri, USA), mixed thoroughly, and incubated at room temperature for 2 h (Morrison & Jacobs, 1976). The resultant mixtures were finally added to the splenocyte cultures (5  106 cells/ ml) from BALB/c mice at age of 10 weeks and incubated at 37 °C in a humidified incubator with 5% CO2 and 95% air for another 72 h. LPPS concentrations in the cultures were 0, and 1250 lg/ ml; LPS concentrations in the cultures were 0, 2.5, and 10 lg/ml; PMB concentrations in the cultures were 0, 3, and 6 U/ml. A combinatorial study was designed to clarify the effects of PMB on LPPS- or LPS-induced proliferation. Viability of splenocytes was determined using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma–Aldrich Co., Missouri, USA). The absorbance was measured at 550 nm on a plate reader (ELISA reader, ASYS Hitech, GmbH, Austria). The cell viability was directly expressed as the absorbance average (Lin et al., 2007).

2.3.3. Effects of LPPS administration on cytokines secretion by primary splenocytes from female NOD/LtJ and BALB/c mice at different ages Splenocytes (5  106 cells/ml) from female NOD/LtJ and BALB/c mice at different ages of 15, 22 and 26 weeks, respectively, with LPPS at 0, 78, 312, and 1250 lg/ml, or LPS at 2.5 lg/ml (as a positive control) were, respectively, cultured in plates. The plates were incubated at 37 °C in a humidified incubator with 5% CO2 and 95% air for 48 h. The cultured plate was centrifuged at 200  g for 10 min. The supernatants in the cell cultures were respectively collected and stored at 80 °C for cytokine assays. 2.4. Measurement of cytokines level secreted by primary splenocyte cultures by an enzyme-linked immunosorbent assay (ELISA) The measurement of cytokines level secreted by primary splenocyte cultures was manipulated as described previously (Lin et al., 2007). Interleukin (IL)-1b, IL-2, IL-4, IL-5, IL-6, IL-10 and tumour necrosis factor (TNF)-a levels in splenocytes culture were determined using sandwich ELISA kits (mouse DuoSet ELISA Development system, R&D Systems, Minnesota, USA). The sensitivity of the ELISA kits used in this study was about <15.6 pg/ml. 2.5. Statistical analysis Values are expressed as means ± SD. Data are analysed statistically using one-way ANOVA followed, if justified by the statistical probability (P < 0.05), by Duncan’s multiple range test. 3. Results and Discussion Lotus plumule has been widely accepted to be a medicinal food daily for anti-inflammation in the eastern Asian countries. It is generally used for making tea. However, bioactive compounds in the hot water extracts of lotus plumule are still unclear. A novel polysaccharide was first isolated from the lotus plumule hot water extracts. The present study has carried out characterising the lotus plumule polysaccharide (LPPS) and assessing its anti-inflammatory effects. 3.1. Approximate chemical compositions and characteristics of lotus plumule polysaccharide (LPPS) The results showed that the isolated LPPS consisted of protein, carbohydrate, and phenolics. Total protein, total sugar (glucose equivalent), phenolics (gallic acid equivalent), and water content in LPPS were 38.3 ± 2.7%, 42.1 ± 4.3%, 3.1 ± 0.4% and 3.0 ± 0.1% (w/w), respectively (Table 1). The LPPS was prepared by alcohol precipitation from lotus plumule hot water extracts. The yield of Table 1 The composition of lotus plumule polysaccharide (LPPS).

a

Chemical compositiona

LPPSb (%, w/w)

Total protein Total sugar (glucose equivalent) Phenolics (gallic acid equivalent) Water content

38.3 ± 2.7 42.1 ± 4.3 3.1 ± 0.4 3.0 ± 0.1

Neutral sugara

Sugar composition in LPPSb (%)

Glucose Galactose Mannose Fructose Xylose Fucose

25.7 ± 1.6 10.5 ± 3.3 –c 22.0 ± 15.0 33.4 ± 10.0 8.1 ± 3.5

Values are means ± SD (n = 3). The LPPS samples used in this study were lyophilised powder and prepared by alcohol precipitation from lotus plumule hot water extracts. c ‘‘–”: not detectable. b

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LPPS from lotus plumule hot water extract was 3.86 ± 1.47% (w/w) (data not shown). According to the approximate chemical composition in LPPS, we suggest that LPPS may be still a crude polysaccharide. In recent, a crude polysaccharide from Turbinaria ornata (Marine Brown Alga) has reported to exhibit both in vitro antioxidant and in vivo anti-inflammatory potential (Ananthi et al., 2010). In this study we first uncover the novel polysaccharide of lotus plumule (LPPS). However, more characteristics of LPPS remain to be further clarified. Although the LPPS may still contain trace impurity, neutral sugar compositions in LPPS was composed of xylose (33.4 ± 10.0%), glucose (25.7 ± 1.6%), fructose (22.0 ± 15.0%), galactose (10.5 ± 3.3%), fucose (8.1 ± 3.5%) and mannose (trace) (Table 1). To further characterise LPPS, the LPPS was subjected to HPLC analysis. Two identified polysaccharides in LPPS were found (Fig. 1). The molecular weights of LPPS were 391 kDa (89.8 ± 0.5%, major component) and 5 kDa (10.1 ± 1.5%, minor component), respectively (Fig. 1). Polysaccharides seem to be one of active ingredients in Chinese herbal medicine. This study first introduced and characterised a novel polysaccharide, lotus plumule polysaccharide (LPPS). It is found that molecular weights of active polysaccharides with b(1-3) and b-(1-6) linkages distributed from 100 to 2000 kDa (Kidd, 2000). The higher molecular weight of b-glucan polysaccharides, the higher of biological function (Kim et al., 2005). Our study suggests that LPPS is an active polysaccharide with 391 kDa molecular weight, which is rich in xylose, glucose and fructose (Table 1 and Fig. 1). Based on the profile of neutral sugar compositions in LPPS, we suggest that LPPS may be not a b-glucan polysaccharide for the highest amount of xylose. It has been suggested that polysaccharides may exert biological response modifiers (BRMs) via the pathway of Toll-like receptors (TLR) or scavenger receptors (SRs) on immune cells to modulate subsequent cell proliferation, cytokine expression and complement activation (Leung et al., 2006). We attempt to correlate neutral sugar compositions in polysaccharides with biological functions, however they are still scarcely discussed. To further unravel the biological function of LPPS, LPPS has been conducted to treat T1D in NOD mice in vivo.

2500 lg/ml significantly (P < 0.05) inhibited splenocytes viability (Fig. 2). To avoid the cytotoxicity of high concentration LPPS, LPPS administration concentrations at 78, 312 and 1250 lg/ml were selected to further conduct cytokines secretion assay. 3.3. Effects of PMB treatments on LPPS- and LPS-induced splenocytes proliferation To determine whether LPPS was contaminated with LPS, this study examined the effects of PMB treatments on LPPS- and LPSinduced proliferation of splenocytes from BALB/c mice. Table 2 shows the addition effects of PMB at the indicated concentrations of 0, 3, and 6 U/ml on LPPS (0 or 1250 lg/ml)- or LPS (2.5 or 10 lg/ml)-induced splenocytes proliferation. At 3 U/ml PMB addition, LPPS-induced proliferation did not significantly change (P > 0.05), however LPS-induced proliferation significantly (P < 0.05) decreased. PMB is a specific inhibitor of LPS. Obviously, our results suggest that LPS did not contaminate LPPS. Polysaccharides, but not lipopolysaccharides, in LPPS were functional components. Interestingly, at 6 U/ml PMB addition, PMB treatment significantly increased spontaneous (TCM medium only) and LPPS-stimulated splenocytes proliferation. LPS-stimulated (2.5 or 10 lg/ml) splenocytes proliferation were also significantly increased by 6 U/ml PMB addition as compared to those of 3 U/ml PMB addition. Our results further suggest that high concentration (6 U/ml) of PMB addition, itself, significantly increased splenocytes proliferation. PMB has been proved a mitogenic activity on B-cells (Smith & Hammarstrom, 1978). This study further confirmed that high concentration of PMB (6 U/ml) caused splenocytes proliferation, possibly through stimulating B-cells in splenocytes. Now, PMB serves as an antibiotic to treat bacterial and fungal infections (Zhai et al., 2010). It neutralises LPS via its high affinity between PMB and lipid A of LPS (Guo et al., 2007). This study selected PMB to inhibit LPS activity and proved that LPS did not contaminate LPPS. 3.4. Effects of LPPS administrations on Th1 and Th2 cytokines secretion by splenocytes from NOD and BALB/c mice at different ages

3.2. Effects of LPPS administrations on splenocytes viability To evaluate effects of LPPS administrations on Th1 and Th2 cytokines secretion, primary splenocytes from NOD and BALB/c mice at viability of splenocytes detected by MTT method (OD550 )

To evaluate possible cytotoxicity of LPPS administrations, splenocytes from BALB/c mice were cultured with LPPS at the indicated concentrations of 0, 20, 39, 78, 156, 312, 625, 1250, and 2500 lg/ml, respectively, for 72 h. The results showed that LPPS administrations at the indicated concentrations of 20, 39, 78, 156, 312, 625, and 1250 lg/ml did not significantly (P > 0.05) affect splenocytes viability (Fig. 2). However, LPPS administration at

0.5

negative control LPPS positive control (lipopolysaccharide at 2.5µg/ml)

0.3

b b

b

b b

0.2

b

b

b c

0.1

0.0 0

Fig. 1. HPLC chromatograms of molecular weight indicating lotus plumule polysaccharide (LPPS). The LPPS was prepared by alcohol precipitation from lotus plumule hot water extracts.

a

0.4

20

39

78 156 312 625 1250 2500 concentration (µg/ml)

2.5

Fig. 2. Effects of lotus plumule polysaccharides (LPPS) on the viability of splenocytes from female BALB/cByJNarl mice. Each cell population (5  106 cells/ml) was respectively treated with LPPS at the indicated concentrations of 39, 78, 156, 312, 625, 1250 and 2500 lg/ml, as well as lipopolysaccharide (LPS, as a positive control) at 2.5 lg/ml for 72 h. Values are means ± SD (n = 6 biological determinations). Bars not sharing a common letter are significantly different (P < 0.05) from each other analysed by one-way ANOVA, followed by Duncan’s multiple range tests.

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Table 2 Effects of the addition with different concentrations of polymyxin B on LPPS- or LPSinduced proliferation of splenocytes from female BALB/c mice. Treatments

n

TCM medium only LPPSd, 1250 lg/ml LPSe, 2.5 lg/ml LPS, 10 lg/ml

5 8 8 7

Addition of polymyxin B (U/ml)a,b,c 0

3

6

0.16 ± 0.02B 0.17 ± 0.02B 0.24 ± 0.04A 0.28 ± 0.09A

0.16 ± 0.02B 0.17 ± 0.02B 0.18 ± 0.03C 0.22 ± 0.03C

0.22 ± 0.04A 0.19 ± 0.03A 0.23 ± 0.03B 0.26 ± 0.03B

a

Values are means ± SD. Values not sharing a common capital letter within same row are significantly different (P < 0.05) from one another analysed by one-way ANOVA, followed by Duncan’s multiple range test. c The original cell density was 5  106 cells/ml; the degree of splenocyte proliferation was assayed by MTT method and expressed as the absorbance at 550 nm. d ‘‘LPPS”: lotus plumule polysaccharide which was prepared by alcohol precipitation from lotus plumule hot water extracts. e LPS: lipopolysaccharide. b

different ages were cultured with LPPS at the indicated concentrations of 78, 312 and 1250 lg/ml for 48 h. LPS administration at 2.5 lg/ml was selected as a positive control. The amounts of Th1 cytokines, IL-1b, IL-2 and TNF-a, as well as Th2 cytokines, IL-4, IL-5, IL-6 and IL-10, in the splenocytes culture from NOD and BALB/c mice were determined. Unfortunately, most of the IL-1b, IL-2, IL-4, and IL-5 secretion levels by LPPS-administered splenocytes were too low to be detectable (data not shown). However, the results showed that LPPS administrations significantly (P < 0.05) increased productions of IL-6, TNF-a and IL-10 by splenocytes from NOD and BALB/c mice at ages of 15, 22, and 26 weeks, respectively (Table 3). LPPS administrations increased the cytokine secretions in a dose dependent manner in NOD mice at ages of 15, and 22 weeks, respectively. The results further showed that LPPS administrations at the indicated concentrations of 78 and 312 lg/ ml markedly (P < 0.05) increased IL-10/IL-6 (anti-inflammatory/ pro-inflammatory cytokines) secretion ratios by splenocytes from female NOD mice at age of 22 weeks, suggesting that LPPS

administrations may have anti-inflammatory potential (Table 3). Interestingly, ageing (particularly the experimental mice at age of 26 weeks) inhibited LPPS-induced IL-6, TNF-a, and IL-10 secretions by splenocytes from NOD and BALB/c mice. This study suggests that early intervention of LPPS for immunomodulation may have the stronger effects, especially NOD mice having chronic inflammation status. Our previous study exhibited that low dose (40 mg/kg bw/day) and a 3-week supplementation of lotus plumule alleviated acute systemic inflammation in vivo via decreasing visceral organ inflammation and increasing production of anti-inflammatory cytokine IL10 from splenocytes (Lin et al., 2007). This study further discovered that LPPS, a novel polysaccharide from lotus plumule, exerted antiinflammatory effects on primary splenocytes from NOD and BALB/ c mice at different ages, particularly NOD mice (Table 3). During the development of T1D, different cytokines are involved in inflammation. A growing body of evidence suggests that locally produced pro-inflammatory cytokines IL-b, TNF-a, and IL-6 play a critical role in the pathogenesis of inflammatory diseases (Antonelli et al., 2009). IL-10 is an immunomodulatory cytokine with potent anti-inflammatory properties (Mandal, Pritchard, & Nagy, 2010), which can decrease the production of pro-inflammatory cytokines, including TNF-a and IL-1b (O’Shea and Murray, 2008). IL-10 can be expressed in different cells including monocytes, Th2 cells, mast cells, regulatory T cells, and also in a certain subset of activated T cells and B cells. Furthermore, cytotoxic T cells may produce IL-10 to inhibit the actions of NK cells during viral infection. We found that cell constituents of mouse splenocytes contained 41.54% of B cells and 47.11% of T cells (Lin, Chiang, & Lin, 2005). This study suggests that the novel polysaccharide, LPPS with a molecular weight of 391 kDa and rich in xylose, glucose and fructose (Table 1 and Fig. 1), may have potent anti-inflammatory activity via increasing relatively higher IL-10 production by immune cells (Table 3). However, the immunomodulatory mechanism of LPPS remains to be further clarified. Recently, water-soluble crude polysaccharide from Turbinaria ornata (Marine Brown Alga) is considered as a potential in vitro

Table 3 Effects of different concentrations of LPPS administrations in vitro on Th1 and Th2 cytokine secretions by splenocytes from female NOD/LtJ and BALB/c mice at different agesa,b,c,d,f. Cytokines

TNF-a (pg/ml)

IL-6 (pg/ml)

IL-10 (pg/ml)

IL-10/IL-6 (pg/pg)

a

LPPSeadministration (lg/ml)

15-wk age NOD mice

BALB/c mice

NOD mice

BALB/c mice

NOD mice

BALB/c mice

0 78 312 1250 LPS (2.5 lg/ml)g 0 78 312 1250 LPS (2.5 lg/ml)g 0 78 312 1250 LPS (2.5 lg/ml)g 0 78 312 1250 LPS (2.5 lg/ml)g

8.1 ± 16.3BC,c 31.4 ± 18.8C,c 145.9 ± 60.2B,b 180.6 ± 88.8BC,b 476.3 ± 88.9BC,a 14.6 ± 4.2A,b 17.4 ± 4.2B,b 26.1 ± 2.2C,b 63.8 ± 21.6BC,b 536 ± 114BC,a 1 ± 2B,b 42 ± 25BC,b 103 ± 33B,b 202 ± 51B,b 1565 ± 298A,a 0.14 ± 0.28AB,b 2.66 ± 1.85B,a 3.90 ± 1.07B,a 3.23 ± 0.37AB,a 2.94 ± 0.32A,a

50.8 ± 17.1A,d 228.0 ± 61.5B,c 331.2 ± 64.9A,b 256.4 ± 31.8AB,c 874.6 ± 134.3A,a 15.9 ± 5.9A,b 27.9 ± 6.7A,b 63.8 ± 10.5A,b 93.3 ± 21.4AB,b 887 ± 188AB,a 10 ± 12AB,d 19 ± 11CD,d 62 ± 24BC,c 139 ± 40BC,b 662 ± 70C,a 0.62 ± 0.71AB,b 0.75 ± 0.42CD,b 0.95 ± 0.28C,b 1.56 ± 0.15BC,a 0.73 ± 0.10C,b

–h 38.2 ± 50.7C,b 149.6 ± 93.9B,b 193.9 ± 117.9AB,b 819.1 ± 265.1AB,a 13.7 ± 3.4A,b 22.2 ± 9.3AB,b 43.5 ± 20.5B,b 110.9 ± 65.1A,b 1109 ± 425A,a 15 ± 19A,d 116 ± 66A,cd 212 ± 120A,bc 339 ± 153A,b 1521 ± 149A,a 0.90 ± 1.08A,c 6.06 ± 4.03A,a 5.20 ± 2.00A,ab 4.56 ± 4.46A,abc 1.54 ± 0.56B,bc

17.7 ± 15.6B,b 319.9 ± 91.1A,a 396.0 ± 126.7A,a 283.9 ± 100.0A,a 391.5 ± 538.2C,a 14.0 ± 3.7A,b 20.4 ± 8.9AB,b 37.8 ± 10.5BC,b 67.1 ± 19.4BC,b 403 ± 527C,a 3 ± 5AB,b 28 ± 15BCD,b 57 ± 27BC,b 133 ± 51BC,ab 265 ± 347D,a –h 1.21 ± 0.66BCD,b 1.22 ± 0.36C,b 1.84 ± 0.45BC,a 0.53 ± 0.30C,c

–h 28.9 ± 32.8C,cd 127.3 ± 111.6B,b 97.3 ± 62.0C,bc 460.9 ± 94.5BC,a 17.8 ± 8.8A,b 22.2 ± 5.8AB,b 44.7 ± 18.5B,b 87.2 ± 26.2AB,b 713 ± 262ABC,a 5 ± 9AB,b 54 ± 12B,b 78 ± 40B,b 110 ± 33CD,b 1244 ± 236B,a 0.48 ± 0.90AB,c 2.50 ± 0.67BC,a 1.79 ± 0.90C,ab 1.35 ± 0.52C,bc 2.02 ± 0.99B,ab

–h 305.3 ± 42.9A,c 417.2 ± 132.4A,b 214.9 ± 46.6AB,c 739.6 ± 172.3ABC,a 6.1 ± 5.6B,b 16.6 ± 7.6B,b 22.8 ± 9.7C,b 46.3 ± 13.9C,b 677 ± 241BC,a –h 5.9 ± 7.2D,c 14.9 ± 10.7C,c 58.7 ± 17.1D,b 478 ± 67CD,a 0.09 ± 0.18AB,c 0.36 ± 0.40D,c 0.81 ± 0.63C,b 1.30 ± 0.31C,a 0.88 ± 0.47C,b

22-wk age

26-wk age

Values are means ± SD (n = 4-10 biological determinations). Values within same row not sharing common capital letters are significantly different (P < 0.05) from one another analysed by one-way ANOVA, followed by Duncan’s multiple range test. c Values within same column under same cytokine item not sharing common lowercase letters are significantly different (P < 0.05) from one another. d The original cell density was 5  1 06 cells/ml medium. e The LPPS was prepared by alcohol precipitation from lotus plumule hot water extracts. f The sensitivity of ELISA kits used in this study was < 15.6 pg/ml. g positive control. h ‘‘-‘‘: not detectable. b

C.-H. Liao et al. / Food Chemistry 125 (2011) 930–935

antioxidant and in vivo anti-inflammatory agent (Ananthi et al., 2010). Li, Lu, Zhang, Lu, and Liu (2008) indicated that Pholiota nameko polysaccharide possesses significant anti-inflammatory activity suggesting its potential as an anti-inflammatory agent for use in the treatment of various inflammatory-related diseases (Li et al., 2008). This study further suggests that LPPS administration may modulate autoimmune diseases, such as T1D, via its potent anti-inflammatory activities. Chen et al. (2008) have demonstrated that the effect of Astragalus polysaccharides (APS) on the prevention of T1D in NOD mice via correcting the imbalance between the Th1/Th2 cytokines (Chen et al., 2008). Besides, a water-soluble polysaccharide from roots of Actinidia eriantha (AEPS) exhibits strong potential to increase both cellular and humoral immune responses and elicits a balanced Th1/Th2 response (Sun, Wang, Xu, & Ni, 2009). Unfortunately, we found that most of the Th1 (IL-1b and IL-2) and Th2 (IL-4 and IL-5) levels secreted by LPPS-administered splenocytes were too low to be calculable in vitro (data not shown). According to the results from this study, we prudently suggested that LPPS administration in vitro might not markedly affect Th1/Th2 immune balance. However, the effect of LPPS on Th1/Th2 immune balance should be further clarified in vivo. Overall, a novel polysaccharide from lotus plumule (LPPS) was isolated and characterised in this study. The LPPS having a molecular weight of 391 kDa consisted of protein, carbohydrate as well as phenolics, and comprised, magnitude in order, xylose, glucose, fructose, galactose, and fucose. The present study suggests that LPPS administration might protect T1D from inflammation via its potent anti-inflammatory activity. Acknowledgment This study was supported by research grants NSC96-2313-B005-009-MY2 and NSC98-2313-B-005-035-MY3 from the National Science Council, Taipei, Taiwan, Republic of China (ROC). References Ananthi, S., Raghavendran, H. R., Sunil, A. G., Gayathri, V., Ramakrishnan, G., & Vasanthi, H. R. (2010). In vitro antioxidant and in vivo anti-inflammatory potential of crude polysaccharide from Turbinaria ornata (Marine Brown Alga). Food and Chemical Toxicology, 48(1), 187–192. Anderson, M. S., & Bluestone, J. A. (2005). The NOD mouse: a model of immune dysregulation. Annual Review of Immunology, 23, 447–485. Antonelli, A., Ferri, C., Ferrari, S. M., Ghiri, E., Goglia, F., Pampana, A., et al. (2009). Serum levels of proinflammatory cytokines interleukin-1beta, interleukin-6, and tumor necrosis factor alpha in mixed cryoglobulinemia. Arthritis & Rheumatism, 60(12), 3841–3847. Chen, W., Li, Y. M., & Yu, M. H. (2008). Astragalus polysaccharides: an effective treatment for diabetes prevention in NOD mice. Experimental and Clinical Endocrinology & Diabetes, 116(8), 468–474. da Silva, B. P., & Parente, J. P. (2001). An anti-inflammatory and immunomodulatory polysaccharide from Orbignya phalerata. Fitoterapia, 72(8), 887–893. Delovitch, T. L., & Singh, B. (1997). The nonobese diabetic mouse as a model of autoimmune diabetes: immune dysregulation gets the NOD. Immunity, 7(6), 727–738. Dubois, M., Gilles, K., Hamilton, J. K., Rebers, P. A., & Smith, F. (1951). A colorimetric method for the determination of sugars. Nature, 168(4265), 167.

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