Polysaccharides Purified from Wild Cordyceps Activate FGF2/FGFR1c Signaling

ZENG Yangyang, HAN Zhangrun, YU Guangli, HAO Jiejie, and ZHANG Lijuan



Polysaccharides Purified from Wild Cordyceps Activate FGF2/FGFR1c Signaling

ZENG Yangyang, HAN Zhangrun, YU Guangli, HAO Jiejie*, and ZHANG Lijuan*

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Land animals as well as all organisms in ocean synthesize sulfated polysaccharides. Fungi split from animals about 1.5 billion years ago. As fungi make the evolutionary journey from ocean to land, the biggest changes in their living environment may be a sharp decrease in salt concentration. It is established that sulfated polysaccharides interact with hundreds of signaling molecules and facilitate many signaling transduction pathways, including fibroblast growth factor (FGF) and FGF receptor signaling pathway. The disappearance of sulfated polysaccharides in fungi and plants on land might indicate that polysaccharides without sulfation might be sufficient in facilitating protein ligand/receptor interactions in low salinity land. Recently, it was reported that plants on land start to synthesize sulfated polysaccharides in high salt environment, suggesting that fungi might be able to do the same when exposed in such environment. Interestingly, Cordyceps, a fungus habituating inside caterpillar body, is the most valued traditional Chinese Medicine. One of the important pharmaceutical active ingredients in Cordyceps is polysaccharides. Therefore, we hypothesize that the salty environment inside caterpillar body might allow the fungi to synthesize sulfated polysaccharides. To test the hypothesis, we isolated polysaccharides from both lava and sporophore of wild Cordyceps and also fromcultured without or with added salts. We then measured the polysaccharide activity using a FGF2/FGFR1c signaling-dependent BaF3 cell proliferation assay and found that polysaccharides isolated from wild Cordyceps activated FGF2/FGFR signaling, indicating that the polysaccharides synthesized by wild Cordyceps are indeed different from those by the cultured mycelium.

Polysaccharide; Cordyceps;; FGF; BaF3

1 Introduction

Cordyceps, a fungus growing out of the head of a mummified caterpillar, has been extensively used as the most valued traditional Chinese medicine and nutraceuticals not only in China but also in many countries of the world. The polysaccharides of Cordycepsare considered to be one of the major pharmaceutical active components with anti-inflammatory (Won, 2005), anti-oxidative (Wang, 2012), anti-viral (Ohta, 2007), immunomodulatory (Song, 1998), hypoglycemic (Zhang, 2006), antitumor (Lin, 2008) and anti-angiogenic (Yoo, 2004) activities.Increased demand has made Cordyceps an endangered species (Cleaver, 2004; Hsu, 2002; Winkler, 2010). Many companies useortocultivate Cordyceps to meet the increased demand. However, unlike the molecular structure-defined pharmaceutical active ingredients in Cordyceps, such as cordycepin, mannitol, and ergosterols, the polysaccharide structures and biological functions from cultured mycelium may not be the same as that in wild Cordyceps since polysaccharides biosynthesis is very sensitive to environmental conditions, especially to high salt environment (Aquino, 2011).

Land animals as well as all living organisms in ocean synthesize sulfated polysaccharides. Fungi split from animals about 1.5 billion years ago (James, 2006). As early fungi (Zheng, 2011) make the evolutionary journey from ocean to land and branch off from animals, the biggest changes in their living environment may be a sharp decrease in salt concentration. It has been established that sulfated polysaccharides interact with hundreds of signaling molecules (Zhang, 2010), such as growth factors, chemokines, and cytokines, and facilitate many important signaling transduction pathways, including fibroblast growth factor (FGF) and FGF receptor (FGFR) pathway (Itoh, 2011) . The disappearance of sulfated polysaccharides in fungi and plants in land might indicate that polysaccharides without sulfation might be sufficient in facilitating protein ligand/receptor interactions in low salinity land. The presence of sulfated polysaccharides in plants in an adaptation to high salt environments was recently reported(Aquino, 2010). Therefore, we hypothesize that the high salt environment inside caterpillar bodies might allow fungi to synthesize sulfated polysaccharides and by adding salt toculturemight make the culturedsynthesize sulfated polysaccharides as well.

To test the hypothesis, we first isolated 10 polysaccharides from both lava and sporophore of wild Cordyceps. We then isolated 26 different kinds of polysaccharides fromcultured without or with different amount of added salts.An established FGF/FGFR signaling-dependent BaF3 cell proliferation assay was then employed to detect the biological activity of the isolated polysaccharides (Pan, 2010).

FGF/FGFR signaling plays a crucial role in animal development and homeostasis including embryonic development (Slack, 1987), angiogenesis (Cross, 2001), tissue regeneration (Kawai, 2000), bone regeneration (Canalis, 1988), development and maintenance of the nervous system (Timmer, 2007), stem cell self-renewal (Levenstein, 2006), and wound healing (Barrientos, 2008). More importantly, sulfated polysaccharides are required to facilitate FGF/ FGFR signaling at both cell (Yayon, 1991; Rapraeger, 1991) and tissue levels (Ornitz, 2000). To take advantage of such a property,Ornitz. transfected a lymphocyte cell line, BaF3, with FGFR1c cDNA to make a stable cell line (Ornitz, 1992). The BaF3 cells make very little sulfated polysaccharides (Zhang, 2006)and normally depend on interleukin 3 (IL-3) for growth. FGFR1c expressing BaF3 cells only proliferate when both FGF and polysaccharide are added to the growth media in the absence of IL-3 (Fig.1). By using this assay, we discovered that polysaccharides isolated from sporophore of wild Cordyceps activates FGF/FGFR signaling, indicating that the polysaccharides synthesized by wild Cordyceps are different from that of the cultured mycelium.

Fig.1 Principle of FGF and polysaccharide-dependent BaF3 FR1c cell proliferation assay.

2 Materials and Methods

2.1 Materials

, strain 11Y-6, was a generous gift from fungal specialist, Prof. X. L. Jiang (Ocean University of China, Qingdao, China). The wild Cordyceps were purchased from a reliable dealer in Qingdao, China. 1640 medium was obtained from Sigma-Aldrich (USA). Fetal bovine serum was purchased from GIBICO (USA). Fibroblast growth factor 2 (FGF2), IL-3 and G418 were purchased from Goldbio (USA). Heparin, resazurin and β-mercaptoethanol were purchased from Sigma (USA). Hyaluronic acid (HA) was purchased from Fluka (USA).

2.2 Liquid Culture of.

.was maintained on potato dextrose agar (PDA) slants and inoculated at the center of a Petri dish. The mycelium was fermentated in 200mL medium containing 6gL−1potato dextrose (PD) and 20gL−1glucose with 0.05mgL−1-500mgL−1Na2SO4, at 27℃ for 10d (130rmin−1). The mycelia and culture medium were rapidly concentrated by rotary evaporation to 100 mL and extracted with 400mL 95% ethanol at 80℃ 3times to remove lipids. The de-lipided power was then used for polysaccharide extraction.

外语学科的美国学，要形成自身特色，可重点关注美国移民、种族、宗教、社会、教育、人口、妇女、少数人群体、多元文化、城市化、工业文明、环保主义、政治文化、外交思想等多领域的议题。运用跨学科理论和方法分析文化因素在相应议题的作用范式与影响途径，挖掘美国文明演变的深层次原因，建构和丰富中国的美国学理论研究视角和问题分析框架，从本质上进一步把握美国文明的本质特征，为中美两国的对话交流提供更多的理论指导。

2.3 Extraction of Polysaccharides from Wild Cordyceps and Cultured Mycelia

The Cordyceps was divided into larva and sporophore. The larva and sporophore were dried at 40℃ and then pulverized. The pulverized powders were extracted three times with 95% ethanol at 80℃ to obtain defatted powders from larva and sporophore, respectively. The enzymatic extraction method added a step of pronase K digestion of de-lipided starting materials. Briefly, the de-lipided powders from wild Cordyceps and cultured mycelia were digested by pronase K solution overnight at 50℃ and then heated at 100℃ to inactivate the enzyme. The polysaccharide-containing supernatants were collected after centrifugation at 4500rmin−1for 4min and then con- centrated by rotary-evaporation. The concentrated solutions were precipitated by adding 4 volumes of ethanol. The polysaccharide-containing precipitates were then dried. The precipitates were re-dissolved in distilled water at 80℃ for 3h and extracted with distilled water 3 more times at 80℃ allowing all polysaccharides to thoroughly dissolve. The polysaccharide-containing supernatants were precipitated again by adding 4 volumes of ethanol. The polysaccharide-containing precipitates were soaked in 2% Na2CO3or 1% NaOH solution to remove covalently linked protein and then extracted three times at 60℃ for 3h with distilled water. The collected polysaccharide-containing supernatants were neutralized, dialyzed, and precipitated with 4 volumes of ethanol. The precipitated polysaccharides were then dissolved in water and dialyzed against water using dialysis bags with 3000 Da molecular weight cut off. After thorough dialysis, each sample was dried, weighed, and used for further tests.

2.4 Fourier Transformed Infrared Spectrometry (FTIR) Analysis

[8][32][48] 范子英、彭飞、刘冲：《政治关联与经济增长：基于卫星灯光数据的研究》，《经济研究》2016年第1期，第114-126页。

Samples (1-2mg) were dried in a P2O5desiccator for 48h, mixed with 100mg KBr, and pressed under 7kgcm−2to make transparent films, followed by placing them in an FTIR instrument (Nicolet Nexus 470, Thermo Electron, USA) and scanning from 400 to 4 000cm−1to obtain the infrared spectrum.

2.51H-NMR Analysis

The polysaccharide sample was dissolved in 500mL D2O and freeze-dried twice to replace all exchangeable protons with deuterium. The1H NMR spectrum was recorded on a Bruker DPX 400 spectrometer; acetone (the signals at 2.09ppm) was used as an external reference.

目前，旅游业存在明显的供需错位现象，表现为：一方面，旅游业人才需求旺盛；另一方面，一些高校旅游类专业学生却出现实习难、就业难的假象。究其原因，除了学生不愿从事服务业等主观原因外，主要在于高校培养的学生与旅游业要求差距较大，学生普遍存在职业意识不强，职业忠诚度较低的现象。形体礼仪课程“理论+实践”教学模式的实施，重视学生外在形象与内在素质的培养，并通过相应的保障措施达到应有的效果，可在一定程度上缓解学生实习难、就业难的现象，有利于提高旅游类专业学生的职业竞争力[4]。

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2.6 BaF3 Cell Proliferation Assay

The FGFs/FGFRs signaling activation by Cordyceps polysaccharides was evaluated according to a reported method by Pan. (2010). 96-well tissue culture plates were dispersed with 100μL of cell culture media (RPMI 1640 supplemented with 10% fetal bovine serum, 100UmL penicillin, 100μgmL−1streptomycin, 50 μmolL−1β-mercaptoethanol, and 600μgmL−1 G418) containing 100μgmL−1samples or controls (positive control: 2μgmL−1heparin; negative control: 100μgmL−1hyaluronic acid). FGFR1c-expressing BaF3 cells (30000 live cells/ well in 50μL media) were then added. FGF-2 in 50μL media was added to each well with a final concentration of 8nmolL−1. The FGFR1c-expressing BaF3 cells were cultured for 40h, followed by adding 20μL of resazurin (2mgmL−1water solution) to the media for 24h. The fluorescent signals were monitored using 544nm excitation wavelength and 595nm emission wavelength by using a Spectramax M5 plate reader (Molecular Devices).

3 Results and Discussion

3.1 Preparation of Polysaccharides by 5 Different Extraction Methods from either Larval or Sporophore of Wild Cordyceps

Fungi usually synthesize both water soluble and insoluble polysaccharides located in intracellular, cell wall, and extracellular spaces serving for energy storage, structure, and communication purposes. According to published reports, the water-soluble polysaccharides are the most active pharmacological components tested in over 300 kinds of polysaccharides extracted from either plants or fungi (Hu, 2013). Therefore, we decided to purify and to study the structure and function of the water soluble polysaccharides from the wild Cordyceps.

护理工作者在对患者进行护理的过程中，一定要注意安全问题，严格按照操作要求对患者进行护理，对患者及其家属尽量使用温和的态度，拉近和患者之间的距离，这样才能促进医护行业的健康发展。

No sulfated polysaccharides have been shown to be present in fungi on land. Since our hypothesis is that sporophore of the wild Cordyceps might contain sulfated polysaccharides, we decided to add a pronase K digestion step to the conventional water extraction procedure for polysaccharides. The reason was that extraction of sulfated polysaccharide from animal-based samples usually requires protease digestion to get rid of both covalently and non-covalently linked proteins to free sulfated polysaccharides. Another conventional way to get rid of covalently linked proteins is to perform ab-elimination reaction by treating the polysaccharide preparation with base. We used both 2% Na2CO3or 1% NaOH solution for theb-elimination reaction. To test if water temperature made any difference in polysaccharide extraction, we used water at 80℃ and at room temperature, respectively.

By using 5 different extraction methods, we obtained 10 polysaccharide samples, A1-A5 from larval and A6-A10 from sporophore. The amount of polysaccharides obtained and the yield of polysaccharides by using different extraction methods are shown in Table 1.

式中： m1——实际计算质量，t；m2——液压千斤顶的设计量程，0～100 t；p——液压千斤顶的设计油压63.7 MPa；p0——液压千斤顶的实际加载油压，MPa； N——千斤顶个数，N=4。

Interestingly, the yield of larval polysaccharides were 4.47% (enzyme), 2.77% (Na2CO3), 1.86% (NaCl), 1.17% (hot water, 80℃), and 0.23% (cold water, room temperature). These data suggest that protease digestion,b- elimination reaction, and higher temperature help to obtain more polysaccharides from the same amount of starting material. However, different extraction methods did not make significant difference in the polysaccharides obtained from sporophore. To confirm the observations, we repeated the above experiment twice with another two wild Cordyceps collected in different regions of China (data not shown). Indeed, improved larval polysaccharide yield was observed constantly after protease digestion,b-elimination reaction, or hot water extraction, whereas all these treatments did not significantly increase the yield of sporophore polysaccharides. Therefore, the polysaccharides in larval might be different from that in sporophore.

Table 1 Polysaccharides purified by 5 different extraction methods from either larval or sporophore of wild Cordyceps

3.2 Preparation of Polysaccharides from.Culture Without or with Added Salts Using two Different Extraction Methods

Our hypothesis was that added salt inculture might induce sulfated polysaccharide production. To see if different salts could make a compact on polysaccharide biological activity, 100mgL−1of Na2SO4, MnSO4, and MgSO4were added to theculture. To further test if there were a salt concentration-dependent effect on polysaccharide production,was also cultured in the presence of 8 different concentrations of Na2SO4as shown in Table 2.

The polysaccharides under each cultural condition were then extracted either with or without pronase K digestion (Materials and Methods). Data in Table 2 indicate that neither salt nor extraction methods made significant impact on the amount of polysaccharides obtained in most of polysaccharide samples except samples D1, D2, D3, and D4. The polysaccharide yield in these four samples was apparently higher than others.

Table 2 Polysaccharides purified from Cordyceps millitaris cultured in media without or with added salts using either water- or enzyme-based extracting methods

3.3 Characterization of Purified Polysaccharides by FTIR and Proton NMR

We used FTIR to test the purity of the isolated polysaccharides. The representative FTIR spectra of polysaccharides A1 and A4 from wild Cordyceps and D1 and D4 from culturedare shown in Fig.2. The absorption peaks at 3300-3400cm−1(nO−Hof-OH group), 1645-1650cm−1(nC=Oof carboxyl group), and 1050-1107cm−1(nC−O−Cof glycoside) indicated the polysaccharide nature of the tested compounds. However, the strong absorption at 1319cm−1(nC−O−Cof ester) of D1 and D4 indicated that the presence of esterified alkyl groups was not typical for polysaccharide samples. Considering that the polysaccharide D1 and D4 had higher than normal yield (Table 2), we suspect the samples might be not pure. Indeed, the strong absorption peaks in D1 and D4 disappeared afterre-dialyzing (data not shown) and the yield of polysaccharides (shown in Table 2) returned to normal.

Ornitz, D. M., 2000. FGFs, heparan sulfate and FGFRs: Complex interactions essential for development., 22 (2): 108-112.

We also performed1H NMR analysis on selected sets of sample to further check the purity of the isolated polysaccharide. Fig.3 shows a typical1H NMR spectrum of the isolated polysaccharides, D17.1H signals from 5.06 to 4.91ppm represented α or β linkages of the glycosidic bonds in polysaccharides. The1H signals from 3.5 to 5.0 ppm were typical1H profile for polysaccharides.

3.4 Polysaccharides Purified from Wild Cordyceps Activated FGF/FGFR Signaling Quantified by the BaF3 Cell Proliferation Assay

Fig.2 FTIR spectra of native (A) and cultural (B) Cordyceps polysaccharides.

Fig.3 1H NMR of the purified polysaccharide D17.

1H signals from 5.06 to 4.91 ppm representing α or β linkages of the glycosidic bonds in polysaccharides. The1H signals from 3.5 to 5.0ppm were typical1H profile for polysaccharides. The1H NMR spectrum indicated D17 was polysaccharide with decent purity.

This work was supported by the National Natural Science Foundation of China (Grant No. 91129706).

四是开发全国的水资源、水环境、水生态的监测信息系统，建立河湖健康评价体系，是执行“三条红线”控制的重要技术支撑。

The data in Fig.4A shows that the pronase K and alkaline treated samples A1, A4, and A5 from larval and A6, A9, and A10 from sporophore stimulated BaF3 cell proliferation and the data had statistical significance. In contrast, the 26 samples from culturedincluding 13 pronase K treated samples (D18-D26) showed very low or no sign in stimulating BaF3 cell proliferation. The polysaccharide/FGF/FGFR dependent-BaF3 cell proliferation usually requires the presence of sulfated polysaccharides. The three samples A6, A9, and A10 from sporophore stimulated BaF3 cell proliferation indicated that the fungal polysaccharides in sporophore had unique structures that were not present in that of cultured. However, further studies are required to test if sulfated polysaccharides are synthesized by the fungi and contribute to the activities shown in Fig.4.

96-well tissue culture plates were dispersed with 100 μL of BaF3 FR1c cell culture media containing 100μgmL−1samples or control (blank control: 8nmolL−1FGF2; positive control: 2μgmL−1heparin; negative control: 100μgmL−1hyaluronic acid). BaF3 FR1c cells expressing FGFR1c (30000 live cells/well in 50μL media) were then added. FGF-2 in 50μL culture media were then added to each well in a final concentration of 8nmolmL−1. Cells were cultured for 40h, followed by adding 20μL of resazurin to the media for 24h. The fluorescent signal was monitored using 544nm for excitation and 595nm for emission in a Spectramax M5 plate reader (Molecular Devices). Each data point presented were average of 6 measurements from two independent experiments.

Kawai, K., Suzuki, S., and Tabata, Y., 2000. Accelerated tissue regeneration through incorporation of basic fibroblast growth factor-impregnated gelatin microspheres into artificial dermis., 21 (5): 489-499.

Fig.4 Polysaccharides from wild Cordyceps (Fig.4A) were more potent than cultured C. millitaris polysaccha- rides (Figs.4B, C and D) in stimulating BaF3 FR1c cell proliferation based on Polysaccharide/FGF2/FGFR1c signal transduction. Hep, heparin; HA, hyaluronic acid.

4 Conclusion

The anti-inflammatory, anti-oxidative, anti-viral, immunomodulatory, hypoglycemic, antitumor and anti-an- giogenic activities of fungal polysaccharides have been well established. Most of polysaccharide activities have been demonstrated by employing either immune cells or animal models and explained on the basis that animal immune cells have a series of receptors on their cell surface to recognize the foreign polysaccharide structures to initiate signaling cascade. However, the most potent polysaccharide structures in activating immune systems, such as lipopolysaccharides in bacteria orb-glucans in fungi, are very rare in Cordyceps. Therefore, developing novel polysaccharide structure-dependent bioassays will be helpful in understanding Cordyceps polysaccharides, an important pharmaceutical active ingredient in Cordyceps. The BaF3 cell proliferation assay presented in this study might serve for such purposes.

Polysaccharides are as essential for life as DNAs, RNAs, proteins, lipids, and metabolites of the biological system (Marth, 2008). It has long been speculated that the dynamic structural and functional information encoded in polysaccharides may be required to collaborate with and to constraint in order to keep the cells or whole system in balance (Springer and Gagneux, 2013). Coincidently, Cordyceps has long been granted as a medicine that can collaborate, constraint, and keep the whole system in balance. Thus understanding the disappearance of sulfated polysaccha- rides in fungi and plants on land and re-appearance in plants and perhaps in Cordyceps in high salt environment at molecular level might lead to new knowledge about the biological systems.

Acknowledgements

FGFR1c-expressing BaF3 cells were used to assay polysaccharide-mediated FGFR signaling. The FGFR1c- expressing BaF3 cells were treated with 8nmolmL−1FGF2 alone as blank control, 2μgmL−1heparin as posi- tive control, and 100μgmL−1hyaluronic acid as negative control. The 36 polysaccharide samples listed in Tables 1 and 2 were tested in triplets along with the controls at 100 μgmL−1. Two independent experiments were performed for each sample and each data point shown in Fig.4 was average of 6 measurements from two independent experiments.

Aquino, R. S., Grativol, C., and Mourao, P. A., 2011. Rising from the sea: Correlations between sulfated polysaccharides and salinity in plants., 6 (4): e18862.

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在传主的自我塑造和传记作者的合力作用下，成就了当时许多具有道德典范意义的遗民志士形象，在同道中引为共鸣，并成为时代精神风貌的代表者。遗民传记精神风貌之传扬，自然少不了传记文作者的重塑之功，作家们在叙述传主生平经历的同时，更多的精力会聚焦、着力于抽象传主符合遗民道德传统的认知，从而实现其精神境界的提升。

Cleaver, P. D., Loomis-Powers, M., and Patel, D., 2004. Analysis of quality and techniques for hybridization of medicinal fungus(Berk.) Sacc. (Ascomycetes)., 6 (2): 152.

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Hsu, T., Shiao, L., and Hsieh, C., 2002. A comparison of the chemical composition and bioactive ingredients of the Chinese medicinal mushroom DongChongXiaCao, its counterfeit and mimic, and fermented mycelium of., 78 (4): 463-469.

锡是我国优势金属资源，锡石多金属硫化矿是我国特色锡矿资源。由于锡石性脆，在选矿生产中存在锡石过磨和硫化矿欠磨的突出矛盾，影响锡的金属回收率和硫化矿精矿质量。考虑到该类矿石的矿物组成复杂，涉及硫化矿、氧化矿和脉石等众多矿物成分，且不同矿物成分存在较大的吸波特性差异，因此，本文从利用微波选择性加热潜力出发，研究传统加热与微波加热预处理对锡石多金属硫化矿助磨的差异，旨在探索锡石多金属硫化矿的微波加热辅助磨矿新途径。

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James, T. Y., Kauff, F., and Schoch, C. L., 2006. Reconstructing the early evolution of fungi using a six-gene phylogeny., 443 (7113): 818-822.

Another observation in this experiment was that the polysaccharides (A2, A3, A6, and A7) obtained by water extraction alone did not shown any activities in the BaF3 cell-based FGF/FGFR signaling system, which suggest that the conventional fungal polysaccharide extraction methods might have failed to extract such biological active polysaccharides.

Levenstein, M. E., Ludwig, T. E., and Xu, R. H., 2006. Basic fibroblast growth factor support of human embryonic stem cell self-renewal., 24 (3): 568-574.

历史文化古街能够吸引历史爱好者进行参观和游览，但是必须以其原貌保持作为前提。江西省近年来颇为重视历史文化街区的保护，将具有浓厚历史背景的县城列入保护区内。为了保证其发展，还应注重景区的真实性和内容的丰富性。经历多年的沧桑，景点完好保存不太可能，政府应与旅游部门进行合作，最大程度上恢复历史街区的原貌，并且保有其气息，拒绝过于现代化的商业买卖，保留该县城曾经的民风民俗，给旅游者真正的历史文化体验。为了增加旅游者的体验，可以在当地开设舞台剧，还原历史本身，还原历史细节。要促进其发展，可以允许单门单户的经营活动，以客栈等形式存在。在项目上，要以功能补充性为主，而不是以功能改造性为主。

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The absorption peaks at 3300-3400cm−1(nO−Hof-OH group), 1645-1650cm−1(nC=Oof carboxyl group), and 1050-1107 cm−1(nC−O−Cof glycoside), indicated that the polysaccharides had decent purity. The strong absorption at 1319cm−1(nC−O−Cof ester) of D1 and D4 indicated that the hydroxyl groups were esterified by alkyl such as methyl group.

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在最后一层灰土压实后，进行垫层放线。为了进一步控制基础水平，在垫层模板支设过程中，技术人员要使用水准仪对模板四角进行标高测量，以保证工程质量。

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1) IOI 2) stack [stæk] n. 3) pretend [prɪ'tend] v.电影中的反派公司的名称 堆叠 假装

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(Edited by Ji Dechun)

DOI 10.1007/s11802-015-2683-0

ISSN 1672-5182, 2015 14 (1): 171-177

© Ocean University of China, Science Press and Springer-Verlag Berlin Heidelberg 2015

(June 9, 2014; revised July 20, 2013; accepted July 24, 2014)

* Corresponding author. Tel: 0086-532-82031615 E-mail:2009haojie@ouc.edu.cn; lijuanzhang@ouc.edu.cn