Molecules 2015, 20, 4162-4179; doi:10.3390/molecules20034162 OPEN ACCESS molecules ISSN 1420-3049 www.mdpi.com/journal/molecules Article Chemical Constituents and Structural Characterization of Polysaccharides from Four Typical Bamboo Species Leaves Cheng-Zhang Wang 1,2,3,4,*, Hong-Yu Zhang 1, Wen-Jun Li 1 and Jian-Zhong Ye 1 1 Institute of Chemical Industry of Forest Products, CAF, Nanjing 210042, China; E-Mails: [email protected] (H.-Y.Z.); [email protected] (W.-J.L.); [email protected] (J.-Z.Y.) 2 National Engineering Laboratory for Biomass Chemical Utilization, Nanjing 210042, China 3 Key Laboratory of Biomass Energy and Material, Nanjing 210042, China 4 Institute of New Technology of Forestry, CAF, Beijing 100091, China * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel./Fax: +86-25-8548-2471. Academic Editor: Derek J. McPhee Received: 1 December 2014 / Accepted: 12 February 2015 / Published: 5 March 2015 Abstract: In order to find bamboo leaves with high contents of bioactive polysaccharides, 32 samples were chosen to analyze their polysaccharide content by GC and sulfuric acid-anthrone colorimetric assays. Purified polysaccharides (BLPS) were separated from the four varieties P. nigra (Lodd.) Munro (PN), P. vivax McClure (PV), Chimonobambusa quadrangularis (Fenzi) Makino (CQ), and P. bambussoides cv. Tanakae (PB) by ultrasound extraction, solution precipitation, ion exchange resin, DEAE-52 and Sephadex G-100 chromatography. BLPS structural characterization was accomplished by HPLC-GPC, Fourier transform infra-red spectroscopy (FTIR) and NaIO4-HIO4 oxidation reactions. The results showed that the total polysaccharides of the bamboo leaves in samples 1–32 ranged between 1.4% and 5.4%, Samples No. 29–No. 32 (PN, PV, CQ, and PB) contained 2–3 fold more polysaccharides than No. 1~No. 28 among the 32 different species, particularly the content of galactose was in a range of 21.5%–34.1% for these four typical bamboo species leaves, which was also more than 2–3 fold higher than in No. 1–No. 28. Sugar analysis indicated that PN-PBLPS-1, PV-PBLPS-1, CQ-PBLPS-1 and PB-PBLPS-1 from the four varieties were homogeneous polysaccharides with molecular weights of 2.04 × 104, 1.15 × 104, 8.75 × 104 and 1.48 × 104 Da, respectively. PB-PBLPS-1 was a mixture of α-galactopyranose and β-D-glucopyranose linkages with Molecules 2015, 20 4163 α-(1→6) or β-(1→6)glycosidic bonds, while PN-PBLPS-1, PV-PBLPS-1, and CQ-PBLPS-1 had α galactopyranose linkages with α-(1→6) glycosidic bonds. Keywords: bamboo leaves; polysaccharides; HPLC-GPC; FTIR; NaIO4-HIO4 1. Introduction Bamboo belongs to the Gramineae Bambusoideae subfamily and is one of the most valuable evergreen perennial plants in China and South-east Asia. China has been known as the “bamboo kingdom”, and bamboo timber is a traditional forest product containing high cellulose, hemicellulose and lignin used as a construction material [1]. Generally hemicelluoses are a family of polysaccharides which contain a backbone of D-xylopyranosyl residues, linked together by β-(1→4)-glycosidic bonds [2], while the bioactive polysaccharides in bamboo are oligosaccharides of β-D-glucan and xyloglucan or p-coumaroyl arabinoxylan [3]. The investigation showed that bamboo shoot polysaccharides are composed of arabinoxylan (1→3,1→4)-β-D-glucan, xyloglucan and glucomannan [4], and other kinds of arabino-glucuronoxylans are also isolated from bamboo shoot with a linear (1→4)-β-D-xylopranosyl backbone to which α-L-arabinofuranose units or short chains of 4-O-methylglucuronic acid. However, these 4 4 hemicellucosic polysaccharides have a average molecular weight (Mr) range of 10 × 10 –40 × 10 Da, and this results in very weak physiological activity. 4 In order to get strongly bioactive polysaccharides with a average Mr range of less than 10 × 10 Da, scientists often use chemical or biological ways to degradate macromolecular polysaccharides. This causes serious environmental pollution and results in high costs, therefore, it is very important to seek highly bioactive polysaccharides with low molecular weight from forestry resources or biomass residues. Apart from bioactive compounds like flavonoids, glycosides and polyphenols, bamboo leaves contain a large amount of active polysaccharides [5–7]. Extract of bamboo leaves (EBL) has been proved very safe and is used widely in China as an antioxidant in food additives [8]. The key bioactive compounds of EBL are polysaccharides and flavonoids, where particularly bamboo oligosaccharides play important functions [9,10]. The polysaccharide of bamboo leaves (BLPS) is a kind of bioactive heteropolysaccharide with medium molecular mass. BLPS was first isolated from Japanese bamboo leaves of Sasa kurilensis and Sasamorpha chiisanensis [11]. The reported average molecular mass Mr of the BLPS was determined to be in a range of 2 × 104–3 × 104 Da, and this BLPS was proven to have good antitumor activity. Kato separated β-D-glucan and xyloglucan bioactive polysaccharides from bamboo shoots [12], while Suzuki also obtained from green bamboo shoots a bioactive hemicellulose polysaccharide with strong inhibition on S180 tumors [13]. Tang extracted and purified polysaccharides with an average molecular weight (Mr) range of 103–104 Da from bamboo leaves of Phyllostachys heterocycla cv. pubescens, and it had very strong physiological activity [9]. The bioactive BLPS is composed of xylose, arabinose, glucose and galactose, as well as lesser amounts of mannose, rhamnose and uronic acids. In recent years, Ye investigated the total content and chemical constituents of polysaccharides from 17 kind species of bamboo leaves from the Nanjing region, Jiangsu Province. The total content of polysaccharides was Molecules 2015, 20 4164 between 1.9%–5.7% [14,15]. Lu found polysaccharide levels approaching 14.55% in bamboo leaves of Sinocalamus latiflorus McClure [16]. The composition and content of BLPS show very large changes for different varieties, growth place, harvest season, and growth ages. However, so far, there has been little investigation concerning the chemical composition and structural characterization of BLPS from different varieties in China with mono-sugar links, and their average Mr and bioactivity. In this paper, 32 varieties of bamboo leaves from Zhejiang Province in South-East China were chosen to investigate the content of polysaccharides obtained by ultrasound-assisted extraction. Phyllostachys nigra (PN), Phyllostachys vivax (PV), Chimonobambusa quadrangularis (Fenzi) Makino (CQ) and Phyllostachys bambusoides (PB) have been screened to investigate the structural characterization of the purified bamboo leaves polysaccharides (PBLPS) obtained after solution precipitation to remove proteins, ion exchange resin bleaching, and finally purification using DEAE-52 and Sephadex G-100 chromatography. This investigation is of significance to develop new healthcare foods or medicines from BLPS by using the processed bamboo residues. 2. Results and Discussion 2.1. The Components and Content of Polysaccharides in Different Bamboo Leave Varieties Six kinds of standard monosaccharides were mixed, derivatized and checked by gas chromatography as seen in Figure 1. 600,000 6.384 6.627 400,000 5.961 11.274 10.861 Intensity 11.925 200,000 0 0 5 10 15 20 25 30 Time/min Figure 1. GC trace of standard monosaccharides. The retention times of the monsaccharides were determined as 5.961 min for rhamnose, 6.384 min for arabinose, 6.627 min for xylose, 10.861 min for mannose, 11.274 min for glucose and 11.925 min for galactose (Figure 1). A standard curve of glucose was obtained from a linear regression of the content of glucose (C, mg) and absorbance value (A) (Equation (1)). A = 5.8137C − 0.00473(R2 = 0.9987) (1) Molecules 2015, 20 4165 The regression was tested and showed a good linear relation in the content range of 0.0–96.0 μg/mL glucose. By comparison with the relative retention times of the six monosaccharide standards, the monosaccharide composition and their relative contents in 32 species of bamboo leaves were then determined by GC. The BLPS of samples No. 1–No. 32 is mainly composed of 42.6%–62.7% of xylose, 10.2%–24.6% of arabinose, 10.2%–21.2% of glucose, 7.7%–15.7% of galactose and very little rhamnose and mannose. Mannose was not detected in 11 samples: Nos. 3, 4, 7, 8, 10, 11, 13, 14, 18, 19, and No. 26, rhamnose and mannose both were not found in 10 samples: Nos. 15–17, 20–22, and 29–32, while other samples just had less than 6.7% of rhamnose and less than 2.2% of mannose. Another discovery was that samples No. 29–No. 32 of P. nigra (Lodd.) Munro (PN), P. vivax McClure (PV), Chimonobambusa quadrangularis (Fenzi) Makino (CQ), and P. bambussoides cv. Tanakae (PB) not only consisted of four kinds of monosaccharides (xylose, arabinose, glucose and galactose), but the content of galactose is also in a range of 21.5%–34.1%, which is 2–3 fold more than in samples No. 1–No. 28. Beside the chemical composition, a sulfuric acid-anthrone colorimetric assay showed the total polysaccharides content of the 32 samples was between 1.4%–5.4%, and the total polysaccharides of PN, PV, CQ and PB ranged between 4.2%–5.4%, meaning that among the 32 different species listed in Table 1 samples No. 29–No. 32 contained 2–3 fold more polysaccharides than No. 1–No. 28. Therefore, these four typical species were chosen to investigate the structural characterization of BLPS. Table 1. The content of total polysaccharides and monosaccharides from 32 different varieties of bamboo leaves. Total Rhamnose Arabinose Xylose Mannose Glucose Galactose No. Varieties of Bamboo Leaves Polysaccharides (%) (%) (%) (%) (%) (%) (%) P. praecox C.D. 1 1.4 3.2 14.0 48.8 4.7 15.9 13.1 Chu et C.S. Chao 2 CV.