Purification and characterization of a novel polysaccharide–peptide complex from Clinacanthus nutans Lindau leaves

Carbohydrate Polymers 137 (2016) 701–708

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Purification and characterization of a novel polysaccharide–peptide complex from Clinacanthus nutans Lindau leaves Danmin Huang a,b,∗,1 , Yunhong Li a,1 , Fengjie Cui a,∗ , Jun Chen a,c,∗ , Jiamin Sun d a

School of Food and Biological Engineering, Jiangsu University, Zhenjiang, Jiangsu 212013, China Bio Nice Food Science Sdn Bhd, No. 5, Jalan Silc 1/4, Perindustrian Silc, Nusajaya, 79200 Johor, Malaysia School of Pharmacy, Jiangsu University, Zhenjiang, Jiangsu 212013, China d Second Teaching Hospital, Fujian Medical University, Longyan City 364000, China b c

a r t i c l e

i n f o

Article history: Received 2 September 2015 Received in revised form 23 October 2015 Accepted 23 October 2015 Available online 19 November 2015 Keywords: Clinacanthus nutans Polysaccharide Structural characterization Macrophage activation

a b s t r a c t A novel polysaccharide–peptide complex CNP-1-2 with molecular weight of 9.17 × 104 Da was obtained from Clinacanthus nutans Lindau leaves by hot water extraction, ethanol precipitation, and purification with Superdex 200 and DEAE-Sepharose Fast Flow column chromatography. CNP-1-2 exhibited the highest growth inhibitory effect on human gastric cancer cells SGC-7901 with inhibition ratio of 92.34% and stimulated activation of macrophages with NO secretion level of 47.53 ␮mol/L among the polysaccharide fractions. CNP-1-2 comprised approximately 87.25% carbohydrate and 9.37% protein. Monosaccharide analysis suggested that CNP-1-2 was composed of l-rhamnose, l-arabinose, d-mannose, d-glucose and d-galactose with a molar ratio of 1.30:1.00:2.56:4.95:5.09. Methylation analysis, FT-IR, and 1 H NMR spectroscopy analysis revealed that CNP-1-2 might have a backbone consisting of 1,4-linked Glcp, 1,3-linked Glcp, 1,3-linked Manp, 1,4-linked Galp, 1,2,6-linked Galp and 1,2,6-linked Galp. Its side chain might be composed of 1-linked Araf, 1,6-linked Galp and 1-linked Rhap residues. AFM (atomic force micrograph) analysis revealed that CNP-1-2 had the molecular aggregation along with branched and entangled structure. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Recently, polysaccharides obtained from plants and fungi have attracted increasing attentions due to their medicinal values, such as anti-cancer, antioxidant, anti-diabetic and immunobiological activities (Chen et al., 2011; Shen et al., 2013; Sun & Liu, 2009; Xie et al., 2013; Zhou, Yu, Zhang, He & Ma, 2012). Furthermore, most of them are proven to be natural and nontoxic, ideal for producing healthcare foods or medicines (Li, Yuan & Rashid, 2009; Wang et al., 2012). Clinacanthus nutans (Burm. F.) Lindau, commonly known as “Sabah snake grass” in Malaysia, Indonesia and “Erzuihua” in China, which is a well-known traditional medicinal plant in Southeast Asia, is used as a dermic, febrifuge and diuretic (Chen, Zhang, Zhang, Zhang & Xiao, 2015). It possesses a wide range of pharmacological effects, such as antibacterial, antioxidant, anti-proliferative, antiinflammatory, and antiviral activities against varicella-zoster virus

and herpes simplex virus type-2 (Arullappan, Rajamanickam, Thevar & Kodimani, 2014; Sakdarat, Shuyprom, Pientong, Ekalaksananan & Thongchai, 2009; Wanikiat, Panthong, Sujayanon, Yoosook, Rossi & Reutrakul, 2008; Yong et al., 2013). C. nutans contains various chemicals including stigmasterol, lupeol, ␤-sitosterol, belutin, C-glycosyl flavones, sulfur-containing glycosides, glycoglycerolipids, a mixture of nine cerebrosides and a monoacylmonogalatosyl glycerol, chlorophyll derivatives entadamides and clinamides (Sakdarat et al., 2009; Tu et al., 2014). However, to date, no investigations are available about purification and structural characterization of bioactive polysaccharides from C. nutans. Therefore, the goal of the current study was set to (i) bioassayguidedly fractionate and purify the bioactive polysaccharides from traditional medicinal plant C. nutans by gel-filtration chromatography and ion exchange chromatography, and (ii) elucidate its structural characterization by a combination of chemical and instrumental methods. 2. Materials and methods

∗ Corresponding authors at: School of Food and Biological Engineering, Jiangsu University, Zhenjiang, Jiangsu 212013, China. E-mail addresses: [email protected] (D. Huang), [email protected] (F. Cui), [email protected] (J. Chen). 1 These authors are contributed equally to this work. http://dx.doi.org/10.1016/j.carbpol.2015.10.102 0144-8617/© 2015 Elsevier Ltd. All rights reserved.

2.1. Plant material C. nutans (Burm. F.) Lindau plant was cultivated by TK MultiHerbs Enterprise located on 3041, Taman Seremban Baru,

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Fig. 1. Summarized extraction scheme of CNP-1-2 from the leaves of C. nutans.

Seremban, N.S.D.K, Malaysia. Plant was identified and authenticated by Prof. Chen Jun with macroscopic and microscopic examinations and kept at the School of Pharmacy, Jiangsu University (Zhenjiang, China). 2.2. Isolation and fractionation of the polysaccharide from C. nutans The fresh C. nutans leaves were oven dried at 60 ◦ C and then ground to fine powder. As shown in Fig. 1, for extracting polysaccharide fraction, the dried leaves were mixed with distilled water (w/v 1:6) at 90 ◦ C for 4 h. After centrifuging at 10,000 × g for 20 min, the supernatant was concentrated under reduced pressure and precipitated with a final concentration of 75% ethanol for 12 h at 4 ◦ C. The precipitation was collected by centrifugation (10,000 × g, 10 min), followed by dialyzing against distilled water, and lyophilized to yield crude polysaccharides, CNP.

CNP (50 mg) were dissolved in distilled water and filtered through a membrane (0.45 ␮m, Nucleopore), and then the solution was applied to a Superdex 200 (1.6 × 60 cm). The polysaccharide fractions were collected, dialysed against distilled water, and lyophilized. The fraction CNP-1 was proven to have a much higher antiproliferative activity and a much higher ability to stimulate the NO production by RAW264.7 macrophages than CNP-2, so that CNP-1 was further investigated here. The fraction CNP-1 (50 mg) were dissolved in distilled water and filtered through a membrane (0.45 ␮m, Nucleopore). Then the solution was applied to a DEAE-Sepharose fast flow column (2.5 cm × 20 cm) pre-equilibrated with distilled water (pH 7.0). Fractions were prepared in a stepwise elution with distilled water, 0.1, 0.3, 0.5 and 1.0 M NaCl solutions at pH 7.0 at a flow rate of 2.0 ml/min, and with collection of 8 ml for each tube. The polysaccharide content was detected by phenol-sulphuric acid method (Dubois, Gilles, Hamilton, Rebers & Smith, 1956). In addition, the

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protein content was determined spectrophotometrically at 280 nm (Li, Fan & Ding, 2011). Four appropriate fractions named CNP-1-1, CNP-1-2, CNP-1-3 and CNP-1-4 were obtained, concentrated, dialyzed against water, and finally lyophilized for subsequent analysis. Among them, CNP-1-2 showed the highest antiproliferative activity against SGC-7901 cells and stimulated the highest NO production by RAW264.7 macrophages. Thus, herein, CNP-1-2 was collected for further identification of structure and monosaccharide compositions. 2.3. Molecular weight analysis by gel permeation chromatography The molecular weight of the C. nutans polysaccharides CNP-1-2 was determined by high performance gel permeation chromatography (HPGPC). The sample solution was applied to High Performance Liquid Chromatography (HPLC) (Waters, MA, USA) equipped with a TSK-GEL G3000 SWXL column (7.8 mm × 300 mm), eluted with 0.1 mol/L Na2 SO4 solution and detected by a Waters 2414 Refractive Index Detector. The column was calibrated with Dextran T-series standards of known molecular weight (200,000, 70,000, 40,000, 10,000, 5000 Da). The molecular weight of CNP-1-2 was estimated by reference to the calibration curve made above. 2.4. Moisture, fat, ash and uronic acid content The moisture, fat, ash contents were determined according to the methods of AOAC (AOAC, 1990, 15th). Total contents of uronic acid were determined by colorimetric methods of mhydroxydiphenol using glucuronic acid as reference (Blumenkrantz & Asboe, 1973). 2.5. Carbohydrate content and monosaccharide composition analysis Carbohydrate content of CNP-1-2 was determined by the phenol-sulfuric acid method as D-glucose equivalents. Monosaccharide composition analysis of CNP-1-2 was conducted by gas chromatography according to the previously described method (Cui et al., 2014; Cui, Li, Yang, Sun, Wu & Ping, 2014). Briefly, 30 mg of CNP-1-2 was hydrolyzed with sulfuric acid, filtered and applied for the monosaccharide composition in a 6890N GC (Agilent Technologies, Santa Clara, CA, USA) using d-xylose, d-fructose, l-rhamnose, d-arabinose, d-mannose, d-galactose and d-glucose as the standards. 2.6. Protein content and amino acid composition analysis Protein content was evaluated by Bradford Protein Assay Kit (Beyotime Biotechnology, Shanghai, China) using bovine serum albumin as the standard. Amino acids of CNP-1-2 were released by hydrolysis under vacuum in 6 M HCl at 110 ◦ C for 24 h and analyzed with a Hitachi 835-50G automatic amino acid analyzer (Hitachi, Tokyo, Japan) (Su et al., 2014). 2.7. Methylation analysis The CNP-1-2 (20 mg) was methylated 3 times according to Needs and Selvendran (Needs & Selvendran, 1993). Complete methylation was confirmed by the disappearance of the OH band (3200–3700 cm−1 ) in the IR spectrum. The methylated products were hydrolyzed, reduced and acetylated as described by Sweet et al. (Sweet, Shapiro & Albersheim, 1975). The partially methylated alditol acetates were analyzed by gas chromatography-mass spectrometry (GC–MS). GC–MS was done on a HP5890 (II) instrument

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(Hewlett-Packard Component, USA) with an HPS quartz capillary column (30 m × 0.32 mm × 0.25 ␮m). 2.8. IR and NMR analysis FT-IR spectroscopic analysis of CNP-1-2 was recorded on a Nicolet Fourier transform infrared spectrometer (Thermo Nicolet, MA, USA) in a range of 650–4000 cm−1 . For NMR measurements CNP-1-2 was dried in a vacuum over P2 O5 , and then exchanged with deuterium by lyophilizing with D2 O for several times. The deuterium-exchanged polysaccharide (50 mg) was put in a NMR tube and dissolved in 0.7 mL 99.96% D2 O. Spectrum was recorded with a Bruker AV-600 spectrometer (Brucker, IL, USA). The 1 H NMR spectrum was recorded fixing the HOD signal at d 4.70 ppm at 30 ◦ C. 2.9. Atomic force microscopy The topographical AFM image of CNP-1-2 was done according to the described method (He et al., 2013) with some modifications. Briefly, CNP-1-2 was dissolved in de-ionized water with constant stirring and was filtered through a 0.45 ␮m membrane. The sample solution was diluted with de-ionized water to 25 ␮g/ml, placed on a mica disk and dried in ambient air. The atomic force microscopy was operated in contact mode on a MultiMode AFM (NanoScope IIIA controller, Bruker). Three dimensional images were generated with NanoScope Analysis data processing software. 2.10. MTT cell viability assay Human gastric cancer cell line SGC-7901 cells were obtained from American Tissue Culture Collection (ATCC, MD, USA), which were cultured in 5% CO2 at 37 ◦ C in RPMI 1640 medium containing polymyxin B (PMB) and 10% fetal calf serum that was supplemented with 100 U/mL penicillin and 100 g/mL streptomycin. Antiproliferative activity of the C. nutans polysaccharide fractions was determined as follows. SGC-7901 Cells (5 × 104 /well) in exponential growth phase were seeded into each well of a 96-well flat bottomed culture plate and incubated for 24 h before addition of different fractions of C. nutans with the concentration of 50, 100 and 200 ␮g/mL. After incubation for another 48 h, 20 ␮L of the combined MTT (5 mg/mL)/PBS solution was added into each well of the 96 well assay plate. The plates were incubated for 4 h at 37 ◦ C in a humidified, 5% CO2 and the formazan product was solubilized with DMSO. After addition of 150 ␮L of dimethyl sulfoxide (DMSO) and shaking for 10 min, the absorbance of each well was read at 490 nm in the microplate reader (Multiskan GO, Thermo Labsystems, Beverly, MA, USA) and measured values were expressed as mean ± SD. 2.11. Macrophage activation by C. nutans polysaccharide fractions Mouse macrophages (RAW264.7), purchased from the American Tissue Culture Collection (ATCC), were cultured in DMEM medium containing 100 U/mL penicillin, 100 ␮g/mL streptomycin, and 10% fetal bovine serum at 37 ◦ C in a 5% CO2 humidified atmosphere. The effect of a series of concentrations of C. nutans (50, 100 and 200 ␮g/mL) on NO production was determined by analysing NO levels with the Griess reagent. PBS and lipopolysaccharide (1 ␮g/mL) served as negative and positive controls, respectively. 100 ␮l of Griess reagent (equal volumes of 1% (w/v) sulphanilamide in 5% (v/v) phosphoric acid and 0.1% (w/v) naphthyl ethylenediamineHCl) was added to each well with 50 ␮l of the collected supernatant, and allowed to stand in the dark for 20 min at room temperature. NO production was determined by measuring nitrite (NO2 − ) levels in cell supernatants using a colorimetric assay based on the

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Griess reaction. Supernatants (100 ␮L) were reacted with 50 ␮L Griess reagent at room temperature for 20 min, and nitrite was determined by measuring the absorbance at 540 nm using sodium nitrite as the standard. The absorbance at 540 nm was measured and sodium nitrite ranging from 0 to 100 ␮M was used to plot the standard curve (He et al., 2013). 2.12. Statistics Each experiment was repeated three times using duplicate samples. The results were expressed as means ± standard deviations. Statistical comparisons were made by one-way analysis of variance (ANOVA), followed by Duncan’s multiple-comparison test. Differences were considered significant when the p-values were <0.05. 3. Results and discussion 3.1. Isolation and purification of CNP-1-2 Crude polysaccharide extracted from C. nutans (CNP) was obtained as a water-soluble brown-colored powder. Then CNP was purified through a Superdex-200 column, giving two overlapping elution peaks: CNP-1 and CNP-2 (Fig. 2(A)), as detected by the phenol-sulfuric acid assay. CNP-1 mainly contained the high molecular weight polysaccharide, and the CNP-2 mostly contained low molecular weight polysaccharide. In addition, the elution peak of CNP-1 was much bigger than CNP-2, indicating that the polysaccharide content of CNP-1 was much more than CNP-2. MTT analysis (Fig. 5) showed that the inhibition ratios of CNP-1 and CNP-2 on SGC-7901 cells reached to 75.81% and 23.46% at 200 ␮g/mL in 48 h, respectively. The CNP-1 fraction had a much higher growth inhibitory effect on SGC-7901 cells. Besides, macrophage activation assay (Fig. 6) showed that the secretion levels of NO in RAW264.7 cells were measured to be 35.16 ␮mol/L and 9.78 ␮mol/L when the dose of CNP-1 and CNP-2 was 200 ␮g/mL within 48 h, respectively. The ability to stimulate activation of macrophages of CNP-1 was much higher than CNP-2. After DEAE-Sepharose Fast Flow column, four fractions of CNP-1-1, CNP-1-2, CNP-1-3 and CNP-1-4 were eluted. As shown in Fig. 2(B), obviously CNP-1-2 had the highest content of polysaccharide among the four fractions. MTT analysis (Fig. 5) showed that the inhibition ratios of CNP-1-1, CNP-1-2, CNP-1-3 and CNP-1-4 on SGC-7901 cells reached to 11.82%, 92.34%, 85.31% and 22.15% at 200 ␮g/mL in 48 h, respectively. Among them, the fraction CNP-1-2 had the highest growth inhibitory effect. In addition, the secretion levels of NO in RAW264.7 cells were measured to be 13.16 ␮mol/L, 47.53 ␮mol/L, 39.56 ␮mol/L and 9.73 ␮mol/L when the dose of CNP-1-1, CNP-1-2, CNP-1-3 and CNP-1-4 was 200 ␮g/mL within 48 h, respectively (Fig. 6). Among them, CNP-1-2 held the highest ability to stimulate the activation of macrophages.

Fig. 2. (A) Gel filtration of crude extract from C. nutans Lindau leaves on Superdex 200 column. (B) Anion-exchange chromatography of CNP-1 from C. nutans Lindauleaves on DEAE-Sepharose Fast Flow column.

(Na et al., 2010). While another polysaccharide from Cynomorium songaricum Rupr., named CSP-DS1, did not contain uronic acid (Wang, Wang, Huang, Liu & Zhang, 2015). 3.4. Carbohydrate content and monosaccharide composition analysis

HPGPC of CNP-1-2 (Fig. 3(A)) showed a single and symmetrically sharp peak, meaning that the protein and the sugar peak appeared at the same time, and suggesting that CNP-1-2 was a polysaccharide–peptide complex. Based on calibration with standard dextrans, the apparent molecular weight of CNP-1-2 would be 9.17 × 104 Da.

Results from phenol-sulfuric acid assay showed that CNP-1-2 contained 87.25 ± 1.24% carbohydrate. Monosaccharide composition analysis by GC showed that CNP-1-2 was composed of l-rhamnose, l-arabinose, d-mannose d-glucose and d-galactose with a molar ratio of 1.30:1.00:2.56:4.95:5.09, indicating that the polysaccharide was heterogeneous. d-galactose and d-glucose were the most abundant monosaccharides with 34.16% and 33.22% (mol/mol) of the total monosaccharides, respectively. Similarly, dgalactose and d-glucose were the most abundant monosaccharides of PGPW1 isolated from the roots of Panax ginseng (Li, Cai, Geng, Li, Wang & Li, 2012).

3.3. Moisture, fat, ash and uronic acid content

3.5. Protein content and amino acid composition analysis

Moisture, ash and uronic acid content of CNP-1-2 was determined as 1.13 ± 0.32%, 0.5 ± 0.21% and 3.62 ± 0.32% respectively. Fat was not detected in CNP-1-2. Similarly, a polysaccharide SPSCF from Korean Capsosiphon fulvescens contained 4.8% uronic acid

The protein content of CNP-1-2 was found to be 9.37 ± 1.24% by the Bradford Assay. The amino acid composition of CNP-12 was analyzed (Table 1). It had a high content of glutamic acid (12.41 ± 1.26%) and asparagic acid (11.45 ± 0.85%), and a low

3.2. Molecular mass

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Fig. 3. (A) HPGPC profile of CNP-1-2. (B) Fourier transform infrared spectrum of CNP-1-2. (C) 1 H NMR spectrum of CNP-1-2. (D) Atomic force microscopy of CNP-1-2.

Table 1 Amino acid composition of CNP-1-2. Amino acid

Mass%

Asp Glu Ser His Gly Thr Arg Ala Tyr

11.45 12.41 4.63 2.66 6.21 9.05 5.22 6.02 3.30

a

± ± ± ± ± ± ± ± ±

0.85 1.26 0.63 0.14 0.15 1.05 0.38 1.02 0.94

Amino acid

Mass%

Cys Val Met Phe Ile Leu Lys Pro Trp

0.53 ± 0.11 6.92 ± 0.77 1.22 ± 0.14 5.70 ± 0.33 5.59 ± 0.89 7.77 ± 1.03 5.06 ± 0.45 5.91 ± 0.79 n.d.a

Not determined.

content of methionine (1.22 ± 0.14%) and cystine (0.53 ± 0.11%). The amino acid compositions of CNP-1-2 is similar to a Hericium erinaceus glycoprotein HEG-5 which also contained a high content of glutamic acid and asparagic acid and a low content of methionine and cysteine (Cui et al., 2014). 3.6. Methylation analysis CNP-1-2 was methylated, hydrolyzed and converted into alditol acetates for GC–MS analysis. The identification and the proportions

of the methylated alditol acetates of CNP-1-2 were given in Table 2. The results showed that the molar ratios of 2,3,5-Me3 -Araf, 2,3,4-Me3 -Galp, 2,3,6-Me3 -Glcp, 2,3,6-Me3 -Galp, 2,4,6-Me3 -Glcp, 2,4,6-Me3 -Manp, 3,4-Me2 -Galp and 2,3,4,6-Me4 -Rhap were 0.92, 2.12, 2.98, 0.88, 2.21, 2.23, 1.91 and 1.18 according to the peak areas. Glactose based sugar residues (including 1,6-linked Galp, 1,4-linked Galp and 1,2,6-linked Galp) and glucose based sugar residues (including 1,4-linked Glcp and 1,3-linked Glcp) were highly enriched in CNP-1-2, which is consistent with monosaccharide composition analysis. It suggested that CNP-1-2 might have a backbone of 1,4-linked Glcp, 1,3-linked Glcp, 1,3-linked Manp, 1,2,6-linked Galp and small amount of 1,4-linked Galp. In addition, it might have some side chains of 1-linked Araf, 1,6-linked Galp and 1-linked Rhap substituted at C-6 of 1,2,6-linked Galp. 3.7. IR and 1 H NMR analysis As is shown in Fig. 3(B), the infrared (IR) spectrum of CNP-12 displayed a broad stretching characteristic intense peak in the region of 3261.96 cm−1 attributed to hydroxyl and amine groups, and a weak band at 2930.17 cm−1 attributed to C H stretching. The bands around 2360.04 cm−1 indicated aliphatic C H bonds (Xie et al., 2010). Further, the bands at 1597.04 cm−1 and

Table 2 Results of methylation analysis of CNP-1-2. Methylated sugars

Linkages

Molar ratios

Major mass fragments (m/z)

2,3,5- Me3 -Araf 2,3,4-Me3 -Galp 2,3,6-Me3 -Glcp 2,3,6-Me3 -Galp 2,4,6-Me3 -Glcp 2,4,6-Me3 -Manp 3,4-Me2 -Galp 2,3,4,6-Me4 -Rhap

1-linked Arap 1,6-linked Galp 1,4-linked Glcp 1,4-linked Galp 1,3-linked Glcp 1,3-linked Manp 1,2,6-linked Galp 1-linked Rhap

1.00 2.30 3.24 0.96 2.40 2.42 2.08 1.28

43,45,71,87,101,117,129,161 43, 87,99,101,117,129,161,173,189,203 57,71,87,101,117,129,161,203 43,45,87,99,101,117,129,161,233 43,45,71,87,101,117,129,159,161,189 43,45,59,87,118,128,161,234,277 43,71,87,99,129,159,173,189,203 43,71,89,101,117,131,145,161

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Fig. 4. Possible structure of CNP-1-2.

1416.72 cm−1 may be due to asymmetric and symmetric stretching vibrations of carboxylate (Na et al., 2010). The small band at around 1386.11 cm−1 was associated with stretching vibration of C H in the carbohydrate ring. In addition, the absorption at 1237.61 cm−1 could ascertain the presence of protein structures (Xie et al., 2010). The strong band at 1022.83 cm−1 was attributed to the stretching vibrations of pyranose ring (Chen, Xie, Nie, Li & Wang, 2008). According to these attributions, we may speculate that CNP-1-2 is a polysaccharide–peptide complex. As shown in Fig. 3(C), the signals in the range of 5.0–5.6 ppm and 4.0–5.0 ppm in the 1 H NMR spectrum indicated that CNP-1-2 contained ␣-glycosidical configuration and ␤-glycosidical configuration (Huang, Li, Li & Wang, 2011; Wang et al., 2015). The strong signal at 4.70 ppm present in the spectrum is due to HDO. The two weaker signals with similar relative intensity at 5.54 and 5.45 ppm may be attributed to the anomeric protons of ␣-larabinose residues (Pastell, Virkki, Harju, Tuomainen & Tenkanen, 2009; Shakhmatov, Toukach, Kuznetsov & Makarova, 2014; Zheng & Mort, 2008). The high intensities at 5.24, 5.15, 5.10 ppm could be attributed to anomeric protons of ␣-d-glucopyranosyl residues, 3,6-anhydro-␣-l-galactose residues and (1,2,6)-linked-␣d-galactopyranosyl residues, respectively (Sun & Liu, 2009; Wang et al., 2007; Zhang, Li, Li, Wang, Zhu & Yang, 2009). In addition, the small signal at 4.98 ppm represents the existence of (1→3)-linked mannose (Guo et al., 2010). Low signal intensity around 5.65 ppm suggested that CNP-1-2 possibly had low content of uronic acid (Wang et al., 2015). The signals between 3.30 and 4.20 ppm represented ring protons highlighting the presence of pyranose (Poli, Anzelmo, Tommonaro, Pavlova, Casaburi & Nicolaus, 2010). The strong signal at 2.11 ppm may be assigned to the binding of acetyl groups (Popov et al., 2014). The signals at 1.53 and 1.39 ppm from methyl protons were also found (Albert, Friebolin, Marten & Yeman, ˛ Kunikowska & 2013; Paszkiewicz, Tokarska-Pietrzak, Gołebiowski, Stepnowski, 2013). On the basis of the results of HPGPC, GC-MS, IR and NMR, CNP1-2 should contain a backbone of 1,4-linked Glcp, 1,3-linked Glcp, 1,3-linked Manp, 1,2,6-linked Galp and small amount of 1,4-linked Galp. The branches are composed of 1,6-linked Galp with arabinose terminals and rhamnose terminals. The possible structure of CNP1-2 was shown in Fig. 4.

3.9. Antiproliferative activity of CNP-1-2 The growth inhibitory effect of CNP-1-2 against SGC-7901 cells at various concentrations was shown in Fig. 5. As the concentration of CNP-1-2 increased from 50 ␮g/mL to 200 ␮g/mL, its inhibitory effect against SGC-7901 cells increased from 37.88 ± 1.28% to 92.34 ± 0.94% in 48 h. Previous studies also found that C. nutans held a strong antiproliferative activity against cancer cells. For instance, chloroform extract of C. nutans leaves exerted antiproliferative effect on K-562 (91.28 ± 0.03%) and Raji cell lines (88.97 ± 1.07%) at 100 ␮g/ml in 72 h (Yong et al., 2013). In addition, many polysaccharides from plants have shown significant inhibitory activity against various cancer cells. For instance, the Gynostemma pentaphyllum acidic polysaccharides GP-B1 and GP-C1 inhibited four tumor cells

3.8. AFM of CNP-1-2 The topographical AFM image of CNP-1-2 (25 ␮g/mL) was presented in Fig. 3(D). It is apparent that CNP-1-2 was an island-like structure and only slightly disassociated rod-shaped lumps could be seen. It had a diameter and height of about 0.6–2.5 nm, rather than 1.0 nm, and the atomic force image suggested that molecular aggregation was involved, and that the structure was branched and entangled (Satitmanwiwat, Ratanakhanokchai, Laohakunjit, Pason, Tachaapaikoon & Kyu, 2012). Similarly, a polysaccharide–peptide complex TFPS1 isolated from tea flowers had the slight disassociated, branched and entangled structure. However, another polysaccharide–peptide complex from Hirsutella sinensis Liu, Guo, Yu & Zeng formed a three-dimensional interwoven network, in which the main chain and their side-chain of the polymer extended on the topography (He et al., 2013).

Fig. 5. The growth Inhibitory effect on proliferation of SGC-7901 cells (5 × 105 cells/mL) incubated with different concentrations of polysaccharide fractions from C. nutans Lindau leaves at 37 ◦ C for 48 h.

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Acknowledgements This work was supported and funded by Bio Nice Industry Sdn Bhd. References

Fig. 6. Effect of different concentrations of polysaccharide fractions from C. nutans Lindau leaves on NO synthesis in murine macrophage-like cells RAW264.7 Cells (5 × 105 cells/mL) stimulated by each polysaccharide fraction for 48 h.

including MCF-7, HT-29, HepG2 and B16 tumor cells with IC50 values below 200 ␮g/mL (Li et al., 2012). 3.10. Macrophage activation by CNP-1-2 Nitric oxide (NO) as a mediator of macrophages is deemed to be toxic to tumor cells and microorganisms (Shi, Shi & Li, 2014). Herein, we tested the ability of different C. nutans polysaccharide fractions to stimulate the production of NO. The effect of CNP-1-2 on the NO production of RAW264.7 cells was determined by Griess assay (Fig. 6). After 48 h incubation in the presence of 1 ␮g/mL LPS or various concentrations of CNP-1-2, large secretion levels of NO in the media was in a dose-dependent manner. The secretion level of NO in RAW264.7 cells were measured to be 15.28, 32.37 and 47.53 ␮mol/L when the dose of CNP-1-2 was 50, 100 and 200 ␮g/mL within 48 h, respectively. 4. Conclusion A novel polysaccharide–peptide complex CNP-1-2 having significant stimulatory activity of macrophages was purified with a molecular weight of 9.17 × 104 Da from C. nutans Lindau leaves. CNP-1-2 has the backbone of 1,4-linked Glcp, 1,3-linked Glcp, 1,3linked Manp, 1,2,6-linked Galp and side chains of 1-linked Araf, 1,6-linked Galp and 1-linked Rhap substituted at C-6 of 1,2,6-linked Galp. AFM analysis proved its branched and entangled structure of CNP-1-2. Our findings are the first report on purification and characterization of bioactivity polysaccharides from C. nutans Lindau and might facilitate a deeper understanding of chemical structures of C. nutans Lindau polysaccharides. Future studies will focus on finding the structure-activity relationships of CNP-1-2 and developing it as an antitumor agent or an ingredient for healthcare foods.

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