Molar Mass Distribution and Chain Conformation of Polysaccharides from Fortunella margarita (Lour.) Swingle①

ZENG Hong-Liang ZHANG Yi LIU Jun ZHENG Bao-Dong

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Molar Mass Distribution and Chain Conformation of Polysaccharides from(Lour.)①

ZENG Hong-Liang ZHANG Yi LIU Jun ZHENG Bao-Dong②

(350002)

In order to study the effects of different extraction methods on the molar mass distribution and chain conformation of(Lour.)polysaccharides (FP), we used extraction by hot water (WFP), ultrasonic-assisted treatment (UFP), microwave-assisted treatment (MFP) and ultrasonic/microwave-assisted treatment (UMFP), and then Fourier transform infrared (FT-IR) spectroscopy as well as1H and13C nuclear magnetic resonance (NMR) spectroscopy to characterize the structural properties of FP extracted. The molar weight (w), polydispersity index (w/n), root-mean-square (RMS) turning radius (g), molar mass distribution and chain conformation of FP were studied systematically using size-exclusion chromatography (SEC), multi-angle laser light-scattering (MALLS) and refractive index (RI). WFP, UFP, MFP and UMFP are all typical carbohydrates according to1H NMR,13C NMR and FT-IR measurements. The type of glycosidic linkage is mainly a-glycosidic bond with a small amount of-glycosidic bond. The results obtained by the SEC-MALLS-RI system showed the molar masses of WFP and UMFP were distributed mainly in the range of 5.0×106～1.0×107g·mol–1and they accounted for 57.80% and 56.84% of total FP, respectively. The molar masses of UFP and MFP were distributed mainly in the 1.0×106～5.0×106g·mol–1range, which accounted for 38.24% and 52.39% of FP, respectively. WFP and UMFP in water were uniform spherical polymers; UFP and MFP were typical highly branched polymers and the degree of branching for MFP was higher compared to UFP. These results indicated the ultrasonic- and microwave-assisted extraction methods caused a significant decrease of the molar mass of FP but the ultrasonic/microwave synergistic extraction method had no effect.

(Lour), polysaccharides, molar mass distribution, chain conformation

1 INTRODUCTION

(Lour.)(known as kumquat or cumquat), the smallest citrus fruit, thri- ves as a native species in central China[1, 2]. It is highly nutritious, containing a variety of vitamins, amino acids, non-starch polysaccharides, essential oils, limonoids, flavonoids and other active substan- ces[3, 4]. It helps to prevent the rupture of blood vessels by reducing the fragility and permeability of blood capillaries and slowing the hardening of arteries. It is beneficial for patients suffering from high blood pressure, hardening of the arteries and coronary heart disease by its two-way adjustment effect on blood pressure. It is used also in traditional herbal medicines, especially for colds and coughs[5-7].

Polysaccharides are one of the major biological active components of(Lour.)Optimization of the extraction technique ofpolysaccharides (FP) via response surface analysis was reported by Zeng[8]. The optimal con- ditions for extracting FP were determined to be a water/material ratio of 38:1 (mL:g), temperature 88 ℃, extraction time 2.5 h and concentration of ethanol 70% (v/v). Under these conditions, the extraction rate of FP could reach 1.81% with three extraction cycles. Removal of protein from FP preparations by the action of protease was reported by Zhang[9]. The optimal conditions for protein removal were an enzyme content of 120 U·mL–1with hydrolysis for 80 min at 40 ℃. Under these conditions, the proportion of protein removed could reach 75.88% accompanied by a loss of 5.45% of total FP. After FP were treated three times with Sevag reagent (CHCl3:BuOH (4:1, v/v)), the extent of protein removal could reach 85.18%; however, the accompanying extent of FP loss was 14.16% of total FP. The antibacterial and antioxidative activi- ties of FP were reported by Zeng[10]. The results showed they have some antimicrobial effects onRosenbach,,and. They have a certain ability to scavenge ·OH, O2–· and DPPH·, and the efficiency of scavenging increased with increased concentration. The molar mass distribution and chain conformation of, however, have not been reported. The yields of polysaccharides extracted by different methods were not the same and molar mass values were different. Average molecular weight is detected often by gel-permeation chromatography, which is not a good method (low degree of effi- ciency and large deviations) for assessing the effects of extraction methods on the molar mass distribution and chain conformation of polysaccharides[11-13].

This study used different methods to extract FP (hot water (WFP), ultrasonic-assisted treatment (UFP), microwave-assisted treatment (MFP) and ultrasonic/microwave-assisted treatment (UMFP)) and then used Fourier transform infrared (FT-IR) spectroscopy as well as1H and13C nuclear magnetic resonance (NMR) spectroscopy to characterize their structural properties. Molecular weight (w), poly- dispersity index (w/n), root-mean-square (RMS) turning radius (g), molar mass distribution and chain conformation were determined systemati- cally by size-exclusion chromatography (SEC), multi-angle laser light-scattering (MALLS) and refractive index (RI). The objective of this work was to study the effects of different extraction methods on the structural properties, molar mass distribution and chain conformation of FPThe results provide theoretical basis for the research on effective extrac- tion, efficacy components and structure-function relationship of FP and its comprehensive use.

2 EXPERIMENTAL

2.1 Extraction of polysaccharides

WFP: After selecting, cleaning and removing seeds, fresh(Lour.)was added to ten volumes of water and crushed in a juicer. The cloudy juice obtained was poured into a beaker and kept at 80 ℃for 2.5 h in a constant temperature water bath (HH-6, Ronghua, Jiangsu, China). The filtrate obtained by passage through nylon cloth (100-mesh sieve), centrifuging (900 g-force, 15 min), removing protein, decoloring and concentrating was placed into four volumes of ethanol and allowed to form the sediment for 24 h (25 ℃). The supernatant was discarded and the precipitate was freeze-dried.

UFP: The cloudy juice obtained as described above for WFP was poured into a beaker, kept at 45 ℃for 70 min, and treated with an ultrasonic processor (ultrasonic power 180 W; KQ-400KDV, Kunshan, Zhejiang, China). The filtrate obtained by passage through nylon cloth was added to four volumes of ethanol, centrifuged, protein removed, decolored and concentrated as described above for WFP to form the sediment for 24 h (25 ℃). The supernatant was discarded and the precipitate was freeze-dried.

MFP: The cloudy juice obtained as described above was poured into a beaker and kept for 20 min in a microwave oven (microwave power 640 W; EG823LC8-NS, Midea, Guangzhou, China). The filtrate was treated as described above and the pre- cipitate was freeze-dried.

UMFP: The cloudy juice obtained as described above was poured into a beaker and kept for 5 min exposed to an ultrasonic power of 125 W and a microwave power of 100 W in a microwave/ultra- sonic combination reaction system (XO-SM200, Xianou, Nanjing, China). The filtrate was treated and the precipitate was freeze-dried as described above.

2.2 FT-IR spectroscopy

The FP samples and KBr were air-dried to cons- tant weight at 105 ℃ in order to remove the free water or crystal water and eliminate the interference of water molecule absorption peaks[14]. 2 mg of sample FP was weighed and placed into an agate mortar, and then 400 mg of dried KBr powder was added. After grinding evenly under the lamp, the powder was placed into a compression mold and formed into tablets at reduced pressure[15, 16]. The organic functional groups of FP were identified with an FT-IR spectroscope (Thermo Nicolet AVATAR 360, Madison, USA) with scanning wavelength range of 4000～400 cm–1, scanning time of 16～32 s and resolution of 4 cm–1.

2.3 NMR spectroscopy

1H NMR and13C NMR spectra were obtained at 35 ℃ with an NMR spectrometer (AVANCE III 500, Bruker, Germany). 30 mg of sample FP was dis- solved in2H2O and freeze-dried three times. The deuterium-exchanged FP was dissolved in2H2O again and analyzed by NMR spectrometry using dimethyl silicon pentane sodium sulfonate as the internal standard.

2.4 Size-exclusion chromatography

A guard column (SB-G, 6×50 mm, particle size 10 μm) and an SEC column (SHODEX OHPAK SB-806 HQ, 8mm × 300mm, particle size 13 μm) were connected sequentially. The mobile phase was 0.1 mol·L–1NaCl ?ltered through a membrane ?lter (0.45 μm pore size; Millipore Corp., MA, USA) and degassed at reduced pressure for ~1 h at room temperature. The refractive index of the eluent was 1.333 at 30 ℃, wavelength 658.0 nm and flow rate 0.5 mL·min–1. The FP solution was filtered through a membrane ?lter (0.45 μm pore size; Millipore Corp., MA, USA) and 1.5 mL of filtered FP solution (0.1 mg·mL–1) was injected into the SEC column.

2.5 SEC-MALLS-RI system

A pump (515 HPLC, Waters, USA), an injector (Waters, USA) with a 100 μL loop (Rheodyne, IDEX Corp.,USA), SEC columns (OHpak SB-G and SB-806M HQ columns, Shodex, Tokyo, Japan), a MALLS instrument (632.8 nm, DAWN DSP, Wyatt Technology, CA, USA) and an RI detector (RI-101, Shodex, Tokyo, Japan) were connected in series. Bovine serum albumin was used to determine the delay volume between MALLS and RI (0.145 mL). The refractive index (d/d) was set as 0.145 mL·g–1according to the literature[17].wandgof FP were determined under light-scattering intensity of different angles. The correction coefficient of RI was measured using a series of standard solutions of NaCl. The 90o photodiode detector of MALLS was corrected by toluene (calibration constant: 1.4462 × 10–4) and the other photodiode detectors of 17 different angles were normalized using dextran[18].

2.6 Statistical analysis

The data obtained by FT-IR spectroscopy were analyzed by OPUS Spectroscopy software. The data obtained by1H NMR and13C NMR were analyzed by MestReNova software. The data obtained by the SEC-MALLS-RI system were analyzed by ASTRA 6.1 software.

3 RESULTS AND DISCUSSION

3.1 FT-IR spectroscopy

The FT-IR spectra of WFP, UFP, MFP and UMFP are shown in Fig. 1. Each of these four kinds of FP had absorption peaks characteristic of carbohydrate in the range of 4000～400 cm–1. Among WFP, UFP, MFP and UMFP, there was a stretching vibration of O–H of a carbohydrate intra- or intermolecular hydrogen bond at 3200～3600 cm–1. The peak at ～2927 cm–1was assigned to the stretching vibration of C–H in a methylene group (-CH2-). The peak at ～1742 cm–1was assigned to the stretching vibration of C=O in an aliphatic group (-O–CO–R), which showed there was some high methoxy pectin present. The strong peak at 1630 cm–1was caused by the stretching vibration of a free carboxylic carbonyl group. The peaks at ～1420 and ～1335 cm–1were caused by the bending vibration of C–H. The peaks in the range of 1300～1000 cm–1were characteristic of carbohydrate; the peaks at 1248 and 1056 cm–1were caused by the stretching vibration of C–O–C on the glycosidic ring and C–OH on the uronic group, respectively. There was a small peak at 890 cm–1and a weak peak at 815 cm–1, indicating the glycosidic bonds of WFP, UFP, MFP and UMFP were mainly-glycosidic bonds with a small amount of-glycosidic ones.

Fig. 1. FT-IR spectra of WFP, UFP, MFP and UMFP

3.2 NMR spectroscopy

1H NMR and13C NMR spectroscopy was used to analyze the glycosidic bond type and the connection position of the carbohydrate chain of polysaccha- rides[19]. According to the FT-IR spectrogram, the functional groups and fingerprint regions of WFP, UFP, MFP and UMFP were fundamentally the same. Therefore, WFP was selected to analyze the structure of FP further by1H NMR and13C NMR spectroscopy.

3.2.11H NMR

The1H NMR spectra of WFP are shown in Fig. 2. The signals from FP were concentrated mostly in the range of3.2～5.4 ppm and those in the3.2～4.5 ppm range were assigned to the proton of a glycosidic ring. The chemical shifts () of hydrogen on the anomeric carbon shown in Fig. 2 were 4.52, 4.70, 5.02, 5.16, 5.25 and 5.35 ppm, indicating the FP contained five kinds of anomeric protons. The proton signal of WFP >5.0 ppm was significantly smaller compared to WFP <5.0 ppm, indicating the type of glycosidic bond in WFP was mainly-glycosidic, consistent with the results of FT-IR spectroscopy. The signals at1.18 and1.20 ppm in the high-field region of0.8～1.4 ppm were caused by the methyl (H6) proton of 6-deoxidized carbo- hydrate.

Fig. 2.1H NMR spectra of WFP

3.2.213C NMR

The13C NMR spectra of WFP are shown in Fig. 3. There were four signals (92.19, 96.13, 98.09 and 103.70 ppm) in the resonance region of anomeric carbon, indicating FP had four kinds of sugar residues and their relative contents were 54.72: 15.07:7.53:42.25, respectively. The proton signal of WFP in the range of100～105 ppm was significantly greater compared to WFP in the range of95～100 ppm, indicating the type of glycosidic bond in WFP was mainlyglycosidic, consistent with the results of FT-IR and1H NMR analyses. The signals at13.58 and 14.15 ppm in the high-field region were caused by the methyl carbon signal of rhamnose and the signal peaks in the1H NMR spectra corresponding to them were1.18 and 1.20 ppm, respectively. The signals at29.42 and 29.68 ppm were caused by the methyl carbon signal of ethanoyl and the signal peaks in the1H NMR spectra corresponding to them were2.01 and 2.10 ppm, respectively. The signals of FP were concentrated mostly in the range of60～85 ppm, indicating the pyranoid ring accounted for a large proportion of FP.

Fig. 3.13C NMR spectra of WFP

3.3 Molar mass distribution of FP

According to the chromatograms of molar mass distribution of WFP, UFP, MFP and UMFP (Fig. 4), their molecular weights were distributed in the range of 1.0×105～1.0×109g·mol–1, revealing the molar mass distribution of FP was wide, thus indicating a high molecular weight polymer. As shown in Fig. 4, the molecular structures characteristics of FP were determined as the calculated mass of 6.13×10–5 g, dn/dc of 0.145 mL·g–1and the Zimm model. The results showed thewvalues for WFP, UFP, MFP and UMFP were 6.512×106(±1.242%), 4.152×106(±2.012%), 3.396×106(±3.476%) and 6.012×106(±3.132%) g·mol–1. Thew/nvalues were 2.961 (±3.243%), 3.128 (±12.203%), 4.486 (±10.415%) and 2.463 (±2.295%) and thegvalues were 44.8 (±2.0%), 62.8 (±1.3%), 121.8 (±2.0%) and 52.8 (±1.5%) nm, suggesting FP was a kind of mode- rately dispersed macromolecule. Thewof UMFP was the same as that of WFP, and thewof UFP and MFP were smaller, showing the ultrasonic- and microwave-assisted extraction methods had dec- reased the molar mass of FP, consistent with the results reported by Zhou[20, 21]. The degradation caused by the microwave-assisted extraction method was greater compared to the ultrasonic-assisted one. By contrast, the ultrasonic/microwave synergistic extraction method had no effect on the molecular weight of FP probably because the ultrasonic/micro- wave synergistic extraction was in low power and at short time, improving the yield of FP without damaging the molecular structure.

On the basis of the analysis of weight-average molecular weight, the molar mass distribution of FP was divided into four intervals; analysis with ASTRA software suggested they were <1.0×106, 1.0×106~5.0×106, 5.0×106~1.0×107and >1.0×107g·mol–1. The molar mass distributions of WFP, UFP, MFP and UMFP are given in Table 1. The molar masses of WFP and UMFP were distributed mainly in the range of 5.0×106～1.0×107g·mol–1, and they accounted for 57.80% and 56.84% of the total FP, respectively. The molar masses of UFP and MFP were distributed mainly in the range of 1.0×106～5.0×106g·mol–1and respectively accounted for 38.24% and 52.39% of the total FP. The results indicated the ultrasonic- and microwave-assisted extraction methods caused significant degradation of the molar mass of FP, which was degraded from 5.0×106～1.0×107to 1.0×106～5.0×106g·mol–1. By contrast, the ultrasonic/microwave synergistic extraction method had no influence upon the molar mass of FP, which was consistent with the measurements ofw.

3.4 Chain conformation of FP

The chain conformation of FP in water was obtained by measuringwandgand the chain conformation was confirmed by examining the plot slope of RMS forgagainst the molar mass. Wyatt[22]showed that when the slope of the line was lower than 0.33, between 0.5 and 0.6, and 1, the polymer in solution was in a tight uniform spherical conformation, a random coil conformation, and a rod-like conformation correspondingly. The chroma- togram of the chain conformation of FP in water is shown in Fig. 5. The slopes of the lines of WFP and UMFP in water were 0.17±0.00 and 0.04±0.00, respectively, indicating they were close uniform spherical polymers. The graphs of UFP and MFP were similar to a U-shaped curve, so they were typical highly branched polymers and the branching degree of MFP was higher compared to UFP. This might be because MFP had greatergcompared to UFP, thereby causing the MFP molecule in water to adopt a U-shaped highly branched conformation.

Table 1. Molar Mass Distribution of WFP, UFP, MFP and UMFP

Values are mean ± standard deviation (= 3).

Fig. 4. Chromatograms of the molar mass distribution of polysaccharides from(Lour.). (a) WFP; (b) UFP; (c) MFP; (d) UMFP

Fig. 5. Chromatogram of chain conformation of polysaccharides from(Lour.)in water. (a) WFP; (b) UFP; (c) MFP; (d) UMFP

4 CONCLUSION

According to the FT-IR spectroscopy results, WFP, UFP, MFP and UMFP had absorption peaks charac- teristic of carbohydrate in the range of 4000～400 cm–1. Their functional groups and fingerprint region were fundamentally the same. The glycosidic bond type of WFP, UFP, MFP and UMFP was mainly-glycosidic bond with a small amount of-glyco- sidic one, consistent with the results of1H and13C NMR spectroscopy.

The FP are moderately dispersed macromolecules. The molar mass of WFP and UMFP are distributed mainly in the 5.0×106～1.0×107g·mol–1range and account for 57.80% and 56.84% of the total FP, respectively. The molar masses of UFP and MFP are distributed mainly in the range of 1.0×106～5.0×106g·mol-1and they account for 38.24% and 52.39% of total FP, respectively. WFP and UMFP in water are close uniform spherical polymers. By contrast, UFP and MFP are highly branched polymers and the branching degree of MFP is higher than that of UFP. The ultrasonic- and microwave-assisted extraction methods have significant degradation effects on the molar mass of FP but the ultrasonic/microwave synergistic extraction method has no effect. The ultrasonic/microwave synergistic extraction is in low power and at short time, causing little damage and improving the yield of FP.

(1) Jayaprakasha, G. K.; Chidambara Murthy, K. N.; Etlinger, M.; Mantur, S. M.; Patil, B. S. Radical scavenging capacities and inhibition of human prostate (LNCaP) cell proliferation by.2012, 131, 184–191.

(2) Barreca, D.; Bellocco, E.; Caristi, C.; Leuzzi, U.; Gattuso, G. Kumquat (Fortunella japonica Swingle) juice: flavonoid distribution and antioxidant properties.2011, 44, 2190–2197.

(3) Peng, L. W.; Sheu, M. J.; Lin, L. Y.; Wu, C. T.; Chiang, H. M.; Lin, W. H.; Lee, M. C.; Chen, H. C. Effect of heat treatments on the essential oils of kumquat (Fortunella margarita Swingle).2013, 136, 532–537.

(4) Lin, C. C.; Hung, P. F.; Ho, S. C. Heat treatment enhances the NO-suppressing and peroxynitrite-intercepting activities of kumquat (Fortunella margarita Swingle) peel.2008, 109, 95–103.

(5) Wang, Y. C.; Chuang, Y. C.; Ku, Y. H. Quantitation of bioactive compounds in citrus fruits cultivated in Taiwan.2007, 102, 1163–1171.

(6) Wang, Y.; Fu, X. Z.; Liu, J. H.; Hong, N. Differential structure and physiological response to canker challenge between ‘Meiwa’ kumquat and ‘Newhall’ navel orange with contrasting resistance.2011, 128, 115–123.

(7) Agócs, A.; Nagy, V.; Szabó, Z.; Márk, L.; Ohmacht, R.; Deli, J. Comparative study on the carotenoid composition of the peel and the pulp of different citrus species.2007, 8, 390–394.

(8) Zeng, H. L.; Lu, X.; Bian, Z. Y.; Lin, Y. F.; Zhang, Y. Optimization of the extraction technique of Fortunella margarita polysaccharides via response surface analysis.2012, 41, 315–319.

(9) Zhang, Y.; Zeng, H. L.; Zeng, S. X.; Lin, Y. F.; Zheng, B. D. Protein removal from Fortunella margarita polysaccharides by protease.2012, 33, 166–170.

(10) Zeng, H. L.. Fujian Agriculture and Forestry University, Fuzhou 2012.

(11) Tian, Y.; Zeng, H.; Xu, Z.; Zheng, B.; Lin, Y.; Gan, C.; Lo, Y. M. Ultrasonic-assisted extraction and antioxidant activity of polysaccharides recovered from white button mushroom (Agaricus bisporus).2012, 88, 522–529.

(12) Zhao, T.; Mao, G.; Feng, W.; Mao, R.; Gu, X.; Li, T.; Li, Q.; Bao, Y.; Yang, L.; Wu, X. Isolation, characterization and antioxidant activity of polysaccharide from Schisandra sphenanthera.2014, 105, 26–33.

(13) Luo, D. Structural investigation of a polysaccharide (DMB) purified from Dioscorea nipponica Makino.2014, 103, 261–266.

(14) Wen, C. R.; Wang, L. X.; Wang, Z. J.; Wu, C. H.; Liu, Y. N.; He, M. X.; Peng, S. H.; Pang, J. Effect of plasma treatment on the structures and properties of Konjac glucomannan film.2013, 32, 17–22.

(15) Wang, L. X.; Xiao, L. X.; Cai, L. G.; Yin, N.; Kou, D. D.; Pang, J. Influence of Konjac glucomannan on the crystallization morphology and structure of calcium oxalate.2013, 32, 831–838.

(16) Wang, L. X.; Wen, C. R.; Wu, J.; Lin, H. D.; Hu, S. G.; Pang, J. Studies on the molecular chain conformation stability of aminated Konjac glucomannan.2013, 32, 1845–1853.

(17) Shakun, M.; Maier, H.; Heinze, T.; Kilz, P.; Radke, W. Molar mass characterization of sodium carboxymethyl cellulose by SEC-MALLS.2013, 95, 550–559.

(18) Sun, Y. J.; Ye, X. Q.; Pang, J.; Li, J.; Lu, Y. Molecular dynamics simulations of the interactions between Konjac glucomannan and carrageenan.2009, 28, 439–444.

(19) Li, J. E.; Cui, S. W.; Nie, S. P.; Xie, M. Y. Structure and biological activities of a pectic polysaccharide from Mosla chinensis maxim. cv. Jiangxiangru.2014, 105, 276–284.

(20) Zhou, C.; Yu, X.; Zhang, Y.; He, R.; Ma, H. Ultrasonic degradation, purification and analysis of structure and antioxidant activity of polysaccharide from Porphyra yezoensis Udea.2012, 87, 2046–2051.

(21) Zhou, C.; Yu, X.; Ma, H.; Liu, S.; Qin, X.; Yagoub, A. E. G. A.; Owusu, J. Examining of athermal effects in microwave-induced glucose/glycine reaction and degradation of polysaccharide from.2013, 97, 38–44.

(22) Wyatt, P. J. Light scattering and the absolute characterization of macromolecules.1993, 272, 1–40.

3 March 2014;

16 May 2014

① Supported by Science and Technology Plan Major Project of Fujian Province (2013Y0003), Agricultural Products (fruits and vegetables) Processing Engineering Technology Research Center Funding Projects of Fujian Province (2009N2002), Scientific and Technological Innovation Team Support Plan of Institution of Higher Learning in Fujian Province ([2012]03), and Scientific and Technological Innovation Team Support Plan of Fujian Agriculture and Forestry University (cxtd12009)

. Born in 1967, professor, major in food science. E-mail: zbdfst@163.com