Extraction, partial characterization and bioactivity of polysaccharides from boat-fruited sterculia seeds

Extraction, partial characterization and bioactivity of polysaccharides from boat-fruited sterculia seeds

International Journal of Biological Macromolecules 51 (2012) 815–818 Contents lists available at SciVerse ScienceDirect International Journal of Bio...

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International Journal of Biological Macromolecules 51 (2012) 815–818

Contents lists available at SciVerse ScienceDirect

International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Extraction, partial characterization and bioactivity of polysaccharides from boat-fruited sterculia seeds Lianzhong Ai a,b , Jinhong Wu a , Na Che c , Yan Wu a,∗ , Steve W. Cui d a

School of Agriculture and Biology, Bor S. Luh Food Safety Research Center, Shanghai Jiao Tong University, Shanghai 200240, China State Key Laboratory of Dairy Biotechnology, Technology Center of Bright Dairy and Food Co. Ltd., Shanghai 200436, China Shanghai Tongji Hospital, Tongji University School of Medicine, Shanghai 200065, China d Guelph Food Research Centre, Agriculture and Agri-Food Canada, Guelph, Ontario N1G 5C9, Canada b c

a r t i c l e

i n f o

Article history: Received 29 June 2012 Received in revised form 23 July 2012 Accepted 5 August 2012 Available online 10 August 2012 Keywords: Boat-fruited sterculia seeds Polysaccharides Extraction Physicochemical characterization Bioactivity

a b s t r a c t Three polysaccharides (water-soluble (WSP), alkali-soluble (ASP) and insoluble (IMP)) from boat-fruited sterculia seeds were obtained using different extraction methods. Moisture, ash, protein and total carbohydrate content of WSP, ASP and IMP were analyzed. WSP was rich in glucose, rhamnose, arabinose and galactose while small amount of xylose was also detected. The monosaccharide composition as well its relative content for WSP and ASP were similar. The intrinsic viscosity results demonstrated that ASP had much lower intrinsic viscosity than WSP, indicating partial polysaccharides were degraded into low molecular weight polymers during alkaline extraction. The acute anti-inflammatory bioactive results of polysaccharides indicated that WSP demonstrated an inhibitive effect toward acute inflammation. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Boat-fruited sterculia seed (Semen Sterculiae Lychnophorae) is traditionally both an edible and medicinal resource, specified as the ripe seed of Sterculia lychnophora Hance in the Chinese pharmacopoeia. The original plant is a tropical herb of the Sterculiaceae family, mainly distributed in Vietnam, Thailand, Malaysia, Indonesia as well as Southern China due to the limitation of region and climate [1]. The seed contains a large amount of mucilaginous substance which is used as a traditional medicine in Southeast Asia as well as in China. The mucilage product can be sweetened and consumed as a dessert or canned juice [2]. The seeds are commonly used to treat hoarseness of voice and sore throat by clearing heat from the lungs to resolve phlegm, and treat constipation by relaxing the bowels to clear away toxic substances [3]. The boat-fruited sterculia seeds are just primarily used for making tea, desserts and healthy buccal tablets; however, to our best knowledge, little systematic study for their bioactive components has been done so far. In the past several years, medicinal polysaccharides from plant cell walls have been widely studied for their physicochemical properties and biological activities such as anti-tumor, free radical scavenging, anti-inflammatory, antioxidant, immunomodulating

∗ Corresponding author. Tel.: +86 21 34205715; fax: +86 21 34206918. E-mail address: [email protected] (Y. Wu). 0141-8130/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2012.08.006

and antimicrobial properties [4–10]. However, attention paid to the polysaccharides from boat-fruited sterculia seeds was rather limited, perhaps due to the limitation of its habitat mainly in Southeast Asia. Only a few studies on the content and chemical compositions of the polysaccharides from boat-fruited sterculia seeds have been reported [2,11,12]. Animal experiments of aqueous extracts from seeds revealed that the active component with the anti-inflammatory activity was polysaccharides, which exhibited the function of promoting the peristalsis of small intestines [13]. In order to explore the traditional pharmacological effects of boat-fruited sterculia seeds, it is significant to investigate the structural features and functional properties of these polysaccharides. The objectives of the current project were to sequentially extract the polysaccharides from boat-fruited sterculia seeds by different solvents, and investigate the physicochemical properties and characterize the anti-inflammatory bioactivity after oral administration to mice. 2. Materials and methods 2.1. Plant material and chemicals The boat-fruited sterculia seeds harvested in Vietnam were provided by Shanhe Pharmaceutical Co. Ltd. (Wuxi, China). Meta-hydroxydiphenyl, galacturonic acid, glucose, rhamnose, arabinose, galactose, xylose and mannose were purchased from

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Sigma–Aldrich Trading Co., Ltd. (Shanghai, China). All other chemicals and solvents used were of analytical grade unless otherwise specified. 2.2. Analytical methods Moisture and ash contents of the seeds and polysaccharides were determined according to AOCS (1997) [14] and AOAC (2005) [15] methods until no further decrease in weight (AOCS, Ba 2a-38; AOAC 942.05), while crude protein (CP) content in the seeds and solid polysaccharides was determined using the Kjeldahl method with a conversion factor of 6.25 (AOCS, Ba 4a-38). Crude fat content was determined by Soxhlet apparatus extraction using petroleum ether according to AOAC 945.16. Crude fiber (CF) content was determined according to AOAC 962.09. The total carbohydrate content (%) in the seeds was estimated as: {100 − (%Moisture + %Ash + %CF + %CP + % Crude fat)} [16]. Total reducing sugars in the seeds were determined by the Munson–Walker method (AOAC 945.66). Total carbohydrate in the polysaccharides was determined by the phenol–sulphuric acid colorimetric method using glucose as a standard at 490 nm [17]. Proteins in the solution were estimated by the method of binding of Coomassie Brilliant Blue G-250 to protein using bovine serum albumin as a standard [18]. The total uronic acid was colorimetrically determined according to Blumenkrantz and Asboe-Hansen’s method by measuring the absorbance at 525 nm with galacturonic acid as a standard [19]. The composition of neutral monosaccharide was analyzed by gas chromatography (GC-14A, Shimadzu, Japan). The polysaccharides were dissolved in 2 M trifluoroacetic acid (TFA) and hydrolyzed at 121 ◦ C for 1 h in a sealed glass tube into monosaccharides. The mixture of monosaccharides was reduced with hydroxylamine hydrochloride dissolved in pyridine, and then acetylated using acetic anhydride at 90 ◦ C for 30 min into alditol acetates. The alditol acetate derivatives were separated by a capillary column at a temperature program and then detected with a flame ionization detector (FID). The percentage of monosaccharides in the sample was calculated by the peak areas using normalization with the correction factor. 2.3. Extraction procedure of polysaccharides from boat-fruited sterculia seeds 2.3.1. Water extraction of polysaccharides Boat-fruited sterculia seeds were pretreated into defatted powder, and removed some colored materials, oligosaccharides and other low molecular weight compounds, as described previously [20]. Subsequently, the dried powder was extracted twice with deionized water at 70 ◦ C under constant stirring for 3 h. The supernatant aqueous extract was separated from insoluble mucilage with nylon cloth, and then concentrated in a rotary evaporator under reduced pressure at 45 ◦ C and finally precipitated with four volumes of 95% ethanol at 4 ◦ C for 12 h. After collection by centrifugation, washing with acetone, and then drying in the vacuum state, water-soluble polysaccharide (WSP) was obtained. 2.3.2. Mild alkaline extraction of polysaccharides from insoluble mucilage To avoid the partial degradation of polysaccharides, alkalisoluble polysaccharide (ASP) was extracted twice from insoluble substance mentioned above with 0.05 mol/L NaOH solution (solution to powder ratio at 40:1, at 40 ◦ C, for 2 h). The supernatant alkali-soluble extract was separated, concentrated, precipitated by ethanol, collected and dried, just as WSP was done, and then ASP was obtained. The filtered insoluble mucilage obtained was dried in the vacuum, and then insoluble polysaccharide (IMP) was obtained.

The three polysaccharides (WSP, ASP and IMP) were purified further to remove the free proteins by Sevag method, and then redissolved in distilled water, dialyzed against distilled water (Molecular weight cutoff 10,000 Da), and concentrated and lyophilized, consecutively. 2.4. Calculating the yield and purity of polysaccharides The yield and purity of polysaccharides were calculated by the following formulas: Y (%) =

M2 × 100, M1

P (%) =

M3 × 100 M2

where Y and P are the yield and purity of polysaccharides, respectively. M1 , M2 and M3 are respectively the weight of seed powder, polysaccharides and purified polysaccharides. 2.5. Intrinsic viscosity measurement of polysaccharides The aqueous solutions of polysaccharides were prepared by dispersing in deionized water for 1 h at 60 ◦ C using a magnetic stirrer, and then filtering through 0.45 ␮m nylon syringe filter (Chromatographic Specialties Inc., USA) to remove any insoluble particulate matter. The intrinsic viscosities were determined by dilute solution viscometry using a Cannon-Ubbelohde semi-micro dilution glass viscometer (size 75, viscometer constant 8.433 × 10−3 mm2 /s2 , Kinematic viscosity range 1.6–8 mm2 /s; Cannon Instrument Co., USA) in a constant temperature water bath at 25 ◦ C. The polysaccharide viscosity was measured in duplicates in a concentration range. While the relative viscosity (r ) was kept from 1.2 to 2.0, the polysaccharide solution was essentially Newtonian fluid without end effect correction. Intrinsic viscosity [] was calculated using the following relationship [21]. [] = lim

c→0

  sp

c

= lim

c→0

 ln   r

c

where c is concentration of polysaccharide, r relative viscosity, and sp specific viscosity defined as r − 1. Huggins–Kramer plots of sp /c and (ln r )/c versus c were then used to estimate the intrinsic viscosity [] by extrapolation to zero concentration. 2.6. Acute anti-inflammatory bioactivity assessment of polysaccharides The acute anti-inflammatory activity was evaluated by dimethylbenzene-induced mice ear edema, as described previously [20,22,23]. Male Kunming (KM) mice weighing (20 ± 2) g (provided and identified by Nanjing Qinglongshan Animal Center) were used for the assessment of the acute anti-inflammatory activity. The experiment was not done until the animals were settled to adapt themselves to the new environment. The mice were divided randomly into five groups (eight mice per group). WSP (200 mg/kg day), ASP (200 mg/kg day) and IMP (200 mg/kg day) solutions were prepared with normal saline, and the dose of polysaccharides was determined by the pre-experiments and each administered to a test group of mice. Positive control group mice were treated orally with aspirin at a dose of 100 mg/kg day dissolved in normal saline. Normal group received the same amount of normal saline. 3. Results and discussion 3.1. Chemical composition analysis of boat-fruited sterculia seeds The chemical composition of boat-fruited sterculia seeds from Vietnam as analyzed is presented in Table 1. 54.54% (w/w) of

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Table 1 Chemical composition of boat-fruited sterculia seeds from Vietnam. Main components

Moisture

Ash

Crude protein

Crude fat

Crude fiber

Carbohydrate

Reducing sugars

Content (w/w, %)a

13.33 ± 1.05

6.64 ± 0.28

12.36 ± 0.45

5.89 ± 0.16

7.24 ± 0.45

54.54 ± 1.11

29.45 ± 1.31

a

Each value was expressed as mean ± SD (SD = standard deviation, n = 3, number of replicates)

Table 2 Yield and purity of WSP, ASP and IMP.

Table 4 Neutral sugar compositions of WSP, ASP and IMP.

Sample

Yield (w/w, %)

Purity (w/w, %)

Sample

Rhamnose

Arabinose

Xylose

Glucose

Galactose

WSP ASP IMP

13.8 9.6 10.5

69.7 44.1 80.2

WSP ASP IMPa

10.00 12.12 12.40

7.88 7.44 7.67

0.77 1.18 1.08

25.65 12.41 0.33

5.04 4.85 4.55

a

carbohydrates and 29.45% (w/w) of reducing sugars were determined in the seeds. It was calculated that the polysaccharides content reached 25% of the seed, which is almost the same as that of the Zizyphus jujuba Mill [24]. It showed that the boat-fruited sterculia seed was an excellent source of polysaccharides. 3.2. Yield and purity of polysaccharides

Table 5 Intrinsic viscosity of WSP and ASP in water. Polysaccharide sample

WSP

ASP

IMP

Intrinsic viscosity (dL/g)

1.71

0.35

n.d.a

a

The yield of the seed residue obtained by degreasing pretreatment was 70.5% indicating that almost 30% of low molecular weight compounds were removed. By removing the proteins using Sevag method, the yield and purity of water-soluble polysaccharides (WSP), alkali-soluble polysaccharides (ASP) and insoluble polysaccharides (IMP) are shown in Table 2. The yield of WSP (13.8%) accounted for 40.7% of the total (33.9%), the yield of IMP (10.5%) was 31.0% of the total, while ASP yield was relatively low and the proportion was only 28.3%, probably because ASP was extracted with alkali solution at low temperature. The purity of ASP was lower than that of WSP which could be caused by alkali-soluble impurities. Finally, nearly 60% polysaccharides were present in water-insoluble fraction, while IMP accounted for 31% of the total polysaccharides, and appeared to present a swollen state in the water solution at high temperature. 3.3. Chemical composition analysis of polysaccharides Moisture, ash, protein and total carbohydrate content of WSP, ASP and IMP are listed in Table 3. All the extracts appeared dark brown. The ash and protein contents (12.34% and 13.54%) of ASP were higher than those of WSP and IMP, while total carbohydrate content (44.14%) in ASP was the lowest among three polysaccharides (WSP 69.74% and IMP 80.18%). The chemical composition of WPS is almost consistent with that of gum obtained by water extraction method of Phimolsiripol et al. [25]. Most of water-insoluble polysaccharides are either covalently linked to other components or physically trapped in the cell wall matrix [2]. Treatment of the cell walls with alkali can cause cellulose to swell and disrupt the hydrogen bonds between hemicellulose and cellulose, resulting in the solubilization of hemicellulose. Hence under alkaline conditions partial polysaccharides could be effectively released and converted into soluble ones; moreover, the

Monosaccharides were given in mass ratio.

Not detected.

extraction ratio of polysaccharides increased. At the same time, proteins and minerals associated tightly with the polysaccharides were also extracted. 3.4. Neutral monosaccharide composition of polysaccharides The neutral sugar compositions of WSP, ASP and IMP are presented in Table 4. These polysaccharides were heteropolymers containing a range of neutral sugars, including rhamnose, arabinose, galactose, glucose and small amount of xylose. Rhamnose, arabinose, galactose and uronic acids were observed in WSP, ASP and IMP indicating probably pectin-like polysaccharides. Glucose was the predominant sugar in WSP and ASP, whereas only a trace amount was observed in IMP, maybe because the hemicellulose like the glucan in the cell walls were dissolved. In addition, the monosaccharide mass ratios (Rha:Ara:Xyl:Glu:Gal) of WSP (1.0:0.8:0.1:2.6:0.5) and ASP (1:0.6:0.1:1.0:0.4) were a little similar. 3.5. Intrinsic viscosity of polysaccharides Intrinsic viscosity [] of WSP and ASP in water were estimated from Huggins–Kramer plots by extrapolation to zero concentration and are shown in Table 5. Intrinsic viscosity of the macromolecules is usually related tightly to molecular weight according to Mark–Houwink–Sakurada equation [26], [] = kMv˛ , for polymers with similar structure, the larger the average molecular weight is, the higher the intrinsic viscosity becomes. Intrinsic viscosity of ASP (0.35 dL/g) is obviously lower than that of WSP (1.71 dL/g), indicating that partial polysaccharides during the alkaline extraction were possibly degraded into low molecular weight ones. Namely, the polysaccharide macromolecular structure was probably disrupted.

Table 3 Chemical composition of WSP, ASP and IMP obtained from boat-fruited sterculia seeds. Polysaccharide Sample

Moisture

Ash

Protein

Total carbohydrate

Uronic acid

WSP (w/w, %) ASP (w/w, %) IMP (w/w, %)

8.21 ± 0.49 8.73 ± 0.61 7.98 ± 0.66

7.37 ± 0.34 12.34 ± 0.22 5.05 ± 0.71

9.12 ± 0.74 13.54 ± 0.63 3.45 ± 0.41a

69.74 ± 1.52 44.14 ± 1.85 80.18 ± 1.74b

14.78 ± 1.01 7.35 ± 1.72 5.35 ± 1.03b

a b

Determined by Kjeldahl method. Sample was first hydrolyzed before measurement.

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Table 6 Effects of polysaccharides on ear edema induced by dimethylbenzene in mice. Group

Dose (mg/kg day)

Edema (X ± S, mg)

Normal control Positive control WSP test group ASP test group IMP test group

0 100 200 200 200

18.02 12.25 13.98 16.32 17.95

a b

± ± ± ± ±

2.34 2.53b 3.01a 4.03a 2.68a

Edema rate (%) 107.51 72.12 88.33 103.05 106.79

± ± ± ± ±

5.65 4.33 4.78 5.15 5.97

Inhibition rate (%) 0 32.92 17.84 4.15 0.67

P < 0.05. P < 0.01 vs. the normal control.

3.6. Anti-inflammatory bioactivity assessment of polysaccharides The faucitis or pharyngitis is a familiar disease occurring easily in the winter and spring dry seasons, and non-steroidal antiinflammatory drugs (NSAIDs) and antibiotics in modern medicine are still the most commonly used remedies. However, NSAIDs can cause several serious adverse effects, the most important being from gastric injury to gastric ulceration and renal damage [27]. In addition, antibiotic medicines can cause drug resistance after being taken by human beings for a long time, and finally affect the body health seriously. Currently man has greater interests in natural products with medicinal properties, particularly those obtained from plants. Boat-fruited sterculia seed used as both medicine and food is commonly used for the treatment of many diseases on the upper respiratory tract, e.g., for clearing phlegm (by “clearing heat from the lungs” as explained in Chinese medicine), and relieving sore throat to restore the voice; and thus they are prospected to exhibit anti-inflammatory bioactivity and resist the faucitis or pharyngitis. Acute anti-inflammatory activity of polysaccharides was tested in a model of ear edema formation in mice induced by dimethylbenzene. The effects of polysaccharides on ear edema are shown in Table 6. The test groups showed significant (P < 0.05) yet differing inhibition of ear edema induced by the stimulus against the normal group. From the inhibition rate, IMP (inhibition rate 0.67%) exhibited extremely low or negligible anti-inflammatory activity and ASP (4.15%) had the bioactivity but very low, whereas WSP (17.84%) presented a fairly significant inhibitory effect. At the same time, the inhibition rate of WSP (17.84%) on the inflammation was 54.2% that of aspirin (32.92%) used in this study. This indicates that WSP could play a key role in inhibiting on the acute inflammation. ASP bioactivity was much lower than that of WSP, there were two possible reasons: (1) the spatial structure of ASP extracted with alkali was destroyed to a certain degree so that the functions were affected; (2) despite their similarity in monosaccharide mass ratio, the uronic acid content of WSP was higher than that of ASP, uronic acid contained polysaccharide might contribute to the bioactivity. As a result, WSP would be subjected to further investigation to delineate its structure–function relationship. 4. Conclusions On the basis of the results stated above, it is concluded that three kinds of polysaccharides (WSP, ASP and IMP) could be obtained by different extraction methods from boat-fruited sterculia seeds. The water-soluble ones (WSP) were approved to possess valuable acute anti-inflammatory bioactivity. Hence, aqueous extracts of boatfruited sterculia seeds, usually consumed as a tea drink in China, may be used as an accessible source of natural anti-inflammation with potential health benefits. Boat-fruited sterculia seeds maybe deserved to be explored by food and pharmaceutical industries

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