Purification and Characterization of Water Soluble Polysaccharides from Sweet Cherries, Raspberries, by Shirley Wang

Research in Health and Nutrition (RHN) Volume 3, 2015

www.seipub.org/rhn

Purification and Characterization of Water Soluble Polysaccharides from Sweet Cherries, Raspberries, and Ginseng: Chemical Composition and Bioactivity Kelly A. Ross*1, David Godfrey1, Lana Fukumoto1 1

Pacific Agri-Food Research Centre, Agriculture and Agri-Food Canada, Summerland, BC, Canada

Kelly.Ross@agr.gc.ca; David.Godfrey@agr.gc.ca; Lana.Fukumoto@agr.gc.ca Abstract Water soluble polysaccharides were isolated from three varieties of sweet cherries, raspberries, and ginseng berry pulp using a hot water extraction regime followed by ethanolic precipitation and resolubilization in water and centrifugation to ensure water solubility of the polysaccharides at conditions relevant for chemical characterization and bioactivity tests. The water soluble polysaccharides were purified into water eluted/neutral and NaCl eluted/acidic fractions using anion exchange chromatography. The water soluble polysaccharides and purified fractions were evaluated for their chemical composition and molecular weight. In terms of chemical in vitro bioactivity tests, the antioxidant activity (FRAP and ABTS assays) and potential of the polysaccharides to serve as α-amylase and α-glucosidase inhibitors were determined. All of the polysaccharides contained protein and phenolics and in most cases the NaCl eluted/acidic fractions contained higher levels of these compounds compared with water soluble polysaccharides and water eluted/neutral fractions. The sugar monomer and uronic acid content of the purified polysaccharides was influenced by both fruit type and fraction. The water eluted/neutral polysaccharide fractions from the cherry and raspberry samples were of higher molecular weights compared to their respective NaCl eluted/acidic fractions while the opposite was seen for the ginseng berry pulp polysaccharides. All of the polysaccharides possessed antioxidant activity. Of the fractions, only the NaCl eluted/acidic cherry polysaccharides demonstrated α-glucosidase inhibition with values exceeding those of the acarbose control. This work provides information necessary to aid in defining the relationship between chemical characteristics of polysaccharide and bioactivity. Results from this study support the concept that polysaccharides may act as health promoting compounds in fruits and serves as an impetus for increased work in this area. Keywords Fruit; Water Soluble Polysaccharides; Fractionation; Chemical Composition; Bioactivity

Introduction A diet high in fruits and vegetables has been linked to the prevention of degenerative diseases such as cardiovascular diseases, diabetes, cancer, and arthritis (Adnan et al., 2011) and it is well established that the phenolic compounds present in fruits and vegetables may be important components of health promoting diets (Herken and Guzel, 2010). Additionally, water extractable polysaccharides which may be pure or conjugated with proteins, lipids, or phenolics, have emerged as an important class of compounds present in plant material that possess biological activities (Pawlaczyk et al., 2011; Ross and Mazza, 2012; Ross et al., 2014). Information documenting the bioactivity of water extractable polysaccharides mainly exists for polysaccharides isolated from non-fruit plant materials such as tea, herbs, and fungi (Chen et al., 2008a; Chen et al., 2008b; Inngjerdingen et al., 2005) and to a limited extent, work characterizing the bioactivity of polysaccharides present in fruits and vegetables has been performed (Ross et al., 2014). It has been reported that water extractable polysaccharides isolated from wolfberry/goji berry (Lycium barbarum) possess important bioactive functions, including hypoglycemic and hypolipidemic activities (Luo et al., 2004), immuno-modulating action (Gan et al., 2004) and antioxidant activity (Fan et al., 2010; Li et al., 2007). Control of postprandial hyperglycemia by retarding absorption of glucose by inhibition of carbohydrate–hydrolysing enzymes, such α-amylase and α-glucosidase enzymes, has been suggested to be important in the treatment of diabetes (Chen et al., 2009). The α-amylase inhibitors work to slow down the rate of starch digestion of food and restrain the increase of post-meal blood sugar (Wang

www.seipub.org/rhn

et al., 2010) while the α-glucosidase inhibitors prevent degradation of disaccharides to monosaccharides (Wang et al., 2010). Therefore inhibition of α-amylase and α-glucosidase enzymes has been investigated as a means for controlling diabetes and many efforts have been made to search for effective and safe α-amylase and α-glucosidase inhibitors natural materials (Chen et al., 2009; Palanuvej et al., 2009; Zhang et al., 2010). Purification of polysaccharides by column chromatography may influence bioactivity. Li et al. (2010) studied the antioxidant activity of purified fractions of water soluble polysaccharides isolated from pumpkin and found that different fractions exhibited different bioactivity levels. The works of Sun and Kennedy (2010) and Sun et al. (2010) also reported on the influence of fractionation using anion exchange chromatography on the antioxidant activity of polysaccharides. Also, the influence of anion exchange fractionation on the anticoagulant and anti-platelet activity of a polysaccharide-phenolic preparation obtained from the hot water extraction of the medicinal plant Erigeron canadensis L. was reported by Pawlacyk et al. (2011). Therefore purified polysaccharide fractions may show different bioactivities compared to crude hot water polysaccharide extracts and should be investigated. The present work examines whether water soluble polysaccharides and corresponding purified fractions contribute to the health effects of fruits such as cherries, raspberries, and ginseng berries. This work provides information on the chemical composition, molecular weight along with the in vitro antioxidant activities, α-amylase, and α-glucosidase activities of water soluble polysaccharides and corresponding purified fractions isolated from these fruits. This work examines the relationships between chemical composition and bioactivities of water soluble polysaccharides and the corresponding purified fractions present in sweet cherries, raspberries, and ginseng berry pulp, which is not well established and is an area of research that requires continued study. Materials and Methods Samples Sweet cherries (Prunus avium L.), including three varieties (Lapins, Skeena and Sweetheart) were harvested at commercial maturity. All cherries were picked from the orchard of the Agriculture and AgriFood Canada, Pacific Agri-Food Research Centre,

Research in Health and Nutrition (RHN) Volume 3, 2015

Summerland, BC. The fresh cherries were de-stemmed and de-pitted by hand and then frozen and stored at 20 °C until use. Ginseng (Panax quinquefolius) berry pulp was obtained from Agriculture and Agri-Food Canada (London, ON) and collected from a cooperating southern Ontario commercial producer. The samples were frozen as soon as possible (about 1 hour) after field collection and kept at -20 °C until use. The raspberries (Rubus idaeus) were purchased from Triple Crown Packers (Langley, BC) and were received in a frozen condition and kept at -20 °C. All of the frozen fruit samples were freeze dried (Virtis Freeze Dryer, Model 50-SRC-5, Gardiner, NY), placed in polyethylene bags and stored at -20 °C until being subjected to extraction. All chemicals for analyses unless explicitly stated otherwise were obtained from Sigma-Aldrich Co.(St. Louis, MO). Extraction and Purification of Water Soluble Fruit Polysaccharides Polysaccharides were extracted from the fruits samples using methods adapted from the works of Ross et al. (2014) and Wu et al. (2007). Figure 1 shows the extraction scheme for obtaining water soluble polysaccharides from sweet cherry, raspberry, and ginseng berry pulp. It was noted that the crude polysaccharides (CPS) were extracted using a water solvent that was at a temperature higher (70 °C) than the temperature of test solutions used for chemical characterization and bioactivity determination. To remove substances potentially insoluble at lower temperatures and thereby ensure solubility at conditions (temperature and concentrations) used for testing, the CPS product was dissolved in deionized distilled water at a concentration of 10 mg/mL and the solution was covered and stirred using a Heidolph stir place for 16-24 h. After stirring, the solution was centrifuged at 11 687 x g at room temperature for 30 min. The supernatant was filtered through a Whatman No. 41 (55mm) paper on a Buchner funnel with vacuum. The precipitate was gel-like (i.e. not solid) and was termed the insoluble polysaccharide (IPS) product. The filtered supernatant was freeze dried to obtain the water soluble polysaccharide (SPS) product. After freeze drying the SPS was vacuum dried overnight at 60 °C to ensure complete dryness. This procedure is in agreement with the method described by Wu et al. (2007). The yield of the SPS was determined by weighing the product amount. The portion of the SPS product that was intended for chemical characterization and bioactivity testing was

Research in Health and Nutrition (RHN) Volume 3, 2015

sub-sampled and cryomilled for 1.5 min (Cryomill, SpexCerti Prep, Model 6800-115) to obtained a product with uniform particle size. After cryomilling, the samples were placed in polyethylene bags and stored at -20 °C until chemical and bioactivity analysis. The tests solutions for the chemical characterization and bioactivity tests were made up at concentrations no greater than 5 mg/ml which is in agreement with concentrations used for polysaccharide tests solution reported in the works of Pawlacyk et al., (2011); Sun and Kennedy, (2010), Sun et al., (2010) and Wang et al. (2010). The SPS products exhibited obtained from the Skeena cherries, Sweetheart cherries, Lapins cherries, ginseng berry pulp, and raspberries exhibited solubilities of 71.3, 63.1, 75.5, 92.1, and 95.2%, respectively. From these results it was inferred that the SPS products would be completely soluble at concentrations of 5 mg/mL at room temperature. The following is the coding for these samples: SPSSK=water soluble polysaccharides from Skeena variety cherries, SPS-SH= water soluble polysaccharides from Sweeheart variety cherries, SPS-LP=water soluble polysaccharides from Lapin variety cherries, SPSRB=water soluble polysaccharides from raspberries, and SPS-GBP=water soluble polysaccharides from ginseng berry pulp. Purification of Polysaccharides

FIG. 2. REPRESENTATIVE PROFILE OF POLYSACCHARIDES FROM SKEENA CHERRIES ON A DEAE SEPHAROSE FAST FLOW COLUMN ELUTED WITH DISTILLED WATER AND A STEPWISE INCREASE OF 0.1M NACL AQUEOUS SOLUTION AT A FLOW RATE OF 5 ML/MIN

www.seipub.org/rhn

Purification of the water soluble polysaccharides (i.e. SPS product) was performed via fractionation using a medium pressure chromatography system (BioLogic DuoFlow Chromatography System, Bio-Rad Laboratories, USA). The samples were made up to 1 mg/mL concentration and fractionated by anion exchange chromatography on a DEAE Sepharose Fast Flow column (inside diameter 50 mm, length 20 cm). The column was first calibrated with water (750mL or 5 column volumes). The 950 mL of sample (1 mg/mL) was loaded onto the column. The column was first eluted with water (750 mL or 5 column volumes) and followed by a stepwise change to 1 M NaCl (1500 mL or 10 column volumes) using a flow rate of 5mL/min. The water eluted/neutral polysaccharide (WEPS) and 1 M NaCl eluted/acidic polysaccharide (SEPS) fractions were collected with a fraction collector. The fractions were collected as 34 mL volumes in each tube. The carbohydrate content of each tube was measured at 490 nm using the phenolic sulfuric acid method (Dubois et al., 1956) and the protein content of each tube was determined using the Bradford’s Coomassie Brilliant Blue method (Bradford, 1976). A representative chromatogram of the profile of the isolated fractions is provided in Figure 2. The fractions containing carbohydrates were pooled and dialysed using dialysis tubing (Spectra/Por) with a molecular weight cut-off (MWCO) of 1000 Da. The tubing dimensions were 45mm x 29 mm. The length of the tubing used was 90 cm as the capacity of the tubing was 6.4 mL/cm. The water was changed every 6-16 h and four water changes were used during the dialysis period. Upon completion of the dialysis the fractions were freeze dried. After freeze drying, the fractions were cryomilled for 1.5 min (Cryomill, SpexCerti Prep, Model 6800-115) to obtain a product with uniform particle size. After cryomilling the samples were placed in polyethylene bags and stored at -20 °C until chemical and bioactivity analysis. The following is the coding for these samples: WEPS-SK=water eluted/ neutral polysaccharide fraction from Skeena variety cherries, WEPS-SH=water eluted/neutral polysaccharide fraction from Sweetheart variety cherries, WEPSSH=water eluted/neutral polysaccharide fraction from Lapins variety cherries, WEPS-RB=water eluted/ neutral polysaccharide fraction from raspberries, WEPS-GBP=water eluted/neutral polysaccharide fraction from ginseng berry pulp, SEPS-SK= 1M NaCl eluted/acidic polysaccharide fraction from Skeena variety cherries, SEPS-SH= 1M NaCl eluted/acidic polysaccharide fraction from Sweetheart variety

www.seipub.org/rhn

Research in Health and Nutrition (RHN) Volume 3, 2015

cherries, SEPS-LP= 1M NaCl eluted/acidic polysaccharide fraction from Lapins variety cherries, SEPS-RB= 1M NaCl eluted/acidic polysaccharide fraction from raspberries, and SEPS-GPB= 1M NaCl eluted/acidic polysaccharide fraction from ginseng berry pulp

chromatography (HPSEC) following the method described by Ross et al. (2014). In accordance with the method of Carnachan et al. (2012), molecular weights of polysaccharides were estimated by comparison of the retention/elution times with those of the dextran and glucose standards by use of a standard curve.

Chemical Analyses 1) Carbohydrate Content and Uronic Acid Determination Total carbohydrates in the water soluble polysaccharides and purified fractions were assayed by phenol-sulfuric acid (Dubois et al., 1956) using glucose as a standard as described by Ross et al. (2014). Uronic acid content of the SPS and purified fractions was quantified by the Scott method (Blakeney et al., 1983) as decribed by Ross et al. (2014). Experiments were performed in duplicate and results are expressed on a dry weight basis 2) Neutral Sugars Monomer Content Determination The neutral sugar monomer content of the water soluble polysaccharides and purified fractions was determined using the methods of Fan et al. (2010) and Blakeney et al. (1984) as described by Ross et al. (2014). Experiments were performed in duplicate and results are expressed on a dry weight basis. 3) Protein Content Determination Protein content was estimated from the nitrogen content of the water soluble polysaccharides and purified fractions using the method of Tamaki and Mazza (2010) as described by Ross et al. (2014). 4) Total Phenolics The total phenolics content of the water soluble polysaccharides and purified fractions was determined by the Folin-Ciocalteu colorimetric method based on the procedure of Singleton and Rossi (1965) as described by Ross et al. (2014). The total amount of phenolic content was expresses at mg gallic acid equivalent per gram polysaccharide sample (mg GAE g-1 polysaccharide sample) 5) Molecular Weight Determination The molecular weight of the water soluble polysaccharides and purified fractions was determined with high performance size exclusion

Bioactivity Tests 1) Antioxidant Activity-Chemical In Vitro Assays The antioxidant activity of the water soluble polysaccharides and purified fractions in terms of 1) reducing power towards Fe(III) and the ability to act as a radical scavenging compound was investigated by the Ferric Reducing Antioxidant Power (FRAP) assay and the ABTS•+ radical scavenging assay, respectively. Details of this method are provided in Ross et al. (2014). The results are expressed as Trolox equivalents (TE)/g polysaccharide on a dry weight basis. 2) Assay for Determination of Inhibition of αAmylase Activity The ability of the water soluble polysaccharides and purified fractions to inhibit α- amylase was determined using an assay based on the hydrolysis of 2-chloro-4-nitrophenol-α-D-maltotrioside (CNPG3) by α-amylase (Kumanan et al., 2010). An αAmylase enzyme from porcine pancreas, Type VIwas used and exhibited an activity level of 450 U/mL in 50 mM phosphate buffer at pH 6.8. A solution of 0.5 mM CNPG3 (2-chloro-4nitrophenol-α-D-maltotrioside) in 50 mM phosphate buffer at pH 6.8 was prepared. Acarbose was used as the positive control inhibitor. An acarbose solution of 5 mg/mL was prepared in Ultrapure water. Aqueous solutions of the polysaccharide samples (5 mg/mL) were prepared. For analysis, 25 µL of the aqueous polysaccharide samples or acarbose control was added to a well on a 96 well plate. To each polysaccharide sample or acarbose control in each well, 25 µL of 450 U/mL αamylase enzyme was added followed by the addition of 200 uL of 0.5 mM CNPG3. After incubating for 9 min at room temperature, 25 uL of 1 M Na2CO3 was added to stop the reaction and the absorbance at 405 nm was measured. A plate reader (SpectraMax M2, Molecular Devices, Sunnyvale, CA) was used to measure spectrophotometric absorbance. Absorbance was

Research in Health and Nutrition (RHN) Volume 3, 2015

measured for each sample in triplicate for each sample. A blank reaction was carried out without the test sample. 3) Assay for Determination of Inhibition of αGlucosidase Activity The inhibition assay for α-glucosidase activity was performed based on the method of Zhang et al. (2010) and Wu et al. (2012) with modifications. The enzyme α-glucosidase from Saccharomyces was used. The enzyme was prepared to exhibit an activity level of 1 U/mL α-amylase in 50 mM phosphate buffer at pH 6.8. A solution of 0.5 mM pnitrophenyl-α-D-glucopyranoside (pNPG) in 50 mM phosphate buffer at pH 6.8 was prepared. Acarbose was used as the positive control inhibitor. An acarbose solution of 5 mg/mL was prepared in Ultrapure water. For this assay, aqueous solutions of the polysaccharide samples (5 mg/mL) were prepared. For analysis, 25 µL of the aqueous polysaccharide samples or acarbose control was added to a well on a 96 well plate. To each polysaccharide sample or acarbose control in each well, 25 µL of 1 U/mL α-glucosidase enzyme was added followed by the addition of 200 uL of 0.5 mM pNPG was added. After incubating for 14 min at room temperature, 25 uL of 1 M Na2CO3 was added to stop the reaction and the absorbance at 405 nm was measured. A plate reader (SpectraMax M2, Molecular Devices, Sunnyvale, CA) was used to measure spectrophotometric absorbance. Absorbance was measured for each sample in triplicate for each sample. A blank reaction was carried out without the test sample. For both α-amylase and α-glucosidase acitivites, the calculation of results is presented as: % Inhibition = [(Absorbance blank-Absorbance sample)/Absorbance blank] × 100 For both α-amylase and α-glucosidase acitivites the overall results are expressed as % Relative Inhibition which is the inhibition provided by the sample compared to the inhibition provided by 5 mg/mL acarbose. The calculation of these results is presented as: % Relative Inhibition = [% Inhibition sample/% Inhibition acarbose] × 100 Statistical Analysis Statistical analysis was conducted using SAS Institute Inc. Software, version 9.1 (SAS Institute, 2001). Data

www.seipub.org/rhn

were subjected to analysis of variance (ANOVA) with replication using the SAS PROC GLM procedure. Least square (LS) means and Least Significant Difference (LSD) at 5 % significance level were generated using the SAS PROC GLM procedure. Results and Discussion Characterization of Water Soluble Polysaccharides and Purified Fractions Table 1 shows the yield of the water soluble polysaccharides extracted with hot water extraction from the Skeena, Sweetheart, and Lapins cherry varieties, ginseng berry pulp, and raspberries and corresponding water eluted/neutral and NaCl eluted/acidic fractions. The highest water soluble polysaccharide yields were obtained from the raspberries with a value of 2.08% (dry matter basis) and the ginseng berry pulp with a value of 1.35 % (dry matter basis). The yields of the water soluble polysaccharides obtained from the Skeena, Sweetheart, and Lapins cherries were lower with values of 0.65, 0.50, and 0.66% (dry matter basis), respectively. All of these yields were slightly lower than the yields reported for hot water extracted crude polysaccharides from Ross et al. (2014). This indicates that resolubilizing the hot water extracted crude polysaccharides at a concentration of 10mg/mL at room temperature followed by centrifugation to remove any potential insolubles did remove a small amount of material (i.e. a small portion of the crude hot water extracted polysaccharides was insoluble at room temperature). In general, the water eluted/ neutral fractions were the main component of the water soluble polysaccharides as proportions ranged from 25-57% while the NaCl/acidic fractions made up a smaller component of the water soluble polysaccharides as proportions ranged from 24 to 29%. The values of the percent of each fraction obtained after purification on DEAE Sepharose column indicate that losses did occur as 100% recovery was not recorded. The total carbohydrate content of the water soluble polysaccharides from all of the fruit samples ranged from ~22 to 42%. The water soluble polysaccharides obtained from the mature Lapins cherries demonstrated the highest total carbohydrate contents of 42.65% while the water soluble polysaccharides obtained from the ginseng berry pulp and raspberries demonstrated the lowest total carbohydrate contents of 21.83 and 23.16%. These results are comparable to

www.seipub.org/rhn

Research in Health and Nutrition (RHN) Volume 3, 2015

those obtained by Ross et al. (2014). The NaCl eluted/acidic fractions contained the highest proportion of carbohydrates ranging from ∼ 43 to 72%. The neutral fractions all presented the lowest carbohydrates contents, ∼ 10 to 28%. The protein contents of the water soluble polysaccharides obtained from the various fruit samples ranged from ~6 to 17 %, also indicating that these water soluble polysaccharides were conjugated

with protein moieties. These results are comparable to those presented for the crude polysaccharides extracted with hot water in the work of Ross et al. (2014). Generally, the protein contents of the water eluted/neutral fractions obtained from the cherry samples and the ginseng berry pulp samples possessed lower protein contents than corresponding NaCl eluted/acidic fractions. The opposite result was observed for the raspberry samples.

Fruit sample, freeze dried and ground

80% ethanol extraction x 3 Separation of solids/liquids with centrifugation

Supernatant, 80% ethanol solubles

Solids, residue 70°C water extraction x 2

Separation of solids/liquids with centrifugation

Supernatant

Residue

Water removal & precipitate with 90% ethanol

Separation of solids/liquids with centrifugation

Residue, crude polysaccharide (CPS) product

Supernatant, 90% ethanol solubles

Make up CPS solution to 10mg/mL & centrifuge Separation of solubles and insolubles with centrifugation Fractionation of SPS on DEAE Fast Flow Sepharose Column (D 50mm x 20cm) via elution with water followed by 1M NaCl to yield WEPS & SEPS, respectively

Supernatant, soluble polysaccharide (SPS) product

Water eluted/neutral (WEPS) polysaccharide product

Residue, insoluble polysaccharide (IPS) product

NaCl eluted/acidic polysaccharide (SEPS) product

FIG. 1. SCHEME OF EXTRACTION OF WATER SOLUBLE POLYSACCHARIDES FROM CHERRY, RASPBERRY, AND GINSENG FRUITS AND PURIFICATION OF FRACTIONS

Research in Health and Nutrition (RHN) Volume 3, 2015

The water soluble polysaccharides along with the water eluted/neutral and NaCl eluted/acidic fractions were analyzed for total phenolics content and the results indicated (Table 1) that the water soluble polysaccharides and corresponding fractions obtained from all of the fruit samples contained phenolics with total phenolics contents ranging from ~3 to 55 mg GAE/g polysaccharide. The phenolics content of the water soluble polysaccharides were in close agreement with the phenolics content of the water extractable crude polysaccharides reported by Ross et al (2014). In all cases, the water eluted/neutral fractions contained the lower amounts of phenolics compared to the corresponding water soluble polysaccharides. Except for those obtained from the Skeena variety cherry and raspberry samples which showed total phenolic content values significantly lower than the corresponding water soluble polysaccharides, all of the NaCl eluted/acidic fractions contained phenolics at levels comparable to those of the corresponding water soluble fractions. To our knowledge this work is the first to characterize the phenolic content of water soluble polysaccharides and corresponding water eluted/neutral fractions and NaCl eluted/acidic fractions isolated from cherries, raspberries, and ginseng berry pulp. The molecular weight data contained in the high performance size exclusion (HPSEC) chromatograms of the water soluble polysaccharides and corresponding water eluted/neutral and NaCl eluted/acidic fractions are presented in Table 1. These molecular weights were associated with multimodal distributions from HPSEC results and spanned the entire molecular weight range of the standards, 6300 to 0.18 kDa. The molecular weight values presented represent the main molecular weight population residing in this class size and values in parenthesis represent the proportion of population associated with the reported molecular weight. Overall of the water soluble polysaccharides, those obtained from the raspberry samples showed the highest molecular weight of 82.3 kDa and the molecular weight of the water soluble polysaccharides obtained from the ginseng berry pulp samples showed the smallest molecular weight of 2.06 kDa. The water soluble polysaccharides obtained from the cherry samples ranged from 56.4 -62.7 kDa. The water eluted/neutral fractions obtained from the cherry and raspberry samples demonstrated higher molecular weight values compared to the corresponding water soluble

www.seipub.org/rhn

polysaccharides while the corresponding NaCl eluted/acidic fractions showed the lowest molecular weights. The opposite results were observed for the water eluted/neutral and NaCl eluted/acidic fractions obtained from the ginseng berry samples as the water eluted/neutral fractions had the smallest molecular weight (0.372 kDa) and the NaCl eluted/acidic fractions demonstrated the highest molecular weight (28.7 kDa). Table 2 shows results for the neutral sugars content, uronic acid content, and sugar monomer profiles of the water soluble polysaccharides and corresponding water eluted/neutral and NaCl eluted/acidic fractions. The neutral sugars content of the water soluble polysaccharides ranged from ~19 to 49% for all of the fruit samples, which as expected, were comparable to the values for total carbohydrates. The NaCl eluted/acidic fractions contained the highest proportion of neutral sugars ranging from â&#x2C6;ź 41 to 79%. The water/neutral fractions all presented the lower neutral sugars contents, â&#x2C6;ź 9 to 39% compared to the NaCl eluted/acidic fractions. The uronic acid content of the water soluble polysaccharides ranged from 431%. The uronic acid content of the water eluted/neutral fractions ranged from 0.92 to 35.73% while the uronic acid content of the NaCl eluted/acidic fractions ranged from 8.23 to 23.32%. Unexpectedly, the uronic acid content of the water eluted/neutral fractions was higher than that of the NaCl eluted/acidic fractions for all samples except those obtained from the ginseng berry pulp. Ni et al. (2010) examined the chemical composition of polysaccharides from the leaves of Panax ginseng C. A. Meyer that was fractionated using anion exchange chromatography and found that the acidic fraction eluted at the highest salt concentration contained lower levels of uronic acid and galacturonic acid compared to fractions eluted at lower salt concentrations, which is in agreement with out work. Also, Pawlaczyk et al. (2011) used anion exchange chromatography to fractionate polysaccharides from isolated from the medicinal plant Erigeron canadensis L. and found that some of the fractions eluted at higher salt concentrations contained less uronic acid. It is possible that the NaCl eluted/acidic fractions obtained from cherries and raspberries contained proteins and phenolic compounds that possessed a stronger negative charge compared to the fraction containing a higher proportion of uronic acid and therefore eluted under the 1 M NaCl condition.

www.seipub.org/rhn

The sugar monomer content of the water soluble polysaccharides obtained from all the fruit was determined. All fruits contained rhamnose, arabinose, xylose, mannose, galactose, and glucose. Generally, the main sugars found in the all of the water soluble polysaccharides from each fruit sample were arabinose, galactose, and glucose. Our results are mostly in agreement with those of the literature (Fan et al., 2010; Ross et al., 2014). For the purified fractions, for all fruit samples the NaCl eluted fractions showed a decrease in glucose and an increase in galactose content. Fucose was detected at a level of 0.41% in the NaCl eluted/acidic fraction of the ginseng berry pulp polysaccharides. The mannose content of the NaCl eluted/acidic fractions obtained from the cherry samples was enhanced while the mannose content of the water eluted neutral fractions of the ginseng berry pulp and raspberry samples was enhanced. The arabinose content of the water eluted/neutral factions obtained from the ginseng berry pulp and raspberry samples was less than the values observed for the corresponding water soluble polysaccharides and NaCl eluted/neutral fractions. Antioxidant Activity of Water Extractable Crude Polysaccharides The results of the antioxidant activity of the water soluble polysaccharides and corresponding water eluted/ neutral and NaCl eluted/neutral fractions in terms of 1) the reducing power towards Fe(III) and 2) the ability to act as a radical scavenging compound was investigated by the Ferric Reducing Antioxidant Power (FRAP) assay and the ABTS•+ radical scavenging assay, respectively, are presented in Table 3. The water soluble polysaccharides obtained from the raspberry and ginseng berry pulp samples demonstrated the highest antioxidant activities, 375.3 and 159.35 µmole Trolox eq/g polysaccharide (PS) , respectively, as determined with the ABTS•+ radical scavenging assay, and 261.28 and 100.14 µmole Trolox eq/g PS , respectively, as determined using the FRAP assay. The water soluble polysaccharides obtained from the Sweetheart, Skeena, and Lapins cherries demonstrated relatively lower antioxidant activities ranging from 57.76 to 96.76 µmole Trolox eq/g PS, respectively, as determined with the ABTS•+ radical scavenging assay, and 40.03 to 72.15 µmole Trolox eq/g PS, respectively, as determined using the FRAP assay. Fan et al. (2010) examined the antioxidant activity of crude polysaccharides from cherries using the ABTS•+ radical scavenging assay and ORAC (oxygen radical

Research in Health and Nutrition (RHN) Volume 3, 2015

absorbance capacity) antioxidant activity assay, and reported a value of 159.3 µmole Trolox eq/g polysaccharide, which is in agreement with the values reported in our work. Also, the values reported for the water soluble polysaccharides for the cherry, ginseng berry pulp and raspberry samples were comparable to the values presented for the hot water crude polysaccharides obtained from cherry, ginseng berry pulp and raspberry samples presented by Ross et al. (2014) which were reported using the ABTS•+ radical scavenging assay as, ∼ 57-94, 438, and 185 µmole Trolox/g PS respectively and ∼31-55, 264, and 96 µmole Trolox/g PS, respectively as obtained using the FRAP assay. This indicates that here is a very small influence of the solubilisation step on the polysaccharide antioxidant activities. In terms of the antioxidant activities of the purified fractions, except for the raspberry samples, all of the water eluted/neutral fractions demonstrated the lowest antioxidant activities. For the raspberry samples the NaCl eluted/acidic fraction possessed the lowest antioxidant activity. Therefore in our work, the water soluble polysaccharides and purified fractions obtained from the various fruit samples exhibited different antioxidant activities along with different chemical characteristics. The bioactivities of polysaccharides can be affected by many factors including chemical components, molecular mass, structure, conformation, extraction and isolation methods (Mateos-Aparico et al., 2010; Li et al., 2010). The polysaccharides obtained from the raspberry and ginseng berry pulp, possessed higher antioxidant activities compared to the polysaccharides obtained from the different cherry varieties. All of the water soluble polysaccharides obtained from the different fruits, except those obtained from the ginseng berry pulp, demonstrated higher antioxidant activities compared to corresponding water eluted/neutral and NaCl eluted/acidic fractions. Antioxidant activity of fruits and vegetables often correlates with total phenolics content (Thetsrimuang et al., 2011) and the polysaccharides all did contain phenolic compounds (Table 1). The water soluble polysaccharides obtained from the ginseng berry pulp and raspberries contained the both highest antioxidant activity and phenolic content while the water soluble polysaccharides obtained from the different cherry varieties possessed the lower antioxidant activities and phenolic contents. This does suggest that phenolic compounds may be contributing to the antioxidant activity. However, for

Research in Health and Nutrition (RHN) Volume 3, 2015

the water soluble cherry polysaccharides and the corresponding NaCl eluted/acidic fractions, the antioxidant activity of the fraction is at least 2.31 X less than the value observed for the water soluble polysaccharides while the phenolic content of the fraction is only at most 1.36 X less than the phenolic content of the water soluble polysaccharides. The work of Chen et al. (2004) reported a direct relationship between the uronic acid contents and the antioxidant activity of tea polysaccharide conjugates which may partially explain these results as the uronic acid content of the NaCl eluted/acidic cherry fractions is less than the uronic acid content of the corresponding water soluble polysaccharides. The water soluble polysaccharides obtained from the ginseng berry pulp and raspberries contained comparable phenolic contents yet the antioxidant activity of the ginseng berry pulp water soluble polysaccharides were at least 2X lower than the raspberry water soluble polysaccharide. Molecular weight of polysaccharides has been shown to affect antioxidant activity (Chen et al., 2008a). Li et al. (2010) noted that low molecular weight polysaccharides extracted from pumpkin demonstrated greater antioxidant activity. The molecular weights of the ginseng berry water soluble polysaccharides were lower than the molecular weights of the water soluble raspberry polysaccharides. This result is opposite to that of Li et al. (2010). However, monosaccharide composition has been related to antioxidant ability (Tsiapali et al, 2001). When comparing the antioxidant activity of the raspberry and ginseng berry water soluble polysaccharides against their respective fractions, arabinose was found to be a sugar positively correlated to antioxidant ability. Inhibition of α-Amylase and α-Glucosidase Activity by Water Soluble Polysaccharides and Purified Fractions Table 4 shows the inhibitory effects of the water soluble polysaccharides and purified fractions on the α-amylase and α-glucosidase activity at a test concentration of 5 mg/mL. Acarbose was used as a positive control at a test concentration of 5 mg/mL and all inhibition measurements are expressed relative to the inhibition of the acarbose which was normalized at 100% at a 5mg/mL concentration. Only the water soluble polysaccharides obtained from the ginseng berry pulp and raspberry samples along with the purified NaCl eluted/acidic ginseng berry pulp fractions inhibited α-amylase activity at levels of ∼ 3, 40 and 3 %, respectively. These samples did possess a

www.seipub.org/rhn

higher total phenolics content compared to the other samples, which may have contributed to the higher αamylase activities. The α-glucosidase inhibition data shows that of the water soluble polysaccharides, only those obtained from the raspberry samples inhibited α-glucosidase at a level of 115% compared to the control acarbose (100% inhibition). However, all of the NaCl eluted/acidic polysaccharide fractions inhibited α-glucosidase activity (∼19-120%). Interestingly, the NaCl eluted/acidic polysaccharide fraction obtained from the raspberry samples presented lower levels of α-glucosidase inhibition compared to the water soluble fraction which was an opposite the results seen for the NaCl eluted/acidic fractions obtained from the cherry and ginseng berry pulp samples. In fact, the NaCl eluted/acidic fractions obtained from the cherry samples displayed levels of α-glucosidase inhibition higher than that of the acarbose positive control. The α-glucosidase inhibition results for the raspberry samples seem to point to the importance of the presence of phenolic compounds as the water soluble polysaccharides contained significantly higher phenolics content than the corresponding fractions. However, for the cherry samples, the α-glucosidase inhibition results cannot be completely explained by the presence of phenolics as the levels in the NaCl eluted/acidic fractons were not greater than the phenolics levels of the corresponding water soluble polysaccharides. The NaCl eluted/acidic cherry fractions contained higher carbohydrate contents, lower uronic acid and glucose contents, higher fucose content and possessed a lower molecular weight compared to the corresponding water soluble polysaccharides. The work of Chen et al. (2009) indicated that the low molecular weight of black tea may have contributed to its high α-glucosidase inhibition activity. The NaCl eluted/acidic fraction from the ginseng berry pulp sample contained less glucose and contained fucose. A clear correlation between chemical composition and α-amylase and αglucosidase activity was not able to be developed for the samples. As such, many factors of chemical characteristics influence bioactive properties and more study is required to define the relationship between polysaccharides and bioactivity including glycosyl linkage analysis and structural elucidation with NMR (Cantu-Jungles et al., 2014). The data indicates that the inhibition activities are influenced by both fruit type and polysaccharide fraction. In all cases the α-amylase inhibition activity was less than the α-glucosidase inhibition activity level which was in agreement with the work of Wang et al. (2010).

www.seipub.org/rhn

Research in Health and Nutrition (RHN) Volume 3, 2015

TABLE 1. YIELD, TOTAL CARBOHYDRATES, PROTEIN, TOTAL PHENOLICS CONTENTS AND MOLECULAR WEIGHTS OF WATER SOLUBLE POLYSACCHARIDES AND PURIFIED FRACTIONS FROM CHERRY, RASPBERRY, AND GINSENG FRUITS

Sample SPS-SK WEPS-SK SEPS-SK SPS-SH WEPS-SH SEPS-SH SPS-LP WEPS-LP SEPS-LP SPS-GBP WEPS-GBP SEPS-GBP SPS-RB WEPS-RB SEPS-RB

Yield (%)* 0.65±0.02a,1 0.33±0.01b,1 (52%)** 0.19 ±0.04b,1 (29%)** 0.50±0.01a,1 0.13±0.02b,2 (25%)** 0.12±0.01b,1 (24%)** 0.66±0.04a,1 0.38±0.07b,1,3 (57%)** 0.18±0.03b,1 (27%)** 1.35±0.03a,2 0.55±0.07b,3 (41%)** 0.34±0.04c,2 (25%)** 2.08±0.04a,3 1.08±0.12b,4 (52%)** 0.56±0.07c,2 (27%)**

Total Carbohydrates (%)* 36.06±0.62a,1 26.92±3.41a,1 64.76±1.82b,1,3 36.83±2.94a,1 28.39±3.41a,1 61.62±0.75b,1 42.65±1.3a,1 27.33±11.9a,1 71.94±11.08b,3 21.83±2.26a,2 10.24±1.06b,2 42.61±4.4c,2 23.16±2.39a,2 27.20±2.81a,1 43.84±4.53b,2

Protein Content (%)* 8.21±1.13a,1 7.15±1.06a,1,3 11.93±1.84b,1 12.19±0.004a,2 6.98±2.35b,1,3 16.78±2.64c,2 6.31±0.36a,1 6.08±0.29a,3 7.15±0.59a,3 16.64±0.49a,3 8.93±0.26b,1 17.06±0.49a,2 7.08±0.21a,1 9.39±0.27a,1 3.81±0.11b,4

Phenolics Content (mg GAE/g PS)* 19.44±3.61a,1 3.15±0.18b,1 14.28±0.67c,1,3 14.11±0.37a,2 2.91±0.59b,1 13.5±1.0a,1 19.33±0.24a,1 3.61±0.88b,1 16.73±2.93a,3 55.4±2.47a,3 32.64±1.46b,2 53.18±2.37a,2 45.07±2.01a,4 9.8±0.44b,3 4.4±0.20c,4

Molecular Weight (kDa) † 56.4 (44%)‡ 111.1 (65%)‡ 13.1 (55%)‡ 62.7 (34%)‡ 70.3 (52%)‡ 12.3 (54%)‡ 59.8 (43%)‡ 70.3 (52%)‡ 17.3 (63%)‡ 2.06 (27%)‡ 0.372 (33%)‡ 28.7 (26%)‡; 13.3 (27%)‡ 83.2 (66%)‡ 175.6 (69%)‡ 33.2 (82%)‡

*Dry matter basis; ** Values in parenthesis represent the percent of each fraction obtained after purification on DEAE Sepharose column and losses did occur as 100% recovery was not recorded; † Molecular weights were associated with multimodal distributions from HPSEC results and the values presented represents main molecular weight population, ‡ Values in parenthesis represent the proportion of population associated with the reported molecular weight.

Sample Nomenclature: SPS=water soluble polysaccharide, WEPS=water eluted/neutral polysaccharide fraction, SEPS= 1M NaCl eluted/acidic polysaccharide fraction; SK=Cherry: Skeena variety, SH=Cherry: Sweetheart variety, LP=Cherry Lapins variety, GBP=Ginseng Berry Pulp, RB=Raspberries Values followed by different letters within column and at common fruit type and/or variety are significantly different (p≤0.05) Values followed by different numbers within column at common polysaccharide type (i.e. SPS, WEPS, and SEPS) are significantly different (p≤0.05) TABLE 2. NEUTRAL SUGARS, URONIC ACID, AND SUGAR MONOMER COMPOSITION OF WATER SOLUBLE POLYSACCHARIDES AND PURIFIED FRACTIONS OBTAINED FROM CHERRY, RASPBERRY, AND GINSENG FRUITS

Sample SPS-SK WEPS-SK SEPS-SK SPS-SH WEPS-SH SEPS-SH SPS-LP WEPS-LP SEPS-LP SPS-GBP WEPS-GBP SEPS-GBP SPS-RB WEPS-RB SEPS-RB

% Neutral Sugars* (% of total neutral and acidic sugars*) 44.05±2.43a,1 (77.44) 26.88±6.01b,1 (68.53) 76.92±4.94c,1 (90.2) 49.34±0.97a,1 (79.66) 33.24±2.13b,2 (76.82) 74.23±3.57c,1 (90.02) 48.35±1.2a,1 (76.54) 39.46±1.2b,3 (70.01) 79.37±3.92c,1 (89.35) 19.08±0.87a,2 (80.48) 8.55±0.39b,4 (90.33) 41.23±1.89c,2 (79.5) 27.36±0.63a,3 (46.92) 24.13±0.55a,1 (40.31) 56.73±1.3b,3 (70.87)

% Uronic Acid* (% of total neutral and acidic sugars*) 12.83±2.23a,1 (22.56) 12.33±2.8a,1 (31.85) 8.35±0b,1 (9.81) 12.6±4.17a,1 (20.20) 10.03±3.6a,c,1 (23.05) 8.23±0.19b,c,1 (9.99) 14.82±0.99a,1 (23.44) 16.91±1.34a,2 (29.99) 9.46±0.15b,1 (10.66) 4.63±0.29a,2 (19.52) 0.92±0.058b,3 (9.67) 10.63±0.674c,1 (20.5) 30.96±1.16a,3 (53.08) 35.73±1.34b,4 (59.69) 23.32±0.872c,2 (29.13)

% Sugar Monomer Content of Neutral Sugars in Crude Polysaccharides* Rha Fuc Ara Xyl Man Gal Glu 5.69 0.49 28.55 3.80 6.16 34.85 20.46 3.02 0.35 37.64 1.81 3.26 25.61 28.31 5.61 0.75 25.55 5.28 8.85 38.97 15.01 5.52 0.37 32.06 2.88 7.16 33.22 18.79 3.35 0.21 34.45 1.26 7.20 17.61 35.92 5.83 0.52 28.53 3.65 8.26 40.65 12.56 6.32 0.56 29.88 3.71 6.13 32.69 20.71 4.49 0.28 38.56 1.73 4.14 28.34 22.46 6.23 0.83 21.36 6.01 9.81 38.38 17.38 5.96 0.00 21.95 5.09 5.56 44.93 16.52 2.86 0.00 6.17 4.53 10.93 45.38 30.14 7.45 0.41 23.12 3.87 3.42 49.36 12.36 6.08 0.37 41.10 5.52 4.66 25.20 17.08 3.33 0.48 31.68 7.67 9.34 20.38 27.11 5.50 0.22 59.63 3.62 1.08 26.75 3.21

Research in Health and Nutrition (RHN) Volume 3, 2015

TABLE 3 ANTIOXIDANT ACTIVITY OF WATER SOLUBLE POLYSACCHARIDES AND PURIFIED FRACTIONS OBTAINED FROM CHERRY, RASPBERRY, AND GINSENG FRUITS

Sample SPS-SK WEPS-SK SEPS-SK SPS-SH WEPS-SH SEPS-SH SPS-LP WEPS-LP SEPS-LP SPS-GBP WEPS-GBP SEPS-GBP SPS-RB WEPS-RB SEPS-RB

Antioxidant Activity (µmol Trolox eq/g PS) ABTS FRAP 96.76±25.56a,1 72.51±15.9a,1 9.02±1.16b,1 5.94±0.32b,1 30.88±3.07c,1,2 29.9±3.16c,1 57.76±3.00a,2 40.03±3.63a,2 8.43±1.78b,1 3.66±1.42b,1 b,1,4 23.54±2.95 21.17±2.94c,1,4 94.62±7.38a,1 67.13±5.94a,1 13.26±0.28b,1 6.57±1.18b,1 41.01±6.15c,2 43.52±6.18c,2 159.35±6.29a,3 100.14±14.69a,3 b,2 51.78±2.04 3.29±0.48b,1 143.92±5.68a,3 106.46±15.62a,3 375.3±14.82a,4 261.28±10.32a,4 69.28±2.74b,3 23.31±3.42b,2 15.09±0.59c,4 14.31±2.1b,4

Sample Nomenclature: SPS=water soluble polysaccharide, WEPS=water eluted/neutral polysaccharide fraction, SEPS= 1M NaCl eluted/acidic polysaccharide fraction; SK=Cherry: Skeena variety, SH=Cherry: Sweetheart variety, LP=Cherry Lapins variety, GBP=Ginseng Berry Pulp, RB=Raspberries Values followed by different letters within column and at common fruit type and/or variety are significantly different (p≤0.05) Values followed by different numbers within column at common polysaccharide type (i.e. SPS, WEPS, and SEPS) are significantly different (p≤0.05) TABLE 4. INHIBITION EFFECTS OF WATER SOLUBLE POLYSACCHARIDES AND PURIFIED FRACTIONS OBTAINED FROM CHERRY, RASPBERRY, AND GINSENG FRUITS ON Α-AMYLASE AND Α-GLUCOSIDASE ACTIVITY

Sample (5 mg/mL test concentration) Acarbose (positive control) SPS-SK WEPS-SK SEPS-SK SPS-SH WEPS-SH SEPS-SH SPS-LP WEPS-LP SEPS-LP SPS-GBP WEPS-GBP SEPS-GBP SPS-RB WEPS-RB SEPS-RB

Inhibition on Activity (%) α-amylase α-glucosidase 100 100 nda,1 nda,1 nda1 nda,1 a,1 nd 120.07±0.19b,1 nda,1 nda,1 nda,1 nda,1 nda,1 120.82±0.77b,1 nda,1 nda,1 a,1 nd nda,1 nda,1 119.77±0.02b,1 3.12±1.1a,2 nda,1 ndb,1 nda,1 3.37±1.19a,2 19.23±0.05b,2 39.62±0.74a,3 115.16±0.14a,2 b,1 nd ndb,1 ndb,1 56.41±0.14c,3

The % inhibition of the different samples and positive control acarbose were determined at a test concentration of 5 mg/mL Sample Nomenclature: SPS=water soluble polysaccharide, WEPS=water eluted/neutral polysaccharide fraction, SEPS= 1M NaCl eluted/acidic polysaccharide fraction; SK=Cherry: Skeena variety, SH=Cherry: Sweetheart variety, LP=Cherry Lapins variety,

www.seipub.org/rhn

GBP=Ginseng Berry Pulp, RB=Raspberries Values followed by different letters within column and at common fruit type and/or variety are significantly different (p≤0.05) Values followed by different numbers within column at common polysaccharide type (i.e. SPS, WEPS, and SEPS) are significantly different (p≤0.05)

Conclusion This work evaluated and compared the chemical, structural, and molecular characteristics along with the bioactive properties of water soluble polysaccharides from sweet cherries, raspberries and ginseng berry pulp and corresponding water eluted/neutral and NaCl eluted/acidic fractions. The water soluble polysaccharides from cherries, raspberries, and ginseng berry pulp all contain between ~22-43% carbohydrate content and an uronic acid component, however they all also contain protein and phenolic compounds. The NaCl eluted/acidic fractions from all of the fruit samples contained higher carbohydrate content compared to the corresponding water soluble polysaccharides and water eluted/neutral fractions. However, both the water eluted/neutral and NaCl eluted/acidic fractions contained uronic acid, protein, and phenolic compounds. All of the water soluble polysaccharides fractions and corresponding fractions demonstrated antioxidant activity while only the water soluble polysaccharides from raspberries and the NaCl eluted/acidic fractions from all sweet cherry varieties tested inhibited α-glucosidase activity at levels comparable to an acarbose control at comparable concentrations. A clear correlation between chemical characteristics of the polysaccharides and bioactivity could not be established based on the present results as many factors of the chemical characteristics of the studied polysaccharides may influence bioactive properties. In order to define the relationship between specific polysaccharides and bioactivity more study is required. However, this work does provide novel information supporting the hypothesis that polysaccharides present small fruits possess bioactivities which may play a role in enhancing human health. REFERENCES

Adnan, L, Osman, A., and Hamid, A.A. “Antioxidant Activity of Different Extracts of Red Pitaya (Hylocereus Polyrhizus)

Seed.”

International

Journal

Food

Properties 14 (2011):1171-1181.

www.seipub.org/rhn

Research in Health and Nutrition (RHN) Volume 3, 2015

Blakeney, A.B., Harris, P.J., Henry, R.J., and Stone, B.A. “A

Gan,

L,;

Zhang,

S.H.,

Yang,

X.L.,

and

Xu,

H.B.

Simple And Rapid Preparation of Alditol Acetates for

“Immunomodulation and Antitumor Activity by a

Monosaccharide Analysis.” Carbohydrate Research 113

Polysaccharide-Protein Complex grom Lycium barbarum.”

(1983): 113, 291-299.

International Immunopharmacology 4 (2004): 563-569.

Bradford, M.M. “A rapid and sensitive method for the

Herken, E.N., and Guzel, S. “Total Antioxidant Capacity

quantification of microgram quantities of protein

And Total Phenol Contents of Selected Commercial Fruit

utilizing

Juices.” International Journal of Food Properties 13(2010):

the

principle

protein-dye

binding.”

1373-1379.

Analytical Biochemistry 72 (1976): 248-252. Cantu-Jungles, T.M., Maria-Ferreira, D., da Silva, L.M.,

Inngjerdingen,

K.T.,

Debes,

S.C.,

Inngjerdingen,

M.,

Hatsuko Baggio, C., de Paula Werner, M.F., Iacomini, M.,

Hokputsa, S., Harding, S.E., Rolstad, B., Michaelsen, T.E.,

Cipriani, T.R., and Cordeiro, L.M.C. “Polysaccharides

Diallo,

from prunes: Gastroprotective activity and Structural

Polysaccharides from Glinus oppositifolius (L.) Aug. DC., a

Elucidation of Bioactive Pectins.” Food Chemistry

Malian

146(2014):492-499.

Characterization.” Journal of Ethnopharmacology 101

Carnachan, S.M., Bootten, S.M., Mishra, S., Monro, J.A, and

D.,

and

Paulsen,

Medicinal

Plant,

B.S.

“Bioactive

Isolation

and

Pectic Partial

(2005): 204- 214.

Sims, I.M. “Effects of Simulated Digestion In Vitro on Cell

Kumanan, R., Manimaran, S., Saleemulla, K., Dhanabal, S.P.,

Wall Polysaccharides from Kiwifruit (Actinidia spp.).

and Nanjan, M.J. “Screening of Bark of Cinnamomum

Food Chemistry 133 (2012:, 132-139.

Tamala (Lauraceae) by Using α-Amylase Inhibition

Chen, H., Qu, Z., Fu, L., Dong, P., and Zhang, X. “Physicochemical properties and Antioxidant Capacity of 3 Polysaccharides From Green Tea, Oolong Tea, and Chen, H., Zhang, M., Zhishuang, and Q., Bijun, X. Activities

Different

of Pharmaceutical Biomedicial Ressearch 1(2010): 69-72. Li, J., Wang, Y., Zhang, D., Hu, X., Zhang, Z., and Xiang, C. “Characterization and Bioactivity of Water-Soluble

Black Tea.” Food Chemistry 74(2009):C469-C474. “Antioxidant

Assay for Anti-Diabetic Activity.” International Journal

Fractions

Polysaccharide Conjugates from Green Tea (Camellia

Polysaccharidesfrom the Fruit of Pumpkin.” Journal of Food Agriculture and Environment 8 (2010): 237- 241. Li, X.M., Li, X.L., and Zhou, A.G. “Evaluation of Antioxidant Activity of the Polysaccharides Extracted from Lycium

sinensis).” Food Chemistry 106(2008a): 559-563. Chen, H.X., Zhang, M., and Xie, B.J. “Quantification of Uronic Acids in Tea Polysaccharide Conjugates and its Antioxidant Properties.” Journal of Agricultural and

Barbarum Fruits In Vitro”. European Polymer Journal 43 (2007): 488-497. Luo, Q., Cai, Y., Yan, J., Sun, M., and Corke, H. Hypoglycemic

Food Chemistry 52(2004): 3333–3336. Chen, Y., Xie, M.Y., Nie, S.P., Li, C., and Wang, Y.X. “Purification, Composition Analysis and Antioxidant Activity from the Fruiting Bodies of Ganoderma atrum.

and

Hypolipidemic

Effects

and

Antioxidant Activity of Fruit Extracts from Lycium barbarum. Life Sciences 76 (2004): 137-149. Mateos-Aparicio, I., Mateos-Peinado, C., Jimenez-Escrig, A., and Ruperez, P. “Multifunctional Antioxidant Activity of

Food Chemistry 107(2008b): 231-241. Dubios, M., Gilles, K.A., Hamilton, J.K., Robers, P.A.,and Smith, F. “Colormetric Method for Determination of Sugars and Related Substances.” Analytical Chemistry

Polysaccharide Fractions from the Soybean By-Product Okara.” Carbohydrate Polymers 82 (2010): 245-250. Ni, X-Z., Wand, B-Q., Zhi, Y., Wie, N-N., Zhang, X., Li, S-S., Tai, G-H., Zhou.,

28 (1956): 350-356.

Yi-F., and Zhao, J-M. “Total

Fan, H., Mazza, G., and Liao, X. “Purification, Composition

Fractionation and Analysis of Polysaccharides from

and Antioxidant Activity of Polysaccharides from

Leaves of Panax ginseng C. A. Meyer.” Chem. Res. Chinese

Wolfberry,

universities. 26(2010): 230—234.

Cherry,

Kiwi,

and

Cranberry

Fruits.”

Croatian Journal of Food Science and Technology 2(2010), 9-17.

Palanuvej, C., Hokputsa, S., Tunsaringkarn, T., and Ruangrungsi, N. “In Vitro Glucose Entrapment and

Research in Health and Nutrition (RHN) Volume 3, 2015

www.seipub.org/rhn

Mucilaginous

Carbohydrates, Lignins and Micro- Components of

Substances from Selected Thai Medicinal Plants. Scientia

Straw and Shives: Effects of Extractives, Particle Size and

Pharmaceutica.” 77(2009):837-849.

Crop Species. Industrial Crops and Products. 31(2010):

Alpha-Glucosidase

Inhibition

Pawlaczyk, I., Czerchawki, L., Kuliczkowski, W., Karolko, B.,

534-541.

Gancarz,

Thetsrimuang, C., Khammuang, S., and Sarnthima, R.

Activity

“Antioxidant Activity of Crude Polysaccharides From

Polyphenolic-Polysaccharide Preparation Isolated from

Edible Fresh and Dry Mushroom Fruiting Bodies of

the Medicinal Plant Erigeron canadensis L.” Thrombosis.

Lentinus sp. Strain RJ-2.” International Journal of

Research. 127(2011): 328-340.

Pharmacology 7(2011): 58-65.

Pilecki,

W.,

Witkiewicz,

“Anticoagulant

and

W.,

and

Anti-Platelet

Ross, K.A., and Mazza, G. “Conjugated Polysaccharides as

Tsiapali, E., Whaley, S., Kalbfleisch, J., Ensley, H.E.,

Potential Health Promoting Compounds in Berries and

Browder, I.W., and Williams, D.L. “Glucans Exhibit

Cherries.” In Berries: Properties, Consumption and Health

Weak Antioxidant Activity, but Stimulate Macrophage

Benefits; Tuberoso, C., Eds.; Nova Science Publishers Inc.:

Free Radical Activity.” Free Radical Biology & Medicine

New York, 2012; 55-81.

30(2011): 393–402.

Ross, KA., Siow, Y., Brown, D., Isaak, C., Fukumoto, L., and

Wang, Y., Yang, Z., and Wei, X. “Sugar Compositions, α-

Godfrey, D. “Characterization of Water Extractable

Glucosidase Inhibitory and Amylase Inhibitory Activities

Crude Polysaccharides from Cherry, Raspberry, and

of Polysaccharides from Leaves and Flowers of Camellia

Ginseng Berry Fruits: Chemical Composition and

Sinensis Obtained by Different Extraction Methods.”

Bioactivity.” International Journal of Food Properties, in

International Journal of Biological Macromolecules 47

press, 2014. DOI:10.1080/10942912.2013.837066

(2010):534-539.

Rossi, J.A. “Colorimetry of Total

Wu, C., Shen, J., He, P., Chen, Y., Li, L., Zhang, L., Li, Y., Fu,

Phenolics with Phosphomolybdic–Phosphotungstic Acid

Y., Dai, R., Meng, W., and Deng, Y. “The α-Glucosidase

Reagents.” American Journal of Enology and Viticulture.

Inhibiting Isoflavones Isolated from Belamcanda chinensis

16(1965): 144-158.

Leaf Extract” Natural. Products. 6(2012): 110-120.

Singleton, V.L.,and

Sun, Y.X. and Kennedy, J.F. “Antioxidant activities of

Wu, Y., Cui, S.W., Tang, J., Wang, Q., and Gu, X.

Different Polysaccharide Conjugates Isolated from the

“Preparation, Partial Characterization and Bioactivity of

Fruiting Bodies of Chroogomphis rutilus (Schaeff.:Fr.) O.K.

Water

Miller.” Carbohydrate. Polymers 82(2010): 510-514.

Stericulia Seeds.” Carbohydrate Polymers 70 (2007):437-

Sun, Y-X., Liu, J-C., and Kennedy, J.F. “Purification,

Soluble

Polysaccharides

from

Boat-Fruited

443.

Composition Analysis And Antioxidant Activity of

Zhang, L., Li, J., Hogan, S., Chung, H., Welbaum, G.E., and

Different Polysaccharide Conjugates (Apps) from the

Zhou, K. “Inhibitory Effect Of Raspberries On Starch

Fruiting Bodies of Auricularia Polytricha. Carbohydrate

Digestive Enzyme and Their Antioxidant Properties And

Polymers.” 82 (2010):299-304.

Phenolic Composition.” Food Chemisty. 119(2010):592-

Tamaki, Y. and Mazza, G. “Measurement of Lignin from

599.

Flax Shives as Affected by Extraction Structural