International Journal of Biological Macromolecules 51 (2012) 1057– 1062
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International Journal of Biological Macromolecules
jo u r n al hom epa ge: ww w.elsev ier .com/ locate / i jb iomac
ntioxidant and immunomodulatory activities of polysaccharides from the rootsf Sanguisorba officinalis
in Zhangb, Sundar Rao Koyyalamudib,a,∗, Sang Chul Jeongb, Narsimha Reddyb, Paul T. Smithb,. Ananthanc, T. Longvahc
Centre for Complementary Medicine Research, University of Western Sydney, Locked Bag 1797, Penrith South DC, NSW 1797, AustraliaSchool of Science and Health, University of Western Sydney, Locked Bag 1797, Penrith South DC, NSW 1797, AustraliaNational Institue of Nutrition, Department of Health Research, Jamai Osmania PO, Hyderabad-500007 (AP), India
r t i c l e i n f o
rticle history:eceived 11 June 2012eceived in revised form 13 August 2012ccepted 19 August 2012vailable online 27 August 2012
The roots of Sanguisorba officinalis are used in traditional Chinese medicine for the treatment of diseasessuch as inflammation and internal haemorrhage. Several scientific investigations involving extractionand pharmacological studies of terpenoids and triterpenoid glycosides from this herb have been carriedout. However, little is known regarding the immunomodulatory and antioxidant properties of polysac-charides from S. officinalis. Hence the polysaccharides from this herb have been investigated here. Thehot water extract of S. officinalis has been fractionated using size-exclusion chromatography to obtainfour polysaccharide fractions designated as SOP-1, SOP-2, SOP-3 and SOP-4. The range of molecularmasses of these fractions were from 280 Da to 2000 kDa, and their sugar compositions consisted mainlyof fructose, glucose, xylose, arabinose, and rhamnose. The antioxidant activities of the crude polysaccha-
ntioxidant activities ride fractions were evaluated in a biological assay using Saccharomyces cerevisiae, whereas the radicalscavenging activity was measured using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. Analysisof the immunomodulatory activities of these polysaccharide fractions were measured by using mousemacrophages. Most of the polysaccharide fractions have stimulated the production of nitric oxide andtumour necrosis factor-� (TNF-�), and also displayed antioxidant activities. These results suggest thatthe roots of S. officinalis are likely to have therapeutic value for the treatment of cancer.
Polysaccharides are present in large quantities in nature andave multiple applications. Several bioactive polysaccharides haveeen isolated from botanical sources such as mushrooms, algae,
ichens and higher plants [1–4] and many are relatively non-toxicith no significant side effects [1–4]. In recent decades, polysac-
harides have attracted a great deal of attention and have beenroven to be promising candidates as therapeutics in biomedicalrena [4–9]. Many studies have demonstrated that botanicalolysaccharides have potential to activate cells involved in innate
mmunity [1–4] and hence have immunomodulatory proper-
ies. Interestingly, the botanical polysaccharides have also beenroven to possess high antioxidant activities [10–13]. Literaturelso indicates that the herbal antioxidants concurrently exhibit
∗ Corresponding author at: Centre for Complementary Medicine Research, Uni-ersity of Western Sydney, Locked Bag 1797, Penrith South DC, NSW 1797, Australia.el.: +61 246203294; fax: +61 246203017.
significant immunomodulatory activities [14–16]. It is therefore ofgreat interest to investigate immunomodulatory effects of herbalpolysaccharides that exhibit antioxidant activity with low toxicity.
Sanguisorba officinalis L. (Rosaceae) is a perennial plant thatoccurs in the northern districts of China. Its roots have haemo-static, analgesic, and astringent properties, and have been usedin traditional Chinese medicine for the treatment of burns, scalds,inflammation and internal haemorrhage [17,18]. Several scientificinvestigations, involving extraction and pharmacological studies ofterpenoids and triterpenoid glycosides from this herb, have beencarried out that demonstrate significant antioxidant and neuropro-tective activities [18–22]. However, there is very limited literature[23] on the polysaccharides from S. officinalis and their bioactivities.
Preliminary studies carried out in authors’ laboratory with waterextracts of S. officinalis have displayed high antioxidant activity[24]. These studies have also revealed high anti-proliferative activ-ities against several cancer cells [24]. Clinical study indicated that
S. officinalis possessed anti-cancer activities [25]. It is important tonote from the literature [3,4,23] that the botanic polysaccharidesdisplaying immunomodulatory effects have also exhibited anti-tumour properties. These important observations prompted us to
058 L. Zhang et al. / International Journal of Bio
nvestigate the immunomodulatory and antioxidant properties ofolysaccharides from S. officinalis in order to study the contribu-ion of polysaccharides for these activities. Very recently, a wateroluble polysaccharide was isolated from the roots of S. officinalishich displayed significant immunomodulatory and anti-tumour
ctivities [23].In this study, polysaccharides from roots of S. officinalis were
xtracted and different molecular weight fractions were sepa-ated by gel filtration chromatography. Their antioxidant andmmunomodulatory activities were investigated. Partial character-sation of these fractions has also been carried out.
. Materials and methods
.1. Materials
500 g of dried roots of S. officinalis were obtained from Beijingong Ren Tang Chinese Herbal Medicine Shop, Sydney, Australia. Aoucher specimen of the plant has been deposited in the laboratory.he plant materials were ground to a fine powder in a grinder beforextraction.
In this study, mouse monocyte-macrophage cell line (J774A.1acrophages) has been used to test immunomodulatory proper-
ies of polysaccharide fractions extracted from S. officinalis. J774A.1ells have been treated with lipopolysaccharides (LPS) and IFN-� tonduce the production of NO and TNF-�. The abilities of polysaccha-ides to produce NO and TNF-� were measured by Griess reagentnd ELISA tests, respectively. DPPH and yeast assays were used forhe analysis of antioxidant activity.
.2. Extraction and fractionation of polysaccharides from S.fficinalis
The roots of S. officinalis were powdered, and then homogenized.he sample was then autoclaved for 2 h at 121 ◦C, followed by cool-ng to room temperature before filtration. The supernatant wasollected and precipitated with 95% ethanol (1:4 v/v) overnight at◦C. The precipitate containing the polysaccharides and proteinsas pelleted by centrifugation at 10,000 × g for 20 min and then
he pellet was resuspended in distilled water. The supernatant wasltered through a filter paper (pore size, 0.45 �m) and lyophilizedo obtain a crude polysaccharide extract with a yield of 8.64 g/kg26,27].
The crude polysaccharide extract was dissolved in 3 mL of dis-illed water at a concentration of 10 mg/mL and was fractionatedy size-exclusion chromatography using Sepharose CL-6B column2.4 cm × 99 cm) equilibrated with distilled water and eluted withistilled water at a flow rate of 0.42 mL/min. The polysaccha-ide elution profile was determined by the phenol–sulphuric acidethod and Lowery’s method [3,28,29]. The relevant fractions were
ollected, pooled, and concentrated by freeze-drying.
.3. Determination of molecular weights of polysaccharideractions
Estimation of molecular weights of the purified polysaccharideractions was done on the basis of the elution volume and the
olecular weight using a standard dextran series that included2000 (2000 kDa), T450 (450 kDa), T150 (150 kDa), T70 (70 kDa),40 (40 kDa) T10 (10 kDa) and glucose at a concentration of
0 mg/mL each for calibration of the Sepharose CL-6B column [1,3].
The linear regression of the data has provided the standardquation, y = −0.2128x + 1.4771 with a high correlation coefficientR2 = 0.937). This standard curve can be used to determine the
olecular weights of extracted polysaccharide fractions.
l Macromolecules 51 (2012) 1057– 1062
2.4. Analysis of mono-saccharides
The total sugar content was determined by thephenol–sulphuric acid method [28] using glucose as a refer-ence standard. Total protein content of polysaccharides wasdetermined by the method of Lowry et al. [29]. Bovine serumalbumin (BSA) was used as a standard to measure the proteincontent [1]. The monosaccharide composition was analysed by aHewlett Packard 5890 gas chromatography (Agilent Technologies,Palo Alto, CA, USA) equipped with a flame-ionization detectorusing a BP-20 capillary column (12 m long, 0.22 mm i.d., 0.25 �mfilm thickness; SGE Analytical Science Pty Ltd., Ringwood, VIC,Australia). Mono-sugars have been produced from polysaccharidefractions by hydrolysis and obtained sugars were acetylated [30].Fructose, glucose, xylose, arabinose, galactose, ribose, rhamnoseand fucose sugars were used as standards.
2.5. Amino acid analysis
Amino acid analysis was carried out by hydrolysing the sam-ples with 6 N HCl in sealed ampoules in an oven at 110 ◦C for 22 h.Excess acid was removed by continuous flash evaporation at 45 ◦Cunder reduced pressure, dissolved in citrate buffer (pH 2.2) andaliquot of the sample was loaded into an automatic amino acid anal-yser (Biochrom-30, Cambridge, UK). Cysteine and methionine weredetermined separately after performic acid oxidation to cysteic acidand methionine sulfone, respectively [31]. Tryptophan was deter-mined after barytic hydrolysis according to the method describedby Landry and Delhaye [32,33]. Each amino acid was identifiedand quantified using externally calibrated standards (50, 100 and150 pmol/�L) and the results have been validated using authenticamino acid standard mixture (National Institute of Standards andTechnology, SRM 2389).
2.6. Bioactivity tests
2.6.1. Antioxidant activityThe DPPH free radical scavenging assay was conducted using
the Blois method [34]. Each fraction (50 �L) was added to a 150 �Lof 62.5 �M DPPH. After 30 min of incubation, the absorbance ofthe reaction mixtures was measured at 492 nm using a microplatereader (Multiskan 141 EX, Thermo Electron, USA). Ascorbate (vita-min C), an antioxidant, was used as a positive control. The blankcontrol was distilled water instead of polysaccharides. The standardconcentrations (0, 15.6, 31.25, 62.5, 125, 250, 500 and 1000 �M)were prepared in 60% methanol. The free radical scavengingactivities of plant-derived polysaccharides were calculated as theascorbic acid equivalent against the calibration standard curve. Thelinear regression equation of y = −0.0001x + 0.1602 was obtainedwith a correlation coefficient (R2 = 0.9186).
The antioxidant capacities of purified polysaccharides weremeasured in vivo using a Saccharomyces cerevisiae based highthroughput assay [35]. S. cerevisiae BY4743 was cultured overnightin a 50 mL volume by inoculation of a single colony. The culturewas then diluted to an optical density at 600 nm (OD 600) of 0.2in media, and 180 �L of each strain was added into a well in a 96-well microtiter plate, where 10 �L per well of each fraction wasalso added in duplicate. Ten microliters of three different oxidants,namely, hydrogen peroxide (H2O2), cumene hydroperoxide (CHP)and linoleic acid hydroperoxide (LAH) were added to a final con-centration of 2 mM, 150 �M and 75 �M, respectively. The initialOD 600 reading was taken using a microplate reader (Multiskan
EX, Thermo Electron, USA), and the plates were then incubated in a30 ◦C incubator with shaking at 750 rpm. Yeast growth was moni-tored by reading OD 600 at the end of 20 h. Ascorbic acid was usedas a positive control. The effect of samples on the net growth of
Monosaccharide composition of isolated fractions was analysedby gas chromatograph and the results are presented in Table 1.All of the fractions consisted primarily of glucose, fructose, xylose,
Table 1Chemical composition (sugar contents) of polysacharide fractions isolated from S.officinalis.
SOP-1 SOP-2 SOP-3
Total carbohydrate (%) 70.41 27.29 19.28Monosaccharide (% ratio)
east cells was measured after inducing oxidative stress with threeifferent oxidants, by using the following equation:
yeast growth =(
Psample − Pcontrol
Pcontrol
)× 100
here, ‘Pyeast growth’ is the net growth of H2O2, CHP and LAH inducedeast cells after treatment with polysaccharides; ‘Psample’ is theptical density of yeast cells with the treatment of polysaccharides;
Pcontrol’ is the optical density of yeast cells with the treatment ofegative control (oxidants).
.6.2. Measurement of NO productionJ774A.1 macrophages were incubated at 37 ◦C in an atmo-
phere of 5% CO2 for 40 h in culture medium containing variousoses of polysaccharide fractions (20 �L) or LPS (20 �L) as a pos-
tive control in wells of 96-well flat-bottom microtiter plates at density of 4 × 105 cells per well. After incubation, 100 �L of theulture supernatant was removed and assayed for NO productionsing a colorimetric method with sodium nitrite as a standard.
n brief, supernatants were mixed with equal volumes of Griesseagent (0.1% (wt./v) naphthyl ethylenediamine and 1% (wt./v)ulfanilamide in 5% (v/v) phosphoric acid in wells of 96-well flat-ottom microtiter plates). After 5 min at room temperature, thebsorbance was measured at 550 nm in a Bio-Rad (Hercules, CA,SA) microplate reader [36]. All measurements are performed in
riplicate.The respective intercept and slope values were used in the mea-
urement of NO production capacity of polysaccharides. The linearegression equation of y = 0.0024x + 0.1941 was obtained with aood correlation coefficient (R2 = 0.9666).
.6.3. Assay for the measurement of TNF- ̨ productionJ774A.1 cells were incubated for 40 h in culture medium with or
ithout various doses of polysaccharide fractions (20 �L) or LPSs a positive control in 96-well flat-bottom microtiter plates at
density of 4 × 105 cells per well. TNF-� secreted in the cultureupernatants was quantified using enzyme-linked immunosorbentssay kits (BD Biosciences, San Jose, CA, USA). Cytokine concen-rations were determined by extrapolation from TNF-� standardurve, according to the manufacturer’s protocol [36]. All measure-ents are performed in triplicate.The respective intercept and slope values were used in the
easurement of TNF-� production capacity of polysaccharides.he linear regression equation of y = 0.0014x + 0.0559 was obtainedith a significant correlation coefficient (R2 = 0.997).
.6.4. Determination of cell viability by Alamar BLUE assayAlamar Blue assay is a calorimetric assay involving the cellular
eduction of resazurin to resorufin. 100 �L of Alamar Blue solution10% Alamar Blue (Resazurin) in DMEM media) was added to eachell and incubated at 37 ◦C for 1–2 h. Fluorescence was measured
excitation @ 545 nm and emission @ 595 nm) and expressed as aercentage of that in control cells after background fluorescenceas subtracted.
.7. Statistical analysis
Data are expressed as mean ± standard deviation (SD) values.he group mean was compared using a one-way analysis of vari-
nce and Duncan’s multiple range tests. The statistical differenceas considered significant at P < 0.05. The analysis was performedsing SPSS Version 20 Software (IBM SPSS, Chicago, IL, USA) andxcel.
Fig. 1. Gel filtration chromatograms of polysaccharides from S. officinalis showingfour polysaccharide fractions (designated as SOP-1, SOP-2, SOP-3 and SOP-4).
3. Results
3.1. Isolation of polysaccharide fractions from S. officinalis
Polysaccharides were extracted from S. officinalis as describedin Section 2.2 and were fractionated by size-exclusion chromatog-raphy on a Sepharose CL-6B column. Four fractions were selectedon the basis of the total carbohydrate and protein elution pro-files obtained by phenol–sulphuric acid test and Lowry’s method,respectively. These crude polysaccharide fractions are designatedas SOP-1, SOP-2, SOP-3, and SOP-4 (Fig. 1).
The results for sugar content and protein content in each ofthe fractions are presented in Tables 1 and 2. Based on the cali-bration curve obtained from analysis of dextran molecular weightstandards (Fig. 2), the average molecular masses of various iso-lated fractions have been determined. The molecular mass of SOP-1was found to be relatively large, with an estimated average molec-ular mass of more than 2000 kDa (Fig. 2 and Table 3). This wasfollowed by SOP-2 and SOP-3 which contained polysaccharideswith average molecular masses of 388 and 46 kDa, respectively. Thesmallest molecular mass was observed for SOP-4 (286 Da) (Fig. 2),which possibly is a monosaccharide. Literature indicates that largermolecular weight polysaccharides (greater than 1.5 kDa) are gen-erally responsible for biological activity [4,11]. Hence, SOP-4 wasnot considered for further studies.
3.2. Monosaccharide and amino acid composition of the fractions
Fig. 2. Calibration curve for the determination of molecular weights of polysaccha-rd
apaeiafi
cell viabilities even at highest concentration of 100 �g/mL used in
TA
A
ides of S. officinalis based on the elution volume and the molecular mass of standardextran series.
rabinose and rhamnose. The amino acid composition of theolysaccharide fractions is given in Table 2. Aspartate, glutamatend glycine constituted 30–45% of the total amino acids. The totalssential amino acid content was highest in SOP-1 and lowestn SOP-2. The different fractions had varying content of essential
mino acids with lysine as the limiting amino acid in all theractions. The results presented in Tables 1 and 2 indicate that thesolated fractions are likely to be protein-bound polysaccharides.
able 3ntioxidant activities of polysaccharide fractions of S. officinalis along with their average
S. No. (1 mg/mL) Average molecularmass (kDa)
DPPH scavenging activity of waextracts (ascorbate equivalent
SOP-1 2266 552 ± 5
SOP-2 388 967 ± 7
SOP-3 46 977 ± 7
ll the values are mean of duplicate determination ± standard deviation (SD).a DPPH free radical scavenging activity was measured as equivalent of ascorbic acid.b Yeast oxidative stress was measured on the basis of survival of yeast cells (yeast grow
l Macromolecules 51 (2012) 1057– 1062
3.3. Anti-oxidant activities of polysaccharide fractions of S.officinalis
3.3.1. DPPH radical scavenging activityThe results of free radical scavenging capacity of the crude
polysaccharide fractions are presented in Table 3. All the polysac-charide fractions exhibited significant scavenging capacity thatranged from 552 �M to 977 �M ascorbate equiv/g. Extremely highantioxidant activity was displayed by polysaccharide fractionsSOP-2 and SOP-3, with scavenging capacities more than 900 �Mascorbate equiv/g (Table 3).
3.3.2. Antioxidant activity using yeast modelAntioxidant activities of S. officinalis polysaccharides measured
in vivo by using yeast model are also presented in Table 3. Theseresults revealed that the fractions SOP-1, SOP-2 and SOP-3 havehigh activity against the three different oxidants which are H2O2,CHP and LAH. It is interesting to note that the fractions SOP-1, SOP-2 and SOP-3 showed significant DPPH radical scavenging activityand also high antioxidant activity as measured by yeast model.
3.4. Effect of polysaccharides derived from S. officinalis onmacrophage TNF- ̨ and NO production
This study was aimed to determine the immunomodulatoryproperties of the isolated polysaccharide fractions. Treatment ofJ774A.1 cells with polysaccharide fractions isolated from S. offici-nalis resulted in a dose-dependent increase in the production ofTNF-� and NO (Fig. 3). Cell viabilities for various polysaccharidefractions are given in Fig. 4.
From the results presented in Fig. 3 it is evident that all theisolated polysaccharide fractions from S. officinalis have displayedsignificant immunomodulatory activity by increasing the TNF-�production in a dose-dependent manner (Fig. 3B and D). As can beseen from Fig. 3D, there is a sharp increase of immunomodulatoryactivity in the concentration range of 10 to 100 �g/mL (P < 0.05).All the isolated polysaccharide fractions stimulated TNF-� produc-tion that was significantly more than the positive control whichwas LPS (P < 0.05) (Fig. 3B). Most of the polysaccharide fractions ofS. officinalis also activated macrophage NO production in a dose-dependent manner (Fig. 3A and C) (P < 0.05).
3.5. Cell viability
Effect of various polysaccharide fractions derived from S. offici-nalis on the viability of mouse macrophages are given in Fig. 4. It isclear from these results that, all of the fractions showed significant
this study (63% or better viability). These results indicate that thepolysaccharides derived from S. officinalis exhibit low toxicity andthis is consistent with literature reports [1–4].
L. Zhang et al. / International Journal of Biological Macromolecules 51 (2012) 1057– 1062 1061
charid
4
iioi
tsadw2atm
Fig. 3. Effects of S. officinalis polysac
. Discussion
Many plant-derived polysaccharides are known to possessmmunomodulating, anti-tumour and other important bioactiv-ties [1–4,23,37–40]. Fractionation of polysaccharides from S.fficinalis was successfully carried out, and their antioxidant andmmunomodulatory activities were evaluated in this study.
A closer observation of the antioxidant activities (Table 3) revealhat most of the polysaccharide fractions of S. officinal showedignificant antioxidant activity. It is interesting to note that thentioxidant activities of the isolated polysaccharide fractions haveisplayed an inverse correlation with their average moleculareights (Table 3). For example, the antioxidant activities of SOP-
and SOP-3 are stronger than that of SOP-1 which has highestverage molecular weight (Table 3). This was true for the activi-ies measured by both methods, namely, DPPH radical scavenging
ethod and yeast oxidative stress model. Literature suggests that,
0
25
50
75
100
100
Cell
via
bil
itie
s (%
)
Conce ntrat ion of po lysac cha ride
Fig. 4. Cell viabilities of isolated polysaccharid
es on murine J774A.1 macrophages.
high molecular weight polysaccharides generally have high vis-cosity and poor water solubility and hence is difficult for thesemolecules to penetrate into the interior of the cell, which limitstheir biological activity [3,10–13,41].
Results on the stimulation of macrophages by variouspolysaccharide fractions isolated from S. officinalis indicate thatthese fractions have significant immunomodulatory activity.Immunomodulatory effects of the three polysaccharide fractions interms of TNF-� production displayed dose dependence with a sharpincrease in the activity at concentrations greater than 10 �g/mL(Fig. 3D). Excellent TNF-� production capacity has been observedfor the polysaccharide fractions SOP-2 and SOP-3 at 100 �g/mLwith more than 8-fold increase in TNF-� production when com-
pared with untreated macrophages (Fig. 3B and D). Also, theinduced NO production capacity of macrophages by polysaccharidefractions SOP-2 and SOP-3 were significant at the concentrationof 100 �g/mL (Fig. 3A and C). These findings are very valuable
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SOP-3
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062 L. Zhang et al. / International Journal of Bio
nd indicate that the polysaccharides of S. officinalis are highlyuitable candidates to modulate the macrophage function and stim-late the immune system. At this point, it is important to notehat immunomodulatory effects play an important role in anti-umour activity [3,4,23,38,39]. It is therefore expected that theolysaccharide fractions isolated from S. officinalis in this study are
ikely to display anti-tumour activity. Preliminary studies on anti-roliferative activities of water extracts of S. officinalis carried out
n the authors’ laboratory also support this hypothesis [24].All the polysaccharide fractions isolated from S. officinalis in this
tudy contain high proportions of glucose and fructose, and averageevels of xylose, arabinose and rhamnose (Table 1). These mono-ugars may be responsible for the activities of SOP-1, SOP-2 andOP-3. This finding is in agreement with the literature suggest-ng that higher proportions of glucose, fructose and xylose lead toetter immunomodulatory capacities [3,4,16]. Further studies areecessary to determine the structural characteristics of polysac-harides responsible for this important activity.
. Conclusion
Three water soluble polysaccharide fractions (SOP-1, SOP-2 andOP-3) were isolated from S. officinalis by gel filtration chromatog-aphy. These polysaccharides have displayed high antioxidantnd immunomodulatory activities. The observed activities werenversely related to the average molecular masses of the isolatedractions. Two of the polysaccharide fractions (SOP-2 and SOP-) have shown highly significant TNF-� production capacity at00 �g/mL, indicating that they are the most suitable candidates forffectively stimulating the immune system. These results suggesthe potential of S. officinalis polysaccharides as natural anti-tumourgents.
Recent studies revealed that certain structural characteristicsf polysaccharides, such as glycosidic linkages, are responsibleor their immunomodulatory activities [37,40]. Further studiesnvolving structural characterisation of these immunomodulatoryolysaccharides are therefore required in order to understand theiriological activities.
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