Research ArticleThe Metabolism of Polysaccharide from Atractylodesmacrocephala Koidz and Its Effect on Intestinal Microflora
Ruijun Wang Guisheng Zhou Mengyue Wang Ying Peng and Xiaobo Li
School of Pharmacy Shanghai Jiao Tong University Shanghai 200240 China
Correspondence should be addressed to Xiaobo Li xblisjtueducn
Received 23 May 2014 Accepted 6 November 2014 Published 19 November 2014
Academic Editor Min Ye
Copyright copy 2014 Ruijun Wang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
An active polysaccharide from the rhizome of Atractylodes macrocephala Koidz (PAM) was identified to improve andadjust disordered intestinal flora High-performance gel permeation chromatography (HPGPC) and gas chromatography-massspectrometry (GC-MS) were employed to identify the components of PAM as rhamnose glucose mannose xylose and galactoseat a ratio of 003 025 015 041 015 PAMmetabolized in gastrointestinal tractwhen incubatedwith artificial gastric and intestinaljuices Anaerobic incubation of PAM on intestinal flora confirmed that PAM promoted the ability of intestinal bacteria to digestreducing sugar Based on the Shannon index and similarity coefficient index of enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) fingerprinting of the total intestinal bacteria DNA we concluded that PAM can significantly improve disorderedintestinal flora and may be used as an oral adjuvant to regulate intestinal flora
1 Introduction
Recent studies reported that the rising incidences of boweldisease were caused by gut microbes [1 2] These microbeswere also associated with many epidemics chronic illnessessuch as diabetes mellitus [3] inflammatory bowel disease [4]and obesity [5] The gut microbes can be affected by manyfactors [6ndash9] including diet body state and environmentamong which dietary was the major factor Currently itremains unclear how gut bacteria respond to active com-ponents from diets or functional foods Studies on therelationship between intestinal flora and active componentsare warranted
Reports indicated that polysaccharides can impact on thecolonic microbiota [10ndash12] but its mechanistic interactionwith intestinal flora remains unknown Further studies onsuch interactions would be useful to understand the physi-ological benefits of polysaccharide and provide instructionsfor proper therapeutic uses
Polysaccharides are important constituent of many well-known functional herbs also in traditional Chinese medicine(TCM) [13] such as Atractylodes [14ndash16] ginseng [17] andPoria cocos [18] At present the rhizome of Atractylodesmacrocephala Koidz the most common functional food is
known to regulate the gut microbes and is widely used in thetreatment of chronic intestinal disease [15]
In this study a novel polysaccharide (PAM) was iso-lated from the rhizome of A macrocephala with improvedmetabolic effect on intestinal microflora and stability in arti-ficial gastricintestinal juice By using anaerobic incubationand ERIC-PCR fingerprinting methods the effect of PAMon intestinal bacteria in vitro and in vivo was evaluated Thisstudy provided novel understanding on physiological andtherapeutic benefits of polysaccharides on intestinal flora
2 Experimental Section
21 Materials Reagents and Preparation of CAE Driedrhizome of Atractylodes macrocephala Koidz (batch numberHX20081102) and the folia of Cassia angustifolia Vahl (batchnumber HX20081103) were bought from Shanghai HuayuPharmaceutical co Ltd (Shanghai China) Their botanicalorigins were identified by the corresponding author andvoucher specimenswere deposited at the School of PharmacyShanghai Jiao Tong University DEAE-52 was bought fromWhatman (Maidstone Britain) and Sephacryl S-100 gelwas supplied by Amersham (Uppsala Sweden) Monosac-charaides were purchased from Sigma (St Louis USA)
Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 926381 7 pageshttpdxdoiorg1011552014926381
2 Evidence-Based Complementary and Alternative Medicine
Primers ERIC 1R (51015840-ATGTAAGCTCCTGGGGATTCAC-31015840) and ERIC 2R (51015840-AAGTAA GTGACTGGGGTGAGCG-31015840) were obtained from Sangon Biotech Co Ltd (ShanghaiChina) All reagents were of analytical grade
The dry powder of the folia of C angustifolia (senna)was extracted (CAE) thrice with boiling water (1 10) for120min (40min each time) The three filtrates were mergedand evaporated by rotary evaporation under vacuum at 60∘CFinally the concentration of extracts was set at 10 gmL
22 Animals Male SD rats (190ndash200 g and 5 weeks old)were purchased from Shanghai Slack Laboratory Animalco Ltd (Shanghai China) All procedures related to theanimals and their care conformed to the internationallyaccepted principles as found in the Guidelines for Keep-ing Experimental Animals issued by the Government ofChina
23 Extraction Isolation and Purification of PolysaccharidesThe dried powder of the rhizome of A macrocephala (200 g)was extracted thrice with 2000mL boiling water for 3 h (1 heach time) The aqueous extracts were filtrated combinedand concentrated to about 200mL with rotary evaporatorsTo precipitate the crude polysaccharide 3-fold volume ofpure ethanol was added to the extracts at 4∘C overnight Theresulting precipitate was collected by centrifugation washedwith pure ethanol (400mL) repeatedly and dried undernitrogen 20 g dry precipitate was obtained reconstituted in1000mL pure water and deproteinized using 3-fold volumeof Sevag reagent (119899-butanolchloroform vv = 1 4) Finally900mL supernatant was collected concentrated to 100mLand lyophilized to 138 g crude polysaccharides
To purify the isolated polysaccharides 1 g crude polysac-charides were dissolved filtered through 045 120583m filters andapplied to a DEAE-52 cellulose chromatography column(26mm times 60 cm) The crude polysaccharides were fraction-ated and eluted with 1000mL distilled water at a flow rateof 1mLmin The water fractions obtained were determinedaccording to the total carbohydrate content by the phenol-sulfuric acid method using glucose as the standard Thewater elutes with polysaccharides were combined concen-trated to 100mL and divided into ten 10mL aliquots Eachaliquots was fractionated by size-exclusion chromatographyon a Sephacryl S-100 gel chromatography column (24mmtimes 45 cm) eluted with 400mL pure water at a flow rate of05mLmin each time The phenol-sulfuric acid method wasalso employed in this process The water eluting fraction wascollected and concentrated to 50mL and lyophilized to 005 gwhite purified polysaccharide (PAM)
24 Structure Analysis of PAM The molecular weight ofPAM was determined by high-performance gel permeationchromatography (HPGPC) [19] The monosaccharide com-position of PAM was determined by GC-MS [20] The IRspectrum of PAM was determined using a Fourier trans-form IR spectrophotometer (FT-IR) (NEXUS-870 NicoletInstrument Co USA) [21] Microstructure was determinedby atomic force microscopy [22]
25 The Stability of PAM in Artificial GastricIntestinal JuiceThe artificial gastric and intestinal juices were preparedreferenced to Chinese Pharmacopoeia [23] Briefly 40mLartificial gastric or intestinal juice was added to 40mL PAMsolutions (1mgmL in pure water) and incubated in waterbath at 37∘C During incubation 08mL mixed solution wastaken at 0 1 2 3 4 5 6 12 and 24 h respectively Theconcentration of polysaccharide was determined in 04mLsolution using phenol-sulfuric acid method [24] and theconcentration of reducing sugar was tested in 04mL solutionusing DNS method [25] The metabolic rate of PAM wascalculated by the following formula
119872 = [119868 minus (119879 minus 119877)]119868
times 100 (1)
where 119872 is metabolic rate 119868 is initial concentration ofpolysaccharide 119879 is total concentration of polysaccharide 119877is concentration of reducing sugar
26 The Effect of PAM on HumanRat Intestinal BacteriaIn Vitro The human intestinal bacteria juice was preparedas previously reported [26] Briefly PAM was blended withhumanrat intestinal flora liquid in anaerobic incubationand incubated at 37∘C During incubation 4mL mixtureincubated was taken at 0 2 4 6 8 10 12 and 24 hrespectively The digestion of reducing sugar was determinedusing DNS method The calculation formula of reducingsugar digestion rate was given as follows
119877 = (119868 minus 119871)119868times 100 (2)
where 119877 is reducing sugar digestion rate 119868 is initial level ofreducing sugar 119871 is remaining level of reducing sugar
27 The Effect of PAM on the Intestinal Flora In Vivo Afterone week of acclimatization the rats were randomly dividedinto 4 groups (with 8 rats in each group) namely control(healthy) model (untreated) and high- (0105 gkg) and low-dose (0035 gkg) PAM treated groups The rats of model andhigh- and low-dose PAM groups were intragastrically givenCAE 10 gkg (10 g crude herbs per 1 kg rat body weight) twicea day for the first 10 d to induce the disordered model ofintestinal flora From the eleventh day the high- and low-dose PAM groups were treated with PAM solution in dose of0105 gkg and 0035 gkg one time each day for 10 d respec-tively The control and model groups were intragastricallygiven the same volume of distilled water
Three or four pieces of fecal pellets (about 10 g) per ratwere directly collected from the anus into sterile plastic tubesand stored atminus20∘C Procedures were repeated after 8 h Fecalpellets were collected at the 1st 10th and 17th days of theanimal experiment
The protocol to extract total DNA of intestinal bacteriafrom feces andmethod to performERIC-PCRwere describedpreviously [27] Statistical analysis was carried out using theShannondiversity index and similarity coefficients (similaritydegree of the two classification units) Shannonrsquos diversity
Evidence-Based Complementary and Alternative Medicine 3
Figure 1 High-performance gel permeation chromatography ofPAM
index was the value to describe the community diversity ofintestinal flora and it was calculated as follows
1198671015840= sum(119901119894) (log
2119901 minus 119894) (3)
where 119901 represents the proportion of a phylotype relative tothe sum of all phylotypes of intestinal flora The similaritycoefficient was calculated as
119862119904() =(2 times 119895)
(119886 + 119887)
times 100 (4)
where ldquo119886rdquo is the number of total bands in the ERIC-PCRpattern for one sample ldquo119887rdquo is the number for the other andldquo119895rdquo is the number of the common bands shared by bothsamples [27]
3 Results and Discussion
31 Structure Characterization Analysis of PAM PAM wasshown as a single and symmetrical peak inHPGPC (Figure 1)From the chromatography PAM was a homogeneouspolysaccharide The FT-IR spectrum of PAM (Figure 2)revealed a major broad stretching peak at 328974 cmminus1 forthe typical hydroxyl group and a weak band at 292969 cmminus1for the CndashH stretching vibration [21] The absorbance at165198 cmminus1 indicated the presence of carbonyl group Themain absorptions of CndashO stretching 112151 cmminus1 suggestedthat the characteristics of sugar structureswere pyranose con-figurationThe band at 87445 cmminus1 indicated the existence ofmannose in PAMThe band at 89746 cmminus1 was the character-istic peak of 120573-configuration in PAM The PAM image takenfrom atomic force microscope was shown in Figure 3 and themode of PAMmolecular chainwaswinding coil and sphericaldistribution and multiple nodular polysaccharide combinedwith each other by intermolecular interactionsThe diameterof the molecular size was 422 nm under the magnificationof the image display The molecular weight of PAM was28773Da as calculated by drawing standard curve (Figure 4)
100
98
96
94
92
90
88
86
84
82
80
78
76
4000 3500 3000 2500 2000 1500 1000 500
Tran
smitt
ance
()
Wavenumber (cmminus1)
328974
292969
164543
112151
89767
87445
102965
Figure 2 FTIR of PAM
500
400
300
200
100
0
5004003002001000
(nm
)
(nm
)
(nm)
000
1339
Figure 3 Atomic force microscopy images of PAM
The monosaccharide composition (Figure 5) of PAM wasrhamnose glucose mannose xylose and galactose andthe mole ratio was 003 025 015 041 015 The result ofmonosaccharide composition indicated that the percentageof xylose was very high in PAM Previous studies reportedthat human intestinal Bifidobacterium can be activated andproliferated by xylose [28] with enhanced benefits for humanhealth Consumption of xylose can improve the microbialenvironment of the human body So it provided the basis forthe following research about PAM function
32 The Stability of PAM in Artificial GastricIntestinalJuice Figure 6 shows the changes of PAM metabolism inartificial gastricintestinal juice in a 12-hour time windowThe concentration of reducing sugar produced increasedsignificantly in the first 1 h followed by a slow increase untilequilibrium At 12 h the concentration of reducing sugar inthe artificial gastric and intestinal juice was 243 and 181respectivelyThe results indicated that PAMwas metabolizedin gastrointestinal tract Moreover the majority part of PAMwas digested in the intestinal juice
4 Evidence-Based Complementary and Alternative Medicine
54
52
50
48
46
44
42
40
38
36
7 8 9 10 11 12 13 14
T (min)
Log W
M
Figure 4 Standard curve of the molecular weight of PAM
33 The In Vitro Activity of PAM on Intestinal Flora WhenPAMwas added and anaerobically incubated with humanratintestinal bacterial mixture from feces it was found that theconsumption rate of reducing sugar was increased Figure 7shows how the content of reducing sugar changed in humanrat and control The changes of PAM content might bedivided into three main periods slow reduction (0sim2 h)sharp reduction (2sim6 h) and smooth reduction (6sim24 h)From 2 to 6 h the consumption rates of reducing sugardecreased significantly and almost linearly (12ndash02 g and 12ndash03 g in human and rat resp) In general the human intestinalflora was similar to rat on the consumption rate of reducingsugar incubated with PAM 779 and 769 respectivelyTherefore PAM might activate and accelerate the growth ofintestinal flora which provided clues for the further study ofPAM in vivo
34 The In Vivo Metabolic Activity of PAM on Intestinal FloraAmodel animal of intestinal flora disordered was establishedby oral administration of CAE [27] The characteristic symp-tom of watery stools was obviously observed in the modelrats and the other symptomswere also observed in this studysuch as humped back narrow eyes listlessness inappetenceand weight loss When treated by PAM intragastrically
25
30
20
15
10
5
0
0 2 4 6 8 10 12
()
T (h)
Artificial intestinal juiceArtificial gastric juice
Figure 6 Metabolism of PAM in artificial intestinal juicegastricjuice
14
13
12
11
10
09
08
07
06
05
04
03
02
01
00
0 2 4 6 8 10 12 14 16 18 20 22 24
Time (h)
(g)
ControlHumanRat
Figure 7 The effect of PAM on humanrat intestinal flora to digestreducing sugar
the characteristic symptom of watery stools was significantlyimproved Their vigor was increased and their weights wereobviously recovered in groups receiving high and low doseof PAM (Figure 8) Compared to the untreated group thesymptoms in all treated groups reduced more efficiently butthere were no significant differences between groups
ERIC-PCR profiles of intestinal flora DNA from feceswere performed on 32 rats (Figure 9) In healthy condition(control group) the Shannon diversity index (1198671015840) of 32 ratswas 181 plusmn 002 while model group (untreated) dropped to16 plusmn 002 (119875 lt 005) with a decline of 20 It was shownthat the ecological balance of intestinal flora in rats wasdestroyed and flora species were declined When the model
Evidence-Based Complementary and Alternative Medicine 5
320
300
280
260
240
220
200
180
Wei
ght (
g)
a b c
ControlHigh-dose group
Low-dose groupModel
Figure 8 The weight of rats at the periods of modeled and treated (a before administration b after administration c after treatment)
25
20
15
10
05
00
Before senna administration
After PAM treatmentAfter senna administration
Control
Shan
nonrsquos
inde
x
Model
(a)
Before senna administrationAfter senna administrationAfter PAM treatment
25
20
15
10
05
00
Shan
nonrsquos
inde
x
High dose of PAM Low dose of PAM
(b)
Figure 9 Shannonrsquos diversity index of ERIC-PCR fingerprinting of the disordered intestinal flora model of rats before and after treatment(a) Shannonrsquos diversity index of control group and model groups (b) Shannonrsquos diversity index of high dose and low dose of PAM groups(Pflt 005 Pff
lt 001 compared with before senna folium administration)
rats were administrated with PAM the 1198671015840 was increased to174 plusmn 004 (119875 lt 005 compared to model group) in high-dose group However Shannonrsquos diversity index of low doseof PAM group was maintained at a constant level and therewas no significant difference compared with model group Acertain content of PAM might be very important to increasethe diversity index of model group
As shown in Figure 10 Sorenson index (Cs) was usedto compare ERIC-PCR fingerprinting of intestinal floraof different condition rats Before and after being treatedby CAE Cs of models (groups model high-dose PAMand low-dose PAM) ranged from 38 to 45 Comparingwith the control group (58) Cs of model group showed
a significant decrease (119875 lt 005) For high and low doseof PAM groups intestinal flora in each treatment grouppresented a certain degree of recovery especially high-dosegroup (54 119875 lt 005 compared to the model group)Both high and low dose of PAM could improve similaritycoefficients of ERIC-PCR fingerprinting which indicatedthat PAM could significantly improve the structure of intesti-nal flora Furthermore it was reported that consumptionof PAM showed an improvement of intestinal function[29] Studies on the change of intestinal flora structureby PAM treatment can provide reference to understandthe underlying mechanisms of polysaccharides on intestinalflora
6 Evidence-Based Complementary and Alternative Medicine
Figure 10 Similarity coefficient index (119862119904) of ERIC-PCR finger-
printings of rat intestinal flora before and after PAM treatmentControl Group 1 received distilled water in both inducement andtreatment phases Model Group 2 received senna administrationbut distilled water during treatment High-dose PAM and Low-dosePAM Group 3 and Group 4 received high and low doses of PAMrepectively during treatment (Pf
lt 005 comparedwith the controlPnlt 005 compared with the model)
4 Conclusions
In this paper an active polysaccharide of PAM was isolatedfrom the rhizome of A macrocephala and its structure wasalso identified ERIC-PCR profile analyses were successfullyemployed to investigate the effect of PAM on the intestinalflora Our results demonstrated that PAM could improve andadjust the disordered intestinal flora suggesting that PAMmay have the potential as an oral adjuvant for disorderedintestinal flora This study also provided a novel conceptionand evidences for future investigation of the metabolic effectof polysaccharides on intestinal flora and their therapeuticeffects
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by the National Natural ScienceFoundation of China (nos 30973962 and 81473318) Theauthors thank SJTULAB group for their research support
References
[1] B Kim S J Park S P Hong T I Kim W H Kim and JH Cheon ldquoProximal disease extension and related predictingfactors in ulcerative proctitisrdquo Scandinavian Journal of Gas-troenterology vol 49 no 2 pp 177ndash183 2014
[2] C M Guinane and P D Cotter ldquoRole of the gut microbiotain health and chronic gastrointestinal disease understandinga hidden metabolic organrdquo Therapeutic Advances in Gastroen-terology vol 6 no 4 pp 295ndash308 2013
[3] A Lyra S Lahtinen K Tiihonen and A C OuwehandldquoIntestinal microbiota and overweightrdquo BeneficialMicrobes vol1 no 4 pp 407ndash421 2010
[4] P J Turnbaugh M Hamady T Yatsunenko et al ldquoA core gutmicrobiome in obese and lean twinsrdquoNature vol 457 no 7228pp 480ndash484 2009
[5] L A David C F Maurice R N Carmody et al ldquoDiet rapidlyand reproducibly alters the human gut microbiomerdquo Naturevol 505 no 7484 pp 559ndash563 2014
[6] C Chassard and C Lacroix ldquoCarbohydrates and the human gutmicrobiotardquo Current Opinion in Clinical Nutrition amp MetabolicCare vol 16 no 4 pp 453ndash460 2013
[7] S K Lin J R Lambert and M L Wahlqvist ldquoNutritionand gastrointestinal disordersrdquo Asia Pacific Journal of ClinicalNutrition vol 1 pp 37ndash42 1992
[8] A Frolkis L A Dieleman H W Barkema et al ldquoEnvironmentand the inflammatory bowel diseasesrdquo Canadian Journal ofGastroenterology vol 27 no 3 pp e18ndashe24 2013
[9] A N Ananthakrishnan ldquoEnvironmental triggers for inflamma-tory bowel diseaserdquo Current Gastroenterology Reports vol 15no 1 article 302 201
[10] M Rajilic-Stojanovic ldquoFunction of the microbiotardquo Best Prac-tice amp Research Clinical Gastroenterology vol 27 pp 5ndash16 2013
[11] A El-Kaoutari F Armougom J I Gordon D Raoult andB Henrissat ldquoThe abundance and variety of carbohydrate-active enzymes in the human gut microbiotardquo Nature ReviewsMicrobiology vol 11 no 7 pp 497ndash504 2013
[12] Y Bertin F Chaucheyras-Durand C Robbe-Masselot et alldquoCarbohydrate utilization by enterohaemorrhagic Escherichiacoli O157 H7 in bovine intestinal contentrdquo EnvironmentalMicrobiology vol 15 no 2 pp 610ndash622 2013
[13] X Jia C Ding S Yuan et al ldquoExtraction purification and char-acterization of polysaccharides from Hawk teardquo CarbohydratePolymers vol 99 pp 319ndash324 2014
[14] Y ChiW Li HWen X Cui H Cai and X Bi ldquoStudies on sep-aration purification and chemical structure of polysaccharidefromAtractylodes macrocephalardquo Zhong Yao Cai vol 24 no 9pp 647ndash648 2001
[15] N Inagaki Y Komatsu H Sasaki et al ldquoAcidic polysaccharidesfrom rhizomes of Atractylodes lancea as protective principle inCandida-infected micerdquo Planta Medica vol 67 no 5 pp 428ndash431 2001
[16] K-W Yu H Kiyohara T Matsumoto H-C Yang and HYamada ldquoIntestinal immune system modulating polysaccha-rides from rhizomes of Atractylodes lanceardquo Planta Medica vol64 no 8 pp 714ndash719 1998
[17] J-P Cai Y-J Wu C Li et al ldquoPanax ginseng polysaccharidesuppresses metastasis via modulating Twist expression in gas-tric cancerrdquo International Journal of Biological Macromoleculesvol 57 pp 22ndash25 2013
[18] K Y Lee and Y J Jeon ldquoPolysaccharide isolated fromPoria cocos sclerotium induces NF-120581B Rel activation andiNOS expression in murine macrophagesrdquo InternationalImmunopharmacology vol 3 no 10-11 pp 1353ndash1362 2003
[19] M Lin B Xia M Yang S Gao Y Huo and G Lou ldquoCharac-terization and antitumor activities of a polysaccharide from therhizoma of Menispermum dauricumrdquo International Journal ofBiological Macromolecules vol 53 pp 72ndash76 2013
Evidence-Based Complementary and Alternative Medicine 7
[20] N Li C Yan D Hua and D Zhang ldquoIsolation purificationand structural characterization of a novel polysaccharide fromGanoderma capenserdquo International Journal of Biological Macro-molecules vol 57 pp 285ndash290 2013
[21] M Kacurakova N Wellner A Ebringerova Z HromadkovaR H Wilson and P S Belton ldquoCharacterisation of xylan-typepolysaccharides and associated cell wall components by FT-IRand FT-Raman spectroscopiesrdquo Food Hydrocolloids vol 13 no1 pp 35ndash41 1999
[22] N I Abu-Lail and T A Camesano ldquoPolysaccharide propertiesprobed with atomic force microscopyrdquo Journal of Microscopyvol 212 no 3 pp 217ndash238 2003
[23] Pharmacopoeia Commission of Peoplersquos Republic of ChinaChinese Pharmacopoeia 2010
[24] Y Yang D Liu J Wu Y Chen and S Wang ldquoIn vitro antiox-idant activities of sulfated polysaccharide fractions extractedfrom Corallina officinalisrdquo International Journal of BiologicalMacromolecules vol 49 no 5 pp 1031ndash1037 2011
[25] A V Gusakov E G Kondratyeva and A P Sinitsyn ldquoCom-parison of two methods for assaying reducing sugars in thedetermination of carbohydrase activitiesrdquo International Journalof Analytical Chemistry vol 2011 Article ID 283658 4 pages2011
[26] WW Huang M Y Wang H M Shi et al ldquoComparative studyof bioactive constituents in crude and processed Glycyrrhizaeradix and their respective metabolic profiles in gastrointestinaltract in vitro by HPLC-DAD and HPLC-ESIMS analysesrdquoArchives of Pharmacal Research vol 35 no 11 pp 1945ndash19522012
[27] Y Peng Z Wang Y Lu C-F Wu J-Y Yang and X-B LildquoIntestinal microflora molecular markers of spleen-deficientrats and evaluation of traditional Chinese drugsrdquoWorld Journalof Gastroenterology vol 15 no 18 pp 2220ndash2227 2009
[28] W F Broekaert C M Courtin K Verbeke T van de WieleW Verstraete and J A Delcour ldquoPrebiotic and other health-related effects of cereal-derived arabinoxylans arabinoxylan-oligosaccharides and xylooligosaccharidesrdquo Critical Reviews inFood Science and Nutrition vol 51 no 2 pp 178ndash194 2011
[29] F Xie K Sakwiwatkul C Zhang Y Wang L Zhai and S HuldquoAtractylodis macrocephalae Koidz polysaccharides enhanceboth serum IgG response and gut mucosal immunityrdquo Carbo-hydrate Polymers vol 91 no 1 pp 68ndash73 2013
2 Evidence-Based Complementary and Alternative Medicine
Primers ERIC 1R (51015840-ATGTAAGCTCCTGGGGATTCAC-31015840) and ERIC 2R (51015840-AAGTAA GTGACTGGGGTGAGCG-31015840) were obtained from Sangon Biotech Co Ltd (ShanghaiChina) All reagents were of analytical grade
The dry powder of the folia of C angustifolia (senna)was extracted (CAE) thrice with boiling water (1 10) for120min (40min each time) The three filtrates were mergedand evaporated by rotary evaporation under vacuum at 60∘CFinally the concentration of extracts was set at 10 gmL
22 Animals Male SD rats (190ndash200 g and 5 weeks old)were purchased from Shanghai Slack Laboratory Animalco Ltd (Shanghai China) All procedures related to theanimals and their care conformed to the internationallyaccepted principles as found in the Guidelines for Keep-ing Experimental Animals issued by the Government ofChina
23 Extraction Isolation and Purification of PolysaccharidesThe dried powder of the rhizome of A macrocephala (200 g)was extracted thrice with 2000mL boiling water for 3 h (1 heach time) The aqueous extracts were filtrated combinedand concentrated to about 200mL with rotary evaporatorsTo precipitate the crude polysaccharide 3-fold volume ofpure ethanol was added to the extracts at 4∘C overnight Theresulting precipitate was collected by centrifugation washedwith pure ethanol (400mL) repeatedly and dried undernitrogen 20 g dry precipitate was obtained reconstituted in1000mL pure water and deproteinized using 3-fold volumeof Sevag reagent (119899-butanolchloroform vv = 1 4) Finally900mL supernatant was collected concentrated to 100mLand lyophilized to 138 g crude polysaccharides
To purify the isolated polysaccharides 1 g crude polysac-charides were dissolved filtered through 045 120583m filters andapplied to a DEAE-52 cellulose chromatography column(26mm times 60 cm) The crude polysaccharides were fraction-ated and eluted with 1000mL distilled water at a flow rateof 1mLmin The water fractions obtained were determinedaccording to the total carbohydrate content by the phenol-sulfuric acid method using glucose as the standard Thewater elutes with polysaccharides were combined concen-trated to 100mL and divided into ten 10mL aliquots Eachaliquots was fractionated by size-exclusion chromatographyon a Sephacryl S-100 gel chromatography column (24mmtimes 45 cm) eluted with 400mL pure water at a flow rate of05mLmin each time The phenol-sulfuric acid method wasalso employed in this process The water eluting fraction wascollected and concentrated to 50mL and lyophilized to 005 gwhite purified polysaccharide (PAM)
24 Structure Analysis of PAM The molecular weight ofPAM was determined by high-performance gel permeationchromatography (HPGPC) [19] The monosaccharide com-position of PAM was determined by GC-MS [20] The IRspectrum of PAM was determined using a Fourier trans-form IR spectrophotometer (FT-IR) (NEXUS-870 NicoletInstrument Co USA) [21] Microstructure was determinedby atomic force microscopy [22]
25 The Stability of PAM in Artificial GastricIntestinal JuiceThe artificial gastric and intestinal juices were preparedreferenced to Chinese Pharmacopoeia [23] Briefly 40mLartificial gastric or intestinal juice was added to 40mL PAMsolutions (1mgmL in pure water) and incubated in waterbath at 37∘C During incubation 08mL mixed solution wastaken at 0 1 2 3 4 5 6 12 and 24 h respectively Theconcentration of polysaccharide was determined in 04mLsolution using phenol-sulfuric acid method [24] and theconcentration of reducing sugar was tested in 04mL solutionusing DNS method [25] The metabolic rate of PAM wascalculated by the following formula
119872 = [119868 minus (119879 minus 119877)]119868
times 100 (1)
where 119872 is metabolic rate 119868 is initial concentration ofpolysaccharide 119879 is total concentration of polysaccharide 119877is concentration of reducing sugar
26 The Effect of PAM on HumanRat Intestinal BacteriaIn Vitro The human intestinal bacteria juice was preparedas previously reported [26] Briefly PAM was blended withhumanrat intestinal flora liquid in anaerobic incubationand incubated at 37∘C During incubation 4mL mixtureincubated was taken at 0 2 4 6 8 10 12 and 24 hrespectively The digestion of reducing sugar was determinedusing DNS method The calculation formula of reducingsugar digestion rate was given as follows
119877 = (119868 minus 119871)119868times 100 (2)
where 119877 is reducing sugar digestion rate 119868 is initial level ofreducing sugar 119871 is remaining level of reducing sugar
27 The Effect of PAM on the Intestinal Flora In Vivo Afterone week of acclimatization the rats were randomly dividedinto 4 groups (with 8 rats in each group) namely control(healthy) model (untreated) and high- (0105 gkg) and low-dose (0035 gkg) PAM treated groups The rats of model andhigh- and low-dose PAM groups were intragastrically givenCAE 10 gkg (10 g crude herbs per 1 kg rat body weight) twicea day for the first 10 d to induce the disordered model ofintestinal flora From the eleventh day the high- and low-dose PAM groups were treated with PAM solution in dose of0105 gkg and 0035 gkg one time each day for 10 d respec-tively The control and model groups were intragastricallygiven the same volume of distilled water
Three or four pieces of fecal pellets (about 10 g) per ratwere directly collected from the anus into sterile plastic tubesand stored atminus20∘C Procedures were repeated after 8 h Fecalpellets were collected at the 1st 10th and 17th days of theanimal experiment
The protocol to extract total DNA of intestinal bacteriafrom feces andmethod to performERIC-PCRwere describedpreviously [27] Statistical analysis was carried out using theShannondiversity index and similarity coefficients (similaritydegree of the two classification units) Shannonrsquos diversity
Evidence-Based Complementary and Alternative Medicine 3
Figure 1 High-performance gel permeation chromatography ofPAM
index was the value to describe the community diversity ofintestinal flora and it was calculated as follows
1198671015840= sum(119901119894) (log
2119901 minus 119894) (3)
where 119901 represents the proportion of a phylotype relative tothe sum of all phylotypes of intestinal flora The similaritycoefficient was calculated as
119862119904() =(2 times 119895)
(119886 + 119887)
times 100 (4)
where ldquo119886rdquo is the number of total bands in the ERIC-PCRpattern for one sample ldquo119887rdquo is the number for the other andldquo119895rdquo is the number of the common bands shared by bothsamples [27]
3 Results and Discussion
31 Structure Characterization Analysis of PAM PAM wasshown as a single and symmetrical peak inHPGPC (Figure 1)From the chromatography PAM was a homogeneouspolysaccharide The FT-IR spectrum of PAM (Figure 2)revealed a major broad stretching peak at 328974 cmminus1 forthe typical hydroxyl group and a weak band at 292969 cmminus1for the CndashH stretching vibration [21] The absorbance at165198 cmminus1 indicated the presence of carbonyl group Themain absorptions of CndashO stretching 112151 cmminus1 suggestedthat the characteristics of sugar structureswere pyranose con-figurationThe band at 87445 cmminus1 indicated the existence ofmannose in PAMThe band at 89746 cmminus1 was the character-istic peak of 120573-configuration in PAM The PAM image takenfrom atomic force microscope was shown in Figure 3 and themode of PAMmolecular chainwaswinding coil and sphericaldistribution and multiple nodular polysaccharide combinedwith each other by intermolecular interactionsThe diameterof the molecular size was 422 nm under the magnificationof the image display The molecular weight of PAM was28773Da as calculated by drawing standard curve (Figure 4)
100
98
96
94
92
90
88
86
84
82
80
78
76
4000 3500 3000 2500 2000 1500 1000 500
Tran
smitt
ance
()
Wavenumber (cmminus1)
328974
292969
164543
112151
89767
87445
102965
Figure 2 FTIR of PAM
500
400
300
200
100
0
5004003002001000
(nm
)
(nm
)
(nm)
000
1339
Figure 3 Atomic force microscopy images of PAM
The monosaccharide composition (Figure 5) of PAM wasrhamnose glucose mannose xylose and galactose andthe mole ratio was 003 025 015 041 015 The result ofmonosaccharide composition indicated that the percentageof xylose was very high in PAM Previous studies reportedthat human intestinal Bifidobacterium can be activated andproliferated by xylose [28] with enhanced benefits for humanhealth Consumption of xylose can improve the microbialenvironment of the human body So it provided the basis forthe following research about PAM function
32 The Stability of PAM in Artificial GastricIntestinalJuice Figure 6 shows the changes of PAM metabolism inartificial gastricintestinal juice in a 12-hour time windowThe concentration of reducing sugar produced increasedsignificantly in the first 1 h followed by a slow increase untilequilibrium At 12 h the concentration of reducing sugar inthe artificial gastric and intestinal juice was 243 and 181respectivelyThe results indicated that PAMwas metabolizedin gastrointestinal tract Moreover the majority part of PAMwas digested in the intestinal juice
4 Evidence-Based Complementary and Alternative Medicine
54
52
50
48
46
44
42
40
38
36
7 8 9 10 11 12 13 14
T (min)
Log W
M
Figure 4 Standard curve of the molecular weight of PAM
33 The In Vitro Activity of PAM on Intestinal Flora WhenPAMwas added and anaerobically incubated with humanratintestinal bacterial mixture from feces it was found that theconsumption rate of reducing sugar was increased Figure 7shows how the content of reducing sugar changed in humanrat and control The changes of PAM content might bedivided into three main periods slow reduction (0sim2 h)sharp reduction (2sim6 h) and smooth reduction (6sim24 h)From 2 to 6 h the consumption rates of reducing sugardecreased significantly and almost linearly (12ndash02 g and 12ndash03 g in human and rat resp) In general the human intestinalflora was similar to rat on the consumption rate of reducingsugar incubated with PAM 779 and 769 respectivelyTherefore PAM might activate and accelerate the growth ofintestinal flora which provided clues for the further study ofPAM in vivo
34 The In Vivo Metabolic Activity of PAM on Intestinal FloraAmodel animal of intestinal flora disordered was establishedby oral administration of CAE [27] The characteristic symp-tom of watery stools was obviously observed in the modelrats and the other symptomswere also observed in this studysuch as humped back narrow eyes listlessness inappetenceand weight loss When treated by PAM intragastrically
25
30
20
15
10
5
0
0 2 4 6 8 10 12
()
T (h)
Artificial intestinal juiceArtificial gastric juice
Figure 6 Metabolism of PAM in artificial intestinal juicegastricjuice
14
13
12
11
10
09
08
07
06
05
04
03
02
01
00
0 2 4 6 8 10 12 14 16 18 20 22 24
Time (h)
(g)
ControlHumanRat
Figure 7 The effect of PAM on humanrat intestinal flora to digestreducing sugar
the characteristic symptom of watery stools was significantlyimproved Their vigor was increased and their weights wereobviously recovered in groups receiving high and low doseof PAM (Figure 8) Compared to the untreated group thesymptoms in all treated groups reduced more efficiently butthere were no significant differences between groups
ERIC-PCR profiles of intestinal flora DNA from feceswere performed on 32 rats (Figure 9) In healthy condition(control group) the Shannon diversity index (1198671015840) of 32 ratswas 181 plusmn 002 while model group (untreated) dropped to16 plusmn 002 (119875 lt 005) with a decline of 20 It was shownthat the ecological balance of intestinal flora in rats wasdestroyed and flora species were declined When the model
Evidence-Based Complementary and Alternative Medicine 5
320
300
280
260
240
220
200
180
Wei
ght (
g)
a b c
ControlHigh-dose group
Low-dose groupModel
Figure 8 The weight of rats at the periods of modeled and treated (a before administration b after administration c after treatment)
25
20
15
10
05
00
Before senna administration
After PAM treatmentAfter senna administration
Control
Shan
nonrsquos
inde
x
Model
(a)
Before senna administrationAfter senna administrationAfter PAM treatment
25
20
15
10
05
00
Shan
nonrsquos
inde
x
High dose of PAM Low dose of PAM
(b)
Figure 9 Shannonrsquos diversity index of ERIC-PCR fingerprinting of the disordered intestinal flora model of rats before and after treatment(a) Shannonrsquos diversity index of control group and model groups (b) Shannonrsquos diversity index of high dose and low dose of PAM groups(Pflt 005 Pff
lt 001 compared with before senna folium administration)
rats were administrated with PAM the 1198671015840 was increased to174 plusmn 004 (119875 lt 005 compared to model group) in high-dose group However Shannonrsquos diversity index of low doseof PAM group was maintained at a constant level and therewas no significant difference compared with model group Acertain content of PAM might be very important to increasethe diversity index of model group
As shown in Figure 10 Sorenson index (Cs) was usedto compare ERIC-PCR fingerprinting of intestinal floraof different condition rats Before and after being treatedby CAE Cs of models (groups model high-dose PAMand low-dose PAM) ranged from 38 to 45 Comparingwith the control group (58) Cs of model group showed
a significant decrease (119875 lt 005) For high and low doseof PAM groups intestinal flora in each treatment grouppresented a certain degree of recovery especially high-dosegroup (54 119875 lt 005 compared to the model group)Both high and low dose of PAM could improve similaritycoefficients of ERIC-PCR fingerprinting which indicatedthat PAM could significantly improve the structure of intesti-nal flora Furthermore it was reported that consumptionof PAM showed an improvement of intestinal function[29] Studies on the change of intestinal flora structureby PAM treatment can provide reference to understandthe underlying mechanisms of polysaccharides on intestinalflora
6 Evidence-Based Complementary and Alternative Medicine
Figure 10 Similarity coefficient index (119862119904) of ERIC-PCR finger-
printings of rat intestinal flora before and after PAM treatmentControl Group 1 received distilled water in both inducement andtreatment phases Model Group 2 received senna administrationbut distilled water during treatment High-dose PAM and Low-dosePAM Group 3 and Group 4 received high and low doses of PAMrepectively during treatment (Pf
lt 005 comparedwith the controlPnlt 005 compared with the model)
4 Conclusions
In this paper an active polysaccharide of PAM was isolatedfrom the rhizome of A macrocephala and its structure wasalso identified ERIC-PCR profile analyses were successfullyemployed to investigate the effect of PAM on the intestinalflora Our results demonstrated that PAM could improve andadjust the disordered intestinal flora suggesting that PAMmay have the potential as an oral adjuvant for disorderedintestinal flora This study also provided a novel conceptionand evidences for future investigation of the metabolic effectof polysaccharides on intestinal flora and their therapeuticeffects
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by the National Natural ScienceFoundation of China (nos 30973962 and 81473318) Theauthors thank SJTULAB group for their research support
References
[1] B Kim S J Park S P Hong T I Kim W H Kim and JH Cheon ldquoProximal disease extension and related predictingfactors in ulcerative proctitisrdquo Scandinavian Journal of Gas-troenterology vol 49 no 2 pp 177ndash183 2014
[2] C M Guinane and P D Cotter ldquoRole of the gut microbiotain health and chronic gastrointestinal disease understandinga hidden metabolic organrdquo Therapeutic Advances in Gastroen-terology vol 6 no 4 pp 295ndash308 2013
[3] A Lyra S Lahtinen K Tiihonen and A C OuwehandldquoIntestinal microbiota and overweightrdquo BeneficialMicrobes vol1 no 4 pp 407ndash421 2010
[4] P J Turnbaugh M Hamady T Yatsunenko et al ldquoA core gutmicrobiome in obese and lean twinsrdquoNature vol 457 no 7228pp 480ndash484 2009
[5] L A David C F Maurice R N Carmody et al ldquoDiet rapidlyand reproducibly alters the human gut microbiomerdquo Naturevol 505 no 7484 pp 559ndash563 2014
[6] C Chassard and C Lacroix ldquoCarbohydrates and the human gutmicrobiotardquo Current Opinion in Clinical Nutrition amp MetabolicCare vol 16 no 4 pp 453ndash460 2013
[7] S K Lin J R Lambert and M L Wahlqvist ldquoNutritionand gastrointestinal disordersrdquo Asia Pacific Journal of ClinicalNutrition vol 1 pp 37ndash42 1992
[8] A Frolkis L A Dieleman H W Barkema et al ldquoEnvironmentand the inflammatory bowel diseasesrdquo Canadian Journal ofGastroenterology vol 27 no 3 pp e18ndashe24 2013
[9] A N Ananthakrishnan ldquoEnvironmental triggers for inflamma-tory bowel diseaserdquo Current Gastroenterology Reports vol 15no 1 article 302 201
[10] M Rajilic-Stojanovic ldquoFunction of the microbiotardquo Best Prac-tice amp Research Clinical Gastroenterology vol 27 pp 5ndash16 2013
[11] A El-Kaoutari F Armougom J I Gordon D Raoult andB Henrissat ldquoThe abundance and variety of carbohydrate-active enzymes in the human gut microbiotardquo Nature ReviewsMicrobiology vol 11 no 7 pp 497ndash504 2013
[12] Y Bertin F Chaucheyras-Durand C Robbe-Masselot et alldquoCarbohydrate utilization by enterohaemorrhagic Escherichiacoli O157 H7 in bovine intestinal contentrdquo EnvironmentalMicrobiology vol 15 no 2 pp 610ndash622 2013
[13] X Jia C Ding S Yuan et al ldquoExtraction purification and char-acterization of polysaccharides from Hawk teardquo CarbohydratePolymers vol 99 pp 319ndash324 2014
[14] Y ChiW Li HWen X Cui H Cai and X Bi ldquoStudies on sep-aration purification and chemical structure of polysaccharidefromAtractylodes macrocephalardquo Zhong Yao Cai vol 24 no 9pp 647ndash648 2001
[15] N Inagaki Y Komatsu H Sasaki et al ldquoAcidic polysaccharidesfrom rhizomes of Atractylodes lancea as protective principle inCandida-infected micerdquo Planta Medica vol 67 no 5 pp 428ndash431 2001
[16] K-W Yu H Kiyohara T Matsumoto H-C Yang and HYamada ldquoIntestinal immune system modulating polysaccha-rides from rhizomes of Atractylodes lanceardquo Planta Medica vol64 no 8 pp 714ndash719 1998
[17] J-P Cai Y-J Wu C Li et al ldquoPanax ginseng polysaccharidesuppresses metastasis via modulating Twist expression in gas-tric cancerrdquo International Journal of Biological Macromoleculesvol 57 pp 22ndash25 2013
[18] K Y Lee and Y J Jeon ldquoPolysaccharide isolated fromPoria cocos sclerotium induces NF-120581B Rel activation andiNOS expression in murine macrophagesrdquo InternationalImmunopharmacology vol 3 no 10-11 pp 1353ndash1362 2003
[19] M Lin B Xia M Yang S Gao Y Huo and G Lou ldquoCharac-terization and antitumor activities of a polysaccharide from therhizoma of Menispermum dauricumrdquo International Journal ofBiological Macromolecules vol 53 pp 72ndash76 2013
Evidence-Based Complementary and Alternative Medicine 7
[20] N Li C Yan D Hua and D Zhang ldquoIsolation purificationand structural characterization of a novel polysaccharide fromGanoderma capenserdquo International Journal of Biological Macro-molecules vol 57 pp 285ndash290 2013
[21] M Kacurakova N Wellner A Ebringerova Z HromadkovaR H Wilson and P S Belton ldquoCharacterisation of xylan-typepolysaccharides and associated cell wall components by FT-IRand FT-Raman spectroscopiesrdquo Food Hydrocolloids vol 13 no1 pp 35ndash41 1999
[22] N I Abu-Lail and T A Camesano ldquoPolysaccharide propertiesprobed with atomic force microscopyrdquo Journal of Microscopyvol 212 no 3 pp 217ndash238 2003
[23] Pharmacopoeia Commission of Peoplersquos Republic of ChinaChinese Pharmacopoeia 2010
[24] Y Yang D Liu J Wu Y Chen and S Wang ldquoIn vitro antiox-idant activities of sulfated polysaccharide fractions extractedfrom Corallina officinalisrdquo International Journal of BiologicalMacromolecules vol 49 no 5 pp 1031ndash1037 2011
[25] A V Gusakov E G Kondratyeva and A P Sinitsyn ldquoCom-parison of two methods for assaying reducing sugars in thedetermination of carbohydrase activitiesrdquo International Journalof Analytical Chemistry vol 2011 Article ID 283658 4 pages2011
[26] WW Huang M Y Wang H M Shi et al ldquoComparative studyof bioactive constituents in crude and processed Glycyrrhizaeradix and their respective metabolic profiles in gastrointestinaltract in vitro by HPLC-DAD and HPLC-ESIMS analysesrdquoArchives of Pharmacal Research vol 35 no 11 pp 1945ndash19522012
[27] Y Peng Z Wang Y Lu C-F Wu J-Y Yang and X-B LildquoIntestinal microflora molecular markers of spleen-deficientrats and evaluation of traditional Chinese drugsrdquoWorld Journalof Gastroenterology vol 15 no 18 pp 2220ndash2227 2009
[28] W F Broekaert C M Courtin K Verbeke T van de WieleW Verstraete and J A Delcour ldquoPrebiotic and other health-related effects of cereal-derived arabinoxylans arabinoxylan-oligosaccharides and xylooligosaccharidesrdquo Critical Reviews inFood Science and Nutrition vol 51 no 2 pp 178ndash194 2011
[29] F Xie K Sakwiwatkul C Zhang Y Wang L Zhai and S HuldquoAtractylodis macrocephalae Koidz polysaccharides enhanceboth serum IgG response and gut mucosal immunityrdquo Carbo-hydrate Polymers vol 91 no 1 pp 68ndash73 2013
Figure 1 High-performance gel permeation chromatography ofPAM
index was the value to describe the community diversity ofintestinal flora and it was calculated as follows
1198671015840= sum(119901119894) (log
2119901 minus 119894) (3)
where 119901 represents the proportion of a phylotype relative tothe sum of all phylotypes of intestinal flora The similaritycoefficient was calculated as
119862119904() =(2 times 119895)
(119886 + 119887)
times 100 (4)
where ldquo119886rdquo is the number of total bands in the ERIC-PCRpattern for one sample ldquo119887rdquo is the number for the other andldquo119895rdquo is the number of the common bands shared by bothsamples [27]
3 Results and Discussion
31 Structure Characterization Analysis of PAM PAM wasshown as a single and symmetrical peak inHPGPC (Figure 1)From the chromatography PAM was a homogeneouspolysaccharide The FT-IR spectrum of PAM (Figure 2)revealed a major broad stretching peak at 328974 cmminus1 forthe typical hydroxyl group and a weak band at 292969 cmminus1for the CndashH stretching vibration [21] The absorbance at165198 cmminus1 indicated the presence of carbonyl group Themain absorptions of CndashO stretching 112151 cmminus1 suggestedthat the characteristics of sugar structureswere pyranose con-figurationThe band at 87445 cmminus1 indicated the existence ofmannose in PAMThe band at 89746 cmminus1 was the character-istic peak of 120573-configuration in PAM The PAM image takenfrom atomic force microscope was shown in Figure 3 and themode of PAMmolecular chainwaswinding coil and sphericaldistribution and multiple nodular polysaccharide combinedwith each other by intermolecular interactionsThe diameterof the molecular size was 422 nm under the magnificationof the image display The molecular weight of PAM was28773Da as calculated by drawing standard curve (Figure 4)
100
98
96
94
92
90
88
86
84
82
80
78
76
4000 3500 3000 2500 2000 1500 1000 500
Tran
smitt
ance
()
Wavenumber (cmminus1)
328974
292969
164543
112151
89767
87445
102965
Figure 2 FTIR of PAM
500
400
300
200
100
0
5004003002001000
(nm
)
(nm
)
(nm)
000
1339
Figure 3 Atomic force microscopy images of PAM
The monosaccharide composition (Figure 5) of PAM wasrhamnose glucose mannose xylose and galactose andthe mole ratio was 003 025 015 041 015 The result ofmonosaccharide composition indicated that the percentageof xylose was very high in PAM Previous studies reportedthat human intestinal Bifidobacterium can be activated andproliferated by xylose [28] with enhanced benefits for humanhealth Consumption of xylose can improve the microbialenvironment of the human body So it provided the basis forthe following research about PAM function
32 The Stability of PAM in Artificial GastricIntestinalJuice Figure 6 shows the changes of PAM metabolism inartificial gastricintestinal juice in a 12-hour time windowThe concentration of reducing sugar produced increasedsignificantly in the first 1 h followed by a slow increase untilequilibrium At 12 h the concentration of reducing sugar inthe artificial gastric and intestinal juice was 243 and 181respectivelyThe results indicated that PAMwas metabolizedin gastrointestinal tract Moreover the majority part of PAMwas digested in the intestinal juice
4 Evidence-Based Complementary and Alternative Medicine
54
52
50
48
46
44
42
40
38
36
7 8 9 10 11 12 13 14
T (min)
Log W
M
Figure 4 Standard curve of the molecular weight of PAM
33 The In Vitro Activity of PAM on Intestinal Flora WhenPAMwas added and anaerobically incubated with humanratintestinal bacterial mixture from feces it was found that theconsumption rate of reducing sugar was increased Figure 7shows how the content of reducing sugar changed in humanrat and control The changes of PAM content might bedivided into three main periods slow reduction (0sim2 h)sharp reduction (2sim6 h) and smooth reduction (6sim24 h)From 2 to 6 h the consumption rates of reducing sugardecreased significantly and almost linearly (12ndash02 g and 12ndash03 g in human and rat resp) In general the human intestinalflora was similar to rat on the consumption rate of reducingsugar incubated with PAM 779 and 769 respectivelyTherefore PAM might activate and accelerate the growth ofintestinal flora which provided clues for the further study ofPAM in vivo
34 The In Vivo Metabolic Activity of PAM on Intestinal FloraAmodel animal of intestinal flora disordered was establishedby oral administration of CAE [27] The characteristic symp-tom of watery stools was obviously observed in the modelrats and the other symptomswere also observed in this studysuch as humped back narrow eyes listlessness inappetenceand weight loss When treated by PAM intragastrically
25
30
20
15
10
5
0
0 2 4 6 8 10 12
()
T (h)
Artificial intestinal juiceArtificial gastric juice
Figure 6 Metabolism of PAM in artificial intestinal juicegastricjuice
14
13
12
11
10
09
08
07
06
05
04
03
02
01
00
0 2 4 6 8 10 12 14 16 18 20 22 24
Time (h)
(g)
ControlHumanRat
Figure 7 The effect of PAM on humanrat intestinal flora to digestreducing sugar
the characteristic symptom of watery stools was significantlyimproved Their vigor was increased and their weights wereobviously recovered in groups receiving high and low doseof PAM (Figure 8) Compared to the untreated group thesymptoms in all treated groups reduced more efficiently butthere were no significant differences between groups
ERIC-PCR profiles of intestinal flora DNA from feceswere performed on 32 rats (Figure 9) In healthy condition(control group) the Shannon diversity index (1198671015840) of 32 ratswas 181 plusmn 002 while model group (untreated) dropped to16 plusmn 002 (119875 lt 005) with a decline of 20 It was shownthat the ecological balance of intestinal flora in rats wasdestroyed and flora species were declined When the model
Evidence-Based Complementary and Alternative Medicine 5
320
300
280
260
240
220
200
180
Wei
ght (
g)
a b c
ControlHigh-dose group
Low-dose groupModel
Figure 8 The weight of rats at the periods of modeled and treated (a before administration b after administration c after treatment)
25
20
15
10
05
00
Before senna administration
After PAM treatmentAfter senna administration
Control
Shan
nonrsquos
inde
x
Model
(a)
Before senna administrationAfter senna administrationAfter PAM treatment
25
20
15
10
05
00
Shan
nonrsquos
inde
x
High dose of PAM Low dose of PAM
(b)
Figure 9 Shannonrsquos diversity index of ERIC-PCR fingerprinting of the disordered intestinal flora model of rats before and after treatment(a) Shannonrsquos diversity index of control group and model groups (b) Shannonrsquos diversity index of high dose and low dose of PAM groups(Pflt 005 Pff
lt 001 compared with before senna folium administration)
rats were administrated with PAM the 1198671015840 was increased to174 plusmn 004 (119875 lt 005 compared to model group) in high-dose group However Shannonrsquos diversity index of low doseof PAM group was maintained at a constant level and therewas no significant difference compared with model group Acertain content of PAM might be very important to increasethe diversity index of model group
As shown in Figure 10 Sorenson index (Cs) was usedto compare ERIC-PCR fingerprinting of intestinal floraof different condition rats Before and after being treatedby CAE Cs of models (groups model high-dose PAMand low-dose PAM) ranged from 38 to 45 Comparingwith the control group (58) Cs of model group showed
a significant decrease (119875 lt 005) For high and low doseof PAM groups intestinal flora in each treatment grouppresented a certain degree of recovery especially high-dosegroup (54 119875 lt 005 compared to the model group)Both high and low dose of PAM could improve similaritycoefficients of ERIC-PCR fingerprinting which indicatedthat PAM could significantly improve the structure of intesti-nal flora Furthermore it was reported that consumptionof PAM showed an improvement of intestinal function[29] Studies on the change of intestinal flora structureby PAM treatment can provide reference to understandthe underlying mechanisms of polysaccharides on intestinalflora
6 Evidence-Based Complementary and Alternative Medicine
Figure 10 Similarity coefficient index (119862119904) of ERIC-PCR finger-
printings of rat intestinal flora before and after PAM treatmentControl Group 1 received distilled water in both inducement andtreatment phases Model Group 2 received senna administrationbut distilled water during treatment High-dose PAM and Low-dosePAM Group 3 and Group 4 received high and low doses of PAMrepectively during treatment (Pf
lt 005 comparedwith the controlPnlt 005 compared with the model)
4 Conclusions
In this paper an active polysaccharide of PAM was isolatedfrom the rhizome of A macrocephala and its structure wasalso identified ERIC-PCR profile analyses were successfullyemployed to investigate the effect of PAM on the intestinalflora Our results demonstrated that PAM could improve andadjust the disordered intestinal flora suggesting that PAMmay have the potential as an oral adjuvant for disorderedintestinal flora This study also provided a novel conceptionand evidences for future investigation of the metabolic effectof polysaccharides on intestinal flora and their therapeuticeffects
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by the National Natural ScienceFoundation of China (nos 30973962 and 81473318) Theauthors thank SJTULAB group for their research support
References
[1] B Kim S J Park S P Hong T I Kim W H Kim and JH Cheon ldquoProximal disease extension and related predictingfactors in ulcerative proctitisrdquo Scandinavian Journal of Gas-troenterology vol 49 no 2 pp 177ndash183 2014
[2] C M Guinane and P D Cotter ldquoRole of the gut microbiotain health and chronic gastrointestinal disease understandinga hidden metabolic organrdquo Therapeutic Advances in Gastroen-terology vol 6 no 4 pp 295ndash308 2013
[3] A Lyra S Lahtinen K Tiihonen and A C OuwehandldquoIntestinal microbiota and overweightrdquo BeneficialMicrobes vol1 no 4 pp 407ndash421 2010
[4] P J Turnbaugh M Hamady T Yatsunenko et al ldquoA core gutmicrobiome in obese and lean twinsrdquoNature vol 457 no 7228pp 480ndash484 2009
[5] L A David C F Maurice R N Carmody et al ldquoDiet rapidlyand reproducibly alters the human gut microbiomerdquo Naturevol 505 no 7484 pp 559ndash563 2014
[6] C Chassard and C Lacroix ldquoCarbohydrates and the human gutmicrobiotardquo Current Opinion in Clinical Nutrition amp MetabolicCare vol 16 no 4 pp 453ndash460 2013
[7] S K Lin J R Lambert and M L Wahlqvist ldquoNutritionand gastrointestinal disordersrdquo Asia Pacific Journal of ClinicalNutrition vol 1 pp 37ndash42 1992
[8] A Frolkis L A Dieleman H W Barkema et al ldquoEnvironmentand the inflammatory bowel diseasesrdquo Canadian Journal ofGastroenterology vol 27 no 3 pp e18ndashe24 2013
[9] A N Ananthakrishnan ldquoEnvironmental triggers for inflamma-tory bowel diseaserdquo Current Gastroenterology Reports vol 15no 1 article 302 201
[10] M Rajilic-Stojanovic ldquoFunction of the microbiotardquo Best Prac-tice amp Research Clinical Gastroenterology vol 27 pp 5ndash16 2013
[11] A El-Kaoutari F Armougom J I Gordon D Raoult andB Henrissat ldquoThe abundance and variety of carbohydrate-active enzymes in the human gut microbiotardquo Nature ReviewsMicrobiology vol 11 no 7 pp 497ndash504 2013
[12] Y Bertin F Chaucheyras-Durand C Robbe-Masselot et alldquoCarbohydrate utilization by enterohaemorrhagic Escherichiacoli O157 H7 in bovine intestinal contentrdquo EnvironmentalMicrobiology vol 15 no 2 pp 610ndash622 2013
[13] X Jia C Ding S Yuan et al ldquoExtraction purification and char-acterization of polysaccharides from Hawk teardquo CarbohydratePolymers vol 99 pp 319ndash324 2014
[14] Y ChiW Li HWen X Cui H Cai and X Bi ldquoStudies on sep-aration purification and chemical structure of polysaccharidefromAtractylodes macrocephalardquo Zhong Yao Cai vol 24 no 9pp 647ndash648 2001
[15] N Inagaki Y Komatsu H Sasaki et al ldquoAcidic polysaccharidesfrom rhizomes of Atractylodes lancea as protective principle inCandida-infected micerdquo Planta Medica vol 67 no 5 pp 428ndash431 2001
[16] K-W Yu H Kiyohara T Matsumoto H-C Yang and HYamada ldquoIntestinal immune system modulating polysaccha-rides from rhizomes of Atractylodes lanceardquo Planta Medica vol64 no 8 pp 714ndash719 1998
[17] J-P Cai Y-J Wu C Li et al ldquoPanax ginseng polysaccharidesuppresses metastasis via modulating Twist expression in gas-tric cancerrdquo International Journal of Biological Macromoleculesvol 57 pp 22ndash25 2013
[18] K Y Lee and Y J Jeon ldquoPolysaccharide isolated fromPoria cocos sclerotium induces NF-120581B Rel activation andiNOS expression in murine macrophagesrdquo InternationalImmunopharmacology vol 3 no 10-11 pp 1353ndash1362 2003
[19] M Lin B Xia M Yang S Gao Y Huo and G Lou ldquoCharac-terization and antitumor activities of a polysaccharide from therhizoma of Menispermum dauricumrdquo International Journal ofBiological Macromolecules vol 53 pp 72ndash76 2013
Evidence-Based Complementary and Alternative Medicine 7
[20] N Li C Yan D Hua and D Zhang ldquoIsolation purificationand structural characterization of a novel polysaccharide fromGanoderma capenserdquo International Journal of Biological Macro-molecules vol 57 pp 285ndash290 2013
[21] M Kacurakova N Wellner A Ebringerova Z HromadkovaR H Wilson and P S Belton ldquoCharacterisation of xylan-typepolysaccharides and associated cell wall components by FT-IRand FT-Raman spectroscopiesrdquo Food Hydrocolloids vol 13 no1 pp 35ndash41 1999
[22] N I Abu-Lail and T A Camesano ldquoPolysaccharide propertiesprobed with atomic force microscopyrdquo Journal of Microscopyvol 212 no 3 pp 217ndash238 2003
[23] Pharmacopoeia Commission of Peoplersquos Republic of ChinaChinese Pharmacopoeia 2010
[24] Y Yang D Liu J Wu Y Chen and S Wang ldquoIn vitro antiox-idant activities of sulfated polysaccharide fractions extractedfrom Corallina officinalisrdquo International Journal of BiologicalMacromolecules vol 49 no 5 pp 1031ndash1037 2011
[25] A V Gusakov E G Kondratyeva and A P Sinitsyn ldquoCom-parison of two methods for assaying reducing sugars in thedetermination of carbohydrase activitiesrdquo International Journalof Analytical Chemistry vol 2011 Article ID 283658 4 pages2011
[26] WW Huang M Y Wang H M Shi et al ldquoComparative studyof bioactive constituents in crude and processed Glycyrrhizaeradix and their respective metabolic profiles in gastrointestinaltract in vitro by HPLC-DAD and HPLC-ESIMS analysesrdquoArchives of Pharmacal Research vol 35 no 11 pp 1945ndash19522012
[27] Y Peng Z Wang Y Lu C-F Wu J-Y Yang and X-B LildquoIntestinal microflora molecular markers of spleen-deficientrats and evaluation of traditional Chinese drugsrdquoWorld Journalof Gastroenterology vol 15 no 18 pp 2220ndash2227 2009
[28] W F Broekaert C M Courtin K Verbeke T van de WieleW Verstraete and J A Delcour ldquoPrebiotic and other health-related effects of cereal-derived arabinoxylans arabinoxylan-oligosaccharides and xylooligosaccharidesrdquo Critical Reviews inFood Science and Nutrition vol 51 no 2 pp 178ndash194 2011
[29] F Xie K Sakwiwatkul C Zhang Y Wang L Zhai and S HuldquoAtractylodis macrocephalae Koidz polysaccharides enhanceboth serum IgG response and gut mucosal immunityrdquo Carbo-hydrate Polymers vol 91 no 1 pp 68ndash73 2013
33 The In Vitro Activity of PAM on Intestinal Flora WhenPAMwas added and anaerobically incubated with humanratintestinal bacterial mixture from feces it was found that theconsumption rate of reducing sugar was increased Figure 7shows how the content of reducing sugar changed in humanrat and control The changes of PAM content might bedivided into three main periods slow reduction (0sim2 h)sharp reduction (2sim6 h) and smooth reduction (6sim24 h)From 2 to 6 h the consumption rates of reducing sugardecreased significantly and almost linearly (12ndash02 g and 12ndash03 g in human and rat resp) In general the human intestinalflora was similar to rat on the consumption rate of reducingsugar incubated with PAM 779 and 769 respectivelyTherefore PAM might activate and accelerate the growth ofintestinal flora which provided clues for the further study ofPAM in vivo
34 The In Vivo Metabolic Activity of PAM on Intestinal FloraAmodel animal of intestinal flora disordered was establishedby oral administration of CAE [27] The characteristic symp-tom of watery stools was obviously observed in the modelrats and the other symptomswere also observed in this studysuch as humped back narrow eyes listlessness inappetenceand weight loss When treated by PAM intragastrically
25
30
20
15
10
5
0
0 2 4 6 8 10 12
()
T (h)
Artificial intestinal juiceArtificial gastric juice
Figure 6 Metabolism of PAM in artificial intestinal juicegastricjuice
14
13
12
11
10
09
08
07
06
05
04
03
02
01
00
0 2 4 6 8 10 12 14 16 18 20 22 24
Time (h)
(g)
ControlHumanRat
Figure 7 The effect of PAM on humanrat intestinal flora to digestreducing sugar
the characteristic symptom of watery stools was significantlyimproved Their vigor was increased and their weights wereobviously recovered in groups receiving high and low doseof PAM (Figure 8) Compared to the untreated group thesymptoms in all treated groups reduced more efficiently butthere were no significant differences between groups
ERIC-PCR profiles of intestinal flora DNA from feceswere performed on 32 rats (Figure 9) In healthy condition(control group) the Shannon diversity index (1198671015840) of 32 ratswas 181 plusmn 002 while model group (untreated) dropped to16 plusmn 002 (119875 lt 005) with a decline of 20 It was shownthat the ecological balance of intestinal flora in rats wasdestroyed and flora species were declined When the model
Evidence-Based Complementary and Alternative Medicine 5
320
300
280
260
240
220
200
180
Wei
ght (
g)
a b c
ControlHigh-dose group
Low-dose groupModel
Figure 8 The weight of rats at the periods of modeled and treated (a before administration b after administration c after treatment)
25
20
15
10
05
00
Before senna administration
After PAM treatmentAfter senna administration
Control
Shan
nonrsquos
inde
x
Model
(a)
Before senna administrationAfter senna administrationAfter PAM treatment
25
20
15
10
05
00
Shan
nonrsquos
inde
x
High dose of PAM Low dose of PAM
(b)
Figure 9 Shannonrsquos diversity index of ERIC-PCR fingerprinting of the disordered intestinal flora model of rats before and after treatment(a) Shannonrsquos diversity index of control group and model groups (b) Shannonrsquos diversity index of high dose and low dose of PAM groups(Pflt 005 Pff
lt 001 compared with before senna folium administration)
rats were administrated with PAM the 1198671015840 was increased to174 plusmn 004 (119875 lt 005 compared to model group) in high-dose group However Shannonrsquos diversity index of low doseof PAM group was maintained at a constant level and therewas no significant difference compared with model group Acertain content of PAM might be very important to increasethe diversity index of model group
As shown in Figure 10 Sorenson index (Cs) was usedto compare ERIC-PCR fingerprinting of intestinal floraof different condition rats Before and after being treatedby CAE Cs of models (groups model high-dose PAMand low-dose PAM) ranged from 38 to 45 Comparingwith the control group (58) Cs of model group showed
a significant decrease (119875 lt 005) For high and low doseof PAM groups intestinal flora in each treatment grouppresented a certain degree of recovery especially high-dosegroup (54 119875 lt 005 compared to the model group)Both high and low dose of PAM could improve similaritycoefficients of ERIC-PCR fingerprinting which indicatedthat PAM could significantly improve the structure of intesti-nal flora Furthermore it was reported that consumptionof PAM showed an improvement of intestinal function[29] Studies on the change of intestinal flora structureby PAM treatment can provide reference to understandthe underlying mechanisms of polysaccharides on intestinalflora
6 Evidence-Based Complementary and Alternative Medicine
Figure 10 Similarity coefficient index (119862119904) of ERIC-PCR finger-
printings of rat intestinal flora before and after PAM treatmentControl Group 1 received distilled water in both inducement andtreatment phases Model Group 2 received senna administrationbut distilled water during treatment High-dose PAM and Low-dosePAM Group 3 and Group 4 received high and low doses of PAMrepectively during treatment (Pf
lt 005 comparedwith the controlPnlt 005 compared with the model)
4 Conclusions
In this paper an active polysaccharide of PAM was isolatedfrom the rhizome of A macrocephala and its structure wasalso identified ERIC-PCR profile analyses were successfullyemployed to investigate the effect of PAM on the intestinalflora Our results demonstrated that PAM could improve andadjust the disordered intestinal flora suggesting that PAMmay have the potential as an oral adjuvant for disorderedintestinal flora This study also provided a novel conceptionand evidences for future investigation of the metabolic effectof polysaccharides on intestinal flora and their therapeuticeffects
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by the National Natural ScienceFoundation of China (nos 30973962 and 81473318) Theauthors thank SJTULAB group for their research support
References
[1] B Kim S J Park S P Hong T I Kim W H Kim and JH Cheon ldquoProximal disease extension and related predictingfactors in ulcerative proctitisrdquo Scandinavian Journal of Gas-troenterology vol 49 no 2 pp 177ndash183 2014
[2] C M Guinane and P D Cotter ldquoRole of the gut microbiotain health and chronic gastrointestinal disease understandinga hidden metabolic organrdquo Therapeutic Advances in Gastroen-terology vol 6 no 4 pp 295ndash308 2013
[3] A Lyra S Lahtinen K Tiihonen and A C OuwehandldquoIntestinal microbiota and overweightrdquo BeneficialMicrobes vol1 no 4 pp 407ndash421 2010
[4] P J Turnbaugh M Hamady T Yatsunenko et al ldquoA core gutmicrobiome in obese and lean twinsrdquoNature vol 457 no 7228pp 480ndash484 2009
[5] L A David C F Maurice R N Carmody et al ldquoDiet rapidlyand reproducibly alters the human gut microbiomerdquo Naturevol 505 no 7484 pp 559ndash563 2014
[6] C Chassard and C Lacroix ldquoCarbohydrates and the human gutmicrobiotardquo Current Opinion in Clinical Nutrition amp MetabolicCare vol 16 no 4 pp 453ndash460 2013
[7] S K Lin J R Lambert and M L Wahlqvist ldquoNutritionand gastrointestinal disordersrdquo Asia Pacific Journal of ClinicalNutrition vol 1 pp 37ndash42 1992
[8] A Frolkis L A Dieleman H W Barkema et al ldquoEnvironmentand the inflammatory bowel diseasesrdquo Canadian Journal ofGastroenterology vol 27 no 3 pp e18ndashe24 2013
[9] A N Ananthakrishnan ldquoEnvironmental triggers for inflamma-tory bowel diseaserdquo Current Gastroenterology Reports vol 15no 1 article 302 201
[10] M Rajilic-Stojanovic ldquoFunction of the microbiotardquo Best Prac-tice amp Research Clinical Gastroenterology vol 27 pp 5ndash16 2013
[11] A El-Kaoutari F Armougom J I Gordon D Raoult andB Henrissat ldquoThe abundance and variety of carbohydrate-active enzymes in the human gut microbiotardquo Nature ReviewsMicrobiology vol 11 no 7 pp 497ndash504 2013
[12] Y Bertin F Chaucheyras-Durand C Robbe-Masselot et alldquoCarbohydrate utilization by enterohaemorrhagic Escherichiacoli O157 H7 in bovine intestinal contentrdquo EnvironmentalMicrobiology vol 15 no 2 pp 610ndash622 2013
[13] X Jia C Ding S Yuan et al ldquoExtraction purification and char-acterization of polysaccharides from Hawk teardquo CarbohydratePolymers vol 99 pp 319ndash324 2014
[14] Y ChiW Li HWen X Cui H Cai and X Bi ldquoStudies on sep-aration purification and chemical structure of polysaccharidefromAtractylodes macrocephalardquo Zhong Yao Cai vol 24 no 9pp 647ndash648 2001
[15] N Inagaki Y Komatsu H Sasaki et al ldquoAcidic polysaccharidesfrom rhizomes of Atractylodes lancea as protective principle inCandida-infected micerdquo Planta Medica vol 67 no 5 pp 428ndash431 2001
[16] K-W Yu H Kiyohara T Matsumoto H-C Yang and HYamada ldquoIntestinal immune system modulating polysaccha-rides from rhizomes of Atractylodes lanceardquo Planta Medica vol64 no 8 pp 714ndash719 1998
[17] J-P Cai Y-J Wu C Li et al ldquoPanax ginseng polysaccharidesuppresses metastasis via modulating Twist expression in gas-tric cancerrdquo International Journal of Biological Macromoleculesvol 57 pp 22ndash25 2013
[18] K Y Lee and Y J Jeon ldquoPolysaccharide isolated fromPoria cocos sclerotium induces NF-120581B Rel activation andiNOS expression in murine macrophagesrdquo InternationalImmunopharmacology vol 3 no 10-11 pp 1353ndash1362 2003
[19] M Lin B Xia M Yang S Gao Y Huo and G Lou ldquoCharac-terization and antitumor activities of a polysaccharide from therhizoma of Menispermum dauricumrdquo International Journal ofBiological Macromolecules vol 53 pp 72ndash76 2013
Evidence-Based Complementary and Alternative Medicine 7
[20] N Li C Yan D Hua and D Zhang ldquoIsolation purificationand structural characterization of a novel polysaccharide fromGanoderma capenserdquo International Journal of Biological Macro-molecules vol 57 pp 285ndash290 2013
[21] M Kacurakova N Wellner A Ebringerova Z HromadkovaR H Wilson and P S Belton ldquoCharacterisation of xylan-typepolysaccharides and associated cell wall components by FT-IRand FT-Raman spectroscopiesrdquo Food Hydrocolloids vol 13 no1 pp 35ndash41 1999
[22] N I Abu-Lail and T A Camesano ldquoPolysaccharide propertiesprobed with atomic force microscopyrdquo Journal of Microscopyvol 212 no 3 pp 217ndash238 2003
[23] Pharmacopoeia Commission of Peoplersquos Republic of ChinaChinese Pharmacopoeia 2010
[24] Y Yang D Liu J Wu Y Chen and S Wang ldquoIn vitro antiox-idant activities of sulfated polysaccharide fractions extractedfrom Corallina officinalisrdquo International Journal of BiologicalMacromolecules vol 49 no 5 pp 1031ndash1037 2011
[25] A V Gusakov E G Kondratyeva and A P Sinitsyn ldquoCom-parison of two methods for assaying reducing sugars in thedetermination of carbohydrase activitiesrdquo International Journalof Analytical Chemistry vol 2011 Article ID 283658 4 pages2011
[26] WW Huang M Y Wang H M Shi et al ldquoComparative studyof bioactive constituents in crude and processed Glycyrrhizaeradix and their respective metabolic profiles in gastrointestinaltract in vitro by HPLC-DAD and HPLC-ESIMS analysesrdquoArchives of Pharmacal Research vol 35 no 11 pp 1945ndash19522012
[27] Y Peng Z Wang Y Lu C-F Wu J-Y Yang and X-B LildquoIntestinal microflora molecular markers of spleen-deficientrats and evaluation of traditional Chinese drugsrdquoWorld Journalof Gastroenterology vol 15 no 18 pp 2220ndash2227 2009
[28] W F Broekaert C M Courtin K Verbeke T van de WieleW Verstraete and J A Delcour ldquoPrebiotic and other health-related effects of cereal-derived arabinoxylans arabinoxylan-oligosaccharides and xylooligosaccharidesrdquo Critical Reviews inFood Science and Nutrition vol 51 no 2 pp 178ndash194 2011
[29] F Xie K Sakwiwatkul C Zhang Y Wang L Zhai and S HuldquoAtractylodis macrocephalae Koidz polysaccharides enhanceboth serum IgG response and gut mucosal immunityrdquo Carbo-hydrate Polymers vol 91 no 1 pp 68ndash73 2013
Evidence-Based Complementary and Alternative Medicine 5
320
300
280
260
240
220
200
180
Wei
ght (
g)
a b c
ControlHigh-dose group
Low-dose groupModel
Figure 8 The weight of rats at the periods of modeled and treated (a before administration b after administration c after treatment)
25
20
15
10
05
00
Before senna administration
After PAM treatmentAfter senna administration
Control
Shan
nonrsquos
inde
x
Model
(a)
Before senna administrationAfter senna administrationAfter PAM treatment
25
20
15
10
05
00
Shan
nonrsquos
inde
x
High dose of PAM Low dose of PAM
(b)
Figure 9 Shannonrsquos diversity index of ERIC-PCR fingerprinting of the disordered intestinal flora model of rats before and after treatment(a) Shannonrsquos diversity index of control group and model groups (b) Shannonrsquos diversity index of high dose and low dose of PAM groups(Pflt 005 Pff
lt 001 compared with before senna folium administration)
rats were administrated with PAM the 1198671015840 was increased to174 plusmn 004 (119875 lt 005 compared to model group) in high-dose group However Shannonrsquos diversity index of low doseof PAM group was maintained at a constant level and therewas no significant difference compared with model group Acertain content of PAM might be very important to increasethe diversity index of model group
As shown in Figure 10 Sorenson index (Cs) was usedto compare ERIC-PCR fingerprinting of intestinal floraof different condition rats Before and after being treatedby CAE Cs of models (groups model high-dose PAMand low-dose PAM) ranged from 38 to 45 Comparingwith the control group (58) Cs of model group showed
a significant decrease (119875 lt 005) For high and low doseof PAM groups intestinal flora in each treatment grouppresented a certain degree of recovery especially high-dosegroup (54 119875 lt 005 compared to the model group)Both high and low dose of PAM could improve similaritycoefficients of ERIC-PCR fingerprinting which indicatedthat PAM could significantly improve the structure of intesti-nal flora Furthermore it was reported that consumptionof PAM showed an improvement of intestinal function[29] Studies on the change of intestinal flora structureby PAM treatment can provide reference to understandthe underlying mechanisms of polysaccharides on intestinalflora
6 Evidence-Based Complementary and Alternative Medicine
Figure 10 Similarity coefficient index (119862119904) of ERIC-PCR finger-
printings of rat intestinal flora before and after PAM treatmentControl Group 1 received distilled water in both inducement andtreatment phases Model Group 2 received senna administrationbut distilled water during treatment High-dose PAM and Low-dosePAM Group 3 and Group 4 received high and low doses of PAMrepectively during treatment (Pf
lt 005 comparedwith the controlPnlt 005 compared with the model)
4 Conclusions
In this paper an active polysaccharide of PAM was isolatedfrom the rhizome of A macrocephala and its structure wasalso identified ERIC-PCR profile analyses were successfullyemployed to investigate the effect of PAM on the intestinalflora Our results demonstrated that PAM could improve andadjust the disordered intestinal flora suggesting that PAMmay have the potential as an oral adjuvant for disorderedintestinal flora This study also provided a novel conceptionand evidences for future investigation of the metabolic effectof polysaccharides on intestinal flora and their therapeuticeffects
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by the National Natural ScienceFoundation of China (nos 30973962 and 81473318) Theauthors thank SJTULAB group for their research support
References
[1] B Kim S J Park S P Hong T I Kim W H Kim and JH Cheon ldquoProximal disease extension and related predictingfactors in ulcerative proctitisrdquo Scandinavian Journal of Gas-troenterology vol 49 no 2 pp 177ndash183 2014
[2] C M Guinane and P D Cotter ldquoRole of the gut microbiotain health and chronic gastrointestinal disease understandinga hidden metabolic organrdquo Therapeutic Advances in Gastroen-terology vol 6 no 4 pp 295ndash308 2013
[3] A Lyra S Lahtinen K Tiihonen and A C OuwehandldquoIntestinal microbiota and overweightrdquo BeneficialMicrobes vol1 no 4 pp 407ndash421 2010
[4] P J Turnbaugh M Hamady T Yatsunenko et al ldquoA core gutmicrobiome in obese and lean twinsrdquoNature vol 457 no 7228pp 480ndash484 2009
[5] L A David C F Maurice R N Carmody et al ldquoDiet rapidlyand reproducibly alters the human gut microbiomerdquo Naturevol 505 no 7484 pp 559ndash563 2014
[6] C Chassard and C Lacroix ldquoCarbohydrates and the human gutmicrobiotardquo Current Opinion in Clinical Nutrition amp MetabolicCare vol 16 no 4 pp 453ndash460 2013
[7] S K Lin J R Lambert and M L Wahlqvist ldquoNutritionand gastrointestinal disordersrdquo Asia Pacific Journal of ClinicalNutrition vol 1 pp 37ndash42 1992
[8] A Frolkis L A Dieleman H W Barkema et al ldquoEnvironmentand the inflammatory bowel diseasesrdquo Canadian Journal ofGastroenterology vol 27 no 3 pp e18ndashe24 2013
[9] A N Ananthakrishnan ldquoEnvironmental triggers for inflamma-tory bowel diseaserdquo Current Gastroenterology Reports vol 15no 1 article 302 201
[10] M Rajilic-Stojanovic ldquoFunction of the microbiotardquo Best Prac-tice amp Research Clinical Gastroenterology vol 27 pp 5ndash16 2013
[11] A El-Kaoutari F Armougom J I Gordon D Raoult andB Henrissat ldquoThe abundance and variety of carbohydrate-active enzymes in the human gut microbiotardquo Nature ReviewsMicrobiology vol 11 no 7 pp 497ndash504 2013
[12] Y Bertin F Chaucheyras-Durand C Robbe-Masselot et alldquoCarbohydrate utilization by enterohaemorrhagic Escherichiacoli O157 H7 in bovine intestinal contentrdquo EnvironmentalMicrobiology vol 15 no 2 pp 610ndash622 2013
[13] X Jia C Ding S Yuan et al ldquoExtraction purification and char-acterization of polysaccharides from Hawk teardquo CarbohydratePolymers vol 99 pp 319ndash324 2014
[14] Y ChiW Li HWen X Cui H Cai and X Bi ldquoStudies on sep-aration purification and chemical structure of polysaccharidefromAtractylodes macrocephalardquo Zhong Yao Cai vol 24 no 9pp 647ndash648 2001
[15] N Inagaki Y Komatsu H Sasaki et al ldquoAcidic polysaccharidesfrom rhizomes of Atractylodes lancea as protective principle inCandida-infected micerdquo Planta Medica vol 67 no 5 pp 428ndash431 2001
[16] K-W Yu H Kiyohara T Matsumoto H-C Yang and HYamada ldquoIntestinal immune system modulating polysaccha-rides from rhizomes of Atractylodes lanceardquo Planta Medica vol64 no 8 pp 714ndash719 1998
[17] J-P Cai Y-J Wu C Li et al ldquoPanax ginseng polysaccharidesuppresses metastasis via modulating Twist expression in gas-tric cancerrdquo International Journal of Biological Macromoleculesvol 57 pp 22ndash25 2013
[18] K Y Lee and Y J Jeon ldquoPolysaccharide isolated fromPoria cocos sclerotium induces NF-120581B Rel activation andiNOS expression in murine macrophagesrdquo InternationalImmunopharmacology vol 3 no 10-11 pp 1353ndash1362 2003
[19] M Lin B Xia M Yang S Gao Y Huo and G Lou ldquoCharac-terization and antitumor activities of a polysaccharide from therhizoma of Menispermum dauricumrdquo International Journal ofBiological Macromolecules vol 53 pp 72ndash76 2013
Evidence-Based Complementary and Alternative Medicine 7
[20] N Li C Yan D Hua and D Zhang ldquoIsolation purificationand structural characterization of a novel polysaccharide fromGanoderma capenserdquo International Journal of Biological Macro-molecules vol 57 pp 285ndash290 2013
[21] M Kacurakova N Wellner A Ebringerova Z HromadkovaR H Wilson and P S Belton ldquoCharacterisation of xylan-typepolysaccharides and associated cell wall components by FT-IRand FT-Raman spectroscopiesrdquo Food Hydrocolloids vol 13 no1 pp 35ndash41 1999
[22] N I Abu-Lail and T A Camesano ldquoPolysaccharide propertiesprobed with atomic force microscopyrdquo Journal of Microscopyvol 212 no 3 pp 217ndash238 2003
[23] Pharmacopoeia Commission of Peoplersquos Republic of ChinaChinese Pharmacopoeia 2010
[24] Y Yang D Liu J Wu Y Chen and S Wang ldquoIn vitro antiox-idant activities of sulfated polysaccharide fractions extractedfrom Corallina officinalisrdquo International Journal of BiologicalMacromolecules vol 49 no 5 pp 1031ndash1037 2011
[25] A V Gusakov E G Kondratyeva and A P Sinitsyn ldquoCom-parison of two methods for assaying reducing sugars in thedetermination of carbohydrase activitiesrdquo International Journalof Analytical Chemistry vol 2011 Article ID 283658 4 pages2011
[26] WW Huang M Y Wang H M Shi et al ldquoComparative studyof bioactive constituents in crude and processed Glycyrrhizaeradix and their respective metabolic profiles in gastrointestinaltract in vitro by HPLC-DAD and HPLC-ESIMS analysesrdquoArchives of Pharmacal Research vol 35 no 11 pp 1945ndash19522012
[27] Y Peng Z Wang Y Lu C-F Wu J-Y Yang and X-B LildquoIntestinal microflora molecular markers of spleen-deficientrats and evaluation of traditional Chinese drugsrdquoWorld Journalof Gastroenterology vol 15 no 18 pp 2220ndash2227 2009
[28] W F Broekaert C M Courtin K Verbeke T van de WieleW Verstraete and J A Delcour ldquoPrebiotic and other health-related effects of cereal-derived arabinoxylans arabinoxylan-oligosaccharides and xylooligosaccharidesrdquo Critical Reviews inFood Science and Nutrition vol 51 no 2 pp 178ndash194 2011
[29] F Xie K Sakwiwatkul C Zhang Y Wang L Zhai and S HuldquoAtractylodis macrocephalae Koidz polysaccharides enhanceboth serum IgG response and gut mucosal immunityrdquo Carbo-hydrate Polymers vol 91 no 1 pp 68ndash73 2013
Figure 10 Similarity coefficient index (119862119904) of ERIC-PCR finger-
printings of rat intestinal flora before and after PAM treatmentControl Group 1 received distilled water in both inducement andtreatment phases Model Group 2 received senna administrationbut distilled water during treatment High-dose PAM and Low-dosePAM Group 3 and Group 4 received high and low doses of PAMrepectively during treatment (Pf
lt 005 comparedwith the controlPnlt 005 compared with the model)
4 Conclusions
In this paper an active polysaccharide of PAM was isolatedfrom the rhizome of A macrocephala and its structure wasalso identified ERIC-PCR profile analyses were successfullyemployed to investigate the effect of PAM on the intestinalflora Our results demonstrated that PAM could improve andadjust the disordered intestinal flora suggesting that PAMmay have the potential as an oral adjuvant for disorderedintestinal flora This study also provided a novel conceptionand evidences for future investigation of the metabolic effectof polysaccharides on intestinal flora and their therapeuticeffects
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by the National Natural ScienceFoundation of China (nos 30973962 and 81473318) Theauthors thank SJTULAB group for their research support
References
[1] B Kim S J Park S P Hong T I Kim W H Kim and JH Cheon ldquoProximal disease extension and related predictingfactors in ulcerative proctitisrdquo Scandinavian Journal of Gas-troenterology vol 49 no 2 pp 177ndash183 2014
[2] C M Guinane and P D Cotter ldquoRole of the gut microbiotain health and chronic gastrointestinal disease understandinga hidden metabolic organrdquo Therapeutic Advances in Gastroen-terology vol 6 no 4 pp 295ndash308 2013
[3] A Lyra S Lahtinen K Tiihonen and A C OuwehandldquoIntestinal microbiota and overweightrdquo BeneficialMicrobes vol1 no 4 pp 407ndash421 2010
[4] P J Turnbaugh M Hamady T Yatsunenko et al ldquoA core gutmicrobiome in obese and lean twinsrdquoNature vol 457 no 7228pp 480ndash484 2009
[5] L A David C F Maurice R N Carmody et al ldquoDiet rapidlyand reproducibly alters the human gut microbiomerdquo Naturevol 505 no 7484 pp 559ndash563 2014
[6] C Chassard and C Lacroix ldquoCarbohydrates and the human gutmicrobiotardquo Current Opinion in Clinical Nutrition amp MetabolicCare vol 16 no 4 pp 453ndash460 2013
[7] S K Lin J R Lambert and M L Wahlqvist ldquoNutritionand gastrointestinal disordersrdquo Asia Pacific Journal of ClinicalNutrition vol 1 pp 37ndash42 1992
[8] A Frolkis L A Dieleman H W Barkema et al ldquoEnvironmentand the inflammatory bowel diseasesrdquo Canadian Journal ofGastroenterology vol 27 no 3 pp e18ndashe24 2013
[9] A N Ananthakrishnan ldquoEnvironmental triggers for inflamma-tory bowel diseaserdquo Current Gastroenterology Reports vol 15no 1 article 302 201
[10] M Rajilic-Stojanovic ldquoFunction of the microbiotardquo Best Prac-tice amp Research Clinical Gastroenterology vol 27 pp 5ndash16 2013
[11] A El-Kaoutari F Armougom J I Gordon D Raoult andB Henrissat ldquoThe abundance and variety of carbohydrate-active enzymes in the human gut microbiotardquo Nature ReviewsMicrobiology vol 11 no 7 pp 497ndash504 2013
[12] Y Bertin F Chaucheyras-Durand C Robbe-Masselot et alldquoCarbohydrate utilization by enterohaemorrhagic Escherichiacoli O157 H7 in bovine intestinal contentrdquo EnvironmentalMicrobiology vol 15 no 2 pp 610ndash622 2013
[13] X Jia C Ding S Yuan et al ldquoExtraction purification and char-acterization of polysaccharides from Hawk teardquo CarbohydratePolymers vol 99 pp 319ndash324 2014
[14] Y ChiW Li HWen X Cui H Cai and X Bi ldquoStudies on sep-aration purification and chemical structure of polysaccharidefromAtractylodes macrocephalardquo Zhong Yao Cai vol 24 no 9pp 647ndash648 2001
[15] N Inagaki Y Komatsu H Sasaki et al ldquoAcidic polysaccharidesfrom rhizomes of Atractylodes lancea as protective principle inCandida-infected micerdquo Planta Medica vol 67 no 5 pp 428ndash431 2001
[16] K-W Yu H Kiyohara T Matsumoto H-C Yang and HYamada ldquoIntestinal immune system modulating polysaccha-rides from rhizomes of Atractylodes lanceardquo Planta Medica vol64 no 8 pp 714ndash719 1998
[17] J-P Cai Y-J Wu C Li et al ldquoPanax ginseng polysaccharidesuppresses metastasis via modulating Twist expression in gas-tric cancerrdquo International Journal of Biological Macromoleculesvol 57 pp 22ndash25 2013
[18] K Y Lee and Y J Jeon ldquoPolysaccharide isolated fromPoria cocos sclerotium induces NF-120581B Rel activation andiNOS expression in murine macrophagesrdquo InternationalImmunopharmacology vol 3 no 10-11 pp 1353ndash1362 2003
[19] M Lin B Xia M Yang S Gao Y Huo and G Lou ldquoCharac-terization and antitumor activities of a polysaccharide from therhizoma of Menispermum dauricumrdquo International Journal ofBiological Macromolecules vol 53 pp 72ndash76 2013
Evidence-Based Complementary and Alternative Medicine 7
[20] N Li C Yan D Hua and D Zhang ldquoIsolation purificationand structural characterization of a novel polysaccharide fromGanoderma capenserdquo International Journal of Biological Macro-molecules vol 57 pp 285ndash290 2013
[21] M Kacurakova N Wellner A Ebringerova Z HromadkovaR H Wilson and P S Belton ldquoCharacterisation of xylan-typepolysaccharides and associated cell wall components by FT-IRand FT-Raman spectroscopiesrdquo Food Hydrocolloids vol 13 no1 pp 35ndash41 1999
[22] N I Abu-Lail and T A Camesano ldquoPolysaccharide propertiesprobed with atomic force microscopyrdquo Journal of Microscopyvol 212 no 3 pp 217ndash238 2003
[23] Pharmacopoeia Commission of Peoplersquos Republic of ChinaChinese Pharmacopoeia 2010
[24] Y Yang D Liu J Wu Y Chen and S Wang ldquoIn vitro antiox-idant activities of sulfated polysaccharide fractions extractedfrom Corallina officinalisrdquo International Journal of BiologicalMacromolecules vol 49 no 5 pp 1031ndash1037 2011
[25] A V Gusakov E G Kondratyeva and A P Sinitsyn ldquoCom-parison of two methods for assaying reducing sugars in thedetermination of carbohydrase activitiesrdquo International Journalof Analytical Chemistry vol 2011 Article ID 283658 4 pages2011
[26] WW Huang M Y Wang H M Shi et al ldquoComparative studyof bioactive constituents in crude and processed Glycyrrhizaeradix and their respective metabolic profiles in gastrointestinaltract in vitro by HPLC-DAD and HPLC-ESIMS analysesrdquoArchives of Pharmacal Research vol 35 no 11 pp 1945ndash19522012
[27] Y Peng Z Wang Y Lu C-F Wu J-Y Yang and X-B LildquoIntestinal microflora molecular markers of spleen-deficientrats and evaluation of traditional Chinese drugsrdquoWorld Journalof Gastroenterology vol 15 no 18 pp 2220ndash2227 2009
[28] W F Broekaert C M Courtin K Verbeke T van de WieleW Verstraete and J A Delcour ldquoPrebiotic and other health-related effects of cereal-derived arabinoxylans arabinoxylan-oligosaccharides and xylooligosaccharidesrdquo Critical Reviews inFood Science and Nutrition vol 51 no 2 pp 178ndash194 2011
[29] F Xie K Sakwiwatkul C Zhang Y Wang L Zhai and S HuldquoAtractylodis macrocephalae Koidz polysaccharides enhanceboth serum IgG response and gut mucosal immunityrdquo Carbo-hydrate Polymers vol 91 no 1 pp 68ndash73 2013
Evidence-Based Complementary and Alternative Medicine 7
[20] N Li C Yan D Hua and D Zhang ldquoIsolation purificationand structural characterization of a novel polysaccharide fromGanoderma capenserdquo International Journal of Biological Macro-molecules vol 57 pp 285ndash290 2013
[21] M Kacurakova N Wellner A Ebringerova Z HromadkovaR H Wilson and P S Belton ldquoCharacterisation of xylan-typepolysaccharides and associated cell wall components by FT-IRand FT-Raman spectroscopiesrdquo Food Hydrocolloids vol 13 no1 pp 35ndash41 1999
[22] N I Abu-Lail and T A Camesano ldquoPolysaccharide propertiesprobed with atomic force microscopyrdquo Journal of Microscopyvol 212 no 3 pp 217ndash238 2003
[23] Pharmacopoeia Commission of Peoplersquos Republic of ChinaChinese Pharmacopoeia 2010
[24] Y Yang D Liu J Wu Y Chen and S Wang ldquoIn vitro antiox-idant activities of sulfated polysaccharide fractions extractedfrom Corallina officinalisrdquo International Journal of BiologicalMacromolecules vol 49 no 5 pp 1031ndash1037 2011
[25] A V Gusakov E G Kondratyeva and A P Sinitsyn ldquoCom-parison of two methods for assaying reducing sugars in thedetermination of carbohydrase activitiesrdquo International Journalof Analytical Chemistry vol 2011 Article ID 283658 4 pages2011
[26] WW Huang M Y Wang H M Shi et al ldquoComparative studyof bioactive constituents in crude and processed Glycyrrhizaeradix and their respective metabolic profiles in gastrointestinaltract in vitro by HPLC-DAD and HPLC-ESIMS analysesrdquoArchives of Pharmacal Research vol 35 no 11 pp 1945ndash19522012
[27] Y Peng Z Wang Y Lu C-F Wu J-Y Yang and X-B LildquoIntestinal microflora molecular markers of spleen-deficientrats and evaluation of traditional Chinese drugsrdquoWorld Journalof Gastroenterology vol 15 no 18 pp 2220ndash2227 2009
[28] W F Broekaert C M Courtin K Verbeke T van de WieleW Verstraete and J A Delcour ldquoPrebiotic and other health-related effects of cereal-derived arabinoxylans arabinoxylan-oligosaccharides and xylooligosaccharidesrdquo Critical Reviews inFood Science and Nutrition vol 51 no 2 pp 178ndash194 2011
[29] F Xie K Sakwiwatkul C Zhang Y Wang L Zhai and S HuldquoAtractylodis macrocephalae Koidz polysaccharides enhanceboth serum IgG response and gut mucosal immunityrdquo Carbo-hydrate Polymers vol 91 no 1 pp 68ndash73 2013
