research article grappling the high altitude for safe edible bamboo...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 289285 11 pageshttpdxdoiorg1011552013289285

Research ArticleGrappling the High Altitude for Safe Edible Bamboo Shoots withRich Nutritional Attributes and Escaping Cyanogenic Toxicity

Sayanika Devi Waikhom1 Bengyella Louis123 Chandradev K Sharma1 Pushpa Kumari4

Bharat G Somkuwar1 Mohendro W Singh1 and Narayan C Talukdar1

1 Institute of Bioresources and Sustainable Development (IBSD) Takyelpat Imphal Manipur 795001 India2Department of Biochemistry University of Yaounde I BP 812 Yaounde Cameroon3Department of Biotechnology Burdwan University Golapbag More West Bengal 713104 India4AJC Bose Indian Botanic Garden Botanical Survey of India Botanic Garden Howrah 711103 India

Correspondence should be addressed to Sayanika Devi Waikhom sayanikawaikhomgmailcom andNarayan C Talukdar nctalukdaryahoocom

Received 18 June 2013 Accepted 3 October 2013

Academic Editor Kuo-Chen Chou

Copyright copy 2013 Sayanika Devi Waikhom et alThis is an open access article distributed under theCreative CommonsAttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

Consumption of bamboo species with high level of total cyanogenic content (TCC) in Asia by many ethnic groups is significantlyassociated with food poisoning and occasionally Konzo (a neurological disorder) Adequate characterization of edible bamboospecies with low level of TCC and high nutritious attributes is required for consumerrsquos safety as well as for the conservation of thegene pool Here we employed morphological descriptors atomic absorption spectrophotometer RAPD and trnL-F intergenicspacer to characterize 15 indigenous edible bamboo species of north-east India The study indicates that morphologically andgenetically evolved edible bamboo species having large and robust bamboo-shoot texture and growing at low altitude containhigh level of TCC low antioxidant properties and low levels of beneficial macronutrients and micronutrients ImportantlyDendrocalamus species are shown to be rich in TCC irrespective of the growing altitude while Bambusa species are found to havemoderate level of TCC The findings clearly demonstrated that Chimonobambusa callosa growing at high altitude represents safeedible bamboo species with nutritious attributes

1 Introduction

Bamboo shoots are popular traditional food delicacies whichare consumed as fresh fermented or canned in many South-East Asian countries Cyanogenic glycosides are inherentlyproduced in cyanogenic plants as defence arsenals and abun-dantly produced in bamboo shoots in the form of taxiphyllin[1 2] High intake of cyanogenic glycosides is life threaten-ing and significantly associated with neurological disordercalled Konzo [3] Under optimal conditions lactic acid fromfermentation of bamboo shoot reduces total cyanide content(TCC) [4] Nonetheless better results can be achieved if theinitial starting material is poor in TCC Therefore ingestionof fresh or inappropriately fermented bamboo shoots can leadto cyanide poisoning

Irrespective of toxicity some young edible bamboo shoots(le30 days) possess enormous nutritious potentials such ashigh fibre content with antioxidant and antitumor properties

[5 6] Consequently the demand for bamboo shoots ishigh and farmers are often challenged to match supply inboth quality and quantity The magnitude of this increasingdemand is exacerbated by nondomestication of edible bam-boo species As a result nonedible bamboo (or poisonous)shoots are made available in the market which are harmful toconsumers In order to effectively outwit this anthropogenicpressure a holistic approach is required to identify the specieswith rich nutritional attributes for safe human consumptionand domestication

Biochemical studies on antioxidant activity cyanogenicglycoside content and nutrient content have been reportedfor a few species [5 7ndash9] without correlation to the altitudesof sample collection equally lacking morphological andmolecular characterization Based on previous work it isdifficult to sort out edible bamboo species requiring low-processing capital inputs to eliminate the poisonous compo-nents Here we exploit the geographic positioning of edible

2 BioMed Research International

Table 1 Principal geographical coordinates mean soil pH and identified edible bamboo species

GenBank accessionbamboo species 1 2 3 4 5 Voucher noKC013282Chimonobambusa callosa Leimaram 51 plusmn 03 24∘441015840 93∘431015840 1843m (hill) IBSDWS019KC013285Bambusa cacharensis Arapti 58 plusmn 03 24∘441015840 93∘511015840 807m (hill) IBSDWS020JX564900Bambusa manipureana Khong Khang 62 plusmn 01 24∘211015840 94∘111015840 240m (valley) IBSDWS008JX564901Bambusa nutans Arapti 56 plusmn 04 24∘441015840 93∘561015840 226m (valley) IBSDWS023JX507132Bambusa tulda Arapti 55 plusmn 01 24∘441015840 93∘561015840 226m (valley) IBSDWS022JX507131Bambusa oliveriana Arapti 55 plusmn 03 24∘441015840 93∘561015840 728m (hill) IBSDWS010JX564902Dendrocalamus giganteus Arapti 54 plusmn 02 24∘441015840 93∘561015840 803m (hill) IBSDWS001JX564903Dendrocalamus hamiltonii Kwatha 62 plusmn 04 24∘191015840 94∘161015840 358m (valley) IBSDWS004JX564904Dendrocalamus hookeri Arapti 49 plusmn 08 24∘441015840 93∘561015840 770m (hill) IBSDWS005JX564905Dendrocalamus manipureanus Arapti 50 plusmn 04 24∘211015840 93∘571015840 769m (hill) IBSDWS002JX507133Melocanna baccifera Lokchao 65 plusmn 04 24∘201015840 94∘141015840 497m (hill) IBSDWS018JX507134Schizostachyum dullooa Arapti 50 plusmn 04 24∘421015840 93∘571015840 728m (hill) IBSDWS003JX564906Bambusa sp Arapti 52 plusmn 03 24∘441015840 93∘561015840 770m (hill) IBSDWS024JX564907Bambusa sp Andro 66 plusmn 03 24∘471015840 93∘031015840 803m (hill) IBSDWS007KC013288Bambusa tuldoides Andro 64 plusmn 05 24∘471015840 93∘031015840 777m (hill) IBSDWS0061Collection site 2mean pH of the soil at the collection site in the months of July-August of 2009 to 2011 3latitude (N) 4longitude (E) and 5altitude in metersshowing either valley or hill where samples were collected

bamboo species in a dynamic biota and their morphologicaldescriptors RAPD and trnL-F intergenic spacer to studythe interrelatedness of coevolved species of genera Bam-busa Dendrocalamus Chimonobambusa SchizostachyumandMelocannaWe also exploit the geographic positioning ofsame bamboo species to shed light on how altitude influencesthe valuable nutritional attributes of bamboo shoots

2 Material and Method

21 Plant Material Fifteen species of edible bamboo-shootsbelonging to the genera Bambusa Dendrocalamus Chi-monobambusa Schizostachyum and Melocanna were col-lected from different altitudes of Manipur India (23∘471015840ndash25∘411015840 NL 92∘581015840ndash94∘471015840 EL) during July-August of 2009-2010 This region often receives an average rainfall of 1320plusmn 3mm and temperature of 23 plusmn 3∘C during the months ofJuly-August This group of samples was used for morpho-logical characterization and authenticated by the BotanicalSurvey of India (BSI) Kolkata The voucher specimens weredeposited at the Central National Herbarium in BSI forfuture references (Table 1) For biochemical analysis 30-day-old bamboo-shoots (from the day of emergence) from thefield were collected in 2009 2010 and 2011 following routinesampling Subsequent to harvest the bamboo-shoots weredirectly frozen in liquid nitrogen in a thermocol box andtransported to the laboratorywhere theywere stored atminus80∘Cfor downstream analysisThis group of samples was collectedat sunset and the soil pH for the sites of collection wasdetermined as previously described [10]

22 Morphological Analysis To evaluate the morphologicalinterrelatedness among the edible bamboo species we used35 morphological descriptors based on 11 culm types 13culm-sheath types and 11 leaf types (Table S1 available onlineat httpdxdoiorg1011552013289285) The morphological

data were analysed using NTSYS-PC version 22 [11] Simplecoefficient matching was performed using SIMQUAL optionfor generating dataset similaritymatrix [12]The best dendro-gram was computed with Unweighted Pair-GroupedMethodArithmetic Averages (UPGMA) [13] All the samples used inthis study were scored in triplicate

23 DNA Extraction To characterize at molecular levelgenomic DNA was isolated as described by Aras et al [14]The quantity and quality of DNA were checked on BioSpecNanoDrop spectrophotometer (Thermo Scientific USA) andon 08 wv agarose gel electrophoresis respectively

24 Random Amplified Polymorphic DNA (RAPD) Analy-sis PCR was performed using standard RAPD PCR kit(Invitrogen USA) in a volume of 50 120583L in C1000 TouchThermal Cycler (BIO-RAD USA)The run was programmedas follows initial denaturation at 95∘C for 5min followedby 35 cycles of amplification (95∘C for 1min 37∘C for 1minand 72∘C for 2min) and a final extension at 72∘C for 7minAmpliconswere profiled on a 18 agarose gel electrophoresisand revealed with ethidium bromide in Gel Doc-it2 Imager(UVP Co Ltd) The primers used for this analysis arerepresented (Table S2) Polymorphism was scored as 10(presence or absence) to generate a primary binary matrixThe primary binary matrix was used for producing similaritydata using Jaccardrsquos similarity coefficient [15] and computedusing UPGMA This analysis was performed in NTSYS-PCsoftware [11]

25 Analysis of trnL-F Intergenic Spacer In order toauthenticate the edible bamboo species the trnL-Fregion was amplified using the primers set (forward51015840-ggttcaagtccctctatccc-31015840 reverse 51015840-atttgaactggtgacacgag-31015840) as described in Taberlet et al [16] PCR products

BioMed Research International 3

of about 350ndash400 bp were purified and sequenced inABI370X1 Cycler Sequencer (ABI USA) using thesame set of primers Sequences were automaticallytrimmed and assembled in DNAbaser 353 software(httpwwwdnabasercom) Following annotation sequen-ces were assigned to molecular species based on 98ndash100 sequence similarity threshold in the GenBank(httpwwwncbinlmnihgov) and in accordancewithmor-phological descriptors The sequences are available in Gen-Bank as accessions JX564900 (Bambusa manipureana)JX564901 (Bambusa nutans) JX564902 (Dendrocalamusgiganteus) JX564903 (Dendrocalamus hamiltonii) JX564904(Dendrocalamus hookeri) JX564905 (Dendrocalamusmanipureanus) JX507131 (Bambusa oliveriana) JX507132(Bambusa tulda) JX507133 (Melocanna baccifera) JX507134(Schizostachyum dullooa) KC013282 (Chimonobambusacallosa) KC013285 (Bambusa cacharensis) JX564906(Bambusa sp) JX564907 (Bambusa sp) and KC013288(Bambusa tuldoides) respectively

Sequences were aligned using Clustal omega program[17] The program BioEdit [18] was used to assess the natureof variability and the entropy of the alignment among thespeciesThe programTOPALi v25 [19] was used to select thebest substitutionmodel and the best method for phylogenetictree reconstruction based on Akaike Information Criterioncorrected AICc

1and AICc

2 and Bayesian Information Cri-

terion (BIC) Phylogenetic analysis was performed usingthe maximum likelihood (ML) method Hasegawa-Kishino-Yano nucleotide substitutionmodel was used [20]The initialtrees for the heuristic search were obtained automatically asfollows When the number of common sites was lt100 or lessthan one-fourth of the total number of sites the maximumparsimony method was used otherwise BIONJ method withMonte Carlo localization (MCL) distance matrix was usedA discrete Gamma (+G) distribution was used to modelevolutionary rate differences among sites (5 categories (+Gparameter = 20000))The analysis was performed inMEGA5[21]

26 Determination of Total Cyanide Content To determinethe poisonous potential of each species the total cyanidecontent (TCC) was evaluated using the picrate method asdescribed in Bradbury et al [22] with some modificationsFirstly bamboo shoot sheaths were removed and the inner-most edible portion was measured using a ruler and aslide calliper The full length was divided into three equalparts that is the tip the middle and the base Woodybamboos generally grow rapidly and their shoots are ofteneaten young [4ndash6] Moreover based on preliminary findingsusing 10- 20- and 30-day-old bamboo shoots (data notshown) revealed that TCC and other nutritional parametersstudied insignificantly varied with only 30-day-old sampleCogently only 30-day-old bamboo shoots were used for thebiochemical analysis The standard curve for determinationof HCN was established using NaCN solution as follows5mL of alkaline picrate solution (14 g of picric acid in 25Na2CO3) and 5mL of NaCN solution (181mg of NaCN in 1 L

sterile milli-Q water) were pooled together to obtain 100 120583g

HCNmL and heated for 5min in boiling water Volumes of01 02 04 06 and 08 and 1mL of the resultant NaCNalkaline picrate solution were adjusted to 5mL with sterilemilli-Q water to obtain 5 10 20 30 40 and 50120583g HCNrespectively

27 Antioxidant Activity Estimation 100 g of sliced bambooshoot was boiled in 300mL of double distilled water for 2 hat 100∘C The crude extract was filtered through Whatmanno 42 filter paper and concentrated in a rotary evaporatorat 100∘C The solid residues were stored at 4∘C till used Thescavenging effect of 22-diphenyl-1-picrylhydrazyl (DPPH)free radical was assayed as previously described in Men-sor et al [23] L-Ascorbic acid (Sigma USA) was used asreference antioxidant control One mL of 03mM DPPHethanolic solution was added to each sample at differentconcentrations of 20120583g 50120583g 100 120583g 200120583g and 400 120583gThe mixture was vortexed for 1min and then left to standat room temperature in the dark After 30min absorbancewas read at 517 nm inUV1700 spectrophotometer (ShimadzuUSA) The scavenging activity of DPPH free radical wascalculated using the following equation

scavenging activity () = 100 times[119860119862minus 119860119878]

119860119862

(1)

119860119862is the absorbance of the control reaction (containing

all reagents except for the test compound) and 119860119878is the

absorbance of the test compound The inhibition concentra-tion (IC

50) is defined as the amount of extract required to

reduce free scavenging activity by 50 The IC50values were

obtained from the inhibition curve by extrapolation

28 Estimation of Macro- and Micronutrients Total nitrogencontents were determined through digestion and distilla-tion of dry bamboo shoots in Kel-Plus digestion system(Pelican India) according to AOAC [24] protocol Crudeprotein was calculated as Kjeldahl N times 625 based on theassumption that nitrogen (N) constitutes 1600 of a pro-tein The element contents such as potassium (K) sodium(Na) calcium (Ca) magnesium (Mg) copper (Cu) iron(Fe) and zinc (Zn) were estimated using atomic absorptionspectrophotometer (AAS) (Perkin Elmer USA) 500mg ofdried weight (dw) bamboo shoot powder was digestedin 10 4 1 (HNO

3 HClO

4 H2SO4) The digested sample

was appropriately diluted with sterile deionized water andfiltered with Whatman no 42 filter paper All the elementswere analyzed with appropriate multielement hollow cathodelamps (Lumina Lamp Perkin Elmer) against a standardreference solution for AAS (Accutrace Reference StandardUSA) Phosphorus was estimated by absorbance measure-ment at 420 nmof the vanadomolybdophosphoric heteropolycomplex formed in the digestate [25] Cellulose (Ce) contentwas estimated at 620 nm using cold anthrone reagent method[26]

29 Statistical Analysis One-way analysis of variance(ANOVA) was implemented to compare the means of


(a) (b) (c) (d) (e) (f)

(g) (h) (i) (j) (k) (l)

(m) (n) (o)

Figure 1 Morphological features of 30-day-old bamboo shoots imaged with Nikon Coolpix S6200 and the nomenclature as submitted in theGenbank NCBI nucleotide database (a) KC013282C callosa (b) KC013285B cacharensis (c) JX564900B manipureana (d) JX564901Bnutans (e) JX507132B tulda (f) JX507131B oliveriana (g) JX564902D giganteus (h) JX564903D hamiltonii (i) JX564906Bambusa sp(j) JX564904D hookeri (k) JX564905D manipureanus (l) JX564907Bambusa sp (m) KC013288B tuldoides (n) JX507133M bacciferaand (o) JX507134S dullooa

different treatments The differences between individualmeans were tested using the least significant difference(LSD) Computation was performed in SPSS software(version 220 SPSS Inc Chicago USA) The relationshipbetween the different biochemical attributes and 13 ediblebamboo species was analysed using principal componentanalysis (PCA) PCA on standardized data was performedin NTSYS-PC version 22 [11] Principal components witheigenvalues (120576 gt 100) were selected and correlation values(119903 gt 030) were considered as relevant for the PCA Threegenera underrepresented in the study set of 15 were excludedfrom principal component analysis

3 Results

31 Morphological Analysis Of the 15 identified edible bam-boo species studied only 13 were morphologically identified

at the species level and deposited in BSI Kolkata (Table 1)The morphological characteristics of the young (lt30 days)bamboo shoots are depicted in Figure 1 In the study set bam-boo shoots of JX564902D giganteus JX564903D hamil-tonii JX564904D hookeri and JX564905D manipureanuswere generally deep green broad based with robust texture(Figures 1(g) 1(h) 1(j) and 1(k)) An overall strong cophe-netic correlation coefficient of 070 based on morphologicalcharacteristics (Table S1) was obtained indicating a faithfullyconstructed dendrogram (Figure 2) The bamboo speciesclustered into two main clades (I and II) with JX507134Sdullooa evolving in a polyphyletic pattern The clusteringpattern was significantly affected by the morphological char-acteristics such as colour shape and presence of hairs in culmsheaths covering the shoots

32 RAPD Analysis High level of polymorphism wasobserved among the 15 species based on the 9 primer sets


B cacharensis KC013285B nutans JX564901B tulda JX507132B manipureana JX564900B oliveriana JX507131 D giganteus JX564902D hookeri JX564904Bambusa sp JX564907Bambusa sp JX564906B tuldoides KC013288D hamiltonii JX564903D manipureanus JX564905

I

II

S dullooa JX507134

034 041 048 056 063Coefficient

C callosa KC013282M baccifera JX507133

Figure 2 A dendrogram based on morphological descriptors showing the relationship between 15 edible bamboo species generated inNTSYS-PC software computed based on simple matching coefficient [11]

C callosa KC013282

M baccifera JX507133

B cacharensis KC013285

B nutans JX564901

B tulda JX507132B manipureana JX564900

B oliveriana JX507131D giganteus JX564902

D hookeri JX564904Bambusa sp JX564906

Bambusa sp JX564907

B tuldoides KC013288

D hamiltonii JX564903

D manipureanus JX564905

IS dullooa JX507134

II

III

009 022 041 058 075Coefficient

Figure 3 A dendrogram based on Jaccardrsquos similarity coefficient obtained from RAPD data showing the relationship between 15 ediblebamboo species

(Table S2) A strong co-phenetic coefficient of 077 basedon UPGMA analysis was obtained A dendrogram (Figure 3)based on band differences revealed three clades (I II and III)with C callosa forming an out group In this analysis RAPDevidence of JX564903D hamiltonii followed a polyphyleticevolutionary pattern (clade III) The gel profile (Figure S1)provides evidence of JX507131B oliveriana (lane 7) andJX564907Bambusa sp (lane 15) with higher numbers ofdominant characters than those of other species as reflectedin the banding pattern

33 DNA Sequence Analysis Based on DNA sequences theestimatedmodel parameters were base frequencies (A = 25TU = 25 C = 25 and G = 25) and substitution model[TU harr A] = 710 [C harr A] = 710 [G harr A] =1080 [C harr TU] = 1080 [G harr TU] = 710 and

[G harr C] = 710 The estimated transition-transversionbias (119877) ratio was at 076 The overall mean Tajima-Nei[27] evolutionary distance among the species was 051 Inthe sequence set the entropy of the alignment (Figure S2)showed 164 patterns (out of a total of 491 sites) and 276sites were without polymorphism (5621) A maximumlikelihood tree with the highest log likelihood (minus44563)supported by 1000 bootstrap test of replicates showing twomain clades (I and II) was generated (Figure 4) It wasobserved that Dendrocalamus spp formed a close complexrelationship with Bambusa spp The tree without branchswapping evidence C callosa has evolved differently from therest of the edible bamboo species thus forming an out group

34 Total Cyanide Content The level of TCC in all the speciesvaried from 300 to 2604 ppm (for the tip portion) 210 to


Table 2 Total cyanide content (TCC) expressed in part per million (ppm) for different portions of bamboo shoots and their net antioxidantactivity expressed as inhibition concentration (IC50) of DPPH

Samples Total cyanide content (ppm)lowast IC50 (mg Lminus1)Tip Middle Base

KC013282Chimonobambusa callosa 300a 210a 199a 009ab

KC013285Bambusa cacharensis 1533d 1221f 735efgh 058h

JX564900Bambusa manipureana 1007b 515b 761fgh 046fg

JX564901Bambusa nutans 1001b 624c 267ab 183i

JX507132Bambusa tulda 1579d 1406hi 779gh 030ef

JX507131Bambusa oliveriana 1280c 1079e 543c 057gh

JX564902Dendrocalamus giganteus 2604g 2243k 920i 060h

JX564903Dendrocalamus hamiltonii 1897e 766d 654cdef 014bc

JX564904Dendrocalamus hookeri 1595d 1322fgh 360b 045fg

JX564905Dendrocalamus manipureanus 1838e 1270fg 600cd 032de

JX507133Melocanna baccifera 1548d 484b 216a 041ef

JX507134Schizostachyum dullooa 1521d 1279fg 727efgh 061h

JX564906Bambusa sp 1582d 1354gh 669defg 024cd

JX564907Bambusa sp 2063f 1557j 635cde 025cd

KC013288Bambusa tuldoides 2528g 1511ij 825hi 045fg

LSD (119875 le 005) 8578 10653 10931 011lowastppm = mg HCN equivalentskg bamboo shoots and each value is the mean of three replicates for 2009 2010 and 2011 The same letter(s) associated withmean values within a column is (are) not significantly different at P le 005 based on LSD

C callosa KC013282



B nutans JX564901

B tulda JX507132

B manipureana JX564900

B oliveriana JX507131

D giganteus JX564902

D hookeri JX564904

Bambusa sp JX564906

Bambusa sp JX564907




S dullooa JX507134

100

5772

01

98

Figure 4 A maximum likelihood tree based on Hasegawa et al[20] nucleotide substitution model The tree is drawn to scale withbranch lengths measured in the number of nucleotide substitutionsper site Evolutionary analyses were conducted in MEGA5 [21]

2243 ppm (for the middle portion) and 199 to 920 ppm (forthe basal portion) These significant differences (at 119875 lt 005)in toxicity level suggest that bamboo shoot tips are generallytoxic On the contrary a low level TCC was observed inC callosa collected from high altitude in all the studiedsegments that is the tip the middle and the base portions

of the bamboo shoots (Table 2) Often all bamboo shootscollected from low altitude were rich in TCC except forM baccifera and B manipureana with respect to C callosacollected from high altitude Overall all Dendrocalamusspecies growing at either high altitude (gt700m) or lowaltitude (lt400m) were rich in TCC in comparison withother genera (Tables 1 and 2)

35 Antioxidant Activity Bamboo shoot extract of C cal-losa showed the highest significant antioxidant activity (of5346 119875 lt 005) at 400120583gmL of DPPH (Figure 5)whereas the lowest antioxidant activity (of 290 119875 lt005) was obtained with extract of B nutans Akin to thispattern the half-inhibition concentration (IC

50) obtained by

linear regression analysis showed a significant variation from009mgL forC callosa to 183mgmL for B nutans (Table 2)Among the three Dendrocalamus species obtained at altitude(gt700m)D giganteus possessed the least scavenging activitywith an IC

50value of 060 L-Ascorbic acid investigated under

the same conditions had an IC50

value of 0003mgmLimplying that bamboo shoots have a moderate antioxidantactivity (Table 2) Based on IC

50 D giganteus is 30-fold

poorer in antioxidant activity than an equivalent weight ofL-ascorbic acid When put together our results suggest thatDendrocalamus species collected at high or low altitudepossess low antioxidant attributes (Tables 1 and 2)

36 Estimation of Macro- and Micronutrients Atomicabsorption spectrometry (AAS) data revealed that bambooshoots are generally rich in nitrogen phosphorous andcontain moderate amount of calcium Remarkably thehighest nitrogen (1153mg100 g dw) and phosphorous(1154mg100 g dw) content was found in C callosa Bycontrast the lowest nitrogen (673mg100 g dw) and phos-


1

11

21

31

41

51

61

71

81

91

20 50 100 200 400

Ant

ioxi

dant

activ

ity (

)

Concentration (120583gmL)

C callosa KC01282B tulda JX564901D hookeri JX564904Bambusa sp JX564906

B cacharensis KC013285B oliveriana JX507131D manipureana JX564900Bambusa sp JX564907

B manipureanus JX564905D giganteus JX564902M baccifera JX507133B tuldoides KC013288

B nutans JX564904D hamiltonii JX564903S dullooa JX507134L-Ascorbic acid

Figure 5 Antioxidant activity of 15 bamboo shoot extracts against DPPH free radical with respect to L-ascorbic acid as reference antioxidantagent

Table 3 The data represented are obtained from atomic absorption spectrometry analysis showing the different mineral concentrations inmilligram per 100 g dry weight (dw) of bamboo shoots

Samples N P Ce K Na Mg Ca Fe Cu ZnKC013282C callosa 1153m 154g 407075ab 3377e 39bc 110f 6343gh 925abcd 612g 72de

KC013285B cacharensis 815g 87ab 315782a 2093abc 45cde 74ab 2960bc 1315def 358de 563bcd

JX564900B manipureana 957k 94bc 593651abc 2343bcd 53e 175i 324bcd 165f 494f 1280i

JX564901B nutans 825gh 104cd 537279abc 2090abc 32b 175i 535fg 116cde 416e 650cde

JX507132B tulda 721c 116de 370159ab 1453abc 51de 92d 42de 258g 319cd 150j

JX507131B oliveriana 763e 85ab 997234cd 2293abc 43cd 123g 6095fg 101bcd 358de 488bc

JX564902D giganteus 673a 70a 704898abcd 3093de 22a 69a 379cd 783abc 247bc 107gh

JX564903D hamiltonii 747d 118de 430522ab 1910abc 50de 121g 244ab 1682f 30cd 787ef

JX564904D hookeri 692b 75a 569705abc 2400cd 45cde 84cd 733hi 608a 264bc 303a

JX564905D manipureanus 894i 83ab 678458abc 3533e 42cd 85cd 1498a 668ab 255bc 480b

JX507133M baccifera 1102l 135f 1012698cd 1757abc 39bc 149h 5188ef 1337ef 196ab 1193hi

JX507134S dullooa 837h 121ef 709887abcd 1310a 42cd 180i 5695fg 1442ef 155a 918fg

JX564906Bambusa sp 785f 73a 830612bcd 1373ab 32b 92d 401cd 115cde 254bc 1133g

JX564907Bambusa sp 940j 129ef 110449d 1523abc 53e 112f 508ef 13def 785h 1103j

KC013288B tuldoides 900i 122ef 498866ab 2060abc 31b 101e 7815j 112cde 27c 110h

LSD (119875 = 005) 1332 1492 285821 87306 851 828 1017 348 064 155The mean values are for replicates obtained in 2009 2010 and 2011 The same letter(s) associated with mean values within column is (are) not significantlydifferent at P le 005 using LSD

phorous content (70mg100 g dw) was observed in Dgiganteus Among the important macronutrients studied (NP and K) all the bamboo shoots were rich in potassiumand poor in sodium (Table 3) Of all the microelementsassessed iron was significantly present in B tulda at therate of 2580mg100 g dry weight (dw) and was lowest in Dhookeri at the rate of 608mg100 g dw We observed a high

level of zinc in B tulda at the rate of 15mg100 g dw and itslowest level in D hookeri at the rate of 303mg100 g dw Wefound a significant amount of copper only in C callosa at therate of 612mg100 g dw (Table 3)

37 Principal Component Analysis (PCA) Using graphicalapproaches to study biological problems can provide an


080

035

minus010

minus055

minus100

minus080 minus035 010 055 100PC-1 (3219)

PC-2

(16

91

)

TCC

6 10

912

8

7

5

4

3

2

1

11K

N

P

Na

Cu

Mg

Fe

CaCe

AA Zn

Figure 6 PCA polygonal biplot analysis showing the interrelat-edness of nutritional attributes of 12 edible bamboos species Thenumbers represent bamboo species and the vectors are biochemicaltraits KC013285B cacharensis (1) JX564900B manipureana (2)JX564901B nutans (3) JX507132B tulda (4) JX507131B olive-riana (5) JX564902D giganteus (6) JX564903D hamiltonii (7)JX564906Bambusa sp (8) JX564904D hookeri (9) JX564905Dmanipureanus (10) JX564907Bambusa sp (11) and KC013288Btuldoides (12) AA antioxidant activity Ca calcium Ce celluloseCu copper Fe iron K potassium Mg magnesium N nitrogenNa sodium P phosphorous TCC total cyanide content and Znzinc

intuitive picture or useful insights for analysing complicatedrelationship in large data set [28] as demonstrated by manyprevious studies on a series of important biological topicssuch as enzyme-catalysed reactions [29ndash31] inhibition ofHIV-1 reverse transcriptase [32] drug metabolism systems[33] and using Wenxiang diagram [34] to study protein-protein interactions [35 36] The interrelatedness of all thebiochemical traits studied and their relationship with thebamboo species based on PCA generated four principalcomponents PC-1 PC-2 PC-3 and PC-4 The four prin-cipal components with eigenvalues (120576 gt 100) accountedfor 7225 of the total variation in nutritional attributes(Table S3) A positive correlation coefficient (119903 gt 030)was observed in PC-1 among phosphorous iron sodiumcopper magnesium zinc nitrogen and antioxidant activityaccountable for 3219 (119875 lt 005) variation in nutritionalcontent Interestingly we observed that PC-2 and PC-3were highly associated with nitrogen cellulose magnesiumcopper and potassium (1691 variation 119875 lt 005) andtotal cyanide content calcium cellulose zinc and copper(1450 variation 119875 lt 005) respectively In PC-2 and PC-3 no significant correlation was observed among nutritionalattributes PC-4 was associated with cellulose antioxidantactivity and accounted for 1165 (119875 lt 005) net variationin nutritional attributes The interrelatedness between the

nutritional attributes is represented in a polygonal biplot(Figure 6) as previously described [37]

The polygonal biplot (Figure 6) is divided into foursectors by values 2 3 4 5 6 7 8 and 10 with the vertexrepresenting the species In this representation the speciesare the best or the poorest contributing for some or all of thebiochemical traits [38] depending on the length of the vectorlines from the origin The biplot indicates that B manipure-ana (from240maltitude) andB nutans (from226maltitude)had the highest magnesium (Mg) copper (Cu) and nitrogen(N) denoted by 2 and 3 respectively Similarly B tulda(from 226m altitude) andD hamiltonii (from 358m altitude)had the highest value for antioxidant activity (AA) iron(Fe) magnesium (Mg) and zinc (Zn) denoted by 7 and4 respectively On the other hand JX564906Bambusa sp(from 770m altitude) and D giganteus (from 803m altitude)had the highest total cyanide content (TCC) denoted by 6and 10 respectively The numbers 5 and 8 representing Boliveriana (from 728m altitude) and D hookeri (from 770maltitude) were found to be tightly linked with high level ofcalcium (Ca) cellulose (Ce) and potassium (K) respectivelyIn relative terms PCA polygonal biplot intuitively evidencesthat D giganteus (from 803m altitude) is rich in TCC Ccallosa M baccifera and S dullooa were excluded fromPCA biplot analysis for reason of underrepresentation of thegenera

4 Discussion

For any meaningful differentiation of edible bamboo speciesfrompoisonous ones the delineating parametersmust show alow level of conflicting signals Using morphological descrip-tors as discerning tools to categorize the edible bamboospecies the clustering pattern was often significantly affectedby dominant morphological characteristics such as colourshape and presence of hairs in the culm sheath coveringshoots (Figure 1) Considering that most bamboo species andtheir shoots are green in colour this produces conflictingsignal and renders thought-provoking judgements This ischallenging because the homogeneity and conflicting het-erogeneity among species are time consuming and require ahigh level of expertise However the benefit ofmorphologicaldescriptors is that it permits the initial discernment ofpoisonous bamboo shoot (which is usually large and hasrobust-deep green colour) from less toxic bamboo shoots

Although a less efficient technique the benefit of RAPDanalysis to delineate bamboo species is its rapidity and costeffectiveness Nevertheless without any public database forRAPD data the technique was not effective to barcode ouredible bamboo speciesThe dendrogram generated by RAPDand morphological descriptors had similarity a copheneticcoefficient is greater than 05 but differs by the inter-species clustering pattern Nonetheless RAPD discriminatedJX564903D hamiltonii as a polyphyletic evolving species inthe data set This might be a false positive grouping sinceRAPD markers are dominant and cannot discriminate if aDNA fragment is amplified from a homozygous locus or aheterozygous locus By contrast the species were discrimi-nated by point mutations observed at the trnL-F intergenic


spacer region (Figure S3) This phenomenon of low variationcan possibly be explained by the unique asexual reproductivelife cycle and also the high rate of transversion transitionat the trnL-F locus Thus using Kumar and Gadagkar[39] disparity test of substitution pattern homogeneity ratewe showed that variations are generally low at the trnL-Fintergenic spacer region (Figure S4) Nonetheless based ontwo molecular tools used in this study we found that Ccallosa collected at high altitude (1843m) had undergone aunique evolving pattern (Figures 3 and 4) Evidently DNAsequence (Figure S3) shows that C callosa has suffered lessfrom point mutation in comparison with Bambusa speciesSchizostachyum species Dendrocalamus species and Melo-canna species

Undoubtedly since few bamboo species have beenadapted for consumption through ancestral customs andnot domesticated for extensive farming no proper studyhas been conducted to identify low level TCC species vis-a-vis their geographical positions in a dynamic biota Inthis era of high demand for bamboo shoots irrespectiveof the health risk (such as Konzo) identifying species withnutritious attributes nonetheless with low level TCC isindispensable A holistic approach encompassing morpho-logical and molecular tools which are required consideringthe three barcoding tools used in this study showed differ-ent interspecies clustering pattern From the present datagrappling the high altitude for safe edible bamboo speciesallowed for low input capital for eliminating TCC duringbamboo shoot processing Nonetheless prolonged and acuteintake of diet containing cyanogen derivatives could lead todeath exacerbate goitre cretinism in iodine deficient regionsmental confusion irreversible paralysis of legs and ultimatelythe neurological disorder of Konzo [3 39 40] Intriguinglyan optimal cooking procedure (at 98ndash102∘C for 148ndash180min)has shown to reduce cyanogens by 97 in D giganteus [4142] Since only D giganteus was studied [42] this cookingprotocol might not be applicable for other bamboo speciesbecause the binding of taxiphyllin may vary from species tospecies and even at the genus level

Accordingly the safest procedure for consuming bam-boo shoots might only be by using species with low TCCas starting material Previously Haque and Bradbury [9]showed that the total cyanide content decreased from the tipto the base of bamboo shoots which agreed with the presentstudy except for the Dendrocalamus species which furthershowed significant variability irrespective of the altitudeof collection This suggests that the Dendrocalamus speciesshouldmost probably not be consumed or considered at all asedible bamboo Although cyanogenic some succulent bam-boo species are shown to possess a free radical scavengingactivity which is considered as good food Explicitly somespecies showed appreciable amount of antioxidant activityexcept for the Dendrocalamus species further confirmingtheir low nutritious value as a whole Recently Park andJhon [7] evaluated the antioxidant activities of Phyllostachyspubescens and Phyllostachys nigra only and concluded thatthe parameter varies between the species Here using a largerdata set we showed that there is an interspecific variation inantioxidant properties of bamboo shoots collected at differentaltitudes

The recommended dietary allowance (RDA) intake formagnesium (Mg) is 320mg for female adults and 400ndash420mgfor male adults [43] Consequently 100 g of the bambooshoots (except the Dendrocalamus species) can supply 28ndash75 of potassium 1ndash4 of sodium 10ndash22 of phosphorousand 22ndash56ofmagnesium for females and 16ndash43 for adultsrespectively The RDA of iron for pregnant women is 27mgand thus 100 g of the bamboo shoots can provide 26ndash96RDA for pregnant women Equally 100 g of the bambooshoots also contained 28ndash136 of zinc and 2ndash7 of copperrequired as per RDA [43] Based on our studies C callosaprovides more of these beneficial RDA intakes of micro-andmacronutrients as well as substantial antioxidant activityPlants with antioxidant properties have been shown to beimportant natural antitumor and antimutagenic reserves [644] When put together results of mineral content obtainedin this study agreed with the previous quantification reports[8]

5 Conclusions

Considering the commercial implications health risks asso-ciated with poisonous bamboo shoots and nonavailabilityof optimized method for removal of TCC we highlight thefollowing for validating safe edible bamboo shoots priorto commercialization (1) barcoding bamboo shoots usingmorphological descriptors and trnL-F intergenic spacer (2)tagging the packed products with at least the genus nameand beneficial nutritional components (3) cogent tagging ofgenus Dendrocalamus with warning such as ldquobamboo shootsare injurious to healthrdquo (4) refining the available edible bam-boo shoots processing technique to minimize the residualquantity of TCC in the final products and normalizing theprocessing technique for stakeholders involved in the trade(5) conserving germplasm domesticating and extensivelyfarming C callosa at high altitude of at least 1843m from sealevel to avoid extinction

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

This research was supported by the Department of Biotech-nology (DBT) Government of India in the form of a Post-doctoral Fellowship to Sayanika Devi Waikhom The authorBengyella louis is grateful to The Academy of Sciences forDeveloping World (TWAS) and DBT Government of India(program no 3240223450) for his fellowship We extend ourgratitude to D K Hore D Biswas and N Mazumder forproofreading the manuscript

References

[1] F Nartey ldquoToxicological aspects of cyanogenrsquos is in tropicalfood stuffrdquo in Toxicology in the Tropics R L Smith and E ABababumni Eds pp 53ndash73 Taylor and Francis London UK1980


[2] U Schwarzmaier ldquoCyanogenesis of Dendrocalamus taxiphy-llinrdquo Phytochemistry vol 16 no 10 pp 1599ndash1600 1977

[3] H Nzwalo and J Cliff ldquoKonzo from poverty cassava andcyanogen intake to toxico-nutritional neurological diseaserdquoPLoS Neglected Tropical Diseases vol 5 no 6 article e1051 2011

[4] M D Thiyam S S Giri D Y Pramoda and D G A Shanti-bala ldquoAn investigation into elimination of cyanide from underprocess soibumrdquoThe Bioscan vol 5 no 4 pp 639ndash642 2010

[5] SDWaikhom SGhoshNC Talukdar and S SMandi ldquoAsse-ssment of genetic diversity of landraces of Dendrocalamushamiltonii using AFLP markers and association with biochem-ical traitsrdquo Genetics and Molecular Research vol 11 no 3 pp2107ndash2121 2012

[6] N Chongtham M S Bisht and S Haorongbam ldquoNutritionalproperties of bamboo shoots potential and prospects forutilization as a health foodrdquo Comprehensive Reviews in FoodScience and Food Safety vol 10 no 3 pp 153ndash168 2011

[7] E J Park and D Y Jhon ldquoThe antioxidant angiotensin con-verting enzyme inhibition activity and phenolic compounds ofbamboo shoot extractsrdquo LWTmdashFood Science and Technologyvol 43 no 4 pp 655ndash659 2010

[8] C Nirmala E David and M L Sharma ldquoChanges in nutrientcomponents during ageing of emerging juvenile bambooshootsrdquo International Journal of Food Sciences and Nutritionvol 58 no 8 pp 612ndash618 2007

[9] M R Haque and J H Bradbury ldquoTotal cyanide determinationof plants and foods using the picrate and acid hydrolysismethodsrdquo Food Chemistry vol 77 no 1 pp 107ndash114 2002

[10] Y P Kalra ldquoDetermination of pH soils by different methodscollaborative studyrdquo Journal of AOAC International vol 78 no2 pp 310ndash321 1995

[11] F J Rohlf NTSYS-pc Numerical Taxonomy and MultivariateAnalysis System Version 220e Exeter Publication New YorkNY USA 2000

[12] P H A Sneath and R R SokalNumerical TaxonomyThe Prin-ciples and Practice of Numerical Classification W H FreemanSan Francisco Calif USA 1st edition 1973

[13] R R Sokal and C D Michener ldquoA statistical method for eval-uating systematic relationshipsrdquo University of Kansas ScienceBulletin vol 28 pp 1409ndash1438 1958

[14] S Aras A Duran and G Yenilmez ldquoIsolation of DNA forRAPD analysis from dry leaf material of some Hesperis LSpecimensrdquo Plant Molecular Biology Reporter vol 21 no 4 pp461ndash462 2003

[15] P Jaccard ldquoNouvelles recherche sur la distribution floralerdquoBulletin de la Societe Vaudoise de Sciences Naturelles vol 44 pp223ndash270 1908

[16] P Taberlet L Gielly G Pautou and J Bouvet ldquoUniversal pri-mers for amplification of three non-coding regions of chloro-plast DNArdquo PlantMolecular Biology vol 17 no 5 pp 1105ndash11091991

[17] F Sievers AWilmDDineen et al ldquoFast scalable generation ofhigh-quality protein multiple sequence alignments usingClustal Omegardquo Molecular Systems Biology vol 7 article 5392011

[18] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program forWindows 9598NTrdquoNuc-leic Acids Symposium Series vol 41 pp 95ndash98 1999

[19] I Milne F Wright G Rowe D F Marshall D Husmeier andG McGuire ldquoTOPALi software for automatic identificationof recombinant sequences within DNA multiple alignmentsrdquoBioinformatics vol 20 no 11 pp 1806ndash1807 2004

[20] M Hasegawa H Kishino and T Yano ldquoDating of the human-ape splitting by a molecular clock of mitochondrial DNArdquoJournal of Molecular Evolution vol 22 no 2 pp 160ndash174 1985

[21] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[22] M J Bradbury S V Egan and J H Bradbury ldquoDeterminationof all forms of cyanogens in cassava roots and cassava productsusing picrate kitsrdquo Journal of the Science of Food andAgriculturevol 79 no 4 pp 593ndash601 1999

[23] L L Mensor F S Menezes G G Leitao et al ldquoScreening ofBrazilian plant extracts for antioxidant activity by the use ofDPPH free radical methodrdquo Phytotherapy Research vol 15 no2 pp 127ndash130 2001

[24] AOAC Official Method of Analysis Association of OfficialAgriculture Chemicals Washington DC USA 10th edition1985

[25] R A Koenig andC R Johnson ldquoColorimetric determination ofphosphorus in biological materialsrdquo Industrial and EngineeringChemistry vol 14 no 2 pp 155ndash156 1942

[26] D M Updegraff ldquoSemimicro determination of cellulose inbi-ological materialsrdquo Analytical Biochemistry vol 32 no 3 pp420ndash424 1969

[27] F Tajima and M Nei ldquoEstimation of evolutionary distancebetween nucleotide sequencesrdquo Molecular Biology and Evolu-tion vol 1 no 3 pp 269ndash285 1984

[28] S X Lin and J Lapointe ldquoTheoretical and experimental biologyin onerdquo Journal of Biomedical Science and Engineering vol 6 pp435ndash442 2013

[29] G P Zhou and M H Deng ldquoAn extension of Choursquos graphicrules for deriving enzymekinetic equations to systems involvingparallel reaction pathwaysrdquo Biochemical Journal vol 222 no 1pp 169ndash176 1984

[30] K C Chou ldquoGraphic rules in steady and non-steady state enz-yme kineticsrdquo Journal of Biological Chemistry vol 264 no 20pp 12074ndash12079 1989

[31] J Andraos ldquoKinetic plasticity and the determination of productratios for kinetic schemes leading to multiple products withoutrate lawsmdashNew methods based on directed graphsrdquo CanadianJournal of Chemistry vol 86 no 4 pp 342ndash357 2008

[32] I W Althaus A J Gonzales J J Chou et al ldquoThe quinoline U-78036 is a potent inhibitor of HIV-1 reverse transcriptaserdquoJournal of Biological Chemistry vol 268 no 20 pp 14875ndash14880 1993

[33] K C Chou ldquoGraphic rule for drug metabolism systemsrdquo Cur-rent Drug Metabolism vol 11 no 4 pp 369ndash378 2010

[34] K C ChouW Z Lin and X Xiao ldquoWenxiang a web-server fordrawing wenxiang diagramsrdquo Natural Science vol 3 no 10 pp862ndash865 2011

[35] G P Zhou ldquoThe disposition of the LZCC protein residues inwenxiang diagram provides new insights into the protein-protein interaction mechanismrdquo Journal of Theoretical Biologyvol 284 no 1 pp 142ndash148 2011

[36] G P Zhou and R B Huang ldquoThe pH-triggered conversion ofthe PrP(c) to PrP(sc)rdquo Current Topics in Medicinal Chemistryvol 13 no 10 pp 1152ndash1163 2013

[37] P M Kroonenberg ldquoIntroduction to biplots for G times E tablesrdquoResearch Report 51 Department of Mathematics University ofQueensland 1995


[38] W Yan M S Kang B Ma S Woods and P L Cornelius ldquoGGEbiplot versus AMMI analysis of genotype-by-environmentdatardquo Crop Science vol 47 no 2 pp 643ndash655 2007

[39] S Kumar and S R Gadagkar ldquoDisparity index a simple statisticto measure and test the homogeneity of substitution patternsbetweenmolecular sequencesrdquoGenetics vol 158 no 3 pp 1321ndash1327 2001

[40] N Mlingi N H Poulter and H Rosling ldquoAn outbreak of acuteintoxications from consumption of insufficiently processedcassava in Tanzaniardquo Nutrition Research vol 12 no 6 pp 677ndash687 1992

[41] O S A Oluwole A O Onabolu I A Cotgreave H Rosling APersson and H Link ldquoIncidence of endemic ataxic polyneu-ropathy and its relation to exposure to cyanide in a Nigeriancommunityrdquo Journal of Neurology Neurosurgery and Psychiatryvol 74 no 10 pp 1417ndash1422 2003

[42] V L Ferreira K Yotsuyanagi and C R Carvalho ldquoEliminationof cyanogenic compounds frombamboo shootsDendrocalamusgiganteus Munrordquo Tropical Science vol 35 no 4 pp 342ndash3461995

[43] Institute of Medicine (IOM) ldquoUS National Academy of Sci-ences dietary reference intakes for individuals food and nutri-tional boardrdquo 2004

[44] M Zahin F Aqil F M Husain and I Ahmad ldquoAntioxidantcapacity and antimutagenic potential of MurrayakoengiirdquoBioMed Research International vol 2013 Article ID 263509 10pages 2013

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


Table 1 Principal geographical coordinates mean soil pH and identified edible bamboo species

GenBank accessionbamboo species 1 2 3 4 5 Voucher noKC013282Chimonobambusa callosa Leimaram 51 plusmn 03 24∘441015840 93∘431015840 1843m (hill) IBSDWS019KC013285Bambusa cacharensis Arapti 58 plusmn 03 24∘441015840 93∘511015840 807m (hill) IBSDWS020JX564900Bambusa manipureana Khong Khang 62 plusmn 01 24∘211015840 94∘111015840 240m (valley) IBSDWS008JX564901Bambusa nutans Arapti 56 plusmn 04 24∘441015840 93∘561015840 226m (valley) IBSDWS023JX507132Bambusa tulda Arapti 55 plusmn 01 24∘441015840 93∘561015840 226m (valley) IBSDWS022JX507131Bambusa oliveriana Arapti 55 plusmn 03 24∘441015840 93∘561015840 728m (hill) IBSDWS010JX564902Dendrocalamus giganteus Arapti 54 plusmn 02 24∘441015840 93∘561015840 803m (hill) IBSDWS001JX564903Dendrocalamus hamiltonii Kwatha 62 plusmn 04 24∘191015840 94∘161015840 358m (valley) IBSDWS004JX564904Dendrocalamus hookeri Arapti 49 plusmn 08 24∘441015840 93∘561015840 770m (hill) IBSDWS005JX564905Dendrocalamus manipureanus Arapti 50 plusmn 04 24∘211015840 93∘571015840 769m (hill) IBSDWS002JX507133Melocanna baccifera Lokchao 65 plusmn 04 24∘201015840 94∘141015840 497m (hill) IBSDWS018JX507134Schizostachyum dullooa Arapti 50 plusmn 04 24∘421015840 93∘571015840 728m (hill) IBSDWS003JX564906Bambusa sp Arapti 52 plusmn 03 24∘441015840 93∘561015840 770m (hill) IBSDWS024JX564907Bambusa sp Andro 66 plusmn 03 24∘471015840 93∘031015840 803m (hill) IBSDWS007KC013288Bambusa tuldoides Andro 64 plusmn 05 24∘471015840 93∘031015840 777m (hill) IBSDWS0061Collection site 2mean pH of the soil at the collection site in the months of July-August of 2009 to 2011 3latitude (N) 4longitude (E) and 5altitude in metersshowing either valley or hill where samples were collected

bamboo species in a dynamic biota and their morphologicaldescriptors RAPD and trnL-F intergenic spacer to studythe interrelatedness of coevolved species of genera Bam-busa Dendrocalamus Chimonobambusa SchizostachyumandMelocannaWe also exploit the geographic positioning ofsame bamboo species to shed light on how altitude influencesthe valuable nutritional attributes of bamboo shoots

2 Material and Method

21 Plant Material Fifteen species of edible bamboo-shootsbelonging to the genera Bambusa Dendrocalamus Chi-monobambusa Schizostachyum and Melocanna were col-lected from different altitudes of Manipur India (23∘471015840ndash25∘411015840 NL 92∘581015840ndash94∘471015840 EL) during July-August of 2009-2010 This region often receives an average rainfall of 1320plusmn 3mm and temperature of 23 plusmn 3∘C during the months ofJuly-August This group of samples was used for morpho-logical characterization and authenticated by the BotanicalSurvey of India (BSI) Kolkata The voucher specimens weredeposited at the Central National Herbarium in BSI forfuture references (Table 1) For biochemical analysis 30-day-old bamboo-shoots (from the day of emergence) from thefield were collected in 2009 2010 and 2011 following routinesampling Subsequent to harvest the bamboo-shoots weredirectly frozen in liquid nitrogen in a thermocol box andtransported to the laboratorywhere theywere stored atminus80∘Cfor downstream analysisThis group of samples was collectedat sunset and the soil pH for the sites of collection wasdetermined as previously described [10]

22 Morphological Analysis To evaluate the morphologicalinterrelatedness among the edible bamboo species we used35 morphological descriptors based on 11 culm types 13culm-sheath types and 11 leaf types (Table S1 available onlineat httpdxdoiorg1011552013289285) The morphological

data were analysed using NTSYS-PC version 22 [11] Simplecoefficient matching was performed using SIMQUAL optionfor generating dataset similaritymatrix [12]The best dendro-gram was computed with Unweighted Pair-GroupedMethodArithmetic Averages (UPGMA) [13] All the samples used inthis study were scored in triplicate

23 DNA Extraction To characterize at molecular levelgenomic DNA was isolated as described by Aras et al [14]The quantity and quality of DNA were checked on BioSpecNanoDrop spectrophotometer (Thermo Scientific USA) andon 08 wv agarose gel electrophoresis respectively

24 Random Amplified Polymorphic DNA (RAPD) Analy-sis PCR was performed using standard RAPD PCR kit(Invitrogen USA) in a volume of 50 120583L in C1000 TouchThermal Cycler (BIO-RAD USA)The run was programmedas follows initial denaturation at 95∘C for 5min followedby 35 cycles of amplification (95∘C for 1min 37∘C for 1minand 72∘C for 2min) and a final extension at 72∘C for 7minAmpliconswere profiled on a 18 agarose gel electrophoresisand revealed with ethidium bromide in Gel Doc-it2 Imager(UVP Co Ltd) The primers used for this analysis arerepresented (Table S2) Polymorphism was scored as 10(presence or absence) to generate a primary binary matrixThe primary binary matrix was used for producing similaritydata using Jaccardrsquos similarity coefficient [15] and computedusing UPGMA This analysis was performed in NTSYS-PCsoftware [11]

25 Analysis of trnL-F Intergenic Spacer In order toauthenticate the edible bamboo species the trnL-Fregion was amplified using the primers set (forward51015840-ggttcaagtccctctatccc-31015840 reverse 51015840-atttgaactggtgacacgag-31015840) as described in Taberlet et al [16] PCR products




1and AICc








119860119862

(1)








3 HClO





(a) (b) (c) (d) (e) (f)

(g) (h) (i) (j) (k) (l)

(m) (n) (o)



3 Results






I

II

S dullooa JX507134

034 041 048 056 063Coefficient



C callosa KC013282



B nutans JX564901




Bambusa sp JX564907




IS dullooa JX507134

II

III

009 022 041 058 075Coefficient

























C callosa KC013282



B nutans JX564901

B tulda JX507132




D hookeri JX564904

Bambusa sp JX564906

Bambusa sp JX564907




S dullooa JX507134

100

5772

01

98





50) obtained by









1

11

21

31

41

51

61

71

81

91

20 50 100 200 400

Ant

ioxi

dant

activ

ity (

)




























080

035

minus010

minus055

minus100

minus080 minus035 010 055 100PC-1 (3219)

PC-2

(16

91

)

TCC

6 10

912

8

7

5

4

3

2

1

11K

N

P

Na

Cu

Mg

Fe

CaCe

AA Zn





4 Discussion








5 Conclusions




Acknowledgments


References



















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




1and AICc








119860119862

(1)








3 HClO





(a) (b) (c) (d) (e) (f)

(g) (h) (i) (j) (k) (l)

(m) (n) (o)



3 Results






I

II

S dullooa JX507134

034 041 048 056 063Coefficient



C callosa KC013282



B nutans JX564901




Bambusa sp JX564907




IS dullooa JX507134

II

III

009 022 041 058 075Coefficient

























C callosa KC013282



B nutans JX564901

B tulda JX507132




D hookeri JX564904

Bambusa sp JX564906

Bambusa sp JX564907




S dullooa JX507134

100

5772

01

98





50) obtained by









1

11

21

31

41

51

61

71

81

91

20 50 100 200 400

Ant

ioxi

dant

activ

ity (

)




























080

035

minus010

minus055

minus100

minus080 minus035 010 055 100PC-1 (3219)

PC-2

(16

91

)

TCC

6 10

912

8

7

5

4

3

2

1

11K

N

P

Na

Cu

Mg

Fe

CaCe

AA Zn





4 Discussion








5 Conclusions




Acknowledgments


References



















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


(a) (b) (c) (d) (e) (f)

(g) (h) (i) (j) (k) (l)

(m) (n) (o)



3 Results






I

II

S dullooa JX507134

034 041 048 056 063Coefficient



C callosa KC013282



B nutans JX564901




Bambusa sp JX564907




IS dullooa JX507134

II

III

009 022 041 058 075Coefficient

























C callosa KC013282



B nutans JX564901

B tulda JX507132




D hookeri JX564904

Bambusa sp JX564906

Bambusa sp JX564907




S dullooa JX507134

100

5772

01

98





50) obtained by









1

11

21

31

41

51

61

71

81

91

20 50 100 200 400

Ant

ioxi

dant

activ

ity (

)




























080

035

minus010

minus055

minus100

minus080 minus035 010 055 100PC-1 (3219)

PC-2

(16

91

)

TCC

6 10

912

8

7

5

4

3

2

1

11K

N

P

Na

Cu

Mg

Fe

CaCe

AA Zn





4 Discussion








5 Conclusions




Acknowledgments


References



















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



I

II

S dullooa JX507134

034 041 048 056 063Coefficient



C callosa KC013282



B nutans JX564901




Bambusa sp JX564907




IS dullooa JX507134

II

III

009 022 041 058 075Coefficient

























C callosa KC013282



B nutans JX564901

B tulda JX507132




D hookeri JX564904

Bambusa sp JX564906

Bambusa sp JX564907




S dullooa JX507134

100

5772

01

98





50) obtained by









1

11

21

31

41

51

61

71

81

91

20 50 100 200 400

Ant

ioxi

dant

activ

ity (

)




























080

035

minus010

minus055

minus100

minus080 minus035 010 055 100PC-1 (3219)

PC-2

(16

91

)

TCC

6 10

912

8

7

5

4

3

2

1

11K

N

P

Na

Cu

Mg

Fe

CaCe

AA Zn





4 Discussion








5 Conclusions




Acknowledgments


References



















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




















C callosa KC013282



B nutans JX564901

B tulda JX507132




D hookeri JX564904

Bambusa sp JX564906

Bambusa sp JX564907




S dullooa JX507134

100

5772

01

98





50) obtained by









1

11

21

31

41

51

61

71

81

91

20 50 100 200 400

Ant

ioxi

dant

activ

ity (

)




























080

035

minus010

minus055

minus100

minus080 minus035 010 055 100PC-1 (3219)

PC-2

(16

91

)

TCC

6 10

912

8

7

5

4

3

2

1

11K

N

P

Na

Cu

Mg

Fe

CaCe

AA Zn





4 Discussion








5 Conclusions




Acknowledgments


References



















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


1

11

21

31

41

51

61

71

81

91

20 50 100 200 400

Ant

ioxi

dant

activ

ity (

)




























080

035

minus010

minus055

minus100

minus080 minus035 010 055 100PC-1 (3219)

PC-2

(16

91

)

TCC

6 10

912

8

7

5

4

3

2

1

11K

N

P

Na

Cu

Mg

Fe

CaCe

AA Zn





4 Discussion








5 Conclusions




Acknowledgments


References



















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


080

035

minus010

minus055

minus100

minus080 minus035 010 055 100PC-1 (3219)

PC-2

(16

91

)

TCC

6 10

912

8

7

5

4

3

2

1

11K

N

P

Na

Cu

Mg

Fe

CaCe

AA Zn





4 Discussion








5 Conclusions




Acknowledgments


References



















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of






5 Conclusions




Acknowledgments


References



















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

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