AcN

USa

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0h

Industrial Crops and Products 50 (2013) 112ndash 117

Contents lists available at SciVerse ScienceDirect

Industrial Crops and Products

journa l h om epa ge wwwelsev ier com locate indcrop

ltitudinal variability in anthraquinone constituents from novelytotypes of Rumex nepalensis Sprengmdasha high value medicinal herb oforth Western Himalayas

mer Farooqa1 Shahzad A Pandithb1 Manjit Inder Singh Saggooalowastlowasturrinder K Lattooblowast

Department of Botany Punjabi university Patiala Punjab 147002 IndiaPlant Biotechnology Indian Institute of Integrative Medicine (CSIR) Canal Road Jammu Tawi 180001 India

r t i c l e i n f o

rticle historyeceived 19 February 2013eceived in revised form 21 June 2013ccepted 28 June 2013

eywordsumex nepalensisnthraquinonesytotypeeiotic analysisPLColyploidy

a b s t r a c t

Rumex nepalensis is a reputed medicinal herb of North Western Himalayas It owes its pharmacologicalsignificance to an active class of compounds known as anthraquinones In the present study four differ-ent intraspecific cytotypes with varying ploidy level ranging from tetraploidy (4times 2n = 40) to octaploidy(8times 2n = 80) were discovered hitherto unreported The investigation also included the evaluation ofchemical variability in four major anthraquinone constituents of different cytotypes from five altitudi-nal habitats of Himalayas using standard HPLC method There was significant increase in the aglyconecomponents of anthraquinones with the increase in ploidy status except for dodecaploid type (12times2n = 120) that showed a decreasing trend in their accumulation However the overall concentrationof anthraquinones both glycone and aglycone constituents was found to be highest (3395) in thedodecaploid type from Lidderwatt location Polyploidy an important driver of eukaryotic evolution is aprevalent feature among angiosperm species providing dynamic genome flexibility to them Polyploids

can undergo rapid structural and functional alterations allowing them to adapt and persist across het-erogeneous landscapes in the long run It is reflected in the robust adaptability of different cytotypes ofR nepalensis These display appreciable chemical variability and ecological plasticity possibly becauseof the intraspecific polyploidization in response to different ecological niches of a geographical regionThe scope of present investigation also extends to identify elite cytotypes in terms of their desirablechemoprofiles for pharmaceutical and commercial purposes

Introduction

Rumex nepalensis Spreng (Polygonaceae) is a perennial ascend-ng herb distributed throughout Himalayas from Bhutan to Kashmirnd also to Turkey Java and South Africa It is a fairly commonlant of higher altitudes and grows between 900ndash4000 m on moists well as dry slopes under shades and even in plains throughoutashmir (India) R nepalensis is highly reputed for various thera-eutic purposes in Indian traditional systems of medicine The root

f R nepalensis is purgative and is used as a substitute for Rheumpecies (Manandhar 2002) A strong decoction is applied to dislo-ated bones Root paste is applied externally to relieve headache

lowast Corresponding author Tel +91 9419203465 fax +91 191 2569019lowastlowast Corresponding author

E-mail addresses msaggoogmailcom (MI Singh Saggoo) sklattooiiimacinSK Lattoo)

1 Authors have contributed equally

926-6690$ ndash see front matter copy 2013 Elsevier BV All rights reservedttpdxdoiorg101016jindcrop201306044

copy 2013 Elsevier BV All rights reserved

Its leaves are antiseptic and are used for the treatment of syphilicand colic ulcers (Kirtikar and Basu 1987) Several anthraquinonesnaphthalenes flavonoids and other phenolic compounds have beenreported from R nepalensis (Liang et al 2010) These compoundsare used for the treatment of bleeding tumour inflammationpain constipation and tinea in the Chinese folk medicine (Zhanget al 2008) R nepalensis has been investigated for antihistaminicanticholinergic antibradykinin antiprostaglandin (Aggarwal et al1986) purgative antibacterial (Ghosh et al 2003) antipyretic(Venkatesh et al 2003) and anti-inflammatory activities (Gautamet al 2010) The antibacterial antifungal and insecticidal proper-ties have also been reported from the extracts of this plant (Hussainet al 2010)

In present investigation our endeavour was to explore chemi-cal diversity in R nepalensis It also included cytological analysis of

various populations from Kashmir Himalayas to determine chro-mosome counts in corroboration with comparative chemoprofilesof the five different cytotypes of R nepalensis employing standardHPLC method (Singh et al 2005) with slight modifications The

U Farooq et al Industrial Crops and Products 50 (2013) 112ndash 117 113

Table 1Chromosome count and ploidy status recorded in different populations of Rumex nepalensis from five locations of Kashmir Himalayas

S No Meiotic chromosome no (lsquonrsquo) Cytotype (ploidy status) Location of plant collection Accession number (PUN)a

1 20b Tetraploid (4x = 40) Zawoora (3343prime N 7514prime E 2000 m) 576912 30b Hexaploid (6x = 60) Zawoora upper reaches (3444prime N 7448primeE 2300 m) 576763 40b Octaploid (Y) (8x = 80) Yusmarg (3347prime N 7439prime E 2600 m) 576774 40b Octaploid (T) (8x = 80) Thajwas (3418prime N 7448prime E 3400 m) 576605 60 Dodecaploid (12x = 120) Lidderwatt (3404 N 7517prime E 3500 m) 57683

ndex

iadic

2

2

dci(lPmcgwowsbt

2

fi14uAestsdm

a Herbarium code of the Botany Department Punjabi University Patiala as per ldquoIb First chromosomal report at world level

nvestigation suggests positive correlation between ploidy levelnd metabolite accumulation in relation to different altitudinal gra-ients This throws a fresh perspective to identify elite cytotypes

n terms of their desirable chemoprofiles for pharmaceutical andommercial purposes

Materials and methods

1 Plant materials and chemicals

The wild growing plants of R nepalensis were collected from fiveifferent locations of the Kashmir valley (JampK state India) The plantollections were identified by Dr S K Srivastava in-charge Herbar-um Botanical Survey of India (BSI) Northern circle DehradunIndia) The voucher specimens of the plants of different popu-ations were deposited in the Herbarium Department of Botanyunjabi University Patiala India (Table 1) Pure standards of twoajor anthraquinones namely emodin and chrysophanol were pur-

hased from SigmandashAldrich (St Louis USA) and their respectivelycosides emodin glycoside and chrysophanol glycoside (Fig 1)ere obtained from Institute of Himalayan Bioresource Technol-

gy Palampur India (kindly provided by A K Sinha) Their puritiesere above 99 as determined by HPLC analysis All chemicals and

olvents used were have analytical and HPLC grade (E Merck Mum-ai India) Fresh ultra-pure distilled water with resistivity greaterhan 18 M was used

2 Meiotic analysis

For meiotic analysis the young floral buds at an ideal stage werexed in freshly prepared carnoyrsquos fixative (6 alcohol 3 chloroform

acetic acid vvv) for 24 h and then preserved in 70 alcohol atC in the refrigerator The cytological preparations were madesing the squash technique in 2 acetocarmine (Rana et al 2012)

total of 45 pollen mother cells (PMC) from temporary slides werexamined to determine the exact chromosome number at differenttages of meiosis Photomicrographs of chromosome counts were

aken from freshly prepared slides using Nikon 80i Eclipse micro-cope For previous chromosome reports in addition to the IPCNatabase (httpwww tropicosorg) various indices to plant chro-osome numbers were consulted (Darlington and Wylie 1995

Fig 1 Chemical structures of major ant

Herbariorumrdquo by Holmgren and Holmgren (1998)

Moore 1967ndash74 Kumar and Subramanian 1986 Khatoon and Ali1993)

23 Chemical evaluation

Leaf samples of five cytotypes of R nepalensis collected fromfive different locations of the Kashmir valley were air-dried andground into fine powder with a mortar and pestle The powderedleaves (50 g of each sample) of five different plant samples wereextracted each with methanol (3 times 300 mL) using Soxchlet extrac-tion The extracts were filtered and solvents were removed underreduced pressure For arbitrary evaluation of the extracted sam-ples TLC with solvent compositions of 5 10 and 15 methanolin dichloromethane were taken This showed marked variation inthe Rf (retention factor) values and intensity of the visible spotsindicating presence of various compounds in different concentra-tions (Fig 2) Moreover TLC spots were non-UV active The stocksolutions (1 mgmL) of each of the four anthraquinones were freshlyprepared in a mixture of methanol acetonitrile (8020 vv) Sim-ilarly the crude extracts of five cytotypes were also dissolved inthe same solvent composition (20 mgmL) and were filter steril-ized with 045 m membrane filters (Millipore Bedford USA) TheHPLC (Shimadzu CLASS-VP V 614 SPI model) equipped with RP-18e column (E-Merck 5 m 46 times 250 nm) a photo-diode arraydetector (SPD-M10A VP model) and a pump (LC-10AT VP model)was used for analysis of hydroxy-anthraquinone derivatives Ace-tonitrile methanol (955 vv) (solvent B) and water acetic acid(99901 vv pH 35) (solvent A) were used as mobile phase witha linear gradient elution as follows 0ndash15 min 20 B 15ndash25 min20 B 25ndash30 min 50 B 30ndash40 min 70 B 40ndash45 min 100 B45ndash50 min 20 B 50ndash55 min 20 B at a flow rate of 08 mlminThe detection wavelength was set at 254 nm Injection volume ofthe sample was 10 l and the column temperature 30 C (Fig 3)

3 Results and discussion

Detailed cytological analysis of various populations of Rnepalensis revealed the existence of four different chromosomal

races with meiotic chromosome numbers n = 20 30 40 and 60 inthe Kashmir region (Table 1) These chromosome counts are thefirst ever records for this species The present chromosome countof n = 60 (2n = 12x= 120) confirms the previous record (Degraeve

hraquinones and their glycosides

114 U Farooq et al Industrial Crops and Products 50 (2013) 112ndash 117

Fig 2 TLC profiles of methanolic extracts of different cytotypes of R nepalensis in 5 (a) 10 (b) and 15 (c) methanol in dichloromethane showing various compoundsfound to be UV inactive Abbreviations 1 = Tetraploid 2 = Hexaploid 3 = Dodecaploid 4 = Octaploid (Y) and 5 = Octaploid (T)

Fig 3 HPLC chromatograms of standard major anthraquinones (a) and methanolic extracts of leaves of tetraploid (b) hexaploid (c) octaploid Y (d) octaploid T (e) anddodecaploid (f) cytotypes of R nepalensis Abbreviations 1 = emodin 2 = chrysophanol 3 = emodin glycoside 4 = chrysophanol glycoside

U Farooq et al Industrial Crops and Products 50 (2013) 112ndash 117 115

Fig 4 Meiotic chromosome numbers in five populations from different altitudinal ranges in R nepalensis A PMC at metaphase- I showing twenty bivalents (n = 20 Zawoora2 b) A

a esis sh

1rih(eipiantd

cgsroitsaocsgflpic(fs

Lupinus argenteus (Carey and Wink 1994) and Ginkgo biloba (Kauret al 2012) It is well documented in literature that metaboliteaccumulation is a function of interaction between genetic consti-tution and environmental variables (Lattoo et al 2006b Kooke

000 m in altitude) (a) A PMC showing thirty bivalents (n = 30 Zawoora 2300 m) (t metaphase-I showing 40 bivalents (n = 40 Thajwas 3400 m) (d) A PMC at diakin

975) The basic chromosome numbers for the genus Rumex areeported as 7 8 9 and 10 (Darlington and Wylie 1995) Conform-ng to x = 10 the present investigated taxa are tetraploid (Fig 4a)exaploid (Fig 4b) octaploid (Fig 4c and d) and dodecaploidFig 4e) respectively Different ploidy levels and wide range ofcological plasticity explains the wide distribution of this groupn North Western Himalayas The occurrence of intraspecific poly-loids in R nepalensis is indicative of the fact that its genome

s still in flux and constant evolution possibly to enhance thedaptive and survival value of the species under varied ecologicaliches of Himalayan region (Lattoo et al 2006a) The new cyto-ypespolyploids in all probability have high chance of survival atifferent altitudinal gradients

Plants produce an enormous diversity of low molecular weightompounds with a wide range of activity However this numberets further increased by multiple decorations of the commonkeleton by acylation methylation hydroxylation or conjugationeactions used for various metabolic activities in the plant Onef these most widespread modifications is glycosylation whichs related to specific plant functions like xenobiotic detoxifica-ion regulation of hormone homeostasis and biosynthesis andtorage of secondary compounds (Gachon et al 2005) The over-ll concentration of glycosides was found to be higher than thatf their respective aglycone forms at all ploidy levels The dode-aploid type from Lidderwatt (3404 N 7517 E 3500 m in altitude)howed the maximum accumulation of emodin and chrysophanollycosides than their corresponding aglycone moieties This shuf-e in the direction of glycosylation like many of the plant naturalroducts may be towards enhancing their solubility and stabil-

ty for facilitating their easy storage and accumulation in plant

ells Among the four major anthraquinones emodin glycoside38071 mgg plusmn 0907) was found to be in highest concentrationollowed by emodin (5631 mgg plusmn 0235) chrysophanol glyco-ide (4524 mgg plusmn 0268) and chrysophanol (4262 mgg plusmn 0175)

PMC at metaphase- I showing forty bivalents (n = 40 Yusmarg 2600 m) (c) A PMCowing sixty bivalents (n = 60 Lidderwatt (3500 m) (e) Bar = 10 m

A well marked increasing trend in the concentration of all fourcompounds from tetraploid to octaploid variants is in conformitywith previous reports wherein it has been shown that increase insecondary metabolite content is positively correlated with ploidylevel and altitude (Zidorn 2010 Lavania et al 2012) Compara-tive chemoprofile of two octaploids collected from two differentlocations designated as octaploid (T) and octaploid (Y) respectively(Table 1) showed an appreciable difference in the concentrationof anthraquinones (Fig 5) This can be ascribed to the effect ofaltitudinal gradients on the secondary metabolite production Sim-ilar pattern of metabolite accumulation has also been reported in

Fig 5 Graphic representation of the concentrations of key anthraquinones in fivedifferent cytotypes of R nepalensis All values obtained were means of triplicate withstandard errors

U Farooq et al Industrial Crops and Products 50 (2013) 112ndash 117 115

Fig 4 Meiotic chromosome numbers in five populations from different altitudinal ranges in R nepalensis A PMC at metaphase- I showing twenty bivalents (n = 20 Zawoora2 b) A

a esis sh

1rih(eipiantd

cgsroitsaocsgflpic(fs

Lupinus argenteus (Carey and Wink 1994) and Ginkgo biloba (Kauret al 2012) It is well documented in literature that metaboliteaccumulation is a function of interaction between genetic consti-tution and environmental variables (Lattoo et al 2006b Kooke

000 m in altitude) (a) A PMC showing thirty bivalents (n = 30 Zawoora 2300 m) (t metaphase-I showing 40 bivalents (n = 40 Thajwas 3400 m) (d) A PMC at diakin

975) The basic chromosome numbers for the genus Rumex areeported as 7 8 9 and 10 (Darlington and Wylie 1995) Conform-ng to x = 10 the present investigated taxa are tetraploid (Fig 4a)exaploid (Fig 4b) octaploid (Fig 4c and d) and dodecaploidFig 4e) respectively Different ploidy levels and wide range ofcological plasticity explains the wide distribution of this groupn North Western Himalayas The occurrence of intraspecific poly-loids in R nepalensis is indicative of the fact that its genome

s still in flux and constant evolution possibly to enhance thedaptive and survival value of the species under varied ecologicaliches of Himalayan region (Lattoo et al 2006a) The new cyto-ypespolyploids in all probability have high chance of survival atifferent altitudinal gradients

Plants produce an enormous diversity of low molecular weightompounds with a wide range of activity However this numberets further increased by multiple decorations of the commonkeleton by acylation methylation hydroxylation or conjugationeactions used for various metabolic activities in the plant Onef these most widespread modifications is glycosylation whichs related to specific plant functions like xenobiotic detoxifica-ion regulation of hormone homeostasis and biosynthesis andtorage of secondary compounds (Gachon et al 2005) The over-ll concentration of glycosides was found to be higher than thatf their respective aglycone forms at all ploidy levels The dode-aploid type from Lidderwatt (3404 N 7517 E 3500 m in altitude)howed the maximum accumulation of emodin and chrysophanollycosides than their corresponding aglycone moieties This shuf-e in the direction of glycosylation like many of the plant naturalroducts may be towards enhancing their solubility and stabil-

ty for facilitating their easy storage and accumulation in plant

ells Among the four major anthraquinones emodin glycoside38071 mgg plusmn 0907) was found to be in highest concentrationollowed by emodin (5631 mgg plusmn 0235) chrysophanol glyco-ide (4524 mgg plusmn 0268) and chrysophanol (4262 mgg plusmn 0175)

PMC at metaphase- I showing forty bivalents (n = 40 Yusmarg 2600 m) (c) A PMCowing sixty bivalents (n = 60 Lidderwatt (3500 m) (e) Bar = 10 m

A well marked increasing trend in the concentration of all fourcompounds from tetraploid to octaploid variants is in conformitywith previous reports wherein it has been shown that increase insecondary metabolite content is positively correlated with ploidylevel and altitude (Zidorn 2010 Lavania et al 2012) Compara-tive chemoprofile of two octaploids collected from two differentlocations designated as octaploid (T) and octaploid (Y) respectively(Table 1) showed an appreciable difference in the concentrationof anthraquinones (Fig 5) This can be ascribed to the effect ofaltitudinal gradients on the secondary metabolite production Sim-ilar pattern of metabolite accumulation has also been reported in

Fig 5 Graphic representation of the concentrations of key anthraquinones in fivedifferent cytotypes of R nepalensis All values obtained were means of triplicate withstandard errors

116 U Farooq et al Industrial Crops and Products 50 (2013) 112ndash 117

Table 2Relative percentage of four key anthraquinone constituents in the leaves of five different cytotypes of Rumex nepalensis

Ploidy Level Tetraploid Hexaploid Octaploid (Y) Octaploid (T) Dodecaploid

Relative percentage ()a 273 849 2898 2585 3395

all cy

abooasiropiIm

uiwmtt

c1wcuawwwiboA

TM

a Concentration of all four anthraquinones in one cytotypetotal concentration in

nd Keurentjes 2012) It is an efficient adaptive strategy employedy the plants to cope up with the effect of many biotic and abi-tic stresses to perpetuate in diverse environments The variabilityf major phyto-constituents within the same species at differentltitudes and temperature ranges present a significant relation-hip between the quality and quantity of active principles Changesn the chemical constituents of Achillea millefolium has also beenecorded from different Himalayan habitats due to the interactionf plant populations to diverse habitats within a geographic area asrimary selection factors give rise to major or minor differences

n the content of chemical constituents (Agnihotri et al 2005)n many cases it may also influence the therapeutic potential of

edicinal plants (Zarinkamar et al 2011)The relative percentage of different chemical constituents is a

seful criterion to evaluate the total content of compounds presentn a given entity In terms of relative percentage anthraquinones

ere found to be maximum (3395) in the dodecaploid type andinimum (273) in the tetraploid one providing a clue towards

he difference in pharmacological significance of these two cyto-ypes (Table 2)

Morphological variations in all the populations of five differentytotypes were evaluated The tetraploid plants which measured20ndash150 cm in size were tallest than the rest of cytotypes Thereas profuse secondary branching in octaploids with higher con-

entration of secondary metabolites Microscopic characters widelysed for the identification of ploidy levels viz pollen grain diameternd stomatal length were also analyzed The average stomatal sizeas more in dodecaploid type Pollen grains in the tetraploid typeere smaller (2129 times 2026 m) as compared to other cytotypesith the largest pollen grains seen in dodecaploid type measur-

ng 2931 times 2872 m (Table 3) There seems a positive correlationetween the pollen and stomatal size with the level of ploidy Thesebservations were in congruence with earlier reports (Inceer andyaz 2010 Pan 1994) Polyploidization is an important process in

able 3orphological comparison of different cytotypes in Rumex nepalensis

S No Characters Tetraploidcytotype 2n = 40

Hexaploidcytotype 2n = 60

1 Plant height (cm) 120ndash150 100ndash130

2 Branchesplant 7ndash10 4ndash7

3 Leavesbranch 12ndash15 3ndash7

4 Ochrea length(mm)

05ndash1 1ndash15

5 Petiole length(cm)

2ndash25 1ndash15

6 Leaf size (cm) LB 25ndash415ndash2 3ndash52ndash3

7 Leaf surface Smooth Smooth

8 Inflorescencelength (cm)

1ndash2 115ndash25

9 Average stomatalsize upperlower(m)

1968 times 878 2001 times 908

2195 times 850 2203 times 875

10 Pollen size (m) 2129 times 2026 2256 times 2175

11 Habit (Herb) Tallest Tall

12 Habitat Grows on moistrich soils

Found in closearea of waterstreams

totypes times 100

the evolution of most of the eukaryotic species It often causes largescale genomic reorganizations and is accompanied by a wide vari-ety of phenotypic alterations in morphology niche preference andfitness characteristics (Riddle et al 2006) The evolutionary suc-cess of polyploids has often been attributed to the consequencesof having multiple genomes with individuals of higher ploidy lev-els considered to be more adaptable to differing conditions due togenetic advantages that facilitate their establishment and persis-tence (Comai 2005) Change in environmental factors could changethe active principle present in the plants (Southwell and Bourke2001) In this relation differences in longitude latitude humiditysoil and temperature have pronounced impact on the production ofsecondary metabolites The metabolite profile of a plant defines itsecological role and is therefore an important factor in understand-ing the influence of environmental variables on the synthesis andaccumulation of secondary compounds

The present investigation thus identifies existence of four novelchromosomal races with chromosome numbers 2n = 40 60 80and 120 wherein ploidy level range from tetraploidy to dode-caploidy These polyploid races are hitherto unreported in Rnepalensis except for 2n = 120 Chemoprofiling based on four keyanthraquinones of different cytotypes from various altitudinalgradients presented an appreciable variability in chrysophanolemodin and their glycoside contents The increasing pattern ofmetabolite accumulation broadly seems to be in conformity withthe increasing altitude and ploidy status The species presentsrobust adaptability and ecological plasticity possibly because ofthe cytological variation This study has a prospect to exploredesirable populations with higher content of chemical constituentsfor commercial and pharmacological utilization Many studies

considering multiple factors can only provide a thorough under-standing of the pattern establishment survival and consequencesof higher ploidy levels across lineages under varying environmentalpressures

Octaploid cytotype(Y) 2n = 80

Octaploid cytotype(T) 2n = 80

Dodecaploidcytotype 2n = 120

80ndash100 85ndash103 40ndash8010ndash15 9ndash13 6ndash94ndash7 5ndash8 4ndash61ndash2 1ndash2 1ndash2

15ndash2 1ndash2 1ndash5

5ndash63ndash35 4ndash635ndash4 7ndash1045ndash6Hairy Hairy Profusely hairy2ndash3 2ndash4 4ndash5

2301 times 1091 2313 times 1102 2598 times 1268

2279 times 935 2188 times 924 2567 times 12732374 times 2223 2298 times 2206 2931 times 2872Medium Medium Small sizedOccurs on moistslopes

Occurs on moistslopes

Occurs in higheraltitudes

ps and

A

(SoSaofiDan

R

A

A

C

C

D

D

G

G

G

HH

I

K

in the study of chemical constituents and bioactivity of Rumex L World Science

U Farooq et al Industrial Cro

cknowledgments

The authors are grateful to the University grants commissionUGC) New Delhi for providing financial assistance under the DRSAP III programme and to the Department of Science and Technol-gy New Delhi for funding FIST programmes One of the authorsAP is highly grateful to UGC for junior research fellowship Theuthors also thank Dr S K Srivastava Director Botanical Surveyf India (BSI) Northern circle (Dehradun) for his help in identi-cation of plants Thanks are also due to Dr Ram Vishwakarmairector IIIM Jammu for providing necessary facilities for chemicalnalysis This manuscript represents institutional communicationumber IIIM15082012

eferences

ggarwal PK Kumar L Garg SK Mathur VS 1986 Effect of Rumex nepalensisextracts on histamine acetylcholine carbachol bradykinin and PGs evoked skinreactions in rabbits Annals of Allergy 56 177ndash182

gnihotri VK Lattoo SK Thappa RK Kaul P Qazi GN Dhar AK SarafA Kapahi BK Saxena RK Agarwal SG 2005 Chemical variability in theessential oil components of Achillea millefolium agg from different Himalayanhabitats (India) Planta Medica 71 280ndash283

arey DB Wink M 1994 Elevational variation of quinolizidine alkaloid contentsin a lupine (Lupinus argenteus) of the Rocky Mountains Journal of ChemicalEcology 20 849ndash857

omai L 2005 Advantages and disadvantages of polyploidy Nature Reviews Genet-ics 6 836ndash846

arlington CD Wylie AP 1995 Chromosome Atlas of Flowering Plants GeorgeAllen and Unwin Ltd London

egraeve N 1975 Contribution a lrsquoetude cytotaxonomique des RumexndashI Le genereRumex L sensu stricto Caryologia 28 187ndash201

achon CMM Meurinne ML Saindrenan P 2005 Plant secondary metabolismglycosyltransferases the emerging functional analysis Trends in Plant Science10 (11) 542ndash549

autam R Karkhile KV Bhutani KK Jachak SM 2010 Anti-inflammatorycyclooxygenase (COX)-2 COX-1 inhibitory and free radical scavenging effectsof Rumex nepalensis Planta Medica 76 1564ndash1569

hosh L Gayen JR Sinha S Pal S Pal M Saha BP 2003 Antibacterial efficacyof Rumex nepalensis Spreng roots Phytotherapy Research 17 558ndash559

olmgren and Holmgren 1998 httpsweetgumnybgorgihussain F Ahmad B Hameed I Dastagir G Sanaullah P Azam S 2010 Antibac-

terial antifungal and insecticidal activities of some selected medicinal plants ofPolygonaceae African Journal of Biotechnology 9 5032ndash5036

nceer H Ayaz SH 2010 Chromosome numbers in Tripleurospermum Sch Bip

(Asteraceae) and closely related genera relationships between ploidy level andstomatal length Plant Systematics and Evolution 285 149ndash157

aur P Chaudharya A Singh RD Gopichand PR Singh B 2012 Spatial andtemporal variation of secondary metabolite profiles in Ginkgo biloba leavesChemistry and Biodiversity 9 (2) 409ndash417

Products 50 (2013) 112ndash 117 117

Khatoon S Ali SI 1993 Chromosome Atlas of the Angiosperms of Pakistan BCC ampT press University of Karachi Karachi

Kirtikar KR Basu BD 1987 Indian Medicinal Plants vol 3 International BookDistributors India pp 2113ndash2114

Kooke R Keurentjes JJB 2012 Multi-dimensional regulation of metabolicnetworks shaping plant development and performance Journal of ExperimentalBotany 63 (9) 3353ndash3365

Kumar V Subramanian B 1986 Chromosome Atlas of Flowering Plants of theIndian Subcontinent vol 1 Dicotyledon BSI Calcutta

Lattoo SK Khan S Bamotra S Dhar AK 2006a Cytomixis impairs meio-sis and influences reproductive success in Chlorophytum comosum (Thunb)Jacqmdashan additional strategy and possible implications Journal of Bioscience 31629ndash637

Lattoo SK Dhar RS Dhar AK Sharma PR Agarwal SG 2006b Dynamics ofessential oil biosynthesis in relation to inflorescence and glandular ontogeny inSalvia sclarea Flavour and Fragrance Journal 21 (5) 817ndash821

Lavania UC Srivastava S Lavania S Basu S Misra NK Mukai Y 2012Autopolyploidy differentially influences body size in plants but facilitatesenhanced accumulation of secondary metabolites causing increased cytosinemethylation Plant Journal 71 539ndash549

Liang HX Dai HQ Fu HA Dong XP Adebayo AH Zhang LX Cheng YX2010 Bioactive compounds from Rumex plants Phytochemistry Letters 3181ndash184

Manandhar NP 2002 Plants and People of Nepal Timber Press Oregon ISBN 0-8192-527-6

Moore RJ 1967ndash74 Index to plant chromosome numbers Regnum Vegetabile909196

Pan JT 1994 Phylogeny classification and geographic distribution of RodgersiaGray Acta Phytotaxonomica Sinica 92 316ndash327

Rana S Dhar N Bhat WW Razdan S Khan S Dhar RS Dutt P LattooSK 2012 A 12-deoxywithastramonolide-rich somaclonal variant in Witha-nia somnifera (L) Dunalmdashmolecular cytogenetic analysis and significance as achemotypic resource In Vitro Cellular and Developmental BiologymdashPlant 48 (5)546ndash554

Riddle NC Kato A Birchler JA 2006 Genetic variation for the response to ploidychange in Zea mays L Theoretical and Applied Genetics 114 101ndash111

Singh NP Gupta AP Sinha AK Ahuja PS 2005 High-performance thinlayer chromatography method for quantitative determination of four majoranthraquinone derivatives in Rheum emodi Journal of Chromatography A 1077202ndash206

Southwell IA Bourke AC 2001 Seasonal variation in hypericin content of Hyper-icum perforatum L (St Johnrsquos wort) Photochemistry 56 437ndash441

Venkatesh S Reddy BM Reddy RD Ramesh M 2003 Antipyretic activity ofRumex nepalensis roots Nigerian Journal of Natural Products and Medicine 753ndash54

Zarinkamar F Tajik S Soleimanpour S 2011 Effects of altitude on anatomy andconcentration of crocin picrocrocin and safranal in Crocus sativus L AustralianJournal of Crop Science 5 831ndash838

Zhang GQ Zhao HP Wang ZY Cheng JR Tang XM 2008 Recent advances

Technology (Modern Trading in China Medicine) 10 86ndash93Zidorn C 2010 Altitudinal variation of secondary metabolites in flowering

heads of the Asteraceae trends and causes Phytochemistry Review 9197ndash203