Industrial Crops and Products 43 (2013) 768–773

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Industrial Crops and Products

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Intra-specific genetic diversity and chemical profiling of different accessions of

Clitoria ternatea L.
Zahid Ali a , Showkat Hussain Ganie a,b , Alka Narula a , Maheshwar Prasad Sharma b ,
Prem Shankar Srivastava a,∗
a
Department of Biotechnology, Jamia Hamdard, Hamdard Nagar, New Delhi 110062, India
b
Department of Botany, Jamia Hamdard, Hamdard Nagar, New Delhi 110062, India

a r t i c l e i n f o a b s t r a c t

Article history: Clitoria ternatea, commonly known as Shankhpushpi is an important drug of Ayurvedic medicine used for
Received 31 March 2012 centuries as a memory enhancer, nootropic, antistress, anxiolytic, anticonvulsant, tranquilizing and seda-
Received in revised form 30 July 2012 tive agent. In the present study, genetic variability and variation in Kaempferol, a chemical constituent
Accepted 31 July 2012
in eleven accessions of C. ternatea collected from different regions of the country was assessed using 25
RAPD primers and HPLC analysis respectively. Only 7 primers amplified a total of 71 reproducible, clear
Keywords:
and scorable bands of which 32 (45%) were polymorphic. The number of RAPD bands per primer was in
Clitoria ternatea
the range of 7–14 with an average of 10 bands per primer. The genetic distance ranged from 0.02 to 0.28. A
Genetic diversity
HPLC
dendrogram based on UPGMA clustering method revealed two major clusters. Cluster 1 comprises of the
Kaempferol accessions of North India while cluster 2 includes accessions of Central and South India. The Kaempferol
RAPD content was 9.31–20.01 mg/g dw, being highest in the accession of Kurukshetra (Haryana).
© 2012 Elsevier B.V. All rights reserved.

1. Introduction Random amplified polymorphic DNA (RAPD), a PCR based tech-

nique is a simple and cost-effective tool for analysis of plant
Successful management and preservation of natural popula- genome. It is technically least demanding and offer a fast method for
tions depend on accurate assessment of genetic diversity to address providing information from a large number of loci, particularly in
questions regarding genetic relationships among individuals as species where no study has previously been undertaken because it
well as levels and structure of genetic variation (Abdel-Mawgood is considered one of the most efficient molecular methods in terms
et al., 2006). In particular, knowledge of population genetic struc- of ability to produce abundant polymorphic markers within a short
ture provides a historical perspective of evolutionary changes that time and limited budget. RAPD has become widely used in various
characterize a species and allow to predict how populations will areas of plant research and it has proved to be a valuable tool in
respond to future events of natural and artificial origin (Wallace, studying inter and intra-specific genetic variation, patterns of gene
2002). Under the climate change scenario, for management and expression, and identification of specific genes using nearly iso-
protection programs, the genetic structure of species at popula- genic variants (Kuddus et al., 2002). More recently, RAPD has been
tion level has received special attention in the past few years used for estimation of genetic diversity in various plant species such
(Pfenninger et al., 2010). The presence of unique genetic charac- as, Dendrobium species (Zha et al., 2009), Zygophyllum populations
teristics distinguishes members of a given population from those (Hammad and Qari, 2010) and Withania somnifera (Dharmar and
of any other. High diversity is an indicator of better adaptability Britto, 2011) collected from different geographical regions.
of a population and therefore more fitness under rapidly changing There are also studies on correlation between genetic diver-
environment. sity and variation in chemical constituents (Deborah et al., 2005;
Different types of molecular markers have been used to ascer- Kuroda et al., 2007; Han et al., 2008; Tori et al., 2008). Similar stud-
tain DNA polymorphism. Considered as one of the most efficient ies, however, are negligible on medicinal plants. Quantitative and
molecular methods in terms of ability to produce abundant poly- qualitative status of active constituents along with genetic diversity
morphic markers within a short time and limited budget, the in a medicinal plant would help to devise conservation strategies
and selection of right sample for maximum yield.
Clitoria ternatea (Family: Fabaceae) commonly known as “But-
terfly pea”, “Shankhpushpi” has a long traditional use in Ayurvedic
∗ Corresponding author. Tel.: +91 011 26059688; fax: +91 011 26059663; mobile:
medicine as a memory enhancer, nootropic, antidepressant, anti-
+91 09818135178.
E-mail address: [email protected] (P.S. Srivastava). convulsant, tranqualizing and sedative agent (Anonymous, 1962;

0926-6690/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.indcrop.2012.07.070
 Z. Ali et al. / Industrial Crops and Products 43 (2013) 768–773 769

Parrotta, 2001; Prajapati et al., 2003; Khare, 2004; Kapoor, 2005). Table 1
Nucleotide sequence and annealing temperatures of the primers.
It is a climber, up to 2–3 m in height. Stem scandent; leaves pin-
nately 5-foliolate, 6–13 cm long; leaflets ovate or oblong, 2–5 cm Primer code Nucleotide sequence Annealing Molecular GC-content
long; flowers papilionaceous, white or bright blue with yellow or (5 –3 ) temperature weight (%)
(◦ C) (g/mol)
orange centre; pods flat, beaked; seeds yellowish brown, subglo-
bose. Two varieties blue and white flowered, are mentioned in OPN-01 CCT CAGCTTGG 36 3308 63.6
Ayurvedic texts. OPN-02 AACCAGGGGCA 36 3375 63.6
OPN-03 AACCAGGGGCA 38 3253 72.7
A wide range of secondary metabolites including triterpenoids,
OPN-04 GGACCGACCCA 38 3311 72.7
flavanol glycosides, anthocyanins, Kaempferol, quercetin, and lac- OPN-05 TACTGAACGCC 34 3301 54.5
tones have been isolated from C. ternatea (Mukherjee et al., 2008; OPN-06 TGAGACGCACA 34 3350 54.5
Sethiya et al., 2009). One of the metabolites, kaempferol exhibits OPN-07 TCAGCCCAGAG 36 3326 63.6
OPN-08 CACCTCAGCTC 36 3237 63.6
a number of activities that likely contribute to its putative chemo-
OPN-09 TTGCCGGCTTG 36 3339 63.6
preventive properties including inhibition of cell growth (Jin et al., OPN-10 GACAACTGGGG 36 3406 63.6
1995), induction of apoptosis (Jin et al., 1995; Braig et al., 2005; G-01 ATGCTCTGCCC 36 3268 63.6
Chen and Kong, 2005; Niering et al., 2005), and inhibition of pro- G-02 CGGTGACGCAG 38 3382 72.7
teosome activity (Chen and Kong, 2005). G-03 ATGGGGGACTC 36 3397 63.6
G-04 CGGTCACCTCA 36 3277 63.6
There seems to be little or no work on molecular character-
G-05 AGAGGGCCTTG 36 3397 63.6
ization and genetic diversity at inter-zonal level of C. ternatea. G-06 TGATGACCGCC 36 3317 63.6
The present study is an effort to analyse genetic diversity and G-07 CAAAGCTGCGG 36 3366 63.6
kaempferol content in C. ternatea collected from different locations G-08 AGTCGCCGTCA 36 3317 63.6
G-09 GCCGCGTCTTG 38 3324 72.7
in India.
G-10 CGGACCTGCTG 38 3333 72.7
G-11 TAGGTGACCGT 34 3372 54.5
2. Materials and methods G-12 ACCTCTCGACA 34 3261 54.5
G-13 ATGTTCGCAGA 32 3356 45.5
2.1. Plant material G-14 GCCCCGATGGT 38 3333 72.7
G-15 ATGAGCGGACA 34 3390 54.5

The samples of C. ternatea were collected from Hamdard Cam-

pus (New Delhi), Kurukshetra (Haryana), Lucknow (Uttar Pradesh),
were centrifuged at 8000 rpm for 15 min at 15 ◦ C. The upper phase
Jaipur and Udaipur (Rajasthan), Bhopal (Madhya Pradesh) and
was transferred again into autoclaved centrifuge tube and 0.5 vol.
Coimbatore (Tamil Nadu) during the months of September and
of 3 M potassium acetate (pH 5.2) was added. For DNA precipitation
October. Voucher specimens of these samples were prepared and
equal volume of chilled isopropanol (chilled absolute ethanol was
kept in the Herbarium, Department of Botany, Hamdard University,
also used) was used and the tubes were kept at −20 ◦ C for 2 h. It
New Delhi. The identified specimens were compared with authen-
recentrifuged at 8000 rpm for 15 min at 4 ◦ C. The supernatant was
ticated voucher specimens preserved in the herbarium of National
discarded and the pellet was washed with 70% EtOH, air dried and
Institute of Science Communication and Information Resources
dissolved in 250 ␮l of sterile water. The DNA thus obtained was
(NISCAIR) New Delhi. The leaves were used for DNA isolation and
purified by DNA purification kit (HiPurA, India).
the rest of the plant material was lyophilized and stored at −80 ◦ C
for further use. A part of the collected sample was dried at 40 ◦ C for
chemical analysis. 2.3. Polymerase chain reaction (PCR) amplification

2.2. DNA isolation The PCR was carried out in 15 ␮l reaction volume containing 2 ␮l
of DNA (25 ng/␮l), 0.5 ␮l of Taq DNA polymerase (1 U/␮l), 1.0 ␮l of
The modified CTAB protocol of Doyle and Doyle (1990) and MgCl2 (25 mM), 0.5 ␮l dNTP’s (10 ␮M), 0.5 ␮l primer (10 ␮M), 1.5 ␮l
purification kit (HiPurA, India) were applied to extract DNA from of Taq buffer (10×) and the final volume was made-up with sterile
the fresh leaves of C. ternatea. Leaves were washed for 5 min with MilliQ water. The amplifications were carried out using Thermal
sterile de-ionized water. The mid rib was removed and the leaf cycler (Eppendorf). After initial step of denaturation at 94 ◦ C for
lamina was ready for use. 4 min, 35 cycles of denaturation at 94 ◦ C for 1 min, annealing at
suitable temperature (primer dependant) for 1 min and extension
2.2.1. Reagents and solutions at 72 ◦ C for 2 min were provided, which was followed by a final
Extraction buffer 3% (w/v) CTAB (2 M sodium chloride; 100 mM extension of 72 ◦ C for 10 min. The amplified DNA was loaded on
Tris–HCl (pH 8.0); 20 mM EDTA); 0.2% ␤-mercaptoethanol; chlo- 1.2% agarose gel in 0.5× TBE containing 5.0 ␮l of ethidium bromide
roform:isoamyl alcohol (24:1); absolute alcohol (ethanol); 3 M (10 mg/ml) and photographed using gel documentation system
potassium acetate (pH 5.2) and isopropanol. (UVP, Germany). The sequence of primers (OPN-01 to OPN-10 and
G-01 to G-15) is listed in Table 1. The data was analysed through
2.2.2. Protocol band scoring of well-marked amplified fragments.
1 g fresh leaves were pulverized to fine powder by adding liquid
nitrogen in a chilled mortar and pestle followed by the addition of 2.4. Data analysis
100 mg of polyvinylpyrrolidone (PVP) and 10 ml pre-heated CTAB
buffer (containing 0.2% ␤-mercaptoethanol). The slurry was trans- PCR products were scored for the presence (1) or absence (0)
ferred into autoclaved 50 ml centrifuge tube and incubated at 60 ◦ C irrespective of band intensity since each product of identical molec-
for 1 h. 10 ml of chloroform, isoamyl alcohol (CHCl3 :IAA, 24:1) was ular weight was supposed to represent a single locus. Genetic
added to centrifuge tubes and mixed gently for 15 min. The con- analysis was carried out using Nei genetic similarity index (Nei
tent was centrifuged at 8000 rpm for 15 min at 15 ◦ C. The upper and Li, 1979) by using the formula Fxy = 2nxy /(nx + ny ), where nxy
phase was transferred into new autoclaved centrifuge tubes. 10 ␮l is the number of common RAPD fragments shared by two samples
of RNase was added and the tubes were incubated at 37 ◦ C for and nx and ny are the total number of bands scored in each sam-
30 min. 10 ml of CHCl3 :IAA (24:1) was added carefully and the tubes ple. The genetic distance was calculated by using Hillis and Moritz
770 Z. Ali et al. / Industrial Crops and Products 43 (2013) 768–773

equation (1990), D = 1 − F, where “F” is species similarity. The den- Table 2

Percentage of polymorphic bands in C. ternatea (blue and white flowered).
drogram was constructed using the NTSYS-pc software (Numerical
Taxonomy and Multivariate system) (Rohlf, 1993). S. no. Primer Total no. Polymorphic Monomorphism Polymorphism
code of bands bands (%) (%)
2.5. Sample preparation for HPLC 1 OPN-01 14 08 42.86 57.14
2 OPN-02 10 0 100.00 0.00
The collected samples were dried at 40 ◦ C for 24 h and ground 3 OPN-03 08 04 50.00 50.00
4 OPN-04 08 06 25.00 75.00
into fine powder; 400 mg of each sample was first defatted by pre- 5 OPN-06 14 07 50.00 50.00
extraction with 20 ml chloroform and refluxed for 1 h with 30 ml 6 OPN-09 10 06 40.00 60.00
95% aqueous methyl alcohol (MeOH) and 9 ml of 25% hydrochloric 7 OPN-10 07 01 85.72 14.28
acid. After filtration with Whatman paper, the samples were
extracted twice with 20 ml of MeOH for 10 min. The combined
hydrolysates were diluted with MeOH to 100 ml and filtered polymorphic and therefore, were considered to be informative loci.
through syringe filter (0.45 ␮m). The filtrate was directly injected Six monomorphic bands were present in all the genotypes with
(20 ␮l) into the HPLC system. Each extract was injected in triplicate. almost uniform intensity. Of the eight polymorphic bands, two
unique bands (arrow marked) were detected in the genotypes of
2.6. HPLC analysis and conditions Haryana (blue flowered) and U. P. (blue flowered) having band
size of 1.5 kb. The presence of these two unique bands would be
HPLC was performed on Waters HPLC system (Binary Pump 600 helpful in the identification of these two genotypes. Primer OPN-
controller), PDA detector (996), Auto sampler (2707) and C18 col- 06 produced a total of 14 amplification products, 6 of which were
umn (125 × 4). Empower software was used for the data collection. polymorphic; thus giving a polymorphism of 50% (Fig. 2). A unique
The mobile phase consisted of solvent A (2% acetic acid) and sol- band of nearly 0.7 kb (arrow marked) was present in the blue flow-
vent B (methanol, acetic acid and water within 18:18:1). The flow ered genotypes of Tamil Nadu. It is also evident from Fig. 2 that the
rate was 1 ml/min. The detection wavelength was set at 370 nm. genotypes of Delhi, U. P. and Haryana show more genetic fidelity
The retention time (RT) of standard Kaempferol was at 2.13. among themselves than the populations of M. P., Tamil Nadu and
Rajasthan.
3. Results Based on the degree of divergence, 14 genotypes of C. ternatea
were grouped into two clusters, cluster I and cluster II, such that
Twenty five 11-mer primers (10 of OPN-series and 15 of G the genotypes within the cluster have lesser divergence values than
series) were screened to analyse genetic diversity and relationship those between clusters. The first cluster contains genotypes col-
among different accessions of C. ternatea collected from different lected from Delhi, Haryana and U. P. The second cluster comprised
geographical localities of India. The primers of OPN series pro- genotypes of Rajasthan, M. P. and Tamil Nadu (Fig. 3).
duced relatively more amplification ranging from 0.5 to 2.0 kb. Kaempferol was selected as a chemical marker and quan-
Reproducibility of amplification profiles was tested for each primer tified in different accessions through HPLC. The results are
and only seven primers of OPN-series could be considered for the presented in Table 4. Maximum concentration of kaempferol
analysis. PCR fragments having same mobility were considered as (20.01 ± 0.006 mg/g dw) was found in C. ternatea (blue flowered)
identical fragments, receiving equal values regardless of their stain- collected from Kurukshetra (Haryana) and minimum concentration
ing intensity. (9.31 ± 0.0277 mg/gm dw) was detected in white variety collected
Seven primers generated a total of 72 clear and reproducible from Bhopal (M. P.) (Fig. 4).
bands, 32 of which were polymorphic. The percentage of polymor-
phism was 45.07. Primer OPN-4 generated 75% polymorphic bands 4. Discussion
followed by OPN-09 (60%) and OPN-01 (57%). Primers OPN-03 and
OPN-06 generated polymorphism of 50% each. The lower polymor- DNA markers are used to assess genetic diversity at various lev-
phism was observed with primer OPN-10 (14.28%). The number of els of taxon-species, inter and intra population and progeny. At the
RAPD bands was in the range of 7–14 with an average of 10 bands species level, the knowledge of genetic diversity helps understand
per primer. The polymorphism within the different populations the features which make it unique and distinct from other species.
ranged from 0 to 0.75 (Table 2). The genetic distance in different The population level studies explore the demographic characteris-
population was 0.02–0.28 (Table 3). tics of artificial and cultivated population, accessions, collections,
The amplification profile obtained with the primer OPN-01 is germplasm and breeding lines. Diversity studies at this level help
represented in Fig. 1. Eight of the fourteen amplified products were in ex situ conservation programmes.

Table 3
Genetic distance for seven primers in C. ternatea (blue and white flowered).

Genotype 01 02 03 04 05 06 07 08 09 10 11

Del. white 01 – 0.00 0.00 0.03 0.03 0.26 0.20 0.0.19 0.23 0.23 0.23
Del. blue 02 0.00 – 0.00 0.03 0.02 0.28 0.20 0.20 0.22 0.22 0.22
U. P. white 03 0.00 0.00 – 0.03 0.03 0.22 0.21 0.20 0.22 0.22 0.22
U. P. blue 04 0.03 0.03 0.03 – 0.00 0.28 0.22 0.20 0.23 0.23 0.23
Har. blue 05 0.03 0.02 0.03 0.00 – 0.28 0.22 0.20 0.23 0.22 0.22
M. P. white 06 0.26 0.28 0.28 0.28 0.28 – 0.06 0.09 0.06 0.06 0.06
M. P. blue 07 0.20 0.20 0.21 0.22 0.22 0.06 – 0.02 0.06 0.02 0.02
T. N. white 08 0.19 0.20 0.20 0.20 0.20 0.09 0.02 – 0.03 0.03 0.03
T. N. blue 09 0.23 0.22 0.22 0.23 0.23 0.06 0.06 0.03 – 0.03 0.03
Raj 1. blue 10 0.23 0.22 0.22 0.23 0.22 0.06 0.02 0.03 0.03 – 0.00
Raj. 2. blue 11 0.23 0.22 0.22 0.23 0.22 0.06 0.02 0.03 0.03 0.00 –

Del = Delhi (Hamdard Campus); U. P. = Uttar Pradesh (Lucknow); Har. = Haryana (Arjun Herbal Park – Kurukshetra); M. P. = Madhya Pradesh (Bhopal); T. N. = Tamil Nadu
(Coimbatore); Raj. 1 = Rajasthan (Udaipur); Raj. 2. = Rajasthan (Jaipur).
 Z. Ali et al. / Industrial Crops and Products 43 (2013) 768–773 771

Fig. 1. RAPD amplifications of eleven accessions of Clitoria ternatea with primers OPN-01 and OPN-06. M = marker digested with Hind III and Eco R1; lane 1 = Clitoria white
Delhi; lane 2 = Clitoria blue Delhi; lane 3 = Clitoria white U. P.; lane 4 = Clitoria blue U. P.; lane 5 = Clitoria blue Haryana; lanes 6 and 7 Clitoria white M. P.; lanes 8 and 9 = Clitoria
blue M. P.; lanes 10 and 11 = Clitoria white Tamil Nadu; lanes 12 and 13 Clitoria blue Tamil Nadu; lanes 14 and 17 = Clitoria blue Rajasthan.

Fig. 2. RAPD amplifications of eleven accessions of Clitoria ternatea with primers OPN-01 and OPN-06. M = marker digested with Hind III and Eco R1; lane 1 = Clitoria white
Delhi; lane 2 = Clitoria blue Delhi; lane 3 = Clitoria white U. P.; lane 4 = Clitoria blue U. P.; lane 5 = Clitoria blue Haryana; lanes 6 and 7 Clitoria white M. P.; lanes 8 and 9 = Clitoria
blue M. P.; lanes 10 and 11 = Clitoria white Tamil Nadu; lanes 12 and 13 Clitoria blue Tamil Nadu; lanes 14 and 17 = Clitoria blue Rajasthan.

Fig. 3. Dendrogram based on RAPD analysis for the estimation of genetic diversity in different populations of Clitoria ternatea collected from different geographical locations
Clit. W. = Clitoria white; Clit. B. = Clitoria blue; Del. = Delhi; U.P. = Uttar Pradesh; Har. = Haryana; M.P. = Madhya Pradesh; T.N. = Tamil Nadu; Raj. = Rajasthan.
772 Z. Ali et al. / Industrial Crops and Products 43 (2013) 768–773

Fig. 4. Chromatograms showing kaempferol content in Clitoria ternatea collected from M. P. = Madhya Pradesh (A and B) and Haryana (C).

The knowledge of genetic variation has utmost importance diversity in this plant. A total of 71 bands were scored with seven
in devising programmes for better management of optimal uti- 11-mer random primers; 45.07% of the scored bands were poly-
lization and conservation of medicinal plant diversity. Due to morphic which was near to the percentage for Iris setosa (46%)
change in environment, the variation in alleles is very necessary (Artyukova et al., 2001) but is higher than Paeonia suffruticosa
for the possible adaptation of particular species to the chang- (22.5%) (Pei et al., 1995), P. rockii (27.6%) (Pei et al., 1995), Cathaya
ing environment. Therefore, species having more genetic diversity argyrophylla (32.0%) (Wang et al., 1996), and Codonopsis lanceolata
possess more genetic variations from which to choose the most (24.9%) (Guo et al., 2006). In our RAPD analysis, not much genetic
fitting allele. RAPD technology has been demonstrated to be use- variation was observed within the populations of C. ternatea. The
ful not only for the study of population genetic variation but genetic variation of 45.07% can be explained because the plant is
also for taxonomic identities, systematic relationships, parent- cultivated throughout India in herbal gardens and nurseries that
age identifications, identification of interspecific hybridization and ensures less impact of changing environmental conditions. In our
introgressive hybridization (Imtiaz et al., 2011). study, the splitting of the dendrogram into two major clusters is in
To the best of our knowledge, this is first successful use of RAPD accordance with the locality of the plants. The first cluster contains
markers to characterize genotypic and genetic variation at inter samples of North India and the second represents the genotypes
zonal level in C. ternatea. For overall genetic diversity, initially 25 collected from Central, Western and Southern regions. The intra-
primers were used out of which only seven gave highly repro- varietal genotypes of C. ternatea show more genetic fidelity than
ducible and clear bands that were sufficient to analyse genetic the inter-varietal genotypes, except white flowered from Delhi,
each particular clad either contains blue flower or white flowered
Table 4
genotype. All the bands amplified by the OPN-02 shows monomor-
Kaempferol concentration in different samples of Clitoria ternatea. phic pattern, hence it can be used as a molecular marker for the
authentication of this plant.
S. no. Plant samples Amount of
The identification of C. ternatea is also crucial; because the drug
kaempferol
(mg/g dw) ± SE from this plant is the source of Shankhpushpi (Moose, 1976). Major-
ity of drugs being sold in the name of Shankhpushpi are either
1 Blue flowered, Jamia Hamdard Herbal Garden, 10.115 ± 0.003
New Delhi adulterated or spurious. Our molecular characterization would help
2 White flowered, Jamia Hamdard Herbal 10.921 ± 0.005 identify of this herb from adulterants and spurious plant sources
Garden, New Delhi sold in the market.
3 Blue flowered, Lucknow, U. P. 11.653 ± 0.032 Irrespective of the plant part used, DNA analysis will remain the
4 White flowered, Lucknow, U. P. 10.149 ± 0.290
5 Blue flowered, Bhopal, M. P. 16.67 ± 0.017
most effective tool for identification because phytochemical con-
6 White flowered, Bhopal, M. P. 9.311 ± 0.0277 tent will vary with the plant part used, physiological status and
7 Blue flowered, Coimbatore, Tamil Nadu 9.876 ± 0.362 environmental conditions (Srivastava and Mishra, 2009). It would
8 White flowered, Coimbatore, Tamil Nadu 11.448 ± 0.052 therefore, be justified to use both DNA and pharmacognosic phy-
9 Blue flowered, Kurukshetra, Haryana 20.018 ± 0.006
tochemical techniques hand in hand rather than in isolation. The
 Z. Ali et al. / Industrial Crops and Products 43 (2013) 768–773 773

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