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BIO D I V E R S IT A S
ISSN: 1412-033X
Volume 20, Number 3, March 2019 E-ISSN: 2085-4722
Pages: 656-663 DOI: 10.13057/biodiv/d200306
Phenolic and flavonoid content in ethanol extract and agro-
morphological diversity of Curcuma aeruginosa accessions growing in
West Java, Indonesia
NURUL KHUMAIDA1, MUHAMAD SYUKUR1, MARIA BINTANG2, WARAS NURCHOLIS2,3,♥
1Departement of Agronomy and Horticulture, Faculty of Agriculture, Institut Pertanian Bogor. Jl. Raya Dramaga, Kampus IPB Dramaga, Bogor 16680,
West Java, Indonesia
2Department of Biochemistry, Faculty of Mathematics and Natural Sciences, Institut Pertanian Bogor. Jl. Raya Dramaga, Kampus IPB Dramaga, Bogor
16680, West Java, Indonesia. Tel./fax.: +62-251-8423267, email: wnurcholis@apps.ipb.ac.id
3Tropical Biopharmaca Research Center, Institut Pertanian Bogor. Jl. Taman Kencana No. 3, Kampus IPB Taman Kencana, Bogor 16128, West Java,
Indonesia
Manuscript received: 13 November 2018. Revision accepted: 12 February 2019.
Abstract. Khumaida N, Syukur M, Bintang M, Nurcholis W. 2019. Phenolic and flavonoid content in ethanol extract and agro-
morphological diversity of Curcuma aeruginosa accessions growing in West Java, Indonesia. Biodiversitas 20: 656-663. Curcuma
aeruginosa is a rhizomatous medicinal plant with beneficial pharmacological activities. The aim of this work was to analyze the agro-
morphological, extract yield, and phenolic content of ten C. aeruginosa accessions which were collected from different locations in
Indonesia. Cultivation was carried out in the open field in West Java of Indonesia using a completely randomized design. Qualitative
and quantitative parameters were used to investigate agro-morphological traits. Total phenolic and total flavonoids contents were
determined in ethanol extracts of samples. The plants were phenotypically diverse, in which there were significant variations among the
ten C. aeruginosa accessions in number of leaves, plant height, number of shoots, fresh weight of rhizome, and dry weight of rhizome
characters. Variability in the total phenolic and total flavonoid contents ranged from 29.08-46.92 mg GAE/g, and 21.31-33.81 mg QE/g,
respectively. Six accessions had high phenolic content and extract yield. Therefore, these accessions could be utilized for commercial
scale and also showed a high potency for medicinal plant breeding programs.
Keywords: Accessions, agro-morphology, Curcuma aeruginosa, flavonoid, phenolic
INTRODUCTION
In industry, the quality of the traditional medicine
product is directly related to the quality of raw materials
(Salgueiro et al. 2010). However, the quality of the raw
material is dependent on the breeding plant program and
also the practices in the agricultural system. Curcuma
aeruginosa Roxb., namely temu ireng or temu hitam in
Indonesia, is one of the most popular rhizomatous medicinal
plant belonging to the family Zingiberaceae and genus
Curcuma (Sasikumar 2005). The previous works had
shown that the rhizome of C. aeruginosa has beneficial
biological activities such as antioxidant (Nurcholis et al.
2015a), antimicrobial (Kamazeri et al. 2012; Akarchariya
et al. 2017), hair-growth and skin lightening (Srivilai et al.
2017), anti-androgenic (Suphrom et al. 2012), uterine
relaxant (Thaina et al. 2009), and anti-dengue
(Moektiwardoyo et al. 2014). In this context, C. aeruginosa
is an important raw material for an industry of traditional
medicine. Presently, there are no identified Indonesian
varieties of C. aeruginosa (MoA 2018). Thus, the
development of C. aeruginosa varieties is needed to
produce rhizome with the highest quality for industrial
purposes.
Phenolics and flavonoids have been reported as the
phytochemicals found in the rhizome of C. aeruginosa
(Nurcholis et al. 2016a). A study has demonstrated that
phenolics and flavonoids possess biological activities such
as antioxidant (Al-Farsi et al. 2018), antimicrobial (Pandey
et al. 2018), anticancer (Alaklabi et al. 2018), anti-
inflammatory and cytotoxic activity (Udavant et al. 2012).
These properties make them particularly helpful for
traditional medicine applications of C. aeruginosa rhizome.
Thus, the quality of C. aeruginosa rhizome can be
determined based on phenolics and flavonoids contents.
Research on the rhizomes of C. aeruginosa harvested
from the different sites in Indonesia showed a high
fluctuation in curcuminoid and cytotoxicity (Nurcholis et
al. 2016b), phytochemical and rhizome color (Nurcholis et
al. 2017), total phenolics and flavonoids contents
(Nurcholis et al. 2016a). These studies suggested that the
variation in bioactive content and biological activity can be
influenced by several factors such as genetic and
geographical variation. Also, some reports show that agro-
morphological traits are controlled by a genetic factor
(Belaj et al. 2011; Bakić et al. 2017). In Indonesia, there is
no comprehensive study of C. aeruginosa accessions for
agro-morphological traits, phenolics and flavonoids
contents, and extractable yield. The results of this study can
form a guide to facilitate a selection scheme for breeding
programs for identified new cultivars of C. aeruginosa.
Therefore, this study evaluated the morphological
attributes, extract yield, and phenolic contents of ten C.
aeruginosa accessions with grown under the same
KHUMAIDA et al. – Phenolic and agro-morphology in Curcuma aeruginosa
657
environmental conditions, so that the results reflect
genetically differences between accessions studied.
MATERIALS AND METHODS
Plant material
Ten rhizomes of C. aeruginosa accessions were
collected from different regions in Indonesia in February
2015 (Table 1). Identification of plant specimens was
conducted by taxonomist expert of the Biopharmaca
Conservation and Cultivation Station, Tropical Biopharmaca
Research Center, Bogor Agricultural University (IPB). A
field experiment was carried out at the Biopharmaca
Conservation and Cultivation Station, West Java of
Indonesia (6°32’25.47” N and 106°42’53.22” E, at 142.60
m altitude), in December 2015. The experiment was
arranged in a completely randomized design with three-
replications. The plants were grown in the same soil
conditions (latosol soil with pH of 4.5-5, organic C of
1.52%, and N of 0.15%) with plants spacing of 50 cm x 50
cm. Two weeks before planting, the soil was treated with 1
kg cow manure per planting hole. Nine months after
planting, in August 2016, the rhizomes were harvested.
Agro-morphological evaluations
Several qualitative and quantitative characters were
measured to characterize C. aeruginosa accessions
morphologically (Table 2). The characters were evaluated
based on the set standards for traits by the Protection of
Plant Varieties and Farmers' Rights Authority turmeric
descriptor ( PPV-FRA 2011) with modification.
Table 1. Curcuma aeruginosa accessions used in this work with code, collection sites, and geographical coordinates
Accession code/ Voucher specimens
Province
Location
Latitude (N)
Longitude (E)
Altitude (m)
KL/BMK0049032015
Central Java
Klewer
7˚35'05.66"
110˚49'45.38"
96
PK/BMK0053032015
Yogyakarta
Pakem
7˚39'55.46"
110˚25'11.30"
424
BH/BMK0054032015
Yogyakarta
Beringharjo
7˚47'56.40"
110˚22'01.56"
115
GK/BMK0055032015
Yogyakarta
Gunung Kidul
7˚58'04.87"
110˚36'09.67"
180
KP/BMK0056032015
Yogyakarta
Kulonprogo
7˚56'25.03"
110˚14'20.30"
20
PW/BMK0058032015
Central Java
Purworejo
7˚44'25.35"
110˚01'59.00"
56
MD/BMK0063032015
East Java
Madura
7˚02'48.90"
112˚43'47.32"
4
LC/BMK0064032015
West Java
Losari Cirebon
6˚48'17.09"
108˚48'06.04"
1
CB/BMK0065032015
West Java
Ciampea Bogor
6˚32'35.89"
106˚41'22.41"
148
MB/BMK0066032015
Jambi
Muara Bungo
1˚37'00.61"
102˚22'16.28"
65
Table 2. List of the qualitative and quantitative (agro-morphological) characters used for the variability of the 10 C. aeruginosa
accessions
Variables
Details
Stage of observation
Qualitative variables
Pseudostem habit (PSH)
1: compact, 9: open
150 DAP
The color on pseudostem habit (CPSH)
1: purple, 9: green
150 DAP
Venation pattern of the leaf (VPL)
3: close, 5: distant
150 DAP
Leaf disposition (LD)
3: erect (< 45°), 5: semi-erect (45°-85°), 7: horizontal (> 85°)
150 DAP
The margin of the leaf (ML)
3: even, 5: wavy
150 DAP
Purple color on midrib (PCM)
5: 50% of midrib, 7: 75% of midrib
150 DAP
Blue color on rhizome (BCR)
3: few, 5: medium, 7: many
At harvest (9 MAP)
The habit of the rhizome (HR)
3: compact, 5: intermediate, 7: loose
At harvest (9 MAP)
The shape of the rhizome (SR)
3: straight, 5: curved
At harvest (9 MAP)
Status of the tertiary rhizome (STR)
1: absent, 9: present
At harvest (9 MAP)
Length of primary rhizome (LPR)
3: short (< 5 cm), 5: medium (5 - 10 cm), 7: long (> 10 cm)
At harvest (9 MAP)
Number of mother rhizome (NMR)
1: one, 3: two-three, 5: more than three
At harvest (9 MAP)
Internode pattern of rhizome (IPR)
3: close (< 1 cm), 5: distant (> 1 cm)
At harvest (9 MAP)
Quantitative variables
Plant height (PH)
Plant height (cm) measured from the soil level to the tip of the leaf
of the main shoot
150 DAP
Pseudostem diameter (PD)
Pseudostem diameter measured in cm
150 DAP
Number of leaves (NL)
Number of leaves per plant (no.)
150 DAP
Leaf length (LL)
Leaf length in cm
150 DAP
Leaf width (LW)
Leaf width in cm
150 DAP
Number of shoots (NS)
Number of shoot per plant (no.)
150 DAP
Fresh rhizome weight (FRW)
Rhizome fresh weight per plant (kg)
At harvest (9 MAP)
Dry rhizome weight (DRW)
Rhizome dry weight per plant (kg)
At harvest (9 MAP)
Note: DAP: Days After Planting, MAP: Months After Planting
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20 (3): 656-663, March 2019
658
Extraction
After harvesting, the fresh rhizome of each accession
was cut, dried and crushed to a powder. The powder was
extracted, and performed by the maceration method of
Nurcholis et al. (2015b). Briefly, 25 g of the powdered
material of each accession were macerated with 70% (v/v)
ethanol (250 ml) at room temperature. After 24 h, the
accession solution was filtered using a Whatman filter
paper (No. 4) and then subjected to evaporation (BUCHI,
R-250, Switzerland) at 50°C. The extract yield of samples
was calculated based on extract content (%, w/w).
Phenolic and flavonoid content
The total phenolics content of the ethanol extract was
determined spectrophotometrically with the Folin-
Ciocalteu method (Wan-Ibrahim et al. 2010). Extract
sample (10 µL) was added to a 96-well microplate
containing 160 µL distilled water. Then 10 µL of Folin-
Ciocalteu reagent (10%) and 20 µL Na2CO3 (10%) solution
were added and the mixture incubated for 30 min at room
temperature. The absorbance of all accessions was
measured at 750 nm using a microplate reader (Epoch
BioTek, USA). All samples were analyzed in triplicate.
Results of the total phenolic contents in extract samples
were determined from a standard curve and expressed as
mg of gallic acid equivalent per g of extract (mg GAE/g).
The total flavonoid content in ethanol extract of
samples was determined spectrophotometrically using
aluminum chloride reagent (Chang et al. 2002) with minor
modification. The calibration curve was established using
standard quercetin. Each extract or quercetin (10 µL) in a
96-well microplate was added with methanol (60 µL), 10%
aluminum chloride (10 µL), 1 M potassium acetate (10 µL)
and distilled water (120 µL), then the solution was
incubated at room temperature for 30 min. Finally, the
absorbance was determined for all samples at a wavelength
of 415 nm using a microplate reader (Epoch BioTek,
USA). Total flavonoid content was calculated from a
standard quercetin curve and expressed as quercetin
equivalents (mg QE/g).
Data analysis
The qualitative characters data were subjected to
ANOVA followed by the Kruskal-Wallis H test. The data
of quantitative characteristic of agro-morphologically traits,
rhizome extract, total phenolics content, and total
flavonoids content were subjected to ANOVA followed by
Duncan's multiple range test (DMRT). Statistical analysis
was performed using the Statistical Tool for Agricultural
Research (STAR) 2.0.1. All of the data were also analyzed
using multivariate analysis, i.e., Bonferroni correlation,
principal component analysis, and cluster analysis,
performed by R software. Prior to analyses, data were
normalized using log transformation and auto-scaling.
RESULTS AND DISCUSSION
Agro-morphological characters
There was no significant difference (p < 0.05) among
ten C. aeruginosa accessions based on the Kruskal Wallis
rank sum test of the qualitative traits ( Table 3). The
qualitative traits observed in the C. aeruginosa accessions
were presented in Figure 1. The leaf disposition, blue color
on the rhizome, number of mother rhizome, and internode
pattern of rhizome characters showed the same pattern in
all accessions. The blue color of rhizomes in this study was
fewer than our previous study using samples from different
geographical origin (Nurcholis et al. 2017). The accession
MB has a difference for pseudostem habit, the color of the
pseudostem habit, the margin of the leaf, venation pattern
of the leaf, and purple color on the midrib when compared
with other accessions. Based on the habit of the rhizome,
the accessions BH, GK, and MD were found to be
intermediate, whereas most accessions were loose.
Regarding the shape of the rhizome, most accessions were
straight, but the accession LC was curved. The majority of
the accessions had a medium length for the primary
rhizome, while the accessions KL, PW, and MD were long.
Most accessions had a tertiary rhizome, but accessions MD
and CB had no tertiary rhizome. The characteristics of
qualitative agro-morphology were similar to the qualitative
traits that were recorded by Setiadi et al. (2017). Jose and
Thomas (2014) reported the presence of different
morphological traits of C. aeruginosa including lateral
spike position, purple color of the calyx, light pink color of
the corolla, greenish blue color of the rhizome, dark purple
color of leaf sheath, and purple-brown color of the midrib.
Table 3. Qualitative agro-morphological characters of the ten C. aeruginosa accessions
Accession code a
Qualitative agro-morphological characters b
PSH
CPSH
LD
VPL
ML
PCM
NMR
BCR
HR
SR
LPR
IPR
STR
KL
9
9
3
5
5
5
5
3
7
3
7
5
9
PK
9
9
3
5
5
5
5
3
7
3
5
5
9
BH
9
9
3
5
5
5
5
3
5
3
5
5
9
GK
9
9
3
5
5
5
5
3
5
3
5
5
9
KP
9
9
3
5
5
5
5
3
7
3
5
5
9
PW
9
9
3
5
5
5
5
3
7
3
7
5
9
MD
9
9
3
5
5
5
5
3
5
3
7
5
1
LC
9
9
3
5
5
5
5
3
7
5
5
5
9
CB
9
9
3
5
5
5
5
3
7
3
5
5
1
MB
1
1
3
3
3
7
5
3
7
3
5
5
9
H
9.0ns
9.0ns
9.0ns
9.0ns
9.0ns
9.0ns
9.0ns
9.0ns
9.0ns
9.0ns
9.0ns
9.0ns
9.0ns
Note: H=value of Kruskal Wallis test; ns=nonsignificant at the 0.05 probability level; a,b For an explanation of accessions code and
character symbols, see Table 1 and Table 2 respectively
KHUMAIDA et al. – Phenolic and agro-morphology in Curcuma aeruginosa
659
Pseudostem habit/ color pseudostem habit
Venation pattern of leaf
Purple color on midrib
Compact/ purple
Open/ green
Close
Distant
75% on midrib
50% on midrib
The margin of the leaf
Leaf disposition
The shape of the rhizome
Even
Wavy
Erect (< 45°)
Straight
Curved
The habit of the rhizome
Number of mother rhizome
Length of primary rhizome
Loose
Intermediate
> 3 mother rhizomes
Long: > 10 cm
Medium: 5-10 cm
Blue color on rhizome
Status of the tertiary rhizome
Internode pattern of rhizome
Few of blue color on rhizome
Absent
Distant: > 1 cm
Figure 1. Variation in the qualitative morphology of C. aeruginosa
The quantitative traits of the C. aeruginosa accessions
significantly varied (p < 0.05) except for pseudostem
diameter, leaf length, and leaf width (Table 4). The
accession PW displayed highest plant height (179.14 cm),
pseudostem diameter (3.51 cm), number of shoots (9.75),
and fresh rhizome weight (3.90 kg/plants). The fresh and
dry rhizome weight in the current study were contrastingly
different from those reported by Setiadi et al. (2017) with
values of 0.31 and 0.18 kg/plants, respectively. Variation in
agro-morphological character is affected by environmental
(Mohammadi and Asadi-Gharneh 2018), developmental
(Anandan et al. 2018), and genetical (Neugart et al. 2018)
factors.
Extract yield
Ethanol extract yield was varied significantly (p < 0.05)
in different accessions (Table 5). The accession PK
exhibited the highest (7.36%, w/w) ethanol extract yield, in
which the accession LC was the lowest one (3.68%, w/w).
The extract yield in this study was considerably lower than
our previous research (7.92 to 19.71%, w/w) using the
sample from a different geographic origin (Nurcholis et al.
2017). The extract yield in this study was also lower than
those reported for C. aeruginosa by Moektiwardoyo et al.
(2014) (24.13%, w/w) using the sample originated from
Bandung. Extract yield is influenced by maceration time
(Petropulos et al. 2014). Moektiwardoyo et al. (2014) used
three days for maceration time, while in this study was two
days.
Furthermore, the accessions in this research were
cultivated in the same condition and extracted by
technically the same method. Thus, the variation of extract
yield is possibly influenced by the genetic factor in this
case. The extract yield affected by genetic factor was also
reported in other plant species such as Camelina sativa
(Kurasiak-Popowska et al. 2018), Vigna radiata (Wang et
al. 2018), Curcuma zanthorrhiza (Nurcholis et al. 2018),
and Curcuma zedoaria (Syahid and Heryanto 2017).
B I O D I V E R S I T A S
20 (3): 656-663, March 2019
660
Table 4. Quantitative agro-morphological characters of the ten C. aeruginosa accessions
Accession code a
Quantitative agro-morphological characters b
PH (cm)
PD (cm)
NL (no.)
LL (cm)
LW (cm)
NS (no.)
FRW (kg/plants)
DRW (kg/plants)
KL
164.99ab
3.42a
9.33ab
77.14a
17.04a
4.83cd
2.50ab
0.49ab
PK
164.39ab
3.12a
9.09ab
76.89a
15.94a
3.91d
1.80b
0.37b
BH
160.33b
3.17a
9.31ab
73.26a
15.58a
4.54d
2.10b
0.40b
GK
165.30ab
3.42a
9.67a
73.89a
16.51a
4.44d
2.13ab
0.41b
KP
172.49ab
3.30a
8.23bc
78.74a
21.37a
6.31bcd
2.90ab
0.55ab
PW
179.14a
3.51a
9.25ab
77.50a
16.89a
9.75a
3.90a
0.67ab
MD
168.99ab
3.19a
8.50abc
75.09a
15.98a
6.33bcd
3.22ab
0.60ab
LC
176.53ab
3.48a
8.75abc
77.27a
16.95a
7.75abc
3.10ab
0.75a
CB
165.49ab
2.86a
7.79c
74.54a
15.29a
5.57bcd
2.17ab
0.43b
MB
176.33ab
2.91a
7.71c
79.41a
16.14a
8.14ab
2.07b
0.38b
Note: Different letters in column indicating statistically differences mean at P < 0.05 by Duncan's multiple range test. a,bFor an
explanation of accessions code and character symbols, see Table 1 and Table 2 respectively
Phenolic and flavonoid content
Significant differences were detected among the
accessions for phenolics and flavonoids contents (Table 5).
The highest total phenolic content (46.92 mg GAE/g) was
recorded in PK, whereas the accession KP had the lowest
(29.08 mg GAE/g). Total flavonoid content ranged from
21.31 mg QE/g (MB) to 33.81 mg QE/g (GK). The
previous study reported various ranges of total phenolic
content (26.70 to 70.83 mg GAE/g) and total flavonoid
content (7.65-21.71 mg QE/g) in the samples collected
from the different geographical origin (Nurcholis et al.
2016a). Environmental factors and plant species can
profoundly affect the metabolite production in medicinal
plants (Oliveira et al. 2013; Moghaddam and Mehdizadeh
2015; Moghaddam and Pirbalouti 2017; Nurcholis et al.
2018). The present study was conducted in the same
environment and cultivation; therefore a possible reason for
total phenolic and flavonoid content variations is the
genetic factor.
Table 5. Variation in ethanol extract yield, total phenolic and total
flavonoid contents of ten C. aeruginosa accessions
Accession
code a
Extract
yield (%)
Total phenolic
(mg GAE/g)
Total flavonoid
(mg QE/g)
KL
6.01ab
32.08ab
22.14b
PK
7.36a
46.92a
23.53b
BH
6.47ab
35.08ab
26.03ab
GK
6.43ab
40.42ab
33.81a
KP
6.54ab
29.08b
23.53b
PW
5.43ab
30.42b
27.42ab
MD
6.84ab
31.08b
23.53b
LC
3.68b
37.92ab
27.42ab
CB
4.85ab
40.08ab
27.14ab
MB
6.26ab
32.58ab
21.31b
Note: Different letters in column indicate statistical differences
mean at P < 0.05 by Duncan's multiple range test. For an
explanation of accessions code, see Table 1
Table 6. Correlation coefficients among agro-morphology traits, extract yield, total phenolic content and total flavonoid content on ten
C. aeruginosa accessions
CPSH
VPL
ML
PCM
HR
SR
LPR
STR
PH
PD
NL
LL
LW
NS
FRW
DRW
Ext.
Phe.
Fla.
PSH
1.00***
1.00***
1.00***
-1.00***
-0.22
0.11
0.22
-0.17
-0.38
0.50
0.54
-0.52
0.13
-0.37
0.27
0.33
-0.089
0.19
0.41
CPSH
1.00***
1.00***
-1.00***
-0.22
0.11
0.22
-0.17
-0.38
0.50
0.54
-0.52
0.13
-0.37
0.27
0.33
-0.089
0.19
0.41
VPL
1.00***
-1.00***
-0.22
0.11
0.22
-0.17
-0.38
0.50
0.54
-0.52
0.13
-0.37
0.27
0.33
-0.089
0.19
0.41
ML
-1.00***
-0.22
0.11
0.22
-0.17
-0.38
0.50
0.54
-0.52
0.13
-0.37
0.27
0.33
-0.089
0.19
0.41
PCM
0.22
-0.11
-0.22
0.17
0.38
-0.50
-0.54
0.52
-0.13
0.37
-0.27
-0.33
0.089
-0.19
-0.41
HR
0.22
-0.048
0.22
0.49
-0.069
-0.40
0.76*
0.30
0.38
0.11
0.18
-0.38
0.0048
-0.42
SR
-0.22
0.17
0.39
0.37
-0.0057
0.15
0.037
0.30
0.27
0.65*
-0.76*
0.15
0.18
LPR
-0.22
0.18
0.41
0.27
0.068
-0.053
0.30
0.64*
0.43
0.068
-0.53
-0.23
STR
0.18
0.49
0.48
0.40
0.35
0.058
-0.084
-0.04
0.07
-0.0013
0.036
PH
0.26
-0.34
0.73*
0.34
0.94***
0.68*
0.66*
-0.45
-0.45
-0.11
PD
0.72*
0.057
0.37
0.19
0.60
0.64*
-0.22
-0.23
0.38
NL
-0.40
-0.10
-0.33
0.064
0.045
0.18
0.19
0.47
LL
0.53
0.56
0.26
0.25
-0.072
-0.36
-0.59
LW
0.16
0.34
0.31
0.065
-0.48
-0.16
NS
0.74*
0.65*
-0.49
-0.56
-0.10
FRW
0.90***
-0.37
-0.63
0.042
DRW
-0.61
-0.42
0.088
Extract
0.027
-0.29
Phenolic
0.35
Note: For explanation character symbols, see Table 2. *, **, *** Significant at 0.05, 0.01, and 0.001 probability levels after Bonferroni-
adjustment, respectively
KHUMAIDA et al. – Phenolic and agro-morphology in Curcuma aeruginosa
661
Multivariate analyses
The determination of selection characters is important
in plant breeding program (Acquaah 2017). Correlations
between the investigated agro-morphology traits, extract
yield, phenolics content of C. aeruginosa accessions were
shown in Table 6. Results showed a positive and significant
(p ≤ 0.001) correlation between pseudostem habit (PSH),
the color on pseudostem habit (CPSH), venation pattern of
the leaf (VPL), and the margin of the leaf (ML); plant
height (PH) and the number of shoots (NS) (r = 0.94).
Fresh rhizome weight (FRW), which is an important
characteristic in C. aeruginosa medicinal plant breeding of
harvestable rhizomes, exhibited a significant (p ≤ 0.05)
positive correlation with NS (r = 0.74), PH (r = 0.68), and
length of the primary rhizome (LPR) (r = 0.64). Moreover,
dry rhizome weight (DRW) was significantly positively
correlated with FRW (r = 0.90, p ≤ 0.001), NS (r = 0.65, p
≤ 0.05), pseudostem diameter (PD) (r = 0.64, p ≤ 0.05), PH
(r = 0.66, p ≤ 0.05), and the shape of the rhizome (SR) (r =
0.65, p ≤ 0.05). Leaf length (LL) was positively correlated
(p ≤ 0.05) with PH (r = 0.73) and the habit of the rhizome
(HR) (r = 0.76). PD and number of leaves (NL) showed
significant (p ≤ 0.05) positive correlation (r = 0.72). Purple
color on midrib (PCM) was significantly (p ≤ 0.001)
negative correlated with PSH, CPSH, VPL, and ML. The
extract yield with SR (r = -0.76) and DRW (r = -0.61)
showed a significant (p ≤ 0.05) negatively correlation, as
for total phenolic content with NS (r = -0.56) and FRW (r =
-0.63) and between total flavonoid content and LL (r = -
0.59). The improvement leading to produce a high yield
extract and metabolite content such as phenolics are the
main objective of plant breeding in C. aeruginosa.
Therefore, SR, DRW, NS, and FRW can be suggested as an
important selection criterion in C. aeruginosa for good
extraction and bioactive yields. Similar result was obtained
by Mishra et al., (2018) who found selection criteria such
as the fresh weight of rhizome, dry weight of rhizome and
plant height for improvement rhizome yield of C. longa.
The principal component analysis (PCA) was
conducted using agro-morphology traits, extract yield, and
phenolic content. The PCA allows the identification of
patterns showing similarities and differences in data of the
accessions studied (Kwarteng et al. 2018; Mirto et al.
2018). The first five principal components accounted for
90.041% of the cumulative contribution (total variability)
(Table 7). PC-1 had 6.963 variance (Eigenvalue) which is
34.813% of total variation explained (Table 7). Purple
color on leaf midrib (0.364), habit of the rhizome (0.153),
status of the tertiary rhizome (0.044), plant height (0.179),
leaf length (0.247), leaf width (0.003), number of shoots
(0.173) and extract yield (0.023) contributed positively to
PC1. In contrast, pseudostem habit (-0.364), color on
pseudostem habit (-0.364), venation pattern of the leaf (-
0.364), margin of the leaf (-0.364), shape of the rhizome (-
0.036), length of primary rhizome (-0.075), pseudostem
diameter (-0.205), number of leaves (-0.261), fresh rhizome
weight (-0.072), dry rhizome weight (-0.100), phenolic (-
0.093) and flavonoid (-0.217) contributed negatively to
PC1. PC-2 contributed 26.139% of the total variation and
demonstrated positively in the various traits such as purple
color on midrib (0.051), number of leaves (0.015), extract
yield (0.235), phenolic (0.262) and flavonoid (0.017).
However, traits which correlated negatively to PC-2 were
pseudostem habit (-0.051), color on pseudostem habit (-
0.051), venation pattern of the leaf (-0.051), margin of the
leaf (-0.051), habit of the rhizome (-0.173), shape of the
rhizome (-0.218), length of primary rhizome (-0.193),
status of the tertiary rhizome (-0.062), plant height (-
0.354), pseudostem diameter (-0.264), leaf length (-0.236),
leaf width (-0.231), number of shoots (-0.345), fresh
rhizome weight (-0.398) and dry rhizome weight (-0.404).
PC-3, PC-4, and PC5 contributed 11.181%, 9.896%, and
8.012% to the total variation, respectively.
Two-dimensional hierarchical cluster analysis (HCA)
was conducted to compare with the results from PCA
(Péroumal et al. 2017). The HCA allowed the relationship
between the accessions studied and the studied traits to
identify which of these are the most powerful (Bakić et al.
2017). Three groups were defined as shown in Figure 2.
Group 1 composed only one accession (MB) with high
PCM and LL traits but also low CPSH, PSH, VPL and ML
traits. Group 2 was composed of three accessions: KP, PW,
and LC. These accessions showed the highest DRW, FRW,
NS, PH, SR and LW traits. Group 3 was comprised of MD,
CB, BH, GK, KL, and PK accessions. These accessions
were characterized by high total phenolic content, total
flavonoid content, and extract yield. Thus, these accessions
have the potential to be deployed in plant breeding
programs on a commercial scale. In the second dimension,
three main clusters were defined. The first group consisted
of PCM, HR, LL, STR, LW, SR, PH, NS, FRW, and DRW
characters. Extract yield and LPR traits were grouped in the
second cluster. The third cluster contained the traits for
total phenolic content, total flavonoid content, ML, VPL,
PSH, CPSH, PD, and NL.
In this study, the C. aeruginosa accessions exhibited
variations of agro-morphological traits, i.e., fresh weight of
the rhizome, number of leaves, plant height, number of
shoots and dry weight of rhizome. The shape of the
rhizome, number of shoots, fresh rhizome weight and dry
rhizome weight could be used as an important selection
criterion in C. aeruginosa breeding for obtaining high
extract and phenolics content. The accessions of MD, CB,
BH, GK, KL, and PK produced high extract yield, total
flavonoid, and total phenolic contents. These accessions are
recommended for breeding programs resulting in high
extract and the bioactive yield for commercial scale.
B I O D I V E R S I T A S
20 (3): 656-663, March 2019
662
Table 7. Eigenvectors, Eigenvalues and proportion of variation
explained by first five components for different agro-morphology
traits, extract yield, total phenolic, and total flavonoid contents of
C. aeruginosa accessions
Variable a
Eigenvectors
PC-1
PC-2
PC-3
PC-4
PC-5
PSH
-0.364
-0.051
0.014
0.026
0.180
CPSH
-0.364
-0.051
0.014
0.026
0.180
VPL
-0.364
-0.051
0.014
0.026
0.180
ML
-0.364
-0.051
0.014
0.026
0.180
PCM
0.364
0.051
-0.014
-0.026
-0.180
HR
0.153
-0.173
-0.158
-0.130
0.493
SR
-0.036
-0.218
-0.486
-0.034
-0.039
LPR
-0.075
-0.193
0.419
0.187
-0.161
STR
0.044
-0.062
-0.052
-0.662
-0.147
PH
0.179
-0.354
-0.088
0.005
-0.092
PD
-0.205
-0.264
0.048
-0.325
-0.246
NL
-0.261
0.015
0.086
-0.355
-0.347
LL
0.247
-0.236
0.053
-0.242
0.268
LW
0.003
-0.231
0.168
-0.325
0.335
NS
0.173
-0.345
-0.041
0.177
-0.165
FRW
-0.072
-0.398
0.138
0.166
-0.108
DRW
-0.100
-0.404
-0.044
0.135
-0.035
Extract yield
0.023
0.235
0.485
-0.164
0.008
Phenolic
-0.093
0.262
-0.409
-0.074
0.078
Flavonoid
-0.217
0.017
-0.292
-0.007
-0.355
Eigen value
6.963
5.228
2.236
1.979
1.602
Proportion of variation
explained (%)
34.813
26.139
11.181
9.896
8.012
Cumulative proportion
of variation (%)
34.813
60.952
72.133
82.029
90.041
Note: aFor an explanation of variable symbols, see Table 2
Figure 2. Heatmap and hierarchical cluster analysis of two-
dimensional relationships among C. aeruginosa accessions and
traits mainly selected for agro-morphology, extract yield, total
phenolic content and total flavonoid content. For an explanation
of variable symbols, see Table 2.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge financial support
from the Ministry of Research, Technology and Higher
Education of the Republic of Indonesia under the PTUPT
(Penelitian Terapan Unggulan Perguruan Tinggi) grant;
No. 1714/IT3.11/PN/2018. We appreciate Dr. G. John
Acton for English proofreading and Mr. Topik Ridwan for
botanical identification of the plant material used.
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