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1. Ghosh Arghya, Ghosh Ajoy, Ghosh Parthadeb, Chatterjee Padma (2012)
Comparison of bioactive potentials between 2, 7, (14), 10 bisabolatriene-
1,9,12 triol, a bisabolene type sesquiterpene isolated from Curcuma longa L.
and its acetylated derivative, Journal of the Botanical Society of Bengal. 66
(2): 119-124.
2. Ghosh Arghya, Ghosh Parthadeb, Chatterjee Padma (2013) Isolation,
purification and characterisation of 2, 7, (14), 10 bisabolatriene- 1,9,12 triol, a
bisabolene type sesquiterpene isolated from Curcuma longa L., Natural
Products- An Indian Journal. 9 (3): 117-122.
3. Ghosh Arghya, Saha Gautam, Ghosh Parthadeb, Chatterjee Padma (2013) In
vitro true to type propagation of Curcuma caesia Roxb. (Zingiberaceae) and
assessment of its genetic fidelity using RAPD marker, Biotechnology- An
Indian Journal. 7 (4): 121-130.
4. Ghosh Arghya, Ghosh Ajoy, Ghosh Parthadeb, Chatterjee Padma (2013)
Antifungal potentialities of 2, 7, (14), 10 bisabolatriene- 1,9,12 triol, a
bisabolene type sesquiterpene and its acetylated derivative isolated from
Curcuma longa L., Journal of the Botanical Society of Bengal. 67 (1): 37-42.
5. Ghosh Arghya, Ghosh Parthadeb, Chatterjee Padma (2013) Formulation of an
antibacterial crop protectant using the acetylated derivative of 2, 7, (14), 10
Bisabolatriene- 1,9,12 triol isolated from Curcuma longa L, Asian Journal of
Plant Science and Research. 3 (3): 95-99.
6. Ghosh Arghya, Ghosh Parthadeb, Chatterjee Padma (2013) A protocol for
rapid propagation of genetically true to type Indian Turmeric (Curcuma longa
L.) through in vitro culture technique, Advances in Applied Science Research.
4 (3): 39-45.
168
7. Ghosh Arghya, Bandyopadhyay Ayan, Ghosh Parthadeb, Chatterjee Padma
(2013) Isolation of (Z)-7-methoxy-1, 5-dihydrobenzo[c] oxepine from
Curcuma caesia Roxb., Journal of Scientific and Innovative Research. 2 (4):
795-801.
8. Ghosh Arghya, Ghosh Parthadeb, Chatterjee Padma (2013) Evaluation of
Antimicrobial and Antifungal potential of (Z)-7-methoxy-1, 5-
dihydrobenzo[c] oxepine, isolated from Curcuma caesia Roxb., Journal of
Scientific and Innovative Research. 2 (4): 745-750.
9. Ghosh Arghya, Bandyopadhyay Ayan, Ghosh Parthadeb, Chatterjee Padma
(2013) Isolation of a novel terpenoid from the rhizome of Curcuma caesia
Roxb., Journal of Scientific and Innovative Research. 2 (4): 777-784.
10. Ghosh Arghya, Ghosh Parthadeb, Chatterjee Padma (2013) Evaluation of the
bioactive potentialities of a diacetaldehyde terpenoid isolated from Curcuma
caesia Roxb., The Journal of Phytopharmacology. 2 (4): 1-7.
11. Ghosh Arghya, Bandyopadhyay Ayan, Ghosh Parthadeb, Chatterjee Padma
(2013) Evaluation of Antibacterial Potentiality of a Cyclopenta Naphthalene
Tetraol Terpenoid Isolated from Curcuma caesia Roxb.; Research & Reviews:
Journal of Botanical Sciences (Accepted for publication).
In vitro true to type propagation of Curcuma caesia Roxb. (Zingiberaceae)
and assessment of its genetic fidelity using RAPD marker
Arghya Ghosh1,2, Gautam Saha2, Padma Chatterjee1, Parthadeb Ghosh2*1Plant Biochemistry, Molecular Biology & Advance Plant Physiology Research Laboratory, Department of Botany,
University of Kalyani, Kalyani 741235, Nadia, West Bengal, (INDIA)2Cytogenetics & Plant Breeding Section, Department of Botany, University of Kalyani,
Kalyani 741235, Nadia, West Bengal, (INDIA)
E-mail: pdgbot@yahoo.co.in
FULL PAPER
ABSTRACT
An efficient plant propagation system through rhizomal explants was es-
tablished in Curcuma caesia Roxb., a medicinally important herbaceous
annual herb belonging to the family Zingiberaceae. Here we report a rapid
and reliable method for high fidelity micro-propagation. Rhizomal explants
from two months old seedlings were cultured on Murashige and Skoog�s
(MS) medium supplemented with different concentrations of N6-
benzyladenine (BA) (0.5 - 5.0 mg/l), Napthalenic acetic acid (NAA) (0.5 -
5.0 mg/l) and Indole 3 butyric acid (IBA) (0.5 - 5.0 mg/l). During the first
culture on 1.5 mg/l of 6-benzylamino purine (BAP) and 1 mg/l of
Napthalenic acetic acid (NAA) maximum 15.40±0.40a shoots with an aver-
age shoot-length of 8.46±0.06a were produced. The elongated shoots pro-
duced a maximum of 12.00±0.00a roots on half-strength MS liquid medium
supplemented with 1 mg/l of Indole 3 butyric acid (IBA). The plantlets
were acclimatized by transferring them first to peat moss: compost (1:1)
mixture followed by sand: soil (2:1) mixture, recording 95% survival.
Genetic fidelity was assessed by DNA fingerprinting using random ampli-
fied polymorphic DNA (RAPD) of in vitro and in vivo plants. Five arbi-
trary decamers displayed same banding profile showed no genomic alter-
ations, indicating homogeneity among the tissue culture regenerates and
genetic uniformity with that of donor plants. The present study provides
high genetic fidelity micropropagated system for efficient and rapid
micropropagation protocol of this important medicinal plant and great
use in conserving without risk of genetic instability.
2013 Trade Science Inc. - INDIA
KEYWORDS
Micropropagation;
Multiple shoot;
Rhizomal explant;
Genetic fidelity.
BTAIJ, 7(4), 2013 [121-130]
BioTechnologyAn Indian JournalTrade Science Inc.
Volume 7 Issue 4
BioTechnologyISSN : 0974 - 7435
INTRODUCTION
The genus Curcuma L. of the family Zingiberaceae
is well known as the turmeric genus, because Of Cur-
cuma longa. C. longa is the most investigated species
of this genus, although there are over 100 others in this
122 In vitro true to type propagation of Curcuma caesia Roxb. (Zingiberaceae)
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BioTechnology
genus[1,2]. Most of the species of this genus are peren-
nials and grow well in tropics and subtropics where it
requires a hot and moist climate and a fairly light soil.
Curcuma species are native to the countries of the
Southeastern Asia and extensively cultivated in Bengal
(Bangladesh and India), China, Taiwan, Sri Lanka, In-
donesia, Peru, Australia, and the West Indies. Although
turmeric is quite common due to its frquent usage as
spices, most of the other species of Curcuma including
Curcuma caesia as well as turmeric posses some active
medicinal constituents. Medicinal plants are important to
the global economy, as well as source of income for rural
people in developing countries. Generally 70% to 80%
of the ayurvedic medicine was derived from plants[3].
Herbal medicines are produced from field-grown plants
and are used to cure several diseases that are caused by
pathogenic bacteria, fungi, and insects[4]. It is difficult to
ensure the quality control as the medicinal preparations
are multiherb preparations and also difficult to identify
and quantify the active constituents[5]. Due to the ab-
sence of side effects in herbal medicines the demand of
herbal drugs are increasing day by day[6]. So people have
to take the synthetic drugs and antibiotics to get cured
from these diseases. Plants provide a major contribution
to the pharmaceutical industry[7]. The major cause of in-
adequacy of inavialability of herbal medicines are due to
excessive human exploitation, non-regulated collection,
unresolved inherent problems of seed viability and seed
germination, and this priority many species have become
threatened or endangered[8,9].
Curcuma caesia Roxb. is a native of northeast In-
dia extending up to the present day Bastar region of
Chattisgarh. The rhizome has a deep bluish black or
grayish black color. It is used in native medicines. This
species occurs mainly in the northeastern and west
coastal regions of India, extending to the hills. It has
also been cultivated in earlier times for extracting ar-
rowroot powder and for the production of Abir. Rhi-
zome is sometime pale yellow or colorless. It is ground
to a powder, which is purified by repeated washing and
dried. Dried powder is mixed with a decoction of sap-
pan wood (Cesalpinia sappan) when the red color is
obtained. It is used in traditional and local medicines as
a stimulant and carminative. This was reported to have
cosmetic properties. Species such as Curcuma
aeruginosa, Curcuma caulina, Curcuma
leucorrhiza, Curcuma pseudomontana, and Cur-
cuma rubescens are also used as sources of arrow
root powder and in local and tribal medicines. Cur-
cuma caesia was in use earlier as a dye, and now as a
cosmetic, often as a substitute for Curcuma aromatica.
Perhaps, India is the only country where there is a strong
R & D base for turmeric. However, research on Cur-
cuma caesia, especially in the area of pharmacology,
is being pursued by many workers in many countries).
The first ever research on Curcuma caesia in India
was initiated at Udayagiri in Orissa in 1944 under the
Imperial Council for Agriculture Research. However,
organized research programs were initiated in indepen-
dent India during the first Five-Year Plan. Based on a
recommendation of the Spices Enquiry Committee
(1953), turmeric research was started in Kandaghat
(Punjab), Targaon (Maharashtra), and Thodupuzha and
Ambalavayal (Kerala). A scheme for turmeric research
was initiated in 1955 at Andhra Pradesh (at
Peddapalem). However, the real impetus for turmeric
research was received with the organization of the All
India Coordinated Spices and Cashew Improvement
Project. In 1975, research programs were started in
two centers, Coimbatore (Tamil Nadu Agriculture Uni-
versity � TNAU) and Pottangi (Orissa University of
Agricultural and Technology, High Altitude Research
Station). In vitro culture techniques represents an ex-
cellent option for the study and conservation of rare,
threatened or endangered medicinal plants[10,11], as well
as tool for efficient rapid clonal propagation of impor-
tant plants allowing production of genetically stable and
true to true type progeny[12]. Therefore, the interest in
using these techniques for rapid and large-scale propa-
gation of medicinal and aromatic plants has been sig-
nificantly increased[13,14]. Till now, there is no report on
micropropagation and assessment of genetic fidelity of
Curcuma caesia Roxb. In this study we represent for
the first time an efficient protocol for rapid large scale
regeneration of plantlets in vitro from rhizomal explants
of Curcuma caesia Roxb. with maintaining stable gene
pool fidelity of the regenerants as assessed by RAPD.
MATERIALS AND METHODS
Explant preparation
Young disease free rhizomal explants (rhizomal buds
Parthadeb Ghosh et al. 123
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of 2.5 - 3 cm) were collected from 2 months old plant
growing in the medicinal and aromatic plant garden of
the Department of Botany, University of Kalyani,
Kalyani, India. Explants were washed thoroughly un-
der running tap water and then treated with 5% (m/v)
Teepol (Qualigen, Mumbai, India) for 20 min, followed
by rinsing three to five times in sterile double distilled
water. The rhizomal segments were then surface
disinfested with 70% alcohol for 5 min followed by im-
mersion in 0.1% (m/v) aqueous mercuric chloride
(HgCl2) solution for 5 - 6 min and finally rinsed with
sterile double distilled water (five to six times) in a flow
chamber. The surface sterilized explants were trimmed
at cut ends and about 1.2-1.5 cm prior to inoculation
on culture media.
Culture media and conditions
Surface sterilized rhizomal segments (1.2 - 1.5 cm)
were cultured on MS[15] basal medium containing 3%
(w/v) sucrose (Himedia, Mumbai, India) for culture ini-
tiation and served as explant sources for subsequent
experiments. The pH of the medium (Supplemented
with respective growth regulators) was adjusted to 5.8
with 1N NaOH or 1N HCl before gelling with 0.8%
(w/v) agar (Himedia, Mumbai, India). In all the experi-
ments, the chemicals used were of analytical grade. The
explants initially were implanted vertically on the cul-
ture medium in test tube (150 × 25 nm) and plugged
tightly with non-absorbent cotton. All the cultures were
kept under cool fluorescent light (16 h photo period 40
ìmol·m�2 s�1, Philips, India at 25°C ± 2°C) and 60%
- 70 % relative humidity (RH).
Induction of multiple shoots
For initial multiple shoot induction, the explants were
cultured on Murashige and Skoog�s medium supple-
mented with various concentrations of BA (0.5 - 5.0
mg/l) in combination with NAA (0.1 - 5.0 mg/l). The
induced shoots were allowed to grow for 27 days. Af-
ter 27 days it was found that maximum number of shoots
(15.40±0.40a) was obtained in the MS medium supple-
mented with 1.5 mg/l of BA and1 mg/l of NAA.
Rooting of shoots
Small micro shoots grown on subculture medium
were transferred to half and full strength MS media sepa-
rately, supplemented with various concentrations of IBA
(0.5 � 5 mg/l) for root developement. Number of roots
(12.00±0.00a) developed from micro shoots with an
average length of 4.00±0.11cd were higher in half
strength of MS medium supplemented with 1 mg/l of
IBA. Where as the number of roots produced in full
strength MS medium supplemented with 2.5 mg/l of
IBA was 7.40±0.24d with an average length of
5.36±0.15a. IBA was filter sterilized and added to the
medium after autoclaving under the sterilized environ-
ment of laminar air flow cabinet. Data were recorded
on the percentage of rooting, the mean number of roots
per shoot and the root length after four weeks of trans-
fer onto the rooting medium.
Acclimatization of regenerated plants
The complete rooted plantlets with 6 - 8 fully ex-
panded leaves were removed from the culture medium
and the roots were washed gently under running tap
water to remove agar. The plantlets were transferred to
plastic pots (5 cm diameter) containing a mixture of
sterilized garden soil and vermiculite in the ratio 2:1 and
covered with transparent plastic bags to ensure high
humidity. Each was irrigated with 1/6 MS basal salt
solution devoid of sucrose and inositol every 4 days for
2 weeks. The growth chamber was maintained at 26°C
± 1°C, 80% - 85% relative humidity with light intensity
of 50 ìmol·m�2·s�1 on a 16 h photoperiod inside the
culture room conditions. The relative humidity was re-
duced gradually and after 30 days the plantlets were
transferred to pots (25 cm diameter) containing garden
soil and kept under green house for another 2 weeks
for further growth and development. Well acclimatized
in vitro raised plants were transferred finally to its origi-
nal habitat for its survivalability. There are no changes
in respect to morphology, growth characteristics and
floral features etc in between tissue culture regenarate
plants and naturally grown field plants.
DNA extraction and pcr amplification
Genomic DNA was extracted from young leaves
of in vitro raised and field grown plants of Curcuma
caesia Roxb. and mother plant by Cytl trimethyl am-
monium bromide (CTAB) procedure[16] with minor
modifications. Quality and quantity of DNA was
checked on 0.8% agarose gel and also from values
obtained by 260/280 nm UV absorbance ratio[17].
124 In vitro true to type propagation of Curcuma caesia Roxb. (Zingiberaceae)
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Twelve arbitrary decamer RAPD primers (Bengalore
Genni Pvt. Ltd., India) were used for polymerase chain
reaction (PCR) for DNA amplification. DNA finger
printing profiles were compared to evaluate clonal fi-
delity and genetic stability. Amplification was performed
in 25 ìL using PCR mixture of consisting of 2.5 ìL Taq
buffer, 1 ìL dNTPs, 0.5 ìL Taq polymerase, 2 ìL
DNA (approximate 50 ng/ìL), 1.0 ìL primer (10
pmol), 2.5 ìL MgCl2, 1 ìL oil and 14.5 ìL Mili Q
water. The PCR reaction conditions were: preheating
for 5 min at 94°C; 40 cycles of 25 sec at 94°C, 20 sec
at 40°C and 1.25 min at 72°C and elongation was com-
pleted by a final extension of 6 min at 72°C. After am-
plification, the PCR product was resolved by electro-
phoresis in 1.4% agarose gel (Himedia, Mumbai, In-
dia) and stained with ethidium bromide (0.5 ìg/ml). 2.0
- 23.1 kb ë DNA di-gested Hind III was used as the
DNA marker, and bands were visualized under UV light
and photographed using the Gel Doc equipment (Bio
Rad). All the PCR reaction was repeated for thrice.
Statistical analysis
Experiments were set up in completely randomized
block design. Each experiment was repeated three times
with 10 - 12 replicates. Data were analyzed by one
way analysis of variance (ANOVA) and the difference
between means were scored using Duncan�s Multiple
Range Test P 0.05[18] on the statistical package of
SPSS (Version 10).
RESULTS AND DISCUSSION
In vitro establishment
For the primary establishment of in vitro culture
from field grown plants surface sterilization of the ex-
plants was essential because of the microorganism can
live or survive in the vascular tissue of the plants, there-
fore contamination attached to the surface of the ex-
plants. The main contaminants that affect tissue cultured
plants are bacteria and fungi. Particularly hairy plants
are a problem because bubble of air became entrapped
in the explants and prevent good contact with the
disinfesting agent. Evenly storage water and its included
microorganism can be a source of some of the internal
(endogenous) contamination observed in vitro[19]. The
duration of exposure of the explants of the sterilizing
agent is most important for any tissue culture studies.
Therefore, to overcome contamination problem, sur-
face sterilization of explants was done with 0.1% aquous
solution of Mercuric chloride (HgCl2) for 2, 4, 6, 8 and
10. Mercuric chloride (HgCl2) is a very strong ster-
ilant[20]. When the explants sterilization was done with
0.1% aquous solution of HgCl2 for less than 5 min, 65%
of the explants get contaminated. Whereas, when the
duration of exposure of 0.1% aquous solution of HgCl2
was above 5 min contamination frequency was signifi-
cantly reduced to 10%. These explants remained green
and showed healthy growth and proliferation of auxillary
shoots. But when the duration of exposure was above
6 min, a death rate of the explant increases significantly
(70%). All of the explants died when the explants were
treated with 0.1% aquous solution of HgCl2 for 7 - 10
min.
Effect of cytokinin and auxin combination on
shooting
The morphogenetic responses of rhizomal segment
explants to various cytokinin (BA) and auxin (NAA)
are summarized in TABLE 2 and Figure 1C, 1D, 1E.
When Explants were cultured on basal MS medium or,
MS medium contains solely cytokinin (BA), or solely
auxin (NAA) failed to produce shoots even after 4
weeks of inoculation, but when the explants cultured
on MS medium supplemented with different concen-
trations and combinations of cytokinins and auxins with
showed variation in the regeneration percentage and
number of shoots formed. Rhizomes cultured on half
strength MS basal medium showed no response. Actu-
ally there was a greater need of nitrogen and potassium
containing compounds which induce greater amount of
new proteins[21]. These components are deficit in half
strength MS basal medium compared to full strength
MS basal medium. Initial induction of shoots was noted
after 10 - 12 days of inoculation (Figure 1B, 1C). Ob-
servations on different growth parameters from differ-
ent treatments were recorded after 4 weeks of culture
initiation following repeated subculturing after 7 days
intervals. Among the different combinations of cytoki-
nin and auxin tested, the best response (78.22±0.12
%) was obtained in the presence of 1.5 mg/l BA and 1
mg/l NAA (Figure 1E, 1F). The maximum number of
multiple shoots was obtained in the same medium con-
Parthadeb Ghosh et al. 125
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BTAIJ, 7(4) 2013
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taining same concentrations of BA and NAA. The num-
ber of shoots developed in this medium was 15.40±0.40
after 3 weeks. The average length of shoot in this me-
dium was 8.46±0.06. The BA and NAA concentra-
tions higher than 1.5 mg/l BA and 1 mg/l NAA, the
number of shoots as well as percent response was re-
duced (TABLE 2). Reduction in the number of shoots
generated from each bud of rhizome at BA concentra-
tion higher than the optimal level was also reported for
several medicinal plants[13,22]. BA was reported to over-
come apical dominance, release lateral buds from dor-
mancy and promote shoot formation[23]. The stimulat-
ing effective of BA and NAA on multiple shoot forma-
tion has been reported earlier for several medicinal plant
species including Ocimum basilicum L., Feronia
limonia L., Mentha piperita L.[14,24-28].
Treatment
Duration
(min) with
0.1% HgCl2
Number
of
explants
Rate of
Contamination
(after day of
treatment) 2
Rate of
contamination
after day of
treatment) 3
Rate of
Contamination
(after day of
treatment) 4
Rate of
Contamination
(after day of
treatment) 5
Rate of
Contamination
(after day of
treatment) 6
Rate of
Contamination
(after day of
treatment) 7
Percentage of
Contamination
free explants
after 10 days
2 12 3 6 7 9 12 12 11
4 12 1 4 5 6 6 8 28
6 12 0 0 0 0 0 1 60
7 12 0 0 2 All explant
becomes dead*,**
All explant
becomes dead*,**
All explant
becomes dead* 85
8 12 1 2 All explant
becomes dead*
All explant
becomes dead*
All explant
becomes dead*
All explant
becomes dead* 97
10 12 All explant
becomes dead*
All explant
becomes dead*
All explant
becomes dead*
All explant
becomes dead*
All explant
becomes dead*
All explant
becomes dead* 100
TABLE 1 : Standardization of HgCl2 treatment period for surface sterilization of the explants.
�*� indicates explant death due to tissue killing, �**� indicates explant death due to microbial contamination.
Concentrations
of growth
Regulators (mg/l)
Percentage
of shoot
proliferation
Number
of shoots/
explant
Average
Shoot
Length (cm)
MS (Control) 0.00±0.00i 0.00±0.00g 0.00±0.00f
MS+BA+NAA (mg/l)
0.5+0.5 67.22±0.16c 7.20±0.20ef 6.48±0.10b
0.5+1 72.40±0.10b 8.00±0.31e 6.42±0.07b
1+1 56.28±0.28f 12.00±0.31cd 5.42±0.13cd
1.5+1 78.22±0.12a 15.40±0.40a 8.46±0.06a
2+1 64.40±0.34d 14.00±0.63b 5.66±0.10c
2.5+1.5 63.88±0.14d 12.20±0.73cd 6.54±0.08b
3+2 67.80±0.50c 13.40±0.60bc 6.30±0.07b
3.5+2 57.52±0.65e 12.60±0.40bcd 6.32±0.16b
4+2.5 57.26±0.10ef 11.20±0.58d 5.22±0.08d
4.5+3 44.82±0.50g 7.60±0.40ef 5.26±0.10d
5+4 28.80±0.48h 6.20±0.58f 4.46±0.15e
TABLE 2 : Effect of cytokinin and auxin combination on
shooting.
Values are means ± SE. n = 10 - 12 (in triplicate); Means
followed by same does not differ significantly according to
Duncan�s Multiple Range Test (p d� 0.05).
Effect of auxin on rooting
Healthy elongated shoots (4 - 9 cm in length) were
excised and placed on full and half strength MS basal
medium supplemented with different concentrations of
auxin (IBA) at the range of 0.5 - 5.0 mg /l for induction
of roots (TABLE 3). The effects of these auxins on
root induction as well as the length of the roots were
examined after 15 days of inoculation in root induction
medium. In the preliminary experiments conducted, no
rooting was observed when the shoots were culture on
basal (Control) MS medium. Full strength MS medium
containing auxins showed very poor response in root-
ing, but well developed roots were achieved on half
strength MS medium supplemented with IBA (1 mg/
ml) with increase sucrose concentration (4%) gave us
well developed roots within 15 - 20 days. This founda-
tion was supported by reports in various species like
Lavandula vera[29], Ocimum kilimandscharicum[26],
rooting frequency was higher when shoots were inocu-
lated on half strength MS medium. The cause behind
the favorable effect of reduced macronutrient concen-
tration is that the increasing concentration of nitrogen
ions reduced root development. So rooting is favourable
in much lower nitrogen ions concentration than that are
required for shoot developement[30]. IBA was more ef-
fective for root induction than any other auxins in both
(half & full strength MS) types to media. The possible
reason could be that IBA is more stable than any other
auxins like IAA etc. to chemical degradation in tissue
culture media, both during auto-claving and at room
126 In vitro true to type propagation of Curcuma caesia Roxb. (Zingiberaceae)
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BioTechnology
temperature[31]. However, NAA nd IAA formed slen-
der roots in half & full strength MS media. Similar re-
sponse was observed by other species[32,33]. Among
various concentrations of IBA tested, 1 mg /l of IBA
proved to be the best in eliciting the highest frequency
of root formation. Shoot formed roots at a high fre-
quency (90.06±0.19 %) on medium containing 1 mg/l
of IBA. In this medium a maximum number of
12.00±0.00 roots attaining a average length of 5.36 ±
0.15 cm were obtained. Further increase in the IBA
concentration to 1.5 - 5 mg/l reduces root initiation. It
has also been reported that shoots contain high level of
endogenous auxins and the addition of exogenous auxin
caused the inhibition of root development, thus resulted
in callusing at the base of the shoots[34]. The stimulatory
effect of IBA for root formation has also been reported
in many medicinal plant species, including Ocimum
basilicum L.[26], Mentha piperita L.[28]; Ocimum
gratissimum L.[35], Tylophora indica[36].
Acclimatization and field establishment
The ultimate success of in vitro propagation lies in
the successful establishment of plants in soil. The well
developed rooted plantlets were taken out gently from
the test tubes and thoroughly washed with sterile water
to remove adhered agar and traces of the medium to
avoid contamination. 60 plantlets were transferred to
plastic pots containing potting a mixture of (2:1) soil
and vermiculite (Figure 1I). In the first week of trans-
plantation, plantlets kept covered in a polythene tent
for providing the condition of high humidity and suffi-
cient light. The polythene cover was removed periodi-
cally and progres-sively whenever leaves appeared
water soaked. Polythene covers were completely with-
drawn after 4 - 5 weeks of hardening. After 5 weeks
plants were then transferred to larger potted filled soil
with organic manure kept under green house for further
growth and development. Finally the acclimated plants
were then shifted to the field conditions having 85%
Figure 1 : (A) Field grown Curcuma caesia Roxb. plant; (B) Shoot induction from rhizomal explant on MS medium
supplemented with 1.5 mg/l BA and 1 mg/l NAA after 10-12 days of inoculation; (C) Shoot proliferation from rhizomal
explant on MS medium supplemented with 1.5 mg/l BA and 1 mg/l NAA after two subculture; (D) Induction of shoot
multiplication on MS medium supplemented with1.5 mg/l BA and 1 mg/l NAA after repeated subculture; (E) More shoot
multiplication on the same medium by repeated subculture.
Parthadeb Ghosh et al. 127
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Values are means ± SE. n = 10 - 12 (in triplicate); Means followed by same does not differ significantly according to Duncan�s
Multiple Range Test (p d� 0.05).
Concentrations
of growth
regulators (mg/l)
Percentage
of
Rooting
Number
of
roots/shoot
Average
root length
(cm)
Concentrations
of growth
regulators (mg/l)
Percentage
of
Rooting
Number
of
roots/shoot
Average
root length
(cm)
MS ½ strength 0.00±0.00p 0.00±0.00j 0.00±0.00g MS full strength 0.00±0.00p 0.00±0.00j 0.00±0.00g
MS½ +IBA (mg/l) MS+IBA (mg/l)
0.5 19.46±0.12m 6.20±0.58e 4.24±0.11bc 0.5 2.88±0.34o 2.00±0.00i 4.54±0.08b
1.0 90.06±0.19a 12.00±0.00a 5.36±0.15a 1.0 15.84±0.32n 2.40±0.24i 4.68±0.11b
1.5 59.46±0.12d 10.80±0.20b 4.32±0.27bc 1.5 56.46±1.12e 4.40±0.24f 4.54±0.06b
2.0 64.18±0.03c 10.60±0.40b 4.16±0.19bc 2.0 53.96±0.21fg 6.20±0.20e 4.46±0.14bc
2.5 53.20±0.05g 8.40±0.24c 4.48±0.10bc 2.5 74.62±0.18b 7.40±0.24d 4.00±0.11cd
3.0 45.42±0.08h 9.00±0.31c 3.48±0.10e 3.0 74.88±0.30b 5.40±0.24e 4.38±0.09bc
3.5 42.54±0.22i 5.60±0.24e 3.54±0.28e 3.5 39.34±0.18j 5.40±0.24e 3.38±0.10de
4.0 54.70±0.48f 7.60±0.40d 2.92±0.27f 4.0 53.10±0.53g 3.60±0.24fgh 3.54±0.12de
4.5 38.42±0.14j 5.80±0.37e 2.80±0.27f 4.5 34.10±0.78k 3.40±0.24gh 2.60±0.08f
5.0 27.36±0.79l 4.00±0.31fg 2.54±0.32f 5.0 26.08±1.27l 2.80±0.37hi 2.50±0.13f
TABLE 3 : Effect of auxin on rooting.
Figure 1 : (F) Root induction from micropropagated shoots on full strength MS medium supplemented with 1 mg/l and 3%
sucrose concentration after 25-35 days of inoculation; (G) Rapid root induction from micropropagated shoots on ½ strength
MS medium supplemented with 1 mg/l and 4% sucrose concentration after 15 days of inoculation; (H) Mature
micropropagated plant having profuse roots; (I) Acclimatization of micropropagated plants; (J) RAPD marker analysis of in
vitro raised field grown plants and mother plant of Curcuma caesia Roxb.: lane M corresponding to ë digested with EcoRI
and HindIII as molecular weight marker (100-500bp), lane 1,2,3 DNA randomly selected regenerated plantlets, lanes 4 and
5 DNA from mother plant.
128 In vitro true to type propagation of Curcuma caesia Roxb. (Zingiberaceae)
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SL.
No.
Primer
Code
Nucleotide
sequence (5�-3�)
Number of
generated bands
1 RAPD1 GTCCTACTCG -
2 RAPD2 GTCCTTAGCG 2
3 RAPD3 CGGGATCCGC -
4 RAPD4 CTTCCGGCAG 3
5 RAPD5 GGTATTACTT 4
6 RAPD6 TGGCTCGGTA 5
7 RAPD7 CTTCGGCAGA -
8 RAPD8 GGTATTACTT -
9 RAPD9 GACAATGGTA -
10 RAPD10 TTAGCTTAGG -
11 RAPD11 CTCTCCGCCA -
12 RAPD12 GCACGCCGGA 3
TABLE 4 : Number of amplification products generated with
the use of RAPD primers to assess genetic fidelity of
micropropagated and field grown plants.
survived. The growth characteristics of in vitro raised
plants did not show any significant morphological dif-
ferences from those of natural occurring field plants.
Molecular analysis
Genetic uniformity is one of the most important pre-
requisites for the successful micropropagation of any
crop species. Nevertheless, a major problem encoun-
tered in cells grown in vitro is the occurrence of ge-
netic variation due to change in either DNA sequences,
in chromosome structure (duplications, translocations)
or in chromosome number (leading to polyploidy). Fur-
thermore, abnormalities in tissue culture particularly
growth regulators (in particular BA, IBA etc)[37], and in
the plants produce from them often increase in frequency
with increasing culture passages[38].
The PCR based RAPD technique does not require
DNA sequence information and species specificity and
hence it is being conveniently used for assessing genetic
stability and clonal fidelity of micropropagated plants in
a number of genera. There are many reports on mo-
lecular characterization of micropropagated plants by
the RAPD technique especially to confirm the clonal
fidelity and genetic stability among tissue culture grown
plants and donor[39]. Because RAPD analysis is par-
ticularly well suited to high output system required for
plant breeding, it is easy to perform, fast, reliable and
of relatively low cost[39]. Keeping this perspective in
mind, in this paper we performed the genetic integrity
of in vitro regenerated plants from rhizomal explants
and respective naturally occurring field grown donor
plant of Curcuma caesia Roxb.
Total 12 primers were initially screened and finally
5 primers produce clear and scorable amplified bands
ranging from 2 - 5 bands per primer (TABLE 4). Each
primer produced a unique set of amplification products
ranging in size from 100 bp - 3 kb (Figure 1J with primer
5� TGGCTCGGTA 3�). All 5 primers produced a total
of 17 bands with an average of 3.40 fragments. All the
17 scorable bands were monomorphic in nature, indi-
cating homogeneity among the culture regenerates and
genetic uniformity with that of the donor plants. The
possible reason may be multiple shoot bud differentia-
tion without intervening callus phase is least vulnerable
to genetic changes. However, no differences were ob-
served between mother plant and plantlets regenerated
from rhizomal segments by any five primers tested in
present RAPD study.
CONCLUSION
In conclusion, the present study, we established an
efficient and reliable micropropagation protocol for in
vitro regeneration of Curcuma caesia Roxb. from
rhizomal explant, which can ensure large scale propa-
gation, as well as protocol can also be used for raising
genetically uni-form plants, which is important for the
sustainable supply of plant materials to the pharmaceu-
tical industries and for conservation of elite germplasm.
As all the micropropagated plants are genetically true
to type with naturally occurring plants so there reduced
chance of genetic manipulations in micropropagated
plants. As either no or reduced chance of genetic ma-
nipulation occurs, so there is also reduced chance of
variability of secondary metabolite contents in
micropropagated plants with that of naturally occurring
plants. Our results also indicate that multiple shoot in-
duction, rooting of shoots and ultimately regeneration
of Curcuma caesia Roxb. regulated by appropriate
cytokinin and auxin concentration and combinations.
As the micropropagated plants are acclamatized to sur-
vive in natural environment and all the micropropagated
plants are genetically true to type, so this protocol can
be used in industry to make large scale plant produc-
tion. Further, our results demonstrate that RAPD markers
Parthadeb Ghosh et al. 129
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can be applied to evaluate the genetic stability of
regenerants for the ex situ conservation of this impor-
tant aromatic and medicinal herb.
ACKNOWLEDGEMENT
This work was supported by Grants in Aid from
the University of Kalyani, Nadia, West Bengal.
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Available online at www.pelagiaresearchlibrary.com
Pelagia Research Library
Asian Journal of Plant Science and Research, 2013, 3(3):95-99
ISSN : 2249-7412
CODEN (USA): AJPSKY
95
Pelagia Research Library
Formulation of an antibacterial crop protectant using the acetylated derivative of
2,7,(14),10-Bisabolatriene-1,9,12-triol isolated from Curcuma longa L.
Arghya Ghosh1,2
, Parthadeb Ghosh2
and Padma Chatterjee*1
1Plant Biochemistry, Molecular Biology & Advance Plant Physiology Research Laboratory, Department of Botany,
University of Kalyani, Kalyani, Nadia, West Bengal 2Cytogenetics & Plant Breeding Section, Department of Botany, University of Kalyani, Kalyani, Nadia, West Bengal
_____________________________________________________________________________________________
ABSTRACT
The paper includes a comparative account of antibacterial assay of 2, 7, (14), 10 Bisabolatriene- 1,9,12 triol and its
acetylated derivative isolated from Curcuma longa L. The results indicated that acetylated derivative of 2, 7, (14),
10 Bisabolatriene- 1,9,12 triol showed positive results in antibacterial assay. The antibacterial assay was also done
with the 2, 7, (14), 10 Bisabolatriene- 1,9,12 triol and no antibacterial acivity was found for this compound.
Key words: Antibacterial assay; Crop protectant; Curcuma longa L.; 2, 7, (14), 10 Bisabolatriene- 1,9,12 triol;
acetylated derivative of 2, 7, (14), 10 Bisabolatriene- 1,9,12 triol.
_____________________________________________________________________________________________
INTRODUCTION
The plant kingdom is a treasure house of potential drugs and there has been an increasing awareness about their
importance of medicinal plants [1]. Plant is man’s friend in survival, giving him food and fuel and medicine from
the days beyond dawn of civilization [2]. Despite tremendous progress in human medicines, infectious diseases
caused by bacteria, fungi, viruses and parasites are still a major threat to public health [3]. According to World
Health Organization, more than 80% of the world's population relies on traditional medicine for their primary
healthcare needs [4]. The pharmaceutical industry has come to consider them as a source of bioactiveagents, which
have gained considerable importance due to their potential as antioxidative, antidiabetic, anticarcinogenic,
antimicrobial, antiallergic, antimutagenic and anti inflammatory activities [5].
The plant pathogenic bacterium studied in this paper mainly causing spots, blight, gall, rot etc of vegetables and crop
plants. The bacterium Serratia marcescens, is a phloem inhabiting bacterium causing yellow vine disease of
Cucurbits [6]. Another bacterium, Erwinia herbicola causes fire blight of pear and apple, Stewart’s wilt in corn, and
soft rot of fleshy vegetables [7]. The genus Xanthomonas causing numerous leaf spots, fruit spots, blights of annual
and perennial plants, vascular wilts and citrus canker [8]. Arthrobacter chlorophenolicus causing bacterial blight of
holly, is the cause of Douglas fir bacterial gall [9]. Turmeric has immuno enhancing properties [10]. The
antimicrobial activity of ethanolic extract of turmeric was evaluated against several strains of bacteria and fungi [11,
12, 13, 14, 15]. The rhizome extract was effective against bacteria Staphylococcus albus, E. coli, and Pseudomonas
yocyanea. The methanolic extract of C. longa inhibited the growth of Helicobacter pylori [16]. Singh et al. 2002
[17] evaluated the antibacterial potential of C. longa rhizome extracts against pathogenic strains of Gram positive
(Staphylococcus aureus, Staphylococcus epidermidis) and Gram negative (E. coli, Pseudomonas aeruginosa,
Salmonella typhimurium) bacteria. Here we report for the first time the antibacterial potentialities of acetylated
derivative of 2, 7, (19), 10 Bisabolatriene- 1,9,12 triol [18], a bisabolane type sesquiterpene isolated from C. longa
L. against four plant pathogenic bacterium.
Padma Chatterjee et al Asian J. Plant Sci. Res., 2013, 3(3):95-99
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MATERIALS AND METHODS
From the shade dried rhizomes of the plant 2, 7, (19), 10 Bisabolatriene- 1,9,12 triol was isolated, characterised and
indentified by usual physicochemo spectroscopic methods [19]. The compound was acetylated and the process of
acetylation was also explained in details [19].
Antibacterial assay of 2, 7, (19), 10 Bisabolatriene- 1,9,12 triol and its acetylated derivative
Microorganisms, culture media and incubating teparatures
The compound and its acetylated derivative were individually tested against a panel of microorganisms including
Gram negative Serratia marcescens (MTCC NO. 7298) incubated at 300C, Erwinia herbicola (MTCC NO. 3609)
incubated at 370C, Xanthomonas sp. (MTCC NO. 7444) incubated at 30
0C and Gram positive Arthrobacter
chlorophenolicus (MTCC NO. 3706) incubated at 280C. All the bacterial strains were obtained from Institute of
Microbial Technology (IMTECH), Chandigarh, India. The reference strains of bacteria were maintained on nutrient
agar medium and LB medium slants at 40C with a subculture period of 30 days.
Composition of the media
Contituents Weight / Volume Nutrient agar medium (pH 7.0)
Beef extract 1.0g
After adjusting the pH, volume of the medium was adjusted to 1 liter by adding double distilled sterile
water.
Nutrient broth medium has the same composition without agar.
Yeast extract 2.0g
Peptone 5.0g
NaCl 5.0g
Agar 15.0g
Contituents Weight / Volume LB agar medium (pH 7.0)
Tryptone 10.0g After adjusting the pH, volume of the medium was adjusted to 1 liter by adding double distilled sterile
water.
LB broth medium has the same composition without agar.
Yeast extract 5.0g
NaCl
Agar
10.0g
15.0g
Preparation of McFarland standard
The turbidity standard was prepared by mixing 0.5 ml of 1.75% (w/v) BaCl2.2H2O with 99.5 mL of 1%
H2SO4.BaSO4 (v/v). The standard was taken in screw cap test tube to compare the turbidity. The bacterial culture of
selected strains were grown for 48- 72 hours and subsequently mixed with physiological saline. Turbidity was
corrected by adding sterile saline until McFarland 0.5 BaSO4 turbidity standard 108 Colony Forming Unit (CFU) per
ml was achieved. These inocula were used for seeding of the nutrient agar medium, LB medium respectively.
Disc diffusion assay
1 mg of the compound and its acetylated derivative were separately dissolved in 1 ml of propylene glycol and then
the volume was adjusted to 10 ml by adding sterile water. The ultimate concentration reaches to 103 µg/ ml and
sterilized by filtration (0.22 µm millipore filter). The concentrations at 100 µg/ ml, 200 µg/ ml, 250 µg/ ml, 300 µg/
ml, 400 µg/ ml, 500 µg/ ml were taken in each case. The sterile paper discs (6 mm diameter) were saturated with 10
µl of the solution of the compound at a concentration of 100 µg/ ml, 200 µg/ ml, 250 µg/ ml, 300 µg/ ml, 400 µg/ ml,
500 µg/ ml and placed on the inoculated agar of 108 cfu/ml. Antibacterial tests were then carried out by disc
diffusion method [20] using 100 µl of suspension containing 108 CFU/ml of bacteria on nutrient agar medium, LB
medium respectively. Negative controls were prepared using propylene glycol. Gentamicin (10 µg/ disc) was used as
positive reference standards to determine the sensitivity of each bacterial species tested. The inoculated plates were
incubated at 300 C, 37
0 C, 30
0 C and 28
0 C respectively for 48 h, 24 h, 48 h and 72 h. Antibacterial activity was
evaluated by measuring the zone of inhibition and the diameters of these zones were measured in millimeters against
the test organisms.
Determination of Minimum inhibitory concentration
The minimal inhibitory concentration (MIC) values were studied for the bacteria strains, being sensitive to the
acetylated derivative in disc diffusion assay. The inocula of the bacterial strains were prepared from 24-72 h broth
cultures and suspensions were adjusted to 0.5 McFarland standard turbidity. The compound and its acetylated
derivative was dissolved in 1 ml of propylene glycol, were first diluted to the highest concentration (500 µg/ml) to
be tested, and then serial dilutions were made in order to obtain a concentration range from 500 to 100 µg/ml in 10
ml sterile test tubes containing nutrient broth and LB broth medium respectively. MIC values of the acetylated
derivative against bacterial strains were determined based on a micro well dilution method as previously described
[21]. The plate was covered with a sterile plate sealer and then incubated at appropriate temperatures for 24 - 72 h at
300 C, 37
0 C, 30
0 C and 28
0 C respectively. Bacterial growth was determined by absorbance at 600 nm and
confirmed by plating 10 µl samples, forming clear wells on nutrient agar medium or LB medium respectively. The
Padma Chatterjee et al Asian J. Plant Sci. Res., 2013, 3(3):95-99
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MIC was defined as the lowest concentration of the compounds to inhibit the growth of microorganisms. Each test
in this study was repeated, at least, thrice.
RESULTS AND DISCUSSION
Antibacterial assay
Antibacterial assay was performed with 2, 7, (14), 10 Bisabolatriene- 1,9,12 triol and its acetylated product. Results
in table number 1 indicate that acetylated derivative of 2, 7, (14), 10 Bisabolatriene- 1,9,12 triol was positive in
antibacterial assay and the MIC value was 220 µg/ml, 232 µg/ml, 245 µg/ml and 353 µg/ml for the bacterium
Serratia marcescens (MTCC NO. 7298), Erwinia herbicola (MTCC NO. 3609), Xanthomonas sp. (MTCC NO.
7444) and Arthrobacter chlorophenolicus (MTCC NO. 3706) respectively (Table 2 & Figure 1), where as 2, 7, (14),
10 Bisabolatriene- 1,9,12 triol was inert in its antibacterial property (Table 1).
Figure 1: MIC values (indicated by arrows) of the acetylated derivative of 2, 7, (14), 10 Bisabolatriene- 1,9,12 triol against four plant
pathogenic bacteria
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Table: 1 Antibacterial potentiality of 2, 7, (14), 10 Bisabolatriene- 1,9,12 triol
Test sample Test bacterial strains
2, 7, (14), 10 Bisabolatriene- 1,9,12 triol Diameter of inhibition zone in mm
Concentration (µg/ml) Serratia marcescens Erwinia herbicola Xanthomonas sp. Arthrobacter chlorophenolicus
500 No activity No activity No activity No activity
400 No activity No activity No activity No activity
300 No activity No activity No activity No activity
250 No activity No activity No activity No activity
200 No activity No activity No activity No activity
100 No activity No activity No activity No activity
Table: 2 Antibacterial potentiality of the acetylated derivative of 2, 7, (14), 10 Bisabolatriene- 1,9,12 triol (Mic values are indicaied within
bracket)
Test sample Test bacterial strains
Acetylated derivative Diameter of inhibition zone in mm
Concentration (µg/ml) Serratia marcescens Erwinia herbicola Xanthomonas sp. Arthrobacter chlorophenolicus
500 20.7 18.3 19.1 16
400 20 17.1 16 14.2
353 19.1 16.9 15.1 12.3 (MIC value)
300 18.2 16.2 13 No activity
253 17.5 13 11.5 No activity
250 14.3 12.7 11 No activity
245 11.2 12 9.2 (MIC value) No activity
232 8 10.5 (MIC value) No activity No activity
220 6.3 (MIC value) No activity No activity No activity
200 No activity No activity No activity No activity
100 No activity No activity No activity No activity
Hence the paper may be cited as a formulation of antibacterial crop protectant against the common pathogens
causing rot, blight and gall diseases in some vegetables as well as some crop plants. So it was concluded that the
acetylated derivative of 2, 7, (14), 10 Bisabolatriene- 1,9,12 triol possess antibacterial property.
Acknowledgement
This work was supported by Grants in Aid from DST- PURSE and University of Kalyani, Nadia, West Bengal.
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[3] Merina PD, Ankita B, Anu, 2013, Asian Journal of Plant Science and Research, 3(2):107-111.
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[5] Farzana R, Nadia S, Ijaz A, Saima S, Fakhar UN, Shagufta N, 2013, Asian Journal of Plant Science and
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[6]Astri W, 2005, Annals of the Entomological Society of America, 98(6):770-774.
[7] Lindow SE, Nickel H, 1978, Phytopathology, 68: 523-527.
[8] Guillaume D Ejean, 2013, New Phytologist, 198: 899–915.
[9] Tanja RS, Johan HJL, 2013, Microbiologyopen, 2(1): 205–213.
[10] Kuttan R, Sudheeran PC, Josph CD, 1987, Tumori genesis 73: 29-31.
[11] Chauhan UK, Soni P, Shrivastava R, Mathur KC, Khadikar PV, 2003, Oxidation Commun 26: 266-270.
[12] Saju KA, Venugopal MN, Methew MJ, 1998, Current Science 75: 660-662.
[11] Bhavani, S, 1979, Indian J. Exp. Biol. 17: 1362-1366.
[13] Banerjee, A, Nigam, SS, 1978, Indian J. Med. Res. 68: 864-866.
[14] Lutomski J, Kedzia B, Debska W, 1974, Planta Medica 26: 9-19.
[15] Ronita D, Parag K, Snehasikta S, Ramamurthy T., Chowdhury A, Balakrish GN, Asish KM, 2002, Anticancer
Res. 22: 4179-4181.
[16] Shahid M, Rahim T, Shahzad A, Tajuddin LA, Fatma T, Rashid. M, 2002, Current Science 83: 737-740.
[17] Ghosh A, Ghosh A, Ghosh PD, Chatterjee P, 2012, Journal of the Botanical Society of Bengal. 66 (2): 119-124.
[18] Bolliger HR, Brenner M, Gänshirt H, Stahl E, 1965, Thin layer chromatography, a laboratory hand book,
Academic Press INC, New york.
[19] Ghosh A, Ghosh PD, Chatterjee P, 2013, Natural Products- An Indian Journal, 9(3): 117-122.
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Manual of clinical microbiology, 6th edition, Washington, DC: ASM.
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[21] Sokmen A, Gulluce M, Akpulat HA, Tepe B, Sokmen M, Sahin F, 2004, Food Control, 15: 627–634.
Available online at www.pelagiaresearchlibrary.com
Pelagia Research Library
Advances in Applied Science Research, 2013, 4(3):39-45
ISSN: 0976-8610
CODEN (USA): AASRFC
39 Pelagia Research Library
A protocol for rapid propagation of genetically true to type Indian turmeric
(Curcuma longa L.) through in vitro culture technique
Arghya Ghosh1,2
, Padma Chatterjee1
and Parthadeb Ghosh2*
1Plant Biochemistry, Molecular Biology & Advance Plant Physiology Research Laboratory , Department of Botany,
University of Kalyani, Kalyani, Nadia, West Bengal 2Cytogenetics & Plant Breeding Section, Department of Botany, University of Kalyani, Kalyani, Nadia, West Bengal
_____________________________________________________________________________________________
ABSTRACT
As the plant Curcuma longa L. is an ethnomedicinally important one and almost all the parts of the plant are
reported to contain curcumin and its structural analogues. Rhizomatous explants from two months old buds were
cultured on Murashige and Skoog’s (MS) medium supplemented with different concentrations of cytokinins and
auxins. During the first culture on 2.5 mg/l of 6-benzylamino purine (BAP) and 1.5 mg/l of α Napthalenic acetic acid
(NAA) yeilds 9.00±0.57a number of shoots with an average shoot length of 7.20±1.01
a cm. The elongated shoots
produced 9.66±1.20a roots on half strength MS liquid medium supplemented with 2 mg/l of Indole 3 butyric acid
(IBA) and showed 86 % survivability after hardening. Genetic fidelity of the micropropagated plantlets was
confirmed using RAPD analysis employing 12 primers. This system provides high fidelity micropropagation system
for efficient and rapid micropropagation of this important medicinal plant.
Key words: Curcuma longa L., micropropagation, genetic fidelity, RAPD
_____________________________________________________________________________________________
INTRODUCTION
The genus Curcuma L. of the family Zingiberaceae is well known as the turmeric genus, because of Curcuma longa
L. C. longa is the most investigated species of this genus, although there are over 100 others in this genus [1].
Turmeric is conventionally propagated vegetatively through rhizome bits carrying one or two buds. Rich
morphological and genetic diversity is observed among the cultivated types of turmeric, probably due to vegetative
mutations accumulated over a period of time. The rarity of seed set hampers recombination breeding. In such
circumstances, biotechnological tools gain relevance in solving many crop specific problems and for crop
improvement. However, these efforts have been seriously constrained due to absence of well characterized germplasm for augmenting the need of gene pool in the genetic improvement programs. In vitro regeneration or
micropropagation is the best alternative to overcome these hurdles and it holds tremendous potential for rapid
multiplication and production of high quality medicines from them [2, 3, 4, 5, 6]. Hence, there is a need to develop
in vitro germplasm of the wild and cultivated species of Curcuma in India. There is also a need to standardize high
fidelity, rapid and reliable protocol for micropropagation of Curcuma longa. Many workers reported successful
micropropagation of turmeric [7] among them Nadgauda was the first to report micropropagation of turmeric. In this paper we have also design an alternative protocol for clonal propagation of C. longa from rhizomal explant and
assessment of its genetic fidelity through RAPD technique.
Parthadeb Ghosh et al Adv. Appl. Sci. Res., 2013, 4(3):39-45
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40 Pelagia Research Library
MATERIALS AND METHODS
Collection of explant Rhizomes of C. longa was collected in the month of July 2010 from experimental garden of Department of Botany,
University of Kalyani, which located at 22057΄ 92 N latitude, 88
022΄ E longitude with an average altitude of 9.75 m
above mean sea level. The plant was identified in the Taxonomy and Plant systematic Unit, Department of Botany,
University of Kalyani, Nadia.
Explant preparation
Young disease free rhizomal explants (rhizomal buds of 2.5 - 3 cm) were collected from 2 months old plant.
Explants were washed thoroughly under running tap water and then treated with 5% (m/v) Teepol (Qualigen,
Mumbai, India) for 15 min, followed by rinsing three to five times in sterile double distilled water. Another round of
disinfestation was done with 70% alcohol for 5 min followed by immersion in 0.1% (m/v) aqueous mercuric
chloride (HgCl2) solution for 5 - 6 min and finally rinsed with sterile double distilled water (five to six times) in a
flow chamber. The surface sterilized explants were trimmed at cut ends and about 1.2-1.5 cm prior to inoculation on
culture media.
Media and culture conditions
Surface sterilized rhizomal segments (1.2 - 1.5 cm) were cultured on MS [8] basal medium containing 3% (w/v)
sucrose (Himedia, Mumbai, India) for culture initiation and served as explant sources for subsequent experiments.
The pH of the medium was adjusted to 5.8 before gelling with 0.8% (w/v) agar (Himedia, Mumbai, India). The
explants initially were implanted vertically on the culture medium in test tube (150 × 25 nm) and plugged tightly
with non absorbent cotton. All the cultures were kept under cool fluorescent light (16 h photo period 40 µmol·m–2
s–1, Philips, India at 25°C ± 2°C) and 60% - 70 % relative humidity (RH).
Multiple shoot induction and elongation
For initial multiple shoot induction, the explants were cultured on MS medium [8] supplemented with various
concentrations of BA (0.5 - 5.0 mg/l) in combination with NAA (0.5 - 5.0 mg/l). The induced shoots were allowed
to grow for 22 days.
Rooting
Small micro shoots grown on subculture medium were transferred to half and full strength MS [8] media separately,
supplemented with various concentrations of IBA (0.5 – 5 mg/l) for root developement. IBA was filter sterilized and
added to the medium after autoclaving under the sterilized environment of laminar air flow cabinet. Data were
recorded on the percentage of rooting, the mean number of roots per shoot and the root length after four weeks of
transfer onto the rooting medium.
In vitro and ex vitro hardening of plantlets
The complete rooted plantlets with 7 - 9 fully expanded leaves were removed from the culture medium and the roots
were washed gently under running tap water to remove agar. The plantlets were transferred to plastic pots (5 cm
diameter) containing a mixture of sterilized garden soil and vermiculite in the ratio 2:1 and covered with transparent
plastic bags to ensure high humidity. Each was irrigated with 1/6 MS basal salt solution devoid of sucrose and
inositol every 4 days for 2 weeks. The growth chamber was maintained at 26°C ± 1°C, 80% - 85% relative humidity
with light intensity of 50 µmol·m–2·s–1 on a 16 h photoperiod inside the culture room conditions. The relative
humidity was reduced gradually and after 30 days the plantlets were transferred to pots (25 cm diameter) containing
garden soil and kept under green house for another 2 weeks. There are no changes in respect to morphology, growth
characteristics and floral features etc in between tissue culture regenarate plants and naturally grown field plants.
Statistical analysis
Experiments were set up in completely randomized block design. Each experiment was repeated three times with 10
- 12 replicates. Data were analyzed by one way analysis of variance (ANOVA) and the difference between means
were scored using Duncan’s Multiple Range Test P ≤ 0.05 [9] on the statistical package of SPSS (Version 10).
DNA isolation and RAPD analysis
Genomic DNA was extracted from young leaves of in vitro raised and field grown plants of Curcuma caesia Roxb.
and mother plant by Cytl trimethyl ammonium bromide (CTAB) procedure [10] with minor modifications. Quality
Parthadeb Ghosh et al Adv. Appl. Sci. Res., 2013, 4(3):39-45
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41 Pelagia Research Library
and quantity of DNA was checked on 0.8% agarose gel and also from values obtained by 260/280 nm UV
absorbance ratio [11]. Twelve arbitrary decamer RAPD primers (Bengalore Genni Pvt. Ltd., India) were used for
polymerase chain reaction (PCR) for DNA amplification. DNA finger printing profiles were compared to evaluate
clonal fidelity and genetic stability. Amplification was performed in 25 µL using PCR mixture of consisting of 2.5
µL Taq buffer, 1 µL dNTPs, 0.5 µL Taq polymerase, 2 µL DNA (approximate 50 ng/µL), 1.0 µL primer (10 pmol),
2.5 µL MgCl2, 1 µL oil and 14.5 µL MiliQ water. The PCR reaction conditions were: preheating for 5 min at 94°C;
40 cycles of 25 sec at 94°C, 20 sec at 40°C and 1.25 min at 72°C and elongation was completed by a final extension
of 6 min at 72°C. After amplification, the PCR product was resolved by electrophoresis in 1.4% agarose gel
(Himedia, Mumbai, India) and stained with ethidium bromide (0.5µg/ml). 2.0 - 23.1 kb λ DNA di-gested Hind III
was used as the DNA marker, and bands were visualized under UV light and photographed using the Gel Doc
equipment (Bio Rad). All the PCR reaction was repeated for thrice.
RESULTS AND DISCUSSION
In vitro establishment of explant
To overcome contamination problem, surface sterilization of explants was done with 0.1% aquous solution of
Mercuric chloride (HgCl2) for 2, 4, 6, 8 and 10 minutes. Mercuric chloride (HgCl2) is a very strong sterilant [12].
When the explants sterilization was done with 0.1% aquous solution of HgCl2 for 5 min, 60 % of the explants get
survived. Whereas, exposure of 0.1% aquous solution of HgCl2 above and below 5 minute prove to allow death or
contamination of explant respectively.
A= Plants in the experimental garden, B= Germination from bud of rhizome, C= Shoot induction, D= Shoot elongation, E= Shoot
multiplication, F= Clonally propagated shoot established in rooting medium, G= Induction of root with multiple shoot
Parthadeb Ghosh et al Adv. Appl. Sci. Res., 2013, 4(3):39-45
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Table 1: Standardization of 0.1% HgCl2 for explant sterilization
Serial
No.
Treatment duration (min) with
0.1% HgCl2
Number of explants
inoculated
Rate of contamination
(after day of treatment)
Percentage of contamination free
explants after 10 days
1 2 10 2 3 4 5 7 10 0
2 4 10 2 3 4 5 7 10 0
3 5 10 2 3 3 5 7 10 10'
4 6 10 2 3 3 6 6 9 60*
5 8 10 1 1 2 3 4 4 80**
6 10 10 0 0 1 1 1 2 90**
' = Death of inoculated explant due to contamination
* = Yeild of contamination free explants
**= Brownish and death of inoculated explant due to long time exposure of 0.1% HgCl2
Induction and elongation of multiple shoots
When Explants were cultured on basal MS medium or, MS medium [8] contains solely cytokinin (BA), or auxin
(NAA) failed to produce shoots even after 4 weeks of inoculation. MS medium supplemented with different
concentrations and combinations of cytokinins and auxins showed variation in the regeneration percentage and
number of shoots formed. Among the different combinations of cytokinin and auxin tested, the best response
(81.66±4.84a %) was obtained in the presence of 2.5 mg/l BA and 1.5 mg/l NAA (Figure E, F, G) after 22 days of
incubation. The average length of shoot in this medium was 7.20±1.01a. The BA and NAA concentrations higher
than above concentrations of BA and NAA, the number of shoots as well as percent response was reduced (Table 2).
This is probably due to higher concentration of nitrogen and potassium [13, 14, 15]. The stimulating effectivity of
BA and NAA on multiple shoot formation has been reported earlier for several medicinal plant [16, 17, 18, 19, 20].
Table 2: Standardization of concentrations and combinations of BA and NAA for shooting in full strength MS media
Conc. of BA and NAA % of response No. of shoots/explant Average shoot length
Basal MS (Control) 0.00±0.00f 0.00±0.00g 0.00±0.00f
MS+BA+NAA (mg/l)
0.5+0.5 69.66±3.71b 7.33±0.33abc 5.33±0.33bc
0.5+1 54.00±7.00cd 7.66±0.88abc 5.16±0.12bcd
1+1 51.00±3.05cd 8.66±1.45ab 5.46±0.24bc
1.5+1 68.00±2.08b 7.33±0.88abc 5.76±0.93b
2+1 68.33±2.33b 6.33±0.66cde 5.80±0.11b
2.5+1.5 81.66±4.84a 9.00±0.57a 7.20±1.01a
3+2 61.33±4.17bc 6.66±0.66bcd 4.23±0.39cd
3.5+2 59.00±3.05bc 6.00±0.57cde 4.76±0.39bcd
4+2.5 61.33±3.48bc 4.33±0.33ef 3.83±0.44d
4.5+3 47.66±2.72d 5.00±0.57def 1.66±0.33e
5+4 34.00±5.56e 3.66±0.66f 2.26±0.37e
**Values are means ± SE. n = 10 - 12 (in triplicate); Means followed by same does not differ significantly according to Duncan’s Multiple Range
Test (p ≤ 0.05).
Induction of rooting
Healthy elongated shoots (4 - 9 cm in length) were excised and placed on both, full and half strength MS basal
medium [8] supplemented with different concentrations of auxin (IBA) at the range of 0.5 - 5.0 mg /l for induction
of roots (Table 3). In the preliminary experiments conducted, no rooting was observed when the shoots were culture
on basal (Control) MS medium. Full strength MS medium containing auxins (IBA) showed very poor response in
rooting even after 25 days, but well developed roots were achieved on half strength MS medium supplemented with
IBA (2 mg/l) with increase sucrose concentration (4%) gave us well developed roots within 15 - 20 days [21, 12,
23]. In this medium shoot formed roots at a high frequency of 86.33±6.11a % and attaining an average length of
8.46±0.37a
cm were obtained. Further increase in the IBA concentration leads to reduces root initiation [24].
Parthadeb Ghosh et al Adv. Appl. Sci. Res., 2013, 4(3):39-45
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H= Elongation of root, I= Mature clonally propagated plant with multiple shoot and root, J= Hardening of clonally propagated plants,
K= RAPD analysis to detect genetic fidelity (lane 1, 2 contains genomic DNA from field plant & lane 3, 4, 5 contains genomic DNA from
in vitro grown plant
Hardening
The well developed rooted plantlets were taken out gently from the test tubes and thoroughly washed with sterile
water to remove adhered agar and traces of the medium to avoid contamination. The micropropagted plantlets were
transferred to plastic pots containing potting a mixture of (2:1) soil and vermiculite (Figure J) in green house. Finally
the acclimated plants were then shifted to the field conditions showing 86 % of survivility. The growth
characteristics of in vitro raised plants were indentical in morphological with naturally occurring field plants.
RAPD analysis
There are many reports on molecular characterization of micropropagated plants by the RAPD technique especially
to confirm the clonal fidelity and genetic stability among tissue culture grown plants and donor [25, 26, 27]. In this
paper we have performed the genetic integrity of in vitro regenerated plants from rhizomal explants and respective
naturally occurring field grown donor plant of Curcuma longa L.
Total 12 primers were initially screened and finally 6 primers produce clear and scorable amplified bands ranging
from 3 - 5 bands per primer (Table 4). Each primer produced a unique set of amplification products ranging in size
from 100 bp - 3 kb (Figure K with primer 5' CGGGATCCGC 3'). All 6 primers produced a total of 23 bands with an
average of 3.84 fragments. All the scorable bands were monomorphic in nature, indicating homogeneity among the
culture regenerates and genetic uniformity with that of the donor plants. The possible reason may be multiple shoot
bud differentiation without intervening callus phase is least vulnerable to genetic changes. However, no differences
were observed between mother plant and plantlets regenerated from rhizomal segments by any five primers tested in
present RAPD study.
Parthadeb Ghosh et al Adv. Appl. Sci. Res., 2013, 4(3):39-45
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44 Pelagia Research Library
Table 3: Standardization of concentrations of IBA for rooting in half and full strength MS media
Rooting in MS half strength
Basal MS ½ (Control) % of response No. of roots/explant Average root length
MS½ +IBA (mg/l) 0.00±0.00k 0.00±0.00i 0.00±0.00j
0.5 15.00±0.57j 2.00±0.57ghi 3.66±0.14efgh
1 23.33±0.88ij 5.66±0.33bcd 5.56±0.21c
1.5 34.66±0.88fgh 4.66±0.33bcde 7.00±1.00b
2 86.33±6.11a 9.66±1.20a 8.46±0.37a
2.5 49.00±3.78cd 5.66±0.33bcd 5.40±0.15cd
3 48.33±6.00cd 5.66±0.66bcd 4.93±0.14cde
3.5 45.66±2.90cde 4.33±0.33cdef 4.26±0.17cdef
4 39.33±2.60efg 6.33±0.33bc 3.96±0.52ef
4.5 32.00±2.64gh 5.00±0.57bcde 2.60±0.26ghi
5 16.33±2.40j 3.33±0.33defg 2.40±0.66hi
Rooting in MS full strength
Basal MS (Control) 0.00±0.00k 0.00±0.00i 0.00±0.00j
MS+IBA (mg/l)
0.5 3.33±0.66k 1.33±0.33hi 4.16±0.26def
1 17.33±1.85j 2.66±0.66efg 4.60±0.23cdef
1.5 51.66±2.84c 5.33±0.88bcd 4.40±0.20cdef
2 51.00±1.15cd 4.66±0.88bcde 4.03±0.31def
2.5 76.66±2.90b 7.00±1.15b 4.90±0.49cde
3 80.33±3.52ab 6.33±0.88bc 3.33±0.33fghi
3.5 51.66±0.88c 4.33±0.88cdef 3.83±0.68efg
4 42.00±4.58def 4.33±0.66cdef 2.33±0.33hi
4.5 35.00±2.30fgh 3.66±1.20def 3.80±0.72efg
5 28.33±2.02hi 4.00±1.00cdef 2.00±0.40i
**Values are means ± SE. n = 10 - 12 (in triplicate); Means followed by same does not differ significantly according to Duncan’s Multiple Range
Test (p ≤ 0.05).
Table 4: Number of amplification products generated with the use of RAPD primers to assess genetic fidelity of micropropagated and
field grown plants
Serial No. Primer Code Nucleotide sequence
(5'-3') Number of generated bands
1 RAPD1 GTCCTACTCG -
2 RAPD2 GTCCTTAGCG 3
3 RAPD3 CGGGATCCGC 5
4 RAPD4 CTTCCGGCAG 4
5 RAPD5 GGTATTACTT 4
6 RAPD6 TGGCTCGGTA -
7 RAPD7 CTTCGGCAGA -
8 RAPD8 GGTATTACTT -
9 RAPD9 GACAATGGTA -
10 RAPD10 TTAGCTTAGG -
11 RAPD11 CTCTCCGCCA 3
12 RAPD12 GCACGCCGGA 4
CONCLUSION
In this present study, we have established an efficient and reliable micropropagation protocol for in vitro
regeneration of Curcuma longa L. from rhizomal explant, which can ensure large scale propagation, as well as
protocol can also be used for raising genetically uniform plants, which is important for the sustainable supply of
plant materials to the pharmaceutical industries and for conservation of elite germplasm.
Acknowledgements
This work was supported by Grants in Aid from the University of Kalyani, Nadia, West Bengal.
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795
Journal of Scientific and Innovative Research 2013; 2 (4): 795-801
Available online at: www.jsirjournal.com
Research Article
ISSN 2230-4818
JSIR 2013; 2(4): 795-801
© 2013, All rights reserved
Received: 10-08-2013
Accepted: 24-09-2013
Arghya Ghosh Plant Biochemistry, Molecular
Biology & Advance Plant Physiology Research Laboratory,
Department of Botany, University of Kalyani, West Bengal, India
Ayan Bandyopadhyay
Department of Chemistry,
University of Kalyani, West Bengal, India
Parthadeb Ghosh
Cytogenetics & Plant Breeding
Section, Department of Botany, University of Kalyani, West Bengal, India
Padma Chatterjee*
Plant Biochemistry, Molecular
Biology & Advance Plant Physiology Research Laboratory,
Department of Botany, University of Kalyani, West Bengal, India
*Correspondence: Prof. Padma Chatterjee
Department of Botany, University
of Kalyani, West Bengal, Indi-
741235
Tel: 033-25828220, 033-25828275
Fax: 0091-033-2582-8282
E-mail:
schatterjeecal2003@yahoo.co.in
Isolation of (Z)-7-methoxy-1, 5-dihydrobenzo[c] oxepine
from Curcuma caesia Roxb.
Arghya Ghosh, Ayan Bandyopadhyay, Parthadeb Ghosh, Padma Chatterjee
Abstract
Isolation, purification and finally Chemical characterisation of (Z)-7-methoxy-1, 5-
dihydrobenzo[c] oxepine was done describe in this paper. This compound seemed to be a
terpenoid isolated from Curcuma caesia Roxb., an endemic and naturally occurring triploid
plant of northeast Asia. For chemical characterization of the compound various spectroscopic
techniques like UV, IR (FT-IR), HRMS and NMR was used. The compound was positive in
Liebermann’s Burchard test and had melting point of 570C. Best of our knowledge this was the
first report of presence of (Z)-7-methoxy-1, 5-dihydrobenzo [c] oxepine in plants.
Keywords: Curcuma caesia Roxb., (Z)-7-methoxy-1, 5-dihydrobenzo [c] oxepine,
Physicochemical characterization
Introduction
Curcuma caesia Roxb. (Black turmeric) of the family Zingiberaceae is a natural
triploid, endemic and ethnomedicinally important plant. This plant was used by the
tribal’s of northeast India for its unique medicinal properties. There was no such report
on the bioactive potentialities of black turmeric. So far eight metabolites have been
isolated and characterised from Curcuma caesia Roxb. like Borneol, Borneol acetate,
1,8-Cineole, α-Curcumene, γ-Curcumene, β-Elemene, (E)-β-Ocimene, ar-Turmerone
etc.1, 2
Here we report for the first time the presence of (Z)-7-methoxy-1,5-
dihydrobenzo[c] oxepine from the shade dried rhizome of Curcuma caesia Roxb. Best
of our knowledge this compound has seem to be a novel one which was not reported
earlier.
Materials and Methods
Collection of Plant Material
Whole plant of C. caesia Roxb. was collected in the month of July 2010 from
experimental garden of Department of Botany, University of Kalyani, and was
identified in the Department of Botany, University of Kalyani, Nadia.
Extraction and Isolation of Crude Secondary Metabolite Content
2.5 kg shade dried rhizomes of black turmeric plant was powdered of approximately
and extracted three times with 1 liter of 95% EtOH at room temperature to give an
extract of 479 gms. The extract was evaporated under reduced pressure and a solid
Journal of Scientific and Innovative Research
796
residual mass was obtained. The above obtained residual
sample was subjected to repeated preparative thin layer
chromatography using different solvent systems, e.g
solvent system 1. Methanol (5%): benzene (95%) and
solvent system 2. Chloroform (60%): benzene (30%):
acetic acid (10%). Three homogeneous spots were
collected in solvent system 2, having Rf values of 0.87,
0.79 and 0.75 respectively. The sample with Rf value 0.75
was taken up for further study. This sample was positive in
Liebermann’s Burchard test3 and gave purple colour
indicating its terpenoid nature. The compound had melting
point of 570C. The sample was then further analysed
through various spectroscopic techniques like UV
spectroscopy (UV- 1601PC, UV-Visible
Spectrophotometer, Shimadzu), FT-IR spectroscopy
(Perkin Elmer Spectrum- 1 Spectrophotometer), High
Resolution Mass spectroscopy (JEOL- JMS 600
Instrument) and Nuclear Magnetic Resonance
spectroscopy, 1H & 13C (Bruker Avance- 400
Spectrometer) for its proper physicochemical
characterization.
Results and Discussions
Chemical Characterization of the Isolated Sample
The compound was reddish yellow amorphous solid and
was readily soluble in methanol. The melting point of the
sample was 57 0C (Rf- 0.75 in solvent system 2) and it
turned purple in Liebermann’s Burchard test.3 The TLC
chromatogram of the compound showed single spot when
visualized under exposure of UV light as well as iodine
vapor.
Baeyer's Test for Presence of Double or Triple Bond
In ~2-3 mg of the isolated compound in methanol was
added in the very much diluted alkaline solution of
potassium permanganate. The purplish pink colour of the
reaction mixture turns to brown indicating presence of an
active unsaturation (double or triple bond) in the
compound.
UV Spectroscopy of the Isolated Sample
The methanolic spectrum of the sample showed λ max at 854.0 nm, 522.0 nm, 476.50 nm, 281.50 nm, 228.0 nm and
absorbance at = 0.0010, 0.0035, 0.0036, 1.8965, 3.0211 respectively. (Spectrum 1)
Figure 1: Spectrum 1
Journal of Scientific and Innovative Research
797
IR (FT-IR) Spectroscopy of the Isolated Sample
The IR spectrum of the sample showed n- (cm-1): 3078, 2939, 2843, 1638, 1612, 1514, 1465, 1432, 1368, 1268, 1234,
1149, 1122, 1035, 996, 817, 793 and 747. (Spectrum 2)
Figure 2: Spectrum 2
Journal of Scientific and Innovative Research
798
High Resolution Mass spectroscopy of the Isolated Sample
The mass of the sample was noted as to be (TOF MS ES+) 528. 9609 (3M
+) (Spectrum 3)
Figure 3: Spectrum 3
Nuclear Magnetic Resonance Spectroscopy of the Isolated Sample
1H NMR (400 MHz, CDCl3):
δ. 6.84 (1H, dd, J = 8.4, 3.6 Hz, Ar-H-8), 6.67 (2H, d, J = 5.2 Hz, Ar-H-9 and Ar-H-6), 5.95 (1H, dt, J = 8.4, 6.8 Hz
olifinic-H-4), 5.63 (1H, s, olifinic-H-3), 5.08-5.03 (2H, m, H-1), 3.82 (3H, s, H-12), 3.30 (2H, d, J = 6.4 Hz, H-5).
(Spectrum 4)
Journal of Scientific and Innovative Research
799
Figure 4: Spectrum 4
13C NMR and DEPT-135 (100 MHz, CDCl3):
δ. 146.6 (C), 144.0 (C), 138.0 (CH), 132.0(C), 121.3 (2CH), 115.6 (CH2), 114.5 (CH), 111.3 (CH), 55.9 (CH3), 39.9
(CH2). (Spectrum 5 & 6)
Journal of Scientific and Innovative Research
800
Figure 5: Spectrum 5
Figure 6: Spectrum 6
Journal of Scientific and Innovative Research
801
Interpretation of the Structure of the Isolated
Compound
The U.V spectrum of the compound showed the intense
absorption peak (λ max) at 281 nm, indicating the presence
of benzene ring with auxochromic functionality. When I.R
spectrum of this compound was recorded, no characteristic
strong absorption peaks in functional group region were
found. The weak absorption at 3078 cm-1 was assigned for
aromatic C-H stretching. Aliphatic C-H stretching was also
found at 2939 cm-1 (asymmetric) and 2843 cm-1
(symmetric). An absorption peak of medium intensity at
1612 cm-1 was also observed indicating the aliphatic C=C
stretching. In addition to that, the quite intense band at
1514 cm-1 indicated the existence of aromatic C=C
stretching.4
1H-NMR spectrum of compound in figure 1 exhibited the
presence of 12 protons. Among which δ = 6.84 ppm (1H,
dd, J = 8.4, 3.6 Hz, H-8) and 6.67 ppm (2H, d, J = 5.2 Hz,
H-9 and H-6), confirmed the presence of three aromatic
protons. Two olefinic protons had been obtained at δ =
5.95 ppm (1H, dt, J = 8.4, 6.8 Hz, H-4) and at 5.63 ppm
(1H, s, H-3). Two different type of -CH2 (Methylene)
protons was also observed at 5.08-5.03 ppm (2H, m, H-1),
and 3.30 ppm (2H, d, J = 6.4 Hz, H-5). Remaining 3
protons of methyl group was located at 3.82 (3H, s, H-12)
in the 1H-NMR-spectrum.
O
MeO6
7
8
910
1 2
3
45
11
12
Figure 7: Structure of (Z)-7-methoxy-1,5-dihydrobenzo[c]
oxepine
The ten peaks in 13C-NMR-spctrum clearly indicated the
presence of 11 different carbon atoms in which one signal
at δ = 121.3 ppm was accounted for two signals with
double intensity both in 13C-NMR and DEPT-135 spectra.
The DEPT-135 spectrum of the compound in figure 7
showed 7 signals out of which two are negative signals (δ
= 115.6 and 39.9 ppm) indicating the presence of two
methylene carbon in the compound. Remaining five
positive signals ware accounted for five methine (δ =
137.9, 121.3, 114.3, 111.2 ppm) and one methyl carbons (δ
= 55.9). Thus based on the 13C-NMR and DEPT-135
spectral data, compounded in figure 7 was accounted to
have one methyl, two methylene, five methane and three
quaternary carbons. The HRMS-spectrum of the isolated
compound was found 528.9609 (3M+). Hence, the
molecular formula of the isolated fraction must be
C11H12O2 and its structure was shown in Figure 7.
Acknowledgements
This work was supported by Grants in Aid from the DST-
PURSE (Grant No. FD/1626, dated: 29.1.2013) and
University of Kalyani (Grant No. Rev/ 1124 of 12-13,
dated: 28.2.2013), Nadia, West Bengal, India.
References
1. Pandey A.K., Chowdhury A.R., Volatile constituents of
the rhizome oil of Curcuma caesia Roxb. from central
India, Flavour and Fragrance Journal, 2003; 18: 463-465.
2. Behura S., Srivastava V.K., Essential oils of leaves of
Curcuma species, J Essential Oil Res, 2004; 16: 109-110.
3. Bolligr H.R., Brenner M., Ganshirt H., Mangoli H.K.,
Seiler H., Stahl E., Waldi D., Thin layer chromatography, a
laboratory hand book, 1965, p. 492.
4. Dyer J.R., Williams T.R., Applications of absorption
spectroscopy of organic compounds., J. Chem. Educ.,
1965; 42(12): 690.
745
Journal of Scientific and Innovative Research 2013; 2 (4): 745-750
Available online at: www.jsirjournal.com
Research Article
ISSN 2230-4818
JSIR 2013; 2(4): 745-750
© 2013, All rights reserved
Received: 10-08-2013
Accepted: 28-09-2013
Arghya Ghosh Plant Biochemistry, Molecular
Biology & Advance Plant Physiology Research Laboratory,
Department of Botany, University of Kalyani, West Bengal, India
Parthadeb Ghosh
Cytogenetics & Plant Breeding Section, Department of Botany,
University of Kalyani, West Bengal, India
Padma Chatterjee*
Plant Biochemistry, Molecular Biology & Advance Plant
Physiology Research Laboratory, Department of Botany, University
of Kalyani, West Bengal, India
*Correspondence: Prof. Padma Chatterjee
Department of Botany, University
of Kalyani, West Bengal, Indi-
741235
Tel: 033-25828220, 033-25828275
Fax: 0091-033-2582-8282
E-mail:
schatterjeecal2003@yahoo.co.in
Evaluation of Antimicrobial and Antifungal potential of
(Z)-7-methoxy-1, 5-dihydrobenzo[c] oxepine, isolated
from Curcuma caesia Roxb.
Arghya Ghosh, Parthadeb Ghosh, Padma Chatterjee
Abstract
The assessment of antimicrobial potentiality of (Z)-7-methoxy-1, 5-dihydrobenzo[c] oxepine
was the primary objective of this study. Assessment of its antimicrobial activity was performed
using agar cup method and disc diffusion assay respectively. The bioactive compound was
terpenoid in nature. The MIC values tested against different plant pathogenic fungi and bacteria.
Antifungal assay showed the MIC values as 22 mg/ ml, 27 mg/ml, and 17 mg/ml for the test
fungi Botrytis cinerea, Fusarium oxysporum and Rhizopus oryzae respectively. The MIC values
of this mono-terpenoid was 267 µg/ml, 291 µg/ml, 345 µg/ml, and 467 µg/ml tested against the
bacterium Serratia marcescens, Erwinia herbicola, Xanthomonas sp. and Arthrobacter
chlorophenolicus respectively. This is the first report of the bioactive nature of (Z)-7-methoxy-
1, 5-dihydrobenzo[c]oxepine. Due to its antimicrobial property it may function in plant defense
or as an ecofriendly crop protectant or in ethnomedicinal purpose.
Keywords: Curcuma caesia Roxb., (Z)-7-methoxy-1,5-dihydrobenzo [c] oxepine,
Antibacterial assay, Antifungal assay, Agar cup method, Disc diffusion assay, Crop
Protectant.
Introduction
Now a day, solving the mystery of bioactive potentiality of natural product is one of the
largest thrust areas of research in life science. Nature possesses all the disease curing
agents (bioactive phytochemicals) that we need to reveal for our healthy life style. The
use of Neem, Turmeric etc. in Indian tradition is found from ancient time, even when
people do not exactly know the actual bioactive potentiality or mode of action of
phytochemicals present there in. The genus Curcuma belongs to the family
Zingiberaceae and contains 49 genera and 1400 species. In addition to Curcuma longa,
C. zedoaria Rosc., C. caesia Roxb. and C. xanthorrhiza Roxb. are also minor sources
of curcumin colour. The antimicrobial activities of methanolic extract were evaluated
against several strains of bacteria and fungi.1, 2, 3, 4, 5
The rhizome extract was effective
against fungi Fusarium oxysporium, Aspergillus niger, A. nidulans, and Alternaria
solani and bacteria Staphylococcus albus, E. coli, and Pseudomonas pyocyanea.
Fungicidal activity of C. longa was also reported against Botrytis cinerea, Erysiphe
graminis, Phytophthora infestans, Puccinia recondita, Pyricularia oryzae, and
Rhizoctonia solani.6 The experimental plant, C. caesia Roxb. (Black turmeric) of the
family Zingiberaceae is a natural triploid, endemic and ethnomedicinally important
plant.
Journal of Scientific and Innovative Research
746
Use of rhizomal extract of C. caesia Roxb. was very
common among the tribal’s of northeast India for its
unique antimicrobial properties. In this paper we report for
the first time the antimicrobial nature of (Z)-7-methoxy-1,
5-dihydrobenzo[c] oxepine isolated from the shade dried
rhizome of Curcuma caesia Roxb.
Materials and methods
Collection of Plant Material
Whole plant of C. caesia was collected in the month of
July 2010 from experimental garden of Department of
Botany, University of Kalyani, and identified in the
Department of Botany, University of Kalyani, Nadia.
Extraction and isolation of crude secondary metabolite
content
2.5 kg shade dried rhizomes of black turmeric plant was
powdered and extracted three times with 1 liter of 95%
EtOH at room temperature to give an extract of 479 gms.
The extract was evaporated under reduced pressure and a
solid residual mass was obtained. The above obtained
residual sample was subjected to repeated preparative thin
layer chromatography using different solvent systems, e.g
solvent system 1. Methanol (5%): benzene (95%) and
solvent system 2. Chloroform (60%): benzene (30%):
acetic acid (10%). Three homogeneous spots were
collected in solvent system 2, having Rf values of 0.87,
0.79 and 0.75 respectively. The sample with Rf value 0.75
was taken up for further study. This sample was positive in
Liebermann’s Burchard test7 indicating its terpenoid nature
and identified as (Z)-7-methoxy-1, 5-dihydrobenzo [c]
oxepine.
Antifungal Assay
Preparation of Sample Solution
Approximately 1g of the sample isolated from C. caesia
and transferred to a 20 ml volumetric flask. The compound
was totally solubilised in 1 ml of propylene glycol and the
total volume of the stock solution of the sample was
adjusted to 10 ml by addition of sterile double distilled
water. So the concentration of the stock solution of the
sample was 100 mg/ml. By diluting the concentration of
stock solution with the help of addition of sterile double
distilled water different concentrations of the isolated
sample like 25 mg/ml to 5 mg/ml was made. Propylene
glycol with sterile double distilled water was loaded into
the agar cup to maintain the control set. The test solutions
were allowed to diffuse into the agar from the cup. All the
dilutions were sterilised by filtration using membrane filter
(0.02µ pore size).
Fungal Strains
The reference strains used in the antifungal assays were:
Fusarium oxysporum, Botrytis cinerea; Rhizopus oryzae.
All the fungal strains were procured from the Plant
Biochemistry, Molecular Biology & Advance Plant
Physiology Research Laboratory, Department of Botany,
University of Kalyani, India. The test fungal strains were
maintained on PDA medium (pH-6.8) slants at 290C.
Assessment of the Antifungal Potentialities
Antifungal activity was screened by agar cup method.8-11
The isolated sample was tested against three plant
pathogenic fungi like Fusarium oxysporum, Botrytis
cinerea; and Rhizopus oryzae to access their antifungal
nature. The PDA medium was poured in to the sterile petri
plates and allowed to solidify under the sterile environment
of the laminar air flow cabinet. The test fungal cultures
were evenly spread over the media by sterile cotton swabs.
Then wells of 9 millimeter were made in the medium using
sterile cork borer. 100 µl of each sample having different
concentrations were transferred into the separate wells
which was made within the PDA medium. Plates
containing the pure cultures of Rhizopus oryzae and
Botrytis cinerea were allowed to incubated at 290C for 48-
72 hours where as plates containing the pure cultures of
Fusarium oxysporum takes incubation periods of 15-20
days at 290C. After the incubation period was over the
plates were observed for formation of clear inhibition zone
around the well indicated the presence of their antifungal
nature. The zone of inhibition was recorded in millimeter
scale. The final measurement was taken when the control
reached the full size within the petridish. If a culture grew
in an irregular shape, two or more measurements were
made and an average was recorded. From the growth of the
diameter of the fungal colony, the effective concentration
for colony growth inhibition was calculated. All the above
observations were taken in triplicate on each fungus/
sample concentration combinations. One control set was
prepared identical to these and taking propylene glycol
instead of different concentration combinations of sample
solutions.
Antibacterial assay
Microorganisms, Culture Media and their Incubating
Environment
Journal of Scientific and Innovative Research
747
The isolated sample were individually tested against a
panel of microorganisms including Gram negative Serratia
marcescens (MTCC NO. 7298) incubated at 300C, Erwinia
herbicola (MTCC NO. 3609) incubated at 370C,
Xanthomonas sp. (MTCC NO. 7444) incubated at 300C
and Gram positive Arthrobacter chlorophenolicus (MTCC
NO. 3706) incubated at 280C. All the bacterial strains were
obtained from Institute of Microbial Technology
(IMTECH), Chandigarh, India. The reference strains of
bacteria were maintained on nutrient agar medium and LB
medium slants at 40C with a subculture period of 30 days.
Composition of the Media
Details of composition of the media in which test microorganisms were grown are given in table 1.
Table 1: Composition of the media for test bacterium
Medium Constituents Weight / Volume Description
A. Nutrient agar medium
(pH 7.0)
Beef extract 1.0g After adjusting the pH, volume of the
medium was adjusted to 1 liter by adding
double distilled sterile water.
Nutrient broth medium has the same
composition without agar.
Yeast extract 2.0g
Peptone 5.0g
NaCl 5.0g
Agar 15.0g
B. LB agar medium (pH
7.0)
Tryptone 10.0g After adjusting the pH, volume of the
medium was adjusted to 1 liter by adding
double distilled sterile water.
LB broth medium has the same
composition without agar.
Yeast extract 5.0g
NaCl 10.0g
Agar 15.0g
Preparation of Mcfarland Standard
The turbidity standard was prepared by mixing 0.5 ml of
1.75% (w/v) BaCl2.2H2O with 99.5 mL of 1%
H2SO4.BaSO4 (v/v). The standard was taken in screw cap
test tube to compare the turbidity. The bacterial culture of
selected strains were grown for 48- 72 hours and
subsequently mixed with physiological saline. Turbidity
was corrected by adding sterile saline until McFarland 0.5
BaSO4 turbidity standard 108 Colony Forming Unit (CFU)
per ml was achieved. These inocula were used for seeding
of the nutrient agar medium and LB medium respectively.
Disc Diffusion Assay
1 mg of the isolated sample was separately dissolved in 1
ml of propylene glycol and then the volume was adjusted
to 10 ml by adding sterile water. The ultimate
concentration reaches to 103 μg/ ml and sterilized by
filtration (0.22 μm filter). The concentrations at 500 to 100
μg/ ml were taken in each case. The sterile paper discs (6
mm diameter) were saturated with 10 μl of the solution of
the compound at a concentration of 500 to 100 μg/ml and
placed on the inoculated agar of 108 CFU/ml. Antibacterial
tests were then carried out by disc diffusion method12
using
100 μl of suspension containing 108 CFU/ml of bacteria on
nutrient agar medium and LB medium respectively.
Negative controls were prepared using propylene glycol.
Gentamicin (10 μg/ disc) was used as positive reference
standards to determine the sensitivity of each bacterial
species tested. The inoculated plates were incubated at
300C, 37
0C, 30
0C and 28
0C respectively for 48 h, 24 h, 48
h and 72 h. Antibacterial activity was evaluated by
measuring the zone of inhibition and the diameters of these
zones were measured in millimeters against the test
organisms.13-17
Determination of Minimum Inhibitory Concentration
The minimal inhibitory concentration (MIC) values were
studied for the bacteria strains, being sensitive to this
compound in disc diffusion assay. The inocula of the
bacterial strains were prepared from 24-72 hr broth
cultures and suspensions were adjusted to 0.5 McFarland
standard turbidity. The compound was dissolved in 1 ml of
propylene glycol, were first diluted to the highest
Journal of Scientific and Innovative Research
748
concentration (500 μg/ml) to be tested, and then serial
dilutions were made in order to obtain a concentration
range from 500 to 100 μg/ml in 10 ml sterile test tubes
containing nutrient broth and LB broth medium
respectively. MIC values of the compound against
bacterial strains were determined based on a micro well
dilution method as previously described.18, 19
The plate was
covered with a sterile plate sealer and then incubated at
appropriate temperatures for 24 - 72 h at 300C, 37
0C, 30
0C
and 280C respectively. Bacterial growth was determined by
absorbance at 600 nm and confirmed by plating 10 μl
samples, forming clear wells on nutrient agar medium or
LB medium respectively. The MIC was defined as the
lowest concentration of the compounds to inhibit the
growth of microorganisms. Each test in this study was
repeated, at least, thrice.
Statistical Analysis
All the data represented in table number 2 and 3 obtained
during in vitro experiments were expressed as mean ±
standard deviation. Calculation was done with the help of
spread sheet software Microsoft Excel 2010. * Indicates
significance at (P<0.05).
Results
Assessment of Antifungal Potentialities
The minimum inhibitory concentration (MIC) values of
(Z)-7-methoxy-1, 5-dihydrobenzo[c] oxepine against
Botrytis cinerea, Fusarium oxysporum and Rhizopus
oryzae were 22 mg/ml, 27 mg/ml and 17 mg/ml
repectively (Table 2).
Table 2: Assessment of antifungal potentialities of (Z)-7-methoxy-1, 5-dihydrobenzo[c] oxepine
Serial number Fungal strains Concentration of the
compound (MIC
values)
Diameter of inhibition
zone in mm
1 Botrytis cinerea 22 mg/ml 12±0.15
2 Fusarium oxysporum 27 mg/ml 7±0.21
3 Rhizopus oryzae 17 mg/ml 6.5±0.15
Assessment of Antibacterial Potentialities
Antibacterial assay was performed with (Z)-7-methoxy-1,
5-dihydrobenzo[c] oxepine against four plant pathogenic
bacterium and the MIC value was 267 µg/ml, 291 µg/ml,
345 µg/ml and 467 µg/ml for the bacterium Serratia
marcescens (MTCC NO. 7298), Erwinia herbicola
(MTCC NO. 3609), Xanthomonas sp. (MTCC NO. 7444)
and Arthrobacter chlorophenolicus (MTCC NO. 3706)
respectively (Table 3).
Table 3: Assessment of antibacterial potentialities of (Z)-7-methoxy-1, 5-dihydrobenzo[c] oxepine
Serial number Bacterial strains Concentration of the
compound (MIC
values)
Diameter of inhibition
zone in mm
1 Serratia marcescens 267 µg/ml 5.4±0.15
2 Erwinia herbicola 291 µg/ml 9±0.20
3 Xanthomonas sp. 345 µg/ml 23±0.30
4 Arthrobacter
chlorophenolicus
467 µg/ml 10±.20
Journal of Scientific and Innovative Research
749
Discussions
The isolated fraction (Z)-7-methoxy-1, 5-dihydrobenzo[c]
oxepine shows antifungal as well as antibacterial activities
against some major plant pathogenic microbes. This
terpenoid may be involved in the host defense mechanism
of black turmeric. Isolation and characterization of this
terpenoid including evaluation of its antimicrobial
potentialities may also help us to use (Z)-7-methoxy-1, 5-
dihydrobenzo[c] oxepine as a crop protactant.
Acknowledgements
This work was supported by Grants in Aid from the DST-
PURSE (Grant No. FD/1626, dated: 29.1.2013) and
University of Kalyani (Grant No. Rev/ 1124 of 12-13,
dated: 28.2.2013), Nadia, West Bengal, India.
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Biomedicine. 2011; 1(5): 409-412.
777
Journal of Scientific and Innovative Research 2013; 2 (4): 777-784
Available online at: www.jsirjournal.com
Research Article
ISSN 2230-4818
JSIR 2013; 2(4): 777-784
© 2013, All rights reserved
Received: 18-08-2013
Accepted: 29-09-2013
Arghya Ghosh Plant Biochemistry, Molecular
Biology & Advance Plant Physiology Research Laboratory,
Department of Botany, University of Kalyani, West Bengal, India
Ayan Bandyopadhyay
Department of Chemistry,
University of Kalyani, West Bengal, India
Parthadeb Ghosh
Cytogenetics & Plant Breeding
Section, Department of Botany, University of Kalyani, West Bengal, India
Padma Chatterjee*
Plant Biochemistry, Molecular
Biology & Advance Plant Physiology Research Laboratory,
Department of Botany, University of Kalyani, West Bengal, India
*Correspondence: Prof. Padma Chatterjee
Department of Botany, University
of Kalyani, West Bengal, Indi-
741235
Tel: 033-25828220, 033-25828275
Fax: 0091-033-2582-8282
E-mail:
schatterjeecal2003@yahoo.co.in
Isolation of a novel terpenoid from the rhizome of
Curcuma caesia Roxb.
Arghya Ghosh, Ayan Bandyopadhyay, Parthadeb Ghosh, Padma Chatterjee
Abstract
Isolation and characterisation of a novel terpenoid from the rhizome of Curcuma caesia Roxb.
(Black turmeric) followed by assessment of its bioactivity. Chemical characterisation of the
sample was done through UV, IR (FT-IR), HRMS and NMR spectroscopic techniques. The
sample was identified as (2Z,2'Z)- 2,2'-(3aR,10aS)- 1,3,5,8,9,9- hexamethyl-1,2,3,3a-
tetrahydrobenzo [f] azulene-4,10 (5H,8H,9H,10aH)- diylidene) diacetaldehyde. This study is
probably the first report of presence of (2Z,2'Z)-2,2'- (3aR,10aS)- 1,3,5,8,9,9- hexamethyl-
1,2,3,3a- tetrahydrobenzo [f] azulene- 4,10 (5H,8H,9H,10aH)- diylidene) diacetaldehyde in
plants.
Keywords: Curcuma caesia Roxb., (2Z,2'Z)-2,2'- (3aR,10aS)-1,3,5,8,9,9- hexamethyl-
1,2,3,3a-tetrahydrobenzo [f] azulene- 4,10 (5H,8H,9H,10aH) -diylidene)
diacetaldehyde, Physiochemical characterisation, Isolation.
Introduction
Curcuma caesia Roxb. (Black turmeric) of the family Zingeberaceae is an important
unexplored plant valued all over the Asia for its medicinal properties. Black turmeric is
an uncommon endemic as well as ethnomedicinally important of South East Asia. It is
a natural triploid plant and has a reduced growth rate. Black turmeric powder is utilised
by several tribals of the district Nadia of West Bengal, India to incerase the mucus
content in gastric juices, to treat fevers, stomach problems, allergies, diarrhea, chronic
cough, heartburn, wind, bloating, colic, bronchial asthma, flatulence, and jaundice and
other liver ailments. Externally, it has been used for reducing inflammation and
swelling due to sprains, cuts, and bruises. So far eight natural products have been
isolated and characterised from Curcuma caesia Roxb. like Borneol, Borneol acetate,
1,8-Cineole, α-Curcumene, γ-Curcumene, β-Elemene, (E)-β-Ocimene, ar-Turmerone
etc. 1, 2
. Here we report for the first time the presence of (2Z,2'Z)-2,2'- (3aR,10aS)-
1,3,5,8,9,9-hexamethyl- 1,2,3,3a -tetrahydrobenzo [f] azulene- 4, 10 (5H,8H,9H,10aH)-
diylidene) diacetaldehyde compound form the plant Curcuma caesia Roxb. As per
thorough literature survey this compound have seem to be a novel one which was not
reported earlier.
Materials and methods
Collection of Plant Material
Journal of Scientific and Innovative Research
778
Whole plant of C. caesia was collected in the month of
July 2010 from experimental garden of Department of
Botany, University of Kalyani, and was identified in the
Department of Botany, University of Kalyani, Nadia, West
Bengal, India.
Extraction and Isolation of Crude Secondary
Metabolite Content
2.5 kg shade dried rhizomes of black turmeric plant was
powdered of approximately and extracted three times with
1 liter of 95% EtOH at room temperature to give an extract
of 479 gms. The extract was evaporated under reduced
pressure and a solid residual mass was obtained. The above
obtained residual sample was subjected to repeated
preparative thin layer chromatography using different
solvent systems, e.g solvent system 1. Methanol (5%):
benzene (95%) and solvent system 2. Chloroform (60%):
benzene (30%): acetic acid (10%). Three homogeneous
spots were collected in solvent system 2, having Rf values
of 0.87, 0.79 and 0.75 respectively. The sample with Rf
value 0.79 was taken up for further study. This sample was
positive in Liebermann’s Burchard test3 and gave purple
colour indicating terpenoid nature of the compound and
had melting point of 780C. This terpenoid sample positive
towards to 2, 4-Dinitrophenylhydrazine test, indicating
presence of aldehyde/ keto group. 4 The sample was then
further analysed through various spectroscopic techniques
like UV spectroscopy (UV- 1601PC, UV-Visible
Spectrophotometer, Shimadzu), FT-IR spectroscopy
(Perkin Elmer Spectrum- 1 Spectrophotometer), High
Resolution Mass spectroscopy (JEOL- JMS 600
Instrument) and Nuclear Magnetic Resonance
spectroscopy, 1H &
13C (Bruker Avance- 400
Spectrometer) for its proper physicochemical
characterization.
Test for Presence of Keto or Aldehydic Group
1 mg of sample was dissolved in 0.4% alcoholic solution
of 2, 4-Dinitrophenylhydrazine with addition of 2N HCL
by capillary to maintain the acidic environment.
Results
Chemical Characterization of the Isolated Sample
The compound was reddish yellow in colour and was
soluble in spectral grade methanol (Brand- Spectrochem).
The melting point of the sample was 780C and it turned
purple in Liebermann’s Burchard test.3
Detection for Presence of Keto or Aldehydic Group
Addition of 2, 4-Dinitrophenylhydrazine acidic (2N HCL)
solution in 1 mg of 0.4% alcoholic solution of sample
yields orange yellow colour. It shows presence of an either
keto or aldehydic group in the sample. 3
UV Spectroscopy of the Isolated Sample
The methanolic spectrum of the sample showed λ max at 879.50 nm, 873.0 nm, 804.0 nm, 536.0 nm, 360.0 nm, 333.50
nm, 312.0 nm, 238.50 nm, 200.50 nm and absorbance at = 0.0014, 0.0010, 0.0005, 0.0007, 0.0001, 0.0004, 0.0033,
0.6561, 0.2520 respectively (Spectrum 1).
H
H
CHO
CHO
Figure 1: Structure of (2Z,2'Z)-2,2'-(3aR,10aS)-1,3,5,8,9,9-hexamethyl-1,2,3,3a-tetrahydrobenzo [f]
azulene-4,10(5H,8H,9H,10aH)-diylidene) diacetaldehyde
Journal of Scientific and Innovative Research
779
IR (FT-IR) Spectroscopy of the Isolated Sample
The IR spectrum of the sample showed n- (cm-1
): 2968, 2924, 2857, 1675, 1633, 1447, 1378, 1220, 1195, 1152, 1121,
843 (Spectrum 2).
Journal of Scientific and Innovative Research
780
High Resolution Mass Spectroscopy of the Isolated Sample
The mass of the sample was noted as to be (TOF MS ES+) 375.1128 (M + Na) (Spectrum 3).
Journal of Scientific and Innovative Research
781
Nuclear Magnetic Resonance Spectroscopy of the Isolated Sample
1H NMR (400 MHz, CDCl3):
δ. 9.99 (1H, d, J = 8.4 Hz, C=CH-CHO), 9.88 (1H, Cl, J = 8.4 Hz, C=CH-CHO), 5.88 (2H, J = 8.4 Hz, C=CH-CHO),
5.12- 5.10 (2H, m, CH=CH), 2.78-2.75 (1H, m), 2.60 (2H, t, J = 7.6 Hz, HC(Me)-CH-CH-(Me)CH), 2.24-2.17 (9H, m),
2.0 (3H, d, J= 10.0 Hz), 1.68 (6H, s), 1.60 (3H, d, J= 6.4 Hz), 1.35-1.28 (2 H, m) (Spectrum 4).
Journal of Scientific and Innovative Research
782
13C NMR (100 MHz, CDCl3):
δ. 191.64 (CHO), 191.10 (CHO), 164.6, 163.4, 133.6, 132.8, 128.5, 127.3, 122.8, 122.2, 63.4, 58.8, 40.6, 37.3, 32.5, 29.4,
28.0, 26.8, 26.6, 26.0, 25.1, 24.7, 18.6, 17.6 (Spectrum 5).
Journal of Scientific and Innovative Research
783
Discussions
Interpretation of the Structure of the Isolated
Compound
UV spectrum shows the presence of absorption peak (λ
max) at 238.50 nm, which indicates the skeleton, should
contain conjugated enone system(s). The reduced carbonyl
stretching frequency (cm-1
) from its actual 4 value also
supports presence of conjugated carbonyl group(s). 1H
NMR spectrum shows the presence of 32 protons. Among
which δ = 9.99 ppm (1H, d, J = 8.4 Hz) and 9.98 ppm (1H,
d, J = 8.4 Hz) confirms the presence of two aldehydic
protons. Two of the four olefinic protons directly attached
to aldehyde functionality had been obtained at δ = 5.88
ppm (2 H, d, J = 8.4 Hz). Remaining 26 protons was
observed in the aliphatic region of the 1H NMR spectrum.
The 24 peaks in 13
C NMR spectrum clearly indicate the
presence of 24 different carbon atoms in which δ = 191.64
ppm and 191.10 ppm indicates the presence of two
aldehydic carbons. The HRMS spectrum of the isolated
compound was found 375.1121 (M + Na). Hence, the
molecular formula of the isolated fraction must be
C24H32O2 and its structure was shown in Figure 1.
Acknowledgements
This work was supported by Grants in Aid from the DST-
PURSE (Grant No. FD/1626, dated: 29.1.2013) and
University of Kalyani (Grant No. Rev/ 1124 of 12-13,
dated: 28.2.2013), Nadia, West Bengal.
References
1. Pandey A.K., Chowdhury A.R., Volatile constituents of
the rhizome oil of Curcuma caesia Roxb. from central
India, Flavour and Fragrance Journal, 2003; 18: 463-465.
2. Behura S., Srivastava V.K., Essential oils of leaves of
Curcuma species, J Essential Oil Res, 2004; 16: 109-110.
Journal of Scientific and Innovative Research
784
3. Bolligr H.R., Brenner M., Ganshirt H., Mangoli H.K.,
Seiler H., Stahl E., Waldi D., Thin layer chromatography, a
laboratory hand book, 1965. p. 492.
4. Dyer J.R., Williams T.R., Applications of absorption
spectroscopy of organic compounds., J. Chem. Educ.,
1965; 42(12): 690.
1
The Journal of Phytopharmacology 2013; 2(4): 1-7
Online at: www.phytopharmajournal.com
Research Article
ISSN 2230-480X
JPHYTO 2013; 2(4): 1-7
© 2013, All rights reserved
Arghya Ghosh
Plant Biochemistry, Molecular Biology & Advance Plant
Physiology Research Laboratory, Department of Botany, University
of Kalyani, West Bengal, India
Parthadeb Ghosh
Cytogenetics & Plant Breeding
Section, Department of Botany, University of Kalyani, West Bengal,
India
Padma Chatterjee
Plant Biochemistry, Molecular Biology & Advance Plant
Physiology Research Laboratory, Department of Botany, University of Kalyani, West Bengal, India
Correspondence: Prof. Padma Chatterjee
Department of Botany, University
of Kalyani, West Bengal, Indi-
741235
Tel: 033-25828220, 033-25828275
Fax: 0091-033-2582-8282
E-mail:
schatterjeecal2003@yahoo.co.in
Evaluation of the bioactive potentialities of a
diacetaldehyde terpenoid isolated from Curcuma caesia
Roxb.
Arghya Ghosh, Parthadeb Ghosh, Padma Chatterjee*
Abstract
This paper represents evaluation of the bioactive potentialities of a diacetaldehyde terpenoid
isolated from Curcuma caesia Roxb. The terpenoid was identified as (2Z,2'Z)-2,2'- (3aR,10aS)-
1,3,5,8,9,9- hexamethyl- 1,2,3,3a-tetrahydrobenzo [f] azulene- 4,10 (5H,8H,9H,10aH) -
diylidene) diacetaldehyde. Assessment of its antitumour activity, antifungal activity and
antibacterial activity was performed using brine shrimp cytotoxicity assay, agar cup method and
disc diffusion assay respectively. The antitumour, antifungal as well as antibacterial activity was
promising and it showed 25 mg/ml (LC 50 value), (25 mg/ml, 67 mg/ml, 39 mg/ml), (235
µg/ml, 257 µg/ml, 210 µg/ml, 322 µg/ml) value respectively. This study represents the first
report of bioactive nature of (2Z,2'Z)-2,2'- (3aR,10aS)- 1,3,5,8,9,9- hexamethyl-1,2,3,3a-
tetrahydrobenzo [f] azulene- 4,10 (5H,8H,9H,10aH)- diylidene) diacetaldehyde isolated from
plants.
Keywords: Curcuma caesia Roxb., (2Z,2'Z)-2,2'- (3aR,10aS)-1,3,5,8,9,9- hexamethyl-
1,2,3,3a-tetrahydrobenzo [f] azulene- 4,10 (5H,8H,9H,10aH) -diylidene)
diacetaldehyde, Antitumour assay, Antifungal assay, Antibacterial assay.
Introduction
Curcuma caesia Roxb. (Black turmeric) of the family Zingeberaceae is an important
unexplored plant valued all over the Asia for its medicinal properties. So far eight
natural products have been isolated and characterised from Curcuma caesia Roxb. like
Borneol, Borneol acetate, 1,8-Cineole, α-Curcumene, γ-Curcumene, β-Elemene, (E)-β-
Ocimene, ar-Turmerone etc.1, 2
None of the phytochemicals isolated from C. caesia
Roxb. have been accessed for their bioactive potentialities. This paper deals with the
bioactive potentialities of (2Z,2'Z)-2,2'- (3aR,10aS)- 1,3,5,8,9,9- hexamethyl-1,2,3,3a-
tetrahydrobenzo [f] azulene- 4,10 (5H,8H,9H,10aH)- diylidene) diacetaldehyde isolated
from C. caesia Roxb. This is the first report of bioactive nature of this terpenoid.
Materials and methods
Collection of plant material
Whole plant of C. caesia was collected in the month of July 2010 from experimental
garden of Department of Botany, University of Kalyani, and was identified in the
Department of Botany, University of Kalyani, Nadia.
The Journal of Phytopharmacology
2
Extraction and isolation of crude secondary metabolite
content
2.5 kg shade dried rhizomes of C. caesia Roxb. was
powdered and extracted three times with 1 liter of 95%
EtOH at room temperature to give an extract of 479 gms.
The extract was evaporated under reduced pressure and a
solid residual mass was obtained. The above obtained
residual sample was subjected to repeated preparative thin
layer chromatography using different solvent systems, e.g.
solvent system 1. Methanol (5%): benzene (95%) and
solvent system 2. Chloroform (60%): benzene (30%):
acetic acid (10%). Three homogeneous spots were
collected in solvent system 2, having Rf values of 0.87,
0.79 and 0.75 respectively. The sample with Rf value 0.79
was taken up for further study. This sample was positive in
Liebermann’s Burchard test3 and gave purple colour
indicating terpenoid nature of the compound and had
melting point of 780C.
Antitumour assay
Brine Shrimp Cytotoxicity assay was done following the
method of B. N. Meyer et al.4-7
Brine shrimp eggs were
hatched in a shallow rectangular dish (22×32×12 cm), one
third of which was filled with saline water. An aluminium
divider with several 2 mm holes was clamped in the dish to
make to unequal compartments. The eggs (50 mg) were
sprinkled into the larger compartment which was darkened
while the smaller compartment was illuminated. The set
was maintained at 300C- 32
0C and after 48 hours the
phototropic nauplii was collected by pipette from the
lighter side, having been separated by the divider from
their shells.
The shrimps were transferred to each sample vial using a
23 cm disposable pipette and saline water was added to
adjust the volume to 5 ml. The nauplii could be counted in
the stem of the pipette against a lighted background. A
drop of dry yeast suspension (3 mg in 5 ml of saline water)
was added as food to each vial. The vials were maintained
under illumination at room temperature. Surviving shrimps
was counted after every 3 hours up to 24 hours and the
percentage of death at each dose and control was
determined.
Death (%) = test-control×100/control
Four replicates were prepared for each dose level and after
24 hours LC50 values were determined.
Antifungal assay
Preparation of sample solution
Approximately 1g of the sample isolated from C. caesia
and transferred to a 20 ml volumetric flask. The compound
was totally solubilised in 1 ml of propylene glycol and the
total volume of the stock solution of the sample was
adjusted to 10 ml by addition of sterile double distilled
water. So the concentration of the stock solution of the
sample was 100 mg/ml. By diluting the concentration of
stock solution with the help of addition of sterile double
distilled water different concentrations of the isolated
sample like 50 mg/ml to 5 mg/ml was made. Propylene
glycol with sterile double distilled water was loaded into
the agar cup to maintain the control set. The test solutions
were allowed to diffuse into the agar from the cup. All the
dilutions were sterilised by filtration using membrane filter
(0.02µ pore size).
Fungal strains
The reference strains used in the antifungal assays were:
Fusarium oxysporum, Botrytis cinerea; Rhizopus oryzae.
All the fungal strains were procured from the Plant
Biochemistry, Molecular Biology & Advance Plant
Physiology Research Laboratory, Department of Botany,
University of Kalyani, India. The test fungal strains were
maintained on PDA medium (pH-6.8) slants at 290C.
Assessment of the antifungal potentiality
Antifungal activity was screened by agar cup method.8-12
The isolated samples and their different derivatives were
tested against three plant pathogenic fungi like Fusarium
oxysporum, Botrytis cinerea; and Rhizopus oryzae to
access their antifungal nature. The PDA medium was
poured in to the sterile Petri plates and allowed to solidify
under the sterile environment of the laminar air flow
cabinet. The test fungal cultures were evenly spread over
the media by sterile cotton swabs. Then wells of 9
millimetres were made in the medium using sterile cork
borer. 100 µl of each sample having different
concentrations were transferred into the separate wells
which was made within the PDA medium. Plates
containing the pure cultures of Rhizopus oryzae and
Botrytis cinerea were allowed to incubated at 290C for 48-
72 hours where as plates containing the pure cultures of
Fusarium oxysporum takes incubation periods of 15-20
days at 290C. After the incubation period was over the
The Journal of Phytopharmacology
3
plates were observed for formation of clear inhibition zone
around the well indicated the presence of their antifungal
nature. The zone of inhibition was recorded in millimetre
scale. The final measurement was taken when the control
reached the full size within the petridish. If a culture grew
in an irregular shape, two or more measurements were
made and an average was recorded. From the growth of the
diameter of the fungal colony, the effective concentration
for colony growth inhibition was calculated. All the above
observations were taken in triplicate on each fungus/
sample concentration combinations. One control set was
prepared identical to these and taking propylene glycol
instead of different concentration combinations of sample
solutions.
Antibacterial assay
Microorganisms, culture media and their incubating
environment
The isolated sample were individually tested against a
panel of microorganisms including Gram negative Serratia
marcescens (MTCC NO. 7298) incubated at 300C, Erwinia
herbicola (MTCC NO. 3609) incubated at 370C,
Xanthomonas sp. (MTCC NO. 7444) incubated at 300C
and Gram positive Arthrobacter chlorophenolicus (MTCC
NO. 3706) incubated at 280C. All the bacterial strains were
obtained from Institute of Microbial Technology
(IMTECH), Chandigarh, India. The reference strains of
bacteria were maintained on nutrient agar medium and LB
medium slants at 40C with a subculture period of 30 days.
Composition of the media
Details of composition of the media in which test microorganisms were grown are given in table 1.
Table 1: Composition of the media for test bacterium
Medium Constituents Weight / Volume Description
A. Nutrient agar medium
(pH 7.0)
Beef extract 1.0g After adjusting the pH, volume of the medium
was adjusted to 1 liter by adding double distilled
sterile water.
Nutrient broth medium has the same
composition without agar.
Yeast extract 2.0g
Peptone 5.0g
NaCl 5.0g
Agar 15.0g
B. LB agar medium (pH
7.0)
Tryptone 10.0g After adjusting the pH, volume of the medium
was adjusted to 1 liter by adding double distilled
sterile water.
LB broth medium has the same composition
without agar.
Preparation of McFarland standard
The turbidity standard was prepared by mixing 0.5 ml of
1.75% (w/v) BaCl2.2H2O with 99.5 ml of 1%
H2SO4.BaSO4 (v/v). The standard was taken in screw cap
test tube to compare the turbidity. The bacterial culture of
selected strains were grown for 48-72 hours and
subsequently mixed with physiological saline. Turbidity
was corrected by adding sterile saline until McFarland 0.5
BaSO4 turbidity standard 108 Colony Forming Unit (CFU)
per ml was achieved. These inocula were used for seeding
of the nutrient agar medium and LB medium respectively.
Disc diffusion assay
1 mg of the isolated sample was separately dissolved in 1
ml of propylene glycol and then the volume was adjusted
to 10 ml by adding sterile water. The ultimate
concentration reaches to 103 μg/ ml and sterilized by
filtration (0.22 μm millipore filter). The concentrations at
500 to 100 μg/ ml were taken in each case. The sterile
paper discs (6 mm diameter) were saturated with 10 μl of
the solution of the compound at a concentration of 500 to
100 μg/ml and placed on the inoculated agar of 108
CFU/ml. Antibacterial tests were then carried out by disc
diffusion method13
using 100 μl of suspension containing
The Journal of Phytopharmacology
4
108 CFU/ml of bacteria on nutrient agar medium and LB
medium respectively. Negative controls were prepared
using propylene glycol. Gentamicin (10 μg/ disc) was used
as positive reference standards to determine the sensitivity
of each bacterial species tested. The inoculated plates were
incubated at 300C, 37
0C, 30
0C and 28
0C respectively for 48
h, 24 h, 48 h and 72 h. Antibacterial activity was evaluated
by measuring the zone of inhibition and the diameters of
these zones were measured in millimetres against the test
organisms.14-18
Determination of minimum inhibitory concentration
The minimal inhibitory concentration (MIC) values were
studied for the bacteria strains, being sensitive to this
compound in disc diffusion assay. The inocula of the
bacterial strains were prepared from 24-72 hr broth
cultures and suspensions were adjusted to 0.5 McFarland
standard turbidity. The compound was dissolved in 1 ml of
propylene glycol, were first diluted to the highest
concentration (500 μg/ml) to be tested, and then serial
dilutions were made in order to obtain a concentration
range from 500 to 100 μg/ml in 10 ml sterile test tubes
containing nutrient broth and LB broth medium
respectively. MIC values of the compound against
bacterial strains were determined based on a micro well
dilution method as previously described.19, 20
The plate was
covered with a sterile plate sealer and then incubated at
appropriate temperatures for 24 - 72 h at 300C, 37
0C, 30
0C
and 280C respectively. Bacterial growth was determined by
absorbance at 600 nm and confirmed by plating 10 μl
samples, forming clear wells on nutrient agar medium or
LB medium respectively. The MIC was defined as the
lowest concentration of the compounds to inhibit the
growth of microorganisms. Each test in this study was
repeated, at least, thrice.
Statistical analysis
All the data represented in table number 1, 2 and 3
obtained during in vitro experiments were expressed as
mean ± standard deviation. Calculation was done with the
help of spread sheet software Microsoft Excel 2010. *
Indicates significance at (P<0.05).
Results
Assessment of antitumour assay
The isolated sample, named (2Z,2'Z)- 2,2'-(3aR,10aS)-
1,3,5,8,9,9-hexamethyl- 1,2,3,3a-tetrahydrobenzo [f]
azulene-4,10 (5H,8H,9H,10aH)-diylidene) diacetaldehyde
was positive in brine shrimp assay [5, 6, 7, 8] and the LC50
value was 25 mg/ml (Table 2).
Table 2: Antitumour assay of (2Z,2'Z)-2,2'- (3aR,10aS)- 1,3,5,8,9,9-hexamethyl- 1,2,3,3 a- tetrahydrobenzo [f] azulene-
4,10 (5H,8H,9H,10aH)- diylidene) diacetaldehyde
Concentration
(mg/ml)
No. of survivals after LC 50
0 hrs 3 hrs 6 hrs 9 hrs 12 hrs 15 hrs 18 hrs 21 hrs 24 hrs
0
18.0 ±
0.0
18.0 ±
0.0
18.0 ±
0.0
18.0 ±
0.0
18.0 ±
0.0
18.0 ±
0.0
18.0 ±
0.0
18.0 ±
0.0
18.0 ±
0.0
1 18.0 ±
0.0
16.0 ±
0.2*
15.7 ±
0.2*
14.5 ±
0.0
14.0 ±
0.3*
12.6 ±
0.4*
12.2 ±
01*
11.5 ±
0.4*
11.1 ±
0.5*
10 18.0 ±
0.0
16.0 ±
0.02*
15.0 ±
0.5*
14.4 ±
0.3*
14.0 ±
0.2*
12.3 ±
0.2*
12.0 ±
0.6*
11.3 ±
0.3*
10.6 ±
0.1*
25
18.0 ±
0.0
15.9 ±
0.4
15.3 ±
0.3*
14.5 ±
0.3*
14.2 ±
0.6*
12.6 ±
0.1*
11.3 ±
0.2*
11.0 ±
0.2*
9.0 ±
0.3*
25mg/ml
50 18.0 ±
0.0
15.3 ±
0.2*
14.8 ±
0.3*
14.1 ±
0.6*
13.7 ±
0.2*
12.1 ±
0.7*
10.3 ±
0.3*
9.4 ±
0.8*
8.2 ±
0.1*
75
18.0 ±
0.0
14.2 ±
0.5*
14.0 ±
0.2*
13.1 ±
0.2*
12.6 ±
0.6*
11.8 ±
0.1*
9.8 ±
0.3*
8.3 ±
0.7*
6 ±
0.5*
100 18.0 ±
0.0
13.4 ±
0.3*
11.7 ±
0.3*
10.3 ±
0.6*
9.2 ±
0.2*
8.3 ±
0.1*
7.1 ±
0.7*
6.3 ±
0.4*
5.2 ±
0.2*
The Journal of Phytopharmacology
5
125
18.0
±0.0
12.6
±0.5*
11.5
±0.2*
9.9
±0.1*
8.1
±0.6*
7.7
±0.3*
6.2
±0.2*
4.4
±0.2*
3 ±
0.5*
250 18.0 ±
0.0
11.2 ±
0.2*
10.5 ±
0.1*
9.1 ±
0.3*
7.3 ±
0.3*
5.9 ±
0.5*
5.3 ±
0.3*
4.1 ±
0.3*
2 ±
0.1*
500 18.0 ±
0.0
10.3 ±
0.4*
9.7 ±
0.3*
8.2 ±
0.3*
6.1 ±
0.5*
5.1 ±
0.1*
4.7 ±
0.2*
3.5 ±
0.5*
2.1 ±
0.6*
750 18.0 ±
0.0
10.2 ±
0.2*
9.2 ±
0.2*
7.2 ±
0.1*
5.3 ±
0.3*
4.1 ±
0.4*
3 ±
0.4*
1.9 ±
0.3* 0
1000 18.0 ±
0.0
9.5 ±
0.5
8.3 ±
0.5*
6 ±
0.2*
4.6 ±
0.1*
3.3 ±
0.3*
1.2 ±
0.4* 0 0
The observed Values were expressed as mean ± standard deviation.
Calculation was done with the help of spread sheet software Microsoft Excel 2010.
* Indicates significance at (P<0.05)
Assessment of antifungal potentialities
The minimum inhibitory concentration (MIC) values of
(2Z,2'Z)-2,2'- (3aR,10aS)- 1,3,5,8,9,9-hexamethyl- 1,2,3,3
a- tetrahydrobenzo [f] azulene- 4,10 (5H,8H,9H,10aH)-
diylidene) diacetaldehyde against Fusarium oxysporum,
Botrytis cinerea and Rhizopus oryzae were 25 mg/ml, 67
mg/ml and 39 mg/ml repectively (Table 3).
Table 3: Antifungal potentialities of (2Z,2'Z)-2,2'- (3aR,10aS)- 1,3,5,8,9,9-hexamethyl- 1,2,3,3 a- tetrahydrobenzo [f]
azulene-4,10 (5H,8H,9H,10aH)- diylidene) diacetaldehyde
Fungal strains Concentration of the
compound (MIC values)
Diameter of inhibition zone in
mm
Fusarium oxysporum 25 mg/ml 2.9±0.15
Botrytis cinerea 67 mg/ml 9.5±0.20
Rhizopus oryzae 39 mg/ml 13±0.30
Assessment of antibacterial potentialities
Antibacterial assay was performed with (2Z,2'Z)-2,2'-
(3aR,10aS)- 1,3,5,8,9,9- hexamethyl-1,2,3,3 a-
tetrahydrobenzo [f] azulene- 4,10 (5H,8H,9H,10aH)-
diylidene) diacetaldehyde against four plant pathogenic
bacterium and the MIC value was 235 µg/ml, 257 µg/ml,
210 µg/ml and 322 µg/ml for the bacterium Serratia
marcescens (MTCC NO. 7298), Erwinia herbicola (MTCC
NO. 3609), Xanthomonas sp. (MTCC NO. 7444) and
Arthrobacter chlorophenolicus (MTCC NO. 3706)
respectively (Table 4).
Table 4: Antibacterial potentialities of (2Z,2'Z)-2,2'- (3aR,10aS)- 1,3,5,8,9,9-hexamethyl- 1,2,3,3 a- tetrahydrobenzo [f]
azulene-4,10 (5H,8H,9H,10aH)- diylidene) diacetaldehyde
Bacterial strains Concentration of the
compound (MIC values)
Diameter of inhibition zone in
mm
Serratia marcescens 235 µg/ml 9±0.47
Erwinia herbicola 257 µg/ml 6±0.35
Xanthomonas sp. 210 µg/ml 5±0.15
Arthrobacter chlorophenolicus 322 µg/ml 3.8±0.21
The Journal of Phytopharmacology
6
Discussions
Evaluation of bioactive potentialities
The isolated fraction (2Z,2'Z)- 2,2'- (3aR,10aS)-
1,3,5,8,9,9- hexamethyl- 1,2,3,3a -tetrahydrobenzo [f]
azulene- 4,10 (5H,8H,9H,10aH) - diylidene)
diacetaldehyde shows antitumour, antifungal as well as
antibacterial activities.
References
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Professor Padma Chatterjee:
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JOURNAL OF BOTANICAL SCIENCES, "Evaluation of Antibacterial Potentiality of
a Cyclopenta Naphthalene Tetraol Terpenoid Isolated from Curcuma caesia
Roxb.".
Our decision is to: Accepted
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editor@rroij.com
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