List of publications - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/72834/18/18_list of publications.pdf167 1. Ghosh Arghya, Ghosh Ajoy, Ghosh Parthadeb, Chatterjee Padma (2012)

166

List of publications

167

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.


(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.


(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: [email protected]

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

mailto:[email protected]

122 In vitro true to type propagation of Curcuma caesia Roxb. (Zingiberaceae)

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BTAIJ, 7(4) 2013

BioTechnologyAn Indian Journal

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].


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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-


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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


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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.


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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.


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BTAIJ, 7(4) 2013


BioTechnology

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


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BTAIJ, 7(4) 2013


BioTechnology

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.

REFERENCES

[1] GRIN Database, USDA, ARS, National genetic

resources program, Germplasm resources informa-

tion network - (GRIN); National germplasm re-

sources laboratory, Beltsville, Maryland; Available

online at: http://www.ars-grin.gov/cgibin/npgs/html/

taxgenform.pl, (2005).

[2] ISI Database, ISI web of knowledge, Thompson-

ISI, London; Available on-line at: http://portalt.

w ok . mi mas . a c .u k /p o r t a l . c g i ?D e s t A pp =

WOS&Func=Frame, (2005).

[3] S.Pei; Ethnobotanical approaches of traditional

medicine studies: Some experiences from Asia,

Pharmaceutical Biology, 39, 74-79 (2001).

[4] S.J.Murch, S.Krishnaraj, P.K.Saxena;

Phytopharmaceuticals: Massproduction, standard-

ization, and conservation, Herbal Med., 4(2), 39�

43 (2000).

[5] K.C.Wen; The turnover rate of marker constitu-

ents in Chinese herbal medicine, J.Food Drug

Analysis, 8, 270�277 (2000).

[6] A.B.Cunningham; African medicinal plants: Setting

priorities at the interface between conservation and

primary health care, People and plant initiative

working paper 1, Nairobi: UNESCO, 92 (1993).

[7] M.W.Flower; Commercial application and economic

aspects of mass cell cultures, In: H.S.Mantel,

H.Smith (Eds); Plant biotechnology, Cambridge Uni-

versity Press, Cambridge, 3-37 (1983).

[8] R.Arora, S.S.Bhojwani; In Vitro propagation and

low temperature storage of Saussurea lappa,

C.B.Clarke (Ed); An endangered, Medicinal plant,

Plant Cell Reports, 8(1), 44-47 (1989).

[9] G.C.Sudha, S.Seeni; In Vitro propagation and field

establishment of Adhatoda beddomei, C.B.Clarke

(Ed); A rare medicinal plant. Plant Cell Reports,

13(3-4), 203-207 (1993).

[10] D.Ajitkumar, S.Seeni; Rapid clonal multiplication

through in vitro axillary shoot proliferation of Aegle

marmelos (L.) Corr., A medicinal tree, Plant Cell

Reports, 17(5), 422-426 (1998).

[11] E.Prakash, P.S.Sha, V.Khan, P.S.Reddy, K.R.Rao;

Regeneration of plants from seed derived callus of

Hybanthus Enneaspermus L. Muell., A rare ethno-

botanical herb, Plant Cell Reports, 18(10), 873-878

(1999).

[12] C.Y.Hu, P.J.Wang; Meristem shoot tip and bud

culture, In: Handbook of plant cell culture,

D.A.Evans, W.R.Sharp, P.V.Ammirato, Y.Yamada

(Eds); New York, Macmillan, 177�227 (1983).

[13] K.A.Vincent, M.K.Mary, M.Hariharan;

Micropropagation of Kaemferia galanga L.-a

medicinal plant, Plant Cell Tissue Organ Cult, 28,

229-230 (1992).

[14] Y.Sahoo, S.K.Pattnaik, P.K.Chand; In Vitro clonal

propagation of an aromatic medicinal herb Ocimum

basilicum L. (Sweet Basil) by axillary shoot prolif-

eration, In Vitro Cellular and Developmental Biol-

ogy Plant, 33(4), 293-296 (1997).

[15] T.Murashige, F.Skoog; A revised medium for rapid

growth and bioassays with tobacco Tissue cultures,

Physiologia Plantarum, 15, 473-497 (1962).

[16] M.G.Murray, W.F.Thompson; Rapid isolation of high

molecular weight plant DNA, Nucl.Acids Res., 8,

4321-4325 (1980).

[17] J.Smbrook, D.W.Russed; Molecular cloning, A labo-

ratory manual, 3rd Edition, Spring harbor laboratory

press, Cold Spring Harbor, (2001).

[18] D.B.Duncan; Multiple range and multiple F test,

biometrics, 11(1), 1-42 (1955).

[19] R.M.Skirvin, O.McMeans, W.L.Wang; Storage

water is a source of latent bacterial contamination

in vitro, Trends of Agricultural Science, 1, 63-63

(1993).

[20] J.Gopal, J.L.Minocha, H.S.Dhaliwal;

Microtuberization in potato (Solanum tuberosum

L.), Plant Cell Reports, 17(10), 794-798 (1998).

[21] S.K.Guru, R.Chandra, S.Khetrapal, A.Raj,

R.Palisetty; Protein pattern in differentiating ex-

plants of chick pea (Cicer arietinum L.), Indian

Journal of Plant Physiology, 4, 147-151 (1999).

[22] J.Sen, A.K.Sharma; Micropropagation of Withania

somnifera from germinating seeds and shoot tips,

Plant Cell Tissue and Organ Culture, 26(2), 71-73

(1991).

[23] E.F.George; Plant propagation by Tissue culture:

Part 1, The technology, Exegetics, Basingstoke,

(1993).

http://www.ars-grin.gov/cgibin/npgs/html/

http://portalt.


FULL PAPER

BTAIJ, 7(4) 2013


BioTechnology

[24] A.Ahuja, M.Verma, S.Grewal; Clonal propagation

of Ocimum species by tissue culture, Indian

J.Exp.Biol., 20, 455-458 (1982).

[25] L.B.Andrade, S.Echeverrugaray, F.Fracaro,

G.F.Pauletti, L.Rota; The effect of growth regula-

tors on shoot propagation and rooting of common

lavender (Lavandula vera DC), Plant Cell Tissue

and Organ Culture, 56(2), 79-83 (1999).

[26] J.A.Driver, G.R.Suttle; Nursery handling of

propagules, In J.M.Bonga, D.J.Durzan (Eds); Cell

and Tissue Culture in Forestry, Dordrecht, Nether-

lands, 320-335 (1987).

[27] S.Cuenca, J.B.Amo-Marco, R.Parra;

Micropropagation from inflorescence stems of the

spanish endemic plant Centaurea paui loscos ex

willk. (Compositae), Plant Cell Reports, 18(7-8),

674-679 (1999).

[28] Y.Sahoo, P.K.Chand; Micropropagation of Vitex

negundo L., A woody aromatic medicinal shrub

through high frequency axillary shoot proliferation,

Plant Cell Reports, 18(3-4), 301-307 (1998).

[29] N.Komalavalli, M.V.Rao; In Vitro micropropagation

of Gymnema Sylvestre, A multipurpose medicinal

plant, Plant Cell Tissue and Organ Culture, 61(2),

97-105 (2000).

[30] H.R.Juliani, Jr.A.R.Koroch, H.R.Juliani, V.S.Trippi;

Micropropagation of Lippia junelliana (Mold.)

trone, Plant Cell Reports, 59(3), 175-179 (1999).

[31] S.Saha, A.Kader, C.Sengupta, P.D.Ghosh; In Vitro

propagation of Ocimum gratissimum L.

(Lamiaceae) and its evaluation of genetic fidelity

using RAPD marker, American Journal of Plant

Sciences, 3, 64-74 (2012).

[32] M.Faisal, M.Anis; Rapid mass propagation of

Tylophora Indica Merrill via leaf callus culture,

Plant Cell Tissue and Organ Culture, 75(2), 125-

129 (2003).

[33] M.K.Smith; A review of factors influencing the

genetic stability of micropropagated Bananas,

Fruits, 43(4), 219-223 (1988).

[34] Y.R.Dutta, G.Gangopadhyay, S.Das, B.K.Dutta,

K.K.Mukherjee; Esterase as a marker to study the

genetic fidelity of micropropagated Banana,

Biologia Plantarum, 47(3), 421-424 (2003).

[35] M.K.U.Chawdhury, J.K.Vasil; Molecular analysis

of plants regenerated from embryogenic cultures

of hybrid sugarcane cultivars (Saccharum spp.),

Theoretical and Applied Genetics, 86(2-3), 181-188

(1993).

[36] V.Rani, A.Parida, S.N.Raina; Random amplified

polymorphic DNA (RAPD) markers for genetic

analysis in micropropagated plants of Populus

deltoides marsh, Plant Cell Reports, 14(7), 459-

462 (1994).

[37] R.K.Chaudhuri, A.Pal, T.B.Jha; Production of ge-

netically uniform plants from nodal explants of

Swertia Chirata buch, Ham.Ex Wall, A critically

endangered medicinal herb, In Vitro cellular and

developmental biology, 43(5), 467-472 (2007).

[38] S.Bhattacharya, T.K.Bondopadhyay, P.D.Ghosh;

Somatic embryogenesis in Cymbopogon Pendulus

and evaluation of clonal fidelity of regenerants us-

ing ISSR marker, Scientia Horticulturae, 123(4),

505-513 (2010).

[39] J.G.K.Williams, A.R.Kubelik, K.J.Livak,

J.A.Rafalski, S.V.Tingey; DNA polymorphisms

amplified by arbitrary primers are useful as genetic

markers, Nucleic Acids Research, 18(22), 6531-

6535 (1990).

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


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

_____________________________________________________________________________

96



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


_____________________________________________________________________________

97


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.


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


_____________________________________________________________________________

98


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






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



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.

REFERENCES

[1] Maharajan M, Rajendran A, Binu Thomas and Aravindhan, V., 2012, Asian Journal of Plant Science and

Research, 2 (5):577-580.

[2] Dickson AM, Fred OCN, Eleojo O, 2011, Asian Journal of Plant Science and Research, 1 (3): 1-10.

[3] Merina PD, Ankita B, Anu, 2013, Asian Journal of Plant Science and Research, 3(2):107-111.

[4] Thevasundari S, Rajendran A, 2012, Asian Journal of Plant Science and Research, 2 (3):330-334.

[5] Farzana R, Nadia S, Ijaz A, Saima S, Fakhar UN, Shagufta N, 2013, Asian Journal of Plant Science and

Research, 3(2):1-5.

[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.

[20] Murray PR, Baron EJ, Anderson, KW, Wadowsky, RM., Reagan, DR, Walker, ES, Kingsley, LA, 1995,

Manual of clinical microbiology, 6th edition, Washington, DC: ASM.


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99


[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


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

_____________________________________________________________________________



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.


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


_____________________________________________________________________________


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.


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


_____________________________________________________________________________


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].


_____________________________________________________________________________


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.


_____________________________________________________________________________


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.

REFERENCES

[1]GRIN Database USDA, National Germplasm Resources Laboratory, 2005, http://www.ars-grin.gov/cgi-

bin/npgs/html/taxgenform.pl

[2] Gupta A, Sharon M, Appl. Sci. Res., 2012, 3(6):4048-4052.


_____________________________________________________________________________


[3] Sahaya S, Chitra D, Servin W, Sarmad M, Appl. Sci. Res., 2012, 3(5):3339-3343.

[4] Sahaya S, Chitra D, Servin W, Sarmad M, Appl. Sci. Res., 2012, 3(3):1727-1732.

[5] Ananya B, Suresh CD, Mohan CK, Priyabrata S, Appl. Sci. Res., 2011, 2 (5):457-464.

[6] Mohammed F, Satyapal S, Babeet ST, Moinuddin K, Anwar S, Appl. Sci. Res., 2011, 2 (1):208-213.

[7] Nadgauda RS, Mascarenhas AF, Hendre RR, Jagannathan V, Ind. J. Exp. Biol., 1978, 16, 120-122.

[8] Murashige T, Skoog F, Physiologia Plantarum, 1962, 15(3), 473-497.

[9] Duncan DB, Biometrics, 1955, 11 (1), 1-42.

[10] Murray MG, Thompson WF, Nucleic Acids Research, 1980, 8 (19), 4321-4325.

[11] Sambrook, JD, Russed W, Molecular Cloning. A Laboratory Manual, 3rd Edition, Spring Harbor Laboratory

Press, Cold Spring Harbor, 2001.

[12] Gopal J, Minocha JL, Dhaliwal HS, Plant Cell Reports, 1998, 17 (10), 794-798.

[13] Guru SK, Chandra R, Khetrapal S, Raj A, Palisetty R, Indian Journal of Plant Physiology, 1999, 4, 147-151.

[14] Vincent K A, Mathew K M, Hariharan M, Plant Cell Tissue and Organ Culture, 1992, 28 (2), 229-230.

[15] Sen J, Sharma AK, Plant Cell Tissue and Organ Culture, 1991, 26 (2), 71-73.

[16] Sahoo Y, Pattnaik SK Chand P K, In Vitro Cellular and Developmental Biology—Plant, 1997, 33 (4), 293-

296.

[17] Ahuja A, Verma M, Grewal S, Indian Journal of Experimental Biology, 1982, 20, 455-458.

[18] Begum F, Amin N, Azad MAK, Plant Tissue Culture and Biotechnology, 2002, 12 (1), 27-35.

[19] Saha S, Ghosh PD, Sengupta C, Indian Journal of Plant Physiology, 2010,15 (2), 168-172.

[20] Hiregoudar LV, Murthy HN, Bhat JG, Nayeem A, Hema BP, Hahn EJ, Paek KY, Biologia Plantarum, 2006, 50

(2), 291-294. [21] Andrade LB, Echeverrugaray S, Fracaro F, Pauletti GF, Rota L, Plant Cell Tissue and Organ Culture, 1999, 56

(2), 79-83.

[22] Driver JA, Suttle GR, Nursery Handling of Propagules, In: J. M. Bonga and D. J. Durzan, Eds., Cell and Tissue

Culture in Forestry, Dordrecht, Netherlands, 1987, 320-335.

[23] Juliani Jr HR, Koroch AR, Juliani HR, Trippi VS, Plant Cell Reports, 1999, 59 (3), 175-179.

[24] Chawdhury MKU, Vasil JK, Theoretical and Applied Genetics, 1993, 86 (2-3), 181-188.

[25] Rani V, Parida A, Raina SN, Plant Cell Reports, 1994, 14 (7), 459-462.

[26] Chaudhuri RK, Pal A, Jha TB, In Vitro Cellular and Developmental Biology, 2007, 43 (5), 467-472.

[27] Bhattacharya S, Bondopadhyay TK, Ghosh PD, Scientia Horticulturae, 2010, 123 (4), 505-513.

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



*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:

[email protected]

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


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)



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)


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)


799


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)


800




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



Research Article

ISSN 2230-4818

JSIR 2013; 2(4): 745-750







Parthadeb Ghosh

Cytogenetics & Plant Breeding Section, Department of Botany,


Padma Chatterjee*

Plant Biochemistry, Molecular Biology & Advance Plant

Physiology Research Laboratory, Department of Botany, University

of Kalyani, West Bengal, India




741235

Tel: 033-25828220, 033-25828275

Fax: 0091-033-2582-8282

E-mail:

[email protected]

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.


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


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







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


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


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


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


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




dated: 28.2.2013), Nadia, West Bengal, India.

References

1. Lutomski J., Kedzia B. and Debska W. Effect of an

alcohol extract and of active ingredients from Curcuma

longa L. on bacteria and fungi. Planta Medica 1974; 26: 9-

19.

2. Banerjee A. and Nigam S.S. Antimicrobial efficacy of

the essential oil of Curcuma longa L. Indian J. Med. Res.

1978; 68: 864-866.

3. Bhavani Shankar, T.N. and Sreenivasa Murthy, V.

Effect of turmeric (Curcuma longa L.) fractions on the

growth of some intestinal & pathogenic bacteria in vitro.

Indian J. Exp. Biol. 1979; 17: 1362-1366.

4. Saju K.A., Venugopal M.N. and Mathew M.J.

Antifungal and insect-repellent activities of essential oil of

turmeric (Curcuma longa L.). Current Science. 1998; 75:

660-662.

5. Chauhan U.K., Soni P., Shrivastava, R., Mathur K.C.

and Khadikar P.V. Antimicrobial activities of the rhizome

of Curcuma longa L. Oxidation Commun. 2003; 26: 266-

270.

6. Kim M.K., Choi G.J. and Lee H.S. Fungicidal property

of Curcuma longa L. rhizome-derived curcumin against

phytopathogenic fungi in a greenhouse. J. Agric. Food

Chem. 2003; 51: 1578-1581.


Seiler H., Stahl E., Waldi D. Thin layer chromatography. a

laboratory hand book. 1965.

8. Perez C., Paul M., Bazerque P. Antibiotic assay by agar

well diffusion method. Acta Bio Med Exp. 1990; 15: 113-

115.

9. Alade P.I., Irobi O.N. Antibacterial activities of crude

extracs of Acalypha wilsiana, J Ethnopharmacol. 1993; 39:

171-174.

10. Abioye AO, Bamiro SB, Adesida SO, Hunpatin VP,

Adeleke TI. Preliminary Phytochemical and Antimicrobial

studies of Phyllantus amarus L. (Euphorbiaceae). Niger

Quart. J. Hosp. Med. 2004; 14: 282-287.

11. Manivannan K, Karthikai devi G, Anantharaman P,

Balasubramanian T. Antimicrobial potential of selected

brown seaweeds from Vedalai coastal waters, Gulf of

Mannar. Asian Pacific Journal of Tropical Biomedicine.

2011; 1(2): 114-120.

12. Murray P.R., Baron E.J., Pfaller M.A., Tenover FC,

Yolke R. Manual of clinical microbiology. 6th edition.

Washington. DC. ASM. 1995.

13. Habbal O., Hasson S.S., El-Hag A.H., A-Mahrooqi Z.,

Al Hashmi N., Al B.Z., MS Balushi A., A Jabri A.A.

Antibacterial activity of Lawsonia inermis Linn (Henna)

against Pseudomonas aeruginosa. Asian Pacific Journal of

Tropical Biomedicine. 2011; 1(3): 173-176.

14. Vijayakumar A., Duraipandiyan V., Jeyaraj B.,

Agastian P., Karunai R.M., Ignacimuthu S. Phytochemical

analysis and in vitro antimicrobial activity of Illicium

griffithii Hook. f. & Thoms extracts. Asian Pacific Journal

of Tropical Disease. 2012; 2(3): 190-199.

15. Ahmed M., Djebli N., Meslem A., Aissat S.

Antibacterial activity of various honey types of Algeria

against Pathogenic Gram Negative Bacilli: Escherichia coli

and Pseudomonas aeruginosa. Asian Pacific Journal of

Tropical Disease. 2012; 2(3): 211-214.

16. Roopa V.P., Vidhi T.T., Patel V.K. Antimicrobial

activity of ginger and honey on isolates of extracted

carious teeth during orthodontic treatment. Asian Pacific

Journal of Tropical Biomedicine. 2011; 1(S): S58-S61.

17. Renisheya J., Jeba Malar T., Johnson M., Mary Uthith

M., Arthy A. Antibacterial activity of ethanolic extracts of

selected medicinal plants against human pathogens. Asian

Pacific Journal of Tropical Biomedicine. 2011; 1(S): S76-

S78.


750

18. Sokmen A., Gulluce M., Akpulat H.A., Daferera D.,

Tepe B., Polissiou M., Sokmen M., Sahin F. The in vitro

antimicrobial and antioxidant activities of the essential oils

and methanol extracts of endemic Thymus spathulifolius.

Food Control. 2004; 15: 627–634.

19. Golam K., Farjana N., Mohammad A.R., Tanzima Y..

Antimicrobial activities of the rhizome extract of Zingiber

zerumbet Linn. Asian Pacific Journal of Tropical

Biomedicine. 2011; 1(5): 409-412.

777



Research Article

ISSN 2230-4818

JSIR 2013; 2(4): 777-784







Ayan Bandyopadhyay

Department of Chemistry,


Parthadeb Ghosh


Section, Department of Botany, University of Kalyani, West Bengal, India

Padma Chatterjee*

Plant Biochemistry, Molecular






741235

Tel: 033-25828220, 033-25828275

Fax: 0091-033-2582-8282

E-mail:

[email protected]

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.




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


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).


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).


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).


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).


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




dated: 28.2.2013), Nadia, West Bengal.

References

1. Pandey A.K., Chowdhury A.R., Volatile constituents of


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.


784


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


Arghya Ghosh


Physiology Research Laboratory, Department of Botany, University

of Kalyani, West Bengal, India

Parthadeb Ghosh


Section, Department of Botany, University of Kalyani, West Bengal,

India

Padma Chatterjee


Physiology Research Laboratory, Department of Botany, University of Kalyani, West Bengal, India

Correspondence: Prof. Padma Chatterjee



741235

Tel: 033-25828220, 033-25828275

Fax: 0091-033-2582-8282

E-mail:

[email protected]

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.


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.







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


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


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


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.


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*


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


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

1. Pandey A.K., Chowdhury A.R. Volatile constituents of


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.


Seiler H., Stahl E., Waldi D. Thin layer chromatography, a

laboratory hand book; 1965, p. 492.

4. Meyer B.N., Ferrigni N.R., Putnam J.E., Jacobson L.B.,

Nichols D.E., McLaughlin J.L. Brine shrimp: a convenient

general bioassay for active plants constituents. Planta Med.

1982; 45: 31- 34.

5. Hossain S, Kader G, Nikkon F, Yeasmin T. Cytotoxicity

of the rhizomes of medicinal plants. Asian Pac. J. Trop.

Biomed. 2012; 2(2): 125-127.

6. Ramalakshmi S, Edaydulla N, Ramesh P, Muthuchelian

K. Investigation on cytotoxic, antioxidant, antimicrobial

and volatile profile of Wrightia tinctoria (Roxb.) R. Br.

flower used in Indian medicine. Asian Pacific Journal of

Tropical Disease. 2012; S(1): D68-S75.

7. Varahalarao V., Kaladhar D.S.V.G.K. Phytochemical

evaluation and molecular characterization of some

important medicinal plants. Asian Pacific Journal of

Tropical Disease. 2012; S(1): S26-S32.

8. Perez C., Paul M., Bazerque P. Antibiotic assay by agar

well diffusion method. Acta Bio. Med. Exp. 1990; 15: 113-

115.

9. Alade P.I., Irobi O.N. Antibacterial activities of crude

extracs of Acalypha wilsiana, J. Ethnopharmacol. 1993;

39: 171-174.

10. Abioye A.O., Bamiro S.B., Adesida S.O., Hunpatin

V.P., Adeleke T.I. Preliminary Phytochemical and

Antimicrobial studies of Phyllantus amarus L.

(Euphorbiaceae). Niger. Quart. J. Hosp. Med. 2004; 14:

282-287.

11. Manivannan K., Karthikai devi G., Anantharaman P.,

Balasubramanian T. Antimicrobial potential of selected

brown seaweeds from Vedalai coastal waters, Gulf of

Mannar. Asian Pacific Journal of Tropical Biomedicine.

2011; 1(2): 114-120.

12. Balakumar S., Rajan S., Thirunalasundari T., Jeeva S.

Antifungal activity of Aegle marmelos (L.) Correa

(Rutaceae) leaf extract on dermatophytes. Asian Pacific

Journal of Tropical Biomedicine. 2011; 1(4): 309-312.

13. Murray P.R., Baron E.J., Pfaller M.A., Tenover F.C.,

Yolke R. Manual of clinical microbiology, 6th edition,

Washington, DC, ASM; 1995.

14. Habbal O., Hasson S.S., El Hag A.H., AMahrooqi Z.,

Al Hashmi N., Al B.Z., MS Balushi A., A Jabri A.A.

Antibacterial activity of Lawsonia inermis Linn (Henna)

against Pseudomonas aeruginosa. Asian Pacific Journal of

Tropical Biomedicine. 2011; 1(3): 173-176.

15. Vijayakumar A., Duraipandiyan V., Jeyaraj B.,

Agastian P., Karunai R.M., Ignacimuthu S. Phytochemical

analysis and in vitro antimicrobial activity of Illicium

griffithii Hook. f. & Thoms extracts. Asian Pacific Journal

of Tropical Disease. 2012; 2(3): 190-199.

16. Ahmed M., Djebli N., Meslem A., Aissat S.

Antibacterial activity of various honey types of Algeria

against Pathogenic Gram Negative Bacilli: Escherichia

coli and Pseudomonas aeruginosa. Asian Pacific Journal of

Tropical Disease. 2012; 2(3): 211-214.

17. Roopa V.P., Vidhi T.T., Patel V.K. Antimicrobial

activity of ginger and honey on isolates of extracted

carious teeth during orthodontic treatment. Asian Pacific

Journal of Tropical Biomedicine. 2011; 1(S): S58-S61.

18. Renisheya J., Jeba Malar T., Johnson M., Mary Uthith

M., Arthy A. Antibacterial activity of ethanolic extracts of

selected medicinal plants against human pathogens. Asian

Pacific Journal of Tropical Biomedicine. 2011; 1(S): S76-

S78.


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19. Sokmen A., Gulluce M., Akpulat H.A., Daferera D.,

Tepe B., Polissiou M., Sokmen M., Sahin F. The in vitro

antimicrobial and antioxidant activities of the essential oils

and methanol extracts of endemic Thymus spathulifolius.

Food Control. 2004; 15: 627–634.

20. Golam K., Farjana N., Mohammad A.R., Tanzima Y.

Antimicrobial activities of the rhizome extract of Zingiber

zerumbet Linn. Asian Pacific Journal of Tropical

Biomedicine. 2011; 1(5): 409-412.

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Professor Padma Chatterjee:

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a Cyclopenta Naphthalene Tetraol Terpenoid Isolated from Curcuma caesia

Roxb.".

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List of publications - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/72834/18/18_list of publications.pdf167 1. Ghosh Arghya, Ghosh Ajoy, Ghosh Parthadeb, Chatterjee Padma (2012)