Micropropagation and anticancer activity of methanolic extract of Plumbago auriculata Lam

Abstract

Plants are one of the potential hotspots for which the yield of high esteem bioactive intensifies that had therapeutic properties may be utilized as pharmacologically active compounds. In this study, Plumbago auriculata was refined on altered MS medium supplemented with individual and diverse mix of plant development controllers (IBA, IAA, GA3
and KIN). The plantlets removed utilizing methanol for assessing for their phytochemical, cancer prevention agent and anticancer activities. GC-MS investigation of concentrate uncovered that it contained Z - 11-octadecen-1-yl-acetic acid derivation, C-sitosterol, heptadecanoic acid,9 methyl, methyl ester, 4,5,7 trihydroxy isoflavone, E, E, Z, 1, 3, 12 nonadecatriene 5, 14 diol, ethanol 2, (9 octadecenyloxyl, (E), 13 docosenoic corrosive Methyl ester, (Z), bis (2 ethyl hexyl) phthalate and 10, 12, 14- nonacosatriynoic corrosive. In vitro antioxidant potential of the methanolic extract for
the DPPH examine, lessening power test uncovered that IC50 esteem at 260μg/ml and 320μg/ml individually. Methanolic extract was tested against lung (A549) and ovarian (PA1) malignancy cell lines for their anti proliferative and apoptotic activities.

Full text

G. Lakshmanan, et a
l.
International Journal of Advanced Biotechnology and Research (IJBR)
ISSN 0976-2612, Online ISSN 2278–599X,
Vol-7, Issue-4, 2016, pp2001-2011
http://www.bipublication.com
Research Article
Micropropagation and anticancer activity of methanolic
extract of Plumbago auriculata Lam.
G. Lakshmanan1, G. Bupesh1,2, A.Vignesh1,
A. Sathiyaseelan1and K. Murugesan1*
1Unit of Plant Molecular Biology and Biotechnology,
Centre for Advanced Studies in Botany,
University of Madras, Guindy Campus, Chennai –600 025.
2Central Research laboratory,
Sree Balaji Medical College and Hospital, Chromepet,Chennai-600044.
1*Corresponding author. Mobile: (K.Murugesan) kmkmn92@gmail.com & bupeshgiri55@gmail.com
ABSTRACT
Plants are one of the potential hotspots for which the yield of high esteem bioactive intensifies that had therapeutic
properties may be utilized as pharmacologically active compounds. In this study, Plumbago auriculata was refined on
altered MS medium supplemented with individual and diverse mix of plant development controllers (IBA, IAA, GA3
and KIN). The plantlets removed utilizing methanol for assessing for their phytochemical, cancer prevention agent and
anticancer activities. GC-MS investigation of concentrate uncovered that it contained Z - 11-octadecen-1-yl-acetic
acid derivation, C-sitosterol, heptadecanoic acid,9 methyl, methyl ester, 4,5,7 trihydroxy isoflavone, E, E, Z, 1, 3, 12
nonadecatriene 5, 14 diol, ethanol 2, (9 octadecenyloxyl, (E), 13 docosenoic corrosive Methyl ester, (Z), bis (2 ethyl
hexyl) phthalate and 10, 12, 14- nonacosatriynoic corrosive. In vitro antioxidant potential of the methanolic extract for
the DPPH examine, lessening power test uncovered that IC50 esteem at 260µ g/ml and 320µg/ml individually.
Methanolic extract was tested against lung (A549) and ovarian (PA1) malignancy cell lines for their anti proliferative
and apoptotic activities.
Keywords: Plumbago auriculata, Micropropagation, Methanolic extract, Antioxidants, Anticancer
1. INTRODUCTION
Cancer is one of the most dreadful diseases
reported Worldwide and it is a life threatening
disease. Cancer generally occurs in breast,
prostrate, lung, liver, colorectal, cervix, uterus and
stomach [7]. Factors such as age, lifestyle and
food habit, consumption of alcohol, tobacco and
lack of physical activities facilitated cancer
development in human beings. In India, about 6-7
lakh people are diagnosed to have cancer every
year [12]. Lung and ovarian cancers are leading
diseases because of tobacco use in both sexes,
according to the international agency for research
[8]. Ovarian cancer was identified in women by
the changes in hormonal imbalance, menopause
and metastasis [22]. For the treatment of cancer
there have been many advanced treatments and
2001

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surgeries available in recent times such as
radiation, chemotherapy, hormonal and
immunotherapy [24] those treatments also induced
adverse side effects. Some treatments may really
cure the disease but the cost is too high. Hence, it
is imperative to look for an alternative medicine
that is less toxic and drug should be of low-cost
for affordability by patients. Recently, medicinal
plants are recognized as an attractive and
promising approach, the plant-derived
biomolecules have high value in biomedical
research and for the development of drugs against
cancer [14]. Without a doubt, some natural
products have been used for anticancer treatment
because of their biological significance [18] and
less side effects.
Plumbago auriculata (family: plumbaginaceae) is
an ornamental and medicinal plant that has rich
source of alkaloids such as plumagain (2-methyl-
5-hydroxyl, 4-naphthoquinone [21] which could
be used as an anti-cancer drug, antibacterial [17]
antioxidant [5] antifungal, anti-inflammatory [6]
and anticoagulant against various diseases such as
rheumatism, piles, diarrhoea and skin diseases
[27, 3]. These biological activities are usually
undergone due to the existence of particular
chemical compounds, such as naphthoquinones.
Plumbagin is a naphthoquinone commonly
available among Plumbago species, specifically
found in their roots [9]. Plant tissue culture is a
technique is not only a mass breeding for
conservation of endangered medicinal plants and
also for the production of biologically active
compounds. Further there was no report on its
anticancer studies so far.In the present study, the
methanolic extract from the plantlets were
screened for their phytochemical profile. Further
the antioxidant and in vitro anticancer activity was
evaluated against PA1 and A549 cell lines.
2. MATERIALS AND METHODS
Murashige Skoog (MS) medium, plant growth
regulators (IAA, GA3, IBA and KIN), 2,2-
diphenyl-1-picrylhydrazyl (DPPH), trichloroacetic
acid (TCA), sodium hydroxide (NaOH), procured
from Hi-Media, India. Tween 20 and mercuric
chloride were obtained from Sisco Research
Laboratories Pvt. Ltd. (SRL), Methanol was
purchased from Rankem. The chemicals used in
this study include minimum essential medium
(MEM), fetal calf serum (FBS), trypsin, trypan
blue, ethanol, penicillin, streptomycin,
gentamycin, dimethyl sulfoxide (DMSO) and
phosphate buffer saline (Merck, Germany). All
other chemicals and reagents were procured Sisco
Research Laboratories Pvt. Ltd. (SRL).
2.1. Plant collection and explants preparation
Young and healthy disease-free plants of P.
auriculata (Fig. 1) were collected from University
of Madras, Guindy Campus and Chennai, India.
During the months of November to December.
Stem segments excised from field grown plants
were used as explants and they were washed
thoroughly for 30 min under running tap water to
remove the dust particles and soil, and treated
with 2-3 drops of Tween-20. The above processes
were done twice or thrice under running tap water
for removal of surfactants. Then, surface
sterilization was carried out under aseptic
conditions with an aqueous solution of freshly
prepared mercuric chloride (0.5%) for 3–5 min
and then explants were washed 3-4 times with
sterile distilled water. Explants were cut into
smaller pieces by using a sterile scalpel blade
under aseptic conditions.
2.2. Media and Culture Conditions
Murashige and Skoog culture medium with 30g/L
sucrose, 8 g/L bacteriological grade agar-agar was
used throughout the experiments. Growth
regulators GA3, IAA, IBA and KIN either alone
and in combination were added to the MS basal
medium and the pH were adjusted to 5.6±0.02
before autoclaving. 20 mL of medium was
dispensed into each culture flask. After
inoculation, the cultures were incubated in a
growth chamber at 25°C with a light and dark
period of 16/8 hours.
2.3. Multiplication of shoots and roots
Wild healthy P. auriculata, plants were excised
and were used as explants for shoot initiation.

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Growth hormones GA3, IAA, IBA and KIN were
supplemented alone in and combination at various
concentrations (0.5-2.5 mg/L) was added to the
MS culture medium for the induction of multiple
shoots. For the control plant, MS medium alone
was given; the plants were subsequently
subcultured every three weeks. After the
experiment, the plantlets were observed for all the
morphological characters.
2.4. Micro propagated plant extraction
Dried in vitro propagated plant 5g powder was
immersed into methanol for 48hrs. Then the
samples were filtered through Whatman no. 1
filter paper to collect the supernatant. Further it
was condensed by using rotary evaporator. The
condensed sample was stored at room temperature
for further work.
2.5. Preliminary phytochemical screening
The preliminary screening of major
phytoconstituents such as Alkaloids (Mayer’s
test), Flavonoids (Aluminium chloride test),
Carbohydrates (Fehling’s test), Saponins
(Frothing test), Tannins, Polyphenols (Ferric
chloride test) and Proteins (Ninhydrin test) was
done in the methanolic extracts of P.auriculata [1,
11].
2.6. GC–MS analysis for extracted micro
propagated plant
We used the gas chromatography–mass
spectrometry (GC–MS) for the analysis because
this technique is very sensitive, simple and fast.
Secondary metabolite profiling was performed
with Agilent 7809A gas chromatograph (Agilent
Technologies) coupled to a Triple-Axis detector
(Agilent 5975C), using a ZB-5 (30 m × 0.25 mm)
capillary column (0.25 mm film thickness) using
helium as a carrier gas at a flow rate of 1 mL/min.
Approximately 5 mg of the extract was transferred
to a 12mL tube and 5 mL of a methanol:
chloroform (1:1) solution was added. The
resulting solution (100 µL) was transferred to a
2mL glass GC vial with a 200µL glass insert. All
analyses were measured using three biological
replicates.
2.7. Antioxidant activity of micropropagated
plants
2.7.1. DPPH assay
Methanolic extracts of in-vitro propagated P.
auriculata was subjected to DPPH radical
scavenging assay using the method [2].
2.7.2. Reducing power assay
Methanolic extracts of in-vitro propagated P.
auriculata was tested for reducing power assay by
following already existing method [15].
2.7.3. In-vitro Cell viability assay
The cell viability of the extract was determined
using 3-(4, 5-dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium bromide assay (MTT)
illustrated by [13]. The human cancer cell lines
PA1 (Ovarian teratocarcinoma cell line) and A549
(Human lung adenocarcinoma) were obtained
from NCCS (Pune, India) and used in this study.
The cells were grown and maintained in MEM,
pH 7.4 supplemented with 10% Fetal calf serum,
glutamine (2mM), Penicillin (100 units/ml), and
streptomycin (100 mg/mL). Both A549 and PA1
cells were seeded in 96 well plates at a density of
1x104cells. After attaining the confluence the cells
were treated with different concentrations of drug
(5-60µg/ml) and incubated at 24 and 48hours.
Then the cells were incubated with 100µL
(0.5mg/mL) of MTT at 37ºC for 3hours after the
treatment period. The formazan crystals were
dissolved by adding 100µL of DMSO. The
intensity was measured using multiplate reader at
570nm.
2.8. Nuclear staining (DAPI) of cancer cells
The morphological changes of both A549 and
PA1 cells were assessed by DAPI nuclear
staining. Briefly, cell density of 3x104cells were
seeded in 6 well plates and subjected to treatment
with the two different IC50 concentrations. At the
end of the treatment, the media was removed from
the plate and washed with PBS. Then the cells
were fixed with 3% paraformaldehyde for 10mins
at RT. The fixed cells were then permeabilized
with 0.2% Triton X 100 in PBS for 10mins at RT.
After this the cells were incubated with 0.5µg/mL
of DAPI for 5mins. Then the cells were viewed

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under fluorescent microscope with an excitation at
359nm and emission at 461nm to observe the
apoptotic nuclei.
2.9. Statistical Analysis
The experimental results were expressed as mean
± SD. Data were assessed by the student P > 0.05
was considered as stastistically signinficant.
3. RESULTS & DISCUSSION
Searching for the alternative medicine of
anticancer drug, the medicinal plants were played
the significant role in the development of
anticancer molecules. In the present study, We
choosen the plumbagin as an anticancer
compound was studied against the breast cancer,
liver cancer, prostate cancer and lung cancer. The
medicinal plant P. auriculata for the isolation of
phytochemicals was well established and it had
the significant amount of steroid compounds [16].
For compromising world population need to the
rapid drug development, by the biological way in
vitro propagation is one of the technique to
producing higher amount of drug in the short span
of time [19]. Natural products have been
increasingly used worldwide to treat various
diseases, including cancer. Herbal medicines and
phytochemicals could be potent agents for
prevention of lung cancer and treatment by
regulating multimolecular targets involved in
angiogenesis and metastasis [23]. Several
medicinal plants are considered toxic and could
cause serious damage to the health of patients.
Therefore, assessment of the toxicity of medicinal
plants, as well as their herbal preparations, is
essential to determine the applicability of the
sample as a pharmacological drug [25].
3.1. Multiplication of shoot and root of
P.auriculata in MS Media
The P. auriculata callus derived stem showed the
plumbagin content was comparable amount of the
normal plant and while the root showed the rich
amount of plumbagin, the combinations of 1 mg/L
IAA and 1.5 mg/L NAA showed the best result of
callus induction [26] this concentration also
showed very close to our result. The
phytochemical analysis of methanolic extract P.
auriclata showed the polyphenols, tannins,
alkaloids, flavonoids, saponins, carbohydrates and
proteins, presence of these major compounds may
be responsible for the significant antioxidant and
also anticancer activity.
Nowadays, in-vitro plant tissue culture technique
is developed to produce biologically significant
medicinal molecules for therapeutic use. In this
study, P. auriculata plant was taken for the in-
vitro studies. However, normal plants can also
grow but the time for growth and biomass may
vary considerably because of the nutrient types
and availability from their environment. Hence,
the plant tissue culture technique provides the
needful amount of nutrition and at a specific
environment. The various hormones (IAA, GA3,
IBA and KIN) were tested alone among the
hormone, 1.5mg/L of IAA showed maximum
shoot length at 4.1±0.15cm and 1.4±0.15cm of
root length. Then IAA taken for constant
concentration and combined with different
concentration of other remaining hormones,
among the combination MS basal medium
supplemented with 1.5 mg/L IAA with 0.5 mg/L
GA3 combination showed the highest shoot length
at 6.2 ± 0.25 cm (Fig. 2) and 1.5 mg/L IAA with
1.0 mg/L GA3 hormonal combination showed the
highest root length at 0.8±0.2cm. Indole-3-acetic
acid is a plant growth hormone usually used in
tissue culture for elongation and induction of
shoot and root length (Fig. 3). And also IAA in
combination with IBA at the concentration of 1.5
mg/L IAA and 1.0 mg/l IBA showed 4.4±0.25cm
shoot length 2.2±0.2cm root length, IAA
combination with kinetin showed 4.3±0.25cm of
shoot length and 0.1±0.05cm of root length in the
concentration of 1.5mg/L IAA and 0.5mg/L
Kinetin. Both the hormone IBA and KIN
combined with IAA showed moderate response
(Table 1).
3.2. Extraction and phytochemical screening of
in-vitro plant
Methanolic extract of P.auriculata revealed that it
contained alkaloids, phenols and flavonoids

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presented in the Table 2.Then the plant extracts
were subjected to profile the presence of bioactive
compounds through Gas Chromatography and
Mass spectroscopy.
3.3. GC-MS profile of methanolic extract P.
auriculata
P. auriculata contained many phytochemicals
such as Z -11-Octadecen –1-yl- acetate, C-
sitosterol, heptadecanoic acid , 9 methyl , methyl
ester, 4,5,7 trihydroxy isoflavone, E, E, Z, 1, 3, 12
nonadecatriene 5,14 diol, ethanol 2, (9
octadecenyloxyl, (E), 13 docosenoic acid, methyl
ester , (Z), bis (2 ethyl hexyl ) phthalate, 10,12,14
nonacosatriynoic acid (Table 3). Similar study
was previously reported in the GC-MS analysis of
P. scandens root ethanolic extract showed the
four major compounds such as plumbagin, epi–
isoshinanolone, palmitic acid and sitosterol, in
which the compound sitosterol presence in
maximum level as compared with our GC-MS
profile also showed the presence of compound
sitoserol at maximum [27].
3.4. Antioxidant activity of P. auriculata in
vitro
Antioxidant radical scavenging activity of in vitro
plant P. auriculata based on the our knowledge
and literature survey we report first time it showed
IC50 value at 260µg/ml, same time the methanolic
root extract of P. zeylanica and P. rosea showed
that IC50 values 134 µg/ml and 147 µg/ml
respectively [10] (Fig. 4). The callus of P.
zeylanica showed the DPPH value at 360µg/ml
[20]. As estimated by the DPPH scavenging
activity, the antioxidant activity of the methanolic
extract 260µg/ml (IC50) and reducing power assay
the methnolic extract and ascorbic acid(100µg/
ml) shows maximum absorbance at 0.321nm and
0.467nm respectively (Fig. 5). In order to increase
concentration the absorbance was increased, it
means reducing the ferrous to ferric ion
concentration. Ascorbic acid was used as standard
antioxidant.
3.5. Antiproliferative activitity in vitro
The leaves of P. auricalata also showed the
significant antioxidant activity when compared to
the methanolic root extract of various plumbago
species. The major reason for the antioxidant
activity of the plant may be which present
sitosterol in higher percentage in methanolic
extract. Because of this compound presence we
analyzed the anticancer activity against in vitro
lung (A549) and ovarian cancer (PA1) it showed
good anticancer at minimum concentration. Beta
sitosterol is a plant sterol which has the substantial
properties to the human beings such as lowering
the blood chloestrol level, antioxidant, treated
against cancer like stomach, breast, lung and
ovarian through the antiproliferative and induction
of apoptosis.
The cytotoxic activity of the extract was assayed
against A549 (human lung cancer) and PA1
(human ovarian cancer) cell lines using MTT
assay. The extract exhibited a significant
reduction in cell viability both the cell lines
experiment was conducted in a concentration and
time dependent manner. The methanolic extract of
showed minimum cytotoxic activity at 45µg/ml
and 10µg/ml for A549 cell line, 10µg/ml and
60µg/ml for PA1 cell line at 24hours and 48hours
respectively because of concentration obtained
from the IC50 value or the respective cancer cell
line.The apoptotic and nuclear morphology was
studied by nuclear staining DAPI, the cells of the
nucleus stained and exhibit blue colored (Figs. 6-
8).
CONCLUSION
Medicinal plants contain several bioactive
molecules and also posses lesser toxicity and
greater ailments to human beings. Traditional
medicines are having the essential place in recent
medication, because fewer side effects, low cost
and bio availability. Plants having rich antioxidant
and anticancer activity therefore the present study
isolated the bioactive molecule for their unique
property. The medicinal plant P. auriculata were
evaluated for their antioxidant and anticancer
activity. The results indicated the significant anti
lung and ovarian cancer activity at minimal
concentration 10-40 microgram concentration and

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also there is no report for isolation of compounds
from micropropagated plants P. auriculata.
Finally, the present study suggests that the P.
auriculata constitutes novel anticancer active
molecules which should be helpful to the
wellbeing of cancer affected patients.
CONFLICT OF INTEREST
The authors have declared no conflict of interest.
COMPLIANCE WITH ETHICS
REQUIREMENTS
This article does not contain any studies with
human or animal subjects.
ACKNOWLEDGEMENT
One of the authors (G.L) thanks The Director,
CAS in Botany for providing the laboratory
facilities University Grants Commission (UGC-
UPE Phase II) for the funding.
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Fig. 1- A twig of Plumbago auriculata.

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Fig. 2- Shoot and Root multiplication of P. auriculata at the concentration of IAA +IBA (1.5+1.0 mg/L) with
different days.
Fig. 3- GC- MS analysis of methanolic extract of in vitro propagated P. auriculata.
Fig. 4- Percentage of scavenging activity of in vitro P. auriculata methanolic extract.

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Fig. 5- Reducing power of in vitro P. auriculata methanolic extract compared with antioxidant molecule ascorbic
acid.
Fig. 6- Effect of methanolic extract on the cell viability of lung (A549) and ovarian (PA1) cancers cell line.
Fig. 7- Anticancer activity of methanolic extract (P. auriculata) against lung cancer cell line A549.

Micropropagation and anticancer activity of methanolic extract of Plumbago auriculata Lam
G. Lakshmanan, et a
l.
2010
2010
2010
2010
Fig. 8- Anticancer activity of methanolic extract P. auriculata against ovarian cancer cell PA1.
Table 1- Effect of growth hormones on root and shoot induction of Plumbago auriculata .
IAA: Indole Acetic Acid; GA3: Gibberllic Acid; IBA: Indole Butyric Acid; KIN: Kinetin

Micropropagation and anticancer activity of methanolic extract of Plumbago auriculata Lam
G. Lakshmanan, et a
l.
2011
2011
2011
2011
Table 2- Phytochemical screening of methanolic extract of Plumbago auriculata.
+: mild presence; ++: fair quantity ; +++: abundant presence
Table 3- Quantitative analysis of phytochemicals in the methanolic extract of P. auriculata.