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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.
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G. Lakshmanan, et a
International Journal of Advanced Biotechnology and Research (IJBR)
ISSN 0976-2612, Online ISSN 2278599X,
Vol-7, Issue-4, 2016, pp2001-2011
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) &
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
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
Micropropagation and anticancer activity of methanolic extract of Plumbago auriculata Lam
G. Lakshmanan, et a
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.
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 35 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.
Micropropagation and anticancer activity of methanolic extract of Plumbago auriculata Lam
G. Lakshmanan, et a
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 (Mayers
test), Flavonoids (Aluminium chloride test),
Carbohydrates (Fehlings test), Saponins
(Frothing test), Tannins, Polyphenols (Ferric
chloride test) and Proteins (Ninhydrin test) was
done in the methanolic extracts of P.auriculata [1,
2.6. GCMS analysis for extracted micro
propagated plant
We used the gas chromatographymass
spectrometry (GCMS) 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
2.7. Antioxidant activity of micropropagated
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
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
Micropropagation and anticancer activity of methanolic extract of Plumbago auriculata Lam
G. Lakshmanan, et a
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.
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
Micropropagation and anticancer activity of methanolic extract of Plumbago auriculata Lam
G. Lakshmanan, et a
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.
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
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
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 4g/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-
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
Micropropagation and anticancer activity of methanolic extract of Plumbago auriculata Lam
G. Lakshmanan, et a
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.
The authors have declared no conflict of interest.
This article does not contain any studies with
human or animal subjects.
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.
1. Aziz, M.A., (2015) Qualitative phytochemical
screening and evaluation of anti-inflammatory,
analgesic and antipyretic activities of Microcos
paniculata barks and fruits. J. Int. Med. 13(3),
2. Blois, M.S., (1958) Antioxidant determinations
by the use of a stable free radical. Nature. 29,
3. Checker, R., Sharma, D., Sandur, S.K.,
Khanam, S., Poduval, T.B., (2009) Anti-
inflammatory effects of plumbagin are
mediated by inhibition of NF Kappa B
activation in lymphocytes. Int.
Immunopharmocol. 9, 949-58.
4. Deshpande, J., Labade, D., Shankar, K., Kata,
N., Chaudhari, M., Wani, M., Khetmalas, M.,
(2014) In vitro callus induction estimation of
plumbagin content from Plumbago auriculata.
Indian. J. Exp. Biol. 52, 1122-1127.
5. Devasagayam, T.P., Tilak, J.C., Boloor, K.K.,
Sane, K.S., Ghaskadbi, S.S., Lele, R.D., (2004)
Free Radicals and Antioxidants in Human
Health: Current Status and Future Prospects. J.
Assoc. Physicians. India. 52, 794-804.
6. Dorni, C., Vidyalakshmi, K.S., Vasanthi, R.H.,
Rajamanickam G.V., (2007) HPTLC method
for the quantification of plumbagin in three
Plumbago species. Res. J. Phytochem. 1, 46-
7. Ferlay, J., Steliarova-Foucher, E., Lortet-
Tieulent, J., Rosso, S., et al., (2013) Cancer
incidence and mortality patterns in Europe:
Estimates for 40 countries in 2012. European J.
Cancer 49, 1374-1403.
8. Ferlay, J., Soerjomataram, I., Dikshit, R., et al.,
(2015) Cancer incidence and mortality
worldwide: sources, methods and major
patterns in GLOBOCAN 2012. Int. J. Cancer.
136(5), 359-386.
9. Galal, A.M., Ramam, V., Avula, B., Wang,
Y.H., et al., (2013) Comparative study of three
Plumbago L. species (Plumbaginaceae) by
microscopy, UPLC-UV and HPTLC. J. Nat.
Med. 67, 554-561.
10.Gayatri Nahak., Rajani Kanta Sahu., (2011)
Antioxidant activity of Plumbago zeylanica
and Plumbago rosea belonging to family
plumbaginaceae. Natural Products: An Indian
J. 7(2), 51-56.
11.Harbone, A.J., (1998) Phytochemical methods.
A guide to modern techniques of plant
analysis. Chapman and Hall, Third edition,
London, UK.
12.Mallath, M.K., Taylor, D.G., Badwe, R.A., et
al., (2014) The growing burden of cancer in
India: epidemiology and social context. Lancet
Oncol. 15(6), 205-212.
13.Mosmann, T., (1983) Rapid colorimetric assay
for cellular growth and survival: application to
proliferation and cytotoxicity assays. J.
Immunol. Methods. 65(1-2), 55-63.
14.Newman, D.J., Cragg, G.M., (2012) Natural
Products as Sources of New Drugs from 1981
to 2016. J. Nat. Prod. 79, 629-661.
15.Oyaizu, M., (1986) Studies on products of
browning reactions: antioxidative activities of
Micropropagation and anticancer activity of methanolic extract of Plumbago auriculata Lam
G. Lakshmanan, et a
products of browning reaction prepared from
glucosamine. Jap. J. Nutri. 44, 307-315.
16.PAIVA, Selma R. de., LIMA, Lucilene A.,
Maria Auxiliadora C., (2004) Plumbagin
quantification in roots of Plumbago scandens
L. obtained by different extraction techniques.
Anais da Academia Brasileira de Ciências.
76(3), 499-504.
17.Patwardhan, A., Harris, J., Leng, N., Bartha,
G., et al., (2015) Achieving high-sensitivity for
clinal applications using augmented exome
sequencing . Genome Med. 7(71), 1- 14.
18.Pezzuto, J.M., (1997) Plant-derived anticancer
agents. Biochem. Pharmacol. 53(2), 121-33.
19.Sathya, S., Sudhagar, S., Vidhya Priya, M.,
Bharathi Raja, R., et al., (2010) 3-Hydroxylup-
20(29)-ene-27,28-dioic acid dimethyl ester, a
novel natural product from Plumbago
zeylanica inhibits the proliferation and
migration of MDA-MB-231 cells. Chem. Biol.
Interact. 188(3), 412420.
20.Satyajit, K., Gayatri, N., Santi Lata, S., Rajani
Kanta, S., (2012) Antioxidant activity and
phytochemical evaluation of Plumbago
zeylanica linn. in vivo and in vitro. Int. J.
Pharm. Pharmaceut. Sci. 4, 522-526.
21.Schmelzer, G.H., Gurib-Fakim, A., Arroo,
R.R.J., et al., (2008) Plant resources of
Tropical Africa 11(1) Medicinal plants 1,
Wageningen. Netherlands. Prota foundation
22.Siegel, R.L., Miller, K.D., Jemal, A., (2016)
Cancer statistics 2016. CA Cancer J. Clin. 66,
23.Su-Jong Jeong., Chang-Hoi Ho., Hyeon-Ju
Gim., Mollye E. Brown., (2011) Phenology
shifts at start vs. end of growing season in
temperate vegetation over the Northern
Hemisphere for the period 1982-2008. Global
Change Biol. 17, 2385-2399.
24.Tan W, Lu J, Huang M, Li Y, et al., (2011)
Anti-cancer natural products isolated from
Chinese medicinal herbs. Chin. Med. 6(1), 27.
25.Valdir F. Veiga Junior., Angelo C. Pinto.,
Maria Aparecida M., (2005) Plantas
Medicinais: Cura Segura? Quim.Nova. 28,
26.Yi Chen., Suping Gao., (2013) Preliminary
Report of PGRs Influence to Multiple Shoots
Induction and Plant Regeneration on Plumbago
auriculata. Amer. J. Plant Sci. 4, 23-29.
27.Zhang, Q.R., (2007) Research on the Chemical
Composition of Plumbago zeylanica.
Traditional Chinese Medicines. 30(5), 558-
Fig. 1- A twig of Plumbago auriculata.
Micropropagation and anticancer activity of methanolic extract of Plumbago auriculata Lam
G. Lakshmanan, et a
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.
Micropropagation and anticancer activity of methanolic extract of Plumbago auriculata Lam
G. Lakshmanan, et a
Fig. 5- Reducing power of in vitro P. auriculata methanolic extract compared with antioxidant molecule ascorbic
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
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
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.
... Methanolic leaf extract of P. auriculata revealed the presence of the following phytochemicals-tannins, flavonoids, phenols, alkaloids, saponins, proteins, and carbohydrates with phenols being the most abundant compound (Lakshmanan et al., 2016). Aerial parts of P. auriculata extracted in the following solvents: acetone, chloroform, petroleum ether, ethanol and ethyl acetate showed positive results when tested for steroids, carbohydrates, phenolics, tannins, saponins, flavonoids and terpenoids, however the aqueous extract only showed positive for the presence of tannins (Tharmaraj and Antonysamy, 2013). ...
... Methanolic leaf extracts also showed significant cytotoxic activity when assayed against human lung cancer (A549) and ovarian cancer (PAI) cell lines using MTT assay (Lakshmanan et al., 2016). For A549, it showed a minimum cytotoxic activity at 45µg/ml and 10µg/ml cell line and for PAI 10µg/ml and 60µg/ml cell line at 24 hours and 48 hours, respectively. ...
... Bioactive compounds extracted from plants are being extensively researched and utilized as a possible anticancer agent. In vitro P. auriculata leaves were dried and extracted using methanol as a solvent (Lakshmanan et al. 2016). The leaf extract was tested against ovarian (PAI), lung (A549) and malignancy cell lines for apoptotic and anti-proliferative activities. ...
... The commercial exploitation of this technique for the propagation of numerous plant species would benefit many medicinal plants. Indeed, large scale in vitro propagation of medicinal plants has been reported (Kumar and Bhavanandan 1988;Verma et al. 2002;Huang et al. 2010;Deshpande et al. 2015;Patidar et al. 2015;Dilshad et al. 2016;Lakshmanan et al. 2016;Silja et al. 2016). ...
... Regrettably, to date, plumbagin has been extracted mainly from Plumbago auriculata, P. indica, and P. zeylanica, while its extraction from C. willmottianum has not been developed (Nitsch and Nitsch 1967;Kumar and Bhavanandan 1988;Das and Rout 2002;Singh et al. 2002;Chen and Gao 2013;Deshpande et al. 2014;Thach et al. 2014;Lakshmanan et al. 2016). Furthermore, due to the scarcity of germplasm resources, low natural multiplication rates or long juvenile phases, the commercial release of a new genotype of C. willmottianum may take several years by conventional vegetative propagation methods, which greatly restricts its use in landscaping and drug development. ...
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Ceratostigma willmottianum Stapf is an endemic species of China. Although it has high ornamental and medical value, its use is greatly limited by the scarcity of germplasm resources. To protect the wild resources and obtain more plumbagin, a protocol for aseptic rapid propagation of C. willmottianum was developed and the plumbagin content was quantified. The results showed that Murashige and Skoog (MS) medium with 6-benzylaminopurine (6-BA, 0.5 mg L− 1) and 1-naphthaleneacetic acid (NAA, 0.1 mg L− 1) was suitable for axillary bud induction with an induction rate of 82.74%, while MS medium with higher 6-BA (2.0 mg L− 1) and NAA (0.1 mg L− 1) was suitable for multiplication with a propagation coefficient of 2.91. Additionally, 1/2 MS medium with indole-3-butyric acid (IBA, 0.5 mg L− 1) was suitable for rooting, with rooting rate of 94.44 ± 1.93%. High-performance liquid chromatography (HPLC) analysis revealed the tendency of the plumbagin content to decrease in the following order: root had the highest content, followed by the whole-plant, stem and leaf. Notably, the plumbagin content in 1-month-old plantlets was higher than that in 1- and 2-year-old plants, especially in the roots (19.94 mg g− 1 DW). Overall, an aseptic rapid propagation system of C. willmottianum was successfully established for the first time and the roots of 1-month-old plantlets were used to obtain plumbagin rapidly.
... Additionally, it has been reported to have pharmacological properties such as antimicrobial, anticancer, antiulcer, antimalaria and antifungal [63]. Tannins, flavonoids, phenols, carbohydrates, proteins, alkaloids, and saponins are the phytochemicals that have been identified in methanolic extracts of P. auriculata leaves [65]. Tharmaraj and Antonysamy [66] reported the presence of phenolics, tannins, saponins, carbohydrates, steroids, flavonoids, terpenoids in organic extracts of the aerial parts while the aqueous extracts only showed the presence of tannins. ...
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Metal oxide nanoparticles (NPs), such as zinc oxide (ZnO), have been researched extensively for applications in biotechnology, photovoltaics, photocatalysis, sensors, cosmetics, and pharmaceuticals due to their unique properties at the nanoscale. ZnO NPs have been fabricated using conventional physical and chemical processes, but these techniques are limited due to the use of hazardous chemicals that are bad for the environment and high energy consumption. Plant-mediated synthesis of ZnO NPs has piqued the interest of researchers owing to secondary metabolites found in plants that can reduce Zn precursors and stabilise ZnO NPs. Thus, plant-mediated synthesis of ZnO NPs has become one of the alternative green synthesis routes for the fabrication of ZnO NPs. This is attributable to its environmental friendliness, simplicity, and the potential for industrial-scale expansion. Southern Africa is home to a large and diverse indigenous medicinal plant population. However, the use of these indigenous medicinal plants for the preparation of ZnO NPs is understudied. This review looks at the indigenous medicinal plants of southern Africa that have been used to synthesise ZnO NPs for a variety of applications. In conclusion, there is a need for more exploration of southern African indigenous plants for green synthesis of ZnO NPs.
... The methanolic extract had a minimal cytotoxic activity at 10 μg/ml (24 hr) and 60 μg/ml (48 hr) on PA1 cell lines of human ovarian cancer. Consequently, of note is the significant morphological changes also observed in PA1 cancer cells ( Table 2) by nuclear staining (4',6-diamidino-2-phenylindole) method [92]. ...
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Ovarian cancer (OC) accounts for the highest tumor-related mortality among the gynecologic malignancies. Most of the OC patients diagnosed with advanced-stage (III and IV) this situation creates panic and provokes an emergency to discover a new therapeutic strategy. Plants that possess medicinal properties are gaining attention as they are enriched with various chemical compounds that are potential to treat various diseases. It is a prolonged process to provide innovative and significant leads against a range of pharmacological targets for a human disease management system. Though challenges and difficulties are faced in the development of a new drug, the emergence of combinatorial chemistry is providing a new ray of hope and also, the executed effort in discovering the drug, and a chemical compound has been remarkably successful. This review discussed the role of medicinal plants that are native of South Africa in treating the Ovarian Cancer and in drug discovery.
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Background Cancer is one of the most critical but ubiquitous causes of death grappled from past decades. Widely used chemotherapy with cytotoxic activity blocks/ kills the cancer cell. The compounds targeted for anticancerous activity are either derived synthetically or naturally (through plants or microbial origin). Current day, versatile role of plants in medicinal field has been attributed to the secondary metabolites it produces, known for their anticancer activity. Therefore, discovery, identification and commercial production of such novel anticancer drugs is escalated and are centerpiece for pharmaceuticals. Main body A biotechnological approach, principally tissue culture, leads the candidacy to be an alternative method for production of anticancer compounds. A wide range of bioactive agents like alkaloids, steroids, phenolics, saponins, flavonoids, and terpenoids are in huge demand commercially. Plant tissue culture applications are constructively more advantageous over conventional methods in terms of their continuous, controlled, aseptic production, large scale and de novo synthesis opportunity. Various bioreactors are used for mass cultivation of bioactive compound at commercial level. For example: stirred tank reactors are used for production of shikonin from Lithospermum erythrorhizon, vincristine from Catharanthus roseus , podophyllotoxin from Podophyllum etc . Strategies like callus culture, suspension culture and hairy root culture are opted for mass cultivation of these bioactives. Conclusions This review summarizes plant tissue culture as a promising strategy proven to be a colossal breakthrough in reliable and continuous production of existing and novel anticancer compounds and help in combating the increasing future demands.
Plumbago auriculata Lam. is an ornamental plant native to South Africa and widely cultivated in China, but the cultivated plants are dominated by a single variety. The development of new varieties is of great commercial interest, and genetic diversity is the foundation of breeding programs. In this study, 85 progenies were obtained by crosses between Plumbago auriculata and Plumbago auriculata f. alba. The genetic diversity of these hybrids was evaluated using horticultural traits and ISSR and SRAP markers. Of the 25 horticultural traits evaluated, the largest variation was found in the beginning of the blooming period, and sepal length was the least variable trait. Correlation analysis showed that the wider the plant, the greater the number of inflorescences and the earlier the flowering. Seven factors explained 65.171% of the total variance; the first factor was leaf morphology, and the second factor was flower morphology. The genetic diversity of the 85 progenies was analyzed using seven pairs of SRAP primers and eight ISSR primers. The average number of effective alleles for 85 hybrids was 1.638, and the average Shannon index value was 0.507. The Nei genetic similarity coefficient indicated that the similarity between WLBS and WSBL was the highest, while that between BLWS and BSWL was the lowest. Analysis of molecular variance (AMOVA) indicated that the main variation was within populations. Cluster analysis based on horticultural traits and molecular markers divided all progenies into seven and five groups, respectively, and there were obvious differences between the two clusters. In this study, we created intermediate materials for future breeding, taking the first step in the cross-breeding of P. auriculata.
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ABstRACt OBJECtIVE: The main objectives of this study were to qualitatively evaluate the profile of phytochemical constituents present in methanolic extract of Microcos paniculata bark (BME) and fruit (FME), as well as to evaluate their anti-inflammatory, analgesic and antipyretic activities. MEtHOds: Phytochemical constituents of BME and FME were determined by different qualitative tests such as Molisch’s test, Fehling’s test, alkaloid test, frothing test, FeCl3 test, alkali test, Salkowski’s test and Baljet test. The anti-inflammatory, analgesic and antipyretic activities of the extracts were evaluated through proteinase-inhibitory assay, xylene-induced ear edema test, cotton pellet-induced granuloma formation in mice, formalin test, acetic acid-induced writhing test, tail immersion test and Brewer’s yeastinduced pyrexia in mice. REsuLts: M. paniculata extracts revealed the presence of carbohydrates, alkaloids, saponins, tannins, flavonoids and triterpenoids. All of the extracts showed significant (P<0.05, vs aspirin group) proteinaseinhibitory activity, whereas the highest effect elicited by plant extracts was exhibited by the BME (75.94% proteinase inhibition activity) with a half-maximal inhibitory concentration (IC50) of 61.31 μg/mL. Each extract at the doses of 200 and 400 mg/kg body weight showed significant (P<0.05, vs control) percentage inhibition of ear edema and granuloma formation. These extracts significantly (P<0.05, vs control) reduced the paw licking and abdominal writhing of mice. In addition, BME 400 mg/kg, and FME at 200 and 400 mg/kg showed significant (P<0.05, vs control) analgesic activities at 60 min in the tail immersion test. Again, the significant (P<0.05, vs control) post-treatment antipyretic activities were found by BME 200 and 400 mg/kg and FME 400 mg/kg respectively. CONCLusION: Study results indicate that M. paniculata may provide a source of plant compounds with anti-inflammatory, analgesic and antipyretic activities
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Whole exome sequencing is increasingly used for the clinical evaluation of genetic disease, yet the variation of coverage and sensitivity over medically relevant parts of the genome remains poorly understood. Several sequencing-based assays continue to provide coverage that is inadequate for clinical assessment. Using sequence data obtained from the NA12878 reference sample and pre-defined lists of medically-relevant protein-coding and noncoding sequences, we compared the breadth and depth of coverage obtained among four commercial exome capture platforms and whole genome sequencing. In addition, we evaluated the performance of an augmented exome strategy, ACE, that extends coverage in medically relevant regions and enhances coverage in areas that are challenging to sequence. Leveraging reference call-sets, we also examined the effects of improved coverage on variant detection sensitivity. We observed coverage shortfalls with each of the conventional exome-capture and whole-genome platforms across several medically interpretable genes. These gaps included areas of the genome required for reporting recently established secondary findings (ACMG) and known disease-associated loci. The augmented exome strategy recovered many of these gaps, resulting in improved coverage in these areas. At clinically-relevant coverage levels (100 % bases covered at ≥20×), ACE improved coverage among genes in the medically interpretable genome (>90 % covered relative to 10-78 % with other platforms), the set of ACMG secondary finding genes (91 % covered relative to 4-75 % with other platforms) and a subset of variants known to be associated with human disease (99 % covered relative to 52-95 % with other platforms). Improved coverage translated into improvements in sensitivity, with ACE variant detection sensitivities (>97.5 % SNVs, >92.5 % InDels) exceeding that observed with conventional whole-exome and whole-genome platforms. Clinicians should consider analytical performance when making clinical assessments, given that even a few missed variants can lead to reporting false negative results. An augmented exome strategy provides a level of coverage not achievable with other platforms, thus addressing concerns regarding the lack of sensitivity in clinically important regions. In clinical applications where comprehensive coverage of medically interpretable areas of the genome requires higher localized sequencing depth, an augmented exome approach offers both cost and performance advantages over other sequencing-based tests.
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With the explants of young nodals and leaves of Plumbago auriculata, we studied multiple shoots induction and plant regeneration in the medium with different PGR proportions. The results showed the nodals of Plumbago auriculata could be used as the explant to induce multiple shoots. After culturing young nodals for 30 d, optimum single shoot formation was achieved on MS medium supplemented with 0.3 mg/L 6-BA and the induction rate was 70.79%. After being transferred to the medium containing 1.0 mg/L 6-BA, 1.0 mg/L NAA and 0.2 mg/L IAA for 1 - 2 generation, single shoot was induced to form multiple shoots with the multiplication rate was 506.45%. After multiple shoots grew to 3 cm high, the multiple shoots were cut into single shoots and transferred onto half-strength MS medium supplemented with 0.5 mg/L NAA and the rooted rate was 94.33%. Finally the intact plants were obtained.
Each year, the American Cancer Society estimates the numbers of new cancer cases and deaths that will occur in the United States in the current year and compiles the most recent data on cancer incidence, mortality, and survival. Incidence data were collected by the National Cancer Institute (Surveillance, Epidemiology, and End Results [SEER] Program), the Centers for Disease Control and Prevention (National Program of Cancer Registries), and the North American Association of Central Cancer Registries. Mortality data were collected by the National Center for Health Statistics. In 2016, 1,685,210 new cancer cases and 595,690 cancer deaths are projected to occur in the United States. Overall cancer incidence trends (13 oldest SEER registries) are stable in women, but declining by 3.1% per year in men (from 2009-2012), much of which is because of recent rapid declines in prostate cancer diagnoses. The cancer death rate has dropped by 23% since 1991, translating to more than 1.7 million deaths averted through 2012. Despite this progress, death rates are increasing for cancers of the liver, pancreas, and uterine corpus, and cancer is now the leading cause of death in 21 states, primarily due to exceptionally large reductions in death from heart disease. Among children and adolescents (aged birth-19 years), brain cancer has surpassed leukemia as the leading cause of cancer death because of the dramatic therapeutic advances against leukemia. Accelerating progress against cancer requires both increased national investment in cancer research and the application of existing cancer control knowledge across all segments of the population. CA Cancer J Clin 2016. © 2016 American Cancer Society.