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Cytotoxic constituents of Abutilon

indicum leaves against U87MG human
glioblastoma cells
a

Rukaiyya Sirajuddin Khan , Mahibalan Senthi , Poorna Chandra

a

Rao , Ameer Basha , Mallika Alvala , Dinesh Tummuri , Hironori

d

Masubuti , Yoshinori Fujimoto & Ahil Sajeli Begum

Department of Pharmacy, BITS-Pilani Hyderabad Campus,

Jawahar Nagar, Shamirpet Mandal, Hyderabad500 078, Telangana,
India

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Regional Agricultural Research Station (ANGRAU), Palem,

Mahabub Nagar, Telangana, India
c

National Institute of Pharmaceutical Education and ResearchHyderabad, Balanagar, Hyderabad500 037, Telanagana, India
d

Department of Chemistry and Materials Science, Tokyo Institute

of Technology, Meguro, Tokyo152-8551, Japan
Published online: 25 Nov 2014.

To cite this article: Rukaiyya Sirajuddin Khan, Mahibalan Senthi, Poorna Chandra Rao, Ameer
Basha, Mallika Alvala, Dinesh Tummuri, Hironori Masubuti, Yoshinori Fujimoto & Ahil Sajeli Begum
(2014): Cytotoxic constituents of Abutilon indicum leaves against U87MG human glioblastoma cells,
Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.976643
To link to this article: http://dx.doi.org/10.1080/14786419.2014.976643

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Natural Product Research, 2014

http://dx.doi.org/10.1080/14786419.2014.976643

SHORT COMMUNICATION
Cytotoxic constituents of Abutilon indicum leaves against U87MG human
glioblastoma cells
Rukaiyya Sirajuddin Khana, Mahibalan Senthia, Poorna Chandra Raoa, Ameer Bashab,
Mallika Alvalac, Dinesh Tummuric, Hironori Masubutid, Yoshinori Fujimotod and Ahil
Sajeli Beguma*

Downloaded by [Mr Senthi Mahibalan] at 20:44 12 January 2015

Department of Pharmacy, BITS-Pilani Hyderabad Campus, Jawahar Nagar, Shamirpet Mandal,

Hyderabad 500 078, Telangana, India; bRegional Agricultural Research Station (ANGRAU), Palem,
Mahabub Nagar, Telangana, India; cNational Institute of Pharmaceutical Education and ResearchHyderabad, Balanagar, Hyderabad 500 037, Telanagana, India; dDepartment of Chemistry and Materials
Science, Tokyo Institute of Technology, Meguro, Tokyo 152-8551, Japan
(Received 16 July 2014; final version received 10 October 2014)

The study was aimed to identify cytotoxic leads from Abutilon indicum leaves for
treating glioblastoma. The petroleum ether extract, methanol extract (AIM),
chloroform and ethyl acetate sub-fractions (AIM-C and AIM-E, respectively) prepared
from AIM were tested for cytotoxicity on U87MG human glioblastoma cells using 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. These extracts
exhibited considerable activity (IC50 values of 42.6 64.5 mg/mL). The most active
AIM-C fraction was repeatedly chromatographed to yield four known compounds,
methyl trans-p-coumarate (1), methyl caffeate (2), syringic acid (3) and pinellic acid
(4). Cell viability assay of 1 4 against U87MG cells indicated 2 as most active (IC50
value of 8.2 mg/mL), whereas the other three compounds were much less active.
Interestingly, compounds 1 4 were non-toxic towards normal human cells (HEK-293).
The content of 2 in AIM-C was estimated as 3% by HPLC. Hence, presence of some
more active substances besides methyl caffeate (2) in AIM-C is anticipated.
Keywords: Abutilon indicum; MTT; U87MG; methyl caffeate; cytotoxicity;
Malvaceae

1. Introduction
Glioblastoma, a life-threatening malignant brain tumour is one of the leading causes of cancerrelated deaths among children and adults. The average life of patients with this aggressive
disease is being reported to be less than 15 months while under treatments such as surgery,

*Corresponding author. Email: sajeli@hyderabad.bits-pilani.ac.in

q 2014 Taylor & Francis

R.S. Khan et al.

radiotherapy and chemotherapy (Tabatabai et al. 2011). Glioblastoma cells are rapidly
proliferative invading the surrounding normal brain tissue and thereby affecting spine as well.
Due to frequent acquisition of drug-resistant phenotypes and occurrence of secondary
malignancies by glioblastoma cells, the existing chemotherapy could not be effective (Liu et al.
2006; Sakariassen et al. 2007). Hence, there is a need for developing new drugs for the treatment
of glioblastoma. In view of this, this study was aimed to discover cytotoxic leads from a
commonly available plant source that inhibit brain tumour cell proliferation.
Abutilon indicum (Malvaceae), a medicinally important shrub, was selected for the study,
since its cytotoxic properties were previously reported (see later sections). It is well known as
Atibala in Hindi and found in outer Himalayan tracts from Jammu to Bhutan up to an elevation
of 1500 m and extending throughout northern and central India (Rajurkar et al. 2009). A. indicum
is used by different tribal and ethnic groups of India, Malaysia, islands of the Philippines and
Indochina for various medicinal purposes as anti-inflammatory, febrifuge, anthelmintic,
antiemetic, uterine disorders, piles and lumbago (Chopra & Chopra 1958; Chatterjee & Prakash
1991; Nadakarni 1995). Conventionally, the plant is used in inflammation, gonorrhoea treatment
and as immune stimulant (Kirtikar & Basu 1980; Khadabadi & Bhajipale 2010). Previous
phytochemical studies on this plant led to the isolation of more than 40 compounds including
phenolic compounds, alkaloids, flavonoids, steroids and terpenoids (Gaind & Chopra 1976; Kuo
et al. 2008; Pandey et al. 2011; Rajput & Patel 2012). Our study to identify natural cytotoxic
leads from the leaves of A. indicum using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay is described in this communication. The study resulted in the isolation and
characterisation of methyl caffeate as an active constituent from the leaf extract.

2. Results and discussion

2.1. Cytotoxicity effect on U87MG cells
The petroleum ether extract (AIP), methanol extract (AIM) and chloroform and ethyl acetate subfractions (AIM-C and AIM-E, respectively) were prepared from A. indicum leaves. These
fractions were evaluated for cytotoxic activity against U87MG human glioblastoma cells. Results
indicated a dose-dependent increase in growth inhibition of the U87MG cells. The IC50 values of
AIP, AIM, AIM-C and AIM-E were found to be 48.6, 64.5, 42.6 and 45.8 mg/mL, respectively
(Figure 1). No cytotoxic effect on the growth of normal cells (HEK-293) was observed for these
samples. AIM-C was more active than the mother-extract AIM, thus indicating an enrichment of

100

% Growth Inhibition

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AIP
AIM
AIM-C
AIM-E

80
60
40
20
0
10

20

30

40

Concentration in g/ml

Figure 1. Cytotoxic effect on U87MG cell line treated for 72 h.

50

Natural Product Research

cytotoxic substances. Hence, AIM-C was subjected to chromatographic purification to elaborate

active constituents.

2.3. Cytotoxic effect of isolated compounds

Compounds 1 4 were tested for cytotoxic activities against U87MG and HEK-293 cells at
various concentrations ranging from 50 to 3.125 mM for 72 h. Methyl caffeate (2) displayed
marked cytotoxicity against U87MG cells (IC50 value of 8.2 mg/mL 42.2 mM) (Figure 2).
However, compounds 1, 3 and 4 did not show significant effect up to 50 mM. Supplementary
Table S1 (online) presents the results at 50 mM. Furthermore, methyl caffeate (2) was found to
be non-toxic to HEK-293 cells (IC50 . 100 mM).
2.4. Quantification of methyl caffeate in AIM-C by HPLC
Methyl caffeate (2) present in AIM-C fraction was quantified by HPLC using the isolated methyl
caffeate as the standard sample. Under the set of given analytical conditions, the peak of
standard methyl caffeate was observed at 7.44 min exhibiting 98% purity (photo diode array
detection; 220 360 nm). The calibration curve for quantification was generated by the results of
the standard sample ranging from 100 to 500 ppm. The amount of methyl caffeate (2) in AIM-C
was calculated as 2.97% w/w.
In agreement with the present findings, it was reported that chloroform-soluble fraction of
the methanol extract of the whole plant of A. indicum showed cytotoxic activity (91 95%
inhibition of cell viability) against MCF-7, NCI-H460 and SF-264 cell lines at 150 mg/mL (Kuo
et al. 2008). Muhit et al. (2010) also reported on the cytotoxic and antimicrobial activities of
A. indicum extract studied by using brine shrimp lethality assay. Methyl trans-p-coumarate (1)
was previously reported to be inactive to HeLa and KB cells (Tip-Pyang et al. 2010), which is
60

% Growth Inhibition

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2.2. Isolation and characterisation of chemical constituents

Repeated column chromatography of AIM-C over silica gel yielded three aromatic compounds,
methyl trans-p-coumarate (1), methyl caffeate (2) and syringic acid (3), and a C-18 trihydroxy
octadecenoic acid, pinellic acid (4). These compounds were characterised by comparison of the
1
H and 13C NMR data with the reported vales (Lee et al. 2010; Touafek et al. 2011; Sunnam &
Prasad 2013; Guzman et al. 2014) and ESI-MS. Compounds 2 4 are reported for the first time
from A. indicum.

40

20

0
0.6

1.2

2.4

4.8

9.7

Concentration in g/ml

Figure 2. Effect of methyl caffeate (2) on U87MG cell line treated for 72 h.

R.S. Khan et al.

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parallel to the present result. This article describes the first isolation of methyl caffeate (2) from
A. indicum, although caffeic acid was previously characterised from this plant (Rajput & Patel
2012). Cytotoxic properties of 2 against various cell lines such as non-small cell lung
adenocarcinoma, ovarian cells, skin melanoma, colon cancer cells (Chang et al. 2009), NCIH187 (Vongvanich et al. 2006) and human leukaemia (Lee et al. 2001) had been established.
Syringic acid (3) had been previously isolated from malvaceous plants (Althaea officinalis and
Sida acuta) (Kirtikar & Basu 1980). Synthetic syringic acid esters such as syringic acid benzyl
ester were reported to show selective dose-dependent anti-mitogenic effect against human
malignant melanoma cell lines HTB66 and HTB68 (Orabi et al. 2013). Isolation of pinellic acid
(4) from the family of Malvaceae is reported for the first time, although it had been reported from
several other families such as Pinellia ternata (Araceae) (Nagai et al. 2002) and Ulmus
davidiana var. japonica (Ulmaceae) (Choi et al. 2013).
3. Conclusion
The study demonstrated cytotoxic effect of A. indicum leaf extracts on U87MG human
glioblastoma cells. Further investigation resulted in the isolation and characterisation of methyl
caffeate as one of the active principle associated with the activity of AIM-C fraction from the
leaves. In addition, the content of methyl caffeate in the AIM-C fraction was analysed to be
approximately 3% (w/w) by HPLC. The findings indicated that methyl caffeate accounted for
approximately one-sixth of the activity that was shown by AIM-C. It is thus suggested that there
exist more minor and more active constituents in AIM-C besides methyl caffeate and/or the
observed activity of AIM-C was exerted by synergetic effect of several constituents. Methyl
caffeate (2) can be used as a pharmacophore for designing more potent analogues for treating
glioblastoma. In this context, it is interesting to note that synthetic propyl and octyl esters of
caffeic acid had been tested for cytotoxicity (IC50 values of 12 and 70 mM) against HeLa
malignant cells (Fiuza et al. 2004), and phenethyl caffeate had been reported to exhibit
significant cytotoxicity (IC50 value of 0.8 mM) against glioma stem cells (Hothi et al. 2012).
Further scale-up bioassay-guided investigations focusing on AIM-C is highly awaited.
Supplementary material
Experimental details relating to this article are available online, alongside Table S1.
Acknowledgements
One of the authors (R.S.K) is thankful to the University Grants Commission, New Delhi, India for Maulana
Azad National Fellowship for carrying research.

Funding
The authors gratefully acknowledge the financial support by the BITS-Research Initiation Grant for this
work.

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