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Cytotoxic constituents of Abutilonindicum leaves against U87MG humanglioblastoma cellsRukaiyya Sirajuddin Khana, Mahibalan Senthia, Poorna ChandraRaoa, Ameer Bashab, Mallika Alvalac, Dinesh Tummuric, HironoriMasubutid, Yoshinori Fujimotod & Ahil Sajeli Begumaa Department of Pharmacy, BITS-Pilani Hyderabad Campus,Jawahar Nagar, Shamirpet Mandal, Hyderabad500 078, Telangana,Indiab Regional Agricultural Research Station (ANGRAU), Palem,Mahabub Nagar, Telangana, Indiac National Institute of Pharmaceutical Education and Research-Hyderabad, Balanagar, Hyderabad500 037, Telanagana, Indiad Department of Chemistry and Materials Science, Tokyo Instituteof Technology, Meguro, Tokyo152-8551, JapanPublished online: 25 Nov 2014.

To cite this article: Rukaiyya Sirajuddin Khan, Mahibalan Senthi, Poorna Chandra Rao, AmeerBasha, 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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SHORT COMMUNICATION

Cytotoxic constituents of Abutilon indicum leaves against U87MG humanglioblastoma cells

Rukaiyya Sirajuddin Khana, Mahibalan Senthia, Poorna Chandra Raoa, Ameer Bashab,

Mallika Alvalac, Dinesh Tummuric, Hironori Masubutid, Yoshinori Fujimotod and Ahil

Sajeli Beguma*

aDepartment 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 Research-Hyderabad, Balanagar, Hyderabad 500 037, Telanagana, India; dDepartment of Chemistry and MaterialsScience, 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 fortreating glioblastoma. The petroleum ether extract, methanol extract (AIM),chloroform and ethyl acetate sub-fractions (AIM-C and AIM-E, respectively) preparedfrom AIM were tested for cytotoxicity on U87MG human glioblastoma cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. These extractsexhibited considerable activity (IC50 values of 42.664.5mg/mL). The most activeAIM-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 14 against U87MG cells indicated 2 as most active (IC50value of 8.2mg/mL), whereas the other three compounds were much less active.Interestingly, compounds 14were non-toxic towards normal human cells (HEK-293).The content of 2 in AIM-C was estimated as 3% by HPLC. Hence, presence of somemore 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 cancer-

related 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,

q 2014 Taylor & Francis

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

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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 1500m 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 sub-

fractions (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 45.8mg/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

10 20 30 40 500

20

40

60

80

100AIPAIMAIM-CAIM-E

Concentration in g/ml

% G

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

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

active constituents.

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 the1H 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 24 are reported for the first time

from A. indicum.

2.3. Cytotoxic effect of isolated compounds

Compounds 14 were tested for cytotoxic activities against U87MG and HEK-293 cells at

various concentrations ranging from 50 to 3.125mM for 72 h. Methyl caffeate (2) displayedmarked cytotoxicity against U87MG cells (IC50 value of 8.2mg/mL 42.2mM) (Figure 2).However, compounds 1, 3 and 4 did not show significant effect up to 50mM. SupplementaryTable S1 (online) presents the results at 50mM. Furthermore, methyl caffeate (2) was found tobe non-toxic to HEK-293 cells (IC50 . 100mM).

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 7.44min exhibiting 98% purity (photo diode array

detection; 220360 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 (9195%

inhibition of cell viability) against MCF-7, NCI-H460 and SF-264 cell lines at 150mg/mL (Kuoet 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

0.6 1.2 2.4 4.8 9.70

20

40

60

Concentration in g/ml

% G

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

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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 lungadenocarcinoma, ovarian cells, skin melanoma, colon cancer cells (Chang et al. 2009), NCI-

H187 (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 70mM) against HeLamalignant cells (Fiuza et al. 2004), and phenethyl caffeate had been reported to exhibit

significant cytotoxicity (IC50 value of 0.8mM) 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 MaulanaAzad National Fellowship for carrying research.

Funding

The authors gratefully acknowledge the financial support by the BITS-Research Initiation Grant for thiswork.

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AbstractAbstract1. Introduction2. Results and discussion2.1. Cytotoxicity effect on U87MG cells2.2. Isolation and characterisation of chemical constituents2.3. Cytotoxic effect of isolated compounds2.4. Quantification of methyl caffeate in AIM-C by HPLC

3. ConclusionSupplementary materialAcknowledgementsReferences