Cytotoxic Carbazole Alkaloid from the Root of Clausena cxcavata on

Hela Cell Line

Tin Myo Thant

1,2

, Nanik Siti Aminah

, Alfinda Novi Kristanti

, Rico Ramadhan

, Hnin Thanda

Aung

and Yoshiaki Takaya

Ph.D. Student of Mathematics and Natural Sciences, Faculty of Science and Technology, Universitas Airlangga,

Komplek Kampus C UNAIR, Jl. Mulyorejo, Surabaya, Indonesia

Dept. of Chemistry, Mandalar Degree College, Mandalay, Myanmar

Dept. of Chemistry, Faculty of Science and Technology, Universitas Airlangga, Komplek Kampus C UNAIR, Jl. Mulyorejo,

Surabaya, Indonesia

Dept. of Chemistry, Mandalay University, Mandalay, Myanmar

Fac. Of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku, Nagoya, 468-8503 Japan

rico.ramadhan@fst.unair.ac.id, hninthandaaung07@gmail.com, ytakaya@meijo-u.ac.jp

Keywords: Clausena excavata, Carbazole Alkaloid, 7-hydroxylheptaphylline, MTT assay, HeLa.

Abstract: In a search for bioactive constituents from Myanmar medicinal plants, a carbazole alkaloid, named 7-

hydroxy heptaphylline (1) was isolated from the root of Clausena excavata. The structure of isolated

compound was elucidated based on spectrophotometric data such as UV-vis, FT-IR, NMR and HRMS data.

The cytotoxicity of the isolated compound (1) was evaluated by MTT assay against on HeLa cancer cells.

The compound (1) exhibited moderate inhibition activity with IC

41.4 µg/ml.

1 INTRODUCTION

Clausena excavata Burm. f. is a wild shrub, a

member of Rutaceae family predominantly

distributed in India, China and Southeast Asia. The

leaves, twigs, and root barks of C. excavata have

long been used in Asian folk medicine for the

treatment of colic, cough, rhinitis, sore, wounds,

malaria, abdominal pain, snake-bite, preliminary

stage of AIDS and dermatopathy, dysentery,

enteritis, and urethra infection (Waziri et al., 2016a)

(Kumar et al., 2012) (Peh et al., 2013).

The constituents of C. excavata have been

frequently studied. Phytochemical analyses in the

past have revealed that C. excavata is a rich source

of coumarins, carbazole alkaloid along with a small

group of flavonoids, limonoids and triterpenoids

(Cheng et al., 2009) (Mohan, 2012)

(Sunthitikawinsakul et al., 2003) (Kumar et al.,

2012) (Peh et al., 2013) (Peng et al., 2013) (Thant et

al., 2019). Many compounds reported from C.

excavata showed diverse therapeutic activities which

are antibacterial, antifungal, antiplatelet,

antiplasmodial, antitumor, antinociceptive,

antimycobacterial, and anti-HIV-1 activities

(Kongkathip and Kongkathip, 2009).

The coumarins isolated from this plant have

attracted attention due to its bioactive properties

such as the furanone-coumarins named clauslactones

A–J isolated from leaves exhibited tumor promotion

inhibitory effects, nordentatin showed antibacterial

properties and a pyranocoumarin clausenidin

isolated from roots displayed anti-HIV-1 activity

(Kumar et al., 2012). Nevertheless, the other potent

bioactivities of the constituents from C. excavata are

still unknown and worthy of exploration (Cheng et

al., 2009). Moreover, four isolated pyranocoumarins

from C. excavata and screened their cytotoxic

potentials in cancer cells. The study revealed that the

pyranocoumarins are good modulators of tumor cell

death (Waziri et al., 2016b) (N. W. Muhd Sharif,

2011).

Cancer is the second leading cause of death

worldwide. Cervical cancer is one of the most dead

list diseases among women and it is occurred when

the abnormal cells are undergoing to the rapid and

uncontrolled growth on the cervix. Current

treatments for cervical cancer may include surgery,

drugs (hormonal therapy and chemotherapy),

Thant, T., Aminah, N., Kristanti, A., Ramadhan, R., Aung, H. and Takaya, Y.

Cytotoxic Carbazole Alkaloid from the Root of Clausena cxcavata on Hela Cell Line.

DOI: 10.5220/0008858101410144

In Proceedings of the 1st International Conference on Chemical Science and Technology Innovation (ICOCSTI 2019), pages 141-144

ISBN: 978-989-758-415-2

141

radiation and/or immunotherapy. Conventional

cancer treatments such as chemotherapy and

radiotherapy have shown some effectiveness for

reducing or eradicating cancers; however, they can

produce unpleasant side effects, e.g. nausea,

vomiting, changes in bowel habits, fatigue and hair

loss. Complementary and alternative medicine

(CAM), herbals and multivitamin supplements, or

herbal medicine is increasingly used as an adjunctive

treatment for cancer patients to reduce or manage

side effects of conventional cancer treatments.

Several studies have confirmed the anti-proliferative

and cell cycle regulatory effects of some plants

which behave as cancer prevention (Amin et al.,

2009) (Azarifar et al., 2015). More than 25% of

drugs used during the last 20 years are directly

derived from plants, while the other 25% are

chemically altered natural products. Still, only 5-

15% of the approximately 250,000 higher plants

have ever been investigated for bioactive

compounds. The advantage of using such

compounds for cancer treatment is their relatively

non-toxic nature and availability in an ingestive

form (Amin et al., 2009).

In continuing our research, isolation of bioactive

constituents from natural product, isolated

compound (1) was tested on his cytotoxicity activity

on HeLa cancer cell by MTT assay.

2 MATERIALS AND METHODS

2.1 Plant Material

The plant sample of C. excavata was collected from

Pyin Ma Nar Township, Mandalay Division,

Myanmar in October 2016. It was identified by Prof.

Soe Myint Aye, botanist from Department of

Botany, Mandalay University, Myanmar.

2.2 MTT Assay

Anticancer activity tests on isolated compound was

carried out using the MTT assay method (3- [4,5-

dimethylthiazol-2-il] -2,5 diphenyl tetrazolium

bromide) following the protocol of Suwito et al

(Suwito et al., 2018). The cancer cells were seeded

in a 96-well plate at a density of 1×10

cells/well

with a phenol red-free RPMI (Roswell Park

Memorial Institute medium) 1640 medium

(containing 10% FBS (fetal bovine serum)) and

maintained for 24 h. Subsequently, the tested

compound (various concentrations) was applied for

24 h. After addition of 0.5% MTT solution, the

incubation was continued for a further 4 h at 37

֯C/5% CO

. The stop solution (0.04 N HCl in

isopropanol) was added to the culture medium to

each well. Then, the spectroscopic measurement was

carried out at 570 nm (peak) and 630 nm (bottom)

using an ELISA (Enzyme-Linked Immunosorbent

Assay) reader. It was conducted in triplicate.

Doxorubicin was used as a positive control. The

value of IC

was determined using a probit analysis

(SPSS 17, IBM Analytics, New York, NY, USA).

2.3 Extraction and Isolation

The dried roots (3.6 kg) were extracted successively

with 95% EtOH (12.0 L) over a period of two weeks

at ambient temperature. After removing the solvent

156 g of ethanolic extract was obtained. Among

them the extract (100g) was partitioned by using

solvents; n-hexane: methanol (1:1, v/v). Then

methanol portion (80.4 g) was subjected to vacuum

liquid chromatography over silica gel (150g) eluted

with different mixtures of n-hexane: ethylacetate by

stepwise increasing gradient polarity gave a total of

7 combined fractions (MF-1 to -7) were obtained.

MF-6.2.1.1 was purified by using n-hexane:

ethylacetate, ethylacetate (0-30%) with gradient

polarity and silica gel column, gave 44 fractions.

Among them 7-21 showed one spot on tlc with three

different solvent eluction system (n-hex: EtOAc; n-

hexane: CHCl

; n-hexane : Acetone) afforded as

pure compound (1).

2.4 Statistical Analysis

Results were presented as mean ± SD in triplicate

experiment. Differences were determined using

SPSS 17, IBM Analytics, New York, NY, USA at

significant difference of 0.05.

3 RESULTS AND DISCUSSIONS

Spectroscopic Data of Compound-1

(7-hydroxyheptaphylline)

Compound-1: greenish yellow solid. UV(MeOH),

max

(logε) 342 (0.629), 303 (2.519), 237 (1.408) nm.

IR (KBr) υ

max

-1

: 3250, 2958, 2922,2852, 1743,

1614, 1454, 1327, 1186, 9966

H NMR (600 MHz,

DMSO-d6) δ 11.53, 11.27, 9.88, 9.49, 8.14, 7.80,

7.78, 6.86, 6.86, 6.66, 6.65, 6.64, 6.64, 5.29, 5.28,

5.28,5.27, 5.27, 5.27, 5.26, 5.26, 5.26, 3.51, 3.50,

1.78, 1.63.

C NMR (151 MHz, DMSO-d6) δ

ICOCSTI 2019 - International Conference on Chemical Science and Technology Innovation

142

196.45, 156.98, 156.59, 145.16, 143.00, 132.15,

124.25, 122.11, 120.96, 117.83, 115.91, 115.01,

109.84, 109.32, 97.80, 25.92, 23.08, 18.39.

Figure 1: Structure of compound-1 isolated from the root

of C. excavata.

Table 1: Cytotoxic activity of isolated compound (1)

against on Hela cell lines.

Compounds

(ug/mL)

7-hydroxy heptaphylline (1)

41.4

Doxorobicin

1.19

Table 2:

H (600 MHz),

C (151 MHz) NMR and HMBC

spectral data of compound-1 (7-hydroxyheptaphylline).

Position

(mult, J Hz)

(ppm)

HMBC

145.2

109.1

157.9

123.7

8.03 (s, 1H)

125.3

C-1a,C-2,C-

3,C-4a, CHO

115.5

140.2

7.97 (dd, J = 7.7

Hz, 1H)

119.8

C-5a,C-8,C-

7.27 (m, 1H)

120.5

C-5a,C-7,C-

7.40 (dt, J = 7.7, 3.8

Hz, 1H)

125.9

C-6

7.41 (dd, J = 7.7,3.8

Hz, 1H)

110.9

C-5a

117.4

3.64 (d, J = 6.9 Hz,

2H,H-1')

22.9

C-1,C-1a,C-

2,C-10,

C-11

5.33 (d, J = 6.9 Hz,

1H,H-2')

121.3

C-1,C-9,C-

12,C-13

134.2

1.91 (s, 3H)

18.1

C-10,C-

11,C-13

1.78 (s, 3H)

25.7

C-10,

C-11,

C-12

-CHO

9.91

195.4

C-1,C-2,C-4

-NH

8.21

-OH

11.66

Compound-1 (7-hydroxyheptaphylline) was afforded

as yellow green crystal UV (MeOH), λ

max

(logε) 342

(0.629), 303(2.519), 237(1.408), 249(1.64) revealed

as basic carbazole alkaloid. The IR spectrum of

compound-1 showed the presence of -OH and -NH

functional groups at (3250cm

-1

) and also carbonyl

and aromatic benzene groups at (1743, 1612 and

1588 cm

-1

). In the

H NMR spectrum the compound-

1 revealed a total of 11 signals representing 13

protons as characteristic of carbazole alkaloid. In the

down field region three singlets; intramolecular

bonding OH-(δ 11.66) with –CHO (δ 9.91,C-3) and

one broad signal due to –NH (δ 8.21) and another

singlet proton H-4 at δ 8.03 (s,1H).In the aromatic

region four signals with δ values 7.26 (td, J=3.8,

7.7Hz, 1H), 7.40 (td, J=3.8, 7.7Hz, 1H) showed the

absence of substituent in ring A. the presence of

prenyl group was revealed by one methylene protons

at δ 3.64 (d, J=6.9Hz,2H), δ 5.33 (t, J= 6.9Hz,1H)

and one dimethyl group (δ 1.89(s,3H) , δ 1.72(s, 3H)

(Table 2, Figure 1).

According to

C NMR and DEPT (90, 135)

spectra it was revealed that the presence of 18

signals and 18 carbons. Furthermore, the DEPT

spectrum showed the presence of one aldehyde

carbon, 8 quaternary carbons, 6 methine carbons,

one methylene carbon and two methyl carbon as

shown in table 2. The 2D NMR spectra such as

DQF-COSY revealed the correlation of two adjacent

protons. The HSQC spectrum gave the direct

connection between protons and carbon. The HMBC

data showed the position of H-4 singlet proton to C-

1a, C-2, C-3, C-4a, -CHO. The presence and

attachment of prenyl group to core carbazole

alkaloid was revealed by the proton H-1' to C-1,C-1a

C-2, C-10, C-11 (Table 2). Finally, the combination

of fragments and attachments of substituents were

confirmed by HMBC spectrum and compound (1)

was elucidated as 7-hydroxyheptaphylline. It was

also agreement with literature values.

The isolated compound was tested for their

cytotoxicity on Hela cell lines (Table 1). Both of

them showed toxicity against to cervix cancer cell

(Hela) with IC

values 41.4 and µg/ml, where

doxorubicin was used as positive control.

4 CONCLUSIONS

Nowadays the demand for anticancer drugs which

are effective and less side effects are increasing. To

fulfill this aim we conducted the isolation of

bioactive compounds from root of C. excavata. The

isolated compound was evaluated for their

cytotoxicity. The compound (1), 7-

hydroxyheptaphylline with IC

41.4 µg/ml. So the

result revealed that the compound-1 should be

further study for a potential natural anticancer

candidate for HeLa cell in future.

Cytotoxic Carbazole Alkaloid from the Root of Clausena cxcavata on Hela Cell Line

143

ACKNOWLEDGEMENTS

Authors would like to thank to Universitas

Airlangga and Ministry of Research, Technology

and Higher Education for research grant. We also

would like to acknowledge Ms. Helda Dwi

Hardiyanti and Universitas Gadjah Mada for

cytotoxicity measurements.

REFERENCES

Amin, A., Gali-muhtasib, H., Ocker, M., Schneider-stock,

R., 2009. Overview of Major Classes of Plant-Derived

Anticancer Drugs 5, 1–11.

Azarifar, Z., Mortazavi, M. M., Farhadian, R., Parvari, S.,

Mohammadi roushandeh, A., 2015. Cytotoxicity

Effects of Aqueous Extract of Purtulaca oleracea on

HeLa cell Line. Pharm. Sci. 21, 41–45.

Cheng, S. S., Chang, H. T., Lin, C. Y., Chen, P. S., Huang,

C. G., Chen, W. J., Chang, S. T., 2009. Insecticidal

activities of leaf and twig essential oils from Clausena

excavata against Aedes aegypti and Aedes albopictus

larvae. Pest Manag. Sci. 65, 339–343.

Kongkathip, N., Kongkathip, B., 2009. Constituents and

bioactivities of Clausena excavata. Heterocycles 79,

121–144.

Kumar, R., Saha, A., Saha, D., 2012. A new antifungal

coumarin from Clausena excavata. Fitoterapia 83,

230–233.

Mohan, D., 2012. Clausena excavata Burm. f. (Rutaceae):

A review of its traditional uses, pharmacological and

phytochemical properties. J. Med. Plants Res.

N. W. Muhd Sharif, 2011. Cytotoxic constituents of

Clausena excavata. African J. Biotechnol. 10, 16337–

16341.

Peh, T. H., Lim, G. K., Taufiq-yap, Y. H., Cheng, G., Ee,

L., 2013. A New Cytotoxic Carbazole Alkaloid

Isolated from the Stem Bark of Malaysian Clausena

excavata. Can. Chem. Trans. 1, 165–172.

Peng, W. W., Zheng, Y. Q., Chen, Y. S., Zhao, S. M., Ji,

C. J., Tan, N. H., 2013. Coumarins from roots of

Clausena excavata. J. Asian Nat. Prod. Res. 15, 215–

220.

Sunthitikawinsakul, A., Kongkathip, N., Kongkathip, B.,

Phonnakhu, S., Daly, J.W., Spande, T. F., Nimit, Y.,

Napaswat, C., Kasisit, J., Yoosook, C., 2003. Anti-

HIV-1 Limonoid: First Isolation from Clausena

excavata. Phyther. Res. 17, 1101–1103.

Suwito, H., Hardiyanti, H. D., Ul Haq, K., Kristanti, A. N.,

Khasanah, M., 2018. (E)-3-[3-(4-morpholinophenyl)

acryloyl]-2H-chromen-2-one. Molbank 2018, 1–5.

Thant, T. M., Aminah, N. S., Kristanti, A. N., Ramadhan,

R., Phuwapraisirisan, P., Takaya, Y., 2019. A new

pyrano coumarin from Clausena excavata roots

displaying dual inhibition against α-glucosidase and

free radical. Nat. Prod. Res. 0, 1–6.

Waziri, P. M., Abdullah, R., Yeap, S. K., Omar, A. R.,

Abdul, A. B., Kassim, N. K., Malami, I., Karunakaran,

T., Imam, M. U., 2016a. Clausenidin from Clausena

excavata induces apoptosis in hepG2 cells via the

mitochondrial pathway. J. Ethnopharmacol. 194, 549–

558.

Waziri, P. M., Abdullah, R., Yeap, S. K., Omar, A. R.,

Kassim, N. K., Malami, I., How, C. W., Etti, I. C.,

Abu, M. L., 2016b. Clausenidin induces caspase-

dependent apoptosis in colon cancer. BMC

Complement. Altern. Med. 16, 1–12.

ICOCSTI 2019 - International Conference on Chemical Science and Technology Innovation

144