Chemical constituents and potential cytotoxic activity of n-hexane fraction from

Myristica fatua Houtt leaves

S. Fajriah, , Megawati, , S. Hudiyono, , S. Kosela, and , and M. Hanafi

Citation: AIP Conference Proceedings 1862, 030087 (2017); doi: 10.1063/1.4991191

View online: http://dx.doi.org/10.1063/1.4991191
View Table of Contents: http://aip.scitation.org/toc/apc/1862/1
Published by the American Institute of Physics
 Chemical Constituents and Potential Cytotoxic Activity of
n-Hexane Fraction from Myristica fatua Houtt Leaves
S. Fajriah1, 2, Megawati2, S. Hudiyono1, a), S. Kosela1, and M. Hanafi2
1
Department of Chemistry, Faculty of Mathematics and Natural Sciences (FMIPA),
Universitas Indonesia, Depok 16424, Indonesia
2
Research Center for Chemistry, Indonesian Institute of Sciences (LIPI)
Kawasan Puspiptek, Serpong, Tangerang Selatan, Banten 15314, Indonesia
a)
Corresponding author: hudiyono@ui.ac.id

Abstract. The aims of this research were to determine the chemical constituents of n- hexane fraction from Myristica
fatua Houtt leaves by Gas Chromatograpy/Mass Spectrometry (GC/MS) and their cytotoxic activities against MCF-7 cell
lines. The results indicated that sesquiterpenes and fatty acids were major compounds of this fraction, there were trans-
calamenene (17.75 %), hexadecanoic acid (11.14 %), caryophyllene (7.49 %), -muurolene (6.99 %), and -muurolene
(6.60 %). In vitro anticancer activity test against breast cancer MCF-7 cell lines showed potential cytotoxic at IC50
2.19 g/mL.

Keywords: Myristica fatua Houtt, GC/MS, sesquiterpenes, fatty acids, MCF-7 cell lines

INTRODUCTION
Medicinal plants for a pharmaceutical purpose have signifantly raised. Based on WHO data, natural based
compounds are promising agent for drug development. More than half of individuals in developed countries benefit
from traditional medicine for their health system [1].
The use of medicinal plants (plant extracts) give a significant influence in therapeutic treatments method [2].
However, medicinal plants produce secondary metabolites which act as a defense system against infections, insects
and herbivores. Thus, an exploration of their cytotoxic potential is essential to find the benefit of medicinal plants
for human health [2, 3].
The Myristica genus (Myristicaceae) as one of the medicinal plants, is widespread in the tropics of Asia,
Australia and Oceania [4], consisting of 19 genera and nearly 440 species [5]. In this genus, Myristica fatua is
endemic from Kalimantan, Sulawesi and Moluccas islands, Indonesia [6]. M. fatua empirically was not used as a
psychoactive substance or spices such as nutmeg (M. fragrans), but often used as impotence drugs (aphrodisiac) and
well known as male nutmeg [7]. In in vitro assays, it was proved that the seed of M. fatua has anti parasitic [8], anti
bacterial and antioxidant activities [9]. However, the chemical investigation of this species was lack. In this paper,
we describe the chemical constituent of n-hexane fraction from the leaves of M. fatua using GC/MS spectroscopy
and report the cytotoxic activity of this fraction against MCF-7 cell lines.

International Symposium on Current Progress in Mathematics and Sciences 2016 (ISCPMS 2016)
AIP Conf. Proc. 1862, 030087-1030087-6; doi: 10.1063/1.4991191
Published by AIP Publishing. 978-0-7354-1536-2/$30.00

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 MATERIALS AND METHODS

Plant Materials
The leaves of M. fatua were collected from Mekonga forest, Southeast Sulawesi, Indonesia in March 2014 and
identifed by botanist from Research Center for Biology, Indonesian Institute of Sciences (LIPI). A voucher of
specimen is deposited at the Herbarium Bogoriense, LIPI.

Methods
Extraction. The dried leaves of M. fatua (1.37 kg) was macerated with methanol at room temperature, filtered
and dried in vacuo to yield a crude extract (99.6 g). The extract (45.6 g) was subjected to Vacuum Liquid
Chromatography on silica gel with n-hexane as a mobile phase. The n-hexane fraction was evaporated to give n-
hexane oily residue (66.1 mg). Following preparation, chemical composition analysis and bioactivity assay were
carried out using GC/MS spectrometer and MCF-7 cell lines, respectively.
Analysis of n-hexane fraction by Gas Chromatography-Mass Spectrometry (GC-MS). GC-MS analysis of n-
hexane fraction was carried out using Agilent Technologies 7890B with a mass detector and DB-5 capillary column
(5 % diphenyl and 95 % dimethylpolysiloxane) with length 30 m x 0.25 mm i.d., and film thickness 0.25 m. Ultra-
pure helium was used as carrier gas (flow rate, 1 mL/min). Injector and interface were 250 C. Column temperature
was programmed from 40-280 C at increasing rate of 10 C/min, held at initial for 1 min and final temperature for 5
min. The major peaks were analyzed by comparing its mass fragments patterns with standard spectra available in
NIST library.
Preparation of Cancer Cells Line. Cancer cells line used in this study is Breast carcinoma (MCF-7) and the cell
line was cultured in DME Medium with FBS 10 %. This cell was cultured at 37 C with moisture content of
95 % and 5 % CO2 for 3 days until the cell cultures undergo confluent 60-70 %. Subsequently, the old media
discharged, replaced with fresh media and incubated for 24 hours. Prior to adding with new media, the culture cells
washed with PBS 1-2 times and suspended using trypsin-EDTA solution.
In Vitro Anticancer Assay. Anticancer assay was used by Alamar Blue method. Cells line of 100 L was
supplemented with 10 L of n-hexane fraction (10 variation concentration begin 0.0391-20 g/mL). Then, it was
incubated for 24 hours at 37 C. Coloring process was done by adding Alamar blue solution for 4 hours. The color
intensity of the cell line was measured by using ELISA plate reader at 560 nm (excitation) and 590 nm (emission)
wavelength. Percent viability is calculated as follows:

Optical Density (cells+ sample)-Optical Density (negative control)

% Viability = ________________________________________________________ x 100 (1)
Optical Density (cells) Optical Density (negative control)
IC50 of n-hexane fraction was calculated by linear regression analysis between percent survival and the
concentration of the extract [10, 11].

RESULTS AND DISCUSSION

The n-hexane fraction of M. fatua leaves has 0.14 % yield after evaporation process. The chemical composition
of this fraction was analyzed using GC-MS technique. The chromatogram of GC-MS experiment result had been
showed in Fig. 1. GC-MS analysis of n-hexane extract has led to the identification and quantification of 17 different
compounds (percent of similarity > 98) represented in Table 1.
Trans-Calamenene, hexadecanoic acid, caryophyllene, -muurolene and -muurolene were major components of
n-hexane fraction from M. fatua leaves. These compounds showed that sesquiterpenes and fatty acid groups were
major compounds of this extract [12]. The major peaks compounds were analyzed by comparing its mass fragments
patterns with standard spectra from NIST library shown in Fig. 2. On other hand, -copaene and caryophyllene are
the major components from aril and seeds of an M. fatua [13].
In our previous report, the n-hexane fraction from methanol extract of M. fatua leaves showed high toxicity
against brine shrimp (LC50 153.1 g/mL), indicating its potential to be developed as an anticancer agent [14].
Antiproliferative activity against MCF-7 cell lines was found in M. fatua [15]. Therefore, the n-hexane further

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examined against MCF-7 breast cancer cell lines. The preliminary experiment of cytotoxicity effect of n-hexane
fraction of M. fatua leaves against MCF-7 cell lines was done by microscopic observation at concentration 40
g/mL (Fig. 3). The results showed that n-hexane fraction has inhibited breast cancer cell lines MCF-7 compared to
negative control characterized by reduced living cells. Living cells appear as group of cells that attached on the
bottom of the well.

FIGURE 1. Chromatogram of GC-MS analysis from n-hexane fraction of Myristica fatua leaves

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(a)

(b)

(c)

(d)

(e)

FIGURE 2. Comparison mass fragments pattern from major peak compounds of n-hexane fraction (upper) with standard spectra
from NIST library (below): a. Trans-calamenene; b. hexadecanoic acid; c. caryophyllene, d. -muurolene and e. -muurolene.

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 TABLE 1. Chemical composition of n-hexane of Myristica fatua leaves
Retention
No Compounds % Content
Time (min)
1 Trans-Calamenene 17.75 14.725
2 Hexadecanoic acid 11.14 18.994
3 Caryophyllene 7.49 13.458
4 -Muurolene 6.99 14.140
5 -Muurolene 6.60 14.422
6 -Copaene 5.56 12.850
7 Squalene 4.89 26.777
8 -Calacorene 4.39 14.980
9 Methyl stearate 3.06 20.891
10 9-Octadecenoic acid 2.91 20.664
11 Humulene 2.73 13.884
12 Cyclotetracosane 2.66 23.172
13 1-Docosene 2.62 24.748
14 Hexacosanoic acid 2.58 27.798
15 Tetracosane 4.04 30.091
16 Docosanoic acid 2.41 24.256
17 1-Docosene 2.46 21.483

= living cells
(a) (b)
FIGURE 3. Microscopic observation of cytotoxic effect of the n-hexane fraction (a) against MCF-7 cell lines at
concentration 40 g/mL compared to negative control (b).

(a) (b)
FIGURE 4. Percent viability of cytotoxic acitivity of n-hexane fraction (a) and antymicin (b) against MCF-7 cell lines.

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 The cytotoxic activity of n-hexane fraction (Fig. 4) showed significant activity at IC50 2.19 g/mL with
antymicin as positive control (IC50 1.24 g/mL). This strong activity may be due to the presence of caryophyllene in
this fraction [16]. Caryophyllene is a sesquiterpene with several biological activities and signifant increased
anticancer activity on MCF-7 cells [17, 18].

ACKNOWLEDGMENTS
This work was supported by Science and Technology Research Grant program from Indonesia Toray Science
Foundation. We are grateful to Dr. Linar Zalinar Udin and Dr. Nina Artanti for cytotoxicity assay, Salahuddin, S.Si
and Eka Dian, S.Si for GC/MS spectra and Dr. Akhmad Darmawan for valuable discussion.

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