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Procedia Engineering 148 (2016) 1402 – 1407

4th International Conference on Process Engineering and Advanced Materials

Extraction of Ethanolic Extract of Red Betel Leaves and Its Cytotoxicity

Test on HeLa Cells
Mai Anugrahwatia,*, Tuti Purwaningsihb, Rustinac, J.A. Manggalarinic, N.B. Alnavisc, D.N.
Wulandaric, H.D. Pranowoc
a
Chemistry Department, Faculty of Mathematics and Natural Sciences, Islamic University of Indonesia, Jl. Kaliurang Km 14.5, Yogyakarta, 55584, Indon esia
b
Statistic Department, Faculty of Mathematics and Natural Sciences, Islamic University of Indonesia, Jl. Kaliurang Km 14.5, Yogyakarta, 55584, Indonesia
c
Chemistry Department, Faculty of Mathematics and Natural Sciences, Gadjah Mada University, Sekip Utara, Bulaksumur, Yogyakarta, 55281, Indonesia

Abstract

Extraction and identification of active compounds in red betel leaves (Piper crocatum Ruiz&Pav.) have been conducted. Ethanolic extract of
red betel leaves was obtained by Soxhlet extraction followed by solvent removal step using rotary evaporator. Compound identification of the
extract was performed by GCMS. The concentrated ethanol extract was then examined through cytotoxicity test on Hela cells. Results from
GCMS spectrum depicted several compounds which were identified in the extract, i.e. neophytadiene, elimicin and propionic acid. Cytotoxicity
test was conducted on Hela cells using in vitro method and calculated using Probit method. LC50 value from the ethanolic extract of red betel
leaf was 0.81 + 0.26 mg/ml. This result showed that the bioactive compounds potentially inhibited proliferation of Hela cells.

© 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
© 2016 The Authors. Published by Elsevier Ltd.
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Peer-review under responsibility
Peer-review responsibilityofofthe
theorganizing
organizingcommittee
committeeofof
ICPEAM 2016.
ICPEAM 2016

Keywords: soxhlet; extraction; chromatography; phytochemical; LC50

1. Introduction

Red betel is one of medicinal plant commonly found in the Indonesian rainforest. This plant is included in Piper genus which
has leaves in heart shape with a silvery red colour. This plant grows well in the present of enough water and moderate sunlight.
Currently, some people cultivate it in their yard and adopt it as herbal medicine. Investigations concerning the benefits contained
in red betel leaf were mentioned due to some phytochemicals such as phenolic, essential oil, flavonoid, and terpene [1][2] which
gave positive bioactivity effects. These compounds are equipped with functional groups which design them to have properties
such as inhibiting oxidation in free fatty acid, encountering free radical activity[3] or inhibiting unwanted cell growth [4].
Therefore, these bioactive compounds have some potencies as an antioxidant [5], antiseptic [6], antimicrobial activity [1], and
antihiperglycemia [7]. In addition, its leaf extract was also indicated to have an anticancer activity for human breast cancer
cells[4].

* Corresponding author.
E-mail address: mai.anugrahwati@uii.ac.id

1877-7058 © 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Peer-review under responsibility of the organizing committee of ICPEAM 2016
doi:10.1016/j.proeng.2016.06.569
 Mai Anugrahwati et al. / Procedia Engineering 148 (2016) 1402 – 1407 1403

Cancer is anomalistic growth of cell tissues, resulting adverse effect of the cell function. There are two major cases of cancer
in Indonesia, causing the highest mortality number of women, i.e. human breast cancer and cervical cancer [8]. Several methods
are available to heal the patient suffered from cancer such as chemoteraphy, consuming medicine or surgery. However, each
method still has its own shortcoming which still needs development in some alternative method such as herbal medicine.
Therefore, aim of this study was to investigate separation process in extraction of red betel leaves using ethanol, followed by
performing cytotoxicity test on Hela cell line from cervix tumor/cancer. The extraction process of majority previous
investigations were performed using maceration method, in room temperature. In contrast with how people commonly consume
it as herbal medicine by macerating its leaf in hot water and drinking the water[9]. Thus, in this study, the extraction process was
performed using Soxhlet extraction which applied higher temperature, closer to reality application.

2. Experiments

2.1. Material

Fresh leaves of red betel (Piper crocatum Ruiz & Pav.) from 2 year old plant were collected from Turi, Sleman Yogyakarta,
Indonesia. HeLa cell as an cytotoxicity test object was obtained from LPPT, Gadjah Mada University.

2.2. Methods

Fresh leaves of red betel were picked, thinly cut and dried in atmospheric air. First, dried leaves of red betel were taken about
25 g and were extracted with ethanol as a solvent using Soxhlet apparatus in temperature of ca. 78 °C. Secondly, this fraction
were then concentrated under reduced pressure using a rotary evaporator to afford a concentrated extract. Then, the extract was
analyzed using GCMS in order to identify its phytochemical contents. As a comparison, extraction using other solvent
(petroleum ether and ethyl acetate) in the same procedure was also performed in order to identify the phytochemicals which were
dissolved in other solvent polarity.

2.3. Cytotoxicity test

In vitro test of ethanolic extract of red betel leaves in Hela cell was performed using cell culture. Before the test was carried
out, four media were prepared, i.e. RPMI media, culture media, washing media and storage media. HeLa cell was vegetated form
frozen cancer cell which was cleaned using washing media after attaining room temperature. After that, the cells were added with
culture media and incubated in temperature of 37 °C with 5% of CO 2 flow. All of these steps were performed in aseptic
environment.
Cytotoxicity assay was conducted using 24-well microtiter plates. Concentrated extract of red betel leaves was diluted to
specific concentrations then was added into certain wells together with the cells in cultured media. After being incubated for 24
hours, 50 ml of 0.5% trypan blue was added in each well. In the next step, cell characteristic was analyzed under microscope,
dead cell was shown by blue color and living cell was shown by the white one. The numbers of dead cells were calculated by
using haemocytometer and the inhibition concentration 50% (LC50) was enumerated by Probit method.

2.4. Statistical analysis

Statistical analysis of the data was carried out using SPSS program.

3. Results and discussion

3.1. Soxhlet extraction

Separation process of ethanolic extract from leaves of red betel using Soxhlet followed by solvent removal using rotary
evaporator produced a light green gum of the concentrated ethanolic extract with extraction efficiency of about 13%. Soxhlet
extraction was used in this experiment as it applies higher temperature analog to hot water process as how people treat this herbal
medicine before consumption. Rotary evaporator was applied in order to purify the extract by removing out the solvent through
evaporation process. Meanwhile, ethanol was selected as the solvent in this study because it has a polar property resembling to
water and both are generally recognized as safe solvents [10].

3.2. GCMS chromatogram

In this study, compounds extracted with ethanol were qualitatively analyzed with GCMS. Analysis spectra from GCMS
showed that ethanolic extract of red betel leaves contained some chemical compounds such as neophytadiene (peak 1 with
1404 Mai Anugrahwati et al. / Procedia Engineering 148 (2016) 1402 – 1407

retention time of 31.567 min), propionic acid (peak 9 with retention time of 49.583) and also elemicin as the highest peak (peak
11 with retention time of 51.335).

Fig. 1. Chromatogram of ethanolic extract

Neophytadiene is categorized as diterpenoid compound which is used in cigarette to give particular odor and flavor [11]. On the
other side, propionic acid [12] and elemicin were reported to have bioactivity i.e. inhibited microbial growth. Elemicin was
reported to have antibacterial activity [13] without any chronic citotoxicity was found for mice. However, its metabolite gave
both favorable and adverse signs [14] .
Moreover, elimicin also appeared in GCMS results from petroleum ether (Fig.2.) and ethyl acetate (Fig. 4) extracts which
was figured peak 31 and peak 27, respectively. Another phytochemical which showed positive bioactivity from petroleum extract
(Fig. 3) was germacrene [15] in retention time of 23.342 while β-pinene [16] in retention time of 7.643 from the chromatogram
of ethyl acetate extract.

Fig. 2. Chromatogram of petroleum ether extract

 Mai Anugrahwati et al. / Procedia Engineering 148 (2016) 1402 – 1407 1405

Fig. 3. Mass spectrum of elemicin and germacrene from extract of petroleum ether

Fig. 4. Chromatogram of ethyl acetate extract

From these results, it could be observed that several compounds extracted from red betel leaves were dissolved in several
solvents in accordance with “like dissolve like” principle. Active compounds with more polar property were more likely to stay
in ethanolic extract. However, previous studies have mentioned that many other active compounds such as flavonoid and alkaloid
were also present in red betel leaves [6][17]. Yet, these were not able to be identified in this analysis based on extracts from the
three solvents.

3.3. Evaluation of cytotoxicity test

Results from microscope observation in cytotoxicity test of ethanolic extract on HeLa cells figured that the dead cells in
culture media were seen in blue color. At the same time, living cells in a culture media which was given with the extract was
figured in white color. Living cells of HeLa as a control system was shown in Fig.5.
1406 Mai Anugrahwati et al. / Procedia Engineering 148 (2016) 1402 – 1407

Fig. 5. HeLa cells under microscope with magnification of 100x as a control system which was untreated with ethanolic extract showing nearly all white color of
cells

In order to investigate effect of the ethanolic extract on the HeLa cells in culture media, LC50 value from this experiment was
necessary to be determined. LC50 value would show concentration in which the extract causing dead of the cells by 50% of the
examined cells. Determination of LC50 value was performed based on the calculation of the dead and the living cells was shown
in Table 1. Results from Probit analysis which was conducted by SPSS predicted that LC50 value of ethanolic extract from red
betel leaves was 0.81 + 0.26 mg/ml. This result showed that bioactive compounds in ethanolic extract of red betel leaves were
potential to inhibit proliferation of HeLa cells.

Table 1. Calculation of living and dead cells in cytotoxicity tetst

Extract Number of cells

concentration
(mg/mL) Living Dead

Control cells 34666.67 -

2.500 1666.67 18166.67
2.250 3666.67 17166.67
2.000 6166.67 15666.67
1.750 8333.33 15000.00
1.500 1033.33 14333.33
1.250 12500.00 12500.00
1.000 14833.33 10833.33
0.750 17166.67 10000.00
0.500 19000.00 9166.67
0.250 22166.67 7666.67
0.125 25833.33 4666.67

4. Conclusion

Several studies have been performed concerning bioactive phytochemicals in red bêtel leaves that were potential for medicinal
purposes. Most people in Indonesia consumed extract of red betel leaf by macerating it in hot water and drinking the water for
diabetes and heart illness treatment. In this study, ethanolic extract from red betel leaves which was obtained by Soxhlet
extraction was investigated. Several compounds in this extract were identified using GCMS i.e. neophytadiene, propionic acid
and elemicin. Cytotoxicity study of this extract on HeLa cells showed that LC50 value predicted from this experiment was 0.81 +
0.26 mg/ml. This result figured phytochemical compounds in ethanolic extract of red betel leaves obtained via Soxhlet extraction
were found to be potential in inhibiting proliferation of HeLa cells. However, further study concerning the separation of these
bioactive compounds, bioactivities of each separated compound and their health risk during consumption must be further studied.

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