Extraction of Ethanolic Extract of Red Betel Leaves and Its Cytotoxicity Test on HeLa Cells

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Procedia Engineering 148 ( 2016 ) 1402 – 1407

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


Available online at www.sciencedirect.com

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 Anugrahwati


*, Tuti Purwaningsih


, Rustina


, J.A. Manggalarini


, N.B. Alnavis


, D.N.



, H.D. Pranowo


aChemistry Department, Faculty of Mathematics and Natural Sciences, Islamic University of Indonesia, Jl. Kaliurang Km 14.5, Yogyakarta, 55584, Indon esia bStatistic Department, Faculty of Mathematics and Natural Sciences, Islamic University of Indonesia, Jl. Kaliurang Km 14.5, Yogyakarta, 55584, Indonesia

cChemistry Department, Faculty of Mathematics and Natural Sciences, Gadjah Mada University, Sekip Utara, Bulaksumur, Yogyakarta, 55281, Indonesia


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.

Peer-review under responsibility of the organizing committee of 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

© 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/).


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 CO2 flow. All of these steps were performed in aseptic


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


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. 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.


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 concentration (mg/mL) Number of cells 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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