Cytotoxicity Effect of the Pericarp Extracts of Garcinia forbesii King on MCF-7 Breast Cancer and HepG2 Liver Cancer Cell Lines

According to the World Health Organization data in 2004, the top five cancer occurrences in the world were lung cancer, breast cancer, colon cancer, stomach cancer and liver cancer. The cancer incidence increased from 12.7 million cases in 2008 to 14.1 million cases in 2012. While the number of deaths from cancer increased from 7.6 million people in 2008 to 8.2 million in 2012. It is estimated that in 2030 the incidence of cancer may reach 26 million people and 17 million of them will die from cancer. In poor and developing countries, deaths from cancer increase faster1. In women, breast cancer has the highest percentage of new cases (43.3%), and the percentage of death is 12.9%1. In men, liver cancer incidences is on the second highest after lung cancer, with 14.5% deaths2.


INTRODUCTION
According to the World Health Organization data in 2004, the top five cancer occurrences in the world were lung cancer, breast cancer, colon cancer, stomach cancer and liver cancer. The cancer incidence increased from 12.7 million cases in 2008 to 14.1 million cases in 2012. While the number of deaths from cancer increased from 7.6 million people in 2008 to 8.2 million in 2012. It is estimated that in 2030 the incidence of cancer may reach 26 million people and 17 million of them will die from cancer. In poor and developing countries, deaths from cancer increase faster 1 . In women, breast cancer has the highest percentage of new cases (43.3%), and the percentage of death is 12.9% 1 . In men, liver cancer incidences is on the second highest after lung cancer, with 14.5% deaths 2 .
Many cancer drugs used today are derived from plants. Some of the results of previous studies found that natural anti-cancer compounds are from flavonoids, alkaloids and terpenoids 3 . Some natural compounds that have been widely used as cancer drugs are Taxol from Taxus brevifolia 4 , and vincristine and vinblastine from Vinca rosea 5 .
The plants from the genus Garcinia of the Clusiaceae tribe are widespread throughout the tropical and subtropical regions. The species from the genus Garcinia has long been used as traditional medicine in many countries, and contains many chemical compounds with many biological activities, including anticancer activity 6,7 . This species are known to contain many compounds such as flavonoids, benzophenone, lanostane, xanthon and terpenoids, which show anticancer activity. 7-epiclusianone, which is a benzophenone group is isolated from Garcinia brasiliensis 8 . Wallichinanes A-E, which is a group of lanostanes, is isolated from Garcinia wallichii Choisy 9 , nujiangexathone A, which is a group of xanthones, is isolated from Garcinia nujiangensis 3 .
Preliminary research on anticancer activity has been carried out on several Garcinia species. Many plants from Garcinia genus have cytotoxic effects, For example: the root of Garcinia cowa 10 , the stem bark of Garcinia ovalifolia 11 and Garcinia cylindrocarpa 12 , the leaves of Garcinia nijuangensis 3 , the fruits of Garcinia wallichii 9 , the pericarps of Garcinia mangostana 13 , Garcinia brasiliensis 8 and Garcinia dulcis 7 . The fruits, flesh and seeds of the Garcinia dulcis are also shown to contain active compounds that are apoptotic to HepG2 liver cell lines 7 . The pericarps of Garcinia mangostana have cytotoxic effect on nasopharyngeal cancer cell lines CNE1, CNE2, SUNE1 and HONE1; lung cancer cell lines A549 and GLC82; MCF-7 breast cancer cell lines; and Bel-74021 14 and Hep-G2 liver cancer cell lines 15 .
Garcinia forbesii King is one of the species that is widely spread on the island of Kalimantan (Indonesia) and parts of Malaysia 16 . In Banjar, South Kalimantan, this plant is known by its local name Mundar. The phytochemical isolation of this plant extract has found xanthones, 1,3,7-trihydroxy-2-(3-methylbut-2-enyl) -xanthone and forbexanthone compounds 17 . Another study has isolated rubraxanthone, which has antibacterial effect, from the stem bark of this plant 18 .
n-butanol and water extracts from the pericarps of G. forbesii King on MCF-7 breast cancer cells and HepG2 liver cancer cells.

Collection of plant materials
The pericarps of G. forbesii King were collected in December 2016 in Banjar, South Kalimantan. This plant was identified by the botanist of the UPT Plant Conservation Center, Purwodadi Botanical Garden, LIPI, East Java.

Extraction
The pericarps of G. forbesii King were cleaned, dried, and then ground into fine powder. The pericarp powder was extracted by maceration using n-hexane solvent for 1x24 hours and the filtrate was collected. The maceration was repeated three times. All the filtrate was collected and evaporated with a rotary evaporator to obtain a n-hexane dry extract.
The residue obtained from the rest of maceration with n-hexane was dried and macerated again using DCM solvent for 1x24 hours, and this process was repeated three times. The collected filtrate was evaporated with a rotary evaporator until the DCM extract was obtained. Further, the maceration was carried out in the same manner successively using ethyl acetate, n-butanol, and water solvents. The extracts of n-hexane, DCM, ethyl acetate, n-butanol and water were analyzed for their phytochemical contents, and their cytotoxic activities were assessed against MCF-7 breast cancer cells and HepG2 liver cancer cells.
Thin Layer Chromatography (TLC) analysis [19][20][21] The TLC was performed using Kiesel Gel GF254 as the stationary phase. The concentration of sample was 1% w/v. Flavonoids screening was conducted using the n-butanol:glacial acetic acid:water (4:1:5) as the mobile phase, and the ammonia vapor as the appearance spots.
Polyphenols screening was conducted using the chloroform:ethyl acetate:formic acid (1:18:1) as the mobile phase, and FeCl 3 as the appearance spots.
Terpenoids screening was conducted using the n-heksana:ethyl acetate (4:1) as the mobile phase, and anisaldehyde sulfuric acid as the appearance spots.
Alkaloids screening was conducted using the chloroform:ethyl acetate (1:1) as the mobile phase and the Dragendorf reagent as the appearance spots.

Cell culture 21
The cancer cells used were MCF-7 cell line and HepG2 cell line obtained from the Parasitology Laboratory, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada. The cells were grown in RPMI media. After the cell condition reached 80% confluent, the cells were harvested according to the harvest protocol. The cells were suspended in RPMI to a concentration of 5x10 3 cells/100 μL. The cell suspension was inserted into the wells, each 100 µL for each well. Every time the cell suspension was inserted into 12 wells, the cells were resuspended again to ensure homogeneity. The cells were incubated in the incubator overnight to allow for cell recovery after harvesting. The plate containing cells was taken from the incubator, and the media was discarded by turning the plate 180°C above the dump. The plate was pressed gently over a tissue paper to drain the remaining liquid. 100 μL PBS was put into all the wells filled with cells, and then the PBS was discarded by turning the plate. The remaining liquid was drained with tissue paper.

Preparation of extracts solutions for cytotoxicity assay 22,23
A total of 10 mg of each extract was dissolved in 100 μL DMSO, and a stock solution of 1x10 5 ppm was obtained. From the stock solution, sample solutions were made with a concentration of 500 ppm, 250 ppm, 125 ppm, 62.5 ppm, 31.25 ppm and 15,625 ppm using culture media as the diluent.

Cytotoxicity assay 21
Each sample concentration was put into a well containing cancer cells, three wells for each concentration (triplo). The plate was incubated in an incubator for 24 hours. After 24 hours the cell media was removed, washed using PBS 1x, and 100 μL MTT reagent was added to each well, including media control. Cells were re-incubated for 2-4 hours in an incubator until the formazan was formed. Cell conditions were examined with an inverted microscope. After the formazan has clearly formed, a 10% SDS stopper in 0.1 N HCl was added. The plate was wrapped in aluminum foil and incubated in a dark room temperature overnight. After the incubation, the plates was inserted into the ELISA reader, and the absorbance of each well was analyzed with λ = 550-600 nm. Absorbance vs. concentration graphs were made to illustrate the profile of living cells, and the percentage of living cells and IC50 value were calculated using linear regression.

Chemical contents of the extracts
The results of TLC analysis of the extracts are shown in Table 1.

Cytotoxic activity
The cytotoxic activity of each extract against MCF-7 and HepG2 cancer cells expressed by the IC50 values is as shown in Table 2.

DISCUSSION
The results of TLC analysis of several extracts of G. forbesii King pericarps showed that the n-hexane extract contained flavonoids and terpenoids which quantities were larger than the other extracts. The contents of flavonoids and terpenoids decreased with increasing polarity of the solvents in the successive in G. forbesii king pericarps. The content of alkaloids was very small in quantity, and it was only detected in n-hexane and DCM extracts, whereas in other extracts there was no detectable alkaloids. Polyphenols were present in relatively     26 . Xanthone flavonoids are contained in the roots of G. ovalifolia 11 . In G. forbesii itself, the bark contains forbexanthone and rubraxanthone 17,18 .
In vitro anticancer potential is indicated by IC 50 values. Smaller IC 50 value indicates that the drug or test material is more potent as an anticancer. In this study, the anticancer potential assessed using the MTT essay method showed that the n-hexane extract ( The results shown by the n-hexane and DCM extracts indicate that the compounds that actively kill or inhibit the growth of HepG2 liver cancer cells and MCF-7 breast cancer cells may be non-polar and semi-polar compounds, especially flavonoids and terpenoids. This is in line with some of the results of previous studies. Sukandar et al. 12 found several flavonoid compounds such as cylindroxanthones A-C isolated from G cylindrocarpa which had cytotoxic activity against MCF-7 cancer cells with IC 50 of 98.54 μM, 168.53 μM and 59.05 μM, respectively, and against HepG2 cancer cells with IC 50 of 10.41 μM, 59.53 μM, and 37.90 μM, respectively. Hongthong et al. 9 found several terpenoid compounds namely wallichinanes A-E isolated from G. wallichii had cytotoxic activity against MCF-7 with IC 50 of 6.19 μM for wallichinanes D compounds, and >50 μM for other compounds.

CONCLUSION
The pericarps of G. forbesii King contains flavonoid, polyphenol, terpenoid and alkaloid compounds.
The decreasing order of potential cytotoxic activity against MCF-7 cancer cells is n-hexane, DCM and ethyl acetate extracts, whereas for the cytotoxic activity against HepG2 cancer cells, the decreasing order was DCM, n-hexane, and ethyl acetate extracts. The n-butanol and water extracts did not show potential cytotoxic activity againts the two cells.
Extracts of n-hexane, DCM and ethyl acetate from pericarps of G.
forbesii King have cytotoxic activity against MCF-7 and HepG2 cancer cells, therefore it has the potential to be developed as an anticancer.

CONFLICTS OF INTEREST
The authors declare that they have no conflicts of interest.