Cytotoxicity of Buah Merah (Pandanus conoideus Lamk.) Extract on Breast Cancer Cell Line (T47D)

Buah Merah (Pandanus conoideus Lamk.) has been extensively used to treat various diseases including cancer. There are many varieties of buah merah and there was no scientifi c study comparing cytotoxicity of different varieties. The objective of this study was to investigate the cytotoxicity of three varieties of buah merah known as Barugum, Maler and Yanggiru on breast cancer cell line (T47D). All samples were collected from Papua, Indonesia. Each sample was extracted consecutively using three solvents chloroform, methanol and water resulted to nine crude extracts. The cytotoxic activities were determined using MTT assay. The crude extract showed the lowest IC50 was selected for further bioassay-guided fractionation. Fractionation was done using vacuum liquid chromatography coupled with preparative TLC to fi nd the active compounds. Several detection reagents were applied to TLC for identifi cation of the class of the potent compounds. The result showed that the potent extracts was obtained from Barugum methanol extract followed by Maler chloroform extract with IC50 value of 132.83 μg/ml and 139.72 μg/ml, respectively. All Yanggiru extracts did not show activity. The bioassay-guided fractionation of Barugum and Maler extracts showed that the most potent fraction eluted by a mixture of hexane:ethyl acetate (75:25), was in Maler variety with IC50 value of 25,7 μg/ml, four times higher than the most potent fraction of Barugum with IC50 value of 104,61 μg/ml. TLC analysis of the most potent fraction showed that the active compounds was class of terpene. Result of this study supported the utilization of buah merah Maler variety for breast cancer treatment.


Introduction
Plants have an almost limitless ability to synthesis bioactive substances.Most are secondary metabolites, of which at least 12.000 have been isolated, a number estimated to be less than 10% of the total.Only a small percentage of the 400.000 to 500.000species in the plant kingdom has been phytochemically investigated (Wink, 2003).It is well established that secondary metabolites becomes one of the most important sources of pharmacologically active compounds including for cancer treatment (Cragg et al., 1999).
Breast cancer is the second leading cause of death in women today and is the most common cancer among women.Though much less common, breast cancer also occurs in men.More than a half of breast cancer cases are caused by unknown factors (Madigan et al., 1995).Nevertheless, some risk factors which increase someone's probability of having breast cancer are postmenopausal hormone therapy, exposed to ionizing radiation, alcohol consumption, and have BRCA1 and BRCA2 mutant genes (Longnecker et al., 1995 andHortobagyi, 1998).The probability of a woman with the mutant genes to has breast cancer is 50-85% (Hedenfalk et al., 2001).
Different techniques can be used for the chemical evaluation of plants.Bioassayguided fractionation using vacuum column chromatography (VLC) coupled with preparative Thin-layer chromatography (TLC) considers as the simplest and cheapest method.Cytotoxic screening models provide important preliminary data to select plant extracts with potential anti-cancer properties.The most commonly cytotoxic assay used is MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide) assay.
Buah merah (Pandanus conoideus Lamk.) grows naturally in Papua, Indonesia at 1,200-2,000 m above sea level (Anonim, 2005).Recently, buah merah has been extensively used to treat HIV/AIDS, diabetes mellitus, gout and rheumatism, osteoporosis, and cancer.According to Moeljopawiro et al. (2007), extracts of buah merah have ability to inhibit breast, cervical and colon cancer cells proliferation.The scientifi c studies of anticancer activity of buah merah on the growth of cancer cells are relatively very few.In Papua, several varieties of buah merah are observed and so far there were no study comparing the cytotoxicity among these varieties.Therefore, the objective of this research was to study the cytotoxicity of three varieties of buah merah known as Barugum, Maler and Yanggiru and determine the group of bioactive compounds.

Materials And Methods
Three varieties of buah merah including Barugum, Maler, and Yanggiru, collected from Sentani, Jayapura, Papua, Indonesia were used in this study.The breast cancer cell line used was T47D.This cell line was obtained from a cell culture continually grown at the Laboratorium Penelitian dan Pengujian Terpadu (LPPT) UGM.Doxorubicin (Ebewe), was used as a reference on cytotoxic assay.

Extraction of Samples
All samples were extracted using three solvents including chloroform (Merck), methanol (Merck), water.Thirty fi ve gram of fresh fruit for each variety was consecutively extracted using Soxhlet apparatus (Pyrex) at 65°C with 250 ml of solvent.Initially, chloroform was used as a solvent and resulted residue was then extracted using methanol.Finally, water was used for the last extraction.Each extract was evaporated to dryness under reduced pressure on rotary evaporator (Rotavapor, Buchi) and was stored at 4°C for further use.(Mosmann, 1983) In preliminary experiments a serial concentration of 0-500 μg/ml of methanol extracts of buah merah showed a clear dose response with cell mortality.Therefore this concentration range was used in later experiments.

Cytotoxic Assay
An aliquot of 100 μl cell suspension (± 1.5 10 4 cells) was loaded into each well of 96-well microtitre plates and incubated for 24 hours at 37˚C, 5% CO 2 to allow for cell attachment.Extracts of buah merah at different concentrations ranging from 0-500 μg/ml were added and then incubated for 24 hours at the same condition.For each concentration tested four replicates were used.Two controls were applied, a control consisting of medium and another control consisting of mixture of DMSO and culture cells.At the end of the treatment, the medium and extracts were then removed and 110 μl MTT (Sigma) was added.MTT was prepared as 10% MTT stock solution in culture medium, sterilized by fi ltration through a 0.2 mm fi lter and stored at 2-8 °C.The plate was incubated in the dark for 4 hours.After 4 hours, 100 μl stop solution (10% SDS (Sigma) in 0.1 N HCl) was added in each well and again the plates were then incubated in the dark for overnight at room temperature.The absorbance was recorded using ELISA plate reader (Bio-Rad) at 595 nm.The value of IC 50 was determined by probit analysis.

Fractionation
The active extracts, methanol extract of Barugum and chloroform extract of Maler, were subjected to fractionation by VLC on Sephadex LH-20 (Sigma) using a mixture of different solvents ranging from non polar to polar including hexane, ethyl acetate, chloroform, methanol and water.TLC was performed to monitor the metabolites contained in each fraction.The fractions with similar TLC profi les were combined.Fractionation of methanol extract of Barugum was done using 15 eluents while the chloroform extract of Maler with 6 eluents.

Preparative TLC
Preparative TLC was performed similarly like the procedure of TLC, except for the stationary phase plates of preparative TLC were silica gel PF 254 .The plates were developed using a mixture of chloroform : ethyl acetate = 9 : 1 v / v .Based on the profi le, preparative TLC produced three portions (upper, middle and lower) portions for Barugum and two (upper and lower) portions for Maler.These portions were then subjected to cytotoxic assay to determine the active portion containing the active compounds

Sequence of bioassay-guided fractionation
Due to different results in the first fractionation of Barugum and Maler, therefore different strategies of fractionation were applied.
The combined fraction of the methanol a.
extract of Barugum were directly subjected to cytotoxic assay at a single dose of 250 μg/ml representing a middle concentration on the crude extracts cytotoxic assay.The most potent fraction was selected for further separation using preparative TLC then followed by cytotoxic assay.The two fractions of the chloroform extract b.
of Maler showed similar TLC profile therefore these fractions were combined again.Separation was further performed using preparative TLC.The results of the preparative TLC were then subjected to cytotoxic assay.Based on this cytotoxic assay, the most potent portion was further fractionated using 6 eluents.The results of this fractionation were monitored using TLC and the fractions that showed similar TLC profi le were combined.This combined fractions were then evaluated the activity using MTT assay.

Identifi cation of Toxic Compounds Classes
The most toxic fraction obtained from preparative TLC was analyzed by TLC and then visualized by spraying reagents including serium (IV) sulfat, Lieberman-Burchard, vanilin sulfat, Sitroborat, Dragendroff , FeCl 3 , jodium and ammonia to identify the groups of compounds.

Identifi cation of Samples
Based on morphological characteristics, buah merah collected from Papua could be classifi ed into three local varieties, Barugum, Maler and Yanggiru with characteristics as described in Table 1.Yanggiru variety has specific yellow color different among the rest varieties.Although both Barugum and Maler have similar color, but the two can be distinguished easiliy by the shape of the fruit.Barugum fruit normally has a bigger fruit and has oblongus triangular in shape while Maler tends to have slimmer oblongus.

Cytotoxicity of Extracts
Cytotoxicity of 9 crude extracts showed that the water extracts of all samples did not have cytotoxic effect on breast cancer cells.The most toxic extracts with the lowest IC 50 value were found on methanol extract of Barugum with IC 50 value of 132,83 μg/ml, followed by chloroform extract of Maler with IC 50 value of 139,72 μg/ml (Table 2).These two crude extracts were chosen for further bioassay guided fractionation.The three extracts of Yanggiru variety with yellow coloured did not show any anti-cancer activity indicating by IC 50 values were higher than 500 μg/ml.

Isolation of active portions from Barugum and Maler extracts
The fractionation of methanol extract of Barugum yielded fi ve combined fractions (Figure 1A) and directly subjected to cytotoxic assay.Whereas the fractionation of the chloroform extract of Maler resulted two combined fractions (Figure 1B).But due to the high similarity between the two combined fractions on the TLC profi le therefore these fractions were again combined.Further separation was done using preparative TLC.
Bioassay guided fractionation of Barugum.Cytotoxic assay of the combined fractions of Barugum showed that the most toxic sample was the fi rst combined fraction having an activity to allow viability of 16.43% of breast cancer cells at 250 μg/ml.Result of the cytotoxic assay could be seen in Table 3.
The potent combined fraction of Barugum was subjected for preparative  B TLC.The TLC profile showed that the fraction could be divided into two portions; upper and lower portions.Each portion was dissolved in chloroform : methanol = 9 : 1 v / v .These two portions were used for further analysis together with other portion resulted from Maler extract.Bioassay guided fractionation of Maler.The result of preparative TLC of Maler extract showed that the profi le can be divided into 3 portions; upper, middle, and lower portions.Based on the bioassay monitoring of these three portions, the middle portion was confi rmed as the potent portion (Figure 2).This was shown by the lower IC 50 value of the middle portion compared to the upper and the lower portions (Table 4).The middle portion of the Maler was further fractionated by VLC using 9 eluents.The TLC profi le of these 9 fractions were documented.The fractions with the similar profi les were combined giving 6 combined fractions (Figure 3).Based on the result of TLC monitoring there were only 4 combined fractions including 2,4,5 and 6 used for further bioassay.
Comparison of cytotoxicity between Barugum and Maler.The two portions of preparative TLC of Barugum and these four combined fractions of Maler as well as the commercial medicine, doxorubicine were evaluated their activities using MTT assay on breast cancer cells (T47D) (Table 5).The result of this cytotoxicity showed that Maler variety possesses potent anticancer activity on breast cancer cell line (T47D) having lowest IC 50 value of 25,7 μg/ ml.This IC 50 was lower than Doxorubicin with IC 50 value of 41,68 μg/ml.The potent combined fraction was further subjected to TLC.

Identifi cation of Toxic Compounds Classes
The most toxic combined fraction was analyzed by TLC followed by spraying using many detector reagents to identify the class of the toxic compounds.According to the spots obtained, it showed that the toxic compounds were terpene (Table 6).

Discussion
Secondary metabolites are known for their diversity.This can be observed between species or even within species.This diversity of secondary metabolites within species was well presented in this study.Three different local varieties of buah merah showed different cytotoxicity on breast cancer cell lines.The order of varieties of buah merah containing the most potent compounds was Maler, Barugum and the last was Yanggiru which have no anti-cancer activity.As Thorne (1975) stated that secondary metabolite could be used as chemotaxonomic characters and had a considerable impact on plant systematics.It could be used for identification and classifi cation of fl owering plants into a taxon because of the distribution of secondary metabolites is specifi c in certain species.Most of bioactive compounds isolated from plants do not have cytotoxic activity, so called chemopreventive compounds.Chemoprevention could be described as a compound that prevents, inhibits, and normalizes carcinogenesis or prevents invasive cancer development (Meiyanto et al., 2005).Even though, according to Moeljopawiro et al. (2007), extract of buah merah have cytotoxic effects on breast cancer cells (T47D) and colon cancer cells (HT-29).Chemopreventive compounds are more selective than cytotoxic compounds on cancer cells because chemopreventive compounds have specifi c molecular targets (Meiyanto et al., 2005).Their targets are to inhibit carcinogenesis, cell cycle and angiogenesis.
Chemopreventive compounds could not be applied solely for cancer therapy.They can only be used as a supplement to prevent cancer development (Middleton and Kandaswami, 1993).These compounds are very useful in increasing immunity, reducing toxic risk, and lowering cytostatic therapy dose, thus lowering side effect of cytostatic therapy.In addition, the chemopreventive compounds also lowering cancer incidence and risk caused by cancer.Therefore, therapy using combination of cytostatic and chemopreventive compounds gives more effective and effi cient results.
Results in this research indicated that extract of buah merah might not only as chemopreventive compounds but also have cytostatic activity.It's showed by the lower IC 50 value of the combined fraction of Maler compared to the medical cancer drug, Doxorubicin.Futher study to understand how bioactive mechanism of the inhibition of the buah merah is needed.
The class of bioactive compounds obtained that have cytotoxic activity on breast cancer cells were terpenes.Terpenoids are the most abundant secondary metabolites that have various molecule structures.Most of the terpenoids are of plant origin; however, they are also synthesized by other organisms, such as bacteria and yeast as part of primary or secondary metabolism.They function as phytoalexins in plant direct defense, or as signals in indirect defense responses which involves herbivores and their natural enemies.Therefore, most of scientists think that terpenes have more ecological than physiological functions.Whereas diterpenes have a physiological function as a plant hormone, gibberellins (Wink, 2003) Many reported studies have demonstrated the cytotoxic effects of various terpenoids against proliferation, growth and invasion of a variety of liver cancer cell lines.The mechanisms of anti-cancer activities varied including the increase of the apoptotic cell death process and reactive oxygen species (ROS) generation, increase in the tumour suppressor gene p53, apoptotic proteins, such as caspase-3, and -9, cytochrome c (cyt.c), and p38 protein expression (Huang et al., 2008), so that in India and China, terpenoids become the largest group of phytochemicals that are currently being explored as anticancer agents in clinical trials (Thoppil and Bishayee, 2011).

Conclusions
The result of this study showed among 3 varieties of buah merah, Maler variety possesses the most potent anti-cancer activity on breast cancer cell line followed by the Barugum variety.The Yanggiru variety having yellow color, did now show any anticancer activity.The class of the toxic compounds on breast cancer cells (T47D) were terpene.

F1Figure 1 .
Figure 1.Representative diagram of TLC profi les obtained from methanol extract of Barugum (A) and chlorform extract of Maler (B).The numbers designated with arrows represent the combined fractions.

Figure 3 .
Figure 3. Representative diagram of TLC profile of 9 toxic fractions of chloroform extract of Maler.The numbers designated with arrow represent the combined fractions.

Table 2 .
The IC 50 value of chloroform and methanol extracts of buah merah on breast cancer cells (T47D)

Table 1 .
Morphological characteristics of fruits of three local varieties of buah merah

Table 4 .
TheIC 50 value of three portions obtained from TLC preparative of chloroform extract of Maler on breast cancer cells (T47D)

Table 5 .
The IC 50 value of two portions derived from TLC preparative of Barugum, fi ve combined fractions of Maler on breast cancer cells (T47D) and Doxorubicin

Table 6 .
The TLC result of the potent combined fraction of Maler