Endophytic, in general, refers to a situation where one organism lives in another. Endophytes reside in the tissues between living plant cells, forming a mutually beneficial relationship with plants from symbiotic to bordering pathogens. In this case, endophytic fungi lives asymptomatically within the living tissue of the host plant and form mutualistic symbiosis. There could be more than one type of endophytic fungi found within one plant (Petrini et al., 1992).
Studies by Carroll (1988) demonstrated that endophytic fungi caused no harmful effects to the living host plant and they can be isolated from various part of the plant with a careful selection and screening process. The isolation method may affect the species composition of the endophyte collection within a given host plant. Large number of isolates can be obtained from host species, however, only few host specific strains are dominant (Petrini et al., 1992). Different endophytic fungi species are found in different part of a plant; this represents an adaptation mechansim of endophyte against microecology and physiology of given host plant. Various studies demostrated that endophytic fungi produce secondary metabolites such as enzyme and growth hormone which are useful for treatment of various diseases. These bioactive metabolites demonstrated potent anti-bacterial, anti-arthritis and anti-cancer activity as well as immunosupressive activity (Petrini et al., 1992; Carroll, 1988; Strobel and Long, 1998).
In developing countries, cancer has been a major cause of death apart from cardiovascular and cerebrovascular disease (Ueda et al., 2002). Currently, Indonesia is in the 6th rank according to survey result by Cancer Registration Board of the association of Indonesian Pathologist and this status may shift up from year to year (Agency for Health Research and Development, 1997; Department of Health, 1977; DR Cipto Mangunkusumo, Cancer in DR Mangunkusumo National Center of Public Hospital, 1999). This evidence encourages scientist to intensively search for newer and more effective anti-cancer agent to minimize this problem. As an alternative, one stratergy is to develop new herbal medicine which would improve the traditional means of treating cancer to the patient with high selectivity and ability to suppress cancer cells metastasis, whenever possible (Filler and Kripke, 1997).
One of the herbal plant found in regional area of Indonesia is a Simaroubaceae
plant, Brucea javanica L. (Merr) with its local name buah makasar. This
plant, empirically, has been reported to be used as traditional medicine for
the treatment of diseases like dysentry, malaria, leukemia and other cancers,
including lung, cervical and skin cancer, by the local society (Department of Health, 1977; Tang and Eisenbrand, 1992; Dalimartha, 2002), however, very few studies were conducted to investigate
that the effectiveness of secondary metabolites produced by its endophytes.
The current study was focussed on the isolation of endophytic fungi from buah makasar [Brucea javanica L. (Merr)] plant and the cytotoxic effect of secondary metabolites in n-butanol extract isolated in leukemia cell L1210.
MATERIALS AND METHODS
Materials: Eagles MEM media, leukemia cell L1210 were a kind gift from Prof Dr. Yasuo Fujimoto, College of Pharmacy, Nihon University, Chiba, Japan for research work carried out in BATAN (National Atomic Energy Agency), Indonesia Chloramphenicol and Glutamine were purchased from Indonesian registered drug store. Ethanol and sodium hypochlorite were purchased from Kimia Farma. Foetal calf serum was purchased from Flow laboratories. Sodium hydrogen carbonate (NaHCO3) was purchased from Kmia Farma.
Endophytic fungi and bacterial culture media: Endophytic fungi culture media is composed of Corn Meal Malt (CMM) from DIFCO (USA) agar. Potato dextros broth from Oxoid (England). Potato Dextrose Agar (PDA) DIFCO and Potato Dextrose Yeast Extract (PDY) were used in bacterial culture. 76% ethanol and 5.3% sodium hypochlorite were used for surface sterilization.
Sampling of Brucea javanica L. (Merr) plant: Fresh and healthy Brucea javanica L. (Merr) plant were sampled from three locations in West Java: Kebon Raya Bogor, Kebun obat P.T. Eisai Indonesia Cianjur and Balai Penelitian Tanaman obat (BPTO) Tawangmangu using random technique from June until Agustus in dry season. Parts of plant used in the study are twigs (diameter 0.8-10 mm), leaves and fruits. The plant samples were characterised in Herbarium Balitbang Botani, Puslitbang Biology Department of LIPI, Bogor, West Java, Indonesia.
Endophytic fungi isolation: Endophytic fungi isolations were carried
out via direct seed method. Surface sterilization methods were adapted
from Petrini et al. (1992) and Carroll (1988). Briefly, samples were washed
under running water for about 10 min to remove dust and dirt; and were cut into
small pieces and air-dried at room temperature. The twigs and fruits were cut
into dimension of 1 cm pieces; while the leaves were cut into square 0.5x0.5
cm dimension. Then, surface sterilization was conducted as follows: specimens
were soaked in 76% ethanol for 1 min, followed by hypochlorite solution 5.3%
for 5 min and finally in ethanol 76% for 30 sec. The surface-sterilized pieces
of twigs and fruits were aseptically sliced lengthwisely. The cut surface of
the twigs and fruits and the squared pieces leaves were immediately placed on
the surface of CMM medium which contained chloramphenicol (0.00 5% w/v). They
were then incubated at temperature of 27-29°C for 5-7 days . Then, a selection
process based on fungi morphology were applied to differentiate endophytic fungi
from other microbes grown in the culture. The selected fungi were transferred
and were cultured in PDA media (Tomita, 1995).
Purification of isolated fungi: Colony identification was done macroscopically by observing colony characteristic such as growth rate, color and colony morphology. Colonies with same morphology from one isolates were pooled together, on the other hand, the unidentified colonies were separated and grouped according to their morphological status. The pure fungi is then cultured in slant agar. Each of the isolated fungi was culture in two different types of culture media (stock and work culture). Stock culture was kept in freeze dried while work culture was kept in slant agar.
Liquid fermentation: In order to obtain secondary metabolites which have the potential as anti cancer substance, liquid fermention using PDY medium, which consists of potato dextros broth 24.0 g L-1; yeast extract 2.0 g L-1 and CaCO3 5.0 g L-1 at pH 6.0 was conducted. Endophytic fungi was cultured in Petri dish., for 5-7 days. Liquid fermentation was carried out in a 250 mL Erlenmeyer flask, containing 50 mL PDY medium and 5 pieces of endophytic fungi (using cor bock) using orbital shaker incubator rotating at 130 rpm for 14 days at room temperature. Separation of cell mass from supernatant which contain the secondary metabolite of interest was carried out in cold centrifuge at -4°C rotating at 2000 rpm for 20 min. pH of the supernatant was adjusted to pH 3-4 using acetic acid 0.1 M and then extracted in n-butanol. This butanol extract was evaporated down to small volume, using rotary evaporator for bioassay.
Preparation for Leukemia L1210
Preparation of Eagles MEM: 4.7 g of Eagles MEM (Nissui)
was dissolved in 475 mL water to make up solution A and then, 1.3 g of NaHCO3
together with 0.3 g of glutamine were dissolved in 50 mL of water to give solution
B. Then, 25 mL of solution B was added to solution A to give a final volume
of 500 mL medium. Medium was filtered aseptically through millipore filter and
stored in the fridge until use. 15% (v/v) foetal bovine serum/MEM was used in
leukemia cell (L1210) culture and for bioassay (Sumatra, 1998).
L1210 leukemia cell culture method: Frozen stock of L1210 was thawed and tranferred into centrifuge tube which contained 4 mL of MEM and were centrifuged at 1000 rpm. Supernatant was removed and cell pellet was washed three times with 2 mL of MEM. After washing, cell pellet was resuspend in 10 mL MEM, transferred into tissue culture flask for 48 h incubation in an incubator filled with 5% CO2.
Following the incubation periods, cell were harvested and counted using heamocytometer to get cell density of 2x106 cells mL-1 used in the bioassay (Sumatra, 1998).
Bioassay using leukemia cell L1210: Anti cancer activity of endophyte fungi secondary metabolite was measured based on its cytotoxic effect on leukemia cell (L1210). One thousand microliter of L1210 cell suspension were added into each of the 24 multi-well plate followed by the addition of 10 μL methanolic solution of fermentation product at three different concentrations 2.5, 5 and 10 μg mL-1 and incubated over 48 h. As a negative control, cells were treated with only Eagles MEM with 10 μL metanol. Each treatment was done in triplicate.
To calculate IC50 values of secondary metabolite butanol extract
from each of 18 endophytic fungi isolates following formula is used:
Endophytic fungi isolation: Endophytic fungi which is isolated from the pieces of twig, fruit and leaf of Buah Makasar (Brucea javanica L. (Merr) ) plant, was cultured in PDA medium treated with 0.005% chloroamphenicol to isolate the endopytic fungi. The isolation result yields 45 endophytic fungi, where 18 of the total 45 isolates were used in cytotoxic assay (Table 1).
Endophytic fungi with cytotoxic activity: In this study, a selection process of endophytic fungi was performed to produce secondary metabolite which has the cytotoxic activity. Anti proliferative test conducted L1210 cell showed that the isolates have IC50 concentration range from 3.29 to 15.90 μg mL-1 (Table 2).
This study is the first to isolate and to demonstrate the effectiveness of
endophytic fungi from B. javanica in leukemia cells (L1210). Furthermore,
this preliminary results may serve as useful template for future study in finding
new anti-cancer substance from endophytic fungi.
The endophytic fungi isolation procedure used in this study generated 45 fungal endophyte. The effectiveness of endophytic fungi, as anti cancer substance was assessed using cytotoxic assay on 18 of the total 45 endophytic fungi isolated. B. javanica was chosen in this study for isolation of fungal endophyte because, of its tradional use for treatment and cure of malaria, lung cancer and leukemia Samples were selected randomly from 3 locations (Bogor, Cianjur and Tawangmangu) to obtain variety of endophytic fungi based on different geographical condition, as different environmental condition may result in different strain of endophytic fungi (Song, 1998). Interestingly, the samples collected from three parts of the plant twigs, leaves and fruits, variety of endophytic fungi strains were found in a given host plant. This results confirmed previous finding by Petrini et al. (1992).
Furthermore, liquid fermentation on endophytic fungi isolates were performed in our study for two weeks in order to test the ability of the these endophytes to produce secondary metabolites, as describe previously (Stanbury and Whitaker, 1987).
Microscopic identification has been done in our study, however almost all of endophytic fungi were sporeless, so that the microscopic identification was not able to be performed; the identificatione was done only by macroscopic morphology.
In our fermentation procedure, secondary metabolite produced by the fungi were
extracted using non-polar (hexane), semi-polar (Dichlormetane) and polar solution
(butanol). Our fermentation results demonstrated that n-butanol fraction
has strong anti-cancer activity against L1210 cells. The secondary metabolites
were extracted from polar solvent such as n-butanol in order to obtain
polar substance. It is likely that bioactive substances extracted using n-butanol
will be more water-soluble. The extraction result using n-butanol solution
was tested by anti proliferation test against leukemia cell. Leukemia cancer
cell were selected as a model for anti-cancer activity test in our study. This
is in accordance with previous evidence on consumption of B. javanica
extract as anti cancer agent in leukemic patient.. Additionally, this is also
important to justify whether endophyte fungi produce bioactive substance that
are similar to that in buah makasar plant extract (Strobel and Long, 1998).
Cytotoxic assay other than the MTT assay were used in this study, because direct
cell counting by haemocytometer is one of the simplest method, inexpensive,
accurate measure of cell number to assess the cytotoxic effect of secondary
metabolites of endophytic fungi. The result of the cytotoxic assay shows IC50
range of 3.29 to 15.90 μg mL-1, suggesting that endophytic fungi
from buah makasar plant generated secondary metabolites with anti-cancer activity.
||Macroscopic morphology of endophytic fungi from B. javanica
Cytotoxic assay result for n-butanol extract of endophytic
fungi from B. javanica collected in Bogor, Cianjur and Tawangmangu
The bioactivity of these secondary metabolites in the n-butanol extracts
was considered to be active when the IC50 of less than 20 μg
mL-1 as described by Swanson (1990). In attempt to search for a new
potent bioactive substance, crude extract of metabolites with IC50
of less than 20 μg mL-1 is potential for further purification
phase and IC50 that is less than 4 μg mL-1 for pure
substance isolated from natural sources has potential as anti-cancer substances.
In this preliminary study, n-butanol extract of secondary metabolite from endophytic fungi has potent anti-cancer activity and will be further purified to isolate the bioactive compound.
Several endophytic fungi such as Taxomyces andreanae, Pestalotiopsis, Altenaria sp., had been isolated from Taxus brevifolia . These fungi can produce taxol (Petrini et al., 1992; Strobel, 2002). In accordance to this study, the endophytic fungi from B. javanica may also produce secondary metabolites, such as anti-cancer bruceantin which has been isolated in B. javanica as the host plant.
We would like to thank Dr. Harmastini, Research Center for Biotechnology, Indonesian Institute of Sciences, who has given us the opportunity and her guidance during conducting the research of isolation of endophytic microbe of B. javanica at her laboratory. We also thank Dr. Made Sumatra for helping to out a cytotoxic assay using leukemia cell L1210 at National Atomic Energy Agency, Pasar Jumat, Jakarta.