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Ficus septica Burm. F. (Moraceae) is a plant which grows widely in some areas of Indonesia and other Southeast Asia countries. Previously, ethanolic extract of the plant leaves exhibit cytotoxic effect on several cell lines including T47D cells. The extract also showed chemoprevention effect in vivo. In the study, the ethanolic extract was fractionated gradually by n-hexane and ethyl acetate to yield four fractions including n-hexane soluble fraction, n-hexane insoluble fraction, ethyl acetate soluble fraction and ethyl acetate insoluble fraction. These fractions were then investigated for their cytotoxic effect on T47D cells. The cell viability were assessed using MTT colorimetric assay. The results showed that the cytotoxic effect of n-hexane insoluble fraction (IC50 : 9.3 µg/mL) was much more potent than this of n-hexane soluble fraction (IC50 : 331.0 µg/mL). Hexane insoluble fraction was then fractionated with ethyl acetate to provide ethyl acetate soluble fraction and ethyl acetate insoluble fraction. The cytotoxic effect of ethyl acetate soluble fraction (IC50 : 13.7 µg/mL) was much more potent than this of ethyl acetate insoluble fraction (IC50 : >700 µg/mL). In conclusion, n-hexane insoluble fraction and ethyl acetate soluble fraction exhibited potent cytotoxic on breasr cancer T47D cell lines. The fractions are potential to be developed as anticancer agent in breast cancer therapy.
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International Journal of Phytomedicine 3 (2011) 216-226
http://www.arjournals.org/index.php/ijpm/index
Original Research Article
Cytotoxic Effect of Ethanolic Extract Fractions of Indonesia Plant Ficus
septica Burm. F. on Human Breast Cancer T47D cell lines
Agung Endro Nugroho,1,3 Muthi Ikawati,2 Adam Hermawan,1 Dyaningtyas Dewi Pamungkas Putri,1
and Edy Meiyanto1,2
*Corresponding author:
Agung Endro Nugroho
Cancer Chemoprevention
Research Center, Faculty of
Pharmacy, Universitas
Gadjah Mada, Sekip Utara
Yogyakarta, Indonesia.
55281,
Telp: (0274)543120,
Fax : (0274)543120.
Email :
agungendronugroho@yahoo.com;
nugroho_ae@ugm.ac.id
Abstract
Ficus septica Burm. F. (Moraceae) is a plant which grows widely in
some areas of Indonesia and other Southeast Asia countries. Previously,
ethanolic extract of the plant leaves exhibit cytotoxic effect on several
cell lines including T47D cells. The extract also showed
chemoprevention effect in vivo. In the study, the ethanolic extract was
fractionated gradually by n-hexane and ethyl acetate to yield four
fractions including n-hexane soluble fraction, n-hexane insoluble
fraction, ethyl acetate soluble fraction and ethyl acetate insoluble
fraction. These fractions were then investigated for their cytotoxic
effect on T47D cells. The cell viability were assessed using MTT
colorimetric assay. The results showed that the cytotoxic effect of n-
hexane insoluble fraction (IC50 : 9.3 µg/mL) was much more potent
than this of n-hexane soluble fraction (IC50 : 331.0 µg/mL). Hexane
insoluble fraction was then fractionated with ethyl acetate to provide
ethyl acetate soluble fraction and ethyl acetate insoluble fraction. The
cytotoxic effect of ethyl acetate soluble fraction (IC50 : 13.7 µg/mL)
was much more potent than this of ethyl acetate insoluble fraction
(IC50: >700 µg/mL). In conclusion, n-hexane insoluble fraction and
ethyl acetate soluble fraction exhibited potent cytotoxic on breasr
cancer T47D cell lines. The fractions are potential to be developed as
anticancer agent in breast cancer therapy.
Keywords : Ficus septica Burm. F., cancer, T47D cells, cytotoxic.
Introduction
Cancer is a disease related to uncontrolled rapid
growth of abnormal cells in the body. They can
invade the healhty surrounding tissue and spread
to other organs. This process is known as
metastatis. Cancer is also named a malignant
tumours and neoplasms [1-3]. Cancer is one of
non-communicable diseases and a public health
problem in the world. Cancer is a major cause of
death in the world, around 13% of all deaths in
2008. More than 70% of all death due to cancer
occured in developing countries. One of main
types of cancer is breast cancer [3]. The
incidence of breast cancer is increasing every
year. Breast cancer is the leading cause of cancer
death. In the United States, breast cancer is a type
of cancer that occur most commonly in women
(28% of total cases) [4]. In Indonesia, percentage
incidence of breast cancer is the second highest
rank after cervical cancer [5].
There are several approaches used for treating
cancer such as surgical excision, irradiation and
chemotherapy. The uses of these methods depend
on the tumor type and the stage of cancer
development [2]. Due to the increasing incidence
ISSN: 0975-0185
This work is licensed under a Creative Commons Attribution 3.0 License.
Nugroho et al. International Journal of Phytomedicine 3 (2011) 216-226
217
of breast cancer every year, the attempt to
discover new anticancer agents is also increasing.
Several study have been done to find out
anticancer agents from natural products
(medicinal plants) for preventing and treating
breast cancer.
Indonesia is the second larger country in the
world after Brazil in terms of biodiversity
including medicinal plants. One of Indonesian
medicinal plant is Awar-awar or Ficus septica
Burm. F. (Moraceae). This plant originates from
and grows widely in some areas of Indonesia and
other Southeast Asia countries. Previously, our
research group named Indonesian Cancer
Chemoprevention Research Center (CCRC) has
selected several Indonesian plants for their
cytotoxic effect on breast cancer T47D cell lines.
One of them with a potent cytotoxic effect was
ethanolic extract of Ficus septica Burm. F.
Treatment of the extract on breast cancer T47D
cell lines for a period of 24 hours resuted in a
cytotoxic effect with IC50 value of 13 µg/mL. The
extract also showed synergistic effect in
combination with chemotherapeutic agent
doxorubicin on the concentration of 4.88 µg/mL
(Ficus septica extract) and 3.75 nM
(Doxorubicin) [6]. Another study, ethanolic
extract of Ficus septica leaves induced apoptosis
in breast cancer cells MCF-7. The extract also
downregulated the expression of Bcl-2 protein
[7]. In vivo study, chemopreventive effect of the
ethanolic extract was studied in 7,12-
dimethylbenz[a]nthracene (DMBA)-induced rat
liver cancer. The extract (750 mg/kg BW)
induced apoptosis throught p53-independent
pathway in 7,12-dimethylbenz[a]nthracene-
induced rat liver cancer [8].
In present study, we investigated the cytotoxic
effect of fractions of ethanolic extract of Ficus
septica Burm. F. leaves on breast cancer T47D
cells line The ethanolic extract was prepared by
macerating the dried-leaves powder with 70%
ethanol. The extract was then fractionated using
n-hexane yielding two fractions of hexane soluble
fraction and insoluble fraction. The insoluble
fraction of n-hexane was then fractionated using
ethyl acetate yielding ethyl acetate soluble
fraction and insoluble fraction. The cytotoxic
activity of fractions were then compared using
IC50 values, a potency parameter of cytotoxic
effect.
Materials and Methods
Materials
Ficus septica Burm. F. was collected from area
around Sumber Arum Moyudan, Yogyakarta,
Indonesia. Ficus septica Burm. F. was identified
by a botanist at Pharmaceutical Biology
Department, Universitas Gadjah Mada, and the
voucher specimen was deposited in herbarium of
the department. Material for cytotoxic assay
were [3 - (4,5-dimetilthiazol-2-yl) -2.5-diphenyl
tetrazolium bromide] (MTT) (Sigma Chemical,
St Loius, MO), H2O2 (Lab Vision Plus),
chromogen 3,3-diaminobenzidin (DAB) (Novo
Castra).
Preparation of Ficus septica ethanolic extract
fractions.
In brief, dried ground powder of fresh leaves of
Ficus septica Burm. F. was extracted using
ethanol 70% with a ratio of 1:5 for 72 hours.
Then, the filtrate obtained was filtered, while the
sediment was re-extracted using ethanol 70% at a
ratio of 1:2 for 72 hours. The re-extraction was
done twice. The extract was then collected, and
evaporated under reduced pressure to give of
viscous ethanolic extract. The extract was added
with 100 mL aquadest, and then mixed to yield
liquid form of ethanolic extract. The extract was
fractionated with n-hexane at a ratio of 140:400
(ethanolic extract:n-hexane) yielding two
fractions of hexane soluble fraction and insoluble
fraction. The insoluble fraction of n-hexane was
then fractionated using ethyl acetate at a ratio of
140:250 (insoluble fraction of n-hexane: ethyl
acetate) yielding ethyl acetate soluble fraction
and insoluble fraction of ethyl acetate. Four
fractions obtained were concentrated by rotary
vacuum evaporator to obtain viscous extract. The
fractions were dried using freeze drying to
eliminate the existence of the remaining traces of
water.
Nugroho et al. International Journal of Phytomedicine 3 (2011) 216-226
218
T47D Cell Culture.
Human breast carcinoma T47D was obtained
from Prof. Masashi Kawaichi (Nara Institute
Sciences and Technology, Japan). The cells were
grown in Dulbecco’s Modified Eagles Medium
(DMEM) containing 10% Fetal Bovine Serum
(Gibco, Grand Island, NY, USA), 1% penicillin-
streptomycin 1% (Gibco), and fungizon 0.5%
(Gibco) in a flask in a humidified atmosphere
(5% CO2) at 37°C.
Cytotoxic Assay.
T47D cell viability were assessed using MTT
colorimetric assay (3-[4,5-diethylthiazol-2-yl]-
2,5-dipheniltetrazolium bromide (Sigma St.
Louis, MO, U.S.A.). The cells were cultured in
96-well plates (Becton Dickinson Co., NJ, USA),
and each well contained 5x103 cells. The culture
cells were incubated in a humidified incubator at
37oC in an atmosphere of 5% CO2 and 95% air
for 24 hours. Cell confluence or crowding of cells
in the plate was about 70-80%. After 24 hours
incubation, culture medium was discarded. The
cells were treated by either Ficus septica Burm.
F. ethanolic extract fractions (treatment groups)
or the vehicle (control group), and then incubated
for 24 hours. The concentrations of the fraction
were 1, 10, 50, 100, 250, 500 and 700 µg/mL in
DMEM. After incubation, the cells were
incubated with 0,5 mg/ml MTT for 4 hours in
37°C. Viable cells react with MTT to produce
purple formazan crystals. After 4 hours, the
stopper 10 % SDS (Sigma Co., St.louis, MO) in
0.01 N HCl (Merck) was added to dissolve the
formazan crystal. The cells were then incubated
for 24 hours in room temperature and protected
from light. After incubation, the cells were
shaken, and cells absorbance was measured by
ELISA reader at λ 595 nm.
Data Analysis
The experimental data was absorbance of each
well, and then converted to percentage of viable
cells as described below.
Percentage of viable cells = [B-C/A-C]x100%. A,
B and C are absorbances of control group,
treatment group and medium, respectively.
The potency of cytotoxic effect is represented by
IC50 value calculated using probit analysis. IC50
value represents a concentration of the fractions
that produce cells death of 50%. Calculation of
IC50 values based on linear regression
relationship between logarithm of concentration
versus probit value of the percentage of cell
viability.
Statistical analysis
All data were expressed as mean ± SEM. One-
way analysis of variance (ANOVA) followed by
the least significant difference (LSD) test were
used for statistical analyses. P-values less than
0.05 were considered significant.
Results
Previous study, ethanolic extract of Ficus septica
Burm. F. leaves showed potent cytotoxic activity
on T47D cell lines. Subsequently, the ethanolic
extract were then fractionated gradually using n-
hexane and ethyl acetate yielding four fractions
of hexane soluble fraction, hexane insoluble
fraction, ethyl acetate soluble fraction, and ethyl
acetate insoluble fraction. The procedure to
obtain these fractions described in Fig 1. In the
study, we investigated these fractions on T47D
cell lines. The potency of cytotoxic effects of the
fractions were then compared using IC50 values.
Effect of n-hexane soluble fraction on T47D
cell viability
Figures 2-3 show the effects of a series
concentration of n-hexane soluble fraction of
Ficus septica Burm. F. leaves on the viability of
breast cancer T47D cell lines for 24 hours
incubation. All concentrations used in the
experiment could decrease the cells viability
significantly (P<0.05) in concentration-dependent
manner (Fig. 2). At highest concentration (700
µg/mL), the fraction decreased the cell viability
by 59.33±2.41 %. IC50 value of n-hexane soluble
fraction of Ficus septica Burm. F. leaves was
331.0 µg/mL (Table 1).
Nugroho et al. International Journal of Phytomedicine 3 (2011) 216-226
219
Fig. 1. Scheme of procedures for obtaining fractions of Ficus septica Burm. F ethanolic extract.
Fig. 2. Effects of hexane soluble fraction of Ficus septica Burm. F ethanolic extract on T47D cell viability. Cells were
incubated for 24 hr with various concentrations of hexane soluble fraction. Cell proliferations were examined by MTT
assay. Results are the mean ± SEM of three experiments.
Nugroho et al. International Journal of Phytomedicine 3 (2011) 216-226
220
Table 1. The IC50 values of cytotoxic effect of ethanolic extract fractions of Ficus septica Burm. F ethanolic extract on
human breast cancer T47D cell.
Treatment IC50 value (µg/mL) Inhibitory effect at highest concentration
(700 µg/mL)
n-hexane soluble fraction 331.0 59.33±2.41 %
n-hexane insoluble fraction 9.3 88.51±2.22 %
ethyl acetate soluble fraction 13.7 83.78±0.96 %
ethyl acetate insoluble fraction >700 38.68±1.55%
Fig. 3. Cells morphology after incubation with vehicle (A), hexane soluble fraction of Ficus septica Burm. F ethanolic
extract at concentration of 50 µg/mL (B), 500 µg/mL (C), and 750 µg/mL (D). Death cell was pointed by black arrow.
Effect of n-hexane insoluble fraction on T47D
cell viability
Figures 4-5 show the effects of a series
concentration of n-hexane insoluble fraction of
Ficus septica Burm. F. leaves on the viability of
breast cancer T47D cell lines for 24 hours
incubation. The n-hexane insoluble fraction
decreased the cells viability potently in
concentration-dependent manner. At
concentration of 10 µg/mL (low concentration)
decreased the the cell viability by 52.33±2.41 %.
A decrease of less than 50% at low concentration
indicates that the fraction has a potent cytotoxic
effect. Whereas at the highest concentration (700
µg/mL), the fraction decreased the cell viability
by 88.51±2.22 %. IC50 value of n-hexane soluble
fraction of Ficus septica Burm. F. leaves was 9.3
µg/mL (Table 1). It indicates that the n-hexane
insoluble fraction is more potent than this of
soluble fraction.
Therefore, n-hexane insoluble fraction was
fractionated by ethyl acetate to yield soluble
fraction and insoluble fraction. Subsequently,
both fraction were evaluated for their cytotoxic
effects. Their effects of a series concentration of
these fractions on the viability of breast cancer
T47D cell lines for 24 hours incubation were
showed in Fig. 6-9.
Nugroho et al. International Journal of Phytomedicine 3 (2011) 216-226
221
Fig. 4. Effects of hexane insoluble fraction of Ficus septica Burm. F ethanolic extract on T47D cell viability. Cells were
incubated for 24 hr with various concentrations of hexane insoluble fraction. Cell proliferations were examined by MTT
assay. Results are the mean ± SEM of three experiments.
Fig. 5. Cells morphology after incubation with vehicle (A), hexane insoluble fraction of Ficus septica Burm. F ethanolic
extract at concentration of 50 µg/mL (B), 500 µg/mL (C), and 750 µg/mL (D). Death cell was pointed by black arrow.
Nugroho et al. International Journal of Phytomedicine 3 (2011) 216-226
222
Fig. 6. Effects of ethyl acetate soluble fraction of Ficus septica Burm. F ethanolic extract on T47D cell viability. Cells
were incubated for 24 hr with various concentrations of ethyl acetate soluble fraction. Cell proliferations were examined
by MTT assay. Results are the mean ± SEM of three experiments.
Fig. 7. Cells morphology after incubation with vehicle (A), ethyl acetate soluble fraction of Ficus septica Burm. F
ethanolic extract at concentration of 50 µg/mL (B), 100 µg/mL (C), and 250 µg/mL (D). Death cell was pointed by black
arrow.
Nugroho et al. International Journal of Phytomedicine 3 (2011) 216-226
223
Fig. 8. Effects of ethyl acetate insoluble fraction of Ficus septica Burm. F ethanolic extract on T47D cell viability. Cells
were incubated for 24 hr with various concentrations of ethyl acetate insoluble fraction. Cell proliferations were
examined by MTT assay. Results are the mean ± SEM of three experiments.
Fig. 9. Cells morphology after incubation with vehicle (A), ethy acetate insoluble fraction of Ficus septica Burm. F
ethanolic extract at concentration of 10 µg/mL (B), 250 µg/mL (C), and 700 µg/mL (D). Death cell was pointed by black
arrow.
Nugroho et al. International Journal of Phytomedicine 3 (2011) 216-226
224
Effect of ethyl acetate soluble fraction on
T47D cell viability
Ethyl actate soluble fraction succeeded to
decrease the cells viability in concentration-
dependent manner. All concentrations used in the
experiment could decrease the cells viability
significantly (P<0.05). At concentration of 50
µg/mL decreased the cells viability up to 60%. A
decrease of less than 50% at low concentration
indicates that the fraction has a potent cytotoxic
effect. At higher concentration (250-700 µg/mL),
the fraction strongly depleted the cell viability up
to 80% (Fig. 6-7). IC50 value of n-hexane soluble
fraction of Ficus septica Burm. F. leaves was 13.7
µg/mL (Table 1).
Effect of ethyl acetate insoluble fraction on
T47D cell viability
Effect of ethyl acetate insoluble on T47D cell
viability was showed in Fig. 8-9. All
concentrations used in the experiment could not
decrease the cells viability up to 50%. At highest
concentration (700 µg/mL), the fraction
decreased the cell viability by 38.68±1.55%. IC50
value of n-hexane soluble fraction of Ficus
septica Burm. F. leaves was more than 700
µg/mL. It indicates that the ethyl acetate
insoluble fraction is much less potent than this of
the ethyl acetate soluble fraction.
Discussion
Attemps to discover anticancer agent from
natural product is increasing along with
increasing cases of cancer. Indonesia has the
second largest biodiversity in the world including
medicinal plants. In Indonesia, exploration of
anticancer agents from medicinal plants have
been widely studied. Kirana et al. have screened
11 main species of Zingiberaceae from Indonesia
for their antitumor effect on human HT-29 colon
cancer and MCF-7 breast cancer cells. In this
study, Curcuma longa, Kaempferia pandurata and
Zingiber aromaticum showed inhibitory activity
against both cell lines [9]. Sugiyanto et al. have
also screened many Indonesia plants for their
anticancer activity. Among the many Indonesian
plants studied, sambung nyawa (Gynura
procumbens), beluntas (Pluchea indica), murbei
(Morus alba) dan tapak doro (Vinca alba) leaves
showed anticarcinogenic activity on lung tumor
growth of mice [10]. Our research group named
Cancer Chemoprevention Research Center
(CCRC) has selected several Indonesian plants
for their anticancer activity in breast cancer. The
plants which is promising to be further
investigated were Brucea janvanica [11],
Thyphonium flagelliforme [12], Areca catechu
[13], Vigna sinensis [14], Piper aduncum [15],
Cosmos caudatus [16] and Ficus septica [7].
Previous study, ethanolic extract of Ficus septica
Burm. F. showed a potent cytotoxic effect on
breast cancer T47D cell lines with IC50 value of
13 µg/mL [6]. The extract also induced apoptosis
in MCF-7 breast cancer cells, and downregulated
Bcl-2 protein [7]. In vivo study of the extract on
dose of 750 mg/kg BW induced apoptosis
throught p53-independent pathway in 7,12-
dimethylbenz[a]nthracene-induced rat liver
cancer [8].
In present study, we investigated the active
fraction of ethanolic extract of Ficus septica
Burm. F on breast cancer T47D cell lines. The
fractionation was carried out in two stages using
n-hexane and ehtyl acetate, respectively, as
described in Fig.1. First step fractionation using
n-hexane aimed to separate polar and non-polar
compounds. The step yielded n-hexane soluble
fraction and n-hexane insoluble fraction. The
results showed that the IC50 value of n-hexane
insoluble fraction (9.3 µg/mL) was much less
than this of n-hexane soluble fraction (331
µg/mL) (Table 1). It indicates that the cytotoxic
effect of n-hexane insoluble fraction was much
more potent than this of n-hexane soluble
fraction. Based on the results, the active
compounds of ethanolic extract of Ficus septica
Burm. F might be partitioned in n-hexane
insoluble fraction. The next step, hexane
insoluble fraction was partitioned with ethyl
acetate to separate polar compounds and semi
polar yielding ethyl acetate soluble fraction and
ethyl acetate insoluble fraction, respectively. The
Nugroho et al. International Journal of Phytomedicine 3 (2011) 216-226
225
results showed that the IC50 value of ethyl acetate
soluble fraction (13.7 µg/mL) was much less than
this of ethyl acetate insoluble fraction (>700
µg/mL) (Table 1). It indicates that the cytotoxic
effect of ethyl acetate soluble fraction was much
more potent than this of ethyl acetate insoluble
fraction. From this result, the active compounds
of n-hexane insoluble fraction might be
partitioned in ethyl acetate soluble fraction, but it
need to be explored more details.
The cytotoxic activity of natural products is
related to presence of anticancer compound in
these plants including Ficus septica Burm. F. Wu
et al. found out the active compounds from Ficus
septica leaves i.e. phenanthroindolizidine
alkaloids. These compounds were ficuseptine,
(+)-tylophorine, and a mixture of (+)-tylocrebrine
and (+)-isotylocrebrine. These compounds
showed potent cytotoxic effect on two human
cancer cell lines (NUGC and HONE-1)[17].
These cell lines are gastric adenocarcinoma and
nasopharyngeal carcinoma cell lines,
respectively. Other types of alkaloids were also
found from methanolic extract of Ficus septica
leaves and evaluated for cytotoxic effect. The
compounds include ficuseptamines A and B
(aminocaprophenone alkaloids) and
ficuseptamine C (pyrrolidine alkaloid) [18]. The
alkaloids were also found in other parts of Ficus
septica Burm. F and exhibited potent cytotoxic
effect. Phenanthroindolizidine alkaloids could be
also isolated from both roots and stems of Ficus
septica. These compounds showed cytotoxic
effect on NUGC and HONE-1 cell lines [19-20].
Based on the facts, phenanthroindolizidine
alkaloids have an important role in the cytotoxic
effect of Ficus septica Burm. F. However, the
extract compounds on each fraction of Ficus
septica leaves ethanolic extract need to be
explored further.
Conclusion
Based on the result dan discussion described
above, we concluded that n-hexane insoluble
fraction and ethyl acetate soluble fraction
exhibited potent cytotoxic on breasr cancer T47D
cell lines. The fractions are potential to be
developed as anticancer agents in breast cancer
therapy.
Author’s Contribution
AEN was responsible to make a research concept
and design of the study, data collection,
acquisition of data, analysis of data, statistical of
data, drafted and corresponding author the
manuscript. MI and AH contributed to providing
the ethanolic extract fractions of Indonesia plant
Ficus septica Burm. F. DD helped to culture the
cells and collect the data. EM participated in
drafted the manuscript. All author have already
read and approved the final revision of this
manuscript.
Acknowledgements
We gratefully thank to DP2M DIKTI (Directorate
of Higher Education) Ministry of Education,
Indonesia through “Hibah Bersaing” Research
Grant 2011 for financial support in the study.
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... The chloroform fractions at pH 7 and 9 were carried out by thin-layer chromatography using silica gel GF 254 as stationary phase and chloroform: methanol: ammonia (18: 15: 1) as mobile phase. Dragendorff solution was used as a sprayer reagent to identify alkaloid compounds [22], [23]. ...
... Wheresoever (4T1 and MCF-7 cells line) were grown in DMEM medium, (T47D and HeLa) were grown in RPMI medium, Raji was grown in IMDM medium completely. The cytotoxic examination was adopted from Satria, et al., (2017) [21], [23], [24]. ...
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Aim: This study was carried out to investigate cytotoxic activity towards T47D, 4T1, MCF-7, HeLa, and Raji cells of alkaloid fractions of Zanthoxylum acanthopodium DC. fruits. Zanthoxylum acanthopodium DC. Methods: The fruit was extracted by maceration. The ethanol extract was fractionated with liquid-liquid extraction using n-hexane, chloroform at pH 3, 7, and 9 to obtained alkaloid fractions. Cytotoxic activity for fraction chloroform at pH 7 and 9 was determined with MTT assay. Results: The IC50 of fraction chloroform at pH 7 and 9 was (92.67 ± 1.37; 71.87 ± 1.04; 159.87 ± 0.63; 123.39 ± 0.81; and 103.09 ± 0.58 µg/mLfor pH 7) and (451.29 ± 25.48; 247.18 ± 2.82; 318.46 ± 5.40; 303.96 ± 8.75; and 181.45 ± 1.35 µg/mL for pH 9) respectively. Conclusion: The results reveal that alkaloid fractions at pH 7 and 9 of Zanthoxylum acanthopodium DC. Fruits have cytotoxic activity. Our further study is to isolate and assesses anticancer activity from alkaloid compounds.
... Tebakak (Ficus septica) traditionally used by the people of Lampung as a food wrapper. The alkaloids found in Ficus septica leaves have cytotoxic effects on cancer cells [15]. ...
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Plastic has become the dominant problems in recent years since it become the dominant material for food packaging. More than 600 thousand tons of plastic becoming waste in 2019. Leaf as food packaging material has been known since the dawn of civilization In Indonesia itself leaf and other plant's organic material commonly used as traditional food wrapper. Commonly known leaves such as banana leaf, waru leaf, tepus leaf, simpur leaf, syzygium leaf and also tembakak leaf are used for wrapping traditional food. Thus triggering for the use of environmentally friendly tebakak leaf plates that are easily biodegradable, easy to produce, hygienic, and have lampung cultural background. One main question can the consumer shift from plastic based disposable plate to greener leaf base plate. Can tebakak leaf plate as an eco-friendly product try to replace plastic or just another plastic alternative. And, the consumer's perspective of tebakak leaf plates as an eco-friendly product. We learn that both leaf plates (tebakak leaf plates and teak leaf plates) can be contender for plastic plates and paper plates while styrofoam plates has the lowest value. The consumer agrees that tebakak leaf plates and the teak leaf plates are green and want to use them. The main hindrance of using the leaf plates is the availability on the market and its higher price.
... Cytotoxic study was conducted using MTT assay against various human cancer cell lines T47D, MCF7, WiDr and HeLa cells. 22 Vero cells were used as normal cell model. The RPMI (for T47D, WiDr and Hela cells) and DMEM media (for MCF7, Vero cells) were used to maintain the growth of cells at 37°C, added with 10% FBS, 1% penicillin-streptomycin and 1% fungizone, supplied with 5% CO2. ...
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Endophytes have drawn the attention of researchers regarding their abilities to produce novel and bioactive compounds having medicinal values. Some studies reported that light could affect fungal secondary metabolite production. This study examined the effect of light exposure to metabolite profiles of an endophytic fungus isolated from the stem of Coleus amboinicus and its consequences on bioactivity. The endophytic fungi identified as Syncephalastrum racemosum was isolated from the stem of C. amboinicus and was cultured in Potato Dextrose Broth for two weeks under dark and light conditions. The metabolite profiles were examined using TLC analysis and gas chromatography. The conducted bioactivity testing included antibacterial, cytotoxic and DPPH antioxidant assays. In this study, two compounds namely methyl hexadecanoate and methyl (Z)-octadec-9-enoate were present in both C. amboinicus stem and S. racemosum extracts under dark condition. These substituents were diminished when the fungus was cultured and subjected to the light. Additional compounds were observed upon light exposure and this affected bioactivity. Antioxidant activity of extract exposed to light was better than that of in the dark condition (41.84 ± 0.38 vs 57.19 ± 0.88 µg/mL). A similar trend was seen in antibacterial activity, although the differences were insignificant. On the contrary, the extract from dark condition was found to be more cytotoxic against T47D cells compared to that of the one exposed to light, with IC50 value of 420.06 ± 12.98 and >500 µg/mL, respectively.
... After incubation, the cells were shaken, and absorbance was measured using ELISA reader at λ 595 nm. The data which were absorbed from each well were converted to the percentage of viable cells [19] [20], [21]. The equation to determine the viability of cells: ...
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BACKGROUND The use of medicinal plants is increasing in several decades for relief many diseases. Indonesia consists of thousands of islands with various plants and the manners of the community using plants as a treatment for every disease traditionally. AIM Cytotoxic activity of ethyl acetate fraction (EAF) of Zanthoxylum acanthopodium fruit was tested towards T47D breast cancer cells. METHODS The in vitro cytotoxic activity was performed by MTT assay, and the result was expressed as the IC50 (Inhibitory Concentration), and cell cycle inhibition was investigated by flow cytometry to assess the inhibition in every phase of cell cycle, and the role of expression cyclin D1 and p53 in cell cycle inhibition were performed by immunocytochemistry. RESULTS EAF was showed to have high activity with a value of IC50 48.94 ± 0.32 µg/mL. EAF of 25 µg/mL caused cell accumulation at G0/G1 (60.48%) and in a control cell (51.69%) and decreased expression of cyclin D1 and increased expression of p53. CONCLUSION The results obtained in this study provided scientific support for further investigation on compounds in Z. acanthopodium fruit which in the future could be used for medication.
... To improve the pharmacological profile (Febriansah et al., 2014;Nugroho et al., 2014a;Sunarwidhi et al., 2014). Interestingly in line with the growing use of medical plants, the development of antihypertensive, anti-allergy, anti cancer and anti-diabetic drugs either as a single agent or combination is increasing (Harwoko et al., 2014;Nugroho et al., 2011a;Nugroho et al., 2011b;Nugroho et al., 2013b). ...
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Background: The effects of tylophorine, a natural alkaloid found in Tylophora indica, administered as a single compound or in combination with doxorubicin on cell cycling and apoptosis were assessed in T47D breast cancer cells, selected as a model system for breast cancer. Methods: Cell cycle distribution and apoptosis were examined by flow cytometry. Caspase 3 and 9 expression was determined by immunocytochemistry.Result: We found that tylophorine did not significantly influence the cell cycle distribution of T47D cells. However, the alkaloid did prevent accumulation of cells in the G2/M phase. In addition, tylophorine increased the number of apoptotic cells. Expression of proapoptotic proteins (caspases 3 and 9) was up-regulated upon administration of tyloporine alone or in combination with doxorubicin. Conclusions: Tylophorine alone or in combination with doxorubicin induced apoptosis in T47D breast cancer cells through modulation of the cell cycle and affecting the expression of caspases 3 and 9.
... After incubation, the cells were shaken, and absorbance was measured using microplate reader at λ 595 nm. The data which were absorbed from each well were converted to percentage of viable cells [8,[10][11][12][13][14]. ...
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Objective: This study was carried out to investigate cell cycle arrest activity toward T47D cells of Litsea cubeba heartwoods and fruits extract.Methods: Dry extracts were prepared from dry-grounded heartwoods and fruits by cold maceration using n-hexane, ethylacetate, and ethanol solvent. Cytotoxic activity were measured by [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide] methods and interpreted with inhibitory concentration 50% (IC50) value. Cell cycle arrest was investigated by flowcytometry method.Results: IC50 values for each n-hexane, ethylacetate, and ethanol of L. cubeba heartwoods and fruits were 76.34±2.61; 67.52±2.45; 71.23±2.37; 75.59±3.24; 162.58±15.08; 63.70±2.67 μg/mL, respectively. Cell cycle arrest for ethylacetate extract of heartwoods and fruits were accumulated in G0/G1 phase (56.70% and 65.56%).Conclusion: These results suggest that L. cubeba heartwoods and fruits has cell cycle arrest activity.
... This step lasted for 24 h at 37 °C and was protected from light. After incubation, the well plates were shaken and the absorbance was measured for each well using ELISA reader at λ 595 nm [9]. ...
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Objective: This research aimed to fractionate the ethyl acetate extract from secondary metabolites of Streptomyces sp. GMY01 and to identify which fraction contains cytotoxic active compounds against human breast cancer MCF7 cell lines.Methods: Secondary metabolites were obtained from fermentation of Streptomyces Sp. GMY01 for 15 d. The supernatant containing these secondary metabolites was extracted through partition using ethyl acetate as the solvent. Fractionation of the ethyl acetate extract was conducted via column chromatography using silica gel as the solid phase while the gradient mobile phase consisted of n-hexane, ethyl acetate, and methanol. The cytotoxicity of each fraction was calculated using MTT-assay.Results: The ethyl acetate extract could be separated into 9 fractions using column chromatography. The cytotoxic effect of each fraction differed from each other. The smallest IC50 value was obtained from fraction 4. Further investigation should be conducted to discover the active anticancer compound. The active compound with cytotoxic effect was found in fraction 4 because of the highest IC50 value.Conclusion: This fraction is potential to be investigated more deeply as anticancer, especially for breast cancer.
... Some of them are being investigated through intensive studies. [41][42][43][44] Conclusion MCLEE and MCFEE or its combinations succeeded in decreasing SBP, DBP, and MABP of dexamethasone-induced hypertensive rats. However, it is necessary to conduct further research to determine the mechanism of action even though it has been reported that Morinda citrifolia is able to reduce hypertension through the activity of ACE inhibitor and antioxidant activity of phenolic compounds including scopoletin and rutin that could capture free radicals. ...
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The effect of ethanolic extract of Morinda citrifolia leaves and fruit on blood pressure in dexamethasone-induced hypertension rat was evaluated. Total phenolic content of Morinda citrifolia leaves ethanolic extract (MCLEE) and Morinda citrifolia leaves ethanolic extract (MCFEE) was 1.789 ± 0.116 and 1.677 ± 0.051 mg of gallic acid equivalents per gram sample, respectively. Rutin level in MCLEE was 0.92 ± 0.19%, and scopoletin level in MCFEE was 0.46 ± 0.05%. MCLEE, MCFEE, and its extract combination significantly decreased the blood pressure of hypertensive rats. The combination group showed highest hypotensive activity by lowering systolic blood pressure by 16.71 ± 3.95%, diastolic blood pressure by 21.49 ± 7.90%, and mean arterial blood pressure by 19.58% ± 6.35. All extract treatments have not been able to repair or inhibit renal damage caused by dexamethasone induction.
... [26][27][28][29] The Southeast Asia countries have the biggest biodiversity in the world including medicinal plants and intensive studies are being carried out on some medicinal plants. [30][31][32][33][34][35] ...
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Licorice (Glycyrrhiza glabra), Pulasari stem bark (Alyxia reinwardtii) and Sembung leaf (Blumea balsamifera) are traditionally used to treat gastrointestinal disorders. The aim of the study was to investigate gastroprotective effect of hot water extracts combination of those herbal against aspirin-induced gastric ulcer model in rats. The combination consisted of fixed doses of Licorice 273 mg/kg BW and Sembung leaf 457.5 mg/kg BW, and also consisted of Pulasari stem in various doses i.e. 100 mg/kg BW (first group), 200 mg/kg BW (second and sixth group) and 300 mg/kg BW (third group). The fourth grup rats received sucralfate 360 mg/kg BW. Ten minute after seven consecutive days of drug administration, the rats were induced with aspirin 450 mg/kg BW except sixth group rats. The fifth group rats only received aspirin without any protective agents. The number and area of gastric ulcers were evaluated macroscopically. Whereas, histopatological observation was used for evaluation of mucosal damage score, and the number of eosinophils and mast cells. In the study, herbal extracts combination markedly exhibited protective effects indicated by less number and smaller area of gastric ulcers in comparison to those of aspirin group (P < 0.05). The score of mucosal damages were also decreased in herbal extracts combination groups. The number of eosinophils and mast cells of herbal combination groups were observed to be smaller than those of aspirin group (P < 0.05). In conclusion, herbal combination of Licorice (Glycyrrhiza glabra), Pulasari stem bark (Alyxia reinwardtii) and Sembung leaf (Blumea balsamifera) is potential to develop as a gastroprotective agent.
... Exploration of the medicinal plants have been done for their pharmacological activities, herbal formulation, phytochemical studies etc. Since the cardiovascular diseases, cancer and diabetes mellitus are the biggest killer in Indonesia, the studies of medicinal plants for the treatment of these diseases are increasing [30][31][32][33][34] . In the study, the results of TLC profile exhibited the presence of andrographolide in EEAP due to the same spot colour and Rf between certain spot in EEAP with a single spot of standard andrographolide. ...
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Hypertension is a main cause of approximately 66% of cardiovascular disease in 15 countries in the Asia Pacific region, including Indonesia. The high cost of treatment and the side effects of drugs are causes of the lack of public awareness in dealing with hypertension. In Indonesia, Andrographis paniculata [Burm.f.] Ness grows abundantly, and is widely used as a traditional medicine such as in treatment of hypertension. This study aimed to evaluate the antihypertensive activity of the ethanolic extract of A. paniculata herbs (EEAP) in Wistar rats with a non-invasive method. In the study, the herb was extracted by 90% ethanol, and identified for its andrographolide, flavonoids and phenolics contents. Antihypertensive activity was evaluated using CODA “Non Invasive Blood Pressure (NIBP) system with Volume Pressure Recording method”. High blood pressure was induced with an alpha adrenergic receptor agonist, phenylephrine (0.9 mg/kgBW). EEAP at doses of 45, 90 and 180 mg/kgBW was evaluated for its antihypertension in comparison to nifedipine (10.8 mg/kgBW). Blood pressure measurements were performed 3 times i.e. before induced (baseline), 15 and 45 minutes after phenylephrine administration. Based on TLC-densitometric data, andrographolide, total flavonoids and total phenolics contents in EEAP were 12.85 ± 0.46%, 0.72 ± 0.01% and 1.66 ± 0.01%, respectively. EEAP exhibited a potent antihypertensive activity in phenylephrine-induced hypertensive rats. EEAP could decrease systolic and diastolic blood pressures up to 120% and 150%, respectively. EEAP fractions are potential to develop as a hypotensive agent in hypertension therapy. © 2015 International Journal of Toxicological and Pharmacological Research. All rights reserved.
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The chemopreventive effect of Ficus septica Burm. f. leaves ethanolic extract (FLEE) was studied in 7,12-dimethylbenz[a]nthracene(DMBA)-induced rat liver cancer. Rats were divided into 5 group, 5 rats (5 wk of age Sprague Dawley rat) in each group. Group 1 was control diet group, administered with 0,5% CMC-Na as vehicle. FLEE was administered 750 mg/kgBW and 1500 mg/kgBW starting 4 wk until 5 wk after DMBA administration at the first until fifth wk to group 2 and group 3. Group 4 was control extract group, administered with 750 mg/kgBW and group 5 was DMBA group. DMBA is a carcinogen to induce liver cancer was also administered in DMBA control group and all animals were necropsied at 6 wk after DMBA administration. Activity of inducing apoptosis was detected using Double Staining method in 750 mg/kgBW FLEE group compared to control group but no in 1500 mg/kgBW FLEE group resulted in 100% dead. Apoptotic cells would have orange flourescence but normal cells would have green flourescence detected by flourescence microscope. To investigate the protein that involved in apoptotic mechanism, we studied p53 expression using Imunohistochemistry (IHC). There was no difference expression of p53 in both tested and control groups. Based on the results, FLEE has a potency as chemoprentive agent because its activity on inducing apoptosis in liver cancer with p53-independent pathway. The mechanism of apoptosis induction of this extract needs to be explored by observing the expression of related proteins. Keywords: apoptosis, Ficus septica, liver cancer, p53 independent pathway
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Chalcones and their derivates have been shown to have potency as anticancer. Chalcone is a flavonoid compound that is found in temu kunci (Kaempferia pandurata) and sirihan (Piper aduncum L.). This study aim is to know the cytotoxic effect of the ethanolic extract of K. pandurata rhizome and P. aduncum L. leaves towards breast cancer cell line T47D. Measurement of ethanolic extract of flavonoid was done by using cellulose for TLC and n-buthanol: acetic acid: water (4:1:5) for the mobile phase and compound cluster detection using citroboric acid reagent and ammonia. Colorimetric test 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay is used to monitor the cytotoxicity effect. The screening for the compound, found in the plants was done using molecular docking. K. pandurata rhizome and P. aduncum L. leaves ethanolic extract shows a cytotoxic effect toward breast cancer cell with it’s IC50 value as much as 66 μM and 68 μM. The cytotoxic effect is then connected to it’s result of molecular docking using ArgusLab among it’s chalcone compound in plants toward tubulin activity and estrogen receptor α. Furthermore, these docking processes were obtained the lowest scoring value in panduratin A both in tubulin and estrogen receptor α. This results were then compared with other experimental ligands, and showed that the compound interaction was stronger than the colchicine and raloxifen. This shows the probability of chalcone having more of an effect toward tubulin and estrogen receptor α in inducing cytotoxicity.
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The cytotoxic effect of secondary metabolites extract of endophytic fungi (1.3.11, 1.1.6,.and 1.2.6) isolated from the fruit of "Tanaman Buah Makassar "on in-vitro T47D and MC-7 intact cells and identification of the fungus 1.3.11 by ITS regions of ribosomal DNA sequence were carried out. The cytotoxic effect of these extract (IC50) was assessed on T47D and MCF-7 cell (concentration 5-200 μg/ml) over 24 and 478 hr in CO2 using MTT methods on ELIZA reader at 595 nm. The results shows IC50 of fungi 1.3.11 and 1.2.6 were 7 and 50 μg/mL respectively. Extracts of fungi 1.1.6, however, showed weak or no cytotoxic effect toward T47D. Incubation of MCF-7 cells with extract of fungi 1.3.11, it showed an IC50 of 31 μg/mL. Furthermore, both T47D and MCF-7 cells showed pronounced morphological changes after 48 hour incubation with extracts of fungi 1.3.11 at 0.5 and 5 μg/mL. Moreover, T47D, but not MCF-7 cells showed morphological changes when incubated with extract of (i) fungi 1.2.6 (40 μg/mL) and (ii) fungi 1.1.6 (232 μg/mL). The results of identification of the fungus based on ribosomal DNA sequence show that it has highest DNA sequence similarity with Botryosphaeria parva.
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The incidence of breast cancer in developing countries showed an increase from year to year. The efforts of cancer prevention or treatment of the more important given the frequency of occurrence is quite high. Several studies have been focuses on natural products as agents of cancer chemoprevention and as co-chemotherapy agent against cancer. One of medicinal plant that is widely used as anticancer is rodent tuber (Thyphonium flagelliforme (Lodd.) Blume). In present study, we investigated the anticancer acitivty of rodent tuber extract (RTE) in vitro. Study of anti-proliferative conducted on human breast cancer primary T47D cells. Furthermore, the present study also investigated the molecular mechanisms of cell cycle arrest and induction of apoptosis. The study of rodent tuber as co-chemotherapy is determined by examining the effects on T47D and its combination with tamoxifen (TAM). The results showed the cytotoxic effects of RTE on T47D cells with IC50 value of 632 μg/mL. Low concentration RTE (below 250 ug/mL) are proliferative, while the concentration above 250 μg/mL indicated cytotoxic effect. Based on the calculation of Combination Index (CI), combination of RTE with TAM yielded a value above 10 indicated a strong antagonistic effect. In observation of apoptosis, low concentration of RTE (63 μg/mL) stimulate apoptosis better than high concentration of 250 μg/mL. However, the combination study with 5 nM TAM reduced the induction of apoptosis. Furthermore, the observation cell cycle arrest by flowcytometry, demonstrated that RTE 63 and 250 μg/mL increase the population of sub G1 phase respectively from 14.8% to 53.19 and 32.90%. These results suggest that RTE able to induce cell cycle to apoptosis, but low concentration of RTE more effective than high concentration. Similar to apoptosis observation, the combination of RTE and TAM also demonstrated the antagonistic effect by reducing the population of Sub-G1 RTE (63 μg/mL) and TAM 5 nM, respectively from 53.19% and 44.50% to 35.86%. All finding results of this study provide information that the use of rodent tuber extract (RTE) alone is better than the combination with TAM. In addition, the use of RTE together with TAM reduced the effectiveness of TAM in the treatment of breast cancer.
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We have screened 11 important species of Zingiberaceae, used as spices and for medicinal purposes in Indonesia, for their antitumor activity using human HT 29 colon cancer and MCF-7 breast cancer cells. They were Amommum cardamomum, Curcuma aeruginosa, C. longa, C. mangga, C. xanthorrhiza, Kaempferia galanga, K pandurata, K rotunda, Z. aromaticum, Z. cassumunar, and Zingiber officinale. Ethanol extracts of eight species showed strong inhibitory effect on the growth of the cancer cells when evaluated using the colorimetric tetrazolium salt assay. Since curcumin, a yellow pigment isolated from C. longa, has shown its potential anticancer activity in vitro and in vivo studies and is currently undergone clinical trial in the US, we used an extract of C. longa as a comparison. Extracts of K. pandurata and Z aromaticum had very strong inhibitory activity against the two cell lines similar to those of C. longa. However, curcumin was not detectable in the extracts of those two plants. The ethanol extracts of the active species had less effect on the growth of a non-transformed human skin fibroblast cell line (SF 3169). Microscopic examination of cancer cells exposed to extracts of active species showed a characteristic morphology of apoptosis. Further study on Z aromaticum and K. pandurata, including identification of bioactive compounds and elucidation of mechanism(s) likely to be operating, has been carried out.
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Phenanthroindolizidine N-Oxide, ficuseptine-A (1), together with eighteen known compounds was isolated from the leaves of Ficus septica. The structures of these compounds were elucidated by spectroscopic analysis. Among them, phenanthroindolizidines, ficuseptine (1), (+)-tylophorine (4) and a mixture of (+)-tylocrebrine (5) and (+)-isotylocrebrine (6), exhibited strong cytotoxic activity against two human cancer cell lines, NUGC and HONE-1.