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www.wjpps.com Vol 9, Issue 5, 2020. 974 Qaromah et al. World Journal of Pharmacy and Pharmaceutical Sciences CYTOTOXICITY OF RAMIE (Boehmeria nivea L. Gaud.) LEAVES EXTRACT ON MCF-7 BREAST CANCER CELL LINE Annisa Abdiwijaya Qaromah 1 *, Asri Peni Wulandari 1 , Desi Harneti 2 1 Department of Biology, Faculty of Mathematics and Natural Sciences, Padjadjaran University. 2 Department of Chemistry, Faculty of Mathematics and Natural Sciences Padjadjaran University. Jl. Raya Bandung-Sumedang KM. 21, West Java, Indonesia. 45363. ABSTRACT The objective of this study was to investigate the cytotoxicity effect of the crude extract and fractions of Ramie (Boehmeria nivea L. Gaud.) leaves extract on in vitro MCF-7 breast cancer cell line. In vitro cytotoxicity were evaluated by MTT-based assays. The percentage of cell inhibition was analyzed using probit analysis to obtain 50% inhibitory concentration (IC 50 ). Morphological alteration of the cell line after exposure with extract were observed under inverted microscope. The cytotoxicity assay revealed that the ethanol extract and the methylene chloride fraction from the ramie leaves exhibited marked anticancer activity and has strong antiproliferative with IC 50 of 3.27 μg/mL and 3.79 μg/mL, respectively, on MCF-7 cancer cell line. Morphological alteration of the cell line after exposure with Boehmeria nivea L. Gaud. extract were observed under inverted microscope in the dose dependent manner. The crude extract and fractions of the Ramie leaves exhibit cytotoxic activity against MCF-7 cell line and showed morphological changes when incubated with the extract. Boehmeria nivea L. Gaud. could be considered as a potential source of the anticancer agent. However, further research to determine the exact mechanism of action needs to be carried out. KEYWORDS: Cytotoxicity, Boehmeria nivea L. Gaud., fractions, MTT assay. WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES SJIF Impact Factor 7.632 Volume 9, Issue 5, 974-987 Research Article ISSN 2278 – 4357 *Corresponding Author Annisa Abdiwijaya Qaromah Department of Biology, Faculty of Mathematics and Natural Sciences, Padjadjaran University, Jl. Raya Bandung-Sumedang KM. 21, West Java, Indonesia. 45363. Article Received on 04 March. 2020, Revised on 25 March 2020, Accepted on 15 April 2020 DOI: 10.20959/wjpps20205-15979

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Markers for Cancer Stem Cells in Endometriosis974
Qaromah et al. World Journal of Pharmacy and Pharmaceutical Sciences
CYTOTOXICITY OF RAMIE (Boehmeria nivea L. Gaud.) LEAVES
EXTRACT ON MCF-7 BREAST CANCER CELL LINE
Annisa Abdiwijaya Qaromah 1 *, Asri Peni Wulandari
1 , Desi Harneti
1 Department of Biology, Faculty of Mathematics and Natural Sciences, Padjadjaran
University.
2 Department of Chemistry, Faculty of Mathematics and Natural Sciences Padjadjaran
University.
ABSTRACT
The objective of this study was to investigate the cytotoxicity effect of
the crude extract and fractions of Ramie (Boehmeria nivea L. Gaud.)
leaves extract on in vitro MCF-7 breast cancer cell line. In vitro
cytotoxicity were evaluated by MTT-based assays. The percentage of
cell inhibition was analyzed using probit analysis to obtain 50%
inhibitory concentration (IC50). Morphological alteration of the cell
line after exposure with extract were observed under inverted
microscope. The cytotoxicity assay revealed that the ethanol extract
and the methylene chloride fraction from the ramie leaves exhibited
marked anticancer activity and has strong antiproliferative with IC50 of
3.27 μg/mL and 3.79 μg/mL, respectively, on MCF-7 cancer cell line.
Morphological alteration of the cell line after exposure with Boehmeria
nivea L. Gaud. extract were observed under inverted microscope in the
dose dependent manner. The crude extract and fractions of the Ramie leaves exhibit cytotoxic
activity against MCF-7 cell line and showed morphological changes when incubated with
the extract. Boehmeria nivea L. Gaud. could be considered as a potential source of the
anticancer agent. However, further research to determine the exact mechanism of action
needs to be carried out.
KEYWORDS: Cytotoxicity, Boehmeria nivea L. Gaud., fractions, MTT assay.
WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
SJIF Impact Factor 7.632
Volume 9, Issue 5, 974-987 Research Article ISSN 2278 – 4357
*Corresponding Author
Annisa Abdiwijaya
DOI: 10.20959/wjpps20205-15979
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Qaromah et al. World Journal of Pharmacy and Pharmaceutical Sciences
INTRODUCTION
Cancer is the leading cause of death worldwide, accounting for around 9.6 million deaths in
2018. The most common cancer is breast cancer with 2.09 million cases. Cancer arises from
the transformation of normal cells into malignant tumor cells. [1]
One type of cancer is breast
cancer that occurs due to disruption of the system of cell growth in breast tissue. [2]
This type
of cancer most commonly suffered by women worldwide. A cancer treatment method widely
used today are surgery, radiation, hormone therapy, chemotherapy, and targeted protein
therapies aimed to remove the cancerous tissue or make cancer cell death, but the method
induces side effects on normal cells located around the cancer cells and other organs. [3]
Until
now, safer cancer treatment still has needed to be developed.
One effort is the cancer treatment by utilizing the compounds contained in natural materials.
The purpose of screening medicinal plants is to look for an excellent anticancer agent. One of
the plants that can be developed as an anticancer is ramie (Boehmeria nivea L. Gaud.). Ramie
is known as a high-quality fiber producing plant. Development of ramie plants as a producer
of fiber for raw materials in the textile industry, leaving more than 40% of leaf biomass,
which until now has not been optimally utilized. [4]
Some secondary metabolites that have been isolated and reported from the genus Boehmeria
have anticancer activity, namely from steroids, terpenoids, flavonoids, alkaloids,
triterpenoids, and polyphenols. From the steroid group there are β-sitosterol which can
prevent breast cancer and inhibit the growth of several types of specific tumor cells carried
out in vitro, also can reduce the size and level of tumor metastases carried out in vivo. [5]
The
terpenoid group is loliolide, this compound has various biological properties, one of which is
as an anticancer. [6]
Oyarzún et al. (1987) reported that there are two triterpenoid compounds
in Boehmeria excelsa namely boehmerone and boehmerol compounds, but both of them are
not yet known for their biological activity.. [7]
Several studies have shown the content of active compounds of the alkaloid group in the
genus Boehmeria, namely boehmeriasin - A which have cytotoxic activity against 12 cancer
cell lines including lung, colon, breast cancer types, prostate, kidney and leukemia. The
results of which can show that the active compound has a higher level of anticancer activity
compared to standard anticancer drugs. [8,9]
While from the flavonoid group there are
flavonoid compounds, routine, quercetin, and catechin.
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Flavonoid compounds promising anticancer agents. Data from laboratory studies, oncology
investigations and clinical trials in humans indicate that flavonoids have important effects on
cancer chemoprevention and chemotherapy. Many mechanisms of action have been
identified, including the inactivation of carcinogens, anti-proliferation, arrested the cell cycle,
induction of apoptosis and differentiation, inhibition of angiogenesis, antioxidants and a
variety of drug resistance reversal or a combination of these mechanisms. [10]
Similarly,
catechins are powerful antioxidants that are thought to provide several health benefits
including cancer chemoprevention. [11]
Cytotoxicity assay needs to be done to determine the initial selection of compounds that
could potentially kill cancer cells by the parameter Inhibitory Concentration (IC50) value.
This method determines the effectiveness of a cytotoxic agent on cell proliferation. MTT-
based cytotoxicity assay or MTT assay can test the ability of a cell that has received exposure
to an agent from surviving in vitro by colorimetry. [12]
The MTT assay can be used reliably to
measure metabolic activity of cell cultures in vitro for the assessment of growth
characteristics, IC50 values and cell survival. [13]
MCF-7 breast cancer cell line, that is
common in vitro study model, is stable and able to mimic most of the characteristics of
invasive human breast cancer. [14]
Based on these descriptions, it is necessary to study to test
new anticancer agents from ramie leaf extract against the proliferation of MCF-7 breast
cancer cell line.
NCI (National Cancer Institute) categorize the crude extract has the strong potential
anticancer if IC50 values of less than 30 µg/mL and has a moderate potential anticancer active
if IC50 values were in the range 30-100 μg/mL. [3]
Concentration is based on the increase in
the log should be made to the initial selection of anticancer agents that concentrations of 1-
1000 μg/mL. The cytotoxic activity of the agent characterized by decreased cell proliferation
along with the addition of concentration. The smaller the IC50 value, the higher the cytotoxic
activity. [12]
difeniltetrazolium bromida (MTT) [Promega] (Madison, USA).
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Plant Materials
The leaves of B. nivea L. Gaud. used was ramie leaves Lembang clones were collected from
ramie plantations in Wonosobo, Central Java Province of Indonesia. Ramie leaves samples
was dried at room temperature (22-25 o C).
The dried leaves (400 g) were maserated with 96% ethanol (EtOH) at room temperature in
macerator during 3x24 hours. The whole extract was filtered and the solvent was evaporated
under reduced pressure at 40-45C, to afford crude ethanol extract (10 g). Ethanol extract (10
g) was then resolved in methanol:water (1:9) and partitioned successively between n-hexane,
methylene chloride (CH2Cl2), ethylacetate (EtOAc), n-butanol (n-BuOH), and finally water
(H2O) based on increasing polarity of the solvent. n-Hexane, CH2Cl2, EtOAc, n-BuOH, and
H2O fractions were evaporated under vacuum to yield the residues of 17.60, 3.75, 0.67, 2.52,
and 19.43 g fraction respectively. Extracts were stored at 4C until analysis. A partitioning
scheme of B. nivea L. Gaud. Ethanol extract is presented in Figure 1. [15]
All of the isolated fractions were dissolved in dimethylsulfoxide (DMSO) and then were
subjected to cytotoxic assay.
Cell Culture and Treatment
MCF-7 human cancer cell line was used to asses anticancer activity provided by Cell and
Tissue Culture Laboratory, Teaching Hospital Universitas Padjadjaran. Human cancer cell
lines were maintained as monolayer cultures in RPMI-1640 medium, supplemented with 10%
Fetal Bovine Serum and 1% of antibiotic solution under an atmosphere of 5% CO 2 at 37
o C.
Cell were trypsinized confluently. Stock solution of crude ethanol extracts and fractions of B.
nivea L. Gaud. were prepared in 0, 0.1, 1, 10, 100, and 1000 µg/mL. The cells were incubated
and dissolved in RPMI RPMI-1640 medium just before use.
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Fig 1: Partitioning scheme using immiscible solvents.
MTT-based reduction assay
To evaluate effects of the crude ethanol extract and fractions of B. nivea L. Gaud. on cell
viability/proliferation, cells were plated in 96-multiwell culture plates at a density of 0.8 x 10 4
– 1 x 10 4 cells/well. Twenty-four hours after plating, the medium was discarded and fresh
medium containing the extracts at different concentrations (0.1, 1, 10, 100, and 1000 µg/ml)
and control (0 µg/ml) was added.
The ethanol extract and fractions of B. nivea L. Gaud. was tested for in vitro cytotoxicity,
using MCF-7 cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay. After 48 hours incubations with extracts, MTT was added at a final concentration of
0,5 mg/ml and incubated for 2 hours. Then, the medium was removed. Cell suspension
absorbance values read at a wavelength of 550 nm with ELISA plate reader.
To evaluate the effect on cell proliferation, the absorbance at the beginning of incubation was
subtracted from all the experimental condition used, including the control. The results are
expressed as the percentage of cell viability/proliferation relative to control (untreated cells)
carried out in triplicated. Percentage of cell viability (CV) was calculated using the formula:
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Positive values (between 0 and 100%) can be interpreted as inhibition of cell proliferation. A
dose-response curve was plotted to enable the calculation of IC50. The IC50 values were
determined using Probit Analysis. IC50 corresponds to the concentration of the extract that
decrease the number of viable cells by 50%. In this case, the absorbance in the control
corresponds to 100% viability.
Morphological observation of cells treated with crude ethanol and fractions B.nivea L. Gaud.
extract from cytotoxicity study was done to determine the changes induced by the extracts
using inverted microscope with a magnification of 200x. Morphological alteration such as
cell shrinkage, membrane blabbing, rounded ad detached cells were observed for
confirmation effect of cell death.
RESULT AND DISCUSSION
Proliferative effects of MCF-7 cell line
In vitro cytotoxic activity of ethanol extracts and fractions of Boehmeria nivea L. Gaud. on
MCF-7 cell line was evaluated through MTT-assay. The multiple concentrations of the
extracts were used, and the IC50 (concentration of the extract that decrease the number of
viable cells by 50%) for each cell line was calculated from the dose-response curves (Fig.2).
Figure 2 showed the percentage of MCF-7 cell viability after ethanol extracts and fractions
treatment. The viability of MCF-7 cell line was shown in dose dependent manner. The higher
concentration resulted the less viability of the cells. The IC50 value of Boehmeria nivea L.
Gaud. ethanol extracts and fractions were performed in Table 1.
Fig 2: The cytotoxicity of B. nivea L. Gaud. exract and fractions against MCF-7 cell line.
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Based on the Table 1, it’s revealed that the crude extract of ethanol and methylene chloride
(CH2Cl2) fraction treatment resulted the lowest IC50 indicated the high cytotoxicity against
MCF-7 cell line. Results showed that the extracts could significantly inhibit the viability of
the cancer cells and have potential anticancer activity.
Table 1: Cytotoxic activity (IC50) of B. nivea L. Gaud. extract and fractions against
MCF-7 cell line.
Extract/Fraction IC50 (µg/mL)
n-Hexane 124.45 ± 0.043
CH2Cl2 3.79 ± 0.037
EtOAc 156.31 ± 0.033
n-BuOH 431.52 ± 0.068
H2O 731.14 ± 0.025
Result represent IC50 ± standar deviation (n=3)
Some Boehmeria genera have been reported to produce various compounds that have
cytotoxic activity also play a role in the induction mechanism of apoptosis as an anticancer
agent; including β-sitosterol compounds showing inhibition of T47D and MCF-7 breast
cancer cell line growth with IC50 values of 0.55 and 0.87 mM, respectively. [16]
Boehmeriacin-
A cytotoxic activity against 12 cancer cell lines, with GI50 values against MCF-7 at 5
ng/mL. [8]
Boehmeriacin-A, which is isolated from Boehmeria siamensis Craib is also
considered an inhibitor of MDA-MB-231 proliferation through the capture of the G1 phase
cell cycle. Boehmeriacin-A results that potentially inhibit breast cancer cell proliferation can
be considered as candidates for chemotherapy and/or chemopreventive agents for breast
cancer. [17]
In vitro experiments showed that quercetin significantly inhibited the growth of
cancer cell cultures with IC50 values ranging from 7 nM to more than 100 µM. [18]
This
compound can also increase the effectiveness of chemotherapy agents. [19]
The IC50 value category that determines the toxicity of an extract according to NCI is divided
into categories, namely strong anticancer potential if the IC50 value is less than 30 μg/mL and
has moderate active anticancer potential if the IC50 value is in the range of 30-100 μg/mL. [4]
Meanwhile, IC50 value categories according to Alley (1988) which determine the toxicity
properties for a compound are divided into categories, namely very active (IC50 < 2 μg/mL),
active (IC50 2-5 μg/mL), and inactive (IC50 > 5 μg/mL). [20]
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IC50 values obtained in this study for crude extracts and CH2Cl2 fraction are included in the
category of active and strong anticancer. As for the other fractions, there is less potential as
an anticancer. The CH2Cl2 fraction was found to be more effective than other fractions from
ramie extract. The CH2Cl2 fraction showed the most potential inhibitory effect on
proliferation of MCF-7 cell line with IC50 values after 48 hours of exposure was 3.79 ± 0.037
μg/mL.
This cytotoxicity effect is also confirmed through the morphological changes of MCF-7 cells
after treatment with ethanol extract and fractions of ramie leaves B. nivea L. Gaud. The
reduction in the number of cells was found to suppress the cell proliferation and indicated
with having damaged its cell structure. Confirmation by observing the morphological changes
of normal cells and their effects after exposure with extracts containing anticancer (Fig. 3).
The living MCF-7 cell line normally observed in epithelial and polygonal shape. Treatment
of the cell line with the ethanol extract and fractions of ramie leaves differ the appearance of
the normal cells found to be irregular, aggregate, spherical in shape indicating damaged cells
and spreading patterns were constrained. It is suspected that the content of the extract is given
causes the release of the bond between the cells and the cells bond with the substrate. The
in vitro anticancer activity of the extracts in MCF-7 cells was mainly due to the induction of
cell death. The characteristic is common to several chemotherapeutic drugs, which reveal an
anticancer activity mainly due to their ability to induce DNA damage; if such DNA damage
is not properly repaired, its accumulation ultimately ensues in cell death. [21]
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Fig 3: Morphological changes of MCF-7 cells after treatment with EtOH extract, n-
Hexane, CH2Cl2, EtOAc, n-BuOH, and H2O fractions of B. nivea L. Gaud. (0, 0.1, 1, 10,
100, and 1000 µg/mL) after 48 hours.
In our study ethanol extract and fractions obtained from B. nivea L. Gaud. has a cytotoxic
activity on MCF-7 cells. Active fraction search is carried out as an initial stage of screening
EtOH
Control (0 μg/mL) 0,1 μg/mL 1 μg/mL 10 μg/mL 100 μg/mL 1000 μg/mL
n-Hexane
CH2Cl2
EtOAc
n-BuOH
H2O
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of compounds that act as anticancer. In order to gain insight into the nature of the active
principles responsible for the cytotoxic activity, the ethanol extract was fractionated using
solvents of increasing polarity. The entire extract of plant is generally a complicated mixture
of several compounds that possess variable chemophysical properties. The major plan for
extrication of these compounds is based on their chemophysical properties that can be
exploited to primarily separate them into various chemical groups. [22]
From the literature search of the related genera, it is possible to predict the cytotoxic
compounds that might be present in B. nivea L. Gaud. extract. Extraction with solvents of
increasing polarity helps to predict specific classes of compounds. [23]
Research on cytotoxicity test of ramie leaves B. nivea L. Gaud. ethanol extract and fractions
against MCF-7 breast cancer cell line has not been reported. Table 2 shows the information
on the results of the cytotoxicity test of some herbal plant extracts on the proliferation of
MCF-7 cell line with the MTT test, supplemented by the results of this study as a
comparison.
Based on Table 2 it can be seen that the ethanol extract of ramie leaves and CH2Cl2 fraction
as the active fraction has the smallest IC50 value compared to other herbal plant extracts. This
shows that the ethanol extract of ramie leaves and CH2Cl2 fraction has enormous potential as
an anticancer agent that has strong cytotoxic properties against the proliferation of MCF-7
breast cancer cell line. An active constituent of intermediate polarity is thus likely to be
responsible for the observed cytotoxicity, and future bioassay guided fractionation needs to
focus on the CH2Cl2 fraction. These results suggest that CH2Cl2 fraction obtained from B.
nivea L. Gaud. could be used as a potential apoptosis inducing agent, and that the CH2Cl2
fraction obtained from B. nivea L. Gaud. consist of a key component for cytotoxic activity.
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Table 2: IC50 Values of Herbal Plant Extracts against MCF-7 Cell Line.
Species
Refrence EtOh MeOH
0.55 - - -
[24]
Cyperus longus - 64,64 ± 1,64 - 25,34 ±
2,01 35,2 ± 2,69 - -
1,10 - - -
[27]
Piper
retrofractum
Vahl
1,25 - - -
Piper
sarmentosum
Roxb.
1,61 - - -
Piper
porphyrophyllum
N.E.Br.
141,35 ±
[29]
Moringa oleifera
[33]
CONCLUSION
The crude extract and CH2Cl2 fraction of Boehmeria nivea L. Gaud. showed potent cytotoxic
activity which demonstrates a high potential anticancer activity in breast adenocarcinoma
(MCF-7). Further, isolation and characterization of compounds responsible for the cytotoxic
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activity and their evaluation as anticancer agents to focus on the CH2Cl2 fraction against
human breast cancer is necessary.
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