Bioactive Content and Anticancer Bioactivity of MCF-7 Cell in Smadan Root Extract from Bitung
City Forest North Sulawesi
Amanda Gratia
Karundeng1*, Revolson Mege2 and Mokosuli Yermia Semuel2
1Magister Biology Program, faculty of mathematics, natural and earth
sciences, Manado State University, Tondano, Indonesia
2Department of Biology, faculty of mathematics, natural and earth
sciences, Manado State University. Tondano, Indonesia
*For correspondence: amandakarundeng3@gmail.com
Received 18 November 2023; Accepted 16 February 2024; Published 18 March 2024
Abstract
Cancer is a disease with the
second largest death rate in the world. Cancer Prevalence has continued to
increase in the last decades. The research was conducted from May to August
2023. The aim of this research was to determine the bioactive content and
anticancer bioactivity of michigan cancer foundation-7 (MCF-7) in Smadan root
extract from the Bitung city forest, North Sulawesi. The bioactive content was
analyzed using the high performance liquid chromatography (HPLC) method, total
flavonoids were determined using ultraviolet and visible light
spectrophotometry, while the anticancer activity was tested in vitro on MCF-7 cell cancer (breast cancer cells). The results
of the high performance liquid chromatography analysis research showed that 26
compounds were at a wavelength of 254 nanometer, while 37 compounds were at a
wavelength of 310 nanometer. The total flavonoid content of dry Smadan root extract
was 62.66 mg QE/g, while the wet extract was 14 mg QE/g and the cytotoxic activity of Smadan root extract
had IC50 = 50.12 µg/mL. These results indicate that Smadan root
extract has strong cytotoxicity. Thus, the ethanol extract of Smadan roots has
the potential to be developed as a source bioactives and drug of anticancer
that can help people to cure cancer. © 2024 Friends Science Publishers
Keywords: Anticancer;
Bioactive content; MCF-7; Smadan root
Introduction
Indonesia is a megabiodiversity
country. There are various endemic species of flora and fauna. Indonesia is
also known as a country that has an ethnomedical and ethnobotanical culture
(Kaunang and Mokosuli 2017; Rahmawaty et al. 2019). The use of plants
and animals as bioactive sources for the treatment of various types of diseases
is carried out by many tribes in Indonesia and is passed down from generation
to generation (Mokosuli et al. 2019). Indonesia has an abundance of
medicinal plants (Ansori et al. 2021).
Medicinal plants originating from forests have a very important role globally as well for society (Kaunang and Mokosuli 2017; Rahmawaty et al.
2019). Medicinal plants can lead to the discovery of new drugs
that have the potential to treat diseases that are considered uncurable (Ansori et al.
2021; Roy et al. 2022).
Cancer is a
chronic disease with the second highest prevalence of death in the world
(Rakhmanovna 2022). World Health Organization (WHO) states that cancer is one
of the main cause of death throughout the world. Around 8.2 million people die
from cancer (Joya et al. 2020). Based on the number of cases and deaths
due to cancer until 2018, 18.1 million cases and 9.6 million deaths in 2018 are
data from the Global Burden of Cancer released by WHO (Pangribowo 2019). Breast
cancer is the most common cancer in women (Rabiee et al. 2023). The
incidence rate is still increasing in Asia (Choi et al. 2023). Breast
cancer is a serious disease faced by the world and Indonesia (Jumaryatno et
al. 2022; Shaluhiyah and Surjoputro 2023). Breast cancer is one of the
types of cancer that has the highest prevalence in the world (Coelho et al.
2023; Hasnita and Meiriza 2023; Kurniawati
2023). Based on data from the World Health Organization in 2020, 2.1 million
women were diagnosed with breast cancer and this cancer cannot be
underestimated (Qodria and Nurrachma 2020).
Cancer
treatment also currently has major obstacles because sideeffects have a big
impact on cancer patients, so that
treatment based on ethnomedical culture is one of the treatments preferred by cancer patients, specifically medicinal plants that have the potential to be
anticancer or cytotoxic to cancer cells. In developing countries, plants from
nature are used as natural sources to maintain public health. Traditional
medicine is now used as alternative medicine because it is considered safer and
has fewer side effects (Wahid and Raudah 2022). The bioactive content in plants
has antioxidant properties that can prevent cancer. Bioactive compounds can
prevent cells from mutating into cancer cells (Nurtiana and Budijanto 2017).
Available therapies for cancer treatment have major hurdles (Jagdale et al.
2023).
Fig. 1: Smadan root
sampling location in Apela Dua urban village, Ranowulu district, Bitung city,
North Sulawesi
Fig. 2: Roots, stem and
leaves of Smadan from Apela Dua
sub-district, Ranowulu sub-district, Bitung city, North Sulawesi (Personal Documentation)
Currently the
treatment that cancer patients can undergo is chemotherapy, radiology,
immunotherapy and in general surgery, however, this treatment can have side
effects on cancer patients, namely hair loss, neurological disorders, bone
marrow suppression and so on (Zein and Hazar 2022), so there is a need for more
treatment strategies and drugs for the survival and quality of life of cancer
patients (Malacrida et al. 2023; Li et al. 2023). For this
reason, the use of natural ingredients will be beneficial (Roy
et al. 2022), namely plants that have the potential to kill/suppress cancer cells.
Bitung City has medicinal plants
that the community uses as traditional medicine, namely those known as Smadan
roots. When the Smadan root is cut first, it will produce clear water. It is
used by the agraris community of Bitung City as a substitute for drinking water
and is believed to have the property of increasing the body's immune system.
Smadan root decoction water is dark red. Local people generally consume water
boiled from Smadan roots to keep the body healthy and to cure various diseases.
Smadan roots originating from the city of Bitung have been used by the
community as an anticancer and antitumor medicine, but have never been studied
scientifically.
Smadan roots
have some similarities with Bajakah in Kalimantan or in Latin Spatholobus
littoralis Hassk (Sianipar et al. 2023) but are different.
Based on the shape of the leaves and stems, are very different from Bajakah,
but the shape of the roots has several similarities. It is found widely in the forests of Bitung city, and also
in several places in the North Sulawesi region. However, with the acceleration
of development and population growth, many forest areas that was home of Smadan's roots have changed due to plantations and/or housing areas, so their
sustainability needs to be maintained. It is hoped that the research carried
out can have a positive impact on the community in protecting forests and not over-exploiting medicinal plants. Therefore,
the aim of this study was to determine the bioactive content and anticancer
activity of MCF-7 in smadan root extract from the Bitung city forest, North
Sulawesi.
Materials
and Methods
Sample
Smadan root samples were taken
in the forest of Apela Dua urban village, Ranowulu sub-district, Bitung city,
North Sulawesi (Fig. 1).
Characteristics of Smadan roots: Smadan roots have brown and rough outer
root skin, light green stems that grow from creeping roots. It has small leaves;
approximately 1 cm. Smadan roots grow in damp forests and live on trees with
their central roots embedded in the ground. Smadan roots can grow very long, up
to approximately 50 m (Fig. 2).
Research
procedure
This research was carried out in
several stages: Making simplicia and extraction, testing the bioactive content
using the HPLC method, testing the total flavonoid content using UV-Vis
Spectrophotometry and testing the anticancer cytotoxic activity of MCF-7. This
research was conducted from May to August 2023. Extraction and testing of total
flavonoid content of Smadan root was carried out at the Biology Laboratory,
Manado State University and the cytotoxic test on MCF-7 cancer cells as well as
the bioactive content test using the HPLC method was carried out at the Central
Laboratory of Padjadjaran University.
Making simplicia
and extraction
Smadan root samples (plants aged
± 30 years) were taken by cutting the hanging roots (not the core roots) as
samples. Smadan roots were taken from tropical forests in summer with
environmental conditions temperature, 26–27°C, humidity 83%, and wind 3,5 km/h (BMKG 2023). Then the Smadan root samples were cleaned
first, then finely cut into dry samples and wet samples. The dry samples are
oven-treated for 60 min. The wet samples are not oven dried. Both samples were blended separately until smooth.
When the samples were smooth, the two samples were weighed at 100 g each and
put into different jars to enter the maceration stage and 400 mL of 95% ethanol
was added to both. Ratio 1:4 (Semuel et al. 2019) was used to macerate for 3 days. Samples that had been macerated
for 3 days were filtered using filter paper. After that, it goes to the evaporation
stage (changing the solvent into steam) to get a thick extract from Smadan root
extract.
Bioactive content
testing HPLC method
The use of the HPLC (High
Performance Liquid Chromatography) method on Smadan root extract (dry samples)
which was tested at the Central Laboratory of Padjadjaran University to analyze
its bioactive content. The working principle of HPLC is to separate analyte
components based on their cappolarity and any mixture that comes out will be
detected with an existing detector and recorded in the form of a chromatogram.
The steps involved weighing 100 mL of the
Smadan root ethanol extract sample, then dissolving it with 2 mL of and 8 mL of
methanol, then sonicating it for 10 min. After that, filtered with a 0.45 μm millipore and put 1 mL into HPLC
vial (Semuel et al. 2019; Farida et al. 2020).
Flavonoid content
testing using UV-Vis spectrophotometry
Determination of the maximum wavelength of quarcetin: To determine
the maximum wavelength of kearcetin, the quercetin solution was analyzed in the
wavelength range of 400–500 nm
(Rogahang et al. 2023). The analysis results show that the maximum
wavelength of the quercetin standard is 435 nm, which is used to measure the
absorbance of the ethanol extract sample.
Making of a quercetin standard curve: In making standard curve, 25 mg of standard quercetin
was used and dissolved in 25 mL of ethanol. The stock solution was pipetted at
1 mL and then added 10 mL of ethanol to reach a concentration of 100 mg. kg-1. Using a standard 100 mg. kg-1
quercetin solution, several concentrations of quercetin were prepared: 6, 8,
10, 12 and 14 mg. kg-1. To
each of these concentrations, 1 mL of 2% AICI3 was added and 1 mL of
potassium acetate of 120 mM. Samples were incubated for 60 min at
room temperature. The UV-Vis spectrophotometry method with a maximum wavelength
of 435 nm was used to measure absorbance (Rogahang
et al. 2023).
Measurement of total flavonoid levels: Total flavonoid
determined by taking 15 mg of
Smadan root extract dissolved in 10 mL of ethanol to reach a concentration of
1500 mg. kg-1. Pipette 1 mL
of solution then add 1 mL of 2% AICI3 solution and 120 mM potassium acetate. Samples were
incubated for 60 min at room temperature. The UV-Vis spectrophotometry method
with a maximum wavelength of 435 nm was used to measure absorbance (Rogahang et al. 2023).
Cytotoxic (anticancer)
testing
Smadan root samples that had
been evaporated were tested for cytotoxic activity (dry samples) on MCF-7
cancer cells. The equipment used in cytotoxic testing: Biosafety
cabinet (BSC) (Thermo scientific 1300 series a2), CO2 incubator
(Thermo scientific 8000DH series), Centrifuge (Thermo scientific micro CL17),
Multimode reader (Tecan Infinite M200 PRO), Microscope (Thermo scientific EVOS
XL Core). Prepared
anti-proliferation assay working solution. The working solution to be used is
Presto Blue™ cell viability reagent (Gusungi
et al. 2020).
Cell preparation: The cells to be used are at least 70% confluent, then
discard the media on a dish, then rinse the cells twice with 1 mL of PBS, add 1
mL of Trypsin-EDTA solution then incubate for 5 min so is dispersed (under an
inverted microscope the cells will appear to float) transferred the cells into
a tube containing media, centrifuged the cells at a speed of 3000 rpm for 5 min
and discarded the supernatant, then the pellet was dissolved into a tube
containing the media (Gusungi et
al. 2020).
Seeding cells into 96 well plate: Determining the number and viability of cells by trypan
blue exclusion and resuspend cells with a final cell density of 170,000
cells/mL in media (17,000 cells/well) where 10 µL of trypan blue was prepared in a sterile microtube, 10 µL of cell suspension was added to the
trypan blue solution and then homogenized and clean the hemacytometer. Cover
the slip using 70% ethanol then dry, slowly insert 10 μL of trypan blue cell solution into one side of the chamber
using a pipette, count the number of healthy cells and determine the number of
(viable) cells per mL. Seeding/culture of cells into 96 well plates is then
incubated for 24 h or until the cells are at least 70% confluent at a
temperature of 37°C and 5% CO2 gas (Gusungi et al. 2020).
Treatment of cells with sample/positive control/negative control: Prepared 8 microtubes 1.5 mL and each microtube is labeled with
the appropriate dilution concentration, then the stock sample is diluted into 8
concentration variants using media solvent, 96 well plates containing cells are
removed from the incubator. Labeled on the plate along the left margin are
which rows will be treated by the standard and which rows will be sample. Then
remove the media from each well. Using a micropipette, transfer 100 μL of
each sample and positive control from the microtube into each appropriate well
on a 96 well plate containing cells, then incubate again for 48 h (Gusungi et al. 2020).
Administration of presto blue reagent and measurement of absorbance: At this stage,
discard the media in each well, then prepare 9 mL of media in a tube to which 1
mL of "Presto Blue™ cell viability reagent" is added (10 µL of reagent for 90 µL of media). Put 100 µL of the solution mixture into each
well of the microplate and then incubate for 1–2 h
until a color change is visible (When entering living cells, the presto blue
reagent will be reduced from the blue compound resazurin with no intrinsic fluorescent
value, to Table 1: Results of
soaking Smadan root extract
Sample |
Type of solvent |
Solvent volume |
Sample weight |
Extract weight |
Rendemen |
Dry extract |
95% ethanol |
400 mL |
100 g |
4.75 g |
4.75% |
Wet extract |
95% ethanol |
400 mL |
100 g |
4.61 g |
4.61% |
Table 2: HPLC results of Smadan root extract, at a wavelength 310
nm
No. |
RT |
Area |
Area (%) |
Height |
1 |
2.269 |
3582 |
0.65 |
283 |
2 |
2.444 |
5907 |
1.07 |
555 |
3 |
2.853 |
95261 |
17.29 |
13118 |
4 |
3.707 |
6585 |
1.20 |
836 |
5 |
4.358 |
2163 |
0.39 |
267 |
6 |
5.913 |
3749 |
0.68 |
305 |
7 |
6.844 |
8354 |
1.52 |
1048 |
8 |
7.071 |
58261 |
10.57 |
3112 |
9 |
9.859 |
16457 |
2.99 |
1712 |
10 |
10.941 |
1560 |
0.28 |
210 |
11 |
11.743 |
1478 |
0.27 |
201 |
12 |
12.301 |
32753 |
5.94 |
3091 |
13 |
14.079 |
2726 |
0.49 |
300 |
14 |
14.325 |
3222 |
0.58 |
361 |
15 |
15.720 |
10282 |
1.87 |
891 |
16 |
16.628 |
27039 |
4.91 |
2689 |
17 |
16.815 |
16028 |
2.91 |
1460 |
18 |
17.730 |
2565 |
0.47 |
296 |
19 |
18.882 |
41883 |
7.60 |
3196 |
20 |
20.385 |
33082 |
6.00 |
3150 |
21 |
21.134 |
21494 |
3.90 |
1959 |
22 |
21.614 |
2845 |
0.52 |
323 |
23 |
21.877 |
57213 |
10.38 |
4881 |
24 |
22.606 |
43974 |
7.98 |
3000 |
25 |
23.585 |
17240 |
3.13 |
1734 |
26 |
28.600 |
35253 |
6.40 |
719 |
Fig. 3: Retention time of 310 nm wavelength extract
Fig. 4: Retention
time of 254 nm wavelength extract
the red and highly colored
resorufin compound fluorescent). The conversion value is proportional to the
number of metabolically active cells and can be measured quantitatively (to
measure absorbance, the absorbance spectrum for resazurin and resorufin is used), then the absorbance is measured at a wavelength
of 570 nm (reference: 600 nm) using a multimode reader (Gusungi et al.
2020).
Data analysis
HPLC data analysis was carried
out descriptively. Data
analysis of total flavonoids was carried out using
linear regression equations, anticancer content analysis was analyzed
descriptively based on the IC50 value and the results obtained.
Results
Sample
extraction
The extraction method used in
this research is the maceration method. Sample that had been macerated
for 3 days was dark red in color and had a fragrant smell like wood mixed with
the smell of ethanol. The type of solvent used during maceration, namely 95%
ethanol and a solvent volume of 400 mL, the weight
of the sample used is 100 g. The final extract weight obtained from solvent
evaporation was 4.75 g for the dry Smadan root sample and the wet Smadan root
sample was 4.61 g. The soaking yield of Smadan root extract for dry samples was
4.75% and for wet samples 4.61% (Table 1). Using the formula% Soaking = Extract
weight/ sample weight x 100%. Thus, the dry Smadan root extract was higher than
the wet Smadan root extract in the yield results carried out.
Testing bioactive content HPLC method
In the test results, 26 active
compounds in Smadan root extract were detected at a wavelength of 310 nm with a
retention time (RT) of 30 min (Fig. 3). Table 2 clearly
portrays retention time, area, % area and height of the 27 bioactive compounds detected
and active compounds with a very high percentage, i.e., 13,118
detected at a retention time of 2.853 min, Area
95,261 and area
17.29%.
At a
wavelength of 254 nm, 30 active compounds were detected with a retention time
(RT) of 30 min (Fig. 4). In (Table 3) the results of retention time, area, % area and height for 30 bioactive compounds are found.
Active compounds with a very high percentage, i.e., 20.1531,
were detected at a retention time of 2.861 min, area
1,543,462 and area 31.26%.
Analysis of total flavonoid compound content of smadan root extract
Table 4: Quercetin absorbance
Concentration (mg.kg-1) Absorbance
(y) |
6 0.580 |
8 0.608 |
10 0.663 |
12 0.682 |
14 0.689 |
Table 5: Total flavonoid
content of Smadan root extract
Sample |
Absorbance (y) |
Total flavonoid content (mg QE/g) |
Dry extract |
1.895 |
62.66 |
Wet extract |
0.882 |
14.00 |
Fig. 5: Quercetin
absorbance curve
The quercetin
standards with concentrations of 6, 8, 10, 12 and 14 mg. kg-1 with the absorbance
values obtained for
each concentration (mg. kg-1).
This value shows that the higher the concentration of the solution used, the
higher the absorbance value obtained (Table 4). The quercetin standard results
obtained are plotted between the levels and absorbance to obtain a linear
regression equation, namely y = 0.014x + 0.498 with a value of R2 =
0.924. The quercetin calibration curve equation can be used as a comparison to
determine the concentration of flavonoid compounds in the total extract of
Smadan root samples (Fig. 5). The calculation results show that dry Smadan root
extract (dry extract) has a total flavonoid content of 62.66 mg QE/g extract and the total flavonoid content of wet
Smadan root extract (wet extract) is 14 mg QE/g extract (Table 5).
MCF-7 anticancer cytotoxicity activity assay
The curve of
the results of testing Smadan root extract on MCF-7 cells shows IC50 value = 50.12 µg/mL, which means that
Smadan root extract has strong cytotoxic properties in killing MCF-7 cancer
cells or breast cancer cells (Fig. 6). The average viable cancer cells were =
104.45% and the average viable MCF-7 cancer cells after being given Smadan root
extract at a sample concentration of 7.81 µg/mL
= 104.73%, 15.63 µg/mL = 99.80%,
31.25 µg/mL= 61.06%, 62.50 µg/mL= 33.17%, 125.00 µg/mL = 31.24%, 250.00 µg/mL = 29.33%, 500.00 µg/mL = 5.43%, 1000.00 µg/mL = 0.66%. Based on these results, Smadan root extract with higher
concentration can potentially kill
cancer cells or had stronger cytotoxicity
effect on cancer cells (Fig. 7). A concentration of 1000.00 µg/mL had the strongest cytotoxicity activity (Table 6). (Note: The cisplatin concentration used in the assay
was 53 μM).
Table 3: HPLC
results of Smadan root extract, wavelength 254 nm
No. |
RT |
Area |
% Area |
Height |
1. |
2.308 |
82636 |
1.67 |
9460 |
2. |
2.439 |
81741 |
1.66 |
9161 |
3. |
2.861 |
1543462 |
31.26 |
201531 |
4. |
3.333 |
223011 |
4.52 |
9162 |
5. |
3.714 |
366637 |
7.43 |
19452 |
6. |
4.343 |
143739 |
2.91 |
6832 |
7. |
4.925 |
97735 |
1.98 |
4509 |
8. |
5.939 |
20625 |
0.42 |
1616 |
9. |
6.870 |
73304 |
1.48 |
4515 |
10. |
7.265 |
259136 |
5.25 |
14674 |
11. |
7.909 |
56673 |
1.15 |
2382 |
12. |
8.555 |
14900 |
0.30 |
1320 |
13. |
9.528 |
6394 |
0.13 |
521 |
14. |
9.864 |
4605 |
0.09 |
490 |
15. |
10.817 |
28561 |
0.58 |
2378 |
16. |
10.966 |
182283 |
3.69 |
4328 |
17. |
12.097 |
29041 |
0.59 |
1533 |
18. |
14.082 |
36996 |
0.75 |
2672 |
19. |
14.799 |
24404 |
0.49 |
1349 |
20. |
15.425 |
26713 |
0.54 |
2682 |
21. |
15.710 |
75659 |
1.53 |
4816 |
22. |
16.637 |
110266 |
2.23 |
11789 |
23. |
16.833 |
148260 |
3.00 |
10205 |
24. |
17.735 |
44089 |
0.89 |
2418 |
25. |
18.383 |
9220 |
0.19 |
579 |
26. |
18.883 |
188503 |
3.82 |
12225 |
27. |
19.585 |
5339 |
0.11 |
527 |
28. |
20.397 |
180532 |
3.66 |
14381 |
29. |
21.141 |
106416 |
2.16 |
8891 |
Fig. 6: Smadan test results curve for MCF-7 cells Fig 7: Documentation of MCF-7 cell morphology from extract
Smadan root test results 30. |
21.885 |
283545 |
5.74 |
21908 |
31. |
22.615 |
243384 |
4.93 |
14031 |
32. |
23.593 |
73411 |
1.49 |
7427 |
33. |
24.731 |
38394 |
0.78 |
3065 |
34. |
25.334 |
9897 |
0.20 |
737 |
35. |
26.093 |
8339 |
0.17 |
836 |
36. |
26.734 |
13301 |
0.27 |
1081 |
37. |
28.584 |
95633 |
1.94 |
1788 |
Discussion
The bioactive
content and anticancer activity test of MCF-7 Smadan root extract from Bitung
city forest, North Sulawesi was carried out extracting Smadan root samples. The
extraction stage aims to extract chemical components or secondary metabolites
contained in the Smadan root samples. Extraction is a method used to extract
compounds from plants using certain solvents (Senewe et al. 2023).
Factors that influence the extraction process include the extraction method,
type of solvent, particle size, and duration of extraction time (Putri et al.
2021). The
extraction process has 2 stages, namely sample maceration and solvent
evaporation. The maceration process is carried out by soaking the crushed
Smadan root powder into a jar and then adding 95% ethanol. Based on previous
research, ethanol solvent was used as an extraction solvent because ethanol has
selective properties and is able to extract compounds contained in the sample
(Chen et al. 2020). So in this study the researchers used ethanol
solvent for use in the sample maceration process. In the maceration process, Smadan roots were soaked in a
jar for 3 days at room temperature, because based on previous research the
maceration process was carried out at room temperature to protect the bioactive
content which is not heat resistant from being damaged (Nur et al.
2020). Maceration method was chosen because it can avoid the destruction of
thermolabile compounds, which may have very important antioxidants
(Setyawardhani and Saputri 2020). Apart from that, the advantage of the
maceration method is that the procedure and equipment are simple and
affordable. After maceration, the sample is filtered using filter paper to
enter the solvent evaporation process. Evaporation of the solvent uses a rotary
evaporator to convert the solvent into steam and the active compound content of
the Smadan root extract (thick extract) remains. The thick extract resulting
from evaporation of the solvent shows the distinctive aroma of the Smadan root
plant, namely the smell of wood mixed with the smell of ethanol. Then the thick
extract of Smadan roots (dry sample) was tested for bioactive content.
Table 6: Absorbance of extract Smadan root results on MCF-7
cells
|
Media |
Media+
Cell |
Ciplatin |
Solvent |
|
Sample
concentration (µg/mL) |
||||||
7.81 |
15.63 |
31.25 |
62.50 |
125.00 |
250.00 |
500.00 |
1000.00 |
|||||
Absorbance
570 nm |
0.4786 |
0.7932 |
0.5217 |
0.7813 |
0.8204 |
0.7947 |
0.6685 |
0.6136 |
0.6015 |
0.6004 |
0.5138 |
0.5209 |
0.4889 |
0.8024 |
0.5303 |
0.7827 |
0.8090 |
0.8027 |
0.7135 |
0.6117 |
0.6045 |
0.5973 |
0.5330 |
0.5338 |
|
Absorbance
600 nm |
0.6127 |
0.2080 |
0.5923 |
0.2228 |
0.2226 |
0.2403 |
0.4199 |
0.5130 |
0.5214 |
0.5427 |
0.6147 |
0.6518 |
0.6261 |
0.2087 |
0.6035 |
0.2241 |
0.2240 |
0.2428 |
0.3856 |
0.5231 |
0.5222 |
0.5191 |
0.6280 |
0.6650 |
|
Difference
absorbance |
-0.1341 |
0.5852 |
-0.0706 |
0.5585 |
0.5978 |
0.5544 |
0.2486 |
0.1006 |
0.0801 |
0.0577 |
-0.1009 |
-0.1309 |
-0.1372 |
0.5937 |
-0.0732 |
0.5586 |
0.5850 |
0.5599 |
0.3279 |
0.0886 |
0.0823 |
0.0782 |
-0.0950 |
-0.1312 |
|
%
living cells |
|
103.84 |
9.37 |
99.99 |
105.65 |
99.40 |
55.35 |
34.03 |
31.08 |
27.85 |
5.01 |
0.68 |
|
105.06 |
9.00 |
100.01 |
103.81 |
100.19 |
66.77 |
32.30 |
31.40 |
30.81 |
5.86 |
0.64 |
|
Average
living cell |
104.45 |
9.18 |
100.00 |
104.73 |
99.80 |
61.06 |
33.17 |
31.24 |
29.33 |
5.43 |
0.66 |
|
SEM |
0.61 |
0.19 |
0.01 |
0.92 |
0.40 |
5.71 |
0.86 |
0.16 |
1.48 |
0.42 |
0.02 |
|
Data
normalization Living
cells (%) |
104.45 |
9.18 |
100.00 |
104.73 |
99.80 |
61.06 |
33.17 |
31.24 |
29.33 |
5.43 |
0.66 |
In
testing the bioactive content of Smadan root extract using the HPLC method, 26
and 37 active compounds were detected in Smadan root extract. The use of the
HPLC method shows good compound separation. This method is suitable for
determining the content of active compounds (Seal 2016). Active compounds are
very important as antioxidants (Gazali et al. 2018). Antioxidants prevent oxidation of compounds which can
cause cancer (abnormal cells). The main cause of cell damage is the formation
of ROS or Reactive oxygen species. Antioxidant compounds can control ROS
(Andarina and Djauhari 2017). Antioxidants function as a defense system against
free radicals (Aditya and Ariyanti 2016). Antioxidants outside the body can be
obtained in synthetic and natural form (Aditya and Ariyanti 2016). The active
compounds (antioxidants) found from the results of this research are very important
in helping prevent cancer. In previous research, the active compounds obtained
could prevent cancer (Ali et al. 2022; Putra et al. 2023;
Permatasari et al. 2023).
In the
total flavonoid content test using UV-VIS spectrophotometry, the total
flavonoid content of dry Smadan root extract was 62.66 mg QE/g extract and wet Smadan root extract was 14 mg QE/g extract. When compared with dry Smadan root extract,
the flavonoid content is greater than wet Smadan root extract. In the results
of research (Dalming 2021) to determine flavonoid levels, it was found that dry
guava leaf extract was higher than wet guava leaf extract. However, in research
(Ristanti 2019), the total flavonoid content of wet binahong leaves was higher
than dry binahong leaves. This is because wet binahong leaves do not undergo a
drying process. Flavonoid compounds can be reduced due to the drying process.
However, if the drying process is carried out correctly you will definitely get
good results (Santoso and Egra 2018). Flavonoids are natural phenolic compounds
found in almost all plants in the flowers, roots, skin, leaves and even seeds.
Flavonoids play an important role as medicine because they have antioxidant,
antibacterial, antiviral and anticancer activities (Fahira 2023).
The
cytotoxic test results of Smadan root extract against MCF-7 breast cancer cells
obtained an IC50 value = 50.12 µg/mL,
which means that Smadan root extract has strong cytotoxic properties to prevent
and kill MCF-7 breast cancer cells. A very strong cytotoxicity test has an IC50
of less than 10 µg/mL, strong
cytotoxicity has an IC50 value between 10-100 µg/mL and moderate cytotoxicity has an IC50 between 100–500 µg/mL
(Tunjung and Sayekti 2019). Based on previous research, Bajakah from Kalimantan tested 4T1 breast cancer cells and found the IC50
value for the hexane fraction was 20.0 mcg/mL and the ethyl acetate fraction
was 7.4 mcg/mL. Based on these results, it means that Bajakah has strong
cytotoxicity and is very strong in killing 4T1 cancer cells (Iskandar et al.
2022). However, Bajakah research conducted by Yuniarti et al. 2021
showed weak anticancer activity against MCF-7 breast cancer (Yuniarti et al.
2021). In this study, Smadan roots were proven to be strong in killing MCF-7
cancer cells. MCF-7 cancer cells are cancer cells taken from the breast tissue
of a 69 year old woman (Chusniasih and Tutik 2020), while 4T1 breast cancer cells are a cancer cell line
originating from tissue in the mammary glands of the BAlB/c mouse strain
(Schrors et al. 2020).
Conclusion
The Smadan root extract has the
potential to prevent and kill MCF-7 breast cancer cells and can be developed as
a cancer drug because it has been proven to be strong. Further research is
needed to identify the DNA and side effects of Smadan root.
Acknowledgements
The author would like to express
his thanks to the Biology Laboratory, Faculty of Mathematics, Natural and Earth
Sciences, Manado State University and the Central Laboratory of Padjadjaran
University which helped carry out this research.
Author Contributions
AGK and MYS were involved
planning the research and AGK performed the data acquisition/collection. AGK,
RM and MYS aided in interpreting the results. AGK performed data analysis. MYS,
RM revised the manuscript. All authors were involved in this research.
Conflict of Interest
All authors declare no conflict
of interest.
Data Availability
Data presented in this research
will be available on a fair request to the corresponding author.
Ethics Approval
Not applicable to this paper.
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