Submitted : 10-04-2020 Revised : 24-12-2020 Accepted : 24-03-2021 Trad.
Med.
January-April 2021 Vol.
, p 49-58 ISSN-p : 1410-5918 ISSN-e : 2406-9086 Antioxidant and Anticancer Potential of Raja Bulu Banana Peel and Blossom (Musa acuminata Colla (AAB grou.
) Ethanol Extracts in MCF-7 Cell Lines Afifah Bambang Sutjiatmo1.
Wahyu Widowati2*.
Ida Sumiati1.
Twice Priestu1.
Seila Arumwardana3.
Hanna Sari Widya Kusuma3.
Alya Mardhotillah Azizah3 1 Faculty of Pharmacy.
Jenderal Achmad Yani University.
Indonesia 2 Faculty of Medicine.
Maranatha Christian University.
Indonesia 3 Biomolecular and Biomedical Research Center Aretha Medika Utama.
Indonesia
ABSTRACT
Breast cancer is an uncontrolled cell growth in breast tissue.
Surgical treatments of breast cancer can reduce breast aesthetics and chemotherapy can cause severe side effects.
It makes the searches for plants as breast anticancer agents intensively carried out.
Several studies have shown that banana peels and blossoms possess antioxidant and anticancer activity.
This study aims to determine the fruit peel and blossom of Raja Bulu banana (Musa acuminata Colla (AAB grou.
), an endemic banana species in Indonesia, potential as antioxidant and anticancer agent in MCF-7 cells.
Antioxidant potential was determined by using 2,2-diphenyl-1picrylhydrazyl (DPPH) and hydrogen peroxide (H2O.
scavenging activity assay.
Anticancer potential was determined by cytotoxic test using MTS .
,5-dimethylthiazol-2-y.
-5-.
-2-.
-sulfopheny.
-2H-tetrazoliu.
The results showed that 70% ethanol extract of fruit peel (PBEE) and blossom (BBEE) of Raja Bulu banana had median inhibition concentration (IC.
for DPPH scavenging activity at 115.
32 AAg/mL and 162.
52 AAg/mL respectively, while for H 2O2 scavenging activity at 624.
80 AAg/mL and 497.
13 AAg/mL respectively.
Anticancer potential was expressed by inhibiting concentration of 50% proliferation (IC.
of MCF-7 cells for PBEE and BBEE were 115.
00 AAg/mL and 338.
AAg/mL respectively.
This study showed that PBEE and BBEE have antioxidant and anticancer properties.
Key words: Anticancer.
Antioxidant.
Musa acuminate.
MCF-7
INTRODUCTION
Breast cancer is a malignancy in breast epithelial tissue that can originate from the breast ducts .
actiferous ductu.
and breast glands .
(National Cancer Institute, 2.
Breast cancer is a disease with high mortality and incidence rate in the world (Houghton & ChamberlainAos, 2.
Besides cancer risk could be post-translational modifications or mutations in cancer-related genes by some reaction like nitration, nitrosation, phosphorylation, acetylation, and poly (ADPribosy.
It occurs as the result of free radicals or lipid peroxidation byproducts presence such as malondialdehyde (MDA) and 4-hydroxynonenal .
HNE).
Free radicals in carcinogenesis can damage DNA and modify the proteins structure and function that maintain cellular integrity and promote angiogenesis.
The DNA damage by free radicals induces chromosome fragmentation.
It has been demonstrated using hydrogen peroxide (H2O.
It means, free radicals can increase tumorigenesis through some way like DNA damage *Corresponding author : Wahyu Widowati Email : wahyu_w60@yahoo.
and mutation, stimulating cell cycle/proliferation, inhibiting apoptosis, and inhibiting DNA repair (Widowati et al.
, 2013.
The lack of selectivity also the appearance of drug-resistant tumor are factors that limited the solid tumor chemotherapy.
Surgery performed on early-stage breast tumors can reduce the aesthetics of the breast.
Finding natural ingredients for chemotherapy agents continues to be the effort focus.
Natural ingredients as cancer prevention is considered an approach to reduce the increasing cancer incidence (Ray et al.
, 2.
Herbal medicine was containing antioxidants that could be the sources for replacing synthetic chemotherapeutic agents.
It has advantages where they are less cytotoxic toward healthy cells thus safer to use (Widowati et al.
, 2013.
Various compounds produced by edible plants like flavonoids and polyphenols have shown anticancer activity.
Flavonoids are a class of compounds that have been known to have antioxidant and anticancer activity.
Polyphenols can inhibit carcinogenesis by influencing molecular pathways at the initiation, promotion, progression, and metastasis stages (Ferguson et al.
Traditional Medicine Journal, 26.
, 2021 | DOI : 10.
22146/mot.
Afifah Bambang Sutjiatmo Based on previous research on antioxidant activity and anticancer screening of banana peels in n-hexane, ethanol, and water extracts against HUVEC.
HCT-116, and MCF-7 cells was shown that the highest ability to inhibit MCF-7 cell growth was found in 12.
07% ethanol extract of banana peels (Dahham et al.
, 2.
The banana blossom is a food rich in nutrients that contains calcium, phosphorus, iron, and some vitamins A.
B1, and C that beneficial for the human body (Sheng et al.
Therefore, the antioxidant and anticancer potential of 70% ethanol extract Raja Bulu banana (Musa acuminata Colla (AAB grou.
) peel and blossom in MCF-7 cells was determined in this METHODOLOGY Materials Raja Bulu banana peel and blossom (M.
acuminata Colla (AAB grou.
) were obtained from Perkebunan Pisang Raja Bulu in Jampang Kulon.
Sukabumi.
West Java and has been identified by Department of Biology.
School of Life Science and Technology.
Bandung Institute of Technology.
Bandung.
West Java.
Indonesia.
The extract was obtained by using 70% ethanol and rotary vacuum evaporator (IKA RV 3 V-C) at 60oC.
Phytochemical screening test was performed using 1% FeCl 3 (Merck, 1.
, acetate acid, absolute sulfuric acid (H2SO.
(Merck 109.
HCl 1 N (Merck 1003171.
, vanillin (Merck 204-465-.
Mg powder (Merck EM105.
, amyl alcohol (Merck 10.
, also DragendorffAos reagent.
The 2,2-diphenyl-1-picrylhydrazil (DPPH) assay was performed using DPPH (Sigma Aldrich D9.
DMSO (Merck 1029522.
, microplate reader spectrophotometer (MultiskanE GO Microplate Spectrophotometer.
Thermo Scientifi.
Hydrogen peroxide (H2O.
scavenging assay was performed by using ferrous ammonium sulphate .
Sigma 7783.
H2O2 5 mM (Merck 1.
, 1,10-phenanthroline 1 mM (Merck 200-629-.
Cytotoxic test was performed by using MTS testing .
,5-dimethylthiazol-2-y.
-5-.
-2- .
-sulfopheny.
-2Htetrazoliu.
(CellTiter 96A AQueous One Solution Cell Proliferation Assay Promega G3.
and MCF7 cell line (ATCCA HTB-22E).
Methods Extract Preparation The preparation of extract begins with the fresh Raja Bulu banana fruit peel and blossom collection was washed and chopped to be small pieces and weighed.
It was oven-dried at 50AC.
g of Raja Bulu banana peel simplicia were macerated using 70% ethanol around 900 mL.
Meanwhile, 500 g of Raja Bulu banana blossom simplicia were macerated using 70% ethanol, filtered, and evaporated using rotary vacuum evaporator to produce a thick peel banana ethanolic extract (PBEE) and blossom banana ethanolic extract (BBEE) The extract was used for further testing and stored at -20AC (Walida et al.
Widowati et al.
, 2017.
Prahastuti et al.
Phytochemical Screening Test Phytochemical tests were carried out on PBEE and BBEE using the modified Farnsworth method to identify the presence of phenols, steroids/triterpenoids, saponins, terpenoids, flavonoids, and alkaloids qualitatively based on color reaction and precipitation that is typical of each secondary metabolite (Harbone, 1987.
Widowati et al.
, 2017.
Widowati et al.
, 2018.
Prahastuti et al.
, 2.
The method was listed Phenols Identification The sample .
was mixed with FeCl 3 in a dropping plate.
The formation of green/red/blue/purple/black color was indicated phenols in the sample.
Steroid/Triterpenoids Identification The sample .
was sinked in acetate acid at the dropping plate.
Then, after 10 Ae 15 minutes, the mixture was mixed with H2SO4 as much as one drop.
If green or blue color was presented that means the sample contain steroid, but if red or orange sediment was presented that means the sample contain triterpenoids.
Saponins Identification The sample .
was inserted into the test tube that contain water.
It was boiled in the water bath for 5 minutes.
After that, it was shaken vigorously and dripped some HCl 1 N.
If stable foam was present on the surface, it means the sample contain saponins.
Terpenoids Identification The sample .
, vanillin, also H2SO4 was mixed in the dropping plate.
The terpenoids was present in sample if purple color was formed in the Flavonoids Identification The sample .
was mixed with Mg powder and HCl 2N in the test tube.
Then it was heated for around 5 to 10 minutes.
After that it was cooled and filtered to collect the filtrate.
The filtrate was mixed was amyl alcohol.
The red or Traditional Medicine Journal, 26.
, 2021 Antioxidant and Anticancer Potential of Raja Bulu Banana Peel and Blossom orange color formation was indicated flavonoids in Alkaloids Identification The sample as much as 10 mg was dissolved in 5 mL of HCl 2 N.
The mixture was separated into two test tube.
The first tube was mixed with HCl 2 N as much as 3 drops and used as blank, while in the other tube the solution was placed in the dropping plate around 1 Ae 2 drops and used as The sample was mixed with DragendorffAos If the color changes to orange it means the extract was positively containing alkaloids.
DPPH Scavenging Assay DPPH assay was used to determine the DPPH scavenging activity of extracts.
Briefly, 50 AAL of PBEE and BBEE at various concentration and 200 AAL of DPPH 0.
077 mmol were added to 96 well plates as the sample well.
The control was created using only DPPH 0.
077 mmol as much as 250 AAL, while the blank well was created using 200 AAL of DMSO and 50 AAL of PBEE and BBEE.
The mixture was incubated in the dark at room temperature for 30 minutes.
The microplate reader was used to analyze the absorbance at 517 nm.
Each sample scavenging activity was determined by using formula below (Widowati et al.
, 2017.
Widowati et al.
, 2018.
Prahastuti et al.
, 2.
Ac Oe As scavenging% = y 100% Ac = Control absorbance.
As = Samples absorbance H2O2 Scavenging Assay This assay was done by using Mukhopadhyay et al.
method with Briefly, ferrous ammonium sulphate as much as 12 AAL, 60 AAL of PBEE and BBEE at various concentration, and 3 AAL of H2O2 5 mM were added to 96 well plate as the sample well.
The mixture was incubated at dark room temperature for 5 minutes.
Briefly, 1,10-phenanthroline 1 mM as much as 75 AAL was added into the mixture and incubated for 10 minutes at room temperature.
The wavelength of 510 nm was used to determine the absorbance and it was used to measuring the scavenging activity by following equation (Utami et , 2017.
Prahastuti et al.
, 2.
yayc H2 O2 Scavenging Activity (%) = y 100% yayca Ac = Control absorbance .
,10-phenanthroline and ferrous ammonium sulphat.
As = Samples Cytotoxic Test MTS test was used to determine the percentage of cell viability.
The MCF-7 cells that Traditional Medicine Journal, 26.
, 2021 used in this study are continuous cell lines.
The cells were planted with a density of 1 y 10 4 into a 96-well plate for 48 hours, then incubated until the cells reach 80% confluent.
After 48 hours of incubation, the cells in each well were contacted with PBEE and BBEE, epicatechin, and quercetin with work solution at various concentrations in 10% DMSO and incubated for 24 h.
Extracts that not added to the cell were used as blanks.
Control cells was contained cells that not added the Whereas DMSO controls was contained cells added with 10% DMSO.
MTS as much as 20 AAL was added into each well and incubated for 3 hours at 37AC and 5% CO2.
The spectrophotometer was used to measuring the cell absorbance.
The wavelength that used to measuring absorbance was 490 nm (Widowati et al.
, 2013a.
Widowati et al.
, 2013b.
Evacuasiany et al.
, 2014.
Widowati et al.
, 2.
The data were presented as percentage of living cells (%) and inhibition of cell proliferation (%).
The following formula were used to calculating the percentage of living cells (%) and inhibition of cell proliferation (%).
The Median Inhibitory Concentration 50 (IC.
was determined using probit analysis.
% cell viability = (As - A.
/ Ac x 100 % inhibition of cell proliferation = 100 - % cell As = absorbance sample.
Ab = absorbance blank.
Ac = correction of the absorbance control average RESULT AND DISCUSSION The Raja Bulu banana peel and blossom have been reported to contain compounds that have potential as antioxidant and anticancer (Dahham et al.
, 2015.
Roobha et al.
, 2.
The banana peel contains epicatechin while the banana blossom contains quercetin (Joseph et al.
, 2014.
Someya et al.
, 2.
Phytochemical screening of PBEE and BBEE were carried out to provide preliminary information regarding secondary metabolites that contained in the extract.
The results of phytochemical screening can be seen in Table 1.
It was shown that PBEE contained flavonoids, polyphenols, and terpenoids.
The results was different with the phytochemical screening from previous studies on the Raja Bulu banana skin.
Someya et al.
confirms a presence of polyphenol compounds in the Raja Bulu banana skin (Someya et al.
, 2.
, while Guntarti et al.
shows a positive results for flavonoid, saponins, and the and negative results for alkaloids, steroids, and triterpenoids compounds.
Meanwhile, results of Raja Bulu banana blossom extracts shown containing flavonoids, polyphenols, and Afifah Bambang Sutjiatmo Figure 1.
Antioxidant activity of PBEE and BBEE at various concentration against DPPH radical *The activity was presented as mean.
The assay was done triplicate for each concentration and sample.
Sample concentrations: 12.
5 ug/mL.
25 g/mL.
50 g/mL.
100 g/mL.
and 200 g/mL.
Figure 2.
Antioxidant activity of PBEE and BBEE at various concentration against H2O2 radical scavenging.
*The activity was presented as mean.
The assay was done triplicate for each concentration and sample.
Sample concentrations: 30 g/mL.
60 g/mL.
120 g/mL.
240 g/mL.
and 480 g/mL.
This result was different with previous studies where in this study shown positive results on alkaloids compounds while the other previous research shown the presence of saponins compounds in the Raja Bulu banana blossom (Joseph et al.
, 2014.
Pane, 2.
The difference in phytochemical screening results is due to the content of secondary metabolites affected by the conditions of the growth place, physical factors .
emperature, light, humidity, etc.
), genetic factors, environmental stress factors .
etal heavy.
UV ligh.
, and type of solvent used (Mahmoo et al.
DPPH and H2O2 scavenging activity were used to determine the antioxidant acitvity.
Active antioxidant in DPPH free radical scavenging is marked with a color change from dark purple to pale yellow or yellowish (Evacuasiany et al.
, 2014.
Prahastuti et al.
, 2.
While H2O2 scavenging activity was based on the ferrous ammonium sulphate and phenanthroline reaction that could form Fe2 -tri-phenanthroline complex with the color of orange, but if H2O2 radicals exists in that reaction.
Fe2 -tri-phenanthroline complex would not be formed.
When the scavenger of H 2O2 presence in the mixture, it will form the Fe2 -triphenanthroline complex (Utami et al.
, 2017.
Prahastuti et al.
, 2.
The results can be seen in Figure 1.
Figure 2.
Table I and Table II.
The Median Inhibitory Concentration 50 (IC.
was determined using linear regression analysis.
Based on the result.
PBEE exhibit high antioxidant activity for DPPH scavenging than BBEE which IC50 PBEE Traditional Medicine Journal, 26.
, 2021 Antioxidant and Anticancer Potential of Raja Bulu Banana Peel and Blossom Table I.
Phytochemical screening results of PBEE and BBEE
Compounds Group
Alkaloids Flavonoids Polyphenols Saponins Steroid/Triterpenoid Terpenoids Extract
PBEE
BBEE
( ) = contains the compounds group.
(-) = does not contain the compounds group Table II.
The linear equation, coefficient of regression (R.
and IC50 for DPPH scavenging activity of PBEE
and BBEE
Samples PBEE
BBEE
Linear Equation 2404x 22.
IC50 (AAg/mL) Note: The assay was performed in triplicate.
The equation was presented from linear regression analysis of the mean scavenging percentage Table i.
The linear equation, coefficient of regression (R.
and IC50 for H2O2 scavenging activity of PBEE
and BBEE
Samples PBEE
BBEE
Linear Equation 0791x 10.
IC50 (AAg/mL) Note: The assay was performed in triplicate.
The equation was presented from linear regression analysis of the mean scavenging percentage 32 g/mL and BBEE 162.
52 g/mL classified moderate activity with IC50 101-250 g/mL (Marjoni and Zulfisa, 2.
In line with DPPH scavenging activity.
PBEE exhibit lower antioxidant activity for H2O2 radicals scavenging than BBEE which IC50 PBEE 624.
80 g/mL classified inactive activity with IC50 >500 g/mL (Marjino and Zulfisa, 2.
and BBEE 497.
g/mL classified weak activity with IC50 <500 g/mL (Marjoni and Zulfisa, 2.
It means PBEE has more active antioxidant activity in DPPH scavenging activity than BBEE.
BBEE has more active antioxidant activity than PBEE in H 2O2 scavenging activity.
It was in line with previous study that unripe plantain banana has IC50 at 0.
ppm while banana blossom has IC50 at 21.
02 ppm (Schmidt et al.
, 2015.
Liyanage et al.
, 2.
Anticancer potential testing was carried out in vitro on MCF-7 breast cancer cell lines using the MTS assay methods.
MTS assay is the reduction of tetrazolium compounds by formazan compounds that are soluble in cell culture medium.
This change was catalyzed by nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphate (NADPH) produced by the Traditional Medicine Journal, 26.
, 2021 enzyme dehydrogenase in the living cell's mitochondrial respiration chain.
The higher the cell viability, the more colored formazan formed which is characterized by the intensity of the color that is formed the more concentrated (Widowati et al.
, 2013a.
Widowati et al.
, 2013b.
Evacuasiany et al.
, 2014.
Widowati et al.
, 2015.
Widowati et al.
The results of MTS assay was shown in Figure 3.
It shown that the sample was influenced the viability of MCF-7 cells.
The higher concentration of the sample could decrease the viability cell which means inhibited the proliferation strongly.
The IC50 value of anticancer activity both PBEE and BBEE the data of inhibition activity was analyzed using probit analysis.
The IC50 values indicates sample concentration inhibit 50% of cell proliferation.
The IC50 value for PBEE and BBEE was found to be 115.
00 AAg/mL and 47 AAg/mL respectively which means PBEE has more active anticancer classified moderate activity (IC50 : 101-250 AAg/mL) than BBEE classified weak activity (IC50 : 250-500 AAg/mL) (Marjoni and Zulfisa, 2.
Based on IC50 values.
PBEE has a moderate anticancer and BBEE has weak Afifah Bambang Sutjiatmo .
Figure 3.
Cytotoxic activity for BBEE and PBEE in terms of (A) MCF-7 cells viability and (B) MCF-7 cells *The activity was presented as mean.
The assay was done triplicate for each concentration and sample.
Sample concentrations: 3.
91 g/mL.
81 g/mL.
63 g/mL.
25 g/mL.
50 g/mL.
125 g/mL.
250 g/mL.
and 500 g/mL.
anticancer toward MCF-7 breast cancer cells.
This research showed that crude extract yielded anticancer in moderate and weak activities, its because the crude extract consists of several compounds had lower bioactivities compared to its compound, this result data was in line previous study that purification yielded fractions, compounds with higher antioxidant and bioactivity than crude extract (Barbosa-Pereira et al.
, 2.
Although the anticancer potential of PBEE and BBEE are moderate and weak activities.
PBEE and HBBE contains secondary metabolites of flavonoid compounds.
Flavonoids are compounds that known to have proliferative inhibitory activity in various cancer cells and able to induce Catechins are belonging to flavonoid compounds that can reduce murine lymphoma cell concentrations above 100 g/mL (Mooney et al.
Epicatechin suppresses human-induced In addition, epicatechin and related compounds have been found to modulate the signaling enzyme activity during angiogenic signaling, control its release and regulate its receptor (Lim, 2.
According to previous research, epicatechin has less significant effect on MCF-7 and T47D breast cancer cell proliferation compared to other catechins or flavan-3-ols such Traditional Medicine Journal, 26.
, 2021 Antioxidant and Anticancer Potential of Raja Bulu Banana Peel and Blossom as (-)-epigallocatechin (EGC), (-)-epigallocatechin gallate (EGCG), and (-)-gallocatechin gallate (GCG) (Evacuasiany et al.
, 2.
Meanwhile, quercetin is also a flavonol group compound that can induce apoptosis of cancer cells by stimulating cytochrome c release from mitochondria to cytoplasm (Lim, 2.
Cytochrome c was known as heme protein that acts as a water-soluble electron carrier in mitochondrial oxidative phosphorylation.
If an electron coil occurs through cytochrome c oxidase, ionic strength would change and causes the matrix The cytochrome c exits into the cytoplasm because the nonspecific inner membrane permeability transition pore was open.
It would binds to Apaf-1 and form a CARD (Caspase Recruitment Domai.
Several CARDs was combined and formed the apoptosome complex that could bind pro-caspase-9 and activated it into Caspase-9 will later activates caspase-3 as the executor that will damage the nucleus and then degrade the DNA chromosomes of cancer cells (National Cancer Institute, 2.
Quercetin also known could modulating various cell cycle regulators including p21, p27 .
egulator protein inhibits the cell cycl.
and Cyclin D1 .
n important regulator of cell cycle development G1 to S phase.
and stop the cancer cell cycle.
Quercetin causes cessation of the cycle cells by increasing p21 expression and decreasing Cyclin D1 expression (Nunez et al.
, 1.
Research on the anticancer potential of banana peel (Musa sapientu.
by Dahham .
states that PBEE has a cytotoxic effect on HCT-116 cells with a percentage inhibition of 85.
32% at a 100 AAg/mL concentration which indicates an antiangiogenesis effect by inhibiting microvascular growth of cancer cells.
Research on anticancer potential of banana blossom (Musa paradisiac.
by Nadumane & Timsina .
states that BBEE has a significant cytotoxic effect on HeLa cells with IC50 20 AAg/mL and extract fraction has maximum antiproliferative effect with IC50 value < 10 AAg/mL.
BBEE can induce apoptosis in HeLa cells .
ervical cancer epithelial cell.
through the activity of caspase-9 initiator.
There was a two-fold increase in caspase activity in fraction HeLa cells compared with control HeLa cells, which confirmed the apoptosis induction of cell death in HeLa cervical cancer cell lines (Emami et al.
, 2.
CONCLUSION
Based on the result.
PBEE and BBEE have moderate - high in DPPH scavenging and inactive weak H2O2 scavenging activities.
PBEE and BBEE have weak Ae moderate anticancer.
PBEE has more active antioxidant activity in DPPH scavenging Traditional Medicine Journal, 26.
, 2021 activity than BBEE.
BBEE more active antioxidant activity than PBEE in H2O2 scavenging activity.
PBEE has more active anticancer than BBEE.
ACKNOWLEDGEMENT
This research was financially supported by research grand 2019 from Research and Community Service Center University of Jenderal Achmad Yani.
Cimahi.
West Java.
Indonesia.
This research also supported, for the research methodology and laboratory facilities, by Aretha Medika Utama Biomolecular and Biomedical Research Center.
Bandung.
Indonesia.
We are thankful to Muhamad Aldi Maulana.
Cintani Dewi Wahyuni.
Cahyaning Riski Wijayanti.
Aditya Rinaldy from Aretha Medika Utama Biomolecular and Biomedical Research Center.
Bandung.
Indonesia for their valuable assistance.
REFERENCES