Submitted : 26-12-2019 Revised : 19-11-2020 Accepted : 24-03-2021 Trad.
Med.
January-April 2021 Vol.
, p 21-27 ISSN-p : 1410-5918 ISSN-e : 2406-9086 Cytotoxicity and Antiobesity Activity of Freeze-Dried Malus domestica.
Canarium sp.
and Averrhoa bilimbi Fruit Sri Utami1*.
Susi Endrini1.
Said Nafik2.
Seila Arumwardana3.
Rizal Rizal3,4.
Dwi Surya Artie3.
Dewani Tediana Yusepany3.
Hanna Sari Widya Kusuma3.
Wahyu Widowati5 1 Faculty of Medicine.
YARSI University.
Central Jakarta.
Indonesia 2 Directorate General of Intellectual Property.
Ministry of Law and Human Rights.
Indonesia 3 Biomolecular and Biomedical Research Center.
Aretha Medika Utama.
Indonesia 4 Biomedical Engineering.
Department of Electrical Engineering.
Faculty of Engineering.
Universitas Indonesia.
Indonesia 5 Medical Research Centre.
Faculty of Medicine.
Maranatha Christian University.
Indonesia.
ABSTRACT
Obesity has a role in the development of diseases such as diabetes, cardiovascular disease, and hyperlipidemia which is characterized by the increase of adipose tissue mass due to an imbalance of energy intake and expenditure.
Freeze-dried fruits are well known to possess antiobesity activity.
In this study, we have evaluated the antiobesity activity of freeze-dried fruit (M.
Canarium sp.
, and A.
using CHOL.
G6PDH.
TG level, and Oil Red O assay.
The viability of 3T3-L1 cell in the Canarium sp.
freeze-dried in the concentration of 12.
50 AAg/ml has a higher value compared to M.
domestica and A.
The measurements of CHOL.
G6PDH.
TG level, and Oil Red O assay of the M.
domestica freeze-dried in the concentration of 75 g/ml has higher inhibitory activity compared to the Canarium sp.
and A.
bilimbi freezedried.
In the CHOL assay.
domestica freeze-dried has a higher value compared to A.
bilimbi and Canarium In the G6PDH assay, the freeze-dried of M.
domestica has the value of 49.
Canarium sp.
22%),
and A.
13%), while in the Oil Red O assay.
domestica has inhibition activity of 62.
01% and Canarium sp.
The level of TG showed that M.
domestica has higher activity with the value of 60.
54%, while Canarium sp.
The freeze-dried of M.
domestica in the concentration of 75 g/ml has good inhibitory activity of lipid compared to A.
bilimbi and Canarium sp.
Keywords: Antiobesity.
freeze-dried.
Malus domestica.
Canarium sp.
Averrhhoa bilimbi INTRODUCTION Obesity is defined as excessive fat accumulation in adipocytes (Jou et al.
, 2.
and triggers various diseases such as Type 2 Diabetes Melitus (T2DM), cardiovascular disease, and cancer (Lois & Kumar, 2.
When a person becomes obese and adipocytes dilatation, the molecular and cellular adipose tissue undergo changes that will affect systemic metabolism (Attie & Scherer, 2.
Various efforts are made to overcome obesity, the most popular method is a low-fat diet and consumption of various drugs.
The use of chemical drugs in the long term can cause a variety of side effects such as headaches, stomach pain, vomiting, and heart attacks (Park et al.
, 2.
Various types of fruits containing Hydroxicinnamic Acid (HCA) and phenol compounds, flavonoids high have antiobesity activities (Pittler & Ernst, 2004.
Hsu & Yen, 2008.
Kim et al.
, 2010.
Kamisoyama et al.
Swick, 2011.
Dzomba & Musekiwa, 2.
Therefore, the consumption of fruits need to be *Corresponding author : Sri Utami Email : uutsuyono@yahoo.
increased, considering the side effects that could occur and the habit of eating fruits is good for health besides reducing the weight of obesity The different types of fruits containing HCA, phenol, and flavonoid (Pittler & Ernst, 2004.
Hsu & Yen, 2008.
Kim et al.
, 2010.
Kamisoyama et al.
, 2008.
Swick, 2011.
Dzomba & Musekiwa.
Canarium sp.
Malus domestica, and Averrhoa bilimbi are fruits that have bioactive compounds and antiobesity properties.
Canarium sp.
some bioactive compounds proanthocyanidins (PA.
, flavan-3-ols, prodelphinidin, epicatechin, and epigallocatechin.
The results of gas chromatography-mass spectrography (GC-MS) has shown that M.
domestica phenolic content is ( )catechin (C) and (O.
-epicatechin (EC), phloridzin .
, cyanidin .
, cyanidin -3 -O -galactoside .
, chlorogenic acid .
henolic acid.
, and hydroxycinnamates .
coumaric aci.
(Vrhovsek et al.
, 2004.
McGhie et , 2005.
Cuthberton et al.
, 2012.
Francini & Sebastiani, 2.
bilimbi contains ascorbic acid 36,68-60,95 mg/100 g, oxalic acid 8,57-9,82 Traditional Medicine Journal, 26.
, 2021 | DOI : 10.
22146/mot.
Sri Utami mg/100 g, amino acid, citrate acid, cyanidin -3 -O Ae D -glucoside, phenols, flavonoids, saponins, and the various of A.
is Averrhoa carambola contains apigenin -6 -C - L -fucopyranoside, apigenin -6- C- .
Ay-O--Lrhamnopyranosy.
--fucopyranoside, apigenin-6C -.
Ay -O - -L -rhamnopyranosy.
- -D glucopyranoside (Lima et al.
, 2001.
Kumar et al.
Moresco et al.
, 2.
In this study, we evaluated the effectivity of Canarium sp.
domestica, and A.
bilimbi as an antiobesity agent in 3T3-L1 adipocyte cell line.
METHODOLOGY
Freeze Dried Samples domestica.
Canarium sp.
, and A.
were collected from production centers.
Canarium in West Java.
domestica in Malang.
East Java, and A.
bilimbi L.
in West Java.
The plant organs were identified by herbarium staff.
Department of Biology.
School of Life Sciences and Technology.
Bandung Institute of Technology.
Bandung.
West Java.
Indonesia.
The plant organs .
eaves, stem bark, and branc.
were washed and milled respectively, and then freeze-dried to reduce the water compound in the sample.
Freeze-dried domestica (FDM), freeze-dried Canarium sp (FDC), freeze-dried A.
bilimbi (FDA) were stored at20 0 C (Utami et al.
, 2.
3T3-L1
Cell Culture Adipocyte Differentiation Induction The mouse pre-adipocytes cells 3T3-L1 (ATCCACL-.
were obtained from Aretha Medika Utama.
The 3T3-L1 cells were grown and maintained in DulbeccoAos Modified Eagle Medium (DMEM) (Gibco, 11995.
supplemented with 10% fetal calf .
serum (FBS) (Gibco, 26140.
and 1% antibiotic/antimycotic (ABAM) (Gibco, 15240-.
then incubated for 24 h at 37 AC, 5% CO2.
After the confluence of the cells, the medium was discharged, and cells were seeded in a 96-well plate .
y 104 cells/wel.
with DMEM supplemented with 10% FBS and then incubated for 48 h.
After cells reached 80% confluence, cells were induced to differentiate using Millipore ECM.
The medium was replaced by an initiation medium (DMEM containing FBS 10% and 1:10000 dexamethason.
and incubated for 48 h.
Insulin medium was replaced with progression medium (DMEM containing FBS 10% and 1:1000 insuli.
and placed in an incubator for 48 h.
The medium was then replaced again with a maintenance medium (DMEM supplemented with 10% FBS) and incubated for 2-4 days in a 37 AC incubator (Lahrita et al.
, 2015.
Hidayat et al.
, 2015.
Widowati et al.
Widowati et al.
, 2.
Measurement of Lipid Differentiated cells were treated with FDM.
FDC.
FDA .
, 75 .
, then incubated for 24 h.
The medium was discarded, washed with Phosphate Buffer Saline (PBS), then added with Oil-red O 500 l, incubated for 15-30 minutes.
Oil-red O was removed, and cells were washed with Wash Solution.
Cells were observed under the inverted light Olympus microscope (Olympus Inverted Microscope CKX41-F32FL).
Cells were extracted with Dye Extraction 500 l, incubated in an orbital shaker for 15-30 min.
Dye extraction was transferred into a 96-well plate, and absorbance was read at 490 wavelengths (MultiskanE GO Microplate Spectrophotomete.
(Hidayat et al.
Widowati et al.
, 2.
Viability Assay Viability assay in this study is using 3 -.
,5 -dimethylthiazol -2 -y.
-5 -.
-2 -.
- sulfopheny.
-2Htetrazolium (MTS) assay (Promega.
Madison.
WI.
USA) which is a colorimetric method for determining the number of viable cells in proliferation or cytotoxicity (Hidayat et al.
, 2015.
Widowati et al.
, 2.
The 3T3-L1 cells were seeded in 96-well plates .
y 103 cells/wel.
in 100 AAl medium (DMEM containing 10% FBS and 100 U/ml penicillin-streptomyci.
for 24 h at 37 AC humidified atmospheres and 5% CO2.
The medium then washed and supplemented with 99 AAl new medium and 1 AAl of FDM.
FDC.
FDA in various concentrations .
, 50, and 100 AAg/m.
and incubated for 48 h at 37 AC and 5% CO 2.
After 48 h medium was replaced by 20 AAl MTS and incubated for 3 h at 37 AC.
The absorbance was measured at 490 nm (Hidayat et al.
, 2015.
Widowati et al.
The viability assay was performed to determine the safe concentrations for the next Cholesterol Assay The cholesterol assay was determined by enzymatic photometric test or CHOD-PAP method, using DiaSys kit protocol (DiaSys 1 1300 99 10 The mix solution was seeded in 24 well plates .
L ddH20, 5 l sample, and 450 l reagent kit.
ddH2O was used as a blank sample.
Briefly, mixed solutions were incubated for 10 min at 37 oC.
The sample absorbance was measured in Miniskan Reader at 500 nm wavelength (Widowati et al.
The absorbance values were used to determine the cholesterol level in each sample using the formula below.
yaycaycycuycycaycaycuycayce ycuyce ycaycoycyycoyce Cholesterol .
mol/L)= yaycaycycuycycaycaycuycayce ycuyce yaycayco 1 ycIycycaycuyccycaycycc yaycuycuycayceycuycycycaycycnycuycu Traditional Medicine Journal, 26.
, 2021 Cytotoxicity and Antiobesity Activity of Freeze Dried Malus domestica Table I.
Cytotoxicity Activity of Freeze.
bilimbi, and Canarium sp.
in 3T3L1 Cell Line Samples Control 00 AAg/ml 00 AAg/ml 00 AAg/ml 50 AAg/ml FDM 00 A 0.
24 A 5.
90 A 2.
34 A 1.
26 A 7.
Triglyceride Assay The total triglyceride level in cells was measured according to colorimetric enzymatic tests using glycerol-3-phospate-oxidase (GPO) using a kit (DiaSys, 1 5760 99 10 .
A 500 AAL
mixed reaction containing 450 AAL reagent with 5 AAL sample .
ell lysate after treatment in the concentration of 10 and 50 AAg/m.
was incubated in 37 AC for 5 minutes.
ddH2O was used for blank well and standard reagent was used for standard Standard reaction was prepared in seven different concentrations us serial dilution .
068 and 0.
034 mmol/L).
The absorbance was measured at 500 nm of wavelength (Widowati et al.
, 2.
Triglyceride concentration was calculated using the formula:
yaycaycycuycycaycaycuycayce ycuyce ycycaycoycyycoyce Triglyceride yaycaycycuycycaycaycuycayce ycuyce ycycycaycuyccycaycycc ycycycaycuyccycaycycc ycaycuycuycayceycuycycycaycycnycuycu Glucose-6-Phospate Dehydrogenase (G6PDH) Assay The 3T3-L1 cells were seeded in 96 well plates .
x 103 cells/ wel.
in 100 AAL medium (DME containing 10% calf serum and Aba.
oC the humidified atmosphere and 5% CO2.
The glucose-6-phospate dehydrogenase (G6PDH) assay is based on the G6PDH kit (Abcam.
Sample .
AAL) of medium from cell culture and G6PD positive kit for positive control was added into the well and then 30 AAl assay buffer and 50 AAl developer work were added.
The assay buffer without samples was used for blank.
The absorbance was measured at 450 nm.
Then the samples were incubated at 37 AC dark room for 30 And then, the samples were measured again using 450 nm of wavelength (Lahrita et al.
, 2015.
Widowati et al.
, 2.
G6PDH concentration was determined by the formula:
yaA G6PDH = [(.
cN2OeycN.
yycO)] y ycycaycoycyycoyce yccycnycoycycycnycuycu B= blank sample.
T1= first absorbance.
T2= second V= Total volume Traditional Medicine Journal, 26.
, 2021 Cells viability (%) FDC 00 A 0.
85 A 4.
16 A 7.
81 A2.
78 A 8.
FDA
00 A 0.
18 A 3.
01 A 3.
56 A 2.
18 A 4.
Statistical analysis Statistical analysis was performed with Statistical Package for the Social Sciences (SPSS) statistics version 17.
0 software.
One-way analysis of variance (ANOVA) was conducted, followed by Duncan post-hoc test, and p<0.
05 was considered to be significant.
Data are presented as mean A SD.
RESULT AND DISCUSSION
The cytotoxicity activity of FDM.
FDC.
FDA.
were determined in Table I, which shows that FDC in the lowest concentration .
50 AAg/m.
has the highest cytotoxicity activity .
78%) compared to FDM and FDA with value 114.
26% and 102.
The FDM.
FDC.
FDA.
in concentration 00 AAg/ml has viability >100%, while in concentration of 100.
00 AAg/ml show viability <100%.
So, the appropriate concentration for the future study in FDM.
FDC.
FDA is using 00 AAg/ml and 25.
00 AAg/ml.
The total cholesterol level of freeze-dried fruit in the 3T3L1 cell line was shown in Table II.
Based on Table II.
the total cholesterol of FDM in concentration 75 g/ml has the lowest level compared with FDC and FDA compared to positive control but not lower than the negative control.
Based on statistical analysis (Duncan Post Hoc Tes.
FDM was significantly in total cholesterol inhibition activity in concentration 75.
00 g/ml compared to FDC and FDA.
This indicated FDM in 00 g/ml more active to reduce total cholesterol compared to FDC and FDA.
Triglyceride level of FDM.
FDC.
FDA in 3T3L1 cell line show in Table i.
FDC in concentration 25 g/ml has the most significant result in total triglyceride .
<0.
(Table .
compared to FDM and FDA, but not higher than the positive control.
In triglyceride inhibitory activity.
FDM .
g/m.
show the highest value compared to FDA and FDC.
FDM has highest in decrease of triglyceride level compared to FDC and FDA in concentration 75 g/ml.
Glucose-6-fosfat dehydrogenase (G6PDH) is the first reaction catalyzing enzyme of the Sri Utami Table II.
Total Cholesterol of Freeze Dried of M.
bilimbi, and Canarium sp.
in 3T3L1 Cell Line Sample Negative Control Positive Control FDM 75.
00 g/ml FDM 25.
00 g/ml FDC 75.
00 g/ml FDC 25.
00 g/ml FDA 75.
00 g/ml FDA 25 g/ml Total Cholesterol .
g/dL) 02 A 30.
23 A 44.
62A13.
23A21.
03A22.
83A3.
02A20.
43A20.
Inhibition Activity (%) 00 A 16.
38 A 11.
29A5.
97A8.
41A8.
14A1.
40A7.
02A7.
Data was presented as mean A SD from 3 replications.
Superscript letter ( a-.
in each column indicates significance different among concentration based on post hoc Duncan test with p < 0.
05 is considered as significantly different.
FDM : freeze dried M.
FDC : freeze dried Canarium sp.
FDA : freeze dried Table i.
Total Triglyceride of Freeze Dried of M.
bilimbi, and Canarium sp.
in 3T3L1 Cell Line Sample Negative Control Positive Control FDM 75.
00 g/ml FDM 25.
00 g/ml FDC 75.
00 g/ml FDC 25.
00 g/ml FDA 75.
00 g/ml FDA 25 g/ml Total Triglyceride .
g/dL) 66A27.
84A39.
26A19.
40A66.
24A33.
44A24.
65A19.
94A43.
Inhibition Activity (%) 73A5.
00A7.
61A3.
41A13.
03A6.
62A4.
54A3.
30A8.
Data was presented as mean A SD from 3 replications.
Superscript letter ( a-.
in each column indicates significance different among concentration based on post hoc Duncan test with p < 0.
05 is considered as significantly different.
FDM : freeze dried M.
FDC : freeze dried Canarium sp.
FDA : freeze-dried phosphate pentose pathway and provides a reduction effect on all cells in the form of NADPH .
educed form of nicotinamide adenine dinucleotide phosphat.
The following can be seen for the total G6PDH assay in Table IV.
FDM has the lowest total G6PDH compared to FDC and FDA in concentration 75 g/ml, this result in line with lipid inhibitory activity.
FDM has the highest inhibition activity compared to FDC.
and FDA Based on statistical analysis not show a significant However.
FDM in concentration 75 g/ml has the highest G6PDH inhibitory activity compared to FDA and FDC.
Based on Table IV.
FDM in concentration 75 g/ml has the lowest absorbance compared to FDA and FDC.
, but FDM shows not lower than control.
The lipid inhibitory activity.
FDM .
n concentration of 75 g/m.
has the highest activity compared to FDC and FDA, but not higher compared to control.
Based on statistical analysis (Duncan Post Hoc Tes.
FDM .
g/m.
shows the most significant result compared to others .
<0.
However.
FDM in concentration 75g/ml has the highest lipid inhibitory activity compared to FDC and FDA.
Obesity may be reduced by preventing immature fat cells from developing into mature cells, by inhibiting population growth and apoptotic induction of programmed cell death in 3T3-L1 preadipocyte cells (Soeng et al.
, 2.
This study aims to examine the cytotoxicity and antiobesity activity of freeze-dried FDM.
FDC.
, and FDA to 3T3 L1 cell line, the variables of this study are TG.
CHOL.
GPDH.
The various fruits such as M.
Canarium sp.
and A.
bilimbi contains phenol compounds.
In the present study, appropriate concentration for the future study in freeze-dried M.
bilimbi, and Canarium using concentration 75.
00 AAg/ml and 25.
AAg/ml based on viability assay in 3T3L1 adipocyte Methyl gallate isolated from herbs is phenol compounds that may be capable of inhibiting form lipid through the staining test of Oil Red O and decreasing TG levels and also be increasing the release of glycerol levels in 3T3L1 adipocyte cells Traditional Medicine Journal, 26.
, 2021 Cytotoxicity and Antiobesity Activity of Freeze Dried Malus domestica Table IV.
Total G6PDH Freeze Dried of M.
bilimbi, and Canarium sp.
in 3T3L1 Cell Line Samples Control Positive Control FDM 75.
00 g/ml FDM 25.
00 g/ml FDC 75.
00 g/ml FDC 25.
00 g/ml FDA 75.
00 g/ml FDA 25 g/ml Total G6PDH .
mol/min/m.
31A0.
80A0.
40A0.
56A0.
44A0.
57A0.
42A0.
63A0.
Inhibition Activity (%) 20A2.
00A4.
56A1.
63A3.
22A2.
08A11.
13A0.
06A4.
Data was presented as mean A SD from 3 replications.
Superscript letter ( a-.
in each column indicates significance different among concentration based on post hoc Duncan test with p < 0.
05 is considered as significantly different.
FDM : freeze dried M.
FDC : freeze dried Canarium sp.
FDA : freeze dried Table V.
Oil Red O Assay Freeze Dried of M.
bilimbi, and Canarium sp.
in 3T3L1 Cell Line Sample Control Positive Control FDM 75.
00 g/ml FDM 25.
00 g/ml FDC 75.
00 g/ml FDC 25.
00 g/ml FDA 75.
00 g/ml FDA 25 g/ml Oil Red O (Absorbanc.
1338A0.
8186A0.
3060A0.
3808A0.
4574A0.
6807A0.
0120 cd 4093A0.
5192A0.
Inhibition Activity (%) 66A2.
00A8.
63A0.
49A1.
13A11.
85A1.
01A16.
57A19.
Data was presented as mean A SD from 3 replications.
Superscript letter ( a-.
in each column indicates significance different among concentration based on post hoc Duncan test with p < 0.
05 is considered as significantly different.
FDM : freeze dried M.
FDC : freeze dried Canarium sp.
FDA : freeze dried so methyl gallate has antiobesity properties (Hsu & Yen, 2.
FDM has the highest in all cholesterol assay (TG.
CHOL, and G6PDH) and Oil Red O in 00 g/ml due to lipid inhibitory activity compared to Canarium sp.
and A.
Polyphenol in M.
domestica has antiobesity properties (Roh et al.
, 2012.
Jelodarian et al.
Therefore, the addition of fruit to the daily diet reduces overall energy consumption and improves energy disequilibrium.
Thus the continuous intake of fruit may restrict weight gains, reduce fat mass, and control obesity (Singh.
Obesity activity in 3T3-L1 cells, among others, can measure antiadipogenesis activity that prevents adipose deposits that will be decomposed into fat and energy.
The antiadipogenesis test on 3T3-L1
IBMX,
dexamethasone, triglycerides so that it undergoes differentiation into adipocytes.
IBMX will increase intracellular cAMP, dexamethasone binding to Traditional Medicine Journal, 26.
, 2021 glucocorticoid receptors, and insulin binding to insulin receptors.
Three stimulation pathways will activate the PPAR and C/EBP genes.
Adipocytes containing PPAR and C / EBP will activate adipocyte-specific genes that encode secretory factors such as insulin receptors, proteins involved in the synthesis of fatty acids, fatty acid-binding Substances that can inhibit PPAR have antiadipogenesis activity (Sharma et al.
, 2.
Fatty acid compositions of lipid droplets that can be stained using Oil Red O.
CONCLUSION
Freeze-dried of M.
Canarium sp.
and A.
bilimbi have antiobesity potential.
domestica shows the highest antiobesity activity compared to A.
bilimbi and Canarium sp via decreasing of cholesterol, triglyceride.
G6PDH.
ACKNOWLEDGEMENT
We are grateful to Directorate General for Higher Education.
Ministry of National Education Sri Utami of Republic Indonesia, for the Research Grant of Hibah Bersaing/Penelitian Produk Terapan for financial support and gratefully acknowledge support from YARSI University.
This research was also supported by the Biomolecular and Biomedical Research Center.
Bandung.
Indonesia.
We are also thankful to Ika Adhani Sholihah.
Alya Mardhotillah Azizah.
Rr.
Anisa Siwi Handayani.
Riyani Lestari.
Kamila Yashfa Gunawan, and Enden Dea Nataya for their valuable assistance.
REFERENCES