487
Indonesian Journal of Science & Technology 8.
487-498 Indonesian Journal of Science & Technology Journal homepage: http://ejournal.
edu/index.
php/ijost/ Antiangiogenesis Activity of Indonesian Local Black Garlic (Allium Sativum AoSolo ): Experiments and Bibliometric Analysis Retno Arianingrum*.
Nurfina Aznam.
Sri Atun.
Senam.
Alya Rizkita Irwan.
Nida Qurbaniah Juhara.
Nadiya Fitri Anisa.
Latifah Kurnia Devani Department of Chemistry Education.
Universitas Negeri Yogyakarta.
Yogyakarta.
Indonesia Correspondence: E-mail: arianingrum@uny.
ABSTRACT
Cancer is still one of the leading causes of death in the world.
Angiogenesis is the formation of new blood vessels, which plays a vital role in the growth of cancer cells.
As a result, inhibition of proangiogenic factors such as vascular epidermal growth factor (VEGF) could be used as a cancer treatment strategy.
Black garlic (BG) is processed fresh garlic (Allium sativum L.
) for several days under controlled high temperature and humidity.
The research aims to investigate the effect of Indonesian local BG on antiangiogenic activity through in vivo and in silico The in vivo test was carried out by observing the formation of new blood vessels using the Chorio-Allantoic Membrane Assay (CAM).
The in silico study was performed by docking analysis of BG bioactive compounds on the VEGF receptor (VEGFR).
The results showed that ethanol extract, ethyl acetate fraction, and n-hexane fraction of BG inhibit new vessel blood formation, with n-hexane fraction having the highest efficacy.
The molecular docking assay indicated some similarities in the amino acids involved in the interaction between the BG bioactive compounds and the native ligand in binding to VEGFR.
This similarity may lead to interference with VEGFR resulting in angiogenesis inhibition.
The study suggests that BG has the potential to be developed as an anticancer agent, which might be through the mechanism of inhibiting the angiogenesis process.
A 2023 Tim Pengembang Jurnal UPI
ARTICLE INFO
Article History:
Submitted/Received 05 Jul 2023
First Revised 28 Aug 2023
Accepted 11 Oct 2023
First Available online 13 Oct 2023
Publication Date 01 Dec 2023
____________________
Keyword:
Antiangiogenic activity.
Black garlic.
In silico.
In vivo.
Arianingrum et al.
Antiangiogenesis Activity of Indonesian Local Black Garlic A | 488
INTRODUCTION
Cancer is one of the primary causes of death and the major impediment to improving life expectancy in every country Based on GLOBOCAN 2020 data, an estimated there were 19.
3 million new cancer cases and about 10.
0 million cancer deaths occurred in 2020 (Sung et.
al, 2.
Cancer is a non-communicable disease that results from uncontrolled cell or tissue One of the characteristics of cancer during the carcinogenesis process of human tumors is induced angiogenesis.
Angiogenesis plays a role in supporting the life of cancer cells through the growth of new blood vessels, particularly through invasion and metastasis (Hanahan & Weinberg, 2.
VEGF is an important proangiogenic factor that regulates endothelial cell sprouting and proliferation during vasculogenesis.
Tumor cells secrete VEGF, which promotes neovascularization resulting in cancer progression (Zhang et al.
, 2.
Therefore, targeting the VEGF receptor signaling is considered a promising strategy for cancer treatment by inhibiting angiogenesis.
(Abuzenadah et al.
, 2.
Black garlic (BG) is a form of aged garlic prepared from fresh garlic (Allium sativu.
by a Millard reaction at high temperature .
Ae90C) and humidity .
Ae90%) for several times (Ahmed & Wang, 2.
Bioactive compounds in BG are more potent than those in fresh garlic.
Some of the bioactive compounds found in BG including DL-lactic 5-hydroxymethyl-2-furfural.
S,3S)-1-methyl-1,2,3,4-tetrahydrob-carboline-3-carboxylic acid.
R, 3S)-1methyl-1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid.
and 2-acetylpyrrole (Lu et al.
Regarding anticancer activity, it has been reported that BG has a dose-dependent chemopreventive effect on several cancers both in vitro and in vivo.
The BG ethanol extract has a cytotoxic effect on several human cancer cells, including AGS gastric cancer cells.
A549 lung cancer cells.
HepG2 liver cancer cells.
MCF-7 breast cancer cells (Purev et al.
, 2.
, and HT29 colon cancer cells (Dong et al.
, 2.
Furthermore, in SGC7901 human gastric cancer cells.
BG inhibited cell growth and caused apoptosis.
BG has also been shown to inhibit motility and invasiveness in AGS.
The hexane extract of BG induced apoptosis in U937 leukemia cells (Park et al.
, 2.
However, the effect of BG on angiogenesis has yet to be reported.
The two main focuses of this research are to investigate the effect of ethanol extract, ethyl acetate fraction, and n-hexane fraction of BG on antiangiogenic activity by CAM assay and to investigate the influence of bioactive compounds in BG on VEGF receptor activity by docking approach.
This study will present an overview of the prospects for using BG as a functional food product with anticancer effects .
ee Figures 1 and .
Figure 1.
The co-word map network visualization of the progress of BG research is divided into six clusters, where there has been little research on BGAos anticancer activity.
DOI: https://doi.
org/10.
17509/ijost.
p- ISSN 2528-1410 e- ISSN 2527-8045 489 | Indonesian Journal of Science & Technology.
Volume 8 Issue 3.
December 2023 Hal 487-498 Figure 2.
Co-word map density visualization of research developments regarding BG.
METHODS
Apparatus and Materials The equipment used for in vivo testing were a freeze dryer, rotary evaporator, laminar airflow (LAF.
Shimadzu SCBAe1000 A), autoclave, mini drill, syringe, digital microscope, and Kitagawa egg incubator.
The tools used for in silico testing were Laptop OS Windows 8.
CPU Intel Core i5 3210M/5Hz.
GPU NVIDIA GEFORCE GT 650M-2GB, memory 4GB DDR3 1600 MHz.
Marvin sketch.
Avogadro.
Mopac2016.
Autodock tools.
Pymol Molecular Visualization.
Pymol for Educational.
Pyrx, and Biovia Discovery Studio.
The material for in vivo study were Black Garlic One Clove Sindoro 171 Temanggung.
Indonesia, fertile chicken eggs (Elb.
, bFGF (Sigm.
, 10mM pH 7,4 phosphate buffer saline (PBS) from Sigm.
, 0.
8% dimethyl sulfoxide (DMSO) form Merck, 0.
1% NaCl (Merc.
, 96% ethanol (Merc.
, ethyl acetate (Merc.
, n-hexana (Merc.
, and paper disc (Oxoi.
The compounds contained in black garlic for docking were DL-lactic acid.
5hydroxymethyl-2-furfural.
S,3S)-1-methyl-1,2,3,4-tetrahydrob-carboline-3-carboxylic acid.
R, 3S)-1methyl-1,2,3,4-tetrahydro-b-car-boline-3carboxylic acid.
and 2-acetyl pyrrole (Lu et al.
Preparation of Black Garlic Extract BG is put into the freeze-dry to remove the moisture content.
A total of 500 g freeze-dry black garlic was macerated using 1,5 L ethanol 96% .
for 3 x 24 hours at room The filtrate was collected and concentrated using a vacuum rotary The black garlic ethanol extract was then fractionated using ethyl acetate .
to obtain the ethyl acetate fraction.
The remaining ethanol fraction was then fractionated using n-hexane .
to obtain the n-hexane fraction.
Each fraction was concentrated by a vacuum rotary A stock solution of each black garlic extract was prepared by dissolving 10 mg of the extract in 10 mL of 0.
8% DMSO.
Then the solution was diluted with ultrapure water .
to a concentration of 20, 40, and 80 AAg/mL.
A stock solution of bFGF .
ng/AAL) was prepared from 25AAg of bFGF dissolved in 500 AAL of 10 mM PBS pH 7.
Then the solution was diluted with 10 mM PBS pH 7.
4 to obtain 1 ng/AAL bFGF.
Antiangiogenic Activity Test The tools used in in vivo testing were sterilized by autoclave.
One-day-old eggs are cleaned of contaminants with 70% alcohol and then incubated at 37oC.
When the eggs DOI: https://doi.
org/10.
17509/ijost.
p- ISSN 2528-1410 e- ISSN 2527-8045 Arianingrum et al.
Antiangiogenesis Activity of Indonesian Local Black Garlic A | 490 are 3-4 days old, candling is carried out to determine the position of an embryo and the air cavity.
The eggshell is at the poles where there is an air cavity, and a needle is used to create a small hole above the embryo.
Suck air through the polar holes with a suction pipette until the embryo shifts and the air cavity seems to be filled.
The hole was then closed again with On the top of the embryo that still has space, mark a 1x1 cm2 square on the surface of the shell using a pencil.
The eggs are then returned to the incubator.
After 8-9 days, the eggshells are cleaned using 70% ethanol, and the 1x1 cm2 square image is opened with a mini drill and scalpel as a hole to insert the sample.
Samples of BG extract and other reagents (Table .
were placed on a paper disc using a micropipette.
Then the paper disc is inserted into the egg through the hole.
This procedure is performed at the LAF.
Then the hole is closed again using paraffin and returned to the incubator.
When the eggs reach the age of 12 days, they are stored in the refrigerator/freezer at <10oC for 24 hours.
The eggs are opened at 13 days by splitting the eggshell into two sections then the egg contents are removed carefully so that the CAM attached to the eggshell is not wasted and damaged.
The CAM section containing the paper disc was washed with a saline solution and fixed using a 1:1 mixture of methanol and acetone.
Then the newly formed blood vessels around the paper disc can be observed macroscopically (West et al.
where a is the number of new blood vessels treated with the sample, and b is the number of new blood vessels in bFGF group.
The amount of inhibition of each concentration of garlic ethanol fraction on the angiogenesis response can be determined by the following Eq.
To support the experiment, we added bibliometric analysis.
Detailed information for this method is explained in the literature (Al Husaeni & Nandiyanto, 2.
Data Analysis Thin blood vessels that come out of the main blood vessels .
xisting vessel.
are counted as new blood vessels.
The angiogenic response of each treatment was calculated using the following Eq.
ycIyceycoycaycycnycyce ycaycuyciycnycuyciyceycuycnyca ycyceycycyycuycuycyce.
cIyaycI) = yca y 100% yca ycnycuEaycnycaycnycycnycuycu ycyyceycycayceycuycycayciyce = 100% Oe ycIyaycI In Silico Procedure This study used VEGF target protein molecules with the codes 2P2I (Arianingrum et al.
, 2.
, 3HNG, and 3VHE (Asthana et al.
These receptors were downloaded from the RCSB PDB (Protein Data Ban.
on the https://w.
org/ page.
There are 7 other compounds contained in black garlic as ligands (Lu et al.
, 2.
Bevacizumab was used as a positive control.
The structure of the compound is drawn using Marvin's Avogadro used visualization and editing of molecules, while optimizing molecular geometry used Mopac2016 with the PM7 semi-empirical method.
The Autodock tool was used to create separate ligand and receptor files.
Pymol Molecular Visualization was used to visualize the docking results of the original ligand with the docking ligand and Pymol for education to calculate the Root Mean Square Deviation (RMSD) value.
The docking process was carried out using Pyrx and the visualization of the interaction of the ligand with the receptor was used by Biovia Discovery Studio for the docking process.
Bibliometric Analysis RESULTS AND DISCUSSION Bibliometric analysis We performed a bibliometric analysis with Vosviewer and Publish or Perish (PoP) (Al Husaeni & Nandiyanto, 2.
on a database DOI: https://doi.
org/10.
17509/ijost.
p- ISSN 2528-1410 e- ISSN 2527-8045 491 | Indonesian Journal of Science & Technology.
Volume 8 Issue 3.
December 2023 Hal 487-498 source from Pub Med with the title word "black garlic" from 2011 to 2023 .
to determine the number of articles regarding with BG.
We found 116 out of 123 articles related to BG, totaling 5.
55 authors/papers.
The number of publications fluctuated in these years but tended to rise from 2016 to 2018, then fall until 2020 before increasing again from 2021 to 2023.
Through network visualization, the research development map on BG is divided into 6 clusters.
Cluster 1 consists of 21 items, cluster 2 consists of 13 items, cluster 3 consists of 9 items, cluster 4 consists of 8 items, cluster 5 consists of 8 items, and cluster 6 consists of 7 items.
Anticancer is one of the items in cluster 1.
However, angiogenesis is not identified in any of the clusters.
The VOSviewer density map demonstrates that there is still minimal research on BG-related anticancer, as shown by a density map with a small diameter and faded yellow color (Figure .
Extraction of Black Garlic This study used BG obtained from Temanggung.
Wonosobo.
Central Java Province.
Indonesia.
In the study, we used 500 kg of freeze-dried black garlic as a All secondary metabolites will be expected to dissolve during the ethanol maceration process.
The fractionation technique is used to separate compounds based on polarity.
Nonpolar compounds are predicted to dissolve in an n-hexane solvent, while semipolar compounds are expected to dissolve in an ethyl acetate solvent.
Table 1 contains data from the extraction and partitioning of BG.
The yields of ethanol extract, ethyl acetate fraction, and n-hexane fraction of BG in the present research were 31.
00%, respectively.
According to Atun et al.
, the ethanol extract and hexane fraction of BG contains many phenolic and flavonoid compounds.
Antiangiogenesis Activity of Black Garlic The CAM assay was used in the study to investigate antiangiogenesis activity.
The approach has been widely utilized to explore the basic principles of blood vessel development, such as endothelial cell migration, proliferation, and differentiation.
CAM is well adapted to studying the pro- or anti-angiogenic effects of compound natural and pharmaceutical stimuli (Laschke et al.
CAM is easier to work with than other media, such as rabbit corneas (Ribatti et al.
Furthermore, the CAM membrane is a highly vascularized area with numerous blood vessels that are easily observed in line with the growth process of the chicken embryo (Ribatti, 2.
We used the basic Fibroblast Growth Factor .
FGF) as an angiogenic inducer in this study, which makes it easier to observe changes in the development of new vascular The bFGF has a mechanism by attaching to particular receptors on blood vessel endothelial cells and triggering growth Endothelial cells produce protease enzymes, then proliferate and migrate, eventually forming a new extracellular The newly generated blood vessels in the last step are structurally stabilized so blood can circulate (Ribatti et al.
, 2.
The dose of bFGF used for each treatment is We used six groups in this study, three control groups and three sample groups.
Each group's treatment was repeated three The control group consisted of PBS solvent, bFGF, and DMSO solvent.
The sample group consisted of ethanol extract, ethyl acetate fraction, and n-hexane fraction of BG with concentrations of 20, 40, and 80 AAg/mL.
CAM macroscopic results and the average number of new blood vessels in each group are shown in Figure 3 and Table 2.
Figure 4 represents the percentage of DOI: https://doi.
org/10.
17509/ijost.
p- ISSN 2528-1410 e- ISSN 2527-8045 Arianingrum et al.
Antiangiogenesis Activity of Indonesian Local Black Garlic A | 492 Table 1.
The result of extract and fraction of BG.
Material Weight 500 g BG 250 mg ethanol extract 250 mg ethanol extract Concentrated Extract Weight Ethanol extract 158.
Ethyl acetate fraction 90 mg n-hexane fraction 20.
01 mg Yield (%) Figure 3.
CAM macroscopic observation of ethanol extract, ethyl acetate fraction, and nhexane fraction of BG.
The green star indicated paper disk, the black arrow indicated main blood vessels and the blue arrow indicated new blood vessels.
DOI: https://doi.
org/10.
17509/ijost.
p- ISSN 2528-1410 e- ISSN 2527-8045 493 | Indonesian Journal of Science & Technology.
Volume 8 Issue 3.
December 2023 Hal 487-498 Table 2.
The number of new blood vessels from the control and the sample group.
Group Number of New Blood Vessels PBS control Negative control .
ng/AAL bFGF ) DMSO control .
ng/AAL bFGF DMSO) BG Ethanol extract 20 AAg/mL 1ng/AAL bFGF BG Ethanol extract 40 AAg/mL 1ng/AAL bFGF BG Ethanol extract 80 AAg/mL 1ng/AAL bFGF BG Ethyl acetate fraction 20 AAg/mL 1ng/AAL bFGF BG Ethyl acetate fraction 40 AAg/mL 1ng/AAL bFGF BG Ethyl acetate fraction 80 AAg/mL 1ng/AAL bFGF BG n-hexana fraction 20 AAg/mL 1ng/AAL bFGF BG n-hexana fraction 40 AAg/mL 1ng/AAL bFGF BG n-hexana fraction 80 AAg/mL 1ng/AAL bFGF 00A2.
00A1.
33A2.
33A3.
00A1.
00 A2.
67A4.
67A4.
00A3.
00A1.
33A1.
33A2.
Figure 4.
The antiangiogenic using ethanol extract, ethyl acetate fraction, and n-hexane fraction of BG.
The study showed that the number of blood vessels with the DMSO treatment .
ng/mL bFGF and DMSO) was not significantly different from the bFGF treatment .
ng/mL).
This demonstrates that the DMSO solvent does not affect blood vessel development.
DMSO at or above 0.
can change the morphology of the capillary In this study, the stock sample solution was dissolved in 0.
8% DMSO and DOI: https://doi.
org/10.
17509/ijost.
p- ISSN 2528-1410 e- ISSN 2527-8045 Arianingrum et al.
Antiangiogenesis Activity of Indonesian Local Black Garlic A | 494 subsequently diluted with distilled water until the final DMSO concentration was less 06%, which did not influence the number of blood vessels.
In this study, bFGF increased the number of blood vessels by 37%.
We discovered that BG ethanol extract at 20, 40, and 80 g/mL effectively reduced angiogenesis .
08%) in a dose-dependent The ethyl acetate fraction suppressed angiogenesis significantly, but its efficacy was lower than that of the ethanol extract .
46%).
The nhexane fraction at the same concentration showed the highest angiogenesis activity .
35%) compared to the ethanol extract and n-hexane fraction.
This study concludes that black garlic has the potential to inhibit angiogenesis.
So far, research on BG's angiogenesis activity has been quite restricted.
Several chemicals in fresh garlic (FG) have been reported to suppress angiogenesis, including Alliin (Mousa & Mousa, 2.
, diallyl trisulfide (DATS) (Xiao et al.
, 2.
, and Allicin .
iallyl-thiosulfinat.
(Sela et al.
Alliin significantly inhibits the secretion of fibroblast growth factor-2 (FGF.
and VEGF in the CAM model (Mousa & Mousa, 2.
Diallyl trisulfide (DATS) influences the angiogenesis properties of endothelial cells (EC) (Xiao et al.
, 2.
, and Allicin .
suppresses VEGF and bFGFinduced blood vessel formation (Sela et al.
The Alliin content of BG decreases during the preparation process, whereas the quantities of flavonoids, pyruvate, alkaloids, organic acids, and phenols increase (Ryu & Kang, 2017.
Ahmed & Wang, 2.
Alkaloids and flavonoids may be present in the BG ethanol extract, ethyl acetate, and n-hexane DATS was also discovered in BG (Ahmed and Wang, 2.
The compound might be able to inhibit angiogenesis caused by BG.
According to Xiao et al.
DAS
inhibits Akt phosphorylation and suppresses the expression of vascular endothelial growth factor (VEGF) receptors on cell membranes .
ndothelial cell.
Several studies have found a synergistic relationship between antioxidants and antiangiogenesis and anticancer (Mousa & Mousa, 2005.
Ahmed & Wang, 2.
Lu et .
discovered DL-lactic acid.
5hydroxymethyl-2-furfural.
S,3S)-1-methyl-1,2,3,4-tetrahydro-bcarboline-3-carboxylic acid.
R, 3S)-1methyl-1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid.
and 2-acetylpyrrole ABCD in BG with high antioxidant activity.
Therefore, in this work, we performed a docking test to investigate how it affected VEGFR.
In Silico Study The results of energy calculations between the ligand and the receptor are shown in Table 3.
In this study, we used bevacizumab as a positive control.
This drug However, bevacizumab can cause severe intraocular inflammation (Li et al.
, 2.
Therefore, for effective anti-angiogenesis treatment, it is necessary to discover potential VEGFR inhibitors from natural products using computer-assisted approaches.
In this study we used PDB ID: 2P2I (Arianingrum, 2.
, 3VHE, and 3HNG (Asthana, 2.
The docking approach was validated by comparing the RMSD score of the target protein PDB with its natural ligand.
The RMSD score between the target protein PDBs and the natural ligand in this study was less than 2 angstroms (Table .
The data indicated that the protocol was acceptable because the validation conditions were completed, and molecular docking could then be performed (Yusuf et al.
, 2.
These findings indicate that Bevacizumab has the lowest binding energy.
Based on the results of molecular docking studies, the active compounds contained in black garlic extract that have low binding energy are .
R,3S)-1-methyl-1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid and .
S,3S)-1DOI: https://doi.
org/10.
17509/ijost.
p- ISSN 2528-1410 e- ISSN 2527-8045 495 | Indonesian Journal of Science & Technology.
Volume 8 Issue 3.
December 2023 Hal 487-498 methyl-1,2,3,4-tetrahydro-b-carboline-3carboxylic acid.
The lower the binding energy level, the stronger the interaction between the ligand and receptor.
The interaction that occurs is the interaction between VEGFR .
PI, 3HVE, and 3HNG) and the active compounds in black garlic.
Even though the active compound in BG has a higher energy than the original ligand and Bevacizumab, the docking results show that there are similar amino acids in the interaction (Figure .
The similarity of amino acids involved in the interaction between ligand and receptor causes inhibition of the activity of the VEGFR which is responsible for angiogenesis.
Inhibition of VEGFR activity causes inhibition of angiogenesis activity.
These docking results show that the active compounds in BG have the potential to inhibit angiogenesis.
Table 3.
The binding activity of ligand and receptor.
Ligand
Native Ligand
Bevacizumab
R,3S)-1-methyl-1,2,3,4-tetrahydro-b-carboline-3carboxylic acid
S,3S)-1-methyl-1,2,3,4-tetrahydro-b-carboline-3carboxylic acid
Adenosine Uridine 2-acetylpyrrole 5-hydroxymethyl-2-furfural DL-lactic acid
2P2I
RMSD: 1.
Binding Affinity 3HVE
3HNG
RMSD: 0.
859 RMSD: 0.
Figure 5.
Visualization of the interaction of compounds in black garlic with 2P2I receptors.
DOI: https://doi.
org/10.
17509/ijost.
p- ISSN 2528-1410 e- ISSN 2527-8045 Arianingrum et al.
Antiangiogenesis Activity of Indonesian Local Black Garlic A | 496
CONCLUSION
ACKNOWLEDGMENT
The results showed that the ethanol extract, ethyl acetate fraction, and n-hexane BG fraction had antiangiogenesis activity.
bioactive chemicals can interfere with the binding of VEGF receptors with other ligands, inhibiting their pro-angiogenic activity.
This study provides information that BG has the potential to be developed as an antiangiogenesis drug for cancer treatment.
We would like to thank Yogyakarta State University for funding this research through a research grant.
AUTHORSAo NOTE The authors declare that there is no conflict of interest regarding the publication of this article.
The authors confirmed that the paper was free of plagiarism.
REFERENCES