127
EVALUATION ON ANTIOXIDANT ACTIVITY AND
ACTIVE COMPONENTS OF TRIGONA ITAMA PROPOLIS EXTRACT AND ITS POTENTIAL AS SARS-CoV2 INFECTION INHIBITORS
Elma Putri Primandasari1*, Agus Susilo2, Khothibul Umam Al-Awwaly2, Miftakhul Cahyati3,Dewi Masyithoh4
1Department of Business and Hospitality - Faculty of Vocational - Universitas Brawijaya Jl. Veteran – Malang 65145
2Department of Animal Husbandry - Faculty of Animal Science - Universitas Brawijaya Jl. Veteran – Malang 65145
3Department of Oral Medicine - Faculty of Dentistry - Universitas Brawijaya Jl. Veteran – Malang 65145
4 Department of Animal Husbandry - Faculty of Animal Husbandry - Universitas Islam Malang Jl. Mayjen Haryono – Malang 65144
Corresponding Author, email : [email protected]
Submitted : 28 June 2022 Revised : 6 August 2023 Accepted : 24 August 2023
ABSTRACT
Propolis Trigona itama has a blackish brown color and has a bitter taste. Propolis contains immunomodulator, antibacterial, antifungal, and antiviral. Efforts made to determine the potential of propolis require an extraction process. The extraction process aims to obtain propolis ready for consumption. Propolis has thermostable properties with a melting point of 60–65 C, so it does not require high temperatures in the extraction process. The extraction process using MAE requires a faster time because the pressure and temperature can be controlled. This study used a factorial completely randomized design with 2 factors with 9 treatments and 3 replications. The data obtained were analyzed by analysis of variant (ANOVA). If there is a difference, it is continued with the DUNCAN multiple distance test. The results of the extraction using the MAE method of propolis have antioxidant activity using the DPPH method of 1,460µg/ml to 1,413 µg/ml which is expressed in IC50. Trigona itama propolis extract had an average antioxidant value of 0.22 µg/ml to 0.57 µg/ml using the FRAP method.There is a unique component in the GCMS results of Trigona itama propolis extract called bisalcofen. Bisalcofen is an active component which has antioxidant properties. The results of the bisalcofen docking score of -7.6 on the ACE2 protein. Bisalcofen has a stronger potency than umifenovir which can be used as an inhibitor of SARS-CoV2 infection
Keywords: Antioxidant; Bisalcofen; Covid-19; Trigona propolis
INTRODUCTION
Propolis is a hard resin produced by bees from shoots, leaves and plant exudates (Rismawati, 2017). One of the bees that can produce propolis is the Trigona itama bee.
The production of Trigona itama bees is estimated at 10-300 g per colony per year.
Propolis has a bitter taste, has a blackish brown color and has active ingredients such as antimicrobial, antioxidant, and anesthetic properties. The content of
antioxidant activity in Trigona itama propolis ranges from 166-987 ppm, flavonoid content 1,000-1,789 mg/g and phenolic content 9,603-15,170 mg/g (Rosyidi et al., 2018).
The content of active substances in different propolis based on the quality of propolis is influenced by several factors, namely, the location of origin of propolis, types of bees and plants, storage methods, solvents and solution concentration, extraction methods and physicochemical
128 propolis. Several things besides the type of solvent that can affect the extraction of propolis, namely the exposure time and temperature used, the longer the extraction time used, the more material that can be extracted because the process of contact between the material and the solvent is getting bigger (Winata et al., 2015). Propolis has the ability as an antibacterial and immunomodulatory (Khadim, 2020).
Propolis has bioactive components and antioxidant properties that can be used as antibacterial, antifungal, and antiviral (Rismawati, 2017). Based on the components contained in propolis, it is necessary to research the ability of propolis components as anti-Covid-19.
This study focuses on identifying the active substance of propolis as a SARS CoV-2 drug that targets ACE2 from SARS CoV-2. ACE2 is an enzyme that inhibits the corona virus and has an important role as a receptor when it infects, making it the main target of SARS CoV-2 drugs (Jin et al., 2020).
Covid-19 (Corona Virus Disease 2019) is a disease outbreak that is capable of infecting the human respiratory tract (WHO, 2020).
The development of drugs and vaccines or antiviral therapy is urgently needed for SARS CoV-2. The drug currently used contains the active substance Umifenovir as an antiviral to inhibit ACE2 (Angiotnsin- Corverting Enzyme 2). ACE2 is used by viruses as receptors to infect the host (Guo, 2020). Based on this, it is necessary to extract and identify bioactive compounds in Trigona itama propolis in East Java and evaluate its potential as an in silico anti- Covid-19 agent.
METHOD
The material used in this research is Trigona itama propolis from Batu City and Mojekerto Regency. The materials used in this study were distilled water, hexane (Merck), DPPH (Merck), FeSO4 (Emsure) phosphate buffer (Merck), potassium hexacyanoferrate (Emsure), TCA (Emsure), FeCl3 (Emsure). The tools used in this research are microwave merk sharp type R21DOand UV-Vis spectrophotometer type 721.
Production of Propolis Extract (Hamzah and Leo, 2015 with modified)
The Trigona itama propolis extraction process uses a ratio propolis to solvent 1:5.
Propolis, which has been cleaned of various impurities, is cut into small pieces and weighed as much as 20 g. Propolis was put into a 250 ml enlemeyer and 100 ml of hexane solvent was added. Extraction was carried out using a modified Soxhler microwave assisted extraction with power levels (low, medium, and high) and exposure time (10, 20, and 30 minutes). The propolis extract obtained was then left to stand overnight in a glass beaker covered with plastic wrap. The next process is filtering using whatman paper No.01 with a diameter of 12.5 mm. The propolis extract was put into cool storage and filtered again to get a clear propolis extract.
Antioxidant Activity by DPPH Method (Segura-Campos et al., 2014)
Analysis of antioxidant activity using DPPH (2.2 diphenyl-1-picrylhydrazyl) with a concentration of 0.11 M was added to the sample as much as 1 ml. The mixture was homogenized and incubated at room temperature for 30 minutes. Measurements were made using a spectrophotometer at a wavelength of 517 nm. Preparation of standard DPPH curve using concentrations of 4, 8, 16 and 32 mg/l.
Antioxidant by FRAP Method (Maesaroh et al., 2018)
Antioxidant levels were tested using the FRAP (Ferric Reducing Antioxidant Power) method using 0.5 ml of propolis extract, 0.5 ml of 0.5 ml of phosphate buffer pH 6.6 added, 0.5 ml of potassium hexacyanoferrate added. The solution was incubated at 50 C for 20 minutes and 0.5 ml of 10% TCA solution was added (if two layers occur, they are separated using a centrifuge). The top layer was taken as much as 0.5 ml and added 0.5 ml of distilled water, 0.1% FeCl3 as much as 0.5 ml. The solution was incubated at room temperature for 5 to 10 minutes. Measured using a spectrophotometer with a wavelength of 700 nm. The standard curve uses Fe2+ with a concentration of 0.88; 0.44;
0.22; 0.11; and 0.05 µm.
129 FTIR (Fourier Transform Indinfrared Spectrometer) (Chong and Chua, 2020)
The liquid propolis extract was separated from the solvent by oven for. All MIR spectra were obtained between 4000 and 400 cm−1 using an optical resolution of 8 cm−1 and 32 accumulations. Horizontal ATR signal, the refractive index (η) of diamond is 2.4 and leads to a penetration depth (Dp) of 1.66 m, with a data interval of 1 cm−1 and a scan speed of 0.2 cm/s.
Approximately 0.05 ml of the sample was placed onto the ATR crystal, and after each analysis, the ATR crystal was washed with deionized water and dried with a non- abrasive cloth.
GC-MS (Gas Crhomatography-Mass Spectrometry
)
(Wyan et al., 2021)Taken as much as 5 ml of distillate propolis extract for use in each test. The Hewlett-Packard 6890 series gas chromatograph coupled with a Hewlett- Packard 5973 mass selective detector with a 30 m x 250 m x 0.28 m HP5-MS column was used for GC-MS analysis. The total analysis walk was 36 minutes. With an injector temperature of 110 C, the program temperature of 110 C was held for two minutes and then raised to 280 C at a rate of 10 C/min, and a hold for 15 minutes at 280 C. Helium was used as the carrier gas, the flow rate was 1.5 ml/min. Upon completion, the peaks were identified through their MS spectra using the system database (NIST Mass Spectral Library).
In Silico Testing using Molecular Docking
Re-docking is carried out, i.e., the separation of the original ligand and protein and then putting it back into place on the protein by adjusting the size and center of the search box (Flamandita et al., 2020). Reinstallation and docking simulation performed by Autodock PyRx.
Automatic docking tool with easy operational use, high processing accuracy, prediction speed and binding mode (Trott et al., 2010). Autodock PyRx was chosen because of its higher accuracy in terms of binding mode prediction (Vieira et al., 2019). Determining a specific search space in a protein involves three criteria for the
number of points in the dimensions x, y, z (square grid size), grid center grid and grid spacing. Download a re-docking simulation of multiple shapes, the first being generally the best form with the lowest score docking. Then the Root Square Mean Deviation (RSMD) between the native ligand and the result of re-docking the first form was evaluated using PyMol. Python- based software that can be used for protein ligand modeling (Yuan et al., 2017). Results are classified as accurate if the RSMD value is less than 2 (Ramirez et al., 2018). The re- docking coordinates are then used to dock propolis compounds into target proteins.
The propolis compound was tethered to each receptor using the parameters and coordinates obtained from the re-docking simulation. The results of the lowest docking score were considered as the best form used to analyze the interaction between propolis compounds, as ligands, and proteins. The interaction profile is then represented in a 2D visualization using the Discovery Studio Visualizer. Discovery Studio Visualizer is an automated program that generates multiple two-dimensional (2D) diagrams of ligand–protein interactions of anchored complexes.
Determination of the ability of active components in propolis was carried out in silico using molecular docking, using PDB ID ACE2 1R42.
Data Analysis
The study used an experimental method with a factorial completely randomized design consisting of 2 factors with 9 treatments and 3 replications. The data obtained were analyzed by analysis of variance (ANOVA) using Microsoft excel 2007. If there is a difference, it is continued with the DUNCAN multiple distance test.
RESULTS AND DISCUSSION Antioxidant Activity of DPPH Method
The result of statistical analysis showed that the power level, exposure time and interaction of extraction had a very significant effect (p<0.01) on antioxidant activity. The IC50 value increases with increasing power level and increasing
130 extraction time. IC50 value is defined as the ability of a component in propolis to inhibit oxidation cellular proliferation by 50%. The lower the IC50 value, the better the ability of the components in propolis to inhibit the oxidation process caused by the presence of free radicals. The best IC50 value in this study was 1.413 in sample P21 with the extraction process using a medium power level for 10 minutes. South Sulawesi propolis has an EC50 antioxidant activity of 69.96 µg/ml (Pratami, et al., 2018). This shows that Trigonna itama propolis has a better inhibition ability with a low IC50
value. The results of antioxidant analysis can be seen in Figures 1, 2, and 3.
The use of a medium power level for extraction using hexane as a solvent resulted in an extract temperature of 50 °C.
The optimal temperature for a short period of extraction and maximum yield of bioactive constituents is 70 °C. Propolis microwave exposure for too long at high temperatures can harm its thermolabile compounds and increase the solubility of propolis wax substances (Paviani et al., 2013). Cavitation causes high temperature and pressure so that it can reduce particle size, increase mass transfer. The high extraction temperature accelerates the extraction rate, while the high pressure prevents boiling at temperatures above the normal boiling point of the solvent.
(Bankova, et al., 2021). Several authors also studied the effect of extraction temperature, the optimal temperature was 60–65 °C (Oroian et al., 2020).
Figure 1. The Results of the Average Difference in the Level of Extraction Power
of Trigona Itama Propolis Antioxidants (µg/ml)
Figure 2. The Results of the Average Difference in the Extraction Time of the Propolis Antioxidant Trigona Itama (µg/ml)
Figure 3. The Results of the Average Difference in the Extraction Interaction
with the Propolis Antioxidant Trigona itama(µg/ml). P11 : low power level and 10
minutes, P12 : low power level and 20 minutes, P13 : low power level and 30 minutes, P21 : medium power level and 10 minutes, P22 : medium power level and 20 minutes, P23 : medium power level and 30
minutes, P31 : high power level and 10 minutes, P32 : high power level and 20 minutes, and P33 : high power level and 30
minutes
Antioxidant Level FRAP Method
The results of statistical analysis showed that the power level, exposure time and interaction of extraction had a very significant effect (p<0.01) on antioxidant levels. Antioxidant levels increase with increasing power level and extraction time.
The highest antioxidant content of propolis extract is 0.57 µg/ml with a highpower extraction process for 30 minutes. The results of the antioxidant analysis can be seen in Figures 1, 2, and 3. South Sulawesi propolis has a FRAP antioxidant level of 32.1 µg/ml (Pratami, et al., 2018). Propolis from other parts of Indonesia derived from Tetragonula bees has a FRAP antioxidant activity of 189.05 mg/g (Farida, et al., 2022).
131 These results indicate that the antioxidant Trigona itama propolis in this study was lower.
The use of highpower levels and hexane as solvent in the extraction process produces extracts with a temperature of 60
°C which is the ideal temperature to maintain the content of active ingredients that are antioxidants in propolis. The FRAP methodology was carried out to determine the ability of a substance to bind iron ions from oxidation (Kumar, 2015). The FRAP method is based on the compound's ability to reduce the induced potassium ferricyanide from ferrous ions (Fe+3) to ferrous ions (Fe+2). Ionic Fe+3 is chelated with a nucleophilic aromatic ring as a certain chelator group present in polyphenolic compounds. The increase in absorbance indicates a high reducing power. There is a relationship between the content of phenolic compounds in propolis and the reduction capacity of iron ions that contribute to its potential as an antioxidant.
FTIR (Fourier Transform Infrared Spectroscopy
)
The results of FTIR analysis in Trigona itama propolis extract showed that Trigona itama propolis had C-H and OH functional groups which were alkanes and carboxylicacids at wave numbers 2920.88 cm-1 and 2853.85 cm-1. The N=O (Nitro) double bond is also found in the wave number 1436.16 cm-1. Carboxylicacids with C=O double bonds were found at wave number 1702.90 cm-1 and alkanes reappeared at wave numbers 1377.72 cm-1 with C-H bonds (-CH3). Amines groups
with C-N bonds are found at wave number 1025.45 cm-1 and there are also functional groups from alcohols, esters and esters with C-O bonds, besides appearing at wave numbers 1025.45 cm-1 these functional groups also appear at wave numbers 1183.76 cm-1, 1275.03 cm-1, 1096.76 cm-1. Aromatic groups appear at wave numbers 888.53 cm-1, 825.78 cm-1, and 721.66 cm-1 with C-H bonds. The functional groups of amides with C=O double bonds, alkenes with C=C double bonds appear at wave number 1605.91 cm-1 and at wave numbers 3421.48 cm-1 there are primary and secondary functional groups of amines and amides. The results of the FTIR analysis can be seen in Figure 4.
GC-MS (Gas Chromatography-Mass Spectrometry
)
The results of the screening of Trigona itama propolis extract components using GCMS such as Nonacosane, Tetracontane, Hexadecane and Bisalcofen.The GC-MS results can be seen in Figure 5.
Bisalcofen that appears in the GC-MS results is Phenol, 2,2'-methylenebis[6-(1,1- dimethylethyl)-4-meth y1l7- with the chemical formula C23H32O2 with CID 8396.
Bisalcofen has not been found in the original Trigona propolis extract which extracted using low power for 30 minutes.
Bisalcofen appears in the extraction using medium power for 30 minutes and can last up to extraction using high power for 30 minutes.
Figure 4. Results of FTIR Analysis of Trigona itama Propolis Extract
132
Figure 5. Results of GC-MS Analysis of Trigona itama Propolis Extract Bisalcofen Result of Molecular Docking
The components in propolis extract can be used orally if they meet the requirements of Lipinski. The requirenment Lipinski i.e.must can be used as an oral drug, molecular weight <500, Log P <0.05, H-Bone Donor <5, H-Bone Acceptor <10 i.e, Rotatable bonds <10 and have high absorption intestines.Bisalcofen is the only component that meets Lipinski's requirements.Bisalcofen has provisions based on Lipinski, namely it can be used as an oral drug "Yes", molecular weight 340 mol, Log P 4.14, H-Bone Donor 2, H-Bone Acceptor 2, Rotatable bonds 4 and have
“High” absorption. Components that can meet these requirements are then carried out molecular docking. The structure of the interaction between ACE2 and Bisalcofen from molecular docking can be seen in Figure 6. The structure of the interaction between ACE2 and umifenovir from molecular docking can be seen in Figure 7.
Figure 6. 3D View of the Interaction Structure of Bisalcofen with ACE-2
Figure 7. 3D View of the Interaction Structure of Umifenovir with ACE-2
The molecular docking results for bisalcofen have a lower binding affinity value than umifenofir. The binding affinity of bisalcofen is -7.6 which means that this component has the ability to bind to ACE2 as an anti-Covid-19 inhibitor (Table 1).
Table 1. Binding affinity and residues produced by bisalcofen and umifenovir.
Component Binding
Affinity Residue Bisalcofen -7.6 TYR127, SER128,
LEU144, GLU145, ASN149, ASP269, MET270, TRP271, ARG273, PHE274, LEU503, PHE504, HIS505.
Umifenovir -5.8 TYR127, LEU144, GLU145,TRP271, ARG273, PHE504, HIS505,
TYR510,TYR515.
Note: The smaller the binding affinity value, the smaller the energy required for the component to bind ACE-2
133 Bisalcofen not only has the ability to inhibit viruses, but also has abilities such as antibacterial, antifungal, antioxidant, anti- inflammatory and also as an immunomodulator. The ability of a compound as an active component is expressed in the value of pa (probability to
be active). A pa value of more than 0.7 indicates that the compound has a potential, the higher the pa value, the better the accuracy of the compound's ability (Filimonov et al., 2014). The potency of bisalcofen can be seen in Figure 8.
Figure 8. The Active Potential of Bisalcofen Contained in Trigona itama Propolis Extract Natural ingredients that have
immunomodulatory properties and various antiviral activities can potentially be inhibitors of SARS-CoV2 infection.
According to Pratami, et al. (2021) propolis extract can be used as an extract of natural ingredients that can be developed for therapy in SARS-CoV2 infection. Propolis originating from the tropics has a variety of chemical components produced from local biological sources. Bancova, at al. (2014) revealed that propolis originating from the Argentine Andes contains essential oils, in contrast to Trigona propolis originating from East Java which contains bisalcofen components. The component in propolis, namely bisalcofen, requires less energy to inhibit SARS-CoV2 compared to umifenofir. The natural products contained in the Trigona propolis, namely the bisalcofen component, have a greater potential to be used as an inhibitor of SARS-CoV2 infection.
CONCLUSIONS
The component contained in Trigona itama propolis in East Java and has the ability as an antioxidant is Bisalcofen which
is produced from the extraction process using MAE with medium power level with a time of 30 minutes. Bisalcofen has potential as an inhibitor of SARS-CoV2 infection with a binding affinity of -7.6.
ACKNOWLEDGMENT
Thanks to Mr. Khothibul Umam Al- Awwaly who has funded the research and PT. Kembang Joyo Sriwijaya Malang who has supported the research.
REFERENCES
Bankova, -V., Popova, -M., Trusheva, -B., 2014. Propolis volatile compounds:
chemical diversity and biological activity: A review. Chemistry Central Journal. 8(1), 1-8.
https://doi.org/10.1186/1752- 153X-8-28
Bankova, -V., Trusheva, -B., Popova, -M., 2021. Propolis extraction methods:
A review. Journal of Apicultural Research. 60(5), 734-743.
https://doi.org/10.1080/00218839.
2021.1901426
134 Farida, -S., Pratami, D, -K., Sahlan, -M.,
Laksmitawati, D, -R., Rohmatin, -E., Situmorang, -H., 2022. In-vitro antioxidant, in-vivo anti- inflammatory, and acute toxicity study of Indonesian propolis capsule from Tetragonula sapiens.
Saudi Journal of Biological Sciences,
29(4), 2489-2500.
https://doi.org/10.1016/j.sjbs.2021 .12.034
Filimonov, D, -A., Lagunin, A, -A., Gloriozova, T, -A., Rudik, A, -V., Druzhilovskii, D, -S., Pogodin, P, -V., Poroikov, V, -V., 2014. Prediction of the biological activity spectra of organic compounds using the PASS online web resource. Chemistry of Heterocyclic Compounds. 50(3), 444- 457.
https://doi.org/10.1007/s10593- 014-1496-1
Flamandita, -D., Lischer, -K., Pratami, D, - K., Aditama, -R., Sahlan, -M., 2020.
Molecular docking analysis of podophyllotoxin derivatives in sulawesi propolis as potent inhibitors of protein kinases. AIP Conference Proceedings. AIP Publishing LLC. 2230 (1), 20010- 20019.
https://doi.org/10.1063/5.0002596 Guo, Y, -R., Cao, Q, -D., Hong, Z, -S.,Tan,
Y, -Y., Chen, S, -D., Jin, H, -J., Tan, K, -S., Wang, D, -Y., Yan, -Y., 2020.
The origin, transmission and clinical therapies on coronavirus disease 2019 (COVID-19) outbreak–
an update on the status. Military Medical Research. 7(1), 1-10.
https://doi.org/10.1186/s40779- 020-00240-0
Hamzah, -N., Leo, C, -P., 2015.
Microwave assisted extraction of Trigona Propolis: The effects of processing parameters. International Journal of Food Engineering. 11 (6), 861-870.
https://doi.org/10.1515/ijfe-2015- 0106
Jin, -Z., Du, -X., Xu, -Y., Deng, -Y.,Liu, - M., Zhao, -Y., Yang, -H., 2020.
Structure of MPro from SARS CoV- 2 and discovery of its inhibitors.
Nature. 582(7811), 289-293.
https://doi.org/10.1038/s41586- 020-2223-y
Kadhim, A, -H., Hadi, N, -R., Abdulhussein, -M., Abdulhussein, - M., Zamil, S, -T., Zamil, S, -T., 2020.
Preprocessing of the candidate antiviral drugs against COVID-19 in models of SARS-CoV2 targets. La Prensa Medica Argentina. 106(2), 1- 10.
https://www.scholarsliterature.co m/article_pdf/4/scientific_4_315_3 1082020024222.pdf
Kumar, S., 2015. Analytical Techniques for Natural Product Research. CABI, India
Maesaroh, -K., Kurnia, -D., Anshori, J, - A., 2018. Perbandingan metode uji aktivitas antioksidan DPPH, FRAP dan FIC terhadap asam askorbat, asam galat dan kuersetin. Chimica et Natura Acta. 6(2), 93-100.
https://doi.org/10.24198/cna.v6.n 2.19049
Oroian, -M., Ursachi, -F., Dranca, -F., 2020. Influence of ultrasonic amplitude, temperature, time and solvent concentration on bioactive compounds extraction from propolis. Ultrasonics sonochemistry.
64, 1-10.
https://doi.org/10.1016/j.ultsonch.
2020.105021
Paviani, L. -C., Fiorito, -G., Sacoda, -P., Cabral, F, -A., 2013. Different solvents for extraction of Brazilian green propolis:composition and extraction yield of phenolic compounds. InIII Iberoamerican Conference on Supercritical Fluid, 1–7.
http://www.nupeg.ufrn.br/prosci ba/prosciba2013/Papers/T2-11.pdf
135 Pratami, D, -K., Desmiaty, -Y.,
Simorangkir, E, -M., Faradhila, -D., 2021. Standardisasi dan uji aktivitas antioksidan ekstrak bahan alam propolis untuk terapi infeksi SARS- CoV2. Jurnal Ilmu Kefarmasian Indonesia. 19(2), 272-280.
https://doi.org/10.35814/jifi.v19i2.
1062
Pratami, D, -K., Mun’im, -A., Sundowo, - A., Sahlan, -M., 2018.
Phytochemical profile and antioxidant activity of propolis ethanolic extract from Tetragonula bee. Pharmacognosy Journal, 10 (1), 128-135.
https://doi.org/10.5530/pj.2018.1.
23
Ramirez, -D., Caballero, -J., 2018. Is it reliable to take the molecular docking top scoring position as the best solution without considering available structural data. Molecules.
23(5), 1038-1043.
https://doi.org/10.3390/molecules 23051038
Rismawati, S, -N., Ismiyati, -I., 2017.
Pengaruh variasi pH terhadap kadar flavonoid pada ekstraksi propolis dan karakteristiknya sebagai antimikroba. Jurnal
Konversi. 6(2), 89-94.
https://doi.org/10.24853/konversi .6.2.89-94
Rosyidi, -D., Radiati, L, -E., Minarti, -S.,.
Mustakim, Susilo, -A., Jaya -F., Azis, -A., 2018. Perbandingan sifat antioksidan propolis pada dua jenis lebah (Apis mellifera dan Trigona itama ) di Mojokerto dan Batu, Jawa Timur, Indonesia. Jurnal Ilmu dan Teknologi Hasil Ternak. 13(2), 108- 117.https://doi.org/10.21776/ub.jit ek.2018.013.02.5
Segura-Campos, -M., Barbosa-Martín, -E., Matus-Basto, -A., Cabrera-Amaro, - D., Murguía-Olmedo, -M., Moguel- Ordo,-Y., Betancur-Ancona, -D., 2014. Comparison of chemical and functional properties of Stevia rebaudiana (Bertoni) varieties cultivated in mexican southeast.
American Journal of Plant Sciences.
5(3), 1-8.
https://doi.org/10.4236/ajps.2014.
53039
Trott, -O., Olson, A, -J., 2010. AutoDock vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading.
Journal of Computational Chemistry.
31(2), 455–461.
https://doi.org/10.1002/jcc.21334 Vieira, T, -F., Sousa, S, -F., 2019.
Comparing autodock and vina in ligand/decoy discrimination for virtual screening. Applied Sciences.
9(21), 4538-4543.
https://doi.org/10.3390/app92145 38
Winata, E, -W., Yunianta, -Y., 2015.
Ekstraksi antosianin buah murbei (Morus alba L.) metode ultrasonik batch (kajian waktu dan rasio bahan: Pelarut. Jurnal Pangan dan Agroindustri. 3 (2), 773-783.
https://jpa.ub.ac.id/index.php/jpa /article/view/199/205
World Health Organization. 2020. Mental Health and Psychosocial Considerations During the COVID-19 Outbreak. WHO
Wyan, L, -O., Charland J, -M., Mojica, E, R, -E., 2021. Comparative study of the extraction methods for the instrumental analysis of bee propolis. Undergraduate Journal of Teaching&Research. 1, 51-62.
https://par.nsf.gov/servlets/purl/
10284126
136 Yuan, -S., Chan, H, C, -S., Hu, -Z., 2017.
Using PyMOL as a platform for computational drug design.
Wiley Interdisciplinary Reviews: Computational Molecular Science. 7 (2), 1298-1311.
https://doi.org/10.1002/wcms.1298
