Construction of Peptide Vaccine Candidate Based on β-Cell Epitopes of Indonesian Monkeypox Virus (MPXV) Virulence Protein:A Reverse Vaccinology

Viol Dhea Kharisma^1,2, A. N. M. Ansori³, Ahmad Affan Ali Murtadlo²,

Muhammad Hermawan Widyananda⁴, Md. Emdad Ullah⁵, Sin War Naw⁶,

Vikash Jakhmola⁷, Kiran Dobhal⁷, Tarun Parashar⁷, Maksim Rebezov^8,9,10,

Rahadian Zainul^11,12*

¹Department of Biology, Faculty of Science and Technology, Universitas Airlangga, Surabaya, Indonesia.

²Computational Virology Research Unit, Division of Molecular Biology and Genetics,

Generasi Biologi Indonesia Foundation, Gresik, Indonesia.

³Professor Nidom Foundation, Surabaya, Indonesia.

⁴Department of Biology, Faculty of Mathematics and Natural Sciences,

Universitas Brawijaya, Malang, Indonesia.

⁵Department of Chemistry,Mississippi State University, Mississippi, United States.

⁶Department of Chemistry, Myitkyina University, Myitkyina, Myanmar.

⁷Uttaranchal Institute of Pharmaceutical Sciences, Uttaranchal University, Uttarakhand, Dehradun, India.

⁸Department of Scientific Research, Russian State Agrarian University –

Moscow Timiryazev Agricultural Academy, Moscow, Russian Federation.

⁹Faculty of Biotechnology and Food Engineering, Ural State Agrarian University,

Yekaterinburg, Russian Federation.

¹⁰Department of Scientific Research, K.G. Razumovsky Moscow State University of Technologies and Management (The First Cossack University), Moscow, Russian Federation.

¹¹Department of Chemistry, Faculty of Mathematics and Natural Sciences,

Universitas Negeri Padang, Padang, Indonesia.

¹²Center for Advanced Material Processing, Artificial Intelligence, and Biophysic Informatics, Universitas Negeri Padang, Padang, Indonesia.

*Corresponding Author E-mail: rahadianzmsiphd@fmipa.unp.ac.id

ABSTRACT:

Infection with a DNA virus called monkeypox virus (MPXV) in humans has been identified in the Congo since 1970. Antiviral drugs are not effective for preventing MPXV infection. MPXV infection cases in Indonesia are very low but MPXV has the potential to become a global pandemic so it is very important to do prevention such as vaccine development. This study aims to construct a B cell epitope-based peptide vaccine candidate in Indonesian MPXV through an in silico approach.The development of the MPXV vaccine can be performed through a computational approach for preliminary studies. In silico-based construction of vaccines using B cell epitopes, antigenicity, allergenicity, docking, and molecular dynamics analysis have been used by researchers and scientists in solving viral infection cases. We recommend Pep A and Pep D as vaccine candidates because they allow recognition by B cells, antigenic peptides, non-allergenic and non-toxin. Peptide vaccine candidate can trigger B-cell activation to produce IgM isotype-specific antibodies through BCR interaction. In summary, the results of this study can be used for an initial study of MPXV vaccine development in Indonesia.

KEYWORDS: B-cell Epitope, Bioinformatics, MPXV, PeptideVaccine.

INTRODUCTION:

Infection with a DNA virus called monkeypox virus (MPXV) in humans has been identified in the Congo since 1970.¹ Research has continued to date, in May 2022 several cases of MPXV infection appeared in the world, scientists worried that an MPXV pandemic would threaten the world's human population.² MPXV can be spread from person to person through closed communication, skin lesions, and droplets.³ Scientific evidence of MPXV transmission through sexual contact such as vaginal or seminal fluids has not been identified.⁴ MPXV drug treatment has been used in various health facilities in the United Kingdom, but the use of antiviral drugs is not effective for preventing MPXV infection.⁵ MPXV infection cases in Indonesia are very low but MPXV has the potential to become a global pandemic so it is very important to do prevention such as vaccine development.⁶

Vaccine is the right solution for prevention of MPXV infection continuity.⁷ Production of traditional vaccines takes a long time and is expensive⁸. The development of the MPXV vaccine can be performed through a computational approach for preliminary studies. In silico-based construction of vaccines using B cell epitopes, antigenicity, allergenicity, docking, and molecular dynamics analysis have been used by researchers and scientists in solving viral infection cases.^9,10 This study aims to construct a B cell epitope-based peptide vaccine candidate in Indonesian MPXV through an in silico approach.

MATERIAL AND METHODS:

Target Retrieval:

MPXV virulence protein genome sequence (EPI_ISL_14615579) obtained from GISAID (https://gisaid.org/).The amino acid sequence in this study was obtained through a seq genomic modification process via MEGA 11 software, target sequence is saved in FASTA format on Notepad software.^11,12

3D Modeling and Validation:

The 3D structure of MPXV virulence protein was modeled via SWISS-MODEL (https://swissmodel.expasy.org/)with the homology modeling method. Assessment of the target 3D structure model was performed with the Ramachandran plot, 3D model visualized through PyMol v2.5 with structural selection.^13,14

B-cell Epitope Mapping:

The prediction of B-cell epitope mapping in this study is based on linear epitope prediction using the IEDB Analysis Resource (http://tools.iedb.org/bcell/). B cell linear epitope prediction works with the HMMs method and a trend scale to produce probability values. Input sample MPXV virulence protein in AA format for analysis running. Peptides from potential epitopes were obtained through this simulation.^15,16

Antigenicity, Allergenicity, and Toxicity Prediction:

This study uses VaxiJen v2.0 (http://www.ddg-pharmfac.net/vaxijen/ VaxiJen/VaxiJen.html), AllerTop v.0.2 (https://www.ddg-pharmfac.net/AllerTOP/),and ToxiPred (https://webs.iiitd.edu.in/raghava/toxinpred/design.php)for the prediction of antigenicity, allergenicity, and toxicity to obtain antigenic peptides with low probability values as allergens and non-toxic.^17,18

Peptide Stability:

Antigenic peptide stability in this study was identified through PortParam (https://web.expasy.org/protparam/) with reference to physicochemical parameters consisting of amino acids number, molecular weight, theoretical pI, instability index, and grand average of hydropathicity (GRAVY).^19,20

Peptide-Protein Docking:

Molecular interaction simulation in this study was performed using the peptide-protein docking method through ClusPro v2.0 (https://cluspro.bu.edu/login.php). The antigenic peptide of MPXV virulence protein acts as a ligand and the target is the B-cell receptor. Docking in this study aims to simulate antigen recognition through the direct pathway by B cells. Visualization of docking results is done through PyMol v2.5 with structural and color selection.²¹

Molecular Dynamic Analysis:

Interactions fluctuative in this study was identified through molecular dynamics simulations on CABS-flex 2.0 (http://biocomp.chem.uw.edu.pl/CABSflex2/index). Molecular dynamic parameters consist of restraints, rigidity, global c-alpha, number of cycles, temperature, and RNG seed. The output data is RMSF as a determinant of interaction stability.^22,23

Immune Simulation:

Simulation of the immune response in B cells was identified through C-ImmSim (https://kraken.iac.rm.cnr.it/C-IMMSIM/index.php?page=1) to simulate the type of antibody produced by B cells when injecting a vaccine into the body. Simulation parameters consisting of random seed: 12345, simulation volume: 10, and simulation steps: 100 are used in this study.^24,25

RESULTS AND DISCUSSION:

MPXV Sample Information:

Monkeypox virus (MPXV) is a zoonotic virus from the genus Orthopoxvirus, they have dsDNA as a genome, WHO explains that MPXV has the potential to trigger an epidemic or pandemic. This virus is 250nm in size, enveloped, brick-shaped, and can bind to glycosaminoglycans to enter the host. MPXV vaccine construction allows for the handling and prevention of viral infections.²⁶ This study used a sample of MPXV virulence protein as a target of vaccine design from GISAID (https://gisaid.org/)with the following information virus name: hMpxV/Indonesia/JK-NIHRD-MP0024/2022, accession ID: EPI_ISL_14615579, host: human, collection date: 2022-08-18, clade/lineage: IIb B.1, location: Asia/Indonesia/Jakarta, originating lab: RSUPN dr. Cipto Mangunkusumo, submitting lab: National Institute of Health Research and Development, specimen source: lesion swab. The MPXV virulence protein protein sequence obtained was 190-mer ('MTIYGLIAYLIFVTSSIASPLYIPVIPPISEDKSFNSVEVLVSLFPDDQKDYTVTSQFNNYTIGTKDWTINVLSTPDGLDIPLTNITYWSRFTIGRALFKSESEDIFQKKMSILGVSIECKKPSTLLTFLTVRKMTRVFNRFPDMAYYRGDCLEAVYVTMTYKNTKTGETDYTYLSNGGLPAYYRNGVDG').

3D Model of MPXV Virulence Protein:

MPXV virulence protein was modeled using the SWISS-MODEL homology modeling method (https://swissmodel.expasy.org/). Homology modeling works for the construction of template-based homologous models with a minimum similarity value of >20%.²⁷ Model validation analysis was carried out through the Ramachandran plot referring to the value of the favoured region which must be >80% with 0 bad bonds. MPXV virulence protein has a similarity value of 21.71% similar to the template, favoured region of 89.55%, and no bad bonds were found referring to the Ramachandran Plot. 3D visualization with transparent surfaces, and color selection (Figure 1).

Revealing of Peptide Vaccine Candidate Against MPXV Infection:

Epitope mapping prediction aims to determine the position of potential epitopes as targets for vaccine design.²⁸ B-cell epitope mapping consists of various method parameters for epitope classification such as BepiPred 2, Emini Surfaces Accessibility, Karplus and Schulz Flexibility, and Parker Hydrophilicity. All methods are used for prediction of B cell epitopes through the physicochemical properties of amino acids, surface position, hydrophilic properties, propensity scales, and Hidden Markov Models (HMMs) calculations.²⁹ The results of B-cell epitope mapping analysis in this study showed four peptide candidates from potential epitopes that could be recognized by B cells were shown in the plot (Figure 2). The epitope must act as an antigen, non-allergen, and non-toxin. Peptides consisting of Pep A and Pep D have followed these three categories (antigenic peptide, non-allergenic, and non-toxin) (Table 1). Peptide stability refers to physicochemical properties such as theoretical pI, instability index, GRAVY, and molecular weight used in this study. The stability prediction results show that Pep A and Pep D are classified as stable peptides (Table 2).

Figure 1. MPXV virulence protein model. (A) 3D visualization (B) Plot Ramachandran.

Table 1: B-cell epitope prediction assessment

Peptide Label	Position	B-cell Epitope Seq Candidate	Length (mer)	Antigenic, Allergenic, and Toxicity Prediction
Peptide Label	Position	B-cell Epitope Seq Candidate	Length (mer)	Vaxi Jen v2.0	Aller Top v.0.2	Toxi Pred
Pep A	48-64	DQKDYTVTSQFNNYTIG	17	Antigenic	Non-Allergen	Non-Toxin
Pep B	100-109	KSESEDIFQK	10	Non-Antigenic	-	-
Pep C	137-149	RVFNRFPDMAYYR	13	Non-Antigenic	-	-
Pep D	175-187	LSNGGLPAYYRNG	13	Antigenic	Non-Allergen	Non-Toxin

Table 2: Peptide molecular stability

Peptide Label

Amino Acids

Number

Molecular Weight

Theoretical pI

Formula

Instability Index

GRAVY

Classifies

Pep A

1994.10

4.21

C₈₇H₁₂₈N₂₂O₃₂

1.32

-1.135

Stable

Pep D

1381.51

8.59

C₆₁H₉₂N₁₈O₁₉

31.21

-0.638

Stable

Figure 2: The result of B-cell epitope predictions. (A)BepiPred 2, (B) Emini Surfaces Accessibility, (C) Karplus and Schulz Flexibility, (D) Parker Hydrophilicity.

Molecular Interaction and Stability of Peptide Vaccine Candidate:

The 3D model of Pep A and Pep D was obtained from PEP-FOLD v3.5 (https://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD3/)through the folding modeling method. The method works by indenting specific points in the primary structure of the protein and then forming a secondary structure or final model. Visualization of vaccine candidate peptide models was carried out using PyMol v2.5 with transparent surfaces, cartoons, and color selection (Figure 3). Peptide interactions with B cell receptors (BCR) were carried out through docking simulations with the molecular superimposed method on ClusPro v2.0 (https://cluspro.bu.edu/login.php). The energy resulting from the docking consists of the center/lowest pose chosen as the most negative and balanced. Pep A_BCR has a more negative docking energy in cluster 8 members 34 which is -539.8 kJ, Pep D_BCR has a more negative docking energy of -652.4 kJ in cluster 8 and members 55 (Table 3). This energy is needed to form a stable complex and trigger a B-cell response via the BCR to produce specific antibodies to tackle MPXV infection. The 3D structure of the docking results is displayed in PyMol v2.5 with surfaces, cartoons, sticks and color selection structures (Figure 4). Pep A_BCR and Pep D_BCR identified the stability of molecular interactions formed by molecular dynamic simulations. The RMSF value of Pep A_BCR and Pep D_BCR indicated by the P1 label on the plot has <3 Å, the RMSF value produced by the two vaccine candidate peptides is stable (Figure 5).

Table 3: The result of peptide-protein docking simulation

Molecular Complex	Cluster	Members	Cluspro Docking Energy (kJ)
Molecular Complex	Cluster	Members	Center	Lowest
PepA_BCR	8	34	-539.8	-539.8
	11	11	-519.1	-519.1
	13	1	-482.7	-482.7
Pep D_BCR	8	55	-652.4	-652.4
	9	34	-641.5	-641.5
	12	11	-616.3	-616.3

Figure 3: Structural visualization of peptide vaccine candidate. (A) Pep A, (B) Pep D.

Figure 4: Molecular interaction of peptide vaccine candidate with BCR. . (A) Pep A_BCR (B) Pep D_BCR.

Figure 5: Structural conformation and RMSF plot. (A) Pep A_BCR (B) Pep D_BCR.

Prediction of B-cell Immune Response:

Pep A and Pep B sequences were then simulated via C-Imm Sim (https://kraken.iac.rm.cnr.it/C-IMMSIM/index.php?page=1) to identify the antibody isotype. The immune response of B cells through the direct antigen recognition pathway can trigger the formation of IgM isotype antibodies for the neutralization process.³⁰ The simulation results showed that the production of IgM isotype antibodies detected on the fifth day increased to the fifteenth day with a B cell population of 450 - 500 cells per mm3 (Figure 6).

Figure 6: The result of immune simulation analysis.

CONCLUSION:

We recommend Pep A and Pep D as vaccine candidates because they allow recognition by B cells, antigenic peptides, non-allergenic and non-toxin. Peptide vaccine candidate can trigger B-cell activation to produce IgM isotype-specific antibodies through BCR interaction. The results of this study can be used for an initial study of MPXV vaccine development in Indonesia.

CONFLICT OF INTEREST:

The authors declare that there is no conflict of interest.

REFERENCES:

1. Ladnyj ID, Ziegler P, Kima E. A human infection caused by monkeypox virus in Basankusu Territory, Democratic Republic of the Congo. Bull World Health Organ. 1972; 46(5): 593-7.

2. Hraib M, Jouni S, Albitar MM, Alaidi S, Alshehabi Z. The outbreak of monkeypox 2022: An overview. Ann Med Surg (Lond). 2022; 79: 104069. DOI: 10.1016/j.amsu.2022.104069.

3. Karbalaei M, Keikha M. Overlapping outbreak of COVID-19 and monkeypox in 2022: Warning for immediate preparedness in Iran. Int J Surg. 2022; 105: 106892. DOI: 10.1016/j.ijsu.2022.106892.

4. Sah R, Abdelaal A, Reda A, Katamesh BE, Manirambona E, Abdelmonem H, Mehta R, Rabaan AA, Alhumaid S, Alfouzan WA, Alomar AI, Khamis F, Alofi FS, Aljohani MH, Alfaraj AH, Alfaresi M, Al-Jishi JM, Alsalman J, Alynbiawi A, Almogbel MS, Rodriguez-Morales AJ. Monkeypox and Its Possible Sexual Transmission: Where Are We Now with Its Evidence?. Pathogens. 2022; 11(8): 924. DOI: 10.3390/pathogens11080924.

5. Vivancos R, Anderson C, Blomquist P, Balasegaram S, Bell A, Bishop L, Brown CS, Chow Y, Edeghere O, Florence I, Logan S, Manley P, Crowe W, McAuley A, Shankar AG, Mora-Peris B, Paranthaman K, Prochazka M, Ryan C, Simons D, Vipond R, Byers C, Watkins NA; UKHSA Monkeypox Incident Management team, Welfare W, Whittaker E, Dewsnap C, Wilson A, Young Y, Chand M, Riley S, Hopkins S; Monkeypox Incident Management Team. Community transmission of monkeypox in the United Kingdom, April to May 2022. Euro Surveill. 2022; 27(22): 2200422. DOI: 10.2807/1560-7917.ES.2022.27.22.2200422.

6. Harapan H, Setiawan AM, Yufika A, Anwar S, Wahyuni S, Asrizal FW, Sufri MR, Putra RP, Wijayanti NP, Salwiyadi S, Maulana R, Khusna A, Nusrina I, Shidiq M, Fitriani D, Muharrir M, Husna CA, Yusri F, Maulana R, Utomo PS, Andalas M, Wagner AL, Mudatsir M. Confidence in managing human monkeypox cases in Asia: A cross-sectional survey among general practitioners in Indonesia. Acta Trop. 2020; 206: 105450. DOI: 10.1016/j.actatropica.2020.105450.

7. Chakraborty C, Bhattacharya M, Ranjan Sharma A, Dhama K. Monkeypox virus vaccine evolution and global preparedness for vaccination. Int Immunopharmacol. 2022; 113(Pt A): 109346. DOI: 10.1016/j.intimp.2022.109346.

8. Plotkin S, Robinson JM, Cunningham G, Iqbal R, Larsen S. The complexity and cost of vaccine manufacturing - An overview. Vaccine. 2017; 35(33): 4064-4071. DOI: 10.1016/j.vaccine.2017.06.003.

9. Bhattacharya K, Shamkh IM, Khan MS, Lotfy MM, Nzeyimana JB, Abutayeh RF, Hamdy NM, Hamza D, Chanu NR, Khanal P, Bhattacharjee A, Basalious EB. Multi-Epitope Vaccine Design against Monkeypox Virus via Reverse Vaccinology Method Exploiting Immunoinformatic and Bioinformatic Approaches. Vaccines (Basel). 2022; 10(12): 2010. DOI: 10.3390/vaccines10122010.

10. Khan S, Irfan M, Hameed AR, Ullah A, Abideen SA, Ahmad S, Haq MU, El Bakri Y, Al-Harbi AI, Ali M, Haleem A. Vaccinomics to design a multi-epitope-based vaccine against monkeypox virus using surface-associated proteins. J Biomol Struct Dyn. 2022; 19: 1-10. DOI: 10.1080/07391102.2022.2158942.

11. Fahmi M, Kharisma VD, Ansori ANM, Ito M. Retrieval and Investigation of Data on SARS-CoV-2 and COVID-19 Using Bioinformatics Approach. Adv Exp Med Biol. 2021; 1318: 839-857. DOI: 10.1007/978-3-030-63761-3_47

12. Ansori ANM, Kharishma VD, Muttaqin SS, Antonius Y, Parikesit AA. Genetic Variant of SARS-CoV-2 Isolates in Indonesia: Spike Glycoprotein Gene. J Pure Appl Microbiol. 2020; 14: 971-978. DOI: 10.22207/JPAM.14.SPL1.35

13. Nikolaev DM, Shtyrov AA, Panov MS, Jamal A, Chakchir OB, Kochemirovsky VA, Olivucci M, Ryazantsev MN. A Comparative Study of Modern Homology Modeling Algorithms for Rhodopsin Structure Prediction. ACS Omega. 2018; 3(7): 7555-7566. DOI: 10.1021/acsomega.8b00721.

14. Proboningrat A, Kharisma VD, Ansori ANM, Rahmawati R, Fadholly A, Posa GAV, Sudjarwo SA, Rantam FA, Achmad AB. In silico Study of Natural inhibitors for Human papillomavirus-18 E6 protein. Res J Pharm Technol. 2022; 15(3): 1251-6. doi: 10.52711/0974-360X.2022.00209

15. Kharisma VD, Ansori ANM. Construction of Epitope-Based Peptide Vaccine Against SARS-CoV-2: Immunoinformatics Study. J Pure Appl Microbiol. 2020; 14: 999-1005. DOI: 10.22207/JPAM.14.SPL1.38

16. Ansori ANM, Fadholly A, Kharisma VD, Nugraha AP. Therapeutic potential of avian paramyxovirus serotype 1 for cancer therapy. Biochem Cell Arch. 2020; 20: 2827-2832. DOI: 10.35124/bca.2020.20.S1.2827

17. Kharisma VD, Widodo N, Ansori ANM, Nugraha AP. A vaccine candidate of zika virus (ZIKV) from polyvalent conserved b-cell epitope on viral glycoprotein: In silico approach. Biochem Cell Arch. 2020; 20: 2785-2793. DOI: 10.35124/bca.2020.20.S1.2785

18. Ansori ANM, Fadholly A, Hayaza S, Susilo RJK, Inayatillah B, Winarni D, Husen SA. A Review on Medicinal Properties of Mangosteen (Garcinia mangostana L.). Res J Pharm Techol. 2020; 13(2): 974-982. doi: 10.5958/0974-360X.2020.00182.1

19. Fadholly A, Ansori ANM, Utomo B. Anticancer Effect of Naringin on Human Colon Cancer (WiDr Cells): In Vitro Study. Research Journal of Pharmacy and Technology. 2022; 15(2): 885-888. DOI: 10.52711/0974-360X.2022.00148

20. Husen SA, Wahyuningsih SPA, Ansori ANM, Hayaza S, Susilo RJK, Winarni D, Punnapayak H, Darmanto W. Antioxidant Potency of Okra (Abelmoschus esculentus Moench) Pods Extract on SOD Level and Tissue Glucose Tolerance in Diabetic Mice. Res J Pharm Technol. 2019; 12(12): 5683. doi: 10.5958/0974-360X.2019.00983.1

21. Husen SA, Setyawan MF, Syadzha MF, Susilo RJK, Hayaza S, Ansori ANM, Alamsjah MA, Ilmi ZN, Wulandari PAC, Pudjiastuti P, Awang P, Winarni D. A Novel Therapeutic effects of Sargassum ilicifolium Alginate and Okra (Abelmoschus esculentus) Pods extracts on Open wound healing process in Diabetic Mice. Research J. Pharm. and Tech 2020; 13(6): 2764-2770. DOI: 10.5958/0974-360X.2020.00491.6

22. Wijaya RM, Hafidzhah MA, Kharisma VD, Ansori ANM, Parikesit AP. COVID-19 In Silico Drug with Zingiber officinale Natural Product Compound Library Targeting the Mpro Protein. Makara J Sci. 2021; 25(3): 5. DOI: 10.7454/mss.v25i3.1244

23. Kharisma VD, Kharisma SD, Ansori ANM, Kurniawan HP, Witaningrum AM, Fadholly A, Tacharina MR. Antiretroviral Effect Simulation from Black Tea (Camellia sinensis) via Dual Inhibitors Mechanism in HIV-1 and its Social Perspective in Indonesia. Res J Pharm Technol. 2021; 14(1): 455-460. DOI: 10.5958/0974-360X.2021.00083.4

24. Fadholly A, Ansori ANM, Kharisma VD, Rahmahani J, Tacharina MR. Immunobioinformatics of Rabies Virus in Various Countries of Asia: Glycoprotein Gene. Res J Pharm Technol. 2021; 14(2): 883-886. DOI: 10.5958/0974-360X.2021.00157.8

25. Ansori ANM, Fadholly A, Proboningrat A, Hayaza S, Susilo RJK, Naw SW, Posa GAV, Yusrizal YF, Sibero MT, Sucipto TH, Soegijanto S. In vitro antiviral activity of Pinus merkusii (Pinaceae) stem bark and cone against dengue virus type-2 (DENV-2). Res J Pharm Technol. 2021; 14(7): 3705-8. DOI: 10.52711/0974-360X.2021.00641

26. Ansori ANM, Kharisma VD, Fadholly A, Tacharina MR, Antonius Y, Parikesit AA. Severe Acute Respiratory Syndrome Coronavirus-2 Emergence and Its Treatment with Alternative Medicines: A Review. Research Journal of Pharmacy and Technology 2021; 14(10): 5551-7. DOI: 10.52711/0974-360X.2021.00967

27. Husen SA, Ansori ANM, Hayaza S, Susilo RJK, Zuraidah AA, Winarni D, Punnapayak H, Darmanto W. Therapeutic Effect of Okra (Abelmoschus esculentus Moench) Pods Extract on Streptozotocin-Induced Type-2 Diabetic Mice. Res J Pharm Technol. 2019; 12(8): 3703-3708. DOI: 10.5958/0974-360X.2019.00633.4

28. Ansori ANM, Kharisma VD, Solikhah TI. Medicinal properties of Muntingia calabura L.: A Review. Res J Pharm Technol. 2021; 14(8): 4509-2. doi: 10.52711/0974-360X.2021.00784

29. Yi Z, Ling Y, Zhang X, Chen J, Hu K, Wang Y, Song W, Ying T, Zhang R, Lu H, Yuan Z. Functional mapping of B-cell linear epitopes of SARS-CoV-2 in COVID-19 convalescent population. Emerg Microbes Infect. 2020; 9(1): 1988-1996. DOI: 10.1080/22221751.2020.1815591.

30. Fadholly A, Ansori ANM, Sucipto TH. An overview of naringin: Potential anticancer compound of citrus fruits. Research Journal of Pharmacy and Technology. 2020; 13(11): 5613-5619. DOI: 10.5958/0974-360X.2020.00979.8