Vol. 15 | No. 2 |1288-1295| April - June | 2022 ISSN: 0974-1496 | e-ISSN: 0976-0083 | CODEN: RJCABP http://www.rasayanjournal.com http://www.rasayanjournal.co.in Rasayan J. Chem., 15(2), 1288-1295(2022) http://doi.org/10.31788/RJC.1526892 This work is licensed under a CC BY 4.0 license. ANTIBACTERIAL PEPTIDE FROM SOLID PHASE EXTRACTION (SPE) FRACTIONATION ON TRYPSIN HYDROLYSIS OF JATROPHA (Ricinus communis) SEED PROTEIN ACID EXTRACT D. R. Atmawati, Z. Andriana, R. T. Swasono and T. J. Raharjo  Department of Chemistry, Faculty Mathematics and Natural Sciences, Universitas Gadjah Mada, Bulaksumur, Yogyakarta, Indonesia, 55281  Corresponding Author: trijr_mipa@ugm.ac.id ABSTRACT The antimicrobial peptide is a new potential antibiotic type that can be isolated not only from free peptides but also peptides from hydrolyzed protein with proteolytic enzymes. The protein of jatropha seed is toxic due to RIP, ribosome- inactivating protein, and a potential source of AMP. Trifluoroacetic acid was used to isolate jatropha seed protein. The protein was hydrolyzed using trypsin and the hydrolysate separated with a cation exchange solid phase extraction (SPE) column. Each fraction was tested for antimicrobial activity and resulted in the pH 3 and pH 4 fractions being able to inhibit bacterial growth against Staphylococcus aureus (Gram-positive bacteria) and Escherichia coli (Gram- negative bacteria). The pH 3 fraction has IC50 value 17.95 µg/mL to E. coli and 17.03 µg/mL to S. aureus, while the pH fraction has IC50 value 37.30 µg/mL and 95.31 µg/mL respectively. There are three peptides with amino acid sequences of GAGLVPKR, MGACCSKEPSFAEGR and VALASLLSQPLPQISDK identified from those fractions. These peptides potentially inhibit bacterial growth through membrane destabilization mechanism, due to their amphipathic and ionic properties. Keywords: TFA Extraction, Anti-Bacterial Peptide, Ricinus Communis, Cationic Exchange, SPE RASĀYAN J. Chem., Vol. 15, No.2, 2022 INTRODUCTION Antibiotic resistance was declared by World Health Organization as a global health threat two decades ago. Genetic mutation of the pathogen is one of the factors that caused this health concern due to antibiotic over- prescribing. 1,2 It is predicted there will be over 10 million deaths predicted by 2050 due to antimicrobial resistance. 3 Unfortunately, the development of new antibiotics was not grown as fast as other medicine. Antimicrobial peptides (AMPs) are potentially developed as a new type of antibiotics. AMP is a small compound consisting of amino acids that have biological activity against bacteria. AMP is an amphipathic compound because it has both hydrophilic and hydrophobic sites. 4 Compared to traditional antibiotics, AMPs' ability to work faster with some mechanisms is an excellent trait to limit antibiotic resistance. 5 Cell membrane destabilization is the most common mechanism of the AMPs to attack bacteria. Cationic AMPs can generate pores by binding on the negative charge of the bacteria cell membrane and causing cell lysis. Nisin is an antimicrobial peptide from Lactococcus lactis that follows this mechanism against gram-positive bacteria. 6 Different from cationic AMPs, anionic AMPs work with the presence of divalent metal ions, to facilitate peptides with the bacterial cell membrane. Kapacins are anionic AMPs from the κ-casein hydrolysate enzyme where is in the presence of Zn 2+ , it was able to reduce > 90% viable cell numbers in the biofilm of S. mutans. Bacteria need to redesign their cell membrane structure as a defense mechanism against AMPs, but it will require a long period over multiple mutations. 3 AMPs can be isolated not only from free peptides but also peptides from hydrolyzed protein with proteolytic enzymes such as trypsin. 7 Moscoso-Mujica, et al. (2021) purified AMP from hydrolysates of kanihua ( Chenopodium pallidicaule Aellen) seed protein fractions that showed antibacterial activity against both Gram-positive and Gram- negative bacteria. 8 AMPs can be hydrolyzed from a toxic protein, like ricin. Jatropha plant ( Ricinus communis L.) is famous for its seed that contains ricin. Ricin is a ribosome-inactivating protein (RIP) for its ability to inhibit protein synthesis. 9 Ricin consists of two RIP chains, ricin toxin A (RTA) connected by