{"id":51120,"date":"2023-09-30T10:48:50","date_gmt":"2023-09-30T10:48:50","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=51120"},"modified":"2023-10-07T11:17:48","modified_gmt":"2023-10-07T11:17:48","slug":"antibacterial-compounds-towards-staphylococcus-aureus-and-escherichia-coli-of-the-stem-bark-of-inocarpus-fagigerus-fosb","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol16no3\/antibacterial-compounds-towards-staphylococcus-aureus-and-escherichia-coli-of-the-stem-bark-of-inocarpus-fagigerus-fosb\/","title":{"rendered":"Antibacterial Compounds Towards Staphylococcus Aureus and Escherichia Coli of the Stem Bark of Inocarpus Fagigerus Fosb"},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

Staphylococcus aureus<\/em> (S. aureus<\/em>) is a gram-positive bacterium.\nIt is facultatively anaerobic, isn’t move, isn’t form spores, is round, and\ntoxic1,2<\/sup>  S. aureus<\/em> is pathogens\nbacteria in food commonly isolated from the environment and the foods, skin,\nnostrils, and respiratory systems of animals and humans3,4<\/sup> Staphylococcus<\/em>\ninfection isn’t occur on healthy skin, but if it to enter the bloodstream or\ninternal tissues able to cause of serious infections5<\/sup>, such as\npneumonia, sepsis, bacteremia, mastitis, syndrome toxic shock, and arthritis6,7<\/sup>.\nCoagulase-positive staphylococci produce a variety of extracellular protein toxins\nand virulence factors to contribute to their pathogenicity and there are\ninvolved in hemolysin, toxic shock syndrome toxin-1 (TSST-1), Panton-Valentine\nleukocidin (PVL), Staphylococcus enterotoxin (SE), exfoliative toxin A (ETA),\nand exfoliative toxin B (ETB)8<\/sup>. Meanwhile, Escherichia coli <\/em>(E. coli<\/em>) is a\ngram-negative bacterium, in the form of a short straight rod, has no capsule or\nspore, is facultatively anaerobic, and grows easily on a simple nutrient medium9<\/sup>.\nE. coli is the main facultative flora in the intestine that resides normally in\nthe intestinal lumen of the host. As it is a normal flora in the body, when the\nhost is in a weak state (immunosuppression) or when the gastrointestinal\nprotective system is disturbed, these normal “nonpathogenic” bacteria\ncan cause infection10<\/sup>. E. coli<\/em> can produce colisin which\nfunctions as a protective agent for the digestive tract from pathogenic\nintestinal bacteria. It can play an important role in the synthesis of vitamin\nK, conversion of bile pigments, bile acids, and absorption of food substances, but\nif the amount exceeds the threshold then this bacterium becomes a pathogen with\ndifferent virulence mechanisms, such as infectious diseases of the skin,\neyelids, breast, urinary tract, dysentery, heart, bone, muscle, diarrheal\ndisease, kidney failure, sepsis, and meningitis11,12<\/sup>. <\/p>\n\n\n\n

Gayam (Inocarpus fagiferus Fosb<\/em>) or in\nBali known as gatep is a plant from the Fabaceae family which has the potential\nas an antibacterial because traditionally the stem bark has been used by the\nBalinese for a long time as a medicine for dysentery, urinary tract infections,\nand inflammation due to insect bites13,14<\/sup> People in Ambon use the stem\nbark of Gayam as medicine for bloody rhinitis. Preliminary research showed that\nthe methanol extract of Gayam stem bark was able to inhibit the growth of S.\naureus<\/em> and E. coli<\/em> with diameters 14.75 mm and 8.50 mm respectively\nat 100% w\/v. This research will investigate to antibacterial of stem bark Inocarpus\nfagiferus <\/em>Fosb toward S. aureus<\/em> and E. coli<\/em>, then determine the compounds contained in active\nisolate.<\/p>\n\n\n\n

Material and Methods<\/strong><\/p>\n\n\n\n

Materials<\/strong><\/p>\n\n\n\n

The material used is the stem bark of Inocarpus fagiferus<\/em> Fosb obtained from the Klungkung area whose taxonomy has been identified at the Center for Plant Conservation of the Bali Botanical Garden. The bacteria used to research were S. aureus<\/em> and E. coli<\/em> which are obtained in Microbiology Laboratory Biology Department. The chemicals used were methanol, n-hexane, chloroform, n-butanol, agar media (Nutrient Agar), silica gel GF254, aluminum foil, filter paper, synthetic cotton, silica gel 60, and silica GF254.<\/p>\n\n\n\n

Equipment<\/strong><\/p>\n\n\n\n

The equipment used includes a set of glassware, scissors, blender, sifter, analytical balance, stir bar, micropipette (Nesco), petri dish, test tube, measuring cup, beaker glass, incubator (Memmert), spirit lamp, loop needle, Laminar Air Flow (LAF), autoclave, rotary vacuum evaporator, vials, a set of thin layer chromatography (TLC), and a set of tools column chromatography, and LCMS\/MS Agilent type 6120.<\/p>\n\n\n\n

Methods<\/strong><\/p>\n\n\n\n

Extraction and Antibacterial Activity Test of Inocarpus fagiferus<\/em> Fosb Stem Bark<\/strong><\/p>\n\n\n\n

As much as 350 g of dried powder of Inocarpus fagiferus Fosb stem bark was macerated with methanol for \u00b1 24 hours repeatedly to obtain a concentrated extract, then the methanol solvent was evaporated by an evaporator. Furthermore, concentrated methanol extract was suspended in methanol water (7:3), and the methanol solvent was evaporated until only the water extract remained. This water extract was successively partitioned respectively with n-hexane, chloroform, and n-butanol to obtain n-hexane, chloroform, n-butanol, and water extracts. The four extracts were evaporated and weighed, and tested for their antibacterial activity toward S. aureus<\/em> and E. coli<\/em>. The diffusion method using to know potent antibacterial activity. Approximately 1 mL of bacterial suspension was added to 20 mL of nutrient agar medium, then vortexed until homogeneous, cooled, and compacted in a sterile petri dish. In a petri dish, a well with a diameter of \u00b1 6 mm is made. Test extract, positive control (antibiotic), and negative control (aqua dest) of 20\u00b5L each were put into the diffusion well which had been preincubated at 37\u00b0C for 24 hours. The inhibitory diameter was observed after the incubation period. <\/p>\n\n\n\n

Separation, Purification, and Identification of Antibacterial Active Fractions<\/strong><\/p>\n\n\n\n

The most active extract with the largest inhibition zone diameter was then separated and purified by gradient column chromatography (silica gel 60; methanol-chloroform (5:5; 6:4; 7:3; 8:4; 9:1; 10:0). The collected eluate was seen for its separation pattern using the TLC technique. The fractions that had the same separation pattern were combined and tested for antibacterial activity. The most active fraction was continued with a purity test, and identification using LCMS\/MS<\/p>\n\n\n\n

Results and Discussion<\/strong><\/p>\n\n\n\n

Maceration of 350 g of Inocarpus fagiferus<\/em> Fosb stem bark with 2000 mL of methanol (4 x 500 mL) yielded 22.97 g of concentrated methanol extract. The results of both S. aureus<\/em> and E. coli<\/em> antibacterial activity tests against 100% (w\/v) methanol extract are presented in Figure 1 and Table 1.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 1: Antibacterial activity test of 100% (w\/v) concentrated methanol extract<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Table 1: Results of antibacterial activity test of 100% (w\/v) methanol extract toward S. aureus<\/em> and E. coli<\/em><\/strong><\/p>\n\n\n\n\n\n\n\n\n\n
\n

Extract\/compound\/ solvent<\/strong><\/p>\n<\/td>\n

\n

Inhibition zone (mm)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

S. aureus (category)<\/strong><\/p>\n<\/td>\n

\n

E. coli (category)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Methanol extracts<\/p>\n<\/td>\n

\n

14.75 (s)<\/p>\n<\/td>\n

\n

8.50 (m)<\/p>\n<\/td>\n<\/tr>\n

\n

Chloramphenicol<\/em> (positive control)<\/p>\n<\/td>\n

\n

41 (vs)<\/p>\n<\/td>\n

\n

32 (vs)<\/p>\n<\/td>\n<\/tr>\n

\n

Methanol (negative control)<\/p>\n<\/td>\n

\n

0 (ni)<\/p>\n<\/td>\n

\n

0 (ni)<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

noted: vs= very strong;  s= strong;  m= medium; w= weak; and ni= not inhibition<\/p>\n\n\n

Table 1 shows that methanol extracts at 100% (w\/v) can strongly inhibit the growth of S. aureus<\/em> with a zone diameter of 14.75 mm and medium to inhibit E. coli<\/em> with a zone diameter of 8.50 mm. This inhibitory power is based on the zone diameter caused by the test extract, which is categorized as follows; very strong (vs) for a diameter of \u2265 20 mm, strong (s) for a diameter of 10-20 mm,  medium (m) for diameter 5-10 mm, weak (w) for 5 mm, and not inhibition (ni) for diameter \u00a3 5 mm15<\/sup>. Thus, the methanol extract of Inocarpus fagiferus<\/em> Fosb stem bark has more potential to inhibit S. aureus<\/em> than E. coli.<\/em> <\/p>\n\n\n\n

The partition of about 15 g concentrated methanol extract which had previously been suspended in water-methanol (7:3) with n-hexane, chloroform, and n-butanol solvents respectively yielded 0.01 g of n-hexane concentrated extract, 0.01 g of chloroform concentrated extract, 2.75 g of n-butanol concentrated extract, and 0.07 g of an aqueous concentrated extract. Antibacterial activity tests for both S. aureus<\/em> and E. coli<\/em> were only carried out on n-butanol extract due to the limited number of other extracts.<\/p>\n\n\n\n

Determining the minimum inhibitory\nconcentration (MIC) of n-butanol extract at various tests of concentrations i.e.;\n50, 10, 5, and 0.5% (w\/v) towards both S. aureus<\/em> and E.\ncoli<\/em> bacteria\nare presented in Figure 2 and Table 2.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 2: Test the antibacterial activity of the butanol fraction<\/strong> at concentrations of 50, 10, 5, and 0.5%<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Table 2: Results of Antibacterial Activity Test and Determination of Minimum Inhibitory Concentration of n-butanol Extract Against S. aureus and E. coli Bacteria<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n
\n

Concentration (% w\/v)<\/strong><\/p>\n<\/td>\n

\n

Zone Inhibition (mm)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

S. aureus<\/em> (category)<\/strong><\/p>\n<\/td>\n

\n

E. coli<\/em> (category)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

50<\/p>\n<\/td>\n

\n

13.50 (s)<\/p>\n<\/td>\n

\n

11.25 (s)<\/p>\n<\/td>\n<\/tr>\n

\n

10<\/p>\n<\/td>\n

\n

8.50 (m)<\/p>\n<\/td>\n

\n

7.50 (m)<\/p>\n<\/td>\n<\/tr>\n

\n

5<\/p>\n<\/td>\n

\n

6.75 (m)<\/p>\n<\/td>\n

\n

6.25 (m)<\/p>\n<\/td>\n<\/tr>\n

\n

0,5<\/p>\n<\/td>\n

\n

6.25 (m)<\/p>\n<\/td>\n

\n

5.875 (w)<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

vs= very strong;  s= strong;  m= medium; w= weak; and ni= not inhibition<\/p>\n\n\n

Determination of MIC aims to\ndetermine the smallest concentration of extract that is still able to inhibit\nbacterial growth. Table 2 shows that the n-butanol extract of Inocarpus fagiferus<\/em> Fosb stems bark with 0.5% (w\/v)\nis a minimum inhibitory concentration which is still able to inhibit the growth\nof both S. aureus<\/em> and E. coli.<\/em><\/p>\n\n\n\n

Furthermore, the separation of\nabout 2 g of n-butanol extract by gradient column chromatography obtained 4\nfraction groups (FA, FB, FC, and FD) with color, spot number, Rf value, and\nweight of each fraction as shown in Table 3.<\/p>\n\n\n\n

Table 3: Results of separation of n-butanol extract to yield fractions by gradient column chromatography<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n
\n

Fractions<\/strong><\/p>\n<\/td>\n

\n

color<\/strong><\/p>\n<\/td>\n

\n

spot<\/strong><\/p>\n<\/td>\n

\n

 Rf<\/strong><\/p>\n<\/td>\n

\n

Weight<\/strong><\/p>\n

(g)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

FA<\/p>\n<\/td>\n

\n

Clear yellow<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n

\n

0,68<\/p>\n<\/td>\n

\n

0,08<\/p>\n<\/td>\n<\/tr>\n

\n

FB<\/p>\n<\/td>\n

\n

Pale yellow<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n

\n

0,88<\/p>\n<\/td>\n

\n

0,07<\/p>\n<\/td>\n<\/tr>\n

\n

FC<\/p>\n<\/td>\n

\n

Dark reddish yellow<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n

\n

0,73<\/p>\n<\/td>\n

\n

0,24<\/p>\n<\/td>\n<\/tr>\n

\n

FD<\/p>\n<\/td>\n

\n

Clear yellow<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n

\n

0,62<\/p>\n<\/td>\n

\n

0,11<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

The result of test antibacterial\nactivity fourth fractions (FA, FB, FC, and FD) toward both S. aureus<\/em> and E. coli<\/em> showed that only FC was active antibacterial with an inhibition zone\ndiameter of 7.25 and 6.25 mm for S. aureus<\/em> and E.\ncoli<\/em> respectively\nat a concentration of 10% which was categorized as medium (m) as shown in\nFigure 3.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 3: Antibacterial activity test of the fraction FA, FB, FC, and FD at the concentration of 10%<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Furthermore, the result identification\nof FC fraction with LCMS\/MS showed that 5 identified compounds namely maltol,\nnicotinamide, bioachanin A, L-proline, and 2,3-diaminopropionic acid, as well\nas one unidentified compound with a molecular weight of 95.8066 g\/mol as shown\nin Table 4 as follows:<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Table 4: Compounds contained in the FC fraction of the stem bark of Inocarpus fagiferus<\/em> Fosb<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

The compounds maltol, nicotinamide, bioachanin A, and L-proline are known to be antibacterial compounds against both S. aureus<\/em> and E. coli<\/em>. Maltol or 3-Hydroxy-2-methyl-4H-pyran-4-one is a phenolic compound derivative that is medium until strong as an antibacterial toward E. coli<\/em> with an inhibition zone of 10 mm16,17<\/sup>. The mechanism of phenol-derived compounds to inhibit bacterial growth through denaturing cell wall proteins and cytoplasmic membranes, therefore, caused the formation of hydrogen bonds between phenols and proteins which results in disrupted permeability of the cell walls and cytoplasmic membranes, causing an imbalance of macromolecules and cell ions so that it become lysed. Nicotinamide or pyridine-3-carboxamide has antibacterial activity toward S. aureus<\/em> at a concentration of 0.0625\u00b5l\/mL but it is not yet known how its mechanism inhibits bacterial growth18<\/sup>. Biochanin A is a compound of the flavonoid group that is active as an antibacterial toward both S. aureus<\/em> and E. coli<\/em> at a concentration of 0.84 mg\/ mL.  It has a mechanism for inhibiting the nucleic acid synthesis in bacteria through the formation of hydrogen bonds between flavonoid compounds and the nitrogenous base of nucleic acid. This formation will also inhibit the formation of DNA and RNA bacteria19<\/sup>. The interaction of flavonoids will also inhibit the function of membrane cells through complex compounds of extracellular and dissolved proteins so that the cell membrane will be damaged and intracellular compounds will come out20<\/sup>. The L-proline compound in a complex with Cu metal known as Cu(L-prolinate)2 was able to inhibit the growth both of S. aureus<\/em> and E. coli<\/em> with diameter zone 16 mm and 15 mm respectively21<\/sup>.<\/p>\n\n\n\n

Conclusion<\/strong><\/p>\n\n\n\n

In this study,\ncompounds have been extracted from the stems bark of Inocarpus fagiferus<\/em>\nFosb using a methanol solution. It was found that there were five compounds\nincluding maltol,\nnicotinamide, bioachanin A, L-proline, and 2,3-diamino propionic acid, as well\nas one unidentified compound with a molecular weight of 95.8066 g\/mol, where compounds that have\nthe potential to inhibit the growth both of S.\naureus<\/em> and E. coli <\/em>are\nmaltol, nicotinamide, bioachanin\nA, and L-proline.<\/p>\n\n\n\n

Conflict of Interest <\/strong><\/p>\n\n\n\n

There is no conflict of interest<\/p>\n\n\n\n

Funding Source <\/strong><\/p>\n\n\n\n

There are no funding sources<\/p>\n\n\n\n

References<\/strong><\/p>\n\n\n\n

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    Introduction Staphylococcus aureus (S. aureus) is a gram-positive bacterium. It […]<\/p>\n","protected":false},"author":15,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[109],"tags":[],"class_list":["post-51120","post","type-post","status-publish","format-standard","hentry","category-vol16no3"],"_links":{"self":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts\/51120","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/users\/15"}],"replies":[{"embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/comments?post=51120"}],"version-history":[{"count":5,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts\/51120\/revisions"}],"predecessor-version":[{"id":52646,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts\/51120\/revisions\/52646"}],"wp:attachment":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/media?parent=51120"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/categories?post=51120"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/tags?post=51120"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}