1. INTRODUCTION:

Indonesia’s marine waters, half part of the Indonesian archipelago, are located in tropical areas. The waters are geographically surrounded by ocean. Consequently, Indonesia has a great potential for the development and utilization of marine resources, especially seaweed. The particular resources, seaweed are a multicellular algae living in the ocean and classified in Thallophyta division.

Its body is not differentiated into roots, stems, and leaves as in higher plants. Types of highly profitable seaweed are Acanthopora, Gracilaria, Gelidiella, Gelidium used for jelly making, Chondrus, Eucheuma, Gigartina used for carrageenan producing, and Furcellaria, Ascophyllum, and Ecklonia used for alginate producing.

Economic value of seaweed can be elevated by the means of further and continuant research on bioactive compounds it contained. The research may help pharmaceutical industries process seaweed into medicine.

Various studies reported that seaweed was able to produce secondary metabolite such as acrylic acid¹. Seaweed belonging to Rhodophyceae (red algae) division such as Gigartinales contains a large number of acrylic acids, dimethylsulfide compounds, alkaloids, and phenols. Acrylic acid is the first active antibiotic component in red algae that can be identified easily, such as from the type of Gigartina acicularis, and Gigartina stellata. Red algae from Gigartina division has many polysaccharides, making it useful for natural medicine. A chemical analysis showed that the red algae contained terpenoid, acetogenic, and aromatic compounds.

Antibacterial substances in plant can be obtained by extraction^2;3. Thus, we can absorb soluble chemical contents, separating it from insoluble material in liquid solvents⁴. Meanwhile antibacterial substances can be extracted using methanol, ethanol, and ether extract.

We used ethanol, hexane, and distilled water extract. According to Sidharta et al. (2003) some seaweeds from the South Coast in the Special Region of Yogyakarta Indonesia extracted using ethanol, hexane, methanol, benzene and distilled water solvents indicated that bacteria activity in seaweeds extracted using ethanol solvent was better^5;6. According to another research guava leaves extracted using ethanol solvent gave a stronger effect to antimicrobial power in Escherichia coli and Staphylococcus aureus⁷.

Antimicrobial compound is used to control the growth of harmful microbes. Antibacterial property of guava leave extract in inhibiting the growth of Escherichia coli and Staphylococcus aureus has been reported in other studies^7,8.

Penicillin and streptomycin antibiotics are used as a control to compare antimicrobial of Gigartina sp extract in inhibiting test microbes. Both antibiotics are mostly used in health and particular medical sectors. Based on Shannon E,(2016) Sargassum sp. extract obtained by ether extract had the same activity as penicillin antibiotic in inhibiting Escherichia coli and Staphylococcus aureus, but Sargassum sp extract obtained by ether extract had a lower ability in inhibiting Escherichia coli and Staphylococcus aureus compared to streptomisin^9;10;11.

Test microbes we used to find antimicrobial power of Gigartina sp extract were Escherichia coli and Staphylococcus aureus. These microbes were selected due to their pathogenic property^12;13.

1.1 Research problems:

What is Gigartina sp powder extract having the strongest power to inhibit the growth of Escherichia coli and Staphylococcus aureus?

Does Gigartina sp extract have more effective antibacterial power than penicillin and streptomycin antibiotics to inhibit the growth of Escherichia coli and Staphylococcus aureus?

1.2 Research objectives:

To find the strongest Gigartina sp extract able to inhibit the growth of Escherichia coli and Staphylococcus aureus.

To compare antibacterial power of Gigartina sp extract to that of penicillin and streptomycin antibiotics.

2. MATERIALS AND METHODS:

The research consisted of two stages: in stage I we used Completely Randomized Factorial Design with treatment on types of extract and test microbes. Extracts used was sterilized distilled water, ethanol, and hexane; while test microbes used were Escherichia coli and Staphylococcus aureus; in stage II we used Completely Randomized Factorial Design consisting of three types of antimicrobial compounds i.e. Gigartina sp extract whose effectiveness was compared to that of penicillin and streptomycin antibiotics. Each treatment was carried out thrice and tested on the same two test microbes in stage I.

This research was performed in several stages: drying material and making Gigartina sp powder, making Gigartina sp extract, testing the purity of test microbes, multiplying pure culture, testing the antibacterial, determining the effect of antibacterial compounds, and analyzing data. The data obtained were analyzed using ANOVA with a 95% confidence level and Duncan’s Multiple Range Test (DMRT).

3. RESULTS:

3.1 Gigartina Sp. Morphology:

Gigartina sp obtained from Sundak Beach had following general features: the thallus shaped leaf or had a simple or dichotomous (having two branches) lush branch, its color was generally dark red, it was soft as gel and had cystocarp in the form of nodules. In addition, it also had holdfast consisting of single cell rooting similar to root and able to hold blade such as leaves. Gigartina sp had an average length of 19 cm and width of 3.5cm.

3.2 Gigartina sp powder extraction:

In this research, the extract liquid consisting of ethanol, hexane, and sterilized distilled water was used to absorb bioactive compounds in Gigartina sp powder. Gigartina sp extract was made from ethanol, hexane, and sterilized distilled water liquid of 100ml for each and Gigartina sp powder of 25gr. The mixture was then macerated for five days using maceration technique. The macerated liquid of Gigartina sp extract was then evaporated using Rotary Evaporator at 90^˚C. And then, the final result, a thick liquid of Gigartina sp extract was obtained.

3.3 Purity test of Escherichia coli and Staphylococcus aureus:

Purity test on test microbes included observation on colony morphology, cell morphology, motility tests, catalase tests, and gran staining and biochemical property tests including carbohydrate fermentation test, nitrate reduction test, peptonization test, indole formation test, and starch hydrolysis test.

3.4 Antibacterial power of Gigartina sp extract based on agar diffusion method:

The result of antibacterial activities using agar diffusion method from Gigartina sp extract with ethanol, hexane, and sterilized distilled water solvents on Staphylococcus aureus and Escherichia coli is indicated in Table I.

Table I: Inhibition zone area (cm²) of the result of antibacterial activity of Gigartina sp extract on test microbes of Escherichia coli and Staphylococcus aureus with extracts variation

Extracts Variation	Inhibition Zone Area (cm²)		Average
Extracts Variation	*E. coli*	*S. aureus*	Average
Ethanol	0.194 ^c	0.379 ^d	0.286 ^C
Hexane	0.092 ^abc	0.148 ^bc	0.120 ^B
Distilled water	0.041 ^ab	0.071 ^abc	0.056 ^AB
Negative control of ethanol	0.073 ^abc	0.088 ^abc	0.081 ^AB
Negative control of hexane	0.018 ^ab	0.042 ^ab	0.029 ^A
Negative control of sterilized distilled water	0.012 ^a	0.031 ^ab	0.021 ^A
Average	0.072 ^A	0.259 ^B

Note: Numbers followed by the same letter in the same column and row present no significant difference on a 95% confidence level.

Based on the data analysis performed using ANOVA in term of inhibition zone area as a result of the antibacterial activity of Gigartina sp extract on test microbes Escherichia coli and Staphylococcus aureus, there was no significant difference between extracts variations and both test microbes. The result of DMRT analysis was used to find significant difference showed by Gigartina sp extract with extracts variations. Based on the test, ethanol extract indicated a significant difference in term of inhibition zone area formed by Escherichia coli and Staphylococcus aureus. The difference was its inhibition zone area 0.286 cm² larger than hexane and distilled water extract. In other words, ethanol extract are more strongly inhibiting the growth of test microbes if compared to other extracts. It was also supported by its inhibition area larger than hexane and distilled water’s that was 0.194cm² on Eschericia coli and 0,379cm²Staphylococcus aureus. In addition, when compared to ethanol, hexane and distilled water did not show any significant difference. It was because each extracts had different ability in extracting Gigartini sp as well as different dielectric level. It was easier for polar chemical compounds to be soluble in polar solvent, while non-polar chemical compounds would also be more soluble in non-polar solvents. Hexane was polar; while distilled water and ethanol were non-polar; it was the same with Gigartina sp extract that was also non-polar. Hence, ethanol extract had a more effective ability in extracting antimicrobial compounds if compared to distilled water.

Based on the result of DMRT analysis test on Gigartina sp on the extract variation, there was a significant difference between ethanol extract that was 0.286cm² and hexane and distilled water extracts that were 0.120 cm²and 0.056cm² respectively. It indicated three significantly different inhibition zones. Based on the result obtained, Gigartina sp with ethanol and hexane extracts more strongly inhibited the growth of test microbes if compared to that with distilled water extract.

Based on the interaction between inhibition zone area of Gigartina sp and test microbes of Escherichia coli and Sthaphylococcus aureus with extracts variation, there was a significant difference among the three extracts. It implied that Gigartina sp extract was able to inhibit the growth of test microbes. Based on extracts variation, Gigartina sp extract with ethanol extracts had a more powerful inhibition ability than that with hexane and distilled water extracts.

We found that distilled water extract was less able to inhibit the growth of Escherichia coli when compared to Staphylococcus aureus. It was because distilled water was the most common solvent and able to dissolve active compounds due to its polar properties. As a result, compounds i.e., phenol, alkaloid, polysaccharides, and compounds that were not functioned as antimicrobials could also be dissolved.

Fig. 1 Antibacterial activity of Gigartina sp. Extract on test microbes Escherechia coli and Staphylococcus aureus on Inhibition Zone Area (cm²) with different extract variation.

Figure 1 shows that ethanol extract had inhibition zone areas that were 0.194cm² on Eschericia coli and 0.379 cm² on Staphylococcus, larger than the inhibition zone area made by hexane and distilled water extracts. The result of treatment on both test microbes indicated a significant difference. It meant that the three extracts had strong inhibition power in inhibiting the growth of Staphylococcus aureus. It was proven by the average of the inhibition zone area that was 0.259cm². It was higher than that on Eschericia coli that was 0.072cm².

The inhibition zone area for negative control in Figure 2 shows that the inhibition zone area produced by Eschericia coli with ethanol, hexane, and distilled water controls were 0.074cm², 0.018cm², 0.012cm²respectively. The average inhibition zone area produced by ethanol control, hexane control, and distilled water control was 0.088cm², 0.042cm², and 0.031cm²respectively.

Fig. 2 Antibacterial activity of Gigartina sp. Extract on Negative Control microbes Escherechia coli and Staphylococcus aureus on Inhibition Zone Area (cm²) with different extract variation.

Based on the result obtained for negative control, there was an inhibition zone. It was because the solvent used had an ability to inhibit test microbes even though evaporation had been performed and Gigartina sp extract was not added. However, the average inhibition zone area produced by each control on both microbes was smaller than the inhibition zone area produced by the addition of Gigartina sp extract. It showed that Gigartina sp extract was able to inhibit the growth of test microbes.

3.5 Comparison of antibacterial activity of Gigartina sp extract with penicillin and streptomycin based on agar diffusion method:

Based on the result of inhibition zone area measurement performed in stage I, the most optimum result to inhibit both test microbes was found that was Gigartina sp extract with ethanol. Gigartina sp extract with ethanol had stronger power in inhibiting the growth of Escherichia coli and Staphylococcus aureus with inhibition zone area of 0.0194 cm² and 0.379 cm²respectively. According to the result of research performed, penicillin and streptomycin had antibacterial activity on Escherichia coli and Staphylococcus aureus (Table II).

Table II: Inhibition zone area (cm2) resulted from penicillin and streptomycin antibiotics as well as Gigartina sp extract with ethanol on the growth of test microbes of Escherichia coli and Staphylococcus aureus

Antibiotic Compound	Inhibition Zone Area (cm²)		Average
Antibiotic Compound	*E. coli*	*S. aureus*	Average
Streptomycin	0.0417^a	0.407^a	0.224^A
Penicillin	2.758^a	11.687^b	7.22^B
Gigartina sp extract*	0.194^a	0.379^a	0.286^A
Average	0.998^A	4.167^B

Note: Numbers followed by the same letter in the same column and row show no significant different on a 95% confidence level.

(*): with ethanol extract

4. DISCUSSION:

Based on the statistical analysis performed (ANOVA), the inhibition zone area on the antibacterial compound of Gigartina sp extract with ethanol extract of 20µL, penicillin and streptomycin on Escherichia coli and Staphylococcus aureus showed a significant difference between treatments of antibacterial compounds with a 95% confidence level. It was because the value of F-count resulted was higher than F-table.

Based on the result of penicillin DMRT analysis, there was a significant difference between streptomycin and Gigartina sp extract with ethanol. The result showed that Gigartina sp ethanol extract had the same ability as streptomycin as antibacterial compound in inhibiting Escherichia coli and Staphylococcus aureus. This was proven by the equal average of inhibition zone area of both test microbes that was 0.224cm² and 0.286cm²for streptomycin and Gigartina sp ethanol extract respectively.

The inhibition zone area resulted from the antibacterial activity of Gigartina sp ethanol extract on Escherichia coli and Staphylococcus aureus were 0.194cm² and 0.379cm² respectively; Streptomycin on Escherichia coli and Staphylococcus aureus were 0.0417cm² and 0.407cm²respectively; while penicillin antibiotic on Escherichia coli and Staphylococcus aureus were 2.758 cm² and 11.687cm² respectively.

The findings showed that antibacterial activity of penicillin on Staphylococcus aureus was higher than that on Escherichia coli. It was because Staphylococcus aureus was a gram-positive bacterium having a simpler cell wall structure than Escherichia coli that was Gram negative bacteria¹⁴. According to Volk and Wheeler (1993), the structure of cell wall in Gram positive bacteria only consisted of peptidoglycan layer; while gram negative bacteria had an outer lipid membrane composed of protein, phospholipids, and lipopolysaccharides in addition to the peptidoglycan layer¹⁵. Hugo and Russell (1998) reported that the cell wall of Staphylococcus aureus was categorized as Gram positive bacteria as it had more peptidoglycan layers and few lipids relatively; while Escherichia coli relatively had more lipids¹⁶.

Antibiotics could be classified based on their selectivity on the mechanism of its antibacterial action that was based on the inhibition of cell wall synthesis, the inhibition of protein synthesis, cell membrane damage, and the inhibition of DNA and RNA synthesis¹⁵. Based on this antibiotics classification, penicillin was classified into the antibiotic whose action mechanism was to inhibit the microbial cell wall (peptidoglycan) synthesis. This mechanism was related to the property of cell wall consisting of two carbohydrate polymers i.e. N-acetylglucosamine and N-acetylmuramic as well as a small amount of amino acid. Any compound inhibiting cell wall synthesis would weaken the cell wall and lead to an opened cell membrane and finally dissipated the cell content. Penicillin had the same structure as terminal D-Alanyl-D-alanine reacting to enzyme transpeptidase. When the cell wall synthesis was inhibited by penicillin, terminal D-Alanyl-D-alanine would be replaced with penicillin and it reacted to enzyme transpeptidase. Due to this reaction, the ability to complete the formation of Pentaglycine Bridge between N-acetylglucosamine and N-acetylmuramic was dismissed. Finally, the cell wall failed to be synthesized because the cell wall of microbes consisted of polymers; such as N-acetylglucosamine and N-acetylmuramic connected by Pentaglycine Bridge made from the reaction of terminal D-Alanyl-D-alanine reacting to enzyme transpeptidase¹⁵.

Based on the antibacterial action, streptomycin was classified into antibiotic inhibiting protein synthesis. Streptomycin affected a number of Gram positive and Gram-negative bacteria. Streptomycin provided non-typical effects such as the release of potassium from the cell¹⁵. Its typical bactericidal effects depended on its ability to be bound specifically on one of proteins in the 30S ribosomal subunit. This binding led to two main effects on protein synthesis i.e. causing incorrect reading on mRNA and preventing ribosomal movement after it was bound to the first amino acid to form protein¹⁵.

Based on the test result on antibacterial activity between Gigartina sp ethanol extract with penicillin and that with streptomycin, the largest inhibition zone area was showed by the extract with penicillin. The inhibition zone area made by penicillin on Staphylococcus aureus and Escherichia coli was 11.687cm² and was 2.758cm²respectively. It was suspected that Gigartina sp extract was one of the extracts able to produce bioactive compounds such as alkaloids and phenol. According to Glombitza and Hansen (1979), seaweed from the division of Rhodophyceae (red algae), Gigartinales contained a large amount of acrylic acid, dimethylsulfide compound, alkaloid, and even phenol^17;18.

The ability of Gigartina sp ethanol extract was weaker if compared to penicillin. This can be due to the target of antibiotic compounds from each compound. Nevertheless, Gigartina sp extract had a potential as a producer of antibacterial compounds due to Gigartina sp ethanol extract that had a larger inhibition zone area than streptomycin. Based on the average of each antibiotic compound, penicillin had a more significant inhibition zone area than the inhibition zone area made by streptomycin or Gigartina sp extract with ethanol. The inhibition zone area made by penicillin was larger than that made by streptomycin and Gigartina sp extract. The average of test microbes showed was significantly different. It was proven by the inhibition zone area made by Staphylococcus aureus that was 4.167 cm² much larger than that made by Eschericia coli that was only 0.988 cm².

5. CONCLUSION:

Based on the research findings on the antibacterial activity of Gigartina sp extract on test microbes i.e. Escherichia coli and Staphylococcus aureus with extracts variation, it can be concluded that, Gigartina sp with ethanol extract had more powerful antimicrobials on Staphylococcus aureus and Escherichia coli than that with hexane and distilled water extract.Gigartina sp with ethanol extract on E.coli had an equal activity to streptomycin’s and showed no significant difference result between penicillin and streptomycin. Gigartina sp with ethanol extract on S.aureus showed a significant difference result between penicillin and streptomycin.

6. ACKNOWLEDGEMENT:

Authors are thankful to all the peer reviewers and editors for their opinions and suggestions.

7. CONFLICT OF INTEREST:

The authors declare that there is no conflict of interest regarding the publication of this article

8. AUTHOR CONTRIBUTIONS:

F.S. Maharini conceived, designed and did the experiments; N. Nambiar and S. Poddar supervised and edited the paper.

9. REFERENCE:

1. Naveed N. Medicinal Uses of Red Algae and Blue-Green Algae. Research Journal of Pharmacy and Technology. 2014; 7(12): 1472-1475.

2. Pawar AR, Vikhe DN, Jadhav RS. Recent Advances in Extraction Techniques of Herbals–A Review. Asian Journal of Research in Pharmaceutical Science. 2020; 10(4):287-292. https://doi.org/10.5958/2231-5659.2020.00050.8

3. Nasser JA. Phytochemicals: Extraction, Isolation & Identification of Bioactive Compounds from Aristolochia bracteolate. International Journal of Advancement in Life Sciences Research. 2020; 3(3):50-54. https://doi.org/10.31632/ijalsr.20.v03i03.005

4. Samydurai P. Saradha M. Effects of Various Solvent on the Extraction of Antimicrobial, Antioxidant Phenolics from the Stem Bark of Decalepis hamiltonii Wight and Arn. Asian Journal of Research in Pharmaceutical Science. 2016; 6(2):129-34.

5. Sidharta BR. Screening of antibiosis activity from green algae (Chlorophyta) from Drini Beach, Yogyakarta: a Preliminary Study. Biota. 2003; VIII (2) : 53-58.

6. Sagar S. Rastogi A. Adsorptive Elimination of an Acidic Dye from Synthetic Wastewater using Yellow Green Algae along with Equilibrium Data Modelling. Asian Journal of Research in Chemistry. 2018;11(5):778-86. https://doi.org/10.5958/0974-4150.2018.00137.2

7. Biswas B. Rogers K. McLaughlin F. Daniels D. Yadav A. Antimicrobial activities of leaf extracts of guava (Psidium guajava L.) on two gram-negative and gram-positive bacteria. International journal of microbiology. 2013; 2013:1-7. https://doi.org/10.1155/2013/746165

8. El-Sayed MA. Kamel MM. El-Raei MA. Osman SM. Gamil L. Abbas HA. Study of antibacterial activity of some plant extracts against Enterohemorrhagic Escherichia coli O157: H7. Research Journal of Pharmacy and Technology. 2013; 6(8):916-919.

9. Shannon E. Abu-Ghannam N. Antibacterial derivatives of marine algae: An overview of pharmacological mechanisms and applications. Marine drugs. 2016; 14(4):81. https://doi.org/10.3390/md14040081.

10. Kumar KR. Gopinath P. Detection of Serum Bactericidal activity of Clinical Isolates of Escherichia coli. Research Journal of Pharmacy and Technology. 2016; 9(10):1588-1590. https://doi.org/10.5958/0974-360X.2016.00313.9

11. Ravichandran H. Gopinath P. Detection of sea and seb genes encoding enterotoxins among clinical isolates of Staphylococcus aureus. Research Journal of Pharmacy and Technology. 2016; 9(10):1632-1634. https://doi.org/10.5958/0974-360X.2016.00326.7

12. Abbas HA. El-Sayed MA. Kamel MM. Gamil L. Allium kurrat and Eruca sativa are Natural agents for Inhibition and Eradication of Enterohemorrhagic Escherichia coli O157: H7 Biofilm. Research Journal of Pharmacy and Technology. 2014; 7(4):425-428.

13. Adak S. Chakraborty D. Maji HS. Basu S. Roy P. Mitra S. Mukherjee N. Barik S. Goswami A. Comparison of the antimicrobial activity of the phyto-constituents obtained from the stem bark and leaf extracts of Phyllanthus emblica L. against different strains of Staphylococcus aureus and Salmonella typhi. Research Journal of Pharmacology and Pharmacodynamics. 2018; 10(2):53-60.

14. Kato Y. Otsuki M. Nishino T. Antibacterial properties of BO-2727, a new carbapenem antibiotic. The Journal of antimicrobial chemotherapy. 1997;40(2):195-203. https://doi.org/10.1093/jac/40.2.195

15. Volk WA, Wheeler MF. Mikrobiologi Dasar, edition 5. Vol. 1. Diterjemahkan oleh Markham. Penerbit Erlangga, Jakarta. 1993; 396.

16. Denyer SP. Hodges NA. Gorman SP. editors. Hugo and Russell's Pharmaceutical Microbiology. edition 7. John Wiley & Sons; 2008 Apr 15.

17. Glombitza KW. Marine Algae in Pharmaceutical Science, herausgeg. von HA Hoppe, T. Levring und Y. Tanaka, XIII, 807 S., Preis DM 170, Walter de Gruyter and Co., Berlin–New York 1979. https://doi.org/10.1002/ardp.19803130516

18. Steffy JA. Parveen MH. Durga V. Manibalan S. Extraction purification of phlorotannins from different species of marine algae and Evaluation of their Anti-Oxidant potential. Research Journal of Engineering and Technology. 2013; 4(4):163-168.

Received on 04.10.2021 Modified on 26.12.2021

Research J. Pharm. and Tech 2022; 15(10):4801-4806.

DOI: 10.52711/0974-360X.2022.00806