Antibacterial compound from Euchema spinosum originated from Tasikmalaya West Java against pathogen bacteria with TLC-bioautography

Streptococcus mutans (Gram-positive) and Shigella dysenteriae (Gram-negative) are two types of pathogen bacteria. The use of synthetic antibiotics against both bacteria is known to impact the bacteria's resistance. E. spinosum from Tasikmalaya is a potential macroalgae as a source of an antibacterial compound for both bacteria. The research aims to determine the antibacterial metabolite compound from E. spinosum originated from Tasikmalaya against S. mutans and S. dysenteriae . The research was conducted through several stages, starting from phytochemical screening, gradual maceration using hexane, ethyl acetate, and methanol, determination of antibacterial activity, and TLC-bioautography. Phytochemical screening showed that both raw material and extracts contained alkaloids, flavonoids, and steroids. The result showed that hexane, ethyl acetate, and methanol extract could inhibit the growth of S. dysenteriae starting from a concentration of 400 µg/mL. However, only ethyl acetate extract can inhibit the growth of S. mutans, starting from a concentration of 20 µg/mL. The chromatogram of the hexane extract showed the presence of 6 spots, ethyl acetate extract showed 5, and the methanol extract showed only 4, resulted from the elution system, respectively. The TLC-bioautography against S. dysenteriae showed that there was the presence of three clear zones on the ethyl acetate extract, detected as flavonoid, and three clear zones on the methanol extract. The TLC-bioautography against S. mutans showed one clear zone on the chromatogram of ethyl acetate extract. According to the AlCl 3 spray reagent confirmation test, the active compound was the flavonoid group.


INTRODUCTION
Methods

Raw material determination
The identification of E. spinosum took place in the Jatinangor Herbarium with identification sheet number 082/HB/02/2019.

Maceration and phytochemical screening
The E. spinosum material was dried first, then macerated gradually using hexane, ethyl acetate, and methanol solvents. Each of the mixtures was then filtrated and concentrated with a rotary vacuum evaporator to produce three viscous extracts, namely hexane, ethyl acetate, and methanol extracts. The raw material and the extracts were then analyzed for their chemical content, including: Alkaloids: 2 grams of raw material and the extracts of E. spinosum were placed in different Erlenmeyer. Each of them was acidified with 2 N HCl and then filtered. The filtrate was then alkalized with 10% NH4OH and was added with chloroform. The chloroform layer was then separated and tested using Dragendorf reagent and Mayer reagent. The characterization of the alkaloids appeared in two different colors; orange color for Dragendorff reagent and white precipitation for Mayer reagent. Flavonoids: 2 grams of raw material were added with water and heated. 5 mL of 2 N HCl was added to the mixture, continued by adding a little Magnesium powder, then filtered. After that, amyl alcohol was put into the filtrate and shaken quickly. The presence of flavonoids was seen from the color appearing on the amyl alcohol layer, where it could be red, yellow, or orange in color.

Monoterpenoids and sesquiterpenoids:
The raw material and each extract were dissolved in ether and then filtered. The filtrates were evaporated and dried on a drop plate porcelain. Then, 10% vanillin reagent in sulfuric acid was added to the dried extract. The appearance of colors was the indication of positive results. Steroids: The raw material and each extract were dissolved in ether and then filtered. The filtrates were then added by Lieberman Burchard's reagent. The appearance of green-blue color was the indication of steroids. Tannins and saponins were analyzed using the method conducted by Safitri et al. (Safitri et al., 2018).

Antibacterial assay from all extracts
The stock solution of E. spinosum extract was prepared by dissolving 50 mg extract in 50 mL of 96% ethanol. The stock solution was firstly diluted with water to obtain a variant extract concentration of 20, 40, 60 µg/mL, and they were called tested extracts. Firstly, S. dysenteriae ATCC 13313 was cultured on SSA media (Alemu et al., 2019), and S. mutans ATCC 25175 was cultured on blood agar (George et al., 2017), both were incubated at 37°C for 24 hours.
The assay of extract activity to S. dysenteriae applied the disk diffusion method. First, a small portion of the bacteria was dissolved in nutrient broth to produce turbidity equivalent to the standard of 0.5 Mcfarland (Alemu et al., 2019). Second, the suspension was then smeared on the surface of SSA media. Third, the paper disks were dipped in each test solution, including the tested extracts, 96% ethanol solvent, and 5 µg/mL of cotrimoxazole as the comparator for S. dysenteriae assay. Fourth, The disks were then affixed to the SSA surface and incubated at 37°C for 48 hours.
The assay of the extract activity to S. mutans was conducted with the agar well diffusion method. First, the S. mutans culture was smeared on the surface of the media. Second, wells were made on the blood agar medium using a 6 mm diameter perforator. Third, approximately 50 µL of each concentration of the tested extract, 96% ethanol solvent, and 5 µg/mL of chlorhexidine as the comparator (Jones, 1997) were put into the well using a micropipette. Fourth, the medium containing a tested sample was incubated at 37°C for 48 hours. Each of those assays was done under a laminar airflow environment.

Thin-layer chromatography
Approximately 30 μL of each of E. spinosum extracts diluted with each solvent (1 μg/mL) were then spotted onto the activated GF254 TLC plate using a micropipette. The plates were then eluted with the selected eluent combination as shown in Table 1, with an optimized composition than the previous ones.

TLC-bioautography
The TLC-bioautography was conducted based on the previous methods (Dewanjee et al., 2015). Each chromatogram was then analyzed using the 254 nm and 366 nm UV lamp then the number of spots that appeared was calculated. The chromatogram containing the well-separated compound was then attached to the surface of the SSA medium for S. dysenteriae and blood agar medium for S. mutans for 30 minutes. After that, the chromatogram plate was withdrawn from the medium. Subsequently, the medium was incubated at 37°C for 24 hours. Then, the results obtained were observed, and the clear zone that appeared was compared with the chromatogram. Lastly, each spot producing a clear zone was sprayed using AlCl3 and Dragendorff spray reagent.

RESULT AND DISCUSSION
The results of raw material identification stated that the material used was Euchema spinosum. The pharmacological activity of E. spinosum likely depends on the content of the active chemical compounds, especially the secondary metabolites. E. spinosum, based on Phytochemical screening analysis, was known to contain flavonoid, alkaloid, and steroid components (Table 2).
It is proven that the continuous maceration method with the three solvents could engage the essential secondary metabolite compounds in the material, shown by the detection of three metabolite components in the three extracts. Moreover, the maceration process was also able to attract monoterpenoids and sesquiterpenoids. They are known as the constituents of essential oils, previously being undetected at the time of phytochemical screening in the raw material. Several components of essential oils are known to eliminate S. mutans and S. dysenteriae actively since they can destroy biofilms made by S. mutans (Khan et al., 2020;Tofiño-Rivera et al., 2016) and by S. dysenteriae (Batista et al., 2018;Kang et al., 2020).
Apart from flavonoids, alkaloids and steroids are signified to have antibacterial activity, especially against S. mutans and S. dysenteriae. Xu et al. successfully isolated several alkaloid groups such as Fructigenine A, Fructigenine B, and Brevicompanine G from Pleosporales sp (marine fungi) that actively inhibit the growth of S. dysenteriae. Xu also successfully isolated the steroid class, namely Ergosta-4,6,8 (14), 22-tetraen-3-one, and (22E) -Ergosta-5,22-dien-3-one that are also known to actively inhibit the growth of S. dysenteriae (Xu et al., 2018). Meanwhile, alkaloids can damage the peptidoglycan constituent components in bacterial cells, causing the cell wall layer to not forming appropriately. In addition, alkaloids also inhibit the activity of the topoisomerase enzyme in bacteria (Singkoh et al., 2019). Some of the alkaloids from Rhodophyta algae are the indole alkaloid group (Pérez et al., 2016).

Antimicrobial activity
Initially, all hexane, ethyl acetate, and methanol extract concentrations did not show the inhibition growth activity against S. dysenteriae. However, after the assay was continued with an increase in the concentration measure, all extracts finally showed the inhibition zone starting from a 400 µg/mL concentration (Figure 1). However, only ethyl acetate extract was particularly active against S. mutans bacteria and gave an inhibition starting from 20 μg/mL (Table 3 and Figure 1).  Although the phytochemical screening showed the same class of compounds present in each extract, the polarity of these compounds was different. Hydrophilicity and lipophilicity are known to affect the antibacterial activity of a compound. Lipophilic extracts from the Rhodophyta division have been proven to inhibit the growth of several bacteria (Cortés et al., 2014). Lipophilic compounds such as flavonoids and terpenoids are known to be able to interact with bacterial cell walls so that they can damage membranes (Dharmautama et al., 2019;Medina-Flores et al., 2016). Lipophilic flavonoids also actively inhibit the growth of both gram-positive and gram-negative bacteria (Farhadi et al., 2019). As an example, Apigenin (4′,5,7-Trihydroxyflavone) could reduce the virulence level of S. mutans (André et al., 2018). Hydrophilic compounds are also known to actively resist several microbes including the glycoside group, such as aminoglycosides (Nweze et al., 2020), flavonoid glycosides (Zou et al., 2016), and surfactants (Anestopoulos et al., 2020). Hence, the three extracts were continued to the examination using the TLC-bioautography analysis against S. dysenteriae bacteria. Meanwhile, the ethyl acetate extract was the only one subjected to the TLCbioautography assay against S. mutans.

TLC-bioautography
TLC analysis of the three extracts resulted in that hexane extract eluted with hexane: ethyl acetate (4:1) showed at least 6 spots. Methanol extract eluted with chloroform: acetone eluent (1:2) showed the presence of 4 spots, and ethyl acetate extract eluted with chloroform: ethyl acetate eluent (2:3) showed 6 spots ( Figure 2). Unfortunately, the elution systems could not reproduce the Rf spot with precision, where the elution at different times produces the same number of spots, but with diverse Rf. Each extract was known to contain many compounds with different polarities. It shows that E. spinosum is rich in compounds that might likely produce pharmacological activity, especially antibacterial.  These compounds were probably flavonoids glycosides that can act as a surfactant capable of penetrating bacterial cell wall membranes. The spray reagent assay on the spot showed negative results ( Figure 4) where none of the compounds reacted with the spray reagents, but the phytochemical screening confirmed the appearance of flavonoids, proving the possibility of flavone glycosides within. As we know, flavone glycosides with sugar at C5 would not react with AlCl3 reagents (Harborne, 1973). As for the ethyl acetate extract, a clear zone appeared on the spot with an Rf between 0.5-0.8. It indicates that the polarity of the compound was semipolar, almost non-polar. The spray reagent assay showed that the spot producing the clear zone reacted with AlCl3 (observed under 366 nm UV lamp lighting). This compound was likely flavonoids of the flavone type, evidenced by its ethyl acetatesoluble nature resulting in a positive outcome in the phytochemical screening and with the AlCl3 reagent (Harborne, 1973). On the other hand, the hexane extract showed no clear zone at all. In this extract, the components of the compound were very non-polar, so it was likely that this compound was unable to penetrate the bacterial cell wall.
The ethyl acetate extract assay against S. mutans showed a clear zone at the starting point. It indicates that the compound has a semipolar relative to polar nature. Analysis with AlCl3 showed a positive result in the spot area ( Figure 4). The information about the specific active compound needs to be further clarified through the isolation stage to determine which flavonoid groups having an activity to inhibit the growth of both S. dysenteriae and S. mutans.

CONCLUSION
It is proven that E. spinosum extract can inhibit the growths of S. dysenteriae and S. mutans bacteria, but only E. spinosum in ethyl acetate extract could inhibit both bacteria. All of the tested extracts might have the ability to inhibit the growth of S. dysenteriae starting from 400 µg/mL, where only ethyl acetate extract could inhibit S. mutans growth starting from 20 µg/mL. The class of compounds predicted to have antibacterial activity against the two tested bacteria were flavonoids.

ACKNOWLEDGEMENT
Thank you to LPPM-UNISBA that has funded this research through a young lecturer research scheme with the number SPK 002/B.04/LPPM/2019 and the author also owes thanks to Prof. I. Sahidin from Halu Oleo University for the important advice to accomplish this manuscript.

CONFLICT OF INTEREST
The author declared that there is no conflict of interest in this research. The data could be published by the author completely.