Bacterial infection Escherichia coli is one of the diseases that attack human digestive organs. Diarrheal disease is still a major public health problem with severe cases of illness and death. Mortality and morbidity caused by this bacteria are 1,7 million children every year, generally attacking children under 5 years of age [1,2].

To cope with this disease caused by bacteria, metronidazole antibiotics are used. Metronidazole is a class of antimicrobial drugs used to treat various infections caused by protozoan microorganisms and anaerobic bacteria. The use of this drug has side effects on the body such as changes in taste on the tongue [3-5].

One alternative that has been done in the prevention of this disease is to use marine natural materials obtained from waters Sabang sourced from sponges. But for the time being the sponge distribution in Sabang is still very limited. The sponge has been widely used for various research studies and can also be used as a medicine for various types of diseases [6,7].

Compound purification using the MPLC Sepacore X50 HPLC Shimadzu C196-E06IR Prominence LC-MS Mariner by using a detector. IR spectra were obtained from FTIR Shimadzu IR Prestige 21.

Tools and materials

Methanol, ethyl acetate, chloroform, dichloromethane, n-hexane, dragendorff reagents, cerium sulfate reagents, nutrient agar medium (NA), bacterial resistant E. coli, chloramphenicol, and H₂O.

Biomaterials

There are three types of Clathria Sp., Xestospongia muta, and Endoctycon delaubenfelsi obtained from the location of the three wells and Anoi Itam, Sabang Island Indonesia in 2018. The third type of sponge is taken at a depth of 20 to 35 meters. Sponge Storage at Marine Chemical Laboratory, Marine Science Study Program, Faculty of Marine and Fisheries. Sponge analysis was performed at the Laboratory of Natural Products for drug discovery, Graduate School / School of Pharmaceutical Science, Osaka University, Japan.

Extraction and isolation

Crude methanol fraction Clathria sp. used as much as 32,8 grams of crude 3x partitioned using n- Hexane: EtOAc: EtOH (1: 1: 1 v/v) yields n-Hexane fraction (1,43 g), EtOAc fraction (9,64 g), and EtOH fraction (21,73 g). Based on bioactivity, EtOH fraction yields [21,73 g (MIC₅₀ = 10 μg/mL)]. The ethanol fraction as much (21,73 g) will then be fractionated using an open chromatography column (OPN-C180 with a gradient eluent EtOH: TFA 0,1% to produce 4 fractions. The activity of the third fraction [7,88 g (MIC₅₀ = 0,75 μg / mL)] and purified using RP-18 HPLC column with MtCN eluent: H₂O: TFA 0,1% gradient resulting in 7 fractions. Activity from the fourth fraction [2,74 g (MIC₅₀ = 0,6 μg/mL)] showed cytotoxic activity of Escherichia coli. (MNZ MIC₅₀ = 0,1 μg/mL).

Xestospongia muta (24,5 grams of crude) was partitioned 3x using n-Hexane: EtOAc: EtOH (1: 1: 1 v / v) resulting in n-Hexane fraction (0,23 g), EtOAc fraction (6,28 g ), and the EtOH fraction (16,53 g). Based on the results of bioactivity, obtained EtOH fraction [16,53 g (MIC₅₀ = 0,9 μg / mL)]. The ethanol fraction (16,53 g) was further fractionated by using open chromatographic column (OPN-C18) with EtOA eluent: EtOH: 0,1% TFA resulting in 4 fractions. The activity of the second fraction [9,82 g (MIC₅₀ = 0,6 μg/mL)] and purified by using the RP-18 MPLC column with the Acetonitrile eluent: MeOH gradient resulting in 7 fractions. The third fraction activity [4,28 g (MIC₅₀ = 0.5 μg/mL)] is then refined using HPP RP-18 column with the MeCN: H₂O gradient eluent and yields 5 fractions. Activity from the fourth fraction [0,15 g (MIC₅₀ = 0,2 μg/mL)] showed cytotoxic activity against Escherichia coli.

Endectyon delaubenfelsi (42,85 gram of crude) was repeated 3x using n-Hexane: EtOAc: EtOH (1: 1: 1 v / v) resulted in fraction of n-Hexane (3,46 g), EtOAc fraction (27,83 g), and the EtOH fraction (11,58 g). Based on the results of bioactivity, the EtOAc fraction obtained was [27.83 g (MIC₅₀ = 0,7 μg / mL)]. The ethyl acetate fraction (27,83 g) was further fractionated by an open chromatographic column (OPN-C18) with EtOAc-graded eluates: Acetonitrile gradient yielded 6 fractions. Activity of the fourth fraction [12,45 g (MIC₅₀ = 0,5 μg / mL)] to be purified using a 5C-18 MS II HPLC column with CHCl3 eluent: MeOH: H₂O low-phase gradient yielded 9 fractions. The activity of the fifth fraction [3,36 g (MIC₅₀ = 0,4 μg / mL)] was further refined using a 5C-18 MS II HPLC column with the MeCN: H₂O: TFA eluent of 0,1% gradient resulting in 5 fractions. The activity of the third fraction [1,13 g (MIC₅₀ = 0,2 μg / mL)] shows cytotoxic activity against Escherichia coli.

Bacterial cultivation and growth conditions

Escherichia coli incubated at 37°C on Middlebrook 7H10 added with 10% Middlebrook OADC and 0,5% gliserol, or Middlebrook 7H9 lsolution with 10% Middlebrook OADC, 0,2% gliserol, and 0,05% Tween 80 [8-11].

Antimicrobial activation of the extract under aerobic and hypoxic conditions

MIC values against Escherichia coli were determined by using the established MTT method. Midlog-phase bacilli (Escherichia coli: 1 x 10⁴ CFU/0,1 mL) were inoculated in a 96-well plate, then serially diluted samples were added. For aerobic conditions, bacteria were incubated at 37°C for 12 h (Escherichia coli). For the hypoxic conditions, the protocol of Rustad et al. was used with minor modifications [12-16]. Mycobacterial were grown in Middlebrook 7H9 broth at 37°C under nitrogen atmosphere containing oxygen (0,2%) until OD₆₀₀=0,8. Subsequently, Mycobacterial were inoculated in a 96-well plate (same density) under aerobic conditions and incubated at 37°C under the nitrogen atmosphere containing oxygen (0,2%) for 12 h (Escherichia coli). After incubation, MTT solution (50 mL, 0,5 mg/mL) was added to each well and incubated at 37°C for an additional 12 h under aerobic or hypoxic conditions. OD₆₀₀ was measured to determine MIC value [17-19].

The curve of death time from extract to bacteria

OD560 was measured Escherichia coli was grown in Middlebrook 7H9 broth at 37°C under anaerobic condition or nitrogen atmosphere containing oxygen (0,2%) until OD₆₀₀=0,8. Escherichia coli culture in Middlebrook 7H9 broth was adjusted to 1 x 10⁶ CFU/mL, then extract (4 x MIC) was added. An aliquot (100 mL) was collected at each time point, and serially diluted cultures were plated on Middlebrook 7H10 agar to measure CFU. The numbers of colonies were counted after four-week incubation determine MIC value [20,21].

Based on the test of several isolated sponges obtained different MIC₅₀ values in Figures 1-4.

Figure 1: The result of MIC₅₀ test.

Figure 2: FTIR Spectrum of Clathria sp.

Figure 3: FTIR Spectrum of Xestospongia muta.

Figure 4: FTIR Spectrum of Endectyon delaubensif.

Clathria sp. [2,74 g (MIC₅₀ = 0,6 μg / mL) that has been isolated as a colorless solid The results of FTIR data show that the functional groups of secondary amine at 3435,56 cm^-1, C-H methyl at 2853,39 cm^-1, C-H methylene at 2769,64 cm^-1 and C-N imine at 1637,27 cm^-1. Based on the results of the data interpretation indicating the active metabolite compounds belong to the group of alkaloid compounds.

Xestospongia muta [0,15 g (MIC₅₀ = 0,2 μg/mL)] which has been isolated as a colorless solid. The results of FTIR data show that the functional group of OH alcohol at 3435,56 cm^-1, CH methyl at 2853,39 cm^-1, CH methylene at 2769,64 cm^-1 and C≡N imine at 2365,28 cm^-1, CN imine fingerprint in 1637, 27 cm^-1, and CO alcohol fingerprint at 1342,24 cm^-1. Based on the results of the data interpretation indicates that the active metabolite compounds belong to the group of alkaloid compounds.

Endectyon delaubenfelsi [1,13 g (MIC₅₀ = 0,2 μg/mL)] that has been isolated as a colorless solid. The results of FTIR data indicate that the functional group of N-H of amine at 3434,6 cm^-1, C-H methyl at 3090,46 cm^-1, and C-N imine fingerprint at 1637,27 cm^-1. Based on the results of data interpretation indicating active metabolite compounds belong to the group of alkaloid compounds.

The results of the three sponges that have been tested show different MIC₅₀. Sponge type Clathria sp. has a MIC₅₀ value = 0,6 μg / mL which is very far from MIC₅₀ MNZ = 0,1 μg/mL which means this type of sponge is not suitable as an alternative to bacterial infection of Escherichia coli. Metronidazole (MNZ) has the highest resistance to antibiotics that is 96,43%. Metronidazole is an effective antiprotozoal and antibacterial agent capable of fighting anaerobic protozoan parasites and anaerobic gram-negative bacilli, including Bacteroides sp., And gram-positive spore-forming anaerobes [22-24].

Metronidazole requires reductive activation of nitro groups by a susceptible organism. Single electron transfer forms a highly reactive nitro radical anion. The anions kill susceptible organisms through the radically mediated mechanism. The sponges of Xestospongia muta and Endectyon delaubenfelsi have an MIC₅₀ value of 0.2 μg/mL which is almost close to the MIC₅₀ MNZ value. So that these two sponge can be used as medicine as a substitute for MNZ [25-27].

Metronidazole (MNZ) is not always well consumed by humans. This is due to the resulting negative effects that affect people who take this drug and suffer from certain diseases. Based on the MIC₅₀ results, the two sponges can be used as Metronidazole (MNZ) replacement drugs [28].

Bioactive compounds obtained from the Indonesian marine sponge contain two types of Xestospongia muta and Endectyon delaubenfelsi which have bioactivity against Escherichia coli with the same MIC₅₀ value that is MIC₅₀ = 0,2 μg / mL. The MIC₅₀ value of both sponges approximates the MIC₅₀ value to MNZ commercial drug with MIC₅₀ value = 0.2 μg/mL.