Bioactivity of Indonesian’s Marine Sponge Xestospongia muta as Anti- dormant Mycobacterium smegmatis

The research of isolation and characterization from the Indonesian’s Marine Sponge Xestospongia muta, Clathria sp. and Endectyon delaubenfelsi as anti-dormant against Mycobacterium smegmatis has been conducted on May 2018. The bioactive compounds were isolated based on bioassay-guided separation with several steps of chromatography. The result of bioactivity showed different activity from Xestospongia muta (MIC50=0.2 μg/mL), Clathria sp. (MIC50=0.9 μg/mL) and Endectyon delaubenfelsi (MIC50=0.4 μg/mL). The results of FTIR spectrum interpretation of Xestospongia muta has vibration at 3435.56 cm-1 indicate as O-H alcohol functional group with the confirmation of the C-O alcohol at 1342.24 cm-1 in the fingerprint region. The active metabolite has C≡N imine at 2365.28 cm-1 with the confirmation of C-N imine at 1637.27 cm-1 in the fingerprint region. Based on MIC50 data shows that Xestospongia muta has potential cytotoxic against Mycobacterium smegmatis.


Introduction
Mortality and morbidity infections caused by Tuberculosis (TB) remains a major global with 1.7 million deaths and an estimated incidence of 10.4 million cases by 2016 [1].
Isoniazid is one type of antibiotic that is used to treat tuberculosis (TBC-TB) to be taken for 6 months, every day for the first two months, and three times a week for four months [2]. Drugs also known as isonicotinylhydrazide (INH) are usually combined with other tuberculosis drugs such as ethambutol, pyrazinamide and rifampin to eradicate bacteria. Mycobacterium tuberculosis in pulmonary tuberculosis or extrapulmonary disease [3]. But the use of this antibiotic drug gives side effects to the body one of hepatitis disease [4].
In addition to antibiotic drugs, tuberculosis treatment can also be overcome using drugs derived from natural marine materials such as sponge. Currently, the sponge has been widely used for medicinal materials because sponges contain antitumor compounds, antiviral, antibacterial, antifungal, antifouling, and antimalarial.

Cultivation of bacterial and growth conditions
M. smegmatis were maintained on Middlebrook 7H10 agar supplemented at 37°C with 10% Middlebrook OADC and 0.5% glycerol [5].

Preparation of the extract under aerobic and hypoxic conditions
MTT method used for determined MIC value of M. smegmatis. Midlog-phase bacilli (M. smegmatis: 1 × 10 4 CFU/0.1 mL) were inoculated and serially diluted samples added in a 96-well plate [6,7]. Mycobacterial were incubated for 36 hours at 37°C (aerobic conditions) [8]. The protocol of Rustad et al. was used with minor modifications (for hypoxic conditions) [9][10][11]. Mycobacterial were grown in Middlebrook 7H9 broth under nitrogen atmosphere containing oxygen (0.2%) at 37°C based on OD 600 =0.8 [12,13]. Mycobacterial were inoculated under aerobic conditions and incubated at 37°C under nitrogen atmosphere containing oxygen 0.2% on a 96-well plate for 96 hours [14]. MTT solution was added to each well after incubation. Then, the well incubated at 37°C for an additional 12 hour under aerobic or hypoxic conditions. Furthermore, the well plate measured to determine MIC value in OD 560 [15,16].

The curve of death time against M. smegmatis
M. smegmatis culture on Middlebrook 7H9 broth was arranged to 1 × 10 6 CFU/mL. Furthermore, added the extract until 4 × MIC [17]. and Middlebrook 7H10 agar were diluted to measuring CFU. The numbers of colonies were counted after 36 hours incubation [18].

Results
Compounds that have been isolated from several sponges have different MIC 50 values ( Figure 1). Clathria sp. ((2.74 g) MIC 50 =0.9 µg/mL) was isolated as a colorless solid. Based on the FTIR spectrum shows that the active metabolite has functional groups at 3435.56 cm -1 as N-H secondary amines, 2853.39 cm -1 as C-H methyl, 2769.64 cm -1 as C-H methylene and the fingerprint region of amine at 1637.27 cm -1 as C-N imine ( Figure 2). The results show that the active compound as an alkaloid.
Xestospongia muta [(0.15 g) MIC 50 =0.2 µg/mL] was isolated as a colorless solid. Based on the FTIR spectrum shows that the active metabolite has functional groups at 3435.56 cm -1 as O-H alcohol, at 2853.39 cm -1 as C-H methyl, at 2769.64 cm -1 as C-H methylene, at 2365.28 cm -1 as C≡N imine, the fingerprint region at 1637.27 cm -1 (as C-N imine) and 1342.24 cm -1 (as C-O alcohol) (Figure 3). Interpretation of FTIR spectrum indicates that the active compound as an alkaloid with oxygenated carbon in the hydrocarbon system.
Endectyon delaubenfelsi [(1.13 g) MIC 50 =0.4 ug/mL] was isolated as a colorless solid. Based on the FTIR spectrum shows that the active metabolite has functional groups at 3434.6 cm -1 as N-H amine, C-H methyl was detected at 3090.46 cm -1 , and the fingerprint region at 1637.27 cm -1 as C-N imine (Figure 4). Interpretation of data indicates that the active metabolite compound as an alkaloid.

Discussion
Based on the results of extraction and isolation of the three sponges,     [19], so that the Xestospongia muta has potential activity against Mycobacterium smegmatis.
The work drugs from alkaloid group are bactericidal that can kill 90% of the germ population after the first few days of use [20]. Isoniazid drugs are highly effective against developing germs. The mechanism of action of isoniazid has an effect on fat, nucleic acid biosynthesis, and glycolysis. The main effect of this drug is to inhibit the biosynthesis of mycolic acid (mycolic acid) which is an important element of Mycobacterium cell wall. INZ works by inhibiting the synthesis of mycolic acid which is an essential element of Mycobacterium tuberculosis cell wall formation [21].
Isoniazid drugs if taken simultaneously with other drugs will cause some interaction. It inhibits anticonvulsant drug metabolism, increases the risk of bleeding if taken with warfarin, reduces the absorption of isoniazid, and increases the risk of peripheral neuropathy.

Conclusion
The alkaloid compounds originating from the Indonesian marine sponges of the Xestospongia muta species have activity against M. smegmatis with MIC 50 =0.2 μg/mL which is almost equivalent to the MIC 50 value of Isoniazid ie 0.1 μg/mL.