*3.5. Cytotoxicity Assay*

The tested results of the extract against cell lines were shown in (See Supplementary Figure S2). The results revealed that the extract showed varying efficacy against cell lines. The highest activity against U-87 at 100 μg/mL was found to be 59.63 ± 1.9%. It also showed significant activity against MDA and HT-29 with cell inhibition was found to be 55.23 ± 1.09% and 52.31 ± 2.4% respectively at 100 μg/mL (Table 5). Overall, the results suggested the potential cytotoxic activity against various cell lines.

**Table 5.** Cytotoxic activity of extract of *Streptomyces* sp.S2A against HT-29, MDA and U-87 MG.


#### *3.6. Gas Chromatography-Mass Spectrometry (GC-MS)*

Analysis of components of the active fraction with the highest activity by GC-MS analysis implied nine peaks at the retention time of (i) 17.239; (ii) 17.309; (iii) 20.811; (iv) 21.311; (v) 21.406; (vi) 21.586; (vii) 22.071; (viii) 22.126; (ix) 24.257. Further examination of MS peaks revealed *m*/*z* at 168, 259, 210 and 350. According to NIST library search, peak retentions at 21.311, 21.406 and 21.586 correspond to single compound i.e., pyrrolo[1–a]pyrazine-1,4-dione,hexahydro-3-(2-methylpropyl) (Figure 3). Other tentatively identified compounds were diphenylmethane, 2-Isopropyl-1-Phenyl-3-Pyrrolidin-1-yl Propane-1,3-Dione and Benzene, 1 1-tetradecyclidenebis (The GC-MS spectrum indicated the ions at 70 and 154 corresponded to molecule C7H10NO2 and C4H8 ions (See Supplementary Figures S3 and S4). The spectrum was similar to that of pyrrolo[1–a]pyrazine-1,4-dione,hexahydro-3-(2-methylpropyl) spectra in the GC-MS library and a study reported by Yang et al. [26]. The FT-IR spectrum of the

partially purified metabolite showed the characteristic functional groups such as NH stretching peak of a primary amine at 3313.20 cm<sup>−</sup>1. The functional group at 2923.91 cm−<sup>1</sup> corresponded to strong C-H stretching in alkanes. The peak at 1646.10 cm−<sup>1</sup> was assigned to C-N stretch in primary amine. The absorption peak at 1516.05 cm−<sup>1</sup> was assigned to C=C stretch in an alkene. The peak at 1454.69 cm−<sup>1</sup> was assigned to C-H bend in alkanes. The peak at 1240.27 cm−<sup>1</sup> was assigned to C-O stretch. The absorption peak at 1033.54 cm−<sup>1</sup> was assigned to the ether. The peak ranging from 605.8 cm−<sup>1</sup> to 701.09 cm−<sup>1</sup> was assigned to strong C-H bend in alkenes (Figure 4).

**Figure 3.** Chemical structure of the compound pyrrolo[1–a]pyrazine-1,4-dione,hexahydro-3-(2-methylpropyl).

**Figure 4.** FT-IR spectrum of the active extract of *Streptomyces* sp.S2A.

#### **4. Discussion**

With this outlook, the present investigation was carried out to identify bioactive compound from marine actinobacteria exhibiting antagonistic activity against bacterial and fungal pathogens, enzyme inhibition activity, antioxidant and cytotoxic activity. *Streptomyces* sp.S2A isolated from marine sediment of Gulf of Mannar produced white aerial mycelium to colorless substrate mycelium on SCA medium. Shirling and Gottileb [27] reported that the pigmentation paradigm could be used for the classification and identification. However, there was no pigment pattern observed with the isolate. Extraction of metabolites with ethyl acetate yielded the dark color residue. Purification by silica gel column chromatography resulted in six fractions, of which one fraction exhibited significant activity. The antagonistic activity of the bioactive compound showed the high zone of inhibition against *Micrococcus luteus* and *Staphylococcus epidermidis* (16 mm). Moderate activity was observed against *Bacillus cereus*, *Klebsiella pneumoniae* and *Staphylococcus aureus* (14 mm). Susceptibility of *Escherichia coli* to the compound was found to be weaker (10 mm). Inhibitory potential of the bioactive compound against fungal pathogens showed good activity against *Fusarium moniliforme* and *Bipolaris maydis*, whereas no zone of inhibition was observed against *Aspergillus flavus* and *Alternaria aleternata*. MIC was

used to determine the efficacy of the compound at different concentration. The reduction in the growth of bacterial pathogens was observed with the concentration ranging from 7.8–31.25 μg/mL. MIC of the compound showed excellent antifungal activity against *Fusarium moniliforme* and *Bipolaris maydis*. Ethyl acetate extract of *Streptomyces* sp.S2A also showed significant α-glucosidase and α-amylase inhibition activity, though IC50 of the extract were less than that of acarbose. Antioxidant and cytotoxic activities of the extract was determined by the DPPH, ABTS, FRAP and metal chelating assays and against HT-29, MDA and U-87 MG cell lines. The results thus obtained showed the presence of potent antioxidant and anticancer agents.

The partial chemical composition relates to the metabolite was detected by GC-MS. The chromatogram of fraction A43 showed a total of nine peaks. Of all, the major constituent was pyrrolo[1–a]pyrazine-1,4-dione,hexahydro-3-(2-methylpropyl)-, constituted 80.7% and this may be the active principle compound. The other chemical compound was identified as diphenylmethane (6%), 2-Isopropyl-1-Phenyl-3-Pyrrolidin-1-yl-Propane-1,3-Dione (2%) and Benzene, 1 1-tetradecyclidenebis (2%). Pyrrolo[1–a]pyrazine-1,4-dione,hexahydro-3-(2-methylpropyl is a peptide derivative of diketopiperazine with the molecular weight as 210 and empirical formula as C11H18N2O2. All the bioassays mentioned in the above paragraph were confirmed with the commercially available purified compound: pyrrolo[1–a]pyrazine-1,4-dione,hexahydro-3-(2-methylpropyl). Antimicrobial and the cytotoxic activity of the purified compound were found to be significantly higher the partially purified compound, whereas the enzyme inhibition potential of the partially purified compound against α-glucosidase and α-amylase were better in comparison with commercial compound. The mass spectrum of the commercial compound corresponds to 70.0315 *m*/*z* and 154.0152, which is same as shown by the compound present in partially purified extract (See Supplementary Figures S5 and S6, Tables S1–S3).

Pyrrolopyrazines are known for their wide range of biological activities such as antioxidant, anti-angiogenesis, anti-tumor and antimicrobial [28]. Manimaram et al. reported the presence of antibacterial metabolite, pyrrolo[1–a]pyrazine-1,4-dione,hexahydro-3-(2-methylpropyl) in the crude extract of *Streptomyces* sp. VITMK1 isolated from mangrove soil [29]. Antifouling potential of pyrrolo[1–a]pyrazine-1,4-dione,hexahydro-3-(2-methylpropyl) against *Vibrio halioticoli* and *Loktanella honkongensis* was studied by Dash et al. [30]. Sponge-derived marine bacteria significantly inhibited the larval settlement of *Balanus amphitrite* and *Hydroides elegans*. Another marine bacteria isolated from the sponge, *Spongia officinalis* showed the potent antibacterial and antifungal activity of pyrrolo[1–a]pyrazine-1,4-dione,hexahydro-3-(2-methylpropyl) [31]. Diketopiperzines derivatives present in marine *Streptomyces* sp. had shown good anti-H1N1 activity [32]. Mithun and Rao also reported the presence of pyrrolopyrazines in *Micrococcus luteus* with anti-cancer activity against HCT-15 cell line [33]. Presence of pyrrolo[1–a]pyrazine-1,4-dione,hexahydro-3-(2-methylpropyl) was detected in *Streptomyces* sp. MUM 256 isolated from the mangrove forest in Malaysia. This compound was reported to possess antioxidant and anticancer activities [34]. Anti-cancer metabolites were also reported from *Streptomyces malaysiense* sp.MUSC 136 isolated from the mangrove ecosystem. The bioactive metabolite exhibited strong antioxidant activity and high cytotoxic activity against HCT-116 cells [35]. The first report on marine *Staphylococcus* sp. derived pyrrolo[1–a]pyrazine-1,4-dione,hexahydro-3-(2-methylpropyl) was reported by Lalitha et al. [36]. Purified metabolite was potentially active against lung (A549) and cervical (HeLa) cancer cells in a dose-dependent manner. Thus, the present study suggested that the pyrrolopyrazines derivative pyrrolo[1–a]pyrazine-1,4-dione,hexahydro-3-(2-methylpropyl) may account for the observed antagonistic, antioxidant, and cytotoxic activities in marine actinobacteria, *Streptomyces* sp.S2A. The results obtained in the current study demonstrate that bioactive metabolites produced by marine actinobacteria have tremendous potential for pharmaceutical product and are a subject of future investigation.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-2607/6/3/72/s1.

**Author Contributions:** Conceptualization, S.S. and R.R.V.; Methodology, S.S. and R.R.V.; Software, S.S. and R.R.V.; Validation, S.S. and R.R.V.; Formal Analysis, S.S. and R.R.V.; Investigation, S.S.; Resources, R.R.V.; Data curation, Writing—Original Draft Preparation, S.S. and R.R.V.; Writing—Review & Editing, S.S. and R.R.V.; Visualization, S.S. and R.R.V.; Supervision, R.R.V.; Project Administration, R.R.V.; Funding Acquisition, R.R.V.

**Funding:** This research was funded by DST-PURSE program, DST, New Delhi, grant number DST-PURSE/ Phase-II/2017-2018." The APC was funded by DST-PURSE program.

**Acknowledgments:** The authors would like to thank SIF, VIT University, Vellore for carrying out GC-MS analysis. The authors would also like to acknowledge the facilities provided by DST-PURSE program, DST, New Delhi to the University of Mysore, Mysuru.

**Conflicts of Interest:** The authors confirm that this article content has no conflict of interest.
