*3.6. Secondary Metabolite Profiling and GC-MS Analysis of Metabolite Extract*

Characterization of bioactivities of the metabolites had demonstrated that the present endophytic strain, *S. californicus* ADR1 was an unexplored source of antimicrobial, antibiofilm and antioxidant agents, which might have potential clinical applications. It was therefore prudent to analyze the composition of the metabolite extract to unravel the class of compounds produced. Therefore, the ADR1 metabolite extract was subjected to chemical profiling using specific reagents. The tests revealed the classes of compounds present in the extract of metabolites produced by the strain ADR1, which included anthraquinones, anthocyanins, terpenoids, terpenes, flavonoids, phenols, alkaloids and glycosides (Table 3). Various compounds, which belonged to these classes have therapeutic significance and are routinely reported from phytochemical screening of plant extracts. But recent literature suggested that actinobacteria too are proving to be excellent sources of such class of compounds [14,71].


**Table 3.** Chemical profiling of ethyl acetate extract of secondary metabolites produced by *S. californicus* strain ADP4.

Note: '+': Present; '−': Absent.

While the results of chemical profiling were primarily qualitative in nature suggesting the presence or the absence of a specific chemical class, it did not offer any clue about the identity of the compounds. But it is important to have a deeper insight into the chemical class and identity of the specific compounds produced by the culture if these are to be considered for drug development. In pursuit of this goal,

GC-MS analysis of the ADR1 metabolites was undertaken to probe the identity of metabolites based on their mass spectrum.

The GC-MS analysis revealed the presence of alkaloids, terpene, terpenoids and glycosides in the ADR1 metabolite extract, thus confirming the findings of chemical profiling. The compounds were assigned probable identities based on similarity index (SI) value, generated from the mass spectral analyses of the test compounds and their comparison with the reference compounds listed in the NIST and Wiley library. An SI of more than 85 was considered for assignment of probable identity to the compounds [72]. Out of the total 30 peaks detected in the chromatogram, 20 peaks were specific to the culture *S. californicus* ADR1 while rest of the peaks were related to the production media and the solvent control (Figure S1). Only six of ADR1 metabolite peaks were found to have SI value greater than 85. Thus, identities could be assigned to only these compounds (Table 4). Majority of the compounds detected in the GC-MS chromatogram showed lower degree of similarity with SI values between 60–80 (Table S1), suggesting a high probability of occurrence of novel compounds among the secondary metabolites produced by *S. californicus* strain ADR1.

The first compound, detected at RT 11.945 min, showed SI of 88 with Methanoazulen-9-ol, decahydro-2, 2, 4, 8-tetramethyl-stereoisomer, which belonged to the class sesquiterpene and possessed antibacterial as well as antioxidant properties [73]. This compound was earlier reported as a major component of plant essential oils [74,75] but there are no reports on this from microbial sources as yet. Another compound in ADR1 metabolite extract was detected at RT 14.519 min, which showed SI of 90 with 5-z-methyl-2-z-hydroxycarbonyl-5-e-ethenyl-4-z-propen-2-ylcyclohexanone, was a terpenoid having structure similar to Asperaculane B, a known GABA-transaminase inhibitor having significance in epilepsy treatment [76]. Terpenes and terpenoids have wide therapeutic potential like antimalarial, antibacterial, antiinflammatory, antioxidant as well as wound healing properties [77]. A flavonoid that was found most abundant among the ADR1 metabolites, showed SI of 85 with 4 -Methoxy-2 -(trimethylsiloxy) acetophenone. Flavonoids are well regarded molecules with broad-spectrum biological activities as well as applications in nutraceuticals and cosmetic industry [78]. However, there are no reports on the activity of this molecule till date. An alkaloid with SI of 88 in comparison to pyridineethanamine, n-methyl-n-[2-(4-pyridinyl) ethyl] was detected among the ADR1 metabolites at RT 16.068 min. The compound is known for its importance in the treatment of vertigo [79]. Alkaloids are well regarded for their wide spectrum of therapeutic activities including antibacterial, antioxidant, anti-inflammatory, anti-diabetic, antimalarial and anticancer properties [80]. The presence of therapeutically important chemical classes of compounds in ADR1 metabolite extract underscored its importance in the discovery of novel compounds with prominent therapeutic potential.


**Table 4.** Similarity Index-based analysis of the compounds in the secondary metabolite extract of *S. californicus* ADR1. The data were obtained from GC-MS chromatogram. The reference compounds were from NIST and Wiley Library.
