## 1 Introduction  
Microalgae are microscopic organisms inhabiting almost every marine environment, including freshwater ecosystems, such as ponds and lakes, and saltwater ecosystems, such as the oceans around the world and even the Antarctic [1; 2]. Numerous studies showed that microalgae contain high level of carotenoids [3], chlorophyll [4], phenolic content and polyunsaturated fatty acids (PUFAs) [5; 6]. Carotenoids and chlorophyll are well recognized as natural antioxidant pigments due to their capability to scavenge free radicals, reduce oxidative stress and enhance immunization [7]. PUFAs might be used for preventing cardiovascular disease [8]. Additionally, the fossilized remains of the microalgal diatom known as diatomic is also commercially valuable as a filter, absorbent, anti-caking agent, and material for insulation [9]. Thus, microalgae exhibit high potential as sources of biological activities in the food, pharmaceutical, environmental and cosmetic industries. _Chaetocors calcitrans_, the species belonging to the family _Bacillusriohyceae_, was used in this study, since it contains high amounts of natural antioxidant pigments (carotenoids and chlorophylls) and PUFAs [10; 11]. Recently, [PERSON] and co-workers [11] identified fucoxanthin from this diatom extract as being influential for antioxidant activity. The group also suggested that the outstanding antioxidative activity of fucoxanthin isolated from the dichloromethane fraction of _C. calcitrans_ may be attributed to the presence of allelic bonds at C-7\({}^{\prime}\) position, conjugated carbonyl, 5,6-monoepoxide and the acetyl groups, in which the excited electrons in radicals are transferred [12]. Another group, [PERSON] et al. [13], discovered that exposing crude ethanol extracts of this indigenous microalgae, _C. calcitrans_ on human breast cancer cell lines, MCF-7, resulted in apoptosis and decreased proliferation of these cells. [PERSON] et al. [14] also reported that through an apoptosis determination test the ethyl acetate (CEA) extract of _C. calcitrans_ showed remarkable cytotoxicity effect on parental cancer cell lines, MDA-MB-231 cells. Other closely related species, _Chaetocors muelleri_ and _Chaetocors lauderi_ displayed antimicrobial and antibacterial effects due to elevated amounts of eicosapentaenoic acids (EPA) [15; 16]. Nevertheless, there is no available information regarding the anti-inflammatory activity of _C. calcitrans_. Aside from potential therapeutic applications for _C. calcitrans_, it has also been reported that this microalga is suitable as a valuable feed supplement or substitute for conventional animal feed sources. Microalgal biotechnology has made it possible to produce this microalga commercially via continuous production even though it is not very easy to grow on a large scale [17]. Metabolite extraction is a key stage in the workflow of a metabolomics study. In this regard, the choice of solvents used for extraction is critical since the main aim of this step is to extract as wide a spectrum of chemical structures as possible from the metabolites [18]. Hence, solvents with polarity indexes closest to the desired polarities of the metabolites are usually selected. To evaluate the contribution of metabolites in microalgae to their antioxidant activity, a mostly targeted approach was used [19; 20]. In the targeted approach, only the desired metabolites were analyzed and, generally, the number of metabolites is low. However, the extraction of microalgae can contain a lot of metabolites and some of the metabolites may contribute to their activity. Thus, comprehensive and holistic analysis (the un-targeted approach) is necessary. \({}^{1}\)H nuclear magnetic resonance (NMR)-based metabolomics have been used widely for un-targeted analysis to evaluate the correlation of the identified metabolites in \({}^{1}\)H NMR spectra of plant extracts to their biological activities, such as antioxidant [21], anti-inflammation [22], and antidiabetic [23] activities. However, there is no data on the metabolic discrimination of _C.