Supercritical Carbon Dioxide Extracts of Cordyceps sinensis: Chromatography-based Metabolite Profiling and Protective Efficacy Against Hypobaric Hypoxia

Abstract

The toxicity and disposal concerns of organic solvents used in conventional extraction purposes has entailed the need for greener alternatives. Among such techniques, supercritical fluid extraction (SFE) has gained popularity by yielding extracts of high purity in a much faster manner. Carbon dioxide (CO2) is generally preferred as a supercritical solvent because of its lower temperature requirements, better diffusivity and easy removal. The present study describes the characterization of supercritical CO2 extracts of Indian variety of Cordyceps sinensis (CS)- a high-altitude medicinal mushroom widely revered in traditional medicine for its extensive anti-hypercholesterolemic, anti-inflammatory, anti-proliferative and energy-enhancing properties. Experimental parameters viz. 300 and 350 bar of extraction pressure, 60°C of temperature, 0.4°L/h CO2 of flow rate and use of 1% (v/v) of ethanol as entrainer were optimized to prepare three different extracts namely, CSF1, CSF2 and CSF3. High-performance thin-layer chromatography (HPTLC) was used for assessing the quality of all the extracts in terms of cordycepin, the pivot biomarker compound in CS. Characterization by HPTLC and GC-MS confirmed the presence of flavonoids and nucleobases and, volatile organic compounds (VOCs), respectively. The chromatographic data acquired from metabolite profiling were subjected to chemometric analysis in an open source R studio which illustrated interrelatedness between CSF1 and CSF2 in terms of two major principal components. i.e. Dim 1 and Dim 2 whose values were 40.33 and 30.52% in variables factor map plotted using the HPTLC-generated retardation factor values. The factor maps based on retention times of the VOCs exhibited a variance of Dim 1 = 43.95% and Dim 2 = 24.85%. Furthermore, the extracts demonstrated appreciable antibacterial activity against Escherichia coli and Salmonella typhi by generation of reactive oxygen species (ROS), protein leakage and efflux pump inhibition within bacterial pathogens. CSFs were elucidated to be significantly cytoprotective (p < 0.05) in a simulated hypobaric hypoxia milieu (0.5% oxygen). CSF2 showed the best results by effectively improving the viability of human embryonic kidney (HEK 293) cells to 82.36 ± 1.76% at an optimum dose of 100 µg/ml. Levels of hypoxia inducible factor-1 alpha (HIF-1α) were modulated four-fold upon supplementation with CSF2. The results collectively evinced that the CSF extracts are substantially bioactive and could be effectively utilized as mycotherapeutics for multiple bioeffects.

Update on potential of edible mushrooms: high‐value compounds, extraction strategies and bioactive properties

“…Other green alternatives to the use of organic solvents can be supercritical carbon dioxide extraction, a technique requiring low temperatures and treatment time, while providing good diffusivity ratio and extraction yields. A recent example can be the extracts from Cordyceps sinensis, containing flavonoids, nucleobases and volatile organic compounds, among other high‐quality components (Mishra et al ., 2021). Ultrasound‐assisted extraction has also revealed as an emerging system for the extraction of potentially mycoactive compounds.…”

“…For this reason, these species could be considered as potential target viral inhibitors (Choengpanya et al ., 2021). The promising myotherapy treatment of hypobaric hypoxia condition by C. sinensis extracts was another example of the wide variety of possibilities that mushroom provides in the human medicinal field (Mishra et al ., 2021). Furthermore, it should be remembered that these resources could also be utilised for the treatment of different animal complications such as the antiparasitic control of gastrointestinal nematodes in order to prevent anthelmintic resistance.…”

The Effect of Green Extraction Technologies on the Chemical Composition of Medicinal Chaga Mushroom Extracts

“…For example, polysaccharide extracts derived from medicinal mushrooms are commercially produced by a combination of ethanol and high-pressure water extraction which is time, labor, and energy-consuming [ 8 ]. In addition, some of these conventional techniques require toxic organic solvents, give low yields, can cause thermal degradation of active compounds, loss of volatile compounds, have low extraction selectivity, and can lead to the presence of residues in the desired compounds and final products [ 9 , 10 , 11 ]. All this has led to an interest in new extraction methods that will be ecologically and economically acceptable [ 12 , 13 ] and are merged under the term Green Extraction Technologies (GETs).…”

“…The authors reported increased recovery of phenolic compounds, carbohydrates, proteins, and minerals when compared with conventional extraction with shaking. Similarly, Mishra et al [ 10 ] optimized the supercritical CO 2 extraction parameters to obtain more bioactive Ophiocordyceps sinensis ( Cordyceps sinensis in the reference paper) extracts. This technique proved to be protective of mushrooms’ thermolabile compounds.…”