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. 2021 Aug 26:12:628924.
doi: 10.3389/fphar.2021.628924. eCollection 2021.

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

Affiliations

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

Jigni Mishra et al. Front Pharmacol. .

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.

Keywords: Cordyceps sinensis (Berk) Sacc.; GC-MS; HPTLC; hypobaric hypoxia (HH); metabolomics; supercritical fluid extract.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Effect of extraction pressure and temperature on the yields of C. sinensis supercritical fluid (CSF) extracts. Combination of various pressures (200, 250, 300 and 350 bar) and temperatures 40, 50, and 60°C were used for the optimization protocol. 1% (v/v) ethanol was used as entrainer. CO2 flow rate was 0.4 L/h.
FIGURE 2
FIGURE 2
HPTLC chromatogram at 254 nm shows bands distinctive of cordycepin, the main marker metabolite used for quality assessment of Cordyceps spp. The retardation factor (Rf) was determined to be 0.45. Lanes labelled Std1, Std2 and Std3 contain 0.25, 0.5 and 1 µg of standard cordycepin.
FIGURE 3
FIGURE 3
HPTLC chromatogram at 254 nm confirms the presence of adenine and cytosine in CSF1 and CSF2 extracts. Respective retardation factor values are written in parentheses (A). Quantities detected from densitometric scanning are also given (B). Lanes labelled Std1, Std2 and Std3 contain 0.5, 0.58 and 0.66 µg of each standard nucleobase. Values for quantification are represented as mean ± SD (n = 3).
FIGURE 4
FIGURE 4
HPTLC chromatogram at 254 nm confirms the presence of ascorbic acid, gallic acid and quercetin in CSF1 and CSF2 extracts, and only quercetin in CSF3 extract. Respective retardation factor values are written in parentheses (A). Quantities detected from densitometric scanning are also given (B). Lanes labelled Std1, Std2 and Std3 contain 0.75, 0.875 and 1 µg of each standard flavonoid. Values for quantification are represented as mean ± SD (n = 3).
FIGURE 5
FIGURE 5
Total ion chromatograms obtained after GC-MS analysis of Cordyceps sinensis supercritical carbon dioxide extracts. Based on area percentages, the most abundant volatile organic compounds in CSF1, CSF2 and CSF3 were revealed to be 9,12-octadecadienoic acid (Z,Z)- (A); 9,12-octadecadienoic acid (Z,Z)-, trimethylsilyl ester (B) and hexadecanoic acid, trimethylsilyl ester (C), respectively.
FIGURE 6
FIGURE 6
Variables factor map depicting variances among CSF1, CSF2 and CSF3 extracts on basis of HPTLC (A) and GC-MS (B) metabolite profiling. Similarities were observed between CSF1 and CSF2, the supercritical extracts prepared using ethanol as co-solvent, owing to their close presence in same quadrant. CSF3 occurred on a separate quadrant from the former. Rf, retardation factor value of metabolites detected by HPTLC; Rt, retention time of VOCs identified by GC-MS.
FIGURE 7
FIGURE 7
Among all the supercritical CO2 extracts of Cordyceps sinensis, CSF1 was discerned to have the highest in vitro free radical (DPPH, ABTS) scavenging activity and ferric ion reducing power (FRAP). Values are represented as mean ± SD (n = 3).
FIGURE 8
FIGURE 8
Distinct zones of inhibition after treatment with CSF1 and CSF2 extracts, against pathogenic Salmonella typhi can be clearly seen (A). Generation of reactive oxygen species after treatment with different doses (50, 100, 125 µg/ml) of Cordyceps sinensis supercritical fluid extracts within E. coli (B) and S. typhi (C) was deduced as a potential antibacterial mode of action. Induction of protein leakage of aforesaid extracts against E. coli (D) and S. typhi (E) was confirmed to be yet another inhibitory mechanism. The supercritical fluid extracts also led to inhibition of efflux pump activities in the bacterial cells (F). Nutrient broth and kanamycin acted as negative and positive controls, respectively. Data are expressed as mean ± SD (n = 3). * represents significant change at level of p < 0.05.
FIGURE 9
FIGURE 9
Restoration of cellular viability by C. sinensis supercritical CO2 extracts. At an optimum dose of 100 µg/ml, CSF1, CSF2 and CSF3 improved HEK 293 cellular viability to 79.53% (A), 82.36% (B) and 68% (C), under hypoxia. N, normoxia; H, hypoxia; N+x, supplementation of various doses (x = 50/75/100/125 µg/ml) of CSF in normoxia; H+x, supplementation of various doses (x = 50/75/100/125 µg/ml) of CSF in hypoxia. Results are expressed as mean ± SD (n = 3). * represents significant change at level of p < 0.05 with respect to N, # represents significant change at level of p < 0.05 with respect to H.
FIGURE 10
FIGURE 10
Western blot showing level of hypoxia inducible factor-1 alpha (HIF-1α) (A), which otherwise elevated under hypoxia stress, was decreased after supplementation with CSF2 (B). Hypoxia+CSF2 indicates supplementation of 100 µg/ml of CSF2 extract in HEK 293 cells being cultured in low oxygen tension (0.5% O2). Results are expressed as mean ± SD (n = 3). * represents significant change at level p < 0.05.

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