(Inonotus Obliquus) Mushroom

antioxidants

Article Effects of pH and Temperature on Water under Pressurized Conditions in the Extraction of Nutraceuticals from Chaga (Inonotus obliquus) Mushroom

Ibrahim M. Abu-Reidah 1,2,*,†, Amber L. Critch 1,2,†, Charles F. Manful 1,2 , Amanda Rajakaruna 1,2, Natalia P. Vidal 1,2 , Thu H. Pham 1,2 , Mumtaz Cheema 1 and Raymond Thomas 1,2,*

1 School of Science and the Environment, Grenfell Campus, Memorial University of Newfoundland, Corner Brook, NL A2H 5G4, Canada; [email protected] (A.L.C.); [email protected] (C.F.M.); [email protected] (A.R.); [email protected] (N.P.V.); [email protected] (T.H.P.); [email protected] (M.C.) 2 The Functional Foods Sensory Laboratory, Grenfell Campus, Memorial University of Newfoundland, Corner Brook, NL A2H 5G4, Canada * Correspondence: [email protected] (I.M.A.-R.); [email protected] (R.T.) † These authors contributed equally.

Abstract: Currently, there is increased interest in finding appropriate food-grade green extraction systems capable of extracting these bioactive compounds from dietary mushrooms for applications in various food, pharmacological, or nutraceutical formulations. Herein, we evaluated a modified Citation: Abu-Reidah, I.M.; Critch, Swiss water process (SWP) method using alkaline and acidic pH at low and high temperature under A.L.; Manful, C.F.; Rajakaruna, A.; pressurized conditions as a suitable green food grade solvent to obtained extracts enriched with Vidal, N.P.; Pham, T.H.; Cheema, M.; Thomas, R. Effects of pH and myco-nutrients (dietary phenolics, total antioxidants (TAA), vitamins, and minerals) from Chaga. Temperature on Water under Ultra-high performance liquid chromatography coupled to high resolution accurate mass tandem Pressurized Conditions in the mass spectrometry (UHPLC-HRAMS-MS/MS) was used to assess the phenolic compounds and Extraction of Nutraceuticals from vitamin levels in the extracts, while inductively coupled plasma mass spectrometry (ICP-MS) was Chaga (Inonotus obliquus) Mushroom. used to determine the mineral contents. Over 20 phenolic compounds were quantitatively evaluated Antioxidants 2021, 10, 1322. in the extracts and the highest total phenolic content (TPC) and total antioxidant activity (TAA) https://doi.org/10.3390/ was observed at pH 11.5 at 100 ◦C. The most abundant phenolic compounds present in Chaga antiox10081322 extracts included phenolic acids such as protocatechuic acid 4-glucoside (0.7–1.08 µg/mL), syringic acid (0.62–1.18 µg/mL), and myricetin (0.68–1.3 µg/mL). Vitamins are being reported for the first Academic Editors: Raffaella Boggia, time in Chaga. Not only, a strong correlation was found for TPC with TAA (r-0.8, <0.0001), but Giosuè Costa and Federica Turrini also, with individual phenolics (i.e., Salicylic acid), lipophilic antioxidant activity (LAA), and total ◦ Received: 7 July 2021 antioxidant minerals (TAM). pH 2.5 at 100 C treatment shows superior effects in extracting the ◦ Accepted: 11 August 2021 B vitamins whereas pH 2.5 at 60 and 100 C treatments were outstanding for extraction of total Published: 23 August 2021 fat-soluble vitamins. Vitamin E content was the highest for the fat-soluble vitamins in the Chaga extract under acidic pH (2.5) and high temp. (100 ◦C) and ranges between 50 to 175 µg/100 g Chaga. ◦ ◦ Publisher’s Note: MDPI stays neutral Antioxidant minerals ranged from 85.94 µg/g (pH7 at 100 C) to 113.86 µg/g DW (pH2.5 at 100 C). ◦ ◦ with regard to jurisdictional claims in High temperature 100 C and a pH of 2.5 or 9.5. The treatment of pH 11.5 at 100 C was the most published maps and institutional affil- useful for recovering phenolics and antioxidants from Chaga including several phenolic compounds iations. reported for the first time in Chaga. SWP is being proposed herein for the first time as a novel, green food-grade solvent system for the extraction of myco-nutrients from Chaga and have potential applications as a suitable approach to extract nutrients from other matrices. Chaga extracts enriched with bioactive myconutrients and antioxidants may be suitable for further use or applications in the Copyright: © 2021 by the authors. food and nutraceutical industries. Licensee MDPI, Basel, Switzerland. This article is an open access article Keywords: chaga (Inonotus obliquus); accelerated solvent extraction (ASE); functional foods; swiss distributed under the terms and water process (SWP); green extraction; total antioxidant activity; total phenolic content; B-complex conditions of the Creative Commons vitamins; fat-soluble vitamins Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Antioxidants 2021, 10, 1322. https://doi.org/10.3390/antiox10081322 https://www.mdpi.com/journal/antioxidants Antioxidants 2021, 10, 1322 2 of 18

1. Introduction Functional foods refer to food products or ingredients that are useful in providing known health benefits beyond basic nutritional requirements [1]. Mushrooms are well rec- ognized as high quality sources of functional ingredients with respect to having enhanced levels of vitamins, proteins, and fiber [2]. Historically, mushrooms have rich traditions in folk medicine in many cultures across the globe, as well as popularity as food ingredient in many different cuisines. For example, the well characterized sporocarp are best known for their medical treatments or therapies in many Asian countries [3]. Recent studies have shown the health-promoting properties of mushroom to be associated with their ability to scavenge reactive oxygen species, decrease inflammation, protect against tumors, provide insulin resistance in type 2 diabetes, and bolster the body’s immune system [3,4]. One of the most common dietary mushrooms used as a functional food ingredient and having several health benefits in traditional and folk medicine is Chaga (Inonotus obliquus)[5]. Chaga mushroom is a member of the Hymenochaetaceae family [6] commonly found on birch trees within the colder (boreal) climates of North America, Northern Europe, and Asia [7]. Morphologically, I. obliquus first develops a white heart rot in the host tree. The chaga spores enters trees or the poorly healed branch stubs through their wounds. During the infection cycle, penetration of the tree wood takes place only around the sterile exterior mycelial mass [8]. The chaga fungus causes decays within the living tree for up to several decades. In the living trees, only the sterile mycelia masses (the black exterior conks) are produced. I. obliquus will begin to produce fertile sporocarp underneath the bark. These masses emerge as a whitey mass that turn to brown with time. The fruiting body is rarely observed, as the sexual stage comes almost about fully beneath the bark. These fruiting bodies produce basidiospores which can spread the infection to other susceptible trees. Inonotus obliquus commonly found on birch trees within the colder (boreal) climates of North America, Northern Europe, and Asia [7]. As such Chaga distribution is reflective of the circumboreal forest ecosystem (Figure1 ) in the Holarctic regions of Canada, the USA, Asia, and Europe (mainly Russia, Japan, Siberia, and Korea). In these regions, Chaga is highly valued for its use as a therapeutic drink and in enhancing the immune system in traditional or folk medicine [8]. The health benefits ascribed to enhancing the immune system functions are associated with beta- glucans activities. Furthermore, Chaga is reported to suppress the growth of cancer cells via decreased expression of the beta-catenin pathway, regulation of gut bacteria, and its high antioxidant activity [5,9,10]. Previous studies have also reported that Chaga polyphenols and superoxide dismutase enzyme form a potent antioxidant system associated with reduced oxidative stress, diabetes, cancer, and cardiovascular disease risks [11,12]. These health benefits of Chaga are also due to its high myco-nutrient content [13]. Chaga is reported to be a rich source of macrominerals such as potassium (K), nitrogen (N), calcium (C), and a reasonable source of magnesium (Mg), chlorine (Cl), phosphorus (P), sodium (Na), rubidium (Rb), sulfur (S), and manganese (Mn) [14], as well as trace or antioxidant minerals such as iron (Fe), copper (Cu), zinc (Zn), selenium (Se), and the micronutrient iodine (I). Regarding vitamins, Chaga contains B vitamins such as vitamin B1 (Thiamine), vitamin B2 (Riboflavin), vitamin B3 (Niacin), vitamin D2 (ergocalciferol), vitamins A, and vitamin K [15]. Besides the abundance of these vitamins, Chaga also contains significant levels of dietary phenolics which also act as antioxidants [16]. Consid- ering the rich myconutrient composition of Chaga, there is great interest in the food and nutraceutical industries to find suitable food-grade, green extraction techniques that will permit the recovery of high contents of these mycochemicals from Chaga for applications in functional foods, nutraceuticals, and pharmacological formulations or innovations. Many different methods have been used historically to extract myco-chemicals from a variety of natural sources [17,18]. To this end, organic solvents such as ethanol, methanol, acetone, ethyl acetate—or their mixture with water, dichloromethane, and hexane—either at ambient temperature or at the boiling point of the chosen solvent(s) are often used. Longer Antioxidants 2021, 10, 1322 3 of 18

treatment times, higher temperatures, large quantities of solvent required, decomposition of thermo-labile myco-chemicals, environmental health and safety risks are challenges Antioxidants 2021, 10, x FOR PEERassociated REVIEW with some of the organic solvents typically employed for extracting3 of bioactive 19

compounds from Chaga [19].

FigureFigure 1. Global 1. Global Geographical Geographical distribution distribution of InonotusInonotus obliquus obliquus in inthe the circumboreal circumboreal regions. regions.

Today,These innovative health benefits extraction of Chaga methods are also aredue exploredto its high continuously myco-nutrient to content overcome [13]. these challenges.Chaga is reported The Swiss to be water a rich extraction source of ma methodcrominerals is an such example as potassium of a green (K), systemnitrogen that is considered(N), calcium food-grade, (C), and a andreasonable represents source an of environmentally magnesium (Mg), friendlychlorine (Cl), technique phosphorus combining water,(P), sodium temperature, (Na), rubidium osmosis, (Rb), pressure, sulfur (S), and and extraction manganese time (Mn) to [14], remove as well primarily as trace or caffeine antioxidant minerals such as iron (Fe), copper (Cu), zinc (Zn), selenium (Se), and the mi- from coffee beans [20]. This method was designed to reduce energy utilization, facilitate cronutrient iodine (I). Regarding vitamins, Chaga contains B vitamins such as vitamin B1 the use of alternative water-based solvents geared at producing high-quality safe extracts (Thiamine), vitamin B2 (Riboflavin), vitamin B3 (Niacin), vitamin D2 (ergocalciferol), vit- andamins isolates A, and from vitamin a range K [15]. of Besides matrices the [ 19abunda]. Currently,nce of these there vitamins, is a paucity Chaga also of information contains in thesignificant scientific levels literature of dietary concerning phenolics how which pH al modulatesso act as antioxidants solvents’ extraction [16]. Considering efficiency the using therich Swiss myconutrient water extraction composition method of Chaga, under there pressurized is great interest conditions in the food and evenand nutraceu- less is known whentical applied industries to extractingto find suitable myco-chemicals food-grade, green from extraction Chaga matrices. techniques Recently, that will boiling permit water extractionthe recovery was of demonstrated high contents of to these be successful mycochemicals in the from extraction Chaga for of bioactiveapplications mycochemicals in func- fromtional Chaga foods, sourced nutraceuticals, from different and pharmacological geographical formulations areas for various or innovations. medicinal applications in oncologyMany [different21]. Boiling methods water have extraction been used method historically represents to extract the myco-chemicals most common from approach to obtaina variety Chaga of natural extracts, sources and [17,18]. tea in To many this end, cultures organic and solvents laboratory such as applications ethanol, metha- currently. Wenol, hypothesize acetone, ethyl that acetate—or pH will enhance their mixture theextraction with water, efficiency dichloromethane, of water-based and hexane— extraction either at ambient temperature or at the boiling point of the chosen solvent(s) are often solvents under pressurized conditions and could be useful in extracting high levels of used. Longer treatment times, higher temperatures, large quantities of solvent required, functional ingredients from Chaga mushrooms. decomposition of thermo-labile myco-chemicals, environmental health and safety risks areHerein, challenges we proposedassociated towith use some a modified of the organic version solvents of the Swisstypically water employed extraction for ex- method undertracting pressurized bioactive compounds conditions from at varying Chaga [19]. pH as a green solvent or potential food-grade approachToday, suitable innovative for recovering extraction highmethods levels are of explored myconutrients continuously from to Chaga. overcome these challenges. The Swiss water extraction method is an example of a green system that is 2. Materialsconsidered andfood-grade, Methods and represents an environmentally friendly technique combining 2.1.water, Chemicals temperature, osmosis, pressure, and extraction time to remove primarily caffeine fromStandards coffee beans of water-soluble [20]. This method vitamins was designe includingd to reduce thiamine energy hydrochloride utilization, facilitate (vitamin B1), riboflavinthe use of (vitamin alternative B2), water-based nicotinamide solvents (vitamin geared B3),at producing pantothenic high-quality acid (vitamin safe extracts B5), pyri- doxineand isolates (vitamin from B6), a range folic acidof ma (vitamintrices [19]. B9), Currently, and cobalamin there is a (vitamin paucity of B12) information were purchased in the scientific literature concerning how pH modulates solvents’ extraction efficiency using from Sigma Aldrich (St. Louis, USA). Likewise, polyphenol standards including quercetin, the Swiss water extraction method under pressurized conditions and even less is known ρ trans-ferulicwhen applied acid, to extracting ellagic acid, myco-chemicals gallic acid, from-hydroxybenzoic Chaga matrices. acid, Recently, salicylic boiling acid, water protocate- chuicextraction acid, caffeic was demonstrated acid, and vanillic to be successful acid were in acquiredthe extraction from of Sigma bioactive Aldrich mycochemicals (St. Louis, MO, USA).from ICP-MS Chaga sourced 43-element from different standard geographic mix (IV-ICPMS-71A)al areas for various was medicinal purchased applications from Inorganic

Antioxidants 2021, 10, 1322 4 of 18

Ventures (Christiansburg, Virginia, USA). Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), xylenol orange, ferrous ammonium sulfate hexahydrate, ortho-dianisidine dihy- drochloride, meta-phosphoric acid, glacial acetic acid (HPLC grade), methanol (HPLC grade), acetone (HPLC grade), acetonitrile (HPLC grade), 2,4,6-tripyridyl-s-triazine (TPTZ), ferric chloride hexahydrate, anhydrous sodium sulfate, ethanol (95% v/v), hydrogen per- oxide (30%, v/v), sulfuric acid (98% v/v), sodium phosphate, hydrochloric acid (36% v/v), ortho-phosphoric acid (85% v/v), methylchroman-2-carboxylic acid (Trolox), sodium hy- droxide, glycerol, sodium carbonate, and Folin–Ciocalteu reagent were purchased from Sigma Aldrich (Oakville, ON, Canada). Nitric Acid (TraceMetal™ Grade) and Dionex ASETM Prep DE diatomaceous earth were obtained from Thermo-Fisher Scientiﬁc (Ottawa, ON, Canada).

2.2. Preparation of Chaga Sporocarp Extracts The samples of Chaga mushroom were collected from the province of Newfound- land, Canada. After collection Chaga was powdered using a mechanical mill (Hamilton Beach, purchased from Amazon.ca). Then 5 g samples were filled into 10 mL stainless- steel accelerated solvent extractor (ASE) cell sample holders and extracted using a Dionex 350 ASE (Thermo Scientific, Waltham, MA, USA). Ionized water at different pH values (pH = 2.5, 7.0, 9.5, and 11.5) was used as extraction solvents (SD501, Enagic, Toronto, Canada). The ASE extractions were conducted based on the following program: (number of cycles = 2; pressure = 1500 psi; static time = 5 min; flush ratio = 50% rinse, and temperature = 60 ◦C and 100 ◦C). The extracts recovered were lyophilized using a freeze dryer (FreeZone freeze dry system, Labconco, Kansas City, MO, USA) and the dried extracts stored in amber-colored vials at −20 ◦C until further chemical analysis.

2.3. Total Antioxidant Activity (TAA) Analysis The TAA of the mushroom extracts was measured colorimetrically using the ferric reducing antioxidant power (FRAP) method [22], with minor modiﬁcations. Brieﬂy, 20 µL of each standard or sample was reacted with 180 µL of freshly made FRAP solution, and the resultant mixture incubated in the dark for 120 min. Absorbance measurements were recorded at 593 nm on a Cytation imaging microplate reader (BioTek, Winooski, VT, USA). The TAA extracts’ values were determined by summing the lipophilic (LAA) and hydrophilic (HAA) antioxidant activity values based on a Trolox standard curve using the following concentrations (0.5, 1, 1.5, 2, 2.5, 3, 3.5 mM). The results for the Chaga extracts were expressed in µmol Trolox equivalent (TE)/g extract (dry weight: DW).

2.4. Total Phenolic Content (TPC) Analysis The TPC of mushroom extracts was measured using the Folin-Ciocalteu reagent as described previously [23], with minor modiﬁcations. Thus, 25 µL of each standard or mushroom extract was mixed with 125 µL of a 10-fold diluted Folin–Ciocalteu reagent in microplate wells. Acidiﬁed ethanol (50 µL, 0.7% v/v) or sodium phosphate buffer (50 µL, 50 mM, pH 7.5) was added to analyze the lipophilic or hydrophilic phenolic content in the extracts, respectively. The resultant mixtures were incubated in darkness for 120 min, and the absorbances measured at 755 nm on a Cytation imaging microplate reader (BioTek, Winooski, VT, USA). The TPC of the mushroom extracts were calculated by summation of the hydrophilic phenolic content (HPC) and lipophilic phenolic content (LPC) and the concentration measured based on quercetin standard curve. The results were expressed as µmol quercetin equivalent/g extract (DW).

2.5. B-Vitamin (B1, B2, B3, B5, B6, B9, and B12) Analysis by Ultra-High-Pressure Liquid Chromatography-Mass Spectrometry (UHPLC-MS) The determination of B vitamins (water-soluble) in freeze-dried mushroom extracts was based on the method of Akhavan et al. [24]. Brieﬂy, a stock solution of vitamin B standard mix (B2, B3, B5, B6, and B9) at a concentration of (1 mg/mL) was diluted serially with distilled water to obtain B vitamin calibration standards in the range of 5–100 µg/mL Antioxidants 2021, 10, 1322 5 of 18

Aliquots of the extracts (1 mg) were diluted with distilled water (10 mL) and spiked with hippuric acid as an internal standard. The resultant mixture was homogenized, then incubated in darkness for 45 min at 4 ◦C, followed by centrifugation at 25,200× g for 15 min at 0 ◦C. The supernatant was collected, pooled, and filtered into amber-colored vials for analysis by UHPLC-HRAMS-MS/MS. For the analysis of Vitamin B, a Luna C18 column (100 × 2.0 mm I.D., particle size: 3 µm, pore diameter: 10 nm) purchased from Phenomenex (Torrance, CA) was employed. The solvent system used was as follows: solvent A consisted of 10 mM of ammonium formate and 0.1% Formic Acid, while solvent B consisted of 100% acetonitrile. Chromatographic separation was performed at 35 ◦C (column oven temperature) with a flow rate of 0.3 mL/min, and 10 µL of the extract was injected into the system. The following solvent gradient was used for separating the vitamins: ramp at 20 min with 55% solvent B; next transition to 22 min. 0% Solvent B and 100% of Solvent A. The Orbitrap ESI source was run in positive ion mode for data acquisition. The following optimized parameters were used for the Orbitrap mass spectrometer: sheath gas flow rate of 51; an auxiliary gas flow rate: 0; ion spray voltage: 3.20 kV; capillary temperature: 300 ◦C; S-lens RF: 100 V; capillary voltage: 35 V; mass range: 50–500 m/z; full scan mode at a resolution of 60,000 m/z; top-3 data-dependent MS/MS at a resolution of 30,000 m/z; and collision energy of 35 (arbitrary unit); injection time 22 min; isolation window: 2.0 m/z; automatic gain control target: 0.250; with dynamic exclusion setting of 30.0 s. The mass spectrometer was externally calibrated to 1 µg/mL using ESI negative and positive calibration solutions (Thermo Scientific, Berkeley, MO, USA) before usage.

2.6. Fat-Soluble Vitamins (A, E, D3, K1, K2, E) Analysis by UHPLC-HRAM-MS/MS The determination of fat-soluble vitamins present in the freeze-dried mushroom extracts was based on the method of Ramadan and coworkers with minor modifications [25]. Briefly, mushroom extracts were homogenized in 1 mL of methanol: chloroform (1:1) containing hippuric acid as an internal standard. The resultant mixture was incubated for 30 min in darkness at 4 ◦C followed by sonication for an additional 30 min at 4 ◦C in a sonication water bath. Thereafter, the samples were homogenized, centrifuged (25,200× g for 15 min at 0 ◦C), then the supernatant was collected into amber-colored vials for LC-MS analysis. A polar acclaim C18 column (150 × 4.6 mm I.D., particle size: 5 µm, pore diameter: 12 nm; Thermo Fisher Scientific, Mississauga, ON, Canada) was used for the resolution of the fat-soluble vitamins. The solvent system used consisted of 5 mM ammonium acetate. The separation was carried out at 40 ◦C (column compartment temperature) with a flow rate of 0.3 mL/min, and 10 µL of the extract was injected into the machine and elution done isocratically for 32 min. The Orbitrap MS was operated in the positive ESI ion mode during data acquisition. The following optimized parameters were used for the Orbitrap MS: sheath gas flow rate: 25; ion spray voltage: 500 kV; an auxiliary gas flow rate: 2; S-lens RF: 80.00 V; mass range: 60–900 m/z; capillary temperature: 320 ◦C; capillary voltage: 10.0 V; full scan mode at a resolution of 60,000 m/z; top-three data-dependent MS/MS at a resolution of 30,000 m/z; and collision energy of 35 (arbitrary unit); injection time of 15 min; isolation window: 2.0 m/z; automatic gain control target: 0.250; with dynamic exclusion setting of 30.0 s. The mass spectrometer system was externally calibrated to 1 µg/mL using ESI positive and negative calibration solutions (Thermo Scientific, Berkeley, MO, USA) before usage.

2.7. Phenolic Compounds Analysis by UHPLC-HRAM-MS/MS Aliquots (1 mL) of 10% Formic acid: water (v/v) was added to 1 mg freeze-dried extracts in centrifuge tubes. The resulting mixture was homogenized, incubated in darkness for 15 min and then centrifuged at 25,200× g for 15 min at 0 ◦C. The supernatant was pooled into amber-colored vials for UHPLC-HRAM-MS/MS analysis. A standard mix of select phenolics were used to create a standard curve covering the following concentration range: 5–100 µg/mL. The UHPLC-HRAMS/MS analysis was conducted on an LTQ Orbitrap XL Antioxidants 2021, 10, 1322 6 of 18

mass spectrometer (Thermo Scientific, Berkeley, MO, USA) with an automated Dionex UltiMate 3000 UHPLC system controlled by the Chromeleon software v. 7.3. Resolution of the phenolics present in the sample was performed on a Luna C18 column (100 × 2.0 mm I.D., particle size: 3 µm, pore diameter: 10 nm) purchased from Phenomenex (Torrance, CA, USA). The solvent system used was as follows: Solvent A consisted of H2O (containing 0.1%, v/v Formic acid), and Solvent B consisted of acetonitrile (consisting of 0.1%, Formic acid). Chromatographic separation was done at 25 ◦C (column temperature) with a flow rate of 0.3 mL/min, and 5 µL of the extract was injected into the machine. The following solvent gradient was used for separating the polyphenols: 0–2 min with 0–10% solvent B; 2–15 min applying 10–45% solvent B; 15–30 min using 45–90% solvent B; 30–35 min using 90–10% solvent B. The Orbitrap MS was operated in the negative ESI ion mode. The optimized parameters used for the Orbitrap mass spectrometer were: sheath gas flow rate: 25; ion spray voltage: 5.0 kV; an auxiliary gas flow rate: 2; capillary temperature: 320 ◦C; capillary voltage: 10 V; S-lens RF:80 V; mass range:50–1500 m/z; capillary voltage: 10 V; full scan mode of 60,000 m/z resolution; top-6 data-dependent MS/MS acquisition at a resolution of 30,000 m/z and collision energy of 35 (arbitrary unit); isolation window: 2.0 m/z, automatic gain control target: 2 e5 with dynamic exclusion setting of 30.0 s and injection time of 15 min. The MS was externally calibrated to 1 µg/mL using ESI positive and negative calibration solutions (Thermo Scientific, Berkeley, MO, USA) before usage.

2.8. Extraction and Analysis of Minerals by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) Aliquots (1 mg) of freeze-dried Chaga mushroom extracts were digested in 10 mL of trace nitric acid (70% v/v) using a Multiwave Go-microwave digestion system (Anton Paar GmbH, Graz, Austria). The microwave digester was operated under the following conditions: temperature ramped to 180 ◦C for 10 min, then held at this temperature (180 ◦C) constantly for an additional 20 min. A calibration curve (ranging from 1 to 500 µL−1) was prepared using IV-ICPMS-71A standard mixture (InorganicTM Ventures, Inc.; Christiansburg, VA, USA). Aliquots of the digestates were diluted with 2% nitric acid and spiked with rhodium-103 (final concentration = 10 µL−1) as the internal standard. The ICP-MS analysis was conducted using the following parameters: auxiliary gas flow of 0.79 L/min, nebulizer gas flow of 1.01 L/min, plasma gas flow of 14 L/min, RF power of 1548 W, detector mode KED, dwell time of 0.01 s using argon gas at 99.99% purity.

2.9. Statistical Analysis ANOVA was used to assess the effects of extraction pH and temperature on the antioxidant activity, phenolic compounds, vitamins, and mineral content in the mushroom extracts. When the means were significantly different between pH and temperature combinations, the means were compared with Fisher’s least significant difference (LSD), α = 0.05. Pearson’s correlation coefficients were used to determine the linear association between subclasses of myco-chemicals (phenolics, vitamins, and minerals) and their contributions to the antioxidant activity of the extracts. Outliers were detected using the Grubbs test. Principal component analysis was used to discern similarities between the extraction conditions (pH and temperatures) and the myco-nutrients recovered. Statistical analyses were performed with XLSTAT (Addinsoft, Paris, France). Figures were prepared using XLSTAT and SigmaPlot 13.0 software programs (Systat Software Inc., San Jose, CA, USA).

3. Results and Discussion The current interest in medicinal mushrooms for their unique and promising health promotive benefits such as anti-inflammatory, anticancer, anti-diabetic, and antioxidant properties is remarkable [26]. Furthermore, consumers have high interests in natural sources of dietary antioxidants for use in their daily food intake. Herein, we demonstrated a modified Swiss water method consisting of elevated temperature and pH under pressurized condition using accelerated solvent extraction as a suitable food grade solvent system Antioxidants 2021, 10, 1322 7 of 18

to extract myco-chemicals from Chaga mushroom. Chaga mushroom is considered an enriched source of natural antioxidants.

3.1. Total Polyphenols Content (TPC) and Total Antioxidant Activity (TAA) Principal component analysis demonstrated the TPC and TAA clustered together with the extracts obtained at pH 11.5 at 100 ◦C. The TPC and TAA were also grouped with the HPC, and HAA under these extraction conditions, while the LPC clustered with extracts obtained at pH 2.5/60 ◦C, pH 2.5/100 ◦C, and pH 7/100 ◦C. This grouping captured 96.22% of the total variability in the data set, with PC1 explaining 81.45%, and PC2 accounting for 14.77% of variability in data. TPC and HPC were grouped with Ph 11.5/100 ◦C treatment, implying that they are best extracted using high pH and temperature from Chaga matrices under pressurized conditions. Furthermore, this pH and temperature combination appears to extract the hydrophilic portion comprising the highly polar phenolic compounds, and this may be noted from the ANOVA analysis, which demonstrates HPC as the major contributor to the TPC. Interestingly, the composition of the solvent system is water based and was not anticipated to extract any significant component of lipophilic polyphenols or antioxidants. However, we observed that although most of the polyphenols and antioxidants extracted were hydrophilic in nature, the lower solvent pH (2.5 and 7) was effective in extracting the lipophilic phenolics from the Chaga matrix, while higher pH (11.5) at 100 ◦C was effective in extracting LAA from Chaga under pressurized conditions using water as an accelerated solvent (Figure2). We observed the TAA varied significantly with treatment and ranged from 98.40 to 381.30 µmol TE/g DW, with the highest TAA present in the extract was acquired at pH 11.5 and a temperature of 100 ◦C. Similarly, the TPC ranged between 102.27 ± 1.39 to 273.06 ± 2.19 µM QE/g Chaga DW, with the highest TPC also observed in the extracts acquired at pH 11.5 and a temperature of 100 ◦C (Figure2; Tables S1 and S2). These results demonstrate extraction with pH 11.5 at 100 ◦C has unique conditions and extraction potential towards the acquisition of hydrophilic and lipophilic antioxidants in Chaga mushroom extract using water as an accelerated solvent under pressurized conditions. Hwang and coworkers (2019) [27] reported that phenolic compounds extracted increased in mushrooms treated at 100 ◦C. Seo and Lee (2010) [28] also noted that phenolic components in Chaga extracts produced by supercritical water increased with temperature, implying that temperature appears to be useful in phenolics extraction from Chaga mushroom. It is worth mentioning that the use of pH, temperature, and pressure can change the physiochemical nature of water to extract organic components that are generally not soluble in aqueous solvents, which is clearly shown from the extraction of the lipophilic phenolic components in our study. The values we observed for TPC and TAA in Chaga extracts are consistent with values observed in the literature [27]. However, huge variation is noted in the literature for the phenolics content, and antioxidant activities measured in Chaga. The variation in antioxidant activities and phenolics content reported in the literature are reported to be influenced by source geography, temperature, carrier birch type, time of harvest, and part used for extraction [29]. The correlational analysis between TAA and TPC showed that a strong antioxidant activity came from the HAA (r = 0.76, p < 0.0001) (Figure3). Interestingly, relatively considerable levels of both LAA and LPC were observed in the extract, considering that an aqueous-based solvent system was used (Figure2). The elevated temperature and pH used for the extraction under pressurized conditions appear to contribute to the extraction of lipophilic antioxidants and phenolics in this extraction system. Finally, the correlation coefficient (r = 0.78) between TAA and TPC, suggested that approximately 78% of the extracts’ total antioxidants may be attributed to the TPC present in the extract, which was driven by the high association of the HPC with the TAA (r = 0.81). These findings suggest that the hydrophilic phenolics are a major contributor to the TAA of the extracts obtained in this extraction system. Antioxidants 2021, 10, x FOR PEER REVIEW 8 of 19

of pH, temperature, and pressure can change the physiochemical nature of water to extract organic components that are generally not soluble in aqueous solvents, which is clearly shown from the extraction of the lipophilic phenolic components in our study. The values we observed for TPC and TAA in Chaga extracts are consistent with values observed in the literature [27]. However, huge variation is noted in the literature for the phenolics content, and antioxidant activities measured in Chaga. The variation in antiox- Antioxidants 2021, 10, 1322 idant activities and phenolics content reported in the literature are reported to be influ- 8 of 18 enced by source geography, temperature, carrier birch type, time of harvest, and part used for extraction [29].

FigureFigure 2. 2.Bar Bar charts charts showing showing the the difference difference in in phenolics phenolics and and antioxidants antioxidants following following ANOVA ANOVA for thefor componentsthe components segregated seg- perregated quadrant per quadrant in the PCA in the biplot. PCA Hydrophilicbiplot. Hydrophilic antioxidant antioxidant activity activity (HAA), (HAA lipophilic), lipophilic antioxidant antioxidant activity activity (LAA), total antioxidant(LAA), total (TAA), antioxidant hydrophilic (TAA), phenolic hydrophilic content phenolic (HPC), conten lipophilict (HPC), phenolic lipophilic content phenolic (LPC), and content total (LPC), phenolic and content total phe- (TPC). Antioxidants 2021, 10, x FOR PEER REVIEW 9 of 19 Thenolic antioxidant content (TPC). activities The areantioxidant reported asactivitiesµmol/g are of reported Trolox equivalents. as μmol/g of The Trolox phenolics equivalents. content The is represented phenolics ascontentµmol/g is of quercetinrepresented equivalents. as μmol/g Values of quercetin accompanied equivalents. by the Values same letters accompanied are not signiﬁcantlyby the same differentletters are (p not< 0.05), significantly Fisher’s different LSD test. (p < 0.05), Fisher’s LSD test. The correlational analysis between TAA and TPC showed that a strong antioxidant activity came from the HAA (r = 0.76, p < 0.0001) (Figure 3). Interestingly, relatively considerable levels of both LAA and LPC were observed in the extract, considering that an aqueous-based solvent system was used (Figure 2). The elevated temperature and pH used for the extraction under pressurized conditions appear to contribute to the extraction of lipophilic antioxidants and phenolics in this extraction system. Finally, the correlation coefficient (r = 0.78) between TAA and TPC, suggested that approximately 78% of the extracts’ total antioxidants may be attributed to the TPC present in the extract, which was driven by the high association of the HPC with the TAA (r = 0.81). These findings suggest that the hydrophilic phenolics are a major contributor to the TAA of the extracts obtained in this extraction system.

FigureFigure 3. Correlation 3. Correlation between between TAA TAA and and CA,CA, TAA and and MEA, MEA, TAA TAA and and TPC, TPC, TAA TAAand TAM, and TAM,DHTA DHTAand TAA, and MCA TAA, and MCA and TAA,TAA, ToPA’s ToPA’s andand TAA, TAA, DHPA DHPA22 and and TAA, TAA, SalA SalA and TA, and ToHBen TA, ToHBen and TPC, and ToCA TPC, and ToCA TAA, andDHPA2 TAA, and DHPA2TAA, DHPA1 and TAA, DHPA1and and TPC, TPC, SalA SalA and andTPC, TPC,ToHBen ToHBen and TAA and content. TAA content.HAA = hy HAAdrophilic = hydrophilic antioxidant activity, antioxidant LAA = activity, lipophilic LAA antioxidant = lipophilic activity, TAA = total antioxidant activity, HPC = hydrophilic phenolic content, LPC = lipophilic phenolic content, TPC = antioxidant activity, TAA = total antioxidant activity, HPC = hydrophilic phenolic content, LPC = lipophilic phenolic content, total phenolic content. TAM = total antioxidant minerals, ToHBen = total hydroxybenzoic acids, ToPA’s = total other phe- TPC =nolic total acids. phenolic ToCA content = total. TAMcinnamic = total acids. antioxidant DHPA1 = 3,4- minerals,dihydroxybenzaldehyde, ToHBen = total hydroxybenzoic DHPA2 = 3,4-dihydroxybenzalacetone, acids, ToPA’s = total other phenolicSalA acids. = Salicylic ToCA acid, = total DHTA cinnamic = dihydroxyterephtalic acids. DHPA1 = acid, 3,4-dihydroxybenzaldehyde, MEA = methyl-gallic acid, CA DHPA2 = caffeic = acid. 3,4-dihydroxybenzalacetone, Antioxidant activity SalA =was Salicylic expressed acid as, DHTA µmol Trolox = dihydroxyterephtalic equivalent/g extract acid, of Chaga MEA and = methyl-gallicphenolic conten acid,t was CA expressed = caffeic as µmol acid. quercetin Antioxidant equiv- activity was expressedalent/g extract as µ molof Chaga. Trolox R-values equivalent/g represent extractPearson ofcorrelation Chaga andcoefficients, phenolic p = content0.05, n = 4 was per expressedreplicate of each as µ molsample quercetin at various pH values and temperatures. equivalent/g extract of Chaga. R-values represent Pearson correlation coefﬁcients, p = 0.05, n = 4 per replicate of each sample at various pH values and3.2. temperatures. Chaga Extracts Composition Mushrooms have a long history in folk medicine and ethnopharmacological uses for their health promotive benefits. The rich myco-nutritional composition is attributed to these health promotive benefits [30]. Evaluation of the extracts obtained in this study demonstrated Chaga is a good source of vitamins, dietary polyphenols, antioxidant, trace, and macro minerals.

Antioxidants 2021, 10, 1322 9 of 18

3.2. Chaga Extracts Composition Mushrooms have a long history in folk medicine and ethnopharmacological uses for their health promotive beneﬁts. The rich myco-nutritional composition is attributed to these health promotive beneﬁts [30]. Evaluation of the extracts obtained in this study demonstrated Chaga is a good source of vitamins, dietary polyphenols, antioxidant, trace, and macro minerals.

3.2.1. Minerals The minerals recovered in the Chaga extracts were grouped as antioxidant minerals, trace, macro, and heavy metals (Table1) according to their dietary or nutritional relevance. The results suggest significant quantities of antioxidant minerals (Mn, Cu, Zn, and Se), macronutrients (Na, Mg, P, S, K, and Ca), trace minerals (B, Cr, and Co) and to a lesser extent toxic heavy metal (As) were present in the extracts (Table1). However, the heavy metals extracted are significantly below the tolerable limits recommended for safe human consumption [31] (Institute of Medicine (US) Panel on Micronutrients, 2001). Principal component analysis was used to discern the extraction conditions’ effectiveness in recovering the different classes of minerals from Chaga matrices using the modified Swiss method (SWP). In the PCA biplot, the total microminerals and trace minerals were grouped with pH 2.5 and pH 9.5 at the higher temperature treatments (100 ◦C). These treatments seem to promote the extraction of macronutrients and trace minerals. The total antioxidant minerals were grouped with a pH of 11.5 and high temperature (100 ◦C). This may indicate that antioxidant minerals can be better extracted by high temperature and pH from Chaga using the SWP. Furthermore, pH 2.5 at 60 ◦C was better for extracting heavy metals like Arsenic. Zn was the predominant antioxidant mineral present in the extract with values ranging from 67.37 ± 1.29 to 105.87 ± 3.05 µg/g DW of Chaga, with the highest concentration observed in the extracts collected at pH 2.5 and 9.5 at a temperature of 100 ◦C (Table1). Mn, Fe, Cu, and Se were the other antioxidant minerals present in the Chaga extracts. A pH of 2.5 at low temperature (60 ◦C) was effective for the extraction of Mn from Chaga (9.85 ± 3.05 µg/g DW). This was several folds higher than the values observed at the other pH and temperature combinations (0.0 to 0.539.85 ± 3.05 µg/g DW). Conversely, a pH of 9.5 at temperatures of 60 ◦C and 100 ◦C, as well as a pH of 2.5 at a temperature of 60 ◦C was the most effective in the recovery of Se from Chaga matrices. This indicate pH is a very important determinant in modulating the level of selenium extracted from Chaga using the SWP. We next assessed the contributions of the antioxidant minerals to the total antioxidant activity measured in the extract using Pearson’s correlation. The total antioxidant minerals were moderately correlated (r = 0.52) with the TAA in the extracts recovered (Figure3 and Table S3A,B). The results of the mineral contents expressed in µg/g DW of Chaga mushroom is presented in Table1 along with the recommended daily allowances (RDA’s) [31]. The antioxidant minerals form a very important part of the total minerals found in Chaga. These minerals are often required in low quantities in human nutrition to meet the RDAs [32] and typically supplemented in many food formulations. The recommended dietary allowance (RDA) for Cu, Mn, and Zn has been established. An infusion of Chaga mushroom made from 10 g dry weight (DW) (teaspoon full) could meet the demand for Cu, Mn, Zn and Se which is set at 5%, 10%, 10%, and 4%, respectively [31]. Arsenic (As) was the only heavy metal observed in the extract and the values ranged between 0.81 ± 0.04 to 1.63 ± 0.05 with the highest-level present at pH 2.5 and a temperature of 60 ◦C. It is important to note the level of As detected in the extract is low. As is lethal in humans in the range of 2–10 mg/kg/day for an average size human [33]. The values reported in these extracts are below the safe levels reported for human consumption. The U.S. Food and Drug Administration (FDA) has established tolerance levels for As to be permissible at levels ranging from 0.5 to 2 µg/mL in different foods [33]. Antioxidants 2021, 10, 1322 10 of 18

Table 1. Mineral content in Chaga (Inonotus Obliquus) extracts as a function of solvent pH and temperature under pressurized conditions.

pH Treatment and Temperature **

Classiﬁcation of pH 2.5 at 60 ◦C pH 7 at 60 ◦C pH 9.5 at 60 ◦C pH 2.5 at 100 ◦C pH 7 at 100 ◦C pH 9.5 at 100 ◦C pH 11.5 at 100 ◦C RDAs * Minerals

Antioxidant minerals

55Mn 9.85 ± 0.04 a 0.53 ± ND b 0.38 ± 0.01 c 0.34 ± 0.01 d 0.28 ± 0.14 e 0.11 ± 0.08 f ND *** 2 (mg) 63Cu 2.10 ± 0.15 f 3.84 ± 0.25 d 4.24 ± 0.08 c 4.27 ± 0.19 c 3.45 ± 0.31 e 4.38 ± 0.78 b 4.82 ± 0.04 a 1 (mg) 64Zn 67.37 ± 1.29 g 91.86 ± 4.90 e 93.20 ± 2.53 d 105.87 ± 3.05 a 79.75 ± 0.42 f 104.75 ± 0.37 b 103.05 ± 2.84 c 10 (mg) 80Se 3.46 ± 0.07 ab 3.05 ± 1.76 c 3.63 ± 0.42 a 3.37 ± 0.93 b 2.42 ± 0.18 d 3.51 ± 0.46 ab 3.04 ± 0.11 c 55 (µg)

Macrominerals

23Na 1439.35 ± 5.91 c 1406.28 ± 5.16 d 1414.55 ± 11.03 cd 1612.63 ± 1.21 a 1228.21 ± 0.68 e 1583.92 ± 2.11 ab 1580.22 ± 23.89 b 800 (mg) 24Mg 787.45 ± 2.15 d 1028.89 ± 1.58 b 1036.58 ± 1.35b 1155.43 ± 10.34 a 891.44 ± 1.24 c 1159.06 ± 7.20 a 1161.53 ± 6.87 a 375 (mg) 31P 896.61± 0.60 a 339.63 ± ND*** c 335.85 ± 15.82 c 370.48 ± 2.40 b 290.58 ± 0.46 d 378.80 ± 9.31 b 335.65 ± 3.00 c 700 (mg) 39K 2004.07 ± 19.96 a 940.47 ± 4.41 e 1026.71 ± 9.75 d 1853.77 ± 27.08 b 902.02 ± 7.62 e 1211.62 ± 2.28 c 899.74 ± 4.81 e 2000 (mg) 44Ca 480.10 ± 7.83 d 660.32 ± 3.67 b 671.57 ± 3.90 b 745.19 ± 6.58 a 577.58 ± 5.58 c 756.34 ± 11.04 a 750.47 ± 3.73 a 800 (mg)

Trace Minerals

11B 64.25 ± 17.44 a 11.25 ± 2.57 c 8.97 ± 3.86 d 12.81 ± 5.68 bc 10.21 ± 0.36 d 15.26 ± 11.78 b 13.84 ± ND b 52Cr 3.94 ± 0.49 ND ND ND ND ND ND 40 (µg)

Toxic Minerals

75As 1.63 ± 0.04 a 0.94 ± 0.01 d 0.97 ± 0.02 c 1.03 ± 0.01 b 0.81 ± 0.03 e 1.04 ± 0.09 b 1.04 ± 0.01 b * RDA’s recommended daily allowances. Values were obtained from “Commission Regulation (EC) No 629/2008”. ** Values represent means of three replicates ± standard errors. For each column, values accompanied by the same letters are not signiﬁcantly different (p < 0.05), Fisher’s LSD test. *** ND = not detected. Macro-mineral chlorine; trace minerals ﬂuoride, uodine, and molybdenum; and toxic mineral. The Chaga extracts are at various pH values (2.5, 7, 9.5, and 11.5) and at temperatures 60 ◦C and 100 ◦C. Fe, Co, Cd, and Pb were not detected at any treatment. Highest values of Mn, P, K, B, Cr, and as were detected in pH 2.5 at 60 ◦C.

Both the macro and trace minerals clustered with pH 2.5 and a temperature of 100 ◦C in the PCA biplot suggesting this condition was optimal for the extraction of these minerals from Chaga using the SWP. Ca, Na, and Mg were the predominant macro minerals present in the extracts and their levels were signiﬁcantly impacted by an extraction pH of 2.5 and 9.5 at 100 ◦C, where the highest levels for these minerals were observed. The other macrominerals Ca and P recovery were highest at a pH of 2.5 and temperature 60 ◦C. We observed Ca, Na, and Mg were the predominant macrominerals present in the extracts with signiﬁcantly higher levels recovered (Table1). Similarly, boron the predominant trace minerals, was more successfully recovered when extracted at pH 2.5 and a temperature of 60 ◦C. These results clearly demonstrate that a pH of 2.5 or 9.5 at 100 ◦C are useful for extracting macro and trace minerals from Chaga using the SWP.

3.2.2. Vitamins (Water- and Fat-Soluble) Water is an interesting and practical solvent in extraction, and its physiochemical properties can change with varying temperatures. These conditions allow water at near-critical conditions to dissolve hydrophobic compounds [34]. Furthermore, an increased move- ment/rotation of water molecules can also be observed at higher temperature which allows for dissolving less polar compounds, analogous to that of methanol and ethanol (slightly polar solvents) which in turn may enable the extraction of nonpolar components [34,35]. In this context, we observed that when water was used as a solvent (pressurized ﬂuid) in the SWP evaluated in this study both fat- and water-soluble vitamins were successfully extracted from Chaga matrices. Vitamin E and K were the predominant vitamins observed in the extracts with values with the average of 112, 460 and 2423 ng/g DW Chaga, respectively. The high alkaline and high-temperature treatment (pH 11.5/100 ◦C) was successful in extracting both classes of vitamins including vitamins D, and K (fat-soluble vitamins), as well as B-vitamins (water-soluble vitamins). On the other hand, total vitamin E (fat- soluble) including Beta-tocopherol analogue was found to be best extracted at the treatment with lower pH (2.5) and higher temperature (100 ◦C). These ﬁndings suggest that high temperature strongly impacts the extraction of the fat and water-soluble vitamins. From the PCA of fat-soluble vitamins (FSV) and water-soluble vitamins (B-vitamins), it was observed that vitamin K2, total vitamin D, total B-vitamins, and total vit. K are grouped with higher temperature and alkaline pH (9.5 and 11.5) at 100 ◦C. Alpha-Tocopherol and Vitamin K1 are grouped with pH7 and temperature of 100 ◦C. On the other hand, vitamin Antioxidants 2021, 10, 1322 11 of 18

A and beta-tocopherol grouped with pH 2.5/60 ◦C, pH 2.5/100 ◦C, and pH 9.5/60 ◦C extraction conditions. Despite the large volume of research on Chaga in the scientific literature, to the best of our knowledge there is no work describing the vitamin content in the literature. Vitamin D2 was mentioned and evaluated in different mushroom genotypes and its quantity ranged from 0.3 to 5.3 µg/100 g. However, our results are in the range of 0.02 to 0.04 µg/100 g DW Chaga. Despite the low content of vitamin D in mushrooms, high levels of sterols could be found in mushrooms like ergosterol, as a potential source and precursor of vitamin D2 [36]. The transformation of ergosterol to vitamin D2 can be achieved by exposure to ultra- violet (UV) irradiation [37]. Sunlight exposure is low in the boreal ecosystem compared to Antioxidants 2021, 10, x FOR PEER REVIEW 12 of 19 other regions, and this, as well as the extraction conditions employed in this study, may account for the low vitamin D levels observed in Chaga extracts. The effect of solvent pH and temperatures on vitamin A, vitamin D, vitamin K1, vitaminThe K2, effect and of solvent vitamin pH E and which temperatures includes on Alpha, vitamin tocopherol A, vitamin is D, shown vitamin in K1, Figure vit- 4. Vitaminamin K2, A and content vitamin in E samples which includes extracted Alpha, with tocopherol pH 2.5 and is shown 9.5 at in 60 Figure◦C are 4. notVitamin signifi- cantlyA content different in samples from eachextracted other. with No pH significant 2.5 and 9.5 difference at 60 °C wasare not found significantly among the different different from each other. No significant difference was found among the different extraction sys- extraction systems for Vitamin D2 B5. tems for Vitamin D2 B5.

FigureFigure 4. Vitamin4. Vitamin contents contents in inInonotus Inonotus obliquus obliquus samplessamples extracted under under different different combin combinationsations of ofpH pH (2.5, (2.5, 7, 9.5, 7, 9.5, 11.5) 11.5) and and temperaturestemperatures (60 (60◦C °C and and 100 100◦ C).°C). Bars Bars represent represent meansmeans ofof fourfour replicates ±± standardstandard errors. errors. Values Values accompanied accompanied by bythe the samesame letters letters are are not not signiﬁcantly significantly different different ( p(p< < 0.05),0.05), Fisher’sFisher’s LSD test. test.

VitaminVitamin B1B1 (Thiamin)(Thiamin) was reported reported to to be be in in the the range range of of 0.02–1.6 0.02–1.6 µg/gµg/g DW, DW, while while vitaminvitamin B3B3 (Niacin)(Niacin) was found in in the the range range of of 1.2–6 1.2–6 µg/gµg/g DW DW in indifferent different kinds kinds of mush- of mush- roomroom [[38].38]. In In Chaga, Chaga, the the best best extract extract contained contained 0.17 ng/100g 0.17 ng/100g of B3 at pH of B39.5 atand pH a temper- 9.5 and a temperatureature of 100 °C. of 100 ◦C. VitaminVitamin B5B5 content of of Chaga Chaga samples samples extracted extracted at pH at pH2.5, 7, 2.5, 9.5 7, at 9.5 both at 60 both °C and 60 ◦ 100C and 100°C ◦areC arenot notsignificantly significantly different different from fromeach eachother, other, whereas whereas the samples the samples extracted extracted at pH at pH11.5, 11.5, 100 100 °C is◦C significantly is significantly different different from fromsamp samplesles at pH at2.5 pH and 2.5 pH and 7 and pH results 7 and in results a high in a highcontent content of vitamin of vitamin B5. Extraction B5. Extraction temperature temperature had no had significant no significant impact impact on vitamin on vitamin B5 B5content. content. The The lowest lowest vitamin vitamin B6 content B6 content was wasfound found in the in mushroom the mushroom sample sample extracted extracted at atpH pH 7, 7,100 100 °C.◦C. The The highest highest and and the thelowest lowest vitamin vitamin B3 content B3 content were wereextracted extracted at pH at9.5, pH 60 9.5, 60°C,◦C, and and pH pH 7, 100 7, 100°C, respectively,◦C, respectively, and these and thesevalues values were significantly were significantly different different from each from eachother. other. Vitamin Vitamin B1 content B1 content is highly is highly elevated elevated in samples in samples extracted extracted at pH 9.5, at pH60 °C 9.5, and 60 ◦C shows the least value at pH 7, 100 °C. Vitamin B2 and Vitamin B12 were undetectable. and shows the least value at pH 7, 100 ◦C. Vitamin B2 and Vitamin B12 were undetectable. Vitamin B3 was found in the range from 3.25 to 11.62 ng/100 g in Chaga extracts, with the Vitamin B3 was found in the range from 3.25 to 11.62 ng/100 g in Chaga extracts, with highest level observed at pH 9.5 and temperature of 100 °C. Although the levels for some of the vitamins recovered using this method are lower than that for vitamins in other mushroom varieties. This work demonstrates for the first time the acquisition of both fat- and water-soluble vitamins simultaneously in Chaga extracts obtained using water as pressurized extraction fluids at varying pH and temperature in the SWP proposed.

3.2.3. Phenolics The phenolic compounds identified and quantified in the extracts obtained using the SWP is illustrated in Table 2. Analysis of the phenolic composition of the different extractions/treatments resulted in the identification and quantification of twenty-four phenolic compounds in Chaga mushroom extracts following LC-HESI-HRAM-MS/MS analysis.

Antioxidants 2021, 10, 1322 12 of 18

the highest level observed at pH 9.5 and temperature of 100 ◦C. Although the levels for some of the vitamins recovered using this method are lower than that for vitamins in other mushroom varieties. This work demonstrates for the ﬁrst time the acquisition of both fat- and water-soluble vitamins simultaneously in Chaga extracts obtained using water as pressurized extraction ﬂuids at varying pH and temperature in the SWP proposed.

3.2.3. Phenolics The phenolic compounds identified and quantified in the extracts obtained using the SWP is illustrated in Table2. Analysis of the phenolic composition of the different extractions/treatments resulted in the identification and quantification of twenty-four phenolic compounds in Chaga mushroom extracts following LC-HESI-HRAM-MS/MS analysis. The results for the composition at the best extraction conditions at pH 11.5 and a temperature of 100 ◦C is presented in Table2. The identification was based on the molecular formula, retention time, and exact molecular mass (Table2). The compounds: 3,4-dihydroxybenzaldehyde, 4-methoxycinnamic acid, dihydroxyterephtalic acid, protocatechuic acid, and caffeic acid were found as the highest phenolics in the Chaga extracts produced using the modified Swiss water method (Table2). It was suggested that phenolics in Chaga are the main components responsible for the antioxidant activity [18], and this is consistent with the high correlations observed between these components in our study. According to the results obtained, 3,4-dihydroxybenzaldehyde was significantly higher (104.851 µ/mL) when extracted at pH 11.5 and a temperature of 100 ◦C compared to other treatments. The content found in our research are consistent with previous reports in the literature as this compound was a major phenolic compound observed, but the levels obtained in our work are much higher. Interestingly, 3,4-dihydroxybenzaldehyde has been reported to have significant biological activities such as antioxidative, antibacterial, anti-aging, and anti-inflammatory [39]. The present study also revealed other phenolics like flavonoid aglycones such as myricetin, quercetin, isorhamnetin, and phenolic acids—such as gallic, vanillic, protocatechuic, ferulic, ellagic acid, methyl ellagic, syringic, and salicylic acids—to be major compounds in the Chaga extracts obtained. Total hydroxycinnamic acids (69.6 µg/mL), total hydroxybenzoic acids (129.20 µg/mL), and total phenolics (58.22 µg/mL) were found at higher levels in the extracts obtained using pH 11.5, 100 ◦C. Phenolic acids like 4-methoxycinnamic_acid (57 µg/mL), 3,4- dihydroxybenzaldehyde (104.85 µg/mL), 3,4-dihydroxybenzalacetone (57.17 µg/mL), and Caffeic acid (11.73 µg/mL) were observed in the extracts. Protocatechuic acid 4- glucoside is the only glycosylated phenolic acid detected along with Syringic acid, which was significantly higher at pH 7 and pH 9.5, and temperature of 60 ◦C compared with the other treatments. Ferulic acid showed similar trend, though the amounts were lower. Myricetin was significantly (p < 0.05) higher in pH 7_60 ◦C than other pH-temperature treatments. Vanillic acid, ferulic acid, isorhamnetin, and salicylic acid were detected in moderate amounts between 0.5–0.6 µg/mL. It is worth mentioning that the flavonoids (isorhamnetin, myricetin), salicylic acid, and protocatechuic acid 4-glucoside are identified in Chaga mushroom for the first time in our study to the best of our knowledge. These phenolics were detected as aglycones, which might be due to the heat/and or pH impact on the aglyconic linkage with sugars’ moieties. Salicylic acid was significantly (p < 0.05) higher at pH 11.5 and temperature of 100 ◦C compared to the other treatments. One other major phenolic compound detected in Chaga mushroom using the extraction conditions was 3,4-dihydroxybenzaldehyde (104.851 µg/mL). Furthermore, 4-methoxycinnamic acid (57.23 µg/mL) is being reported in Chaga for the first time. This compound was found to have antioxidant, antihyperglycemic, and hypolipidemic activities [40–42]. Other new compounds that were identified and quantified in for the first time in Chaga include methyl-ellagic acid and ellagic acid. Both these compounds were found to exist at higher amounts in extracts obtained using pH 11.5–100 ◦C treatment. These compounds were reported to have anti-radical and strong neuroprotective and antioxidants properties [43]. Antioxidants 2021, 10, x FOR PEER REVIEW 13 of 19 Antioxidants 2021, 10, x FOR PEER REVIEW 13 of 19 Antioxidants 2021, 10, x FOR PEER REVIEW 13 of 19

The results for the composition at the best extraction conditions at pH 11.5 and a temper- The results for the composition at the best extraction conditions at pH 11.5 and a temper- Theature results of 100 for °C the is compositionpresented in atTable the best 2. The extraction identification conditions was atbased pH 11.5 on andthe moleculara temperature of 100 °C is presented in Table 2. The identification was based on the molecular atureformula, of 100retention °C is presentedtime, and exactin Table molecular 2. The identificationmass (Table 2). was The based compounds: on the molecular 3,4-dihy- formula, retention time, and exact molecular mass (Table 2). The compounds: 3,4-dihy- Antioxidants 2021, 10, 1322 formula,droxybenzaldehyde, retention time, 4-methoxycinnamic and exact molecular acid, mass dihydroxyterephtalic (Table 2). The compounds: acid, protocatechuic 3,4-dihy-13 of 18 droxybenzaldehyde, 4-methoxycinnamic acid, dihydroxyterephtalic acid, protocatechuic droxybenzaldehyde,acid, and caffeic acid 4-methoxycinnamicwere found as the high acid,est dihydroxyterephtalic phenolics in the Chaga acid, extracts protocatechuic produced acid, and caffeic acid were found as the highest phenolics in the Chaga extracts produced acid,using and the caffeic modified acid Swiss were foundwater asmethod the high (Tableest phenolics 2). It was in thesuggested Chaga extractsthat phenolics produced in using the modified Swiss water method (Table 2). It was suggested that phenolics in usingChaga the are modifiedthe main componentsSwiss water responsiblemethod (Table for the2). antioxidantIt was suggested activity that [18], phenolics and this inis ChagaQuercetin are the has main previously components been responsible reported in for Chaga the antioxidant and it was also activity detected [18], inand our this present is Chagaconsistent are withthe main the high components correlations responsible observed for between the antioxidant these components activity [18], in our and study. this is consistentwork but with at lowerthe high amounts correlations [27]. observed between these components in our study. consistent with the high correlations observed between these components in our study. −1 Table 2. Phenolics content (µg·g−1 dry weight) in Inonotus obliquus extracts obtained under different pH and temperature Table 2. Phenolics content (µg·g−1 dry−1 weight) in Inonotus obliquus extracts obtained under different pH and temperature TableTable 2. Phenolics 2. Phenolics content content (µg·g (µg−1· gdry dryweight) weight) in Inonotus in Inonotus obliquus obliquus extractsextracts obtained obtained under under different different pH pHand and temperature temperature Tableconditions 2. Phenolics content (µg·g−1 dry weight) in Inonotus obliquus extracts obtained under different pH and temperature conditionsconditions. conditions MW Amount Recovered MWMW AmountAmount Recovered Recovered CompoundsCompounds Formula MW m/zm/z [M [M-H]-− H]− RTRT (min) (min) Amount Recovered StructureStructure Compounds Formula (g/mol)MW m/z [M-H]- RT (min) Amount(µg/mL(µg/mL PARecovered PA eq.) eq.) Structure Compounds Formula (g/mol) m/z [M-H]- RT (min) (µg/mL PA eq.) Structure HydroxybenzoicHydroxybenzoic acidsacids (g/mol) (µg/mL PA eq.) Hydroxybenzoic acids Hydroxybenzoic acids GallicGallic acid_1 acid_1 11.1211.12 0.260.26 Gallic acid_1 11.12 0.26 GallicGallic acid_1 acid_2 C7H7 6O65 5 170.12 169.0142 11.1213.35 0.200.26 Gallic acid_1acid_2 C7H6O5 170.12 169.0142 11.1213.35 0.260.20 GallicGallic acid_2 acid_3 C7H6O5 170.12 169.0142 13.3514.74 0.110.20 Gallic acid_2 C7H6O5 170.12 169.0142 13.35 0.20 Gallic acid_2acid_3 C7H6O5 170.12 169.0142 13.3514.74 0.200.11 Gallic acid_3 14.74 0.11 Gallic acid_3 14.74 0.11

Protocatechuic acid C7H6O4 154.12 153.0193 16.90 32.21 ProtocatechuicProtocatechuic acidacid CC7H7H6O64O4 154.12154.12 153.0193 153.0193 16.90 16.90 32.21 32.21 Protocatechuic acid C7H6O4 154.12 153.0193 16.90 32.21 Protocatechuic acid C7H6O4 154.12 153.0193 16.90 32.21

Salicylic acid C7H6O3 138.122 137.0244 29.71 1.44 SalicylicSalicylic acid acid C CH7HO6O3 138.122138.122 137.0244 137.0244 29.71 29.71 1.44 1.44 Salicylic acid C7 7H6 63O3 138.122 137.0244 29.71 1.44 Salicylic acid C7H6O3 138.122 137.0244 29.71 1.44

Vanillic acid_1 0.47 Vanillic acid_1 0.47 Vanillic acid_1acid_2 0.470.45 VanillicVanillic acid_2 acid_1 8 8 4 0.470.45 Vanillic acid_2 C8H8O4 168.148 167.0350 19.48 0.45 VanillicVanillic acid_2acid_3 acid_2 C8H8O4 168.148 167.0350 19.48 0.450.450.33 Vanillic acid_3 CC8H8H8O84O4 168.148 167.0350 167.0350 19.48 19.48 0.33 VanillicVanillic acid_3 acid_3 C8H8O4 168.148 167.0350 19.48 0.330.33 Vanillic acid_3acid_4 0.330.18 VanillicVanillic acid_4 acid_4 0.180.18 Vanillic acid_4 0.18

2,3-Dihydroxybenzal- 2,3-Dihydroxybenzal- 2,3-Dihydroxybenzal- 7 6 3 dehyde2,3- C7H6O3 138.12 137.0244 18.28 104.85 dehyde C7H6O3 138.12 137.0244 18.28 104.85 DihydroxybenzaldehydeDHBA1dehyde CC7H7H6O63O3 138.12138.12 137.0244 137.0244 18.28 18.28 104.85 104.85 DHBA1DHBA1 DHBA1

2,5-Dihydroxytereph- 2,5-Dihydroxytereph- 8 6 6 2,5-Dihydroxytereph- C8H6O6 198.13 197.0092 16.00 9.83 2,5-Dihydroxytereph-thalic acid2,5- (DHTA) C8H6O6 198.13 197.0092 16.00 9.83 thalic acid (DHTA) C8H6O6 198.13 197.0092 16.00 9.83 thalicDihydroxyterephthalic acid (DHTA) CC8H8H6O66O6 198.13198.13 197.0092 197.0092 16.00 16.00 9.83 9.83 thalic acidacid (DHTA) (DHTA)

Hydroxycinnamic Hydroxycinnamic HydroxycinnamicHydroxycinnamicacids acidsacids acids

Antioxidantso-Coumaric 2021, 10 acid, x FOR PEER REVIEW 18.11 0.22 14 of 19 o-Coumarico-Coumaric acid 9 8 3 18.1118.11 0.220.22 o-Coumaric acid CC9H9H8O83O3 164.16164.16 163.0401 163.0401 18.11 0.22 o-Coumaricp-Coumaric acid C9H8O3 164.16 163.0401 18.1122.91 0.240.22 p-Coumarico-Coumaric acid C9H8O3 164.16 163.0401 18.1122.91 0.220.24 p-Coumaric acid C9H8O3 164.16 163.0401 22.91 0.24 p-Coumaric acid 22.91 0.24

Caffeic acid C9H8O4 180.16 179.0349 19.99 11.72 Caffeic acid C9H8O4 180.16 179.0349 19.99 11.72

4-methoxycinnamic C10H10O3 178.187 177.0557 21.11 0.57 acid

Hispidin C13H10O5 246.22 245.0455 18.81 0.45

Ferrulic acid (trans) 21.90 0.40 C10H10O4 194.18 193.0506 Ferrulic acid_1 (cis) 17.10 0.18

Flavonoid derivatives

Isorhamnetin C16H12O7 316.26 315.0510 23.34 0.47

Myricetin_1 22.41 0.30 C15H10O8 318.24 317.0303 Myricetin_2 30.10 1.32

Quercetin C15H10O7 302.236 301.0354 30.27 0.54

Other phenolic acids

Syringic acid C9H10O5 198.174 197.0455 17.30 1.14

Ellagic acid C14H6O8 302.197 300.9989 22.61 0.25

Antioxidants 2021, 10, x FOR PEER REVIEW 14 of 19 AntioxidantsAntioxidants 20212021,,, 1010,,, xxx FORFORFOR PEERPEERPEER REVIEWREVIEWREVIEW 1414 ofof 1919 Antioxidants 2021, 10, x FOR PEER REVIEW 14 of 19

Antioxidants 2021, 10, 1322 14 of 18

Table 2. Cont. Caffeic acid C9H8O4 180.16 179.0349 19.99 11.72 Caffeic acid C9H8O4 180.16 179.0349 19.99 11.72 CaffeicCaffeic acidacid C C99HH88OO44 180.16180.16180.16 179.0349179.0349179.0349 19.99 19.99 19.99 11.72 11.72 11.72 MW Amount Recovered Compounds Formula m/z [M − H]− RT (min) Structure (g/mol) (µg/mL PA eq.)

4-methoxycinnamic 4-methoxycinnamic4-methoxycinnamic4-methoxycinnamic acid CC1010HH10O103O3 178.187178.187 177.0557 177.0557 21.11 21.11 0.57 0.57 C1010H1010O33 178.187 177.0557 21.11 0.57 acid CC10HH10OO3 178.187178.187178.187 177.0557 177.0557 177.0557 21.11 21.11 21.11 0.57 0.57 0.57 acidacid

HispidinHispidin CC13HHO10O5 246.22246.22 245.0455 245.0455 18.81 18.81 0.45 0.45 Hispidin C1313H10 105O5 246.22 245.0455 18.81 0.45 HispidinHispidin CC1313HH1010OO55 246.22246.22246.22 245.0455 245.0455 245.0455 18.81 18.81 18.81 0.45 0.45 0.45

Ferrulic acid (trans) 21.90 0.40 FerrulicFerrulicFerrulic acidacid acid (trans)(trans) (trans) C10H10O4 194.18 193.0506 21.9021.9021.90 0.400.400.40 CC101010HH1010O104O44 194.18194.18 193.0506 193.0506 FerrulicFerrulic acid_1 acid_1 (cis)(cis) CC10HH10OO4 194.18194.18194.18 193.0506 193.0506 193.0506 17.1017.10 0.180.18 FerrulicFerrulic acid_1acid_1 (cis)(cis) 17.1017.10 0.180.18

Flavonoid derivatives FlavonoidFlavonoidFlavonoid derivativesderivatives derivatives

Isorhamnetin C16H12O7 316.26 315.0510 23.34 0.47 IsorhamnetinIsorhamnetin CC1616HH12O127O7 316.26316.26 315.0510 315.0510 23.34 23.34 0.47 0.47 IsorhamnetinIsorhamnetin CC1616HH1212OO77 316.26316.26316.26 315.0510 315.0510 315.0510 23.34 23.34 23.34 0.47 0.47 0.47

Myricetin_1 22.41 0.30 Myricetin_1Myricetin_1 15 10 8 22.4122.41 0.300.30 Myricetin_1Myricetin_1 C15H10O8 318.24 317.0303 22.4122.41 0.300.30 CC151515HH1010O108O88 318.24318.24 317.0303 317.0303 Myricetin_2Myricetin_2 C15H10O8 318.24318.24 317.0303 317.0303 30.1030.10 1.321.32 Myricetin_2Myricetin_2 30.1030.10 1.321.32

Quercetin C15H10O7 302.236 301.0354 30.27 0.54 Quercetin C1515H1010O77 302.236 301.0354 30.27 0.54 QuercetinQuercetinQuercetin CC1515HHH10O107OO7 302.236302.236 301.0354301.0354 301.0354 30.27 30.27 30.27 0.54 0.54 0.54

Other phenolic acids OtherOtherOther phenolicphenolic phenolic acidsacidsacids

Syringic acid C9H10O5 198.174 197.0455 17.30 1.14 SyringicSyringic acid acid C C9H9H10O105O5 198.174 197.0455 197.0455 17.30 17.30 1.14 1.14 SyringicSyringic acidacid C C99HH1010OO55 198.174198.174198.174 197.0455 197.0455 197.0455 17.30 17.30 17.30 1.14 1.14 1.14

Ellagic acid C14H6O8 302.197 300.9989 22.61 0.25 EllagicEllagic acid acid CC1414HH6O68O8 302.197302.197 300.9989 300.9989 22.61 22.61 0.25 0.25 EllagicEllagic acidacid CC1414HH66OO88 302.197302.197 300.9989300.9989 22.61 22.61 0.25 0.25

Antioxidants 2021, 10, 1322 15 of 18

AntioxidantsAntioxidants 20212021,, 1010,, xx FORFOR PEERPEER REVIEWREVIEW Table 2. Cont. 1515 ofof 1919

MW Amount Recovered Compounds Formula m/z [M − H]− RT (min) Structure (g/mol) (µg/mL PA eq.)

Hispolon C H O 220.224 219.0663 18.72 0.03 HispolonHispolon CC121212HH12 12124OO44 220.224220.224 219.0663 219.0663 18.72 18.72 0.03 0.03

3,4-3,4- Dihydroxyben-Dihydroxyben- 3,4- Dihydroxyben-3,4- Dihydroxybenzalacetone C H O 178.187 177.0557 19.03 57.17 zalacetonezalacetone CC101010HH10 10103OO33 178.187178.187 177.0557 177.0557 19.03 19.03 57.17 57.17 (DHBA2) (DHBA2)(DHBA2)

1515 88 88 3-O-Methylellagic3-O-Methylellagic3-O-Methylellagic acid acidacid C C1515HHH8O88OO8 316.22316.22 315.0146 315.0146 315.0146 25.55 25.55 25.55 0.25 0.25 0.25 Antioxidants 2021, 10, x FOR PEER REVIEW 16 of 19

TotalTotalTotal cinnamatescinnamates cinnamates 69.6569.6569.65 Total Benzoate 129.20 TotalTotalTotal BenzoateBenzoate ﬂavonoids previously been reported in Chaga and it 129.20 129.201.80 was also detected in our present work but at

TotalTotalTotal flavonoidsflavonoids other phenolics lower amounts [27]. 58.24 1.80 1.80 TotalTotal otherother phenolicsphenolics 58.24 58.24 3.2.4. Correlations 3.2.4. Correlations AccordingAccording toto thethe resultsresults obtained,obtained, 3,4-3,4-dihydroxybenzaldehydedihydroxybenzaldehyde waswas significantlysignificantly AccordingToTo getget ato better the resultsunderstanding understanding obtained, of of the3,4- the dihydroxybenzaldehydeantioxidant antioxidant potential potential of ofthe was the Chaga significantly Chaga extracts extracts ob- higherhigher (104.851(104.851 µ/mL)µ/mL) whenwhen extractedextracted atat pHpH 11.511.5 andand aa temperaturetemperature ofof 100100 °C°C comparedcompared higherobtainedtained (104.851 as asa function aµ/mL) function when of ofitsits extractedchemical chemical compositat composition,pH 11.5ion, and we a we assessedtemperature assessed the the correlations of correlations100 °C compared between between the toto otherother treatments.treatments. TheThe contentcontent foundfound inin ourour reresearchsearch areare consistentconsistent withwith previousprevious reportsreports to othertheTAA TAA treatments. and and total total antioxidant The antioxidant content minerals, found minerals, in ourTPC, TPC, re vitamins,search vitamins, are and consistent and dietary dietary with phenolics phenolics previous (Figure reports (Figure 5). 5 As). inin thethe literatureliterature asas thisthis compoundcompound waswas aa mamajorjor phenolicphenolic compoundcompound observed,observed, butbut thethe lev-lev- in theAspreviously literature previously mentioned,asmentioned, this compound a ahigh high was correlation correlation a major phenolicbetween between compoundantioxidant antioxidant observed, capacity capacity but and the TPCTPC lev- waswas elsels obtainedobtained inin ourour workwork areare muchmuch higher.higher. InInterestingly,terestingly, 3,4-dihydroxybenzaldehyde3,4-dihydroxybenzaldehyde hashas els found,obtainedfound, whichwhich in our indicates indicates work are that that much phenolic phenolic higher. compounds compou Interestingly,nds could could 3,4-dihydroxybenzaldehyde be thebe the foremost foremost contributors contributors has to the to beenbeen reportedreported toto havehave significantsignificant biologicalbiological activitiesactivities suchsuch asas antioxidative,antioxidative, antibacterial,antibacterial, beenantioxidantthe reported antioxidant to activity have activity significant of the of Chaga the biologicalChaga extracts extracts activities obtained. obtained. such This asThis is consistentantioxidative, is consistent with antibacterial, with several several reports re- anti-aging,anti-aging, andand anti-inflammatoryanti-inflammatory [39].[39]. TheThe prpresentesent studystudy alsoalso revealedrevealed otherother phenolicsphenolics anti-aging,inports the in literature andthe literatureanti-inflammatory demonstrating demonstrating [39]. high The associationhigh pr asesentsociation betweenstudy between also phenolics revealed phenolics and other theand phenolics antioxidant the antioxi- likelikeactivity dantflavonoidflavonoid activity [1]. aglyconesaglycones [1]. suchsuch asas myricetin,myricetin, quercetin,quercetin, isorhamnetin,isorhamnetin, andand phenolicphenolic acids—acids— suchsuch asas gallic,gallic, vanillic,vanillic, protocatechuic,protocatechuic, ferulic,ferulic, ellagicellagic acid,acid, methylmethyl ellagic,ellagic, syringic,syringic, andand salicylicsalicylic acids—toacids—to bebe majormajor compoundscompounds inin thethe ChagaChaga extractsextracts obtained.obtained. TotalTotal hy-hy- Antioxidant minerals comparison droxycinnamicdroxycinnamic acidsacids (69.6(69.6 µg/mL),µg/mL), totaltotal hydroxybenzoichydroxybenzoic acidsacids (129.20(129.20 µg/mL),µg/mL), andand totaltotal phenolicsphenolics (58.22(58.22 µg/mL)µg/mL) werewere foundfound atat higherhigher levelslevels inin thethe extractsextracts obtainedobtained usingusing pHpH Inonotus obliquus 11.5,11.5, 100100 °C.°C. PhenolicPhenolic acidsacids likelike 4-methoxycinnamic_acid4-methoxycinnamic_acid (57(57 µg/mL),µg/mL), 3,4-dihydroxyben-3,4-dihydroxyben- Suillus luteus zaldehydezaldehyde (104.85(104.85 µg/mL),µg/mL), 3,4-dihydroxybenza3,4-dihydroxybenzalacetonelacetone (57.17(57.17 µg/mL),µg/mL), andand CaffeicCaffeic acidacid (11.73(11.73 µg/mL)µg/mL) werewere observedobserved inin thethe extracts.extracts. ProtocatechuicProtocatechuic acidacid 4-glucoside4-glucoside isis thethe onlyonly Boletus subtomentosus glycosylatedglycosylated phenolicphenolic acidacid detecteddetected alongalong withwith SyringicSyringic acid,acid, whichwhich waswas significantlysignificantly Armillaria mellea higherhigher atat pHpH 77 andand pHpH 9.5,9.5, andand temperaturetemperature ofof 6060 °C°C comparedcompared withwith thethe otherother treatments.treatments. Lactarius deliciosus FerulicFerulic acidacid showedshowed similarsimilar trend,trend, thoughthough ththee amountsamounts werewere lower.lower. MyricetinMyricetin waswas signif-signif- icantlyicantly ((pp << 0.05)0.05) higherhigher inin pHpH 7_607_60 °C°C thanthan otherother pH-temperaturepH-temperature treatments.treatments. VanillicVanillic Tricholoma portentosum acid,acid, ferulicferulic acid,acid, isorhamnetin,isorhamnetin, andand salicylicsalicylic acidacid werewere detecteddetected inin moderatemoderate amountsamounts be-be- 0tweentween 0.5–0.60.5–0.6 50 µg/mL.µg/mL. 100 ItIt isis worthworth mentioningmentioning 150 thatthat 200 thethe flavonoidsflavonoids 250 (isorhamnetin,(isorhamnetin, 300 myrice-myrice- 350 Cu µg/g DW tin),tin), salicylicsalicylic Mn µg/g DW acid,acid, andand protocatechuicprotocatechuicSe µg/g DW acidacid 4-glucoside4-glucosideZn µg/g DW areare identifiedidentifiedTotal inin Antioxidant ChagaChaga minerals mushroommushroom forfor thethe firstfirst timetime inin ourour studystudy toto thethe bestbest ofof ourour knowledge.knowledge. TheseThese phenolicsphenolics werewere detecteddetected Figure 5. Antioxidant minerals comparison from different species [44]. asas aglycones,aglycones, whichwhich mightmight bebe duedue toto thethe heatheat/and/and oror pHpH impactimpact onon thethe aglyconicaglyconic linkagelinkage withwithFigure sugars’sugars’ 5. Antioxidant moieties.moieties. minerals SalicylicSalicylic comparison acidacid waswas significantlysignificantly from different ((p pspecies << 0.05)0.05) [44]. higherhigher atat pHpH 11.511.5 andand tem-tem- with sugars’Likewise, moieties. the Salicylic total antioxidant acid was significantly minerals (TAM) (p < 0.05) were higher moderately at pH 11.5 correlated and tem- with peratureperature ofof 100100 °C°C comparedcompared toto thethe otherother trtreatments.eatments. OneOne otherother majormajor phenolicphenolic compoundcompound the TAALikewise, (r = 0.52; the totalp = 0.016), antioxidant so too minerals was vitamin (TAM) E were with themoderately LPC (r =correlated 0.64). Vitamin with the E detecteddetected inin ChagaChaga mushroommushroom usingusing thethe extracextractiontion conditionsconditions waswas 3,4-dihydroxybenzal-3,4-dihydroxybenzal- existsTAA (r in = different 0.52; p = forms0.016), calledso too ‘Tocopherols’was vitamin E which with the are LPC known (r = antioxidants0.64). Vitamin [36 E]. exists In our in dehydedehyde (104.851(104.851 µg/mL).µg/mL). Furthermore,Furthermore, 4-methox4-methoxycinnamicycinnamic acidacid (57.23(57.23 µg/mL)µg/mL) isis beingbeing presentdifferent work, forms we called have ‘Tocopherols’ determined threewhich types are known of these antioxidants antioxidant [36]. vitamins, In our namely,present reportedreported inin ChagaChaga forfor thethe firstfirst time.time. ThisThis compoundcompound waswas foundfound toto havehave antioxidant,antioxidant, anti-anti- Alpha-,work, we Beta-, have and determined Delta-Tocopherols three types in theof Chagathese antioxidant extracts using vitamins, the SWP. namely, These Alpha-, results hyperglycemic,hyperglycemic, andand hypolipidemichypolipidemic activitiesactivities [40–42].[40–42]. OtherOther newnew compoundscompounds thatthat werewere demonstrateBeta-, and Delta-Tocopherols that phenolic acids in are the highly Chaga associated extracts using with the antioxidantSWP. These activity results presentdemon- identifiedidentified andand quantifiedquantified inin forfor thethe firstfirst timetime inin ChagaChaga includeinclude methyl-ellagicmethyl-ellagic acidacid andand strate that phenolic acids are highly associated with the antioxidant activity present in the ellagicellagic acid.acid. BothBoth thesethese compoundscompounds werewere foundfound toto existexist atat higherhigher amountsamounts inin extractsextracts ob-ob- Chaga extracts obtained using the modified Swiss water method evaluated in this study. tainedtained usingusing pHpH 11.5–10011.5–100 °C°C treatment.treatment. ThTheseese compoundscompounds werewere reportedreported toto havehave anti-anti- Collectively, pH 11.5, 100 °C appears to be very useful for extracting individual dietary radicalradical andand strongstrong neuroprotectiveneuroprotective andand antioxidantsantioxidants propertiesproperties [43].[43]. QuercetinQuercetin hashas phenolics, TPC, and TAA from Chaga matrices (Figure 3). The correlation results between antioxidant activity, TPC, phenolics, vitamins, and antioxidant minerals suggest the Chaga extracts obtained appears to be a promising source of myconutrients with func-

tional and antioxidant properties. Additionally, the correlation between the TAA and TPC together with individual phenolics and total phenolic acids were also established. Notably, 3,4-dihydroxybenzaldehyde (DHBA) showed strong significant (p < 0.0001) correlation with TAA (r = 0.89) and TPC (r = 0.61). Similarly, the salicylic acid (SalA) content in the extract was strongly correlated with the TAA (r = 0.89) and TPC (r = 0.88). The total hydroxybenzoic acids (ToHBen) was also significantly (p < 0.0001) correlated with TAA (r = 0.83) and TPC (0.54), respectively. That may explain the contributions of hydroxybenzoic acids to the total antioxidative capacity of the extract obtained using this solvent extraction system. On the contrary, the ferulic acid (FA) content in the extract was moderately, but significantly (p = 0.0001) correlated with TPC (r = 0.74) as was caffeic acid (CA) with TAA (r = 0.70). This trend in moderate association was consistent between the other phenolics and the TAA or TPC evaluated (Figure 3).

Antioxidants 2021, 10, 1322 16 of 18

in the Chaga extracts obtained using the modified Swiss water method evaluated in this study. Collectively, pH 11.5, 100 ◦C appears to be very useful for extracting individual dietary phenolics, TPC, and TAA from Chaga matrices (Figure3). The correlation results between antioxidant activity, TPC, phenolics, vitamins, and antioxidant minerals suggest the Chaga extracts obtained appears to be a promising source of myconutrients with functional and antioxidant properties. Additionally, the correlation between the TAA and TPC together with individual phenolics and total phenolic acids were also established. Notably, 3,4-dihydroxybenzaldehyde (DHBA) showed strong significant (p < 0.0001) correlation with TAA (r = 0.89) and TPC (r = 0.61). Similarly, the salicylic acid (SalA) content in the extract was strongly correlated with the TAA (r = 0.89) and TPC (r = 0.88). The total hydroxybenzoic acids (ToHBen) was also significantly (p < 0.0001) correlated with TAA (r = 0.83) and TPC (0.54), respectively. That may explain the contributions of hydroxybenzoic acids to the total antioxidative capacity of the extract obtained using this solvent extraction system. On the contrary, the ferulic acid (FA) content in the extract was moderately, but significantly (p = 0.0001) correlated with TPC (r = 0.74) as was caffeic acid (CA) with TAA (r = 0.70). This trend in moderate association was consistent between the other phenolics and the TAA or TPC evaluated (Figure3).

4. Conclusions The SWP method of extraction using hot water with different pH values has been very useful in obtaining high-quality food-grade and green extracts from Chaga enriched with myconutrients. These extracts represent a good source of myconutrients such as phenolics, fat- and water-soluble vitamins, and minerals. The highest TPC and TAA levels were found in extracts using pH 11.5 and a temperature of 100 ◦C. This was the case also for most of the individual phenolics detected in Chaga. Altogether, 24 phenolic compounds were detected in the extract where 3,4-dihydroxybenzaldehyde (104.851 µg/mL) was found as the major phenolic, which was highly correlated with the TAA and TPC. Vitamins are first analyzed in Chaga, being the Vitamin E and K with the highest amounts found. Furthermore, Other phenolics like 4-methoxycinnamic acid, isorhamnetin, protocatechuic acid 4-glycoside, among others are described for the first time in Chaga using the modified Swiss water method evaluated in this study. The Chaga extracts contained elevated TPC content and antioxidant properties at higher pH and temperature under pressurized extraction conditions. Treatment using pH 11.5 at 100 ◦C was useful for recovering the phenolics (total hydroxycinnamic acids, hydroxybenzoic acids, and total other phenolics) and total antioxidants. The antioxidant minerals were also best extracted at pH 11.5 at 100 ◦C, while the macro and trace minerals extraction, were more efficient using either pH 2.5 or 9.5 at 100 ◦C. This work demonstrates that water as pressurized fluid at high pH and temperature were very effective in extracting myconutrients from Chaga matrices and may also be useful for the recovery of bioactive nutrients from different functional food matrices. This work presents a modified Swiss water method at high pH (11.5) and temperature (100 ◦C) under pressurized conditions as a suitable green or food grade solvent for the extraction of antioxidant myconutrients (i.e., phenolics, Zn, K, Mn, and vitamin E) from Chaga matrices. These results suggest Chaga extracts obtained using the SWP could be promising sources of myconutrients or functional components that may have applications in producing nutraceuticals (i.e., Chaga capsules), or functional foods abundant with the antioxidants.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/antiox10081322/s1, Table S1: Hydrophilic (HAA), Lipophilic (LAA) and total antioxidant (TAA) activities in Chaga (Inonotus obliquus) at Various pH values and Temperature treatments, Table S2: Hydrophilic (HPC), Lipophilic (LPC)and total phenolic content (TPC) in Chaga (Inonotus obliquus) at various pH values and Temperature treatments, Table S3A: p-values (Pearson), Table S3B, Coefﬁcients of determination (Pearson). Antioxidants 2021, 10, 1322 17 of 18

Author Contributions: Conceptualization, R.T.; methodology, R.T., T.H.P. and N.P.V.; software, I.M.A.-R. and A.L.C.; validation, I.M.A.-R., T.H.P. and R.T.; formal analysis, C.F.M., A.R. and N.P.V., investigation, I.M.A.-R.; resources, R.T.; data curation, C.F.M., A.R. and N.P.V.; writing—original draft preparation, I.M.A.-R.; writing—review and editing, All; visualization, All; supervision, R.T. and I.M.A.-R.; project administration, R.T. and M.C.; funding acquisition, R.T. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data is contained within the article and supplementary material. Acknowledgments: I. Abu-Reidah would like to thank the MITACS Accelerate for the postdoctoral fellowship funding. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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