Recovery of ergosterol and vitamin D2 from mushroom waste - potential valorization by food and pharmaceutical industries

Abstract

Vitamin D is an essential vitamin for human health and plays a vital role in the regulation and maintenance of calcium homeostasis.Vitamin D deficiencies have also been linked to an increased risk of cancer, hypertension, autoimmune diseases, and diabetes (Jäpelt & Jakobsen, 2013).The main reasons for failing to meet vitamin D requirements are i) low dietary intake, since only a few food products naturally contain vitamin D and ii) limited vitamin D synthesis due to inadequate exposure to sunlight (Bailey et al., 2010;Jäpelt & Jakobsen, 2013).
Vitamin D is a fat-soluble vitamin (non-polar compound) which is found in two major forms, namely D2 and D3 (Dawson-Hughes et al., 2010).The D3 form is mainly contained in animal products such as egg, meat, and fish, while the D2 form is mostly present in mushroom fruiting bodies.Mushrooms contain high levels of ergosterol, a precursor of vitamin D2.The transformation of ergosterol to vitamin D2 can be achieved by the application of artificial or natural ultraviolet (UV) irradiation (Wen et al., 2020).
Ergosterol, the precursor of vitamin D2 is the most abundant sterol found in fungal cell membranes where it is critical for maintaining fluidity, permeability, carrying out various kinds of endocytosis and trafficking and for the cytoskeletal organization (Abe & Hiraki, 2009).In yeast mating, it is also linked with pheromone signalling and membrane fusion (Jin, McCaffery, & Grote, 2008).Recently, it has been suggested that ergosterol also has an immunoactivity role, as it is involved in triggering programmed cell death in host cells (Rodrigues, 2018).
High amounts of mushroom waste are generated during mushroom production (account for up to 20% of total production).Mushroom waste is mainly composed of mushrooms that their caps and/or stalks are misshapen and do not meet the specifications set by retailers (Aguiló-Aguayo, Walton, Viñas, & Tiwari, 2017).These mushroom by-products have high nutritional value.
Moreover, their disposal is linked to managing costs and high environmental impact including global warming, abiotic depletion, acidification, ozone layer depletion, eutrophication, human toxicity, eco-toxicity, depletion of natural resources, and energy consumption (Leiva, Saenz-Díez, Martínez, Jiménez, & Blanco, 2015).Therefore, new alternative and profitable solutions need to be explored (Royse, 2014).Mushroom waste could be utilized for the preparation of extracts enriched in vitamin D that could be used either by the pharmaceutical industry as nutritional supplements or by the food industry as an additional ingredient in foods, adding value to the agriculture sector.
Extraction method might have a significant impact on the recovery of both ergosterol and vitamin D2, therefore, the extraction conditions should be carefully selected and optimized.The application of some non-conventional methods such as UAE and MAE may facilitate the avoidance of saponification step reducing the total extraction time.Apart from the extraction, sample preparation may impact the retention of vitamin D and ergosterol in samples (Gąsecka et al., 2019;Tian, Zhao, Huang, Zeng, & Zheng, 2016).
The current review summarizes the steps from sample preparation until the detection of ergosterol and vitamin D from mushrooms, as well as their potential application in the food and pharmaceutical industries.

Ergosterol and vitamin D2 in mushrooms
Vitamin D occurs in two main forms in nature: Vitamin D2 (ergocalciferol) and Vitamin D3 (cholecalciferol).Vitamin D3 is only found in animal sources such as fish liver oils, eggs and milk products.Vitamin D2 found in limited plant sources in far fewer amounts, putting in vegetarians and vegans being at risk of having vitamin D deficient diets (Ho-Pham, Vu, Lai, Nguyen, & Nguyen, 2012).Mushroom qualify for such a diet and while they are deficient in vitamin D, widely available ones such as shiitake and button mushrooms, are rich sources of ergosterol, a precursor of vitamin D2.Ergosterol can be converted into vitamin D2 by natural or artificial UV irradiation which is a process that has been widely studied (Black, Lucas, Sherriff, Bjorn, & Bornman, 2017;Taofiq, Fernandes, Barros, Barreiro, & Ferreira, 2017).
Mushrooms and other kinds of fungi have high concentrations of ergosterol in their cell membranes where it performs a role similar to that of cholesterol in animals and sitosterol in plants, i.e., strengthening and maintaining fluidity of cell membranes, protein functionality, and modulating intracellular transport (Lingwood & Simons, 2010;Roncero-Ramos & Delgado-Andrade, 2017).It is produced by a fungal specific pathway.Similar to animals, fungi convert acetyl-CoA to lanosterol via the formation of squalene epoxide, however, fungi have a longer and more energy-expensive conversion final process in which 11 oxygen atoms are added to lanosterol to produce ergosterol whereas animals add 10 oxygen atoms in a more efficient process to produce cholesterol (Dupont et al., 2012).
Figure 1 shows the process of conversion of ergosterol in fungal sources into vitamin D2.
On exposure to UV radiation, ergosterol in exposed portions of mushrooms undergoes photochemical cleavage in the B ring, leading to the formation of an intermediate called previtamin D2.On subjection to heat, this intermediate undergoes thermal isomerization to form ergocalciferol, i.e., vitamin D2.The yield of the final product is heavily dependent on the balance between thermal and photochemical reactions (Jasinghe, Perera, & Sablani, 2007), hence the temperature of radiation is critical.Higher temperatures direct the reaction towards the formation of by-products such as lumisterol and tachysterol, in addition to adversely affecting the texture and colour of the mushrooms.Moisture content is also important since excessive dryness leads to an increase in surface area and hence exposure to oxygen, which leads to oxidation of vitamin D2.On the other hand, excess moisture may have a dilution effect on ergosterol content, which might result in a lower conversion rate to vitamin D2 (Jasinghe et al., 2007).However, the total amount of ergosterol produced per gram of mushroom might not be affected by the excess moisture content.Hence, the vitamin D2 fortification process in mushrooms should go hand in hand with minimizing moisture loss and maintaining hardness.The optimal conditions vary with types of mushrooms and there have been many studies to determine them (Ahn, 2018;Lee & Aan, 2016;Won et al., 2018).

Extraction and determination of ergosterol and vitamin D2 from mushrooms
Ergosterol and vitamin D2 extraction requires long determination times.Several steps are involved in the extraction and determination of ergosterol and vitamin D2 from mushrooms including sample preparation, saponification, extraction, cleaning, as well as detection and quantification using HPLC or GC.The first step for the extraction of ergosterol and vitamin D2 is sample preparation.Although samples can be either fresh or dried, most of the studies have determined sterols in lyophilized samples (Heleno et al., 2016a;Heleno et al., 2016b;Teichmann, Dutta, Staffas, & Jägerstad, 2007;Wittig, Krings, & Berger, 2013).To date, not much work has been done on the bulk recovery of ergosterol and vitamin D2 from mushrooms but that there is much to be learned from work done on its analytical determination.

Sample preparation
Sample preparation precedes extraction and determination steps.Studies have shown that sample preparation may have a significant impact on the sterol content of different mushroom species (Gąsecka et al., 2019;Nölle, Argyropoulos, Müller, & Biesalski, 2018).Recently, Gąsecka et al. (2019) reported that the ergosterol content of two mushroom species (Hericium erinaceus and Leccinum scabrum) was significantly affected by the processing method employed.
Specifically, the authors noted that the ergosterol content was higher in the fresh mushroom samples while it declined as the drying temperature increased from 20 to 70 °C (Gąsecka et al., 2019).Slawinska et al. (2016) investigated the effect of hot-air drying (the initial drying temperature was 40 °C, and in the final stage of drying the temperature was raised to 60 °C) and freeze-drying on ergosterol and vitamin D2 contents of three different mushroom species (Agaricus bisporus, Pleurotus ostreatus and Lentinula edodes).The authors reported that the vitamin D2 content was higher in the mushrooms dried by freeze-drying than those dried by hot-air drying, while drying treatment had a slight significant effect only on the ergosterol content of A. bisporus (Slawinska et al., 2016).Similar results were reported by Bernas and Jaworska (2017) where ergosterol content was higher in freeze-dried A. bisporus than its air-dried counterpart (30% loss).Tian et al. (2016) examined the effect of different drying methods (microwave vacuum-drying (microwave power density of 15 W/g, vacuum degree of −80 kPa, for 11 min), hot-air drying (60-70 °C), microwave-drying (539 W for 18 min), vacuum-drying (−90 kPa at 60 °C for 15 h)) on shiitake mushroom nutrient retention, including vitamin D2.The authors noted that drying resulted in the degradation of vitamin D2, with the hot-air and microwave-drying resulting in greater vitamin D2 loss, compared to the other two drying techniques (Tian et al., 2016).These results show the sensitivity of vitamin D2 to degradation in the presence of oxygen.Nölle et al. (2018) investigated the effect of a high precision drying (temperatures: 40, 60, and 80 °C, specific humidity: 10 g/kg, and air velocity: 0.6 m/s) and freezing-drying on the retention of vitamin D2 of different mushroom species, including shiitake (L.edodes), oyster (P.ostreatus), as well as white and brown button mushrooms (A.bisporus) treated with UV-B light (1.5 J/cm 2 ).In the case of oyster and white button mushrooms, the highest vitamin D2 content was found in the freeze-dried samples, while for shiitake and brown mushrooms there was insignificant difference between the freeze-dried and hot-air dried samples.Therefore, it can be concluded that the drying process should be carefully selected according to the mushroom species.

Extraction of ergosterol and vitamin D2
Conventional (e.g., Soxhlet extraction) and non-conventional (e.g., microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE), deep eutectic solvents (DES), supercritical fluid extraction (SFE)) methods have been investigated and developed for the recovery of ergosterol and vitamin D2 from mushrooms (Heleno et al., 2016a;Heleno et al., 2016b;Patil et al., 2018) (Figure 2).The current literature portrays different definitions for conventional extraction.For instance, according to Barba et al. (2017) conventional extraction should be considered as the extraction protocol using conventional solvents which are toxic to the environment and human health.According to Heleno et al. (2016b) conventional extraction is a method that is time-consuming and requires large quantities of hazardous solvents.On the other hand, as a non-conventional is considered the method that requires a lower temperature and shorter treatment times compared to conventional methods (Roselló-Soto et al., 2016).Non-conventional methods have been employed by studies for the recovery of sterols using organic solvents such as methanol.In this case, even though a non-conventional method is employed the whole process cannot be considered as environmentally and human friendly since the principles of 'green extraction' are not satisfied (Chemat, Vian, & Cravotto, 2012).According to Chemat et al. (2012) 'green extraction' is an extraction process that involves: i) reduced energy consumption, ii) the use of alternative non-toxic solvents and renewable natural products, and iii) results in a safe and highquality extracts.Table 1 presents the summary of studies conducted to date extracting vitamin D2 and ergosterol from mushroom matrices.The extraction methods are divided considering the technology used for the extraction rather than the solvent.

Saponification (hydrolysis)
Saponification is a hydrolysis reaction where free hydroxide breaks the ester bonds between the fatty acids and glycerol of a triglyceride, resulting in free fatty acids and glycerol (Prabu, Suriya Prakash, & Thirumurugan, 2015).In ergosterol and vitamin D2 extraction from mushrooms, saponification can be conducted either on samples or on extracts derived from different extraction methods.When hydrolysis is applied on mushroom samples, it breaks the complex structure of mushrooms and improves the recovery of ergosterol and vitamin D2 from the surrounding matrix.Moreover, when conjugated sterols are hydrolyzed to free sterols, they have similar polarity and can be extracted with a single solvent (Han & Zhou, 2015).The saponification may take place at temperatures varying from 60 and 80 °C and times varying from 15 to 60 min (Barreira et al., 2014;Gil-Ramirez et al., 2013;Heleno et al., 2016a).During the hydrolysis, ascorbic acid solution is used to avoid any thermal degradation of sterols.Recent studies have shown that saponification step can be avoided when some non-conventional extraction techniques such as UAE and MAE are employed (Heleno et al., 2016a;Heleno et al., 2016b).

Ultrasound-assisted extraction (UAE)
UAE has been employed for the extraction of different classes of bioactive compounds from mushrooms such as polyphenols, sugars, and vitamins from different mushrooms species (Aguiló-Aguayo et al., 2017;Alzorqi, Sudheer, Lu, & Manickam, 2017;Heleno et al., 2016a;Xu et al., 2016).Ultrasound devices use ultrasound waves above human hearing (>20 kHz) in which pressure fluctuation leads to the cavitation with the resultant unstable bubbles imploding and damaging cell membranes allowing for mass transfer out of the cell (Chemat et al., 2017).In sterol extraction from mushroom, ultrasonic baths have been mainly used with a frequency ranging between 20 to 65 kHz (Heleno et al., 2016a;Patil et al., 2018;Villares et al., 2012;Villares et al., 2014).Several mechanisms are involved in ultrasound extraction such as fragmentation, erosion, capillarity, detexturation, and sonoporation and have been described in the depth in the recent review of Chemat et al. (2017) and summarized in Figure 3.
The extraction of sterols from mushrooms can be influenced by different parameters, such as solvent type, extraction time and ultrasound power, while liquid-to-solid ratio seems to have no significant effects (Heleno et al., 2016a).Patil et al. (2018) employed UAE (ultrasonic bath) in conjunction with deep eutectic solvents (DES) for the extraction of vitamin D2 from A. bisporus.
The vitamin D synthesis was achieved by exposing the extracts to UV light.Villares et al. (2012) used UAE (ultrasonic bath) for the recovery of ergosterol from Τuber melanosporum and T. aestivum using chloroform/methanol mixture (2:1, v/v).After UAE, a clean-up stage of the extracts was conducted with Oasis MAX column preconditioned with 8 mL of hexane.Sterol separation from other lipids was conducted by passing chloroform.The solvent in the final extracts was evaporated under a nitrogen stream and redissolved in 2 mL of chloroform.Heleno et al. (2016a) optimized the UAE of ergosterol from Agaricus bisporus using response surface methodology (RSM).In this study the effects of different parameters were investigated including the type of extraction solvent, liquid-to-solid ratio, extraction time, and ultrasound power.Among the different solvents investigated, ethanol resulted in the highest ergosterol yields followed by limonene and n-hexane.Both ergosterol and vitamin D2 are amphiphilic molecules with small hydroxyl polar heads which allow them to be extracted by a variety of polar and non-polar solvents (Heleno et al., 2016a;Hsueh et al., 2007).The authors noted that at the optimal UAE conditions (extraction time of 15 min and ultrasound power of 375W) ergosterol yields were higher than those obtained by a time consuming Soxhlet extraction method in significantly shorter times (from 4h to 15 min).The authors also claimed that the saponification step can be avoided when the UAE is employed (Heleno et al., 2016a).However, the efficiency of the UAE on purifying the extracts is influenced by the solvent used.For instance, the authors showed that the purity of the extracts obtained by n-hexane and limonene was similar to the extracts obtained after saponification, while ethanol during UAE resulted in less pure extracts.This was attributed to the higher polarity of ethanol than the other two solvents which may lead to the extraction of various compounds (polar and non-polar).In summary, UAE can be used for the extraction of sterols from mushrooms facilitating shorter extraction times.

Supercritical fluid extraction (SFE) and pressurized liquid extraction (PLE)
SFE and PLE are sustainable extraction techniques that have been used for the recovery of various bioactive compounds, including polyphenols, pigments, oils, and sterols from different materials (Aladić et al., 2016;Valadez-Carmona, Ortiz-Moreno, Ceballos-Reyes, Mendiola, & Ibáñez, 2018).In SFE the use of supercritical CO2 (non-polar) facilitates the recovery of non-polar and mid-polar compounds (Gallego, Bueno, & Herrero, 2019).SFE has been employed for the recovery of both ergosterol and vitamin D2 from mushrooms (Gil-Ramirez et al., 2013;Morales et al., 2017).Morales et al. (2017) employed SFE for the production of extracts enriched in vitamin D2.Specifically, SFE was employed for the extraction of ergosterol from shiitake mushrooms and subsequently, extracts were exposed to UV light for the conversion of ergosterol to vitamin D2.In this study, two extraction parameters were examined including extraction temperature (°C) and pressure (bar).Both extraction parameters had a significant effect on the recovery of ergosterol.
The highest ergosterol yields (180 mg/g dw) were obtained at a pressure of 350 bar and a temperature of 70 °C (Morales et al., 2017).Gil-Ramirez et al. (2013) optimized and compared the extraction conditions of SFE and PLE for the recovery of sterols from Agaricus bisporus fruiting bodies.In SFE, extraction pressure and use of co-solvent (10% ethanol) were investigated.
Lower sterol yields were obtained when a co-solvent was used, while there was no significant effect of the extraction pressure.Fractions containing 60% of sterols were obtained at 40 °C and 30 MPa.In the case of PLE, several parameters were investigated including extraction time, the number of cycles, ratio mushroom powder/sand and temperature.The optimal PLE conditions using ethanol as the pressurized solvent were a pressure of 10.7 MPa, a temperature of 50 °C, 5 cycles of 5 min and mushroom/sand ratio of 1:4 (Gil-Ramirez et al., 2013).Between SFE and PLE, PLE found to extract all the sterols from mushrooms, however, SFE was found to be more selective.SFE has the potential of being upscaled for the extraction of both ergosterol and vitamin D2 since it has been upscaled for the extraction of other compounds (e.g., caffeine to make decaffeinated products) whereas PLE remains a small-scale batch technique with mostly analytical applications.

Microwave-assisted extraction (MAE)
MAE uses microwaves which are electromagnetic irradiation ranging in frequency from 300 MHz to 300 GHz.MAE has been employed for the extraction of different classes of bioactive compounds, such as polyphenols, polysaccharides, lipids, and fatty acids (Kumar, Sivacumar, & Ruckmani, 2016;Maeng, Muhammad Shahbaz, Ameer, Jo, & Kwon, 2017;Sinanoglou et al., 2015).However, only few studies to date have employed and optimized the MAE of ergosterol from mushrooms while there is no study optimizing the MAE of vitamin D2 from mushrooms (Young, 1995;Heleno et al., 2016b;Taofiq et al., 2019).Heleno et al. (2016b)

Conventional extraction methods
Currently, the extraction of ergosterol and vitamin D2 from mushrooms is mainly conducted using conventional extraction methods (Table 1).Soxhlet extraction is used for the extraction of both sterols, followed by saponification and purification.Heleno et al. (2016a) employed Soxhlet extraction for the recovery of ergosterol from A. bisporus.Specifically, 4.5 g of samples were extracted with 150 mL of solvent (the effect of n-hexane, ethanol and limonene was examined) during 4 h Soxhlet extraction.After the extraction the solvent was rotary evaporated, and the extracts were saponified using 2 M KOH and 0.1 M ascorbic acid solution at 60 °C for 45 min.Subsequently, the samples were mixed with NaCl solution and n-hexane.Sterols due to their lipophilic nature were transferred from the aqueous to n-hexane phase.The remained aqueous layer was reextracted again using n-hexane.Finally, the n-hexane fraction was dried and the residue was dissolved in methanol (1 mL) before HPLC analysis (Heleno et al., 2016a).
In most of the studies that have been conducted so far, saponification of mushroom samples precedes extraction and purification.A protocol that is usually reported in the literature is saponification of mushroom samples for 30 min to 1 h at 80 or 85 °C, followed by liquid-liquid extraction using non-polar solvents (i.e., n-pentane).Subsequently, the non-polar phase is washed with potassium hydroxide 3% (w/v) in 5% (v/v) ethanol and neutralized using deionized water.

High-performance liquid chromatography (HPLC)
HPLC is the most commonly employed technique for the identification and quantification of vitamin D2 and ergosterol from mushrooms (Table 2).Before injection, samples are filtered using disposable filter discs to remove small particles and thus protect HPLC columns.For the detection of vitamin D2 and ergosterol, reverse phases are usually employed containing carbon chains of 18 carbon atoms, while there is one study used a column with carbon chains of 30 carbon atoms (Table 2).The temperature of the stationary phase (column) is a critical parameter that may affect the retention times of bioactive compounds.In the case of ergosterol and vitamin D2 detection, the temperatures of the stationary phase may vary between 25 and 50 °C.For instance, in the studies of Yuan, Wang, Liu, Kuang, and Zhao (2007) the stationary phase was maintained at ambient temperature, while in the study of Morales et al. (2017) the stationary phase was maintained at 50 °C.For HPLC analysis an injection volume of 10 or 20 μL is usually used, however, when UHPLC (Ultra-high performance liquid chromatography) is employed, an injection volume of 1 μL can be used (Slawinska et al., 2016).Either isocratic or gradient elution can be implemented in HPLC analysis.The combination of methanol and acetonitrile is mainly used as a mobile phase in isocratic elution while in gradient elution two or three mobile phases can be used (Ahlborn et al., 2018).The flow rate that is usually employed is 1 mL/min (Ahlborn et al., 2018;Heleno et al., 2016a;Heleno et al., 2016b;Huang, Lin, & Tsai, 2015;Morales et al., 2017).UV-Vis and photodiode array detectors have been employed for the detection and the wavelengths that are usually used for the detection of both ergosterol and vitamin D are 264 and 13 280 (Table 2).Mass spectrometers (MS) directly or coupled with a PDA detector have also been used for detecting sterols, including vitamin D2, and ergosterol in mushrooms.
It is known that mushrooms have the potential to be the only food source of vitamin D (non-animal and unfortified), while providing a substantial amount of vitamin D2 in a single serving (Cardwell, Bornman, James, & Black, 2018).Fresh mushrooms sold in UK retailers provide 3-5 μg (60-100 % of the RI) per 100 g, which equals to 4-5 chestnut mushrooms or 1-2 portobello mushrooms (Food Manufacture, 2016).
Sun or hot-air dried mushrooms have about 15% of the original weight of fresh mushrooms and will retain about 5% of water, while freeze dried mushrooms will have close to zero moisture and 8-10% of the weight of the original mushroom.While dried mushrooms are cheaper to transport and might represent a cheaper source of vitamin D2, several variables (time of exposure, temperature, and exposure to UV-B radiation) are known to influence their vitamin D2 production (Cardwell et al., 2018).
The potential application of waste mushroom extracts (such as vitamin D2 extracts) in new foods could have regulatory implications.In the European Union, mushroom extracts might fall under the Novel Food Regulation, which relates to foods not widely consumed by people in the EU before May 1997.Mushroom extracts could fall under the category "food consisting of isolated from or produced from plants or their parts" (EFSA, 2016).Only UV treated mushrooms (A.bisporus) and an aqueous extract from shiitake mushroom with the glucan lentinan (L.edodes) have so far been authorized as novel foods through the lengthy authorization process.
Practical applications of vitamin D2 from mushroom waste are limited.The concept of extracting chitin and chitosan from mushroom waste is not new (Wu et al., 2004).Bilbao-Sainz et al. (2017) developed this concept further, creating vitamin D-fortified chitosan films from mushroom waste.The authors treated mushroom stalk bases with UV-B light, obtaining about 90 μg of vitamin D2 per gram on a dry weight basis (4.5 to 6 times the RDA/g).They then prepared fungal chitosan films with characteristics similar to animal derived chitosan.Correa et al., (2018) obtained and extract rich in ergosterol from commercially discarded A. Blazei fruits and used it as a fortifier ingredient in yogurts.The ergosterol extract in yogurts had antioxidant properties and did not alter the nutritional profile of the yogurt.
The use of vitamin D extracts from mushroom waste in new foods could benefit the food industry, as several nutritional and health claims could be made on food packaging.According to the European Food Safety Authority (EFSA, 2006), vitamin D can be added to foods as cholecalciferol (D3) or ergocalciferol (D2).The daily Reference Intake (RI) value for vitamin D is 5 μg, previously known as Recommended Daily Allowances (RDAs), and this is the reference value used on food labels (EU Regulation, 2011).According to the EFSA register of nutrition and health claim (EFSA, 2012), there are several health claims that can be made on foods that are a "source of" or "high" in vitamin D. "Source of" and "high" refer to foods that contain respectively at least 15% or 30% of the vitamin D RDA (15%=0.8μg, 30%=0.16μg) per 100 g of solid food.
The allowed health claims on vitamin D adapted from the EFSA register of nutrition and health claims are shown in Table 3.

Potential application in the pharmaceutical industry
Ergosterol, a vital component of the fungal and protozoal cell membranes plays a fundamental role in membrane fluidity and integrity while acting as a drug target of several antifungal agents (Rodrigues, 2018).Interestingly, ergosterol and its derivatives isolated from mushroom and other natural sources have been reported to have several therapeutic properties.A review by (Picotto, Liaudat, Bohl, & Talamoni, 2012) emphasized the importance of vitamin D (ergocalciferol and cholecalciferol) in anticancer research.Table 4 represents the major preclinical studies portrayed in the current literature underlining the bioactivity of ergosterol and its natural, semi-synthetic and synthetic derivatives.In particular, ergosterol peroxide, a natural ergosterol derivative has been studied more extensively and shown to confer a greater anti-proliferative and cytotoxic potential compared to ergosterol against breast, colorectal, ovarian and renal cancer cell lines (He, Shi, Liu, Zhao, & Zhang, 2018;Kang et al., 2015;L. M. Kuo et al., 2005;Martínez-Montemayor et al., 2019;Russo et al., 2010;Tan et al., 2017).
Mechanistically, ergosterol in its pure or derivative forms were reported to induce caspasemediated intrinsic apoptosis, arrest cell cycle and inhibit migration and invasion of cancer cells while regulating several signalling pathways including the STAT3 and the IGFR/IRS-1/2-MEKras-ERK1/2 (W.J. Chen et al., 2008;Li et al., 2015;Martínez-Montemayor et al., 2019;Tan et al., 2017).Downregulation of β-catenin has also been identified as one of the key molecular mechanisms of action against colorectal and renal cancer cells (He et al., 2018;Kang et al., 2015).
The AK3/STAT3/NF-κB pathway was found to play an important role in mediating the anti-inflammatory activity of ergosterol and ergosterol peroxide in cellular and animal models (Huan, Tianzhu, Yu, & Shumin, 2017;Kobori, Yoshida, Ohnishi-Kameyama, & Shinmoto, 2007; C.-F. Kuo, Hsieh, & Lin, 2011;Zhang, Xu, Li, & Wang, 2015).The anti-inflammatory activity of ergosterol has also been attributed to the suppression of the proinflammatory cytokines including TNF-α, IL-6 and IL-1β (Huan et al., 2017).Although these reports demonstrated the potential therapeutic benefits of ergosterol and its derivatives against cancer and inflammatory diseases, further in vivo mechanistic studies to evaluate their bioavailability and potential toxicity are warranted to lay the foundation for future clinical trials.Moreover, preclinical studies investigating the interactions of ergosterol (and its derivatives) with common anticancer and anti-inflammatory drugs could also open up exciting new avenues for prospective combination therapies especially for rare cancers and inflammatory diseases with limited therapeutic options.

Future directions
Drying may precede extraction and may affect the retention of both ergosterol and vitamin D2.Due to the controversial results and limited knowledge regarding ergosterol and vitamin D2 stability during drying, further studies are required in order to elucidate the mechanism of vitamin D2 formation at different drying temperatures.-Omics technologies, including metabolomics, genomics, and proteomics may facilitate in better understanding of sterol retention in mushrooms after the application of different drying techniques.Apart from the conventional drying technologies that have been applied to date, future studies should be conducted investigating the effect of ohmic heating (also known as Joule heating) on dried mushroom bioactive compounds.
Ohmic heating is a process of heating the food by passing electric current and may result in faster dehydration of food, avoiding any color deterioration and nutritional value degradation (Kaur & Singh, 2016).The use of vitamin D extracts from mushroom waste in new foods could benefit the food industry, yet, the practical applications of vitamin D2 from mushroom by-products are limited.
Therefore, potential food applications of mushroom extracts enriched in ergosterol and vitamin D2 is a field that requires further work.Nonetheless, the legislation should be considered when using vitamin D2 extracts from mushroom waste in new foods.Similarly, despite its potential especially in anticancer and anti-inflammatory drug development, the pharmaceutical application of ergosterol and vitamin D2 from mushroom needs further in vivo mechanistic studies for future clinical trials.As novel drug development entails large resources and time, the approach of combining pre-existing drugs with natural product-based adjuvants to increase their efficiency is promising and economical.Therefore, studies investigating the potential synergistic interactions of ergosterol and vitamin D2 with pre-existing anticancer and anti-inflammatory drugs can provide interesting insights for their future pharmaceutical applications.(Huan et al., 2017) inflammatory response via the AK3/STAT3/NF-κB pathway.

Scleroderma Polyrhizum
In vivo Female BALB/c mice Ergosterol pretreatment at 25 and 50 mg/kg decreased lipopolysaccharide (LPS)-induced lung histopathological changes and lung wet-to-dry weight ratio Suppressed inflammatory cells and proinflammatory cytokines including TNF-α and IL-6.Blocked the activation of NF-κB, COX-2 and iNOS pathways.(Zhang et al., 2015) Ergosterol

Cordyceps militaris and chemical standard
In vitro BV2 microglia cells Dose-dependent reduction of NO production ranging from 13% to 48% at 1 μg/mL -10 μg/mL with no cytotoxicity at 0.1-10 μg/mL.Commercial ergosterol was not effective.

Inonotus obliquus
In vitro RAW 264.7 murine macrophage cells Inhibited the production of nitric oxide (NO).

Saireito
In vitro Mucosal-type murine bone marrow-derived mast cells (mBMMCs) The degranulation of mBMMCs was significantly suppressed at ≥32 µM in a dose-dependent manner.
Not reported.

Cordyceps cicadae
In vitro

Primary human T lymphocytes
Inhibited T-cell proliferation for about 24 h after stimulation with phytohemagglutinin (PHA).
Suppressed the expression of cyclin E, IFN-γ, IL-2, and IL-4, and by arrested cell cycle progression from the G1 to the S phase in T lymphocytes.Downregulated AP-1 proteins including c-Fos and c-Jun in activated T lymphocytes.(Kuo, Weng, Chou, Chang, & Tsai, 2003) Ergosterol and ergosterol peroxide

Naematoloma fasciculare
Normal human serum Ergosterol and ergosterol peroxide displayed anti-complimentary activity on the classical pathway with IC50 values of 5 and 1 μM, respectively.

Source
investigated and optimized three parameters named extraction time, extraction temperature and solid-to-liquid ratio using RSM for the recovery of ergosterol from A. bisporus.The optimal MAE conditions were extraction time of 19.4 ± 2.9 min, extraction temperature of 132.8 ± 12.4 °C and the solid-to-liquid ratio of 1.6 ± 0.5 g/L, yielding 5.56 ± 0.26 mg of ergosterol per g of mushroom by-products.The authors noted that the extraction yields increased as the extraction time and extraction temperature increased, while extraction yields decreased as the solid-to-liquid ratio increased.The authors also reported that the purity of extracts in ergosterol reduced by increasing temperature.This could be either due to the extraction of other compounds than ergosterol as the extraction temperature increased, or ergosterol was degraded as the temperature increased(Heleno et al., 2016b).Recently, Taofiq et al. (2019) optimized the MAE (extraction time (2-25 min) and temperature (60-150 °C)) of ergosterol (dependent variables examined: extraction yields, purity and total extraction yields) from A. blazei and compared the dependent variables with those obtained at the optimal UAE and heat-assisted extraction (HAE).Authors noted that among the different extraction methods MAE was the most efficient in terms of extraction yields (25.44 ± 5.1 mg/ 100g dw) compared to the UAE and HAE 21.49 ± 0.9 and 18.84 ± 2.3 mg/ 100g dw, respectively.However, UAE resulted in extracts with higher ergosterol purity which could be attributed to the combined mechanisms such as fragmentation, erosion and sonoporation that take place through UAE.MAE is a promising extraction technique that can significantly reduce the extraction time of ergosterol recovery from mushrooms.Future studies are encouraged to investigate and optimize the MAE of vitamin D2 (extraction yields and purity) from different mushroom varieties.
The extraction of sterols from mushrooms requires long extraction times when conventional extraction protocols are applied.The application of non-conventional extraction technologies such as UAE and MAE may result in pure extracts without saponification decreasing the extraction times.Future studies should focus on the development of sustainable extraction protocols on a commercial scale for the recovery of ergosterol and vitamin D2 from mushrooms.This could be achieved by the combination of two or more different non-conventional extraction techniques.Pulsed electrical fields (PEF) which is a non-thermal technique has the potential of being employed for the preparation of pure extracts enriched in vitamin D2 and ergosterol.PEF may result in an increase mass transfer by electroporation which induces cell membrane permeabilization (Barbosa-Pereira, Guglielmetti, & Zeppa, 2018).

Figure 1 .
Figure 1.Process of conversion of ergosterol into vitamin D2 in fungal sources.

Figure 2 .
Figure 2. Extraction and determination of ergosterol and vitamin D2 by (a) conventional and (b) non-conventional extraction methods.
hepatic stellate and HL-7702 human hepatic cell lines Ergosterol inhibited activated LX-2 and HL-7702 cells in a dosedependent manner.It protected the Upregulated expressions of permeability of the lysosomal membrane and downregulated the levels of EdU, F-actin, and -SMA.

Table 2 .
Summary of high-performance liquid chromatography (HPLC) conditions been for the detection 911 of ergosterol and vitamin D2 in mushrooms.

Table 4 .
Preclinical studies underlining the bioactivity of ergosterol and its derivatives and possible molecular mechanisms of action.