(Agaricus bisporus) using Deep Eutectic Solvent and Ultrasonication

Research Article Cite This: ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

Extraction of Vitamin D from Button Mushroom (Agaricus bisporus) Using Deep Eutectic Solvent and Ultrasonication Jyoti Patil,† Sharwari Ghodke,‡ Ratnesh Jain,*,‡ and Prajakta Dandekar*,† †

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Department of Pharmaceutical Sciences & Technology, Institute of Chemical Technology, Nathalal Parekh Marg, Matunga, Mumbai−400 019, India ‡ Department of Chemical Engineering, Institute of Chemical Technology, Nathalal Parekh Marg, Matunga, Mumbai−400 019, India ABSTRACT: Deep eutectic solvents (DESs) are emerging as green and sustainable solvents for efficient extraction of bioactive compounds or drugs. In this study, DES composed of glycerol and choline chloride (3:1) was used as a potential solvent for the extraction of vitamin D from button mushrooms. This eliminated the requirement of organic solvents, thus reducing the environmental harm. Use of DES, along with ultrasonication, proved to be an efficient technique for extraction of vitamin D. Also, substantial increase in vitamin D2 content, with simultaneous decrease in ergosterol (vitamin D2 precursor) was observed due to photochemical reaction, when the extract was exposed to ultraviolet light (UV-C). While most of the ergosterol was transformed into vitamin D2, formation of a small amount of overirradiated product was also observed upon irradiation with UV-C. KEYWORDS: Deep eutectic solvent (DES), Vitamin D2, Ergosterol, UV irradiation, Sonication

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insulin secretion by pancreatic β cells, brain and fetal development, etc.10−12 Depending on the species of mushroom and the duration of UV exposure, vitamin D2 content may be as high as 204.7 μg/g of golden oyster mushroom.13 Thus, irradiated cultivated mushrooms are a potential alternative to fatty fish or other natural food sources of vitamin D, particularly for the vegans and vegetarians whose diet is otherwise extremely limited in vitamin D.14 Vitamin D deficiency is widespread in individuals, irrespective of their age, gender, race, and geography. Thus, it is important to develop an efficient extraction method for utilizing vitamin D from the widely available sources, so as to promote its maximum utilization by the population. Conventionally, liquid−liquid extraction has been used for extracting vitamin D and its metabolites from different sources. Organic solvents such as acetonitrile, ethanol, chloroform, methanol, diethyl ether, tetrahydrofuran (THF), hexane, etc. are commonly used during these procedures.15−19 Liquid−liquid extraction involves different types of pure and mixed solvents, having broad solubility and selectivity. Also, the separation steps involved in this type of extraction are simple. However, the large amount of organic solvents utilized during these extraction processes lead to generation of waste having low biodegradability and high volatility, thus polluting the environment.

INTRODUCTION Mushrooms contain various polyphenolic compounds that act as excellent antioxidants due to their free radicals scavenging effect, by single-electron transfer. Numerous researchers have correlated the antioxidant activity of edible mushrooms such as Leucopaxillus giganteus, Sarcodonimbricatus, Agaricus arvensis, and Lentinula edodes with the amounts of phenolic compounds, ascorbic acid, beta-carotene, and lycopene contained in them.1,2 Agaricus bisporus, or the white button mushroom (WBM), is one of the most economic varieties of edible mushrooms, having high content of polyphenols, ergothioneine, selenium, and polysaccharides.3 Recently, the antitumor and immune modulating properties of Agaricus bisporus was demonstrated by researchers.4 Polysaccharides with antioxidant properties have also been extracted from mushrooms.5,6 In addition to the antioxidants, mushrooms are also rich in ergosterol (precursor of vitamin D). Mattila and associates found that wild mushrooms are a source of vitamin D, with this source being acceptable to vegetarians; a high intake of mushrooms should be incorporated in the diet.7 Researchers have found that wild mushrooms contain abundant amounts of ergosterol, which can be converted to vitamin D2, tachysterols, or lumisterols, by UV irradiation.8,9 UV irradiation of the cultivated mushrooms produces a significant amount of the bioavailable vitamin D2 that is efficiently metabolized to 1,25-hydroxyvitamin D2. It is functional in promoting bone mineralization and also in various biological activities in innate and adaptive immunity system, heart functioning and blood pressure regulation, © XXXX American Chemical Society

Received: April 27, 2018 Revised: June 7, 2018 Published: June 8, 2018 A

DOI: 10.1021/acssuschemeng.8b01915 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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ACS Sustainable Chemistry & Engineering

purchased from Himedia Lab. Pvt. Ltd., Mumbai, India. All other chemicals and reagents were used as received from the manufacturers, without further processing. Preparation and Characterization of Deep Eutectic Solvent (DES). Deep eutectic solvent (DES) was prepared by mixing choline chloride, as the hydrogen bond acceptor (HBA), and glycerol, as the hydrogen bond donor (HBD), in a reagent bottle. Glycerol and choline chloride were mixed in different molar ratios (1:1, 3:1, 5:1), on a magnetic stirrer at 200 rpm, for 2 h, at 50 °C, until a clear and homogeneous liquid was formed.24 The viscosity of the resulting DES was measured using an Ostwald viscometer from J-SIL Scientific Industries (Mumbai, India), according to a reported protocol.25 The viscometer tube was filled with 20 mL of the liquid. The upper meniscus of the liquid in the capillary tube was adjusted to the level of the top graduation mark with the aid of vacuum. The time in seconds required for the liquid to flow from the upper mark to the lower mark in the capillary tube was recorded. Similarly, the viscosity of water was recorded as the control. The procedure was repeated three times and the average time was considered for calculation. Viscosity was calculated using formula 1,

Therefore, there is an increasing demand for newer extraction techniques, demonstrating reduced solvent consumption and shortened extraction times. Thus, various advanced extraction techniques, such as supercritical fluid extraction (SFE), microwave assisted extraction (MAE), ultrasound assisted extraction (UAE), and solid phase extraction (SPE), have been investigated. These techniques can be used over conventional methods to reduce the operation time and intensify the extractable yields.18 Green technology explores newer solvents to replace common organic solvents such as carbon tetrachloride, chloroform, benzene, etc. that present inherent toxicity and low biodegradability and have high volatility, thus polluting the environment due to evaporation of volatile organic compounds into the atmosphere.20 The conventional organic solvents can be replaced by green solvents such as ionic liquids (ILs), deep eutectic solvents (DESs), supercritical carbon dioxide (scCO2), and water etc. These solvents possess high biodegradability and low volatility, which reduces the environmental pollution.21 This work was focused on exploration of DES as a green solvent for extracting vitamin D from button mushrooms. DES can be used as a green alternative to the organic solvents as it possesses properties such as very low vapor pressure, low flammability, and easy reusability. Further, the synthesis of DES involves no byproduct generation; hence, there is 100% atom economy involved, facilitating large scale availability and cost effectiveness of the process.22 The DES used in this study was a eutectic mixture of choline chloride (ChCl) and glycerol (Gly). According to Kristina Radošević and co-workers, the DES showed the highest level of biodegradability of 96%. The level of biodegradability of the DES reached 84% after 7 days. The authors also suggested that the highest level of biodegradability could be attributed to a high level of biodegradability of individual components (viz. ChCl and glycerol). Further, the toxicity study of the ChCl based DES gave EC50 values for the inhibition of wheat germination by ChCl of >20000 mg L−1, thus indicating a low toxicity. This suggested that ChCl-based DESs have a potential green profile due to its low toxicity and high level of biodegradability. Choline chloride (ChCl), used as the hydrogen bond acceptor in the DES, is a nontoxic salt and has been approved as a nutritional additive.23 Thus, the nontoxicity of both the components of the DES used in this study suggested its safety. Further, the effect of UV-C exposure was assessed on the conversion of vitamin D precursor (ergosterol) to vitamin D2 (ergocalciferol). Also, the extraction yield was intensified using ultrasonication. It was observed that DES could be used as an alternative to the organic solvents for extracting vitamin D. Furthermore, the study established a green technique for extracting vitamin D from button mushrooms, this source being important for the vegan population who cannot consume most of the commercially available vitamin D supplements that contain vitamin D from animal sources.

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η=

η1ρ2 t 2 ρ1t1

(1)

where t1 and t2 are the time (sec) taken by liquid to flow from the starting point to the end point, η1 is the coefficient of viscosity of water (poise), and ρ1 and ρ2 are the respective densities (kg/m3) of water and sample liquid. Optimizing Extraction of Vitamin-D Using DES. Effect of Cosolvent on the Yield of Vitamin D. Various organic solvents such as ethanol, chloroform, hexane, and methanol have been used for the extraction of vitamin D and its derivatives or precursors.26 Among the organic solvents, only ethanol was miscible with DES (glycerol to choline chloride in a ratio of 3:1) and was thus used as a cosolvent to determine if it exhibited any advantage for improvising the extraction of vitamin D from mushroom powder. Three different proportions of ethanol and DES were investigated. One gram of the mushroom powder was added to a mixture of ethanol in DES (20 mL) containing 10%, 30%, and 50% (v/v) of ethanol and mixed using a magnetic stirrer at 300 rpm for 3 h at 25 °C. The mixture was centrifuged at 8000 rpm for 10 min at 25 °C to remove the debris of mushroom from the extract and the supernatant was used for further analysis. Addition of acetonitrile to DES led to extraction of the product in the acetonitrile layer (organic layer) as the affinity of product is more toward acetonitrile. The formation of two immiscible layers of DES and acetonitrile led to easier separation of solute from DES to the acetonitrile layer (organic layer). This layer was then dried over a rotary evaporator to get the solid content of the extract. This solid content was diluted with acetonitrile (HPLC grade) and filtered using a 0.2 μm syringe filter, for quantifying vitamin D2 by HPLC-UV. Microwave Assisted Extraction (MAE). The extract was subjected to microwave irradiations for different time intervals to increase the yield of vitamin D2. The time period for microwave assisted extraction was varied between 10 and 60 s at the power of 750 W. After microwave extraction, the mixture was centrifuged at 8000 rpm for 10 min at 25 °C, to remove the debris of mushroom from the extract and the supernatant was used for further analysis. Separation of vitamin D from the DES was done as per the above said method. The dried solid content was diluted with acetonitrile (HPLC grade) and filtered using a 0.2 μm syringe filter for quantifying vitamin D2 by HPLC-UV. The experiments were performed in triplicate. Ultrasound Assisted Extraction (UAE). The UAE was carried out using an ultrasonic device (Aczet, Mumbai, India) equipped with a digital timer at the frequency of 65 kHz. The lyophilized powder (1 g) was extracted with 20 mL of DES for different time intervals (10, 20, 30, and 60 min). After ultrasonic extraction, the extracts were centrifuged at 8000 rpm for 10 min at 25 °C to remove the debris and the supernatant was collected. Separation of vitamin D from the DES was done as per the above said method. The dried solid content was diluted with acetonitrile (HPLC grade) and filtered through a 0.2 μm

MATERIALS AND METHODS

Materials. Standard vitamin D2 (ergocalciferol) was procured from Sigma-Aldrich, Bellefonte, PA, USA. Button mushrooms, Agaricus bisporus (White), of commercial grade were purchased from a local market (S. K Agro farm), Mumbai, India. Glycerol AR grade (99.5%), choline chloride extra pure (99%), and absolute ethanol (99.9%) were from S. D. Fine Chemicals Limited, Mumbai, India. Acetonitrile (HPLC grade) and methanol (HPLC grade) were B


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ACS Sustainable Chemistry & Engineering Scheme 1. Photochemical Conversion of Ergosterol (provitamin D2) to Ergocalciferol (vitamin D2)

nylon filter for vitamin D2 quantification by HPLC-UV analysis. Each extraction was performed in triplicate. Effect of Volume of Extracting Solvent. Different volumes of DES were employed to determine the optimum volume facilitating maximum extraction. One gram of the lyophilized powder was added in 10 mL, 20 mL, and 30 mL of DES and ultrasonicated for 30 min, at the frequency of 65 kHz. The reaction was conducted in a glass beaker. After ultrasound assisted extraction, the mixture was centrifuged at 8000 rpm for 10 min, at 25 °C, to remove the debris of mushroom from extract and the supernatant was used for further analysis. Separation of vitamin D from the DES was done as per the above said method. The dried solid content was diluted with acetonitrile (HPLC grade) and filtered using a 0.2 μm syringe filter, for quantifying vitamin D2 by HPLC-UV. Photochemical Reaction: Exposure of Provitamin D to UV Light. Provitamin D2 (ergosterol), a precursor of vitamin D2, gets converted to vitamin D2 (ergocalciferol) when exposed to UV light by photochemical reaction27 as indicated in Scheme 1. Vitamin D extract obtained according to the optimized conditions was exposed to UV−C lamp, covering the spectrum of 100−280 nm and with the intensity 0.25 mW/cm2, for different time periods, viz. 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, and 48 h as shown in Scheme 2, during which the sample was kept at a distance of 10 cm from the lamp. The UV source showed a stable intensity and spectral distribution over the entire period. The exposed extract was filtered using a 0.2 μm syringe filter for vitamin D2 quantification by HPLC-UV analysis. Purification Methods To Isolate Ergocalciferol from Extract. Purification studies were conducted after scaling up the extraction process up to 5 g of the mushroom powder in 100 mL of DES. The extraction was carried out according to the optimized extraction parameters of the ultrasound assisted extraction (UAE). Treatment with Water. 25 mL of the acetonitrile extract was mixed with 25 mL of water and stirred using a magnetic stirrer at 200 rpm for 10 min at 25 °C. 25 mL of n-hexane was added in the resulting aqueous layer and again stirred at 200 rpm for 5 min. The resulting layer was separated using a separating funnel (250 mL capacity). The organic layer (n-hexane) thus obtained was washed thrice with water and then treated with 1 g of anhydrous calcium carbonate to remove the excess of water. The solution was further filtered using Whatmann filter paper (11 μm pore size) to remove the salt. Further, the filtrate was dried by evaporating n-hexane under reduced pressure on a rotary evaporator (Medica instrument Mfg. Co., Mumbai, India). The analyte was diluted with 5 mL of acetonitrile and was filtered into an amber colored HPLC vial, using a 0.2 μm syringe filter for quantifying vitamin D2 by HPLC-UV. Treatment with Hot Water. 25 mL of hot water was dropwise added along the walls of the beaker containing 25 mL of acetonitrile extract. The resulting mixture was stirred on a magnetic stirrer at 300 rpm until the solution attained room temperature (25 °C). 25 mL of n-hexane was added to the resulting aqueous layer, and the mixture was stirred at 200 rpm for 5 min. The resulting layer was separated using a separating funnel (250 mL). The organic layer (n-hexane) thus obtained was washed thrice with water and then treated with 1 g

Scheme 2. Flowchart of Experiments for Vitamin D2 Extraction

of anhydrous calcium carbonate to remove the excess water. The solution was further filtered using Whatman filter paper (11 μm pore size) to remove the salt. Further, the filtrate was dried by evaporating n-hexane under reduced pressure on a rotary evaporator. The analyte was diluted with 5 mL of acetonitrile and was filtered into an amber C


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ACS Sustainable Chemistry & Engineering colored HPLC vial using a 0.2 μm syringe filter for quantifying vitamin D2 by HPLC-UV. Adsorption of Impurities Using Activated Charcoal. The extracted acetonitrile layer was treated with hot water as mentioned for treatment in hot water. After separating the organic layer, 10 mg of activated charcoal was added and the mixture was stirred for 30 min. The charcoal was removed by filtering through a Whatmann filter paper (11 μm pore size). Further, the filtrate was dried by evaporating n-hexane under reduced pressure on a rotary evaporator. The analyte was diluted with 5 mL of acetonitrile and was filtered using a 0.2 μm syringe filter for quantifying vitamin D2 by HPLC-UV. Adsorption of Impurities Using Bentonite. The extract was first treated with water to remove the water-soluble impurities, as mentioned for treatment in hot water. It was further stirred with 10 mg and 100 mg of bentonite for 1 h at 200 rpm on a magnetic stirrer. The bentonite was removed by filtering with a Whatmann filter paper (11 μm pore size). Further, the filtrate was dried by evaporating nhexane under reduced pressure at 40°C on a rotary evaporator. The analyte was diluted with 5 mL of acetonitrile and was filtered using a 0.2 μm syringe filter for quantifying vitamin D2 by HPLC-UV. Saponification. The extract was treated with water to remove all the water-soluble impurities, as mentioned for treatment in hot water. 25 mL of n-hexane was added into the resulting aqueous layer and again stirred at 200 rpm for 5 min. The resulting layers were separated using a separating funnel (250 mL). The organic layer (n-hexane) thus obtained was washed thrice with water and then treated with 1 g of anhydrous calcium carbonate to remove the excess water. Thereafter, the hexane layer was mixed with 10 mL of 50% KOH in acetonitrile and refluxed at 50 °C for 1 h to facilitate saponification.28 The analyte was then filtered using a 0.2 μm syringe filter for quantifying vitamin D2 by HPLC-UV. HPLC Analysis of Vitamin D. HPLC analysis was performed on an Agilent 1290 LC apparatus (California, USA) consisting of an Agilent Quaternary pump equipped with a diode array detector (DAD), an automatic injector, an automatic column temperature control oven, and ChemStation Open Laboratories Data System Software (C.01.07 SR2 [255]). Separation was performed on a reversed-phase C18 column (264 × 4.6 mm), at the dual wavelength channel of 265 nm. The mobile phase, consisting of 95% acetonitrile and 5% methanol, was pumped at the flow rate of 1.5 mL/min at 35 °C for a run time of 10 min. The product obtained was diluted with 5 mL of acetonitrile. The sample was filtered in an amber colored HPLC vial using a 0.2 μm syringe filter and analyzed using HPLC. Standard Vitamin D2 (ergocalciferol) was used for plotting the standard calibration curve. Identification of Ergosterol using Mass Spectroscopy. Mass spectrometry was performed on an Agilent 1260 Infinity series (California, USA) apparatus equipped with Agilent quadrupole mass spectrometer using both positive and negative ionization modes. The analysis was done using a direct injection of the product. Statistical Analysis. Statistical analysis of the results was performed using GraphPad Prism 5.03. One-way or two-way ANOVA was utilized as per the data sets available, and this was used to understand the variance row-wise and column-wise. Further, Bonferroni post-test was used to study the significance level in the different experiments. A P value of >0.05 was considered statistically significant.

Table 1. Characterization and Extraction of DES HBA to HBD ratio

Density (g/mL)

Viscosity (cP) at 25 °C

1:1

1.1812

299

1:3

1.36

302

1:5

1.452

312

Appearance Crystal formation Transparent clear liquid Transparent clear liquid

Extraction efficiency (μg/g DW)

4.25 ± 0.74 1.75 ± 0.98

Although the viscosity of the solvent prepared at 1:1 molar ratio of reactants was the lowest, the solvent exhibited formation of crystals. The extraction efficiency of the DES prepared at the molar ratio of 1:3 was found to be considerably greater as compared to that of the DES prepared using the molar ratio of 1:5, as shown in Table 1. Hence, the DES prepared using the molar ratio of 1:3 was utilized for further investigations. Further, since the viscosity of glycerol at room temperature (25°C) is approximately 1011 cP, which is very high as compared to that of DES, the extraction efficiency was found to be very low.30 Optimization of Extraction Parameters. Effect of DES to Ethanol Ratio on Vitamin D Concentration. Various organic solvents such as ethanol, hexane, chloroform, and methanol were analyzed for their miscibility with DES. Among all the solvents, only ethanol was found to be completely miscible and thus was used to determine whether inclusion of a cosolvent had any synergistic effect on extraction of vitamin D from mushroom powder. The sample was stirred at 300 rpm with different ratios of ethanol viz. 10%, 30%, and 50% v/v, respectively. As the volume of ethanol was increased, a decrease in the yield of vitamin D2 was observed, as shown in Figure 1.

Figure 1. Optimization of solvent (p > 0.05).

High ethanol percentage in DES increases the polarity of the solvent and decreases the interaction between the solvent and solute. Also, the salt content in DES may be responsible for effective cell breakage, as the salt concentration governs the osmotic pressure at the cell wall, which helps in cell lysis and thus increasing the yield of vitamin D.31 So as the ethanol concentration increased, the salt content in the mixture may have decreased, thus leading to low extraction of vitamin D2. Microwave Assisted Extraction of Vitamin D from Mushrooms. The sample was submitted to microwave assisted extraction at 750 W power, for a duration of 1 min. Further increase in the extraction time led to degradation of the solvent. The degradation of the solvent may have occurred due

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RESULTS AND DISCUSSION Preparation of DES and Characterization. DES was prepared using different ratios of HBA (choline chloride) and HBD (glycerol). DESs obtained at all the ratios appeared transparent. Crystal formation was observed in the DES prepared at 1:1 molar ratio, after a period of 1 week. The physical properties of the synthesized DESs were studied and have been stated in Table 1. It is a known fact that the lower the viscosity of the extracting solvent, the more efficient is the extraction.29 D


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ACS Sustainable Chemistry & Engineering to the presence of glycerol in the DES, which imparted a higher viscosity to the solvent. As per the hole theory applicable for ionic liquids, conductivity and viscosity of these liquids is controlled by ion mobility and the availability of voids of suitable dimensions. Since glycerol is a viscous solvent, it was hypothesized to impart a restricted mobility and hence a decreased conductivity to the DES used in this study. The use of microwave energy was thus anticipated to form trimethylamine radicle ion as a degradation product and thus may have accelerated a chain reaction for further degradation of DES.32−34 The maximum yield obtained after microwave assisted extraction was found to be 3.14 ± 0.378 μg/g of dry weight of mushroom powder (DW). The content of vitamin D 2 extracted after different time periods of microwave assisted extraction has been represented in Figure 2.

Figure 3. Ultrasound assisted extraction (p > 0.05).

Figure 4. Optimization of solvent volume (p > 0.05).

the solvent volume above 20 mL did not significantly affect (p > 0.05) the extraction efficiency. Therefore, the solvent volume of 20 mL was considered to be optimum for extracting vitamin D2 from 1 g of powdered mushroom, with the yield with this volume being 3.63 ± 1.65 μg/g DW. Effect of UV Exposure on Vitamin D Content. The powdered A. bisporus (button mushroom) strain employed in this investigation contained ergosterol but a much smaller amount of ergocalciferol (vitamin D2). Irradiation of mushroom powder within a medium has been reported to be more effective than dry irradiation or irradiation of the direct fruiting body.37 Thus, the irradiation of the ergosterol-enriched extract was carried out using acetonitrile as the solvent. Acetonitrile has an absorption wavelength cut off of 190 nm, along with a high dielectric constant of 37.5, which enables it to absorb high energy UV−C wavelength as compared with other solvents.38 Also, the use of organic solvent was anticipated to enhance the yield of vitamin D due to the high solubility of sterol and vitamin D/its derivatives in the organic solvent. The use of an organic solvent was more effective than the solvent-free reaction or employing water to induce vitamin D2 biosynthesis. Another reason to employ the organic solvent was to solubilize the oily fractions obtained after extraction which would be difficult to remove using aqueous medium.37 The extract was dissolved in acetonitrile and exposed to UV−C lamp (0.25 mW/cm2). A significant increase (p < 0.05) in vitamin D was observed with increase in exposure time from 2 to 48 h. The content of Vitamin D2 was increased by 4.25-fold, within 2 h, from 11.75 ± 2.15 to 49.99 ± 1.53 μg/g DW and by 31-fold over the next 48 h, from 11.75 ± 2.15 to 364.2 ± 2.60 μg/g DW (Table 2). Thus, a significant increase in vitamin D content and a simultaneous decrease in ergosterol content was observed when the extract was exposed to UV light, as shown in Figure 5. After exposing the extract for 48 h, formation of an over-radiated product was observed as seen in Figure 5A. Thus, irradiation was terminated at 48 h to avoid formation of overirradiated products. When the mushroom powder was directly exposed to UV− C irradiation, a 16% decrease in the ergosterol level and a

Figure 2. Microwave assisted extraction of vitamin D2, after exposure to microwave irradiations for different time intervals (p < 0.05).

No significant difference (p < 0.05) was observed between the amounts extracted at different time intervals. Thus, microwave assisted extraction was found to be unsuitable for the extraction of vitamin D using DES. Ultrasound Assisted Extraction of Vitamin D from Mushrooms. The increase in the extraction yield of vitamin D2 using ultrasound waves may be partially attributed to the cavitation phenomena but also to the molecular breakdown effects. Ultrasonic power is greatly responsible for the molecular breakdown during extraction of target compounds.26 A prolonged exposure (60 min) of mushroom powder mixed in the solvent to ultrasound did not show decomposition phases. Therefore, exposure duration of 10 to 60 min was selected. Also, exposure beyond 30 min did not result in significant improvement in extraction yields (p > 0.05). Thus, for ultrasound assisted extraction, the time duration of 30 min was considered to be optimum for the maximum extraction of vitamin D2. The yield of vitamin D2 obtained at this time duration was 12 ± 3.04 μg/g of DW, as shown in Figure 3. Effect of Solvent Volume on Vitamin D Extraction. Generally, a larger volume of solvent can dissolve constituents more effectively, leading to an enhancement in the extraction efficiency. Furthermore, an increase in the solvent volume increases the diffusivity, enhancing the mass transfer and improving the extraction efficiency.35 However, too high solvent volumes can lead to wastage, thus decreasing the economic feasibility.36 On the other hand, insufficient amount of solvent can result in a lower extraction yield. Therefore, the choice of a proper solvent volume is a significant parameter for maximizing the extraction efficiency. The extracted amount of vitamin D increased with increase in solvent volume, as shown in Figure 4. However, increase in E


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DES is composed of a hydrogen bond acceptor and a hydrogen bond donor, the extract was suspected to contain residues of the solvent and various other polar as well as nonpolar substances.28 Various purification strategies such as treatment with water/hot water, treatment with adsorbent, and saponification of the extract were performed to remove the impurities present in the extract. The purification was carried out after extracting vitamin D from DES into an acetonitrile layer, with vitamin D having a higher affinity toward the latter. Thus, water could be used during the subsequent purification steps without affecting the hydrogen bonding network of DES. Water used in the purification would remove the extra watersoluble impurities present in the acetonitrile extract. The retention times of water-soluble impurities, fat soluble impurities, and vitamin D2 were found to be 2 min, 3−6 min, and 7.9 min, respectively, as represented in Figure 6.

Table 2. Vitamin D2 Content upon Exposure to UV Radiation UV exposure time

Vitamin D2 content (μg/g DW)

0h 2h 4h 6h 8h 12 h 24 h 48 h

11.75 ± 2.15 49.99 ± 1.53 93.23 ± 1.36 118.08 ± 3.22 142.60 ± 0.3 235.86 ± 1.52 359.5 ± 1.57 364.2 ± 2.60

Figure 6. Waterfall view of HPLC chromatogram of various purification methods. Figure 5. (A) HPLC chromatogram showing increase in vitamin D2 content with increase in UV exposure time. (B) Graphical representation of decrease in ergosterol content with increase in vitamin D content after UV exposure (p < 0.05).

Purification efficiencies of different methods were analyzed based on the percentage area composition. In hot water treatment, which effectively eliminated the water-soluble impurities at retention peak time of 2 min, the percentage area composition was found to be 75.46%. Further, saponification was used to remove fat soluble impurities, where the maximum separation efficiency corresponded to the 95.37% area composition. Moreover, on the commercial scale, since adsorption is a widely used technique, in the present study, adsorbents such as charcoal and bentonite were used for purification which accounted for 23.97% and 76.72% area of the product, respectively. But complete purification was not obtained. Thus, in this investigation, the saponification was regarded as the most effective technique, justifying the commonly used method for removal of impurities from fat soluble vitamins, as it removes the bulk of fat and ester impurities, facilitating the purification of vitamin D2.28,41 The purity of the product was confirmed by 13C NMR, which depicted that the impurities present in the NMR of crude extract were absent in that of the purified sample (Figure 7). Characteristic peaks in the 13C NMR spectrum of the purified vitamin D2 and the crude extract matched with those of the standard vitamin D2.42 Identification of Ergosterol (vitamin D precursor) Using Mass Spectroscopy. ESI is commonly used for the

corresponding increase in vitamin D2 was observed. In an earlier study, when the powder was suspended in methanol and irradiated, a 27% decrease in ergosterol level was observed.37 However, in this investigation, when acetonitrile was used as a solvent for the extract, approximately 59.2% decrease in ergosterol level was observed after 24 h of UV irradiation. Thus, the UV irradiation time for maximum conversion of ergosterol to vitamin D2 was found to depend on the concentration of ergosterol in the extract and the solvent used to dissolve the extract. Liquid−liquid extraction of vitamin D2 from shiitake and button mushrooms after UV−B treatment yielded 106.4 ± 14.7 and 36 μg/g DW.39 Button mushrooms when exposed to sunlight yielded 3.9 ± 0.8 μg/g DW of vitamin D2.40 Comparing these results with the current study, the amount of vitamin D2 increased by 3-fold after 24 h of UV−C exposure. Purification Methods To Isolate Ergocalciferol from the Extract. Purification was conducted to remove the impurities present in the extract due to the use of DES. As F


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Figure 7. 13C NMR spectrum of (A) purified vitamin D2 and (B) crude extract.

Figure 8. Mass spectra of (A) vitamin D2 standard (B) ergosterol.

analysis of several vitamins. Traditionally [M−H2O+H]+ ions are used for monitoring and quantification of all vitamin Drelated compounds. During the fragmentation of vitamin D and vitamin D related compounds, there was a loss of one water molecule, which was in compliance with literature reports.41 As the molecular weight of both the compounds (vitamin D2 and its precursor) is the same, the mass spectra remained the

same (Figure 8). The mass spectra depicted that no major impurities were present in the extract and also confirmed the identity of the product.

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CONCLUSION Deep eutectic solvent (DES), a green solvent, was successfully used to extract vitamin D from button mushrooms. Ultrasonic extraction, coupled with DES, further enhanced the extraction G


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efficiency. Purification of vitamin D2 from the extract was effectively carried out using hot water treatment and saponification. Exposure to UV led to an increase in vitamin D2 content by 31-fold, over a period of 48 h. The amount of vitamin D2 observed after 48 h of UV−C exposure was found to be 364.2 ± 2.60 μg/g DW. This is almost thrice the maximum amount reported by liquid−liquid extraction, in the literature. UV irradiated mushrooms thus represent an excellent source of dietary vitamin D. This is especially important because only a few food sources, such as salmon, herring, and egg yolk, are naturally rich in vitamin D, which cannot be used by vegans. Thus, mushrooms with UV irradiation can serve as a potential source of vitamin D for the vegan population.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail address: [email protected] Phone: +91-223361-2221 Fax: +91-22-3361-1020 (Dr. Prajakta Dandekar). *E-mail address: [email protected]. Phone: +91-223361-2029 Fax: +91-22-3361-1020 (Dr. Ratnesh Jain). ORCID

Prajakta Dandekar: 0000-0001-5968-1072 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are thankful for a Ramanujan fellowship research grant (SR/S2/RJN-139/2011) and for a Ramalingaswami fellowship research grant (BT/RLF/Re-entry/51/2011) for financial support. The authors would like to thank Vijay D. Yadav for his technical advice and help during the project.

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ABBREVIATIONS DESs, Deep eutectic solvents; WBM, White button mushroom; THF, Tertahydrofuran; SFE, Supercritical fluid extraction; MAE, Microwave assisted extraction; UAE, Ultrasound assisted extraction; SPE, Solid phase extraction; ILs, Ionic liquids; scCO2, Supercritical carbon dioxide; ChCl, Choline chloride; HBA, Hydrogen bond acceptor; HBD, Hydrogen bond donor; DAD, Diode array detector; DW, Dry weight of mushroom powder obtained after lyophilization; ESI, Electro spray ionization; EC50, Half maximal effective concentration

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REFERENCES

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(Agaricus bisporus) using Deep Eutectic Solvent and Ultrasonication

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