Mihai-Vlad Vălu¹, Marian Petre¹, Liliana Cristina Soare¹, Lucian Hrițcu2, Răzvan Ștefan Boiangiu², Simone Carradori3 [310137]

[anonimizat] A [anonimizat]¹, Marian Petre¹, Liliana Cristina Soare¹, Lucian Hrițcu2, Răzvan Ștefan Boiangiu², Simone Carradori3*

¹[anonimizat], [anonimizat]., 110040 Pitesti, Romania

2[anonimizat], Bd. Carol I, No. 11, Iasi 700506 Romania

3Università degli Studi G. d'Annunzio Chieti e Pescara, [anonimizat], Italy

*corresponding author: [anonimizat]; [anonimizat] “G. d’Annunzio” [anonimizat], Via dei Vestini 31, 66100 Chieti, Italy

Abstract

Hericium erinaceus (HE) is a medicinal fungus that produces the active biological metabolite erinacine A with a strong antioxidant activity. Classical extraction techniques used to date to obtain metabolites from this fungus require a [anonimizat], especially on a biomedical scale. [anonimizat] (UAE) for the identification and extraction of biological compounds with high antioxidant activity from the mycelium of HE that was developed through a solid cultivation process in the bioreactor. [anonimizat] (RSM). [anonimizat], flavonoids and the diterpenoid erinacine A. It is highlighted that there is a very good correlation between the concentration of polyphenols and flavonoids in the extracts studied with the diterpenoid erinacin A. Also, [anonimizat], since toxic solvents have not been used in the developed extraction procedure. The HE fungus can be used both as a food source and as a possible phytotherapeutic medicine in the prevention or treatment of various neurodegenerative disorders that require drugs with a strong antioxidant activity.

Keywords: HPLC, ultrasound-[anonimizat], [anonimizat] A, bioreactor.

[anonimizat], due to their consistency [1]. [anonimizat] / [anonimizat]: Parkinson's disease, Alzheimer's disease, cancer, etc. [2,3]. The investigation of fungi and extracts obtained from mushrooms to highlight their content of antioxidant compounds and other metabolites has received special attention recently [4,5]. [anonimizat]'s [anonimizat] a progressive neurodegenerative disorder whose etiopathogenesis involves the participation of numerous risk factors [6]. [anonimizat]. Considerable efforts have been made by researchers to find the most effective extraction methods with better efficiency and high bioactivity [7,8]. It should be noted that there is still an attempt to discover an extraction method that is suitable for mushrooms, applying different parameters for high efficiency [7]. Many published studies state that ultrasonic assisted extraction can improve the extraction rate of active biological heat-sensitive ingredients under low temperature processing conditions, which is more effective than conventional extraction techniques [9]. The mechanical effect of ultrasound causes the penetration of solvents into tissue cells, improved mass transfer and tearing of the cell wall which is favorable to the release of tissue cell content, which can reduce the processing time and amount of solvents compared to traditional extraction methods [10]. Hericium (hedgehogs in Latin) is a genus of edible mushrooms of Hericiaceae family. The genus Hericium produces phytochemical substances, erinacin and hericenone, with antioxidant activity, but also various polysaccharides [11]. Erinacine A (Figure 1) was isolated from the fungus Hericium erinaceus, its chemical structure being deposited in PubChem with the indication: 9867477.

Figure 1. Chemical structure of Erinacine A. Chemical Formula: C25H36O6 (432.5 g/mol.)

Mainly, the antioxidant activity of the species HE is due to the presence of polysaccharides (β-glucans), diterpenoids (hericenons, erinacines), but a number of authors have stated that there is a close link between the antioxidant activity of the HE fungus and the presence of flavonoids and polyphenols [12,13]. In this research, the HE fungus was developed on a solid culture medium to obtain the diterpenoid erinacine A and the antioxidant activity was investigated. It has been noted that the biosynthesis of erinacin A in submerged culture can lower production costs, because the chemical synthesis of ciatin diterpenoids is a long, multi-stage process and provides poor yield. This is the first report on the determination of erinacine A, as well as on the optimization of the ultrasound extraction conditions of metabolites with antioxidant activity. The aim of this work was to assess the possibility to enhance the phenolic flavonoid and erinacine A content and antioxidant activity of HE under ultrasound assisted extraction. HE contains unsaturated fatty acids, proteins, carbohydrates and a variety of trace elements [14]. These nutrients have many important roles on the body. [14]. Most scientific studies on the antioxidant properties of HE described classical extractions techniques, although in the literature there are different modern methods of extraction. However, few previous studies have investigated them. Therefore, the objective of this study was to analyze the antioxidant properties of HE extracts obtained by ultrasonic-assisted extraction. At the same time, given that studies are contradictory about the presence of flavonoids and polyphenols with antioxidant activity in mushroom species [15,16], this has aroused our interest to investigate by various physico-chemical methods the possible presence of these biocompounds. The methodology comprehended the development of the HE fungus in a controlled medium bioreactor and subsequently its submission to the ultrasonic extraction technique can enrich the isolation of antioxidants in Hericium erinaceus, in particular polyphenols and flavonoids correlated with the diterpenoid Erinacine A, known for its high antioxidant activity. In addition, the amount of components with antioxidant effect was determined. It has been shown that the antioxidant effects of several mushrooms are also related to their total phenolic and flavonoid content [17]. Further, it is expected that the antioxidant activity is due to the high yield of extracted erinacine A as identified by HPLC. The extraction variables (extraction time, ethanol concentration and solvent/material ratio) were also investigated to maximize the extraction yield in terms of the amount of metabolites and to establish optimal extraction conditions. The response surface method (RSM) is a statistical method used to optimize processing parameters. Therefore, RSM is used to speed up and optimize the operating process to reduce time, energy and raw materials. The response surface methodology was used to optimize ultrasound-assisted extraction conditions for obtaining the maximum yield and content of antioxidants compounds of HE. The most positive thing about RSM is that it can reduce the number of experimental batches and highlight the relationship between response and variables.

Materials and methods

Chemicals

The chemicals and reagents used were purchased from Sigma-Aldrich (Steinheim, Germany). All other substances were of analytical quality.

Solid state cultivation of Hericium erinaceus biomass

The HE mushroom was developed in the laboratory (USAMV Bucharest) for 21 days after an adapted protocol [18]. In a 9 l bioreactor, the culture medium was formed by casein peptone 12.16 g/L, glucose 66.88 g/L, NaCl 1.46 g/L, and KH2PO4 1.0 g/L with a pH of 4.5. Moreover, to enrich the yield of formation of the compounds of interest, namely erinacine A, flavonoids and polyphenols, a content of 0.5% yeast extract, 4% glucose, 0.5% soy powder and 1% oats was added to the culture medium. In view of the optimized protocol we can suggest that a carbon-nitrogen ratio (C/N) of 6 and a pH value of 4 to 5 in the culture medium may be important parameters in promoting the biosynthesis of erinacine A in he mycelium. Finally, a microscopic analysis (Olympus BX 43) was performed to confirm the development of HE in the bioreactor (Olympus BX 43) to observe the presence of mycelial hyphae (Figure 2).

Figure 2. Mycelial hyphae present in Hericium erinaceus observed under the Olympus BX43 fluorescence microscope. Scale bar: 20 µm. Magnification: 40x.

Ultrasound-assisted extraction procedure

The technology for obtaining and concentrating bioactive products of the species HE has been developed on the basis of the principles of sonochemistry, with a modern extraction process based on radiative sources in inert natural environments, such as ultrasonic radiation, using an ultrasonic processor (Hielscher UIP1000hdT), with a probe with a frequency of 20 kHz and a power of 80W (surface intensity of the peak of 26.5 W/cm2), followed by sample freezing to achieve a powder. After completion of the extraction, the samples were filtered through a sintered glass filter of porosity. Lastly, the samples were centrifuged (2.500 x g for 5 min, room temperature), and from the supernatant obtained was aimed at the elimination of water and alcohol by means of the rotary evaporator (Heidolph Hei-VAP Core).

Lyophilization of samples

The last stage consisted of the freeze-making of samples (Christ Alpha 1-2 Ldplus) for obtaining a powder (permanently prepared, shredded as particles) of the fungal material. Lyophilization was aimed at obtaining high-quality extracts to improve extraction efficiency, with a higher content in active principles due to freeze-driedness [19]. The working procedure was optimized and consisted of subjecting the liquid samples that were placed in the Petri dishes (7mm) and freezing the samples at the vaporization temperature of -70 °C for 2 hours and freeze-off at -550 °C under vacuum for 48 hours. The vacuuming of the enclosure was achieved at a constant pressure of 400 μBar. Finally, the substance was restored to room temperature, resulting in a powder extract. An important aspect of freeze-making is that it limits oxidative changes in metabolites since the oxygen concentration is very low under vacuum. Important to note is also the fact that not all extracts will produce a dry product in powder form [19]. Some extracts, in particular non-polar extracts, might lead to an oily product, so consideration should be given to optimize the extraction method. In our experiments, we can state that our procedure led to a powdered fungal material with the maintenance and preservation of the analyzed biocompounds. It is known that the lyophilization technique maintains the highest amount of antioxidant compounds compared to other methods of drying extracts [20].

Optimization of the extraction procedure using RSM model

Response surface methodology (RSM) was used for investigating the influence of three independent variables on total phenolics and yield of HE. The main factors are affecting the extraction efficiency including the ethanol concentration (%, X1), extraction time (min, X2) and the solvent-to-material ratio (mL/g, X3). Temperature was not taken into account in this paper, as the extraction time began once the desired temperature was reached, although there would inevitably be extraction before the nominal temperature was reached. The advantage of RSM is the reduced number of experimental trials needed to evaluate multiple parameters and their interactions. In the study, the experiments were performed on the Box-Behnken Design (BBD). Three variables were used to optimize the best combination of extraction variables for HE yield and total phenolic content. The complete design was carried out in random order and consisted of 17 experiments including five replicates at central point (Table 1). The data from BBD were analyzed by multiple regressions to fit the following quadratic polynomial model Equation 1:

where Y is the predicted response, β0 is a constant bi, bii and bij are the linear, quadratic and interactive coefficients of the model, respectively. Accordingly, Xi and Xj represent the levels of the independent variables, respectively. The quality of the fitted model was expressed by the coefficient of determination (R). A central composite design (CCD) was adopted to investigate the effects of three independent variables.

Proximate composition

The samples were analysed for moisture, proteins, fat, carbohydrates and ash, using the AOAC procedures with minor modifications [21]. Total carbohydrate content was calculated by subtracting the contents of moisture, ash, fat, and protein from 100 and expressed as a percentage of dry mass. Moisture was removed by oven dehydration at 105 °C for 72 h from sample (1 g). Ash was determined by weighing the incinerated residue obtained at 550 °C after 10 h. The dietary fiber content was calculated by combining enzymatic and gravimetric methods. The crude protein content (N × 4.38) of the samples was estimated by the macro-Kjeldahl method; the crude fat was determined by extracting a known weight of powdered sample with petroleum ether, using a Soxhlet apparatus; the ash content was determined by incineration at 600 ± 15 °C.

Macro and microelements

The mineral content (ash) and the analysis of the mineral elements were made on dry samples with the AOAC method no. 930.05. 400 mg of each sample was subjected to dry gray mineralization at 460 ° C. The incineration residue was extracted with 0.5 ml / ml HCl and 0.5 ml / ml HNO3 and made up to an appropriate volume with distilled water to which Fe, Cu, Mn and Zn were weighed directly [22,23]. Standard solutions were used to compare absorption responses with standard analytical solutions of purity >99.9%.

Reducing power

The reducing power of ultrasound extract was measured according to the method of [24]. K4Fe(CN)6 was generated after the antioxidants reacted with K3Fe(CN)6. Then, K4Fe(CN)6 reacted with FeCl3 to produce Perl's Prussian blue (Fe4[Fe(CN)6]3) which had the maximum absorbance at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing power. The evaluation of the potency reduction was performed by mixing each extract in 2 ml of ethanol with 2 ml of 200 mM sodium phosphate buffer (pH 6.5) and 2.5 ml of 1% potassium ferricyanide: the mixture was incubated at 50 ° C for 20 min. Finally, 2.5 ml of 10% trichloroacetic acid was added and the mixture was centrifuged at 3000 g for 10 min. The absorbance was measured at 700 nm (Ocean Optics HR2000+ Spectrometer). The extract concentration providing 0.5 of absorbance (IC50) was calculated from the graph of absorbance at 700 nm plotted against the extract concentration. Ascorbic acid was used as positive controls.

Determination of total phenol and flavonoid contents of HE extract

The flavonoid content was measured according to the method described by Delcour and Varebeke (1985) [25], and quercetin was used for the analytical curve. The results were expressed as mg of quercetin equivalent per gram of dry matter of mushrooms extract (mg QE/g DM). To 1 mL of appropriately diluted extracts, 5 mL of chromogen reagent (0.1% cinnamaldehyde solution in a cooled mixture of 75 mL methanol and 25 mL concentrated HCl) were added. After an incubation period of 10 min, the absorbance was recorded at 640 nm. The total phenolic contents of the extracts were determined using Folin-Ciocalteu reagent, in accordance with the method of [26] Velioglu, Mazza, Gao, and Oomah (1998), where gallic acid (mg GAE/g DM) was used as a standard antioxidant. Absorbance was measured at 750 nm with Ocean Optics HR2000+ Spectrometer.

DPPH-Radical scavenging (antioxidant) activity

The free radical scavenging rate was evaluated by measuring the 2,2-diphenyl-1-picrylhydrazyl (DPPH) with L-ascorbic acid as reference standard. The DPPH assay used a modified procedure from a previously described study [27]. The lyophilized powder extract from Hericium erinaceus was dissolved in methanol and mixed with 260 μL of a 0.2 mM DPPH radical solution (Sigma-Aldrich, USA). After 40 min at room temperature, the absorbance of the resulting solutions and a blank were recorded against 0.1 mg/mL butylated hydroxyanisole (BHA) and L-ascorbic acid (Vitamin C; Sigma-Aldrich, USA) as positive controls. The mixture was shaken vigorously and left to stand for 30 min, and the absorbance was measured at 517 nm. The percentage inhibition was calculated according to the formula: [(A0-A1)/A0] × 100, where A0 was the absorbance of the control and A1 was the absorbance of the sample.

Determination of AChE and BChE inhibitory activities

Acetylcholinesterase and BChE inhibitory activities were measured by slightly modifying the spectrophotometric method developed [28] by (Ellman et al., 1961). Electric eel AChE (Type-VI-S, EC 3.1.1.7, Sigma) and horse serum BchE (Sigma Aldrich/MERK) were used, while acetylthiocholine iodide and butyrylthiocholine chloride (Sigma Aldrich/MERK) were employed as substrates of the reaction. Briefly, in this method, 140 mL of 0.1 mM sodium phosphate buffer (pH 8.0), 20 mL of DTNB, 20 mL of test solution and 20 mL of AChE/BChE solution were added by multichannel automatic pipette. Galantamine (Sigma Aldrich/MERK), was used as the reference drug.

HPLC/DAD-UV analysis

HPLC-DAD (high-performance liquid chromatography with diode-array detector) investigation was done utilizing a High-Performance Liquid Chromatography Systems L-3000 (RIGOL TECHNOLOGIES, INC Beijing, China). HPLC analysis of erinacine A was executed according to the previous study with minor modifications [29]. Identification and quantification analyses were performed by comparison with standard spectra at each retention time. By using the analytical Kinetex EVO C18, the retention time of erinacine A was approximately ~1.9 mins at a flow rate of 1.0 mL/min at 340 nm. Erinacine A was used as standard [30,31]. Stock solution (1 mg/mL) of erinacine A was prepared in 70% ethanol. Standard solutions with the final concentration range of 1–25 µg/mL for erinacine A were obtained. Linear least-square regression analysis for the calibration curves showed correlation coefficients of 0.9968 for erinacine A with respect to the peak area, demonstrating a good linear relationship in the different ranges tested. The yield rate of erinacine A in the H. erinaceus with ethanol extraction is ~4 mg/kg, which was confirmed and quantified by HPLC [29] as shown in Figure 6.

Statistical analysis

The experimental results of the RSM were analyzed using Matlab2018b software. p-values <0.05 were considered to be statistically significant. All experiments were conducted in triplicate unless otherwise noted in the text. The data were analyzed using GraphPad Prism 7 software, and the HPLC data were analyzed with OriginPro software. Analysis of variance (ANOVA) and Duncan’s multiple range test (DMRT) was used to analyze the differences between scavenging activity with a least significance difference (LSD) of p<0.05 as the level of significance.

Results and discussion

Effect of different ultrasonic parameters on the yield of HE

The parameters tracked during ultrasonic extraction by sonochemistry were approximately (extraction time being different) similar for each sample performed and were constantly monitored from the operating system of the device (Figure 3), as follows: 80% sonotrode amplitude, with 27 W per hour, with a net extraction power of 170 W and maintaining a constant temperature in the ultrasound bath.

Figure 3. Processing parameters. Level of extraction parameters to ultrasonication of biological samples of HE (Hielscher UIP1000hdT).

Figure 3 shows the parameters followed during the ultrasonic extraction experiment and a constant maintenance in terms of pressure (bar), temperature (℃), amplitude (%), extraction power (W). It is to be specified that temperature and time have been controlled in order not to degrade biological compounds in the HE fungus. The heat generated during sonication did not have a significant effect on extraction. However, the temperature of the ultrasonic bath on the entire sonication period has been kept as constant as possible by the addition of ice sheets or by taking breaks in the experiments. In general, ultrasound increases the speed of a chemical reaction, reaction conditions are milder, and the energy consumed decreases significant. We used ultrasound to improve the efficiency of extraction.

To obtain a more exhaustive procedure, it was necessary to investigate the process variables. Preliminary trials enabled the range of ethanol concentrations (40-80%), extraction time (20-45 min) and the solvent-to-material ratio (10-30 mL/g) to be fixed. It can be seen in Tables 1-2 that extraction yield was affected most significantly by ethanol (X1) (p<0.05) and solvent-to-material ratio (X3) followed by extraction time (X2) (p<0.05). It was evident that the quadratic parameter (X12) was significant at the level of p<0.05, whereas two quadratic parameters (X12) and interaction quadratic parameters were non-significant (X1X2, X2X3, X1X3) (p>0.05).

Table 1. BBD experimental design with the independent variables.

The values are given as mean ± standard deviation of triplicate determinations. Different letters within the same row mean statistically different at p< 0.05. Total polyphenols were expressed in milligrams of gallic acid equivalents, and total flavonoids as quercetin equivalents. DPPH: 2,2-diphenyl-1-picrylhydrazyl.

Table 2. Estimated regression model of relationship between response variables (yield, TPC, TFC, and DPPH of HE) and independent variables (X1, X2, X3).

*p>0.001; **p>0.0001; ***p<0.0001.

The ANOVA outcome shown in Table 2 revealed that the first order terms of independent variables (X1, X2 and X3), quadratic terms (X12, X32 and X22) and the interaction terms (X1X2, X1X3 and X2X3) significantly affected the content of total phenolics of HE (p<0.05). This could be explained by the increasing extraction time accelerating the chemical decomposition of bioactive compounds in the extraction process, resulting in lower extraction yield.

Response surface analysis (RSA)

In Figure 4, the three-dimensional response surface were very helpful to predict the type of the interaction betwen the two measured variables, the relationship between response and experimental levels of each variable, and locate the optimum conditions for maximum yield of HE. Figure 4A–C shows the interactions between the ethanol concentration and each of the two other factors, namely extraction time, and solvent-to-material ratio and their mutual interaction on the extraction yield of HE can be seen.

Figure 4. Response surface analysis for the extract yield (%) with UAE with respect to: (A) extraction time and ethanol concentration; (B) ethanol concentration and solvent-to-material ratio; (C) extraction time and solvent-to-material ratio. The data were analyzed by Matlab software.

Figure 4A shows the interaction between ethanol concentration (X1) and extraction time (X2) on the extraction yield. Increasing percentages of ethanol from 60 to 80% with extraction time from 30 to 45 min enhanced the extraction yield. It can be seen in Figure 4B that by varying the ethanol concentration from 60 to 80% and decreasing the solvent-to-material ratio from 30 to 10 mL/g, the extraction yield of target compounds was improved. Figure 4C presents the interaction of extraction time and the solvent-to-material ratio. It was found that maximum yield (17.50%) was achieved when the extraction time was 45 min and the solvent-to-material ratio was 20 mL/g. This result was in accordance with previous studies [32,33].

Effect of extraction parameters on TPC, TFC and DPPH

A calibration curve with different concentrations of gallic acid was plotted. The total phenolic content in the extract was expressed in milligram equivalent of gallic acid, and the total flavonoid content in milligram equivalent of quercetin. All samples were analyzed in triplicate.

In this study, however, the total phenolics yield decreased when the ethanol concentration was above 80% (Table 1). The total phenolics yield increased with prolonged extraction time from 20 to 45 min. This observation was understandable because an extended extraction time favors the extraction of phenolic compounds. The total phenolics yield increased with an increase in ethanol concentration from 60% to 80%. It should be noted that, although in a very small amount, flavonoids are also present in the extract of HE. However, we can say that HE has excellent free radical scavenging properties, with a considerable content of polyphenols, but also flavonoids, even if in a smaller amount, this being probably due to the environment in which it was developed in the bioreactor. The differences from other authors [15,16] are due to the different conditions of development in the bioreactor of the biological material and the extraction process with ultrasound. Thus, this experiment clearly concluded that HE has the ability to control oxidative damage and can act as an antioxidant.This is probably due to the increased solubility of phenolic compounds in the mixture of ethanol and water. Also, an extended extraction time favors the extraction of phenolic and flavonoid compounds. The ethanol extracts exhibited high content of phenolics and high radical scavenging property (DPPH scavenged%). The results strongly suggest that phenolics and flavonoids are important components of the HE extracts and this could explain their high radical scavenging activity. The total flavonoid (TFC) and phenolic (TPC) contents for HE were quantified using the Folin-Ciocalteu reagent (Table 1). It was found that the total phenolic content of HE reached 25.22 mg/mg, and total flavonoid content of 3.25 mg/g, which is in accordance with the results (15.76-64 mg/mg) by [12, 34]. Generally the antioxidant activities of several edible mushrooms have been found to be correlated with their total phenolic and flavonoid contents [35]. These results are confirmed by previously published studies that have shown that the lyophilization process is the most effective way to maintain a high amount of flavonoids in fungi [36]. Our results indicated that a shorter extraction time can induce the degradation of biological compounds and the corresponding decrease in the yield of biological compounds. Previous reports suggested that the biological activity of several mushrooms was mainly attributable to polysaccharides, especially the high molecular weight glucans [34]. In the DPPH assay, the maximum antioxidant activity of HE was 92.43%. The results of the present study showed that a dose-dependent increase in free radical extinction is due to the increase in alcohol concentration (80%) with the lowest IC50 value in this assay, as shown in Table 1. The data obtained clearly indicate that the increased DPPH scavenging ability of the alcoholic extract may be attributed to its potent hydrogen donating ability. Gursoy et al [37] reported that the radical scavenging activities of methanol extracts from M. rotunda at 2 mg/mL were at concentration 33.94 ± 0.96% The ultrasound assisted extraction with ethanol of HE showed relatively good antioxidant activity compared to the extracts described in these previous reports. The variation of the antioxidant activity in many scientific articles can be also attributed to the solvent used, because it depends on its type and polarity and on the procedures for isolation and quantification [36].

Antioxidant capacities of ultrasound-assisted HE extracts in relation to total phenolics and other compounds

Total phenolic content of the mushroom extracts can be related to their antioxidant capacities. Our findings showed that there was a good correlation between total phenolics and DPPH free radical scavenging activity of the mushroom extracts. This was in accordance to the findings by several authors who reported that total phenolic content correlated with the free radical scavenging activity of other mushrooms [38,39]. Research published in the past on the biological properties of fungi has used different solvents to obtain extracts, and it is difficult to make comparisons. The evaluation of several species of fungi using the same methods is very important to study biological activities. Determined by biochemical methods, the antioxidant activity expressed by the percentage inhibition of the lipid peroxidation reaction is the highest in HE 80% ethanolic extract. The results confirmed a correlation between these remarkable antioxidant activities and the high content in compounds with free radical scavenging properties. This parallel relationship proves that the total phenolic content directly affects the antioxidant capacity of HE extracts.

Previous reports suggested that the biological activity of several mushrooms was mainly attributable to polysaccharides, especially the high molecular weight glucans and polysaccharide protein complexes exhibiting anticancer properties [40], as well as polyphenols and flavonoids, which are responsible for the cytotoxic and antioxidant activities [41]. In view of the follow-up, it can be said that a large amount of HE powder has been obtained from a small amount of fungal material. Also, the improvement of the extraction of polyphenols and flavonoids by ultrasound is mainly attributed to the effect of acoustic cavitations produced in the solvent by the passage of ultrasonic waves. Therefore, the structure of the cell wall is interrupted and the diffusion through the membranes is accelerated. The efficient extraction period for achieving the maximum yield was approximately in 45 min. However, other parameters could be considered for optimizing the extraction yields of diterpenoids and polyphenols, such as ultrasonic amplitude or precipitation time. Several studies need to be performed to fully understand the extraction and study of all parameters. Based on our results, we can say that the HE fungus mycelium developed in the bioreactor after our optimized process can improve the extraction of polyphenols, flavonoids and diterpenoids. This can also be attributed to ultrasonic assisted extraction. This study also established a good relationship between radical-scavenging activity and phenolic/flavonoid compounds, thus corroborating a potential neuroprotective effect in AD.

Proximate composition

Chemical composition of mushrooms varies according to species [42, 43]. Among the Hericium spp., H. erinaceus is the most studied. Its fruiting body is known as a good source of carbohydrates (76.5% DW, dry weight), protein (18.8% DW), ash (7.52% DW), fiber (7.10% DW), and fats (2.01% DW). These mushrooms also contain several amino acids, substantial amounts of potassium and phosphorus, and aroma substances [43]. The results of Hericium erinaceus proximate composition, free sugars and fatty acids contents are shown in Table 3. H. erinaceus presented a double content of proteins with high energetic value, but slightly lower contents of TAC, fibers and fat. HE have good values of total free sugars due to the highest concentration of arabinose and mannitol. In literature, arabinose has been reported as one of the minor sugars present in mushrooms [44], but for the HE studied herein, arabinose was the major sugar, and this can be attributed to the protocol followed for the development of the fungus in the bioreactor. High levels of arabinose seem to be a characteristic of the genus Hericium [45,46]. The nutritional composition of HE mushrooms can be also related to the composition in fat, protein, and carbohydrates used for cultivation in the bioreactor.

Table 3. Proximate composition (g/100 g dw), free sugars and fatty acids in the HE.

Values are expressed as mean ± SD of carefully conducted triplicate experiments.

Macro and microelements

Mushrooms have been reported in literature as sources of mineral elements [16,44]. In the present study, the macroelements Ca, Mg, Na and K, and the microelements Fe, Cu, Mn and Zn were identified in HE extract powder (Table 4Other researchers have said that K is the main macroelement in mushrooms [47], and this is according to our research.

Table 4. Macro and microelements in Hericium erinaceus.

Values were expressed as mean ± standard deviation of three replicate determinations.

Reducing power

This assay compares antioxidants based on their ability to reduce ferric (Fe3+) to ferrous (Fe2+) ion through the donation of an electron, with the resulting ferrous ion (Fe2+) formation monitored spectrophotometrically at 700 nm. In reducing power assay, reducing ability of a compound depends on the electron donor and free radical quenching capacity [48, 49]. In this study, the scavenging effect of H. erinaceus was found to be 0.349% to 1.166% at 1 to 5 mg/mL (Table 5). The IC50 value was found to be 1.70 mg/mL.

Table 5. Reducing power of Hericium erinaceus extract.

Values were expressed as mean ± SD of three replicate determinations. Absorbance values for positive controls were measured at the concentration of 0.50 mg/mL.

The investigated mushroom dry extract possessed reductive capabilities, although inferior than ascorbic acid, as standard antioxidant. Conversely in the DPPH radical investigation the results suggested that some other antioxidant compounds, not only total phenolics, are responsible for reductive capabilities. From this it could be concluded that investigated mushroom extracts are primary antioxidant products, but they posses reductive capabilities which are still much lower than of standard antioxidant compound ascorbic acid. The reducing powers of the extracts that were produced by ultrasound with ethanol yielded the high absorbance levels. Thus, it may be deduced that the alcoholic extracts with ultrasound possess discrete levels of antioxidant compounds.

AChE and BChE Activity

In view of the previous experiments that revealed a high antioxidant activity of Hericium erinaceus extract obtained with an ethyl alcohol concentration of 80%, we wanted to further check the possible effect on acetylcolinesterase and butylcolinesterase to achieve a multi-target action for the treatment of AD. The outstanding results obtained with this extract indicate the need for further work on the isolation, purification and investigation of the active principles responsible for such activity. Table 6 shows the acetylcholinesterase and butyrylcholinesterase inhibitory activities of the extracts of HE compared with that of galantamine. As shown in Table 6, all extracts showed mild butyrylcholinesterase inhibitory activity. They also showed inhibitory activity against the enzyme acetylcholinesterase. The 80% alcohol extracts of HE demonstrated a higher inhibitory activity, giving IC50 values of 52.1±1.1 mg/mL against AChE, and 48.5±1.4 against BChE, respectively. It appears that ACE inhibitory activity increased when extraction time was prolonged.

Table 6. Acetylcholinesterase and butyrylcholinesterase inhibitory activities of the 80% ethanolic HE extracts.

Values are expressed as the means±SD of triplicate measurements (p<0.05). IC50 values represent the means ± standard deviation of three parallel measurements (p<0.05).

However, the inhibitory activity of the ethanol extract of HE toward AChE was significantly (p < 0.001) lower than that of galantamine, the positive control, at all concentrations tested.

A previous study reported that acetylcholinesterase inhibition activity in the fungus species Pleurotus pulmonarius is 68.60% [50], results consistent with ours. Phenolic acids and flavonoid derivatives have been reported to be potent inhibitors of AchE [50], therefore also in the case of our study due to the presence of polyphenols and flavonoids the inhibitory activity of acetylcholinesterase increased significantly.

HPLC/DAD-UV analysis

Erinacine A was obtained in the glucose-containing medium, as described by [51]. Our article describes the validation of an HPLC/DAD-UV method for the determination of the biocompound erinacine A, from the extract of Hericium erinaceus (solid state cultivation) obtained by ultrasound-assisted extraction. The representative HPLC chromatogram of the powder extract of HE mycelium is shown in Figure 6. Retention time at 1.846 is represented by erinacine A, one of the major compounds found in general in Hericium species [52]. The linear least-square regression analysis for the calibration curve showed a correlation coefficient of r = 0.9968. We quantified it by comparing the peak areas with those of the standard. The level of erinacine A present in mycelium of HE calculated by the calibration curve was 150 µg/g. Our results are in accordance to literature data for other Hericium species [51]. HPLC analysis indicated erinacine A as the major component of our ethanolic extracts, suggesting that it may be the component responsible for antioxidant effects.

Figure 6. The HPLC sample chromatogram of the erinacine A (from HE mycelium powder extract) UV detection at 340 nm. The retention time of diterpenoid erinacine A peak remained within the range of 1.846-1.852 min (top) and erinacine A molecule standard (bottom).

Conclusions

This is the first report on the antioxidant potential of ultrasound-assisted extraction of Hericium erinaceus extract with ethanol developed in bioreactor. We can say that the development of the ultrasonic extraction technique from plant and fungal materials is of great potential and interest in the field of research but also in the bioeconomic field, since conventional techniques for extracting active constituents consume time and solvents, are thermally unsafe and sometimes have a lower efficiency. Moreover, many natural products are thermally unstable causing the degradation of unsaturated compounds or esters by thermal or hydrolytic effects. Flavonoids and polyphenols are frequently found in plants and, presently, edible mushrooms appear to be good candidates as a potential source. The importance of these compounds is related to their demonstrated benefits in ameliorating age-related diseases. Most of the effects focus on their antioxidant properties. In consequence, mushrooms seem to be a potential natural source of dietary flavonoids and polyphenols displaying a great range of compounds in significant concentrations and showing healthy properties. These results indicate the medicinal and antioxidant properties of HE fungus in alternative medicine. Therefore, the results indicate that the UAE of HE provides an extract enriched in antioxidant compounds. Other authors [53-57] also documented positive correlations between total phenolics and DPPH radical scavenging activity, but also the antioxidant potential of mushrooms. Our future studies are ongoing and will focus on researching the possible beneficial effect on the brain of this HE extract.

Funding

This work was supported by a grant from the Romanian National Authority for Scientific Research and Innovation, CNCS – UEFISCDI, project number PN-III-P1-1.2-PCCDI-2017-0662.

Acknowledgments

We thanks Conf. Univ. Dr. Ducu Catalin and CS III. Dr. Moga Sorin for critically reviewing the manuscript and CS III. Dr. Negrea Denis for English editing of the manuscript.

Conflict of interest

The authors declare that they have no potential conflicts of interest to disclose.

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