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Introduction
Ganoderma lucidum is the most popular medicinal mushroom also known as Reshi in Japan. It is one of the most studied fungi due to the great variety of bioactive compounds that have been reported in the mycelium, the mature fruiting body contains more than 400 different bioactive compounds, mainly triterpenoids, polysaccharides, nucleotides, sterols, steroids, fatty acids, proteins/peptides and trace elements that have been reported to have various medicinal effects (Cör et al., 2017, 129). Within these compounds we can find more than 150 antioxidants and phytonutrients, responsible for the most important immunomodulatory, anticarcinogenic and anti-inflammatory properties, hypoglycemic, hypolipid and hepatoprotective. Among the most active components are the polysaccharides that help strengthen the immune system, intensifying the activity of T cells, applied in the treatment of a variety of diseases, including cancer (Pai-Feng et al., 2012, 1). The importance of polysaccharides lies in the fact that they can provide many benefits related to their intervention in biological metabolisms in humans, immunostimulant activity, inhibition of tumor growth, as well as anti-inflammatory action, which are generally related to their structure, molecular weight and degree of branching. Some studies indicate that the polysaccharides of Ganoderma lucidum could promote the release of serum insulin, regulate the activity of enzymes with participation in glucose metabolism and the decrease in plasma glucose (Teng et al., 2012, 173).
There is a wide variety of polymeric compounds that form the structure of cells, the mycelium and the fruitful body of micro and macromycetes, mainly glucans that is a monosaccharide (glucose) and exopolysaccharides, however their major composition are various monosaccharides chains of sizes. Determine the exact structure of fungal glucans many analytical methods have been applied such as chemolysis methods that include methylation analysis, oxidation methods (periodate, IV lead acetate), Smith degradation, NMR spectroscopy is the non-destructive method of analysis more effective and updated structural (Synytsya et al., 2015, 720; Ruthes et al., 2015, 754).
It is important to determine the chemical composition that provides the fungal diversity to optimize the use of their properties, to have simple methods that generate a correlation with other results and make analysis, simpler, faster and less expensive, that is why it has techniques already standardized the new ones offered by the analytical area.
The aim of this study was to investigate the chemical composition of polysaccharides in standardized extract of the fungus Ganoderma lucidum when it is stimulated to increase the production of its functional and medicinal compounds by the addition of acetylsalicylic acid (ASA) under controlled conditions, comparing three techniques.
Method
Chemicals and reagents
Acetylsalicylic acid ≥ 99.0%, dimethyl sulfoxide (DMSO) for HPLC, ≥ 99.7% were purchased from Sigma-Aldrich, U.S.A., all other chemicals used were of analytical purity and grade HPLC.
Obtaining basidiocarps
The biological material used was Ganoderma lucidum CP-145, belonging to the Centre for Genetic Resources of Edible, Functional and Medicinal Mushrooms, Campus Puebla, Mexico of the Colegio de Posgraduados, cultivated in solid sterile substrates based on oak sawdust, two types of basidiocarps were obtained: those added on their substrate with ASA, (10 mM) and control basidiocarps cultured in the traditional way.
Obtaining hydroalcoholic extracts
Obtaining two types of hydroalcoholic extracts: An extract from added basidiocarps and another from basidiocarps from control fungi both extracts were carried out with water-alcohol (80:20) by 24 h, hydroalcoholic extracts were obtained according to patented procedure, each extract was taken at a concentration of 1 g/mL with rotary evaporator at 19°C (Meneses et al. 2016, 5).
Determination of polysaccharides by quantum magnetic resonance analyzer MARSIII of Bruce Copen
The sample is placed directly in the sample holder of the equipment obtaining a reading at 10 seconds, without the use of solvents, reagents or other treatment.
The analizer has a database where the compounds of interest are selected to be monitored in this case polysaccharides, generating a table of data comparing the results of the sample against the references of the software, the answer is given in abundance (http://copen.com.mx/nueva/mars3-analisis.php; https://www.copen.us).
Determination of polysaccharides by the Fehling´s method
This technique was used to determine the content of polysaccharides in the form of glucose, polymers of glucopyranosides and polyalcohols present in the extracts of Ganoderma lucidum. In this test the presence of aldehydes but not ketones are detected by reduction of the deep blue solution of copper (II) to a red precipitate of insoluble copper oxide. Two solutions are required: Fehling's "A" uses 7 g of CuSO4.5H2O dissolved in distilled water containing 2 drops of dilute sulfuric acid. Fehling's "B" uses 35 g of potassium tartrate and 12 g of NaOH in 100 mL of distilled water.
The mixture was made with 15 mL of solution "A" with 15 mL of solution "B", 2 ml of this mixture added to an empty test tube and 3 drops of the compound to be tested added to the tube. It was placed in a water-bath at 60°C. A positive test is indicated by a green suspension and a red precipitate. The test was so sensitive enough that even 1 mg of glucose produced the characteristic red colour of the compound (Ávila et al., 2012, 131; Laine et al., 2002, 608).
Extraction with DMSO for polysaccharides and polyalcohols by GC-MS
Derivatization was performed to obtain them as ester by methylating the polysaccharides, 2 mL of alcohol-alkalinized with sodium hydroxide (10:2) were added to 5 mL of each control extract and to 5 mL with the addition of ASA separately, 0.5 mL of DMSO were added, the sample was maintained 30 min in an ultrasonic bath at room temperature, subsequently it was heated for 1 h at 90°C, cooled, 25 mL of distilled water were added, the aqueous phase was extracted with 10 mL of dichloromethane, the filtrate was centrifuged at 1400 rpm, filtered with a bed of dry anhydrous sodium sulfate (Synytsya and Novák, 2013, 3; Kao et al., 2012, 2).
Results
Determination of polysaccharides by quantum magnetic resonance analyzer MARSIII of Bruce Copen
The results were obtained by the MARSIII analyzer were with the selection of four types of sugars in the database, the variations of their standard deviations are large and there is no significant difference to 95% confidence between the control extract and the added extract. This technique only generates comparison results between the two samples without giving a value of the total content of the sugars present in each sample (Figure 1).
Content of reducing sugars
The sugar reducers Fehling´s method is adequate because it takes advantage of the reduction properties of the sugars present in the extracts with a value of 8.784 ± 2.019% in the control basidiocarps extract and 41.326 ± 1.430% in the extract of basidiocarps added, increasing the concentration of these 4.7 times more, there is a significant difference between the control extract and the added extract to 95% de reliability. The result obtained from saccharides is expected by the presence of the ASA (Meneses et al., 2016, 12).
Determination of polysaccharides and polyalcohols
Ganoderma lucidum is a mushroom with substantial number of active biological compounds present in the form of polysaccharides when analyzed by GC-MS. The extraction with DMSO is recommended for the detection of polysaccharides wich can be seen in Figure 2, the compound with the highest concentration is β-glucopyranoside methyl 41.53 y 36.44 abundance respectively for the added extract and the control extract, along with this we find other polysaccharides and polyalcohols as the expected composition characteristic of this fungus.
Figure 2 Sugars determined by GC-MS, * indicates p<0.05 compared to control and extract added with ASA.
The quantum magnetic resonance analyzer MARSIII by Bruce Copen for the characterization of agricultural area materials such as plants and fungi was evaluated through the determination of polysaccharides in extracts of Ganoderma lucidum, even though it has characteristics that make it an attractive method since it does not require reagents or sample handling (http://copen.com.mx/nueva/mars3-analisis.php; https://www.copen.us), it was not possible to detect a significant diference between the control extracts and those added with ASA.
When the determinations were made with the methods of sugar reduction and GC-MS that are already standardized methods, significant differences were obtained between both extracts as shown in Figure 3, the differences that these last two methods show in their detections suggest Interferences due to the chemical composition of the extracts. In the Fehling´s method the presence of calcium is relevant to generate results that are not affected by this element, being a non-specific technique that yields a total value of reducing sugars and in the chromatograph method the manipulation of samples to make the reaction of methylation generates decrease in the recovery of each of its compounds, although its greatest advantage is to generate very specific results by identifying compounds that can be made through the comparison of the NIST library in the operating software.
Discussion and Conclusion
The increase of the presence of a compound in the added extract shows the effect of the addition of the ASA that stimulates the production of more functional molecules with respect to the control extract, the polysaccharides found are compounds of significant importance and although they have already been reported, their detection is relevant since these compounds are presented in large quantity as a characteristic, in this work a higher concentration of β-methyl glucopyranoside is detected followed by DL-arabinitol and ribitol for the added extract. However, xylitol and L-lyxose are present with greater presence in the control extract, all these compounds with antioxidant activity (Ruthes et al., 2015, 756; He et al., 2006708-709,).
The main composition of the extract is the polysaccharides that stand out: dianhydromannitol, mannose, sorbitol, β-methyl glucopyranose, DL-arabinitol, ribitol, deoxy-d-altritol, Allo-inositol, as can be seen in Figure 2 where the results obtained are compared in the control extract and the extract added with ASA. Detection of these compounds are common in both extracts: D-arabinitol, ribitol, mannitol, β-D-glucopyranoside, methyl β-D-ribopyranoside, which are alcohols and sugars of five carbon atoms. These polysaccharides with demonstrated activity in the stimulation of the immune system also have potential in the treatment of human diseases (Synytsya and Novák, 2013, 805; He et al., 2006, 708-709). The carbohydrate content of edible fungi varies with species and ranges between 35 and 70% DW. It is believed that edible fungi contain a high level of oligosaccharides and only a low level of total soluble sugars (Rathee et al., 2012, 460). Unlike works where the addition of SA and ASA in plants allows to obtaining of crops that develop greater vigor and tolerance to various environmental factors to face the problem due to climate change (Soliman et al., 2018,7), there are few works where these compounds are added to edible and medicinal mushroom, research has been carried out to reduce its browning, such as the case of Agaricus bisporus, where treatment with SA at 250 μM can be used as a useful technology to alleviate post-harvest browning of the fungus carphophore by maintaining the integrity of the membrane due to the improvement of the activity of the antioxidant system and also the accumulation of phenols (Magdziak et al., 2016, 771; Dokhanieh and Aghdamb, 2016,147), another research is aimed at increasing the content of polysaccharides and triterpenes with the addition of SA to Ganoderma lucidum where 12.41 ± 0.32 and 12.62 ± 0.31 mg / g of control polysaccharides and sample additioned with SA were obtained respectively (Ye et al., 2018,7), the combined induction increased polysaccharide 9.02% of polysaccharides being lower concentrations than those detected in this study, this research sought to modulate the properties of Ganoderma lucidum with ASA, in a previous quantification of the extract 0.78% of carbohydrates and glucose were obtained using the ion chromatography and colorimetric method (Meneses et al., 2016, 18) which demonstrated that it is necessary to have more specific techniques for the characterization of these extracts.
There were differences between the polysaccharide profiles of the control extract and the added extract, these differences were originated by the addition of ASA (10 mM) to the substrate, since all the other variables remained constant during the culture of basidiocarps, species, strains, substrate and environmental conditions (Meneses et al., 2016, 15).
The results obtained indicate that it is necessary to continue with the search for new tools that facilitate the analysis of the extract of Ganoderma lucidum to maximize the use of the properties of this medicinal fungus.
