{'fraction_non_alphanumeric': 0.03740238388820386, 'fraction_numerical': 0.02770242498972462, 'mean_word_length': 4.832214765100671, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Phylogenetic and Biochemical Analysis of the Reishi Mushroom (Ganoderma lucidum) Populations from Altai

Fungi are widely used in traditional medicine. Ganoderma lucidum (reishi) has a long history in the pharmacy of China, Korea, Japan, and other countries of Southeast Asia [1]. It is mentioned in the 2000 year old “Holy Book of Miraculous Medicinal Plants” from China. G. lucidum contains many biologically active componds including triterpenes, polysaccharides, peptidoglycans, and ganoderic acids [2,3]. Reishi has much more importance for pharmacy than as a food product; it has a wide range of activities including antitumor, antioxidant, immunomodulating, antiviral, antibacterial, antidiabetic, lipid-lowering, hepatoprotective, gastroprotective, and renoprotective ones [4-8]. Chemical composition of fruit bodies of various G. lucidum strains depends on their origin, rearing, and other conditions, and genetic affinity does not correlate with metabolic groupings [9].


Determining the composition of biologically active substances
Chemical analysis of Biologically Active Substances (BAS) in fruit bodies of G. lucidum included estimation of water and ash content, and obtaining extracts with water and organic solvents (hexane, ethyl acetate, and ethanol). These extracts were used for qualitative and quantitative estimates of various BAS using conventional phytochemical techniques; the results were compared to similar studies.

Water content
To determine water content, chopped reishi were dried at 70°С in an air thermostat until constant weight was achieved.

Ash content
Samples of ~1 g were placed in hot porcelain crucibles and kept in a muffle furnace at 700°С for 2 h, then transferred to a dessicator, cooled and weighed. These procedures were repeated until the difference between two consecutive weighing did not exceed 0.0005 g.

Extraction
Total extraction: Powdered fruit bodies (20.0 g) were extracted in a Soxhlet apparatus for 6 h by ethyl acetate and ethanol, obtaining EA+ and EtOH+ extracts, respectively. Water extraction was performed using 1 l of sodium phosphate buffer (pH 7.0) at 62°C for 16 h in an air thermostat. After cooling, samples were centrifuged at 3000 rpm for 15 min, and the extraction procedure was twice repeated with the sediment. The supernatants were combined, concentrated in a rotor evaporator and adjusted to 200 ml by water, resulting in W+ extract.
Stepwise extraction: Powdered fruit bodies (10.0 g) were extracted by hexane in a Soxhlet apparatus for 6 h, then dried to constant weight in a vacuum dessicator with phosphoric anhydride, and weighed. The same operation was repeated with ethyl acetate, and again with ethanol. Each extract was concentrated in vacuum at 40°С to 100 ml in a Buchi R-210 rotor evaporator (Buchi R-210, Flawil, Switzerland), resulting in G, EA-, and EtOH-extracts. The sediment remaining after ethanol extraction was placed in a 2 l flask containing 1 l of sodium phosphate buffer (pH 7.0), incubated in an air thermostat at 62°С for 16 h, cooled and centrifuged at 3000 rpm for 15 m. This procedure was repeated twice; pooled supernatants were concentrated in a rotor evaporator and adjusted to 100 ml by water, resulting in W-extract.
The remaining insoluble sediment was weighed, and the total mass of extracted material was determined.

Isolation of acid polysaccharides
Acids polysaccharides were isolated as described in Sone et al. [21]. According to this method, 100 ml of 0.1 M NaOH and 20 mg of sodium borohydride were added to 10 g of dried insoluble sediment after water extraction and incubated for 2 h on a magnetic stirrer at room temperature, then centrifuged, and the same procedure was repeated again. The obtained supernatants were pooled, adjusted to pH=7.0 with HCl, concentrated to 50 ml on a Buchi R-210 rotor evaporator (Flawil, Switzerland), and dialyzed against double distilled water for seven days using the Visking (Serva, Germany) dialysis membrane; water was replaced three times at 8 h intervals, then once a day. The dialysate was concentrated to 20 ml using a rotor evaporator; 5 ml aliquotes were used to determine polysaccharide concentration.

Ergosterol extraction
Ergosterol was extracted from the hexane extract as following: 1 ml of methanol was added to 150 mg of the evaporated extract, incubated for 7 days, then filtered. The resulting sediment was dissolved in 5 ml diethyl ether, washed three times with 2 ml of 3% NaOH and three times with 2 ml of sodium bicarbonate, then dried over anhydrous sodium sulfate, filtered, distilled to remove ethers, and recrystallized from methanol.

Polysaccharide content
Polysaccharide content was determined using the modified phenol-sulfur acid method of Dubois et al. [25]: 5.0 ml of concentrated water extract was combined with 20.0 ml of 87.5% ethanol, incubated overnight at 4°С, and centrifuged at 7000 rpm for 30 min. The sediment was washed twice with absolute ethanol, dried in vacuum, and dissolved in distilled water. The reaction was initiated by mixing 1 ml of polysaccharide solution with 0.5 ml 5% phenol and 2.5 ml concentrated sulfuric acid, incubated in a water bath at 100°С for 15 min, and cooled to room temperature. Optical density was determined at the wavelength of 490 nm; concentration of polysaccharides was calculated using a calibration curve built for D-glucose. Results were expressed as µg of glucose equivalent per 1 mg of extract.

Phenol content
Total content of phenols in water and ethanol extracts was determined according to the Folin-Ciocalteu colorimetric method with some modifications [26]: 0.5 ml of solution was mixed with 0.5 ml of Folin-Ciocalteu reagent (Sigma, USA) and incubated for 3 min. Then we added 0.5 ml of 20% sodium carbonate, adjusted the mixture to 5 ml with distilled water, and incubated in the dark for 90 min. Optical density was determined at the wavelength of 725 nm; concentration of phenols was calculated using a calibration

Gr up SM
Copyright  Peltek SE curve built for gallic acid. Results were expressed as µg of gallic acid equivalent per 1 mg of extract.

Flavonoid content
Total flavonoid content was determined according to Park et al. [27]: 1.0 ml of water-ethanol extract solution containing about 1.0 mg/ml of dried substance was mixed with 4.3 ml of solution containing 4.1 ml of 80% ethanol, 0.1 ml of 10% aqueous solution of Al(NO 3 )3х9H 2 O and 1.0 ml of 1M aqueous potassium acetate (CH 3 CO 2 K). Reaction mixture was incubated for 40 min at room temperature. Optical density was measured at the wavelength of 415 nm. Flavonoid content was determined based on the calibration curve built using quercetin; results were expressed in µg of quercetin equivalent per 1 mg of extract.

Fatty acids content
Fatty acids were detected in hexane and ethyl acetate extracts as methyl esters, obtained by treatment with hydrogen chloride [28]. Separation was performed by gas chromatography-mass spectrometry (GC-MS) on an Agilent 7890N/5975C device (Agilent Technologies, USA) with a quadrupol 5973N mass spectrometer (Agilent Technologies, USA) and a DB-1 capillary column. Both total ion current in the 10-800 Da range and selected ion monitoring analysis were performed. Methyl esters of fatty acids were identified using the NIST Mass Spectral Search Program for the NIST / EPA / NIH Mass Spectral Library Version 2.0a. Quantitation was performed using the internal 1 µg/ml tetrafluorobenzobarenel standard.

Results and Discussion
We analyzed nine specimens of G. lucidum from various regions of Altai (Table 1). Fragments of the nuclear ribosomal spacer about 300 bp long containing the ITS1 spacer were sequenced and aligned; all nine sequences turned out to be identical. We constructed a phylogenetic tree based on the sequences listed in Table 1; the tree from Hseu et al. was used as a reference [14]. Differentiation and grouping of isolates of the Ganoderma lucidum complex by random amplified polymorphic DNA-PCR compared with grouping on the basis of internal transcribed spacer sequences [14], and Geastrum pectinatum (EU784241) was taken as the out-group.
The results of phylogenetic analysis are shown on Figure 1. Altai samples of G. lucidum were clearly distinct from other specimens. In addition to G. lucidum, the group containing Altai reishi also included G. oerstedii, G. tsugae, G. valesiacum, G. oregonense, G. ahmаdii, and G. carnosum. According to Hseu et al. [14] this is group I of the G. lucidum complex, which is widespread in Argentina, Canada, USA, Pakistan, Britain, France, Norway, and Western Siberia. Therefore, we found that all studied Altai specimens of G. lucidum have identical ITS1 sequences and belong to group I of this species. Our results accord with the data of Zhang et al., who also found that four reishi samples from Altai belong to this group [13].
Results of the screening suggest that ethanol fractions contain mostly flavonoids, phenols, and coumarins; water fractions are dominated by tannins, carbohydrates, and coumarins; and hexane and ethyl acetate extracts, by terpenoids.
Fatty acid composition of the EA+ extract is given in Table  3. The main fatty acids were palmitic (7.37%), oleic and linoleic (41.40 %), and linolenic (13.95%) acids, as well as traces of myristic, pentadecanoic, palmitoelaidic, and stearic acids, which conforms to

Gr up SM
Copyright  Peltek SE

Gr up SM
Copyright  Peltek SE the composition of G. lucidum from China [31] also found 189.1-1453.3 µg/g of ergosterol in G. lucidum.
Ergosterol isolated from the hexane extract had melting point at 160-162°, which accords with the value of 160° determined by Pruess et al., litas. 161°С [32]; its spectral characteristics were identical to those taken from the Biological Magnetic Resonance Data Bank (bmse000494) [33], and it had no melting point depression with a known sample. In an earlier study we found 7.17% ergosterol in methanol extracts of G. lucidum (Shevelev OB, Seryapina AA, Goryachkovskaya TN, Tatarova LE, Slynko NM, Peltek SE, Markel AL, Moshkin MP Hypotensive and Neurometabolic Effects of Intragastric Reishi (Ganoderma lucidum) Administration in Hypertensive Rat Strain article in preparation). W+ and W-extracts were used to determine protein concentration using the Quick Start ™ Bradford Protein Assay Kit 4 (Bio-Rad, США). In two experimental replicates we found protein concentration of 202 and 203 µg/ml for W+, and 103 and 101 µg/ml for W-, which corresponds to 2.03 and 1.02 mg/g of soluble proteins, respectively. Twofold reduction of protein concentration in W-is probably correlated with partial insolubilization of proteins during ethanol treatment.
According to published studies, ethyl acetate and ethanol extracts contained mostly phenols, flavonoids, and triterpenic acids [34]. We found that fruit bodies of Altai G. lucidum contained 32.4 mg of phenols per 1 g of extract (in pyrocatechol equivalent), while flavonoids made up 11.1 mg per g (in quercetin equivalent).
Polysaccharide content in water extract of the sediment was 752 mg/g after extraction by organic solvents, as determined by the phenol-sulfur acid method, which corresponds to 4.55% of the absolutely dry substance. Extraction of chopped fruit bodies with hot water after concentration and drying yields 2.66 g of dry sediment (10.6% of the absolutely dry substance), and polysaccharides make up 735 mg/g of this extract (calculated based on glucose), which corresponds to 4.44% of the absolutely dry substance.
However, certain studies suggest that BAS yield can be substantially increased using aqueous alkaline solution, which facilitates the extraction of acid polysaccharides [21]. We performed this procedure for the insoluble sediment formed after stepwise extraction and dialysis. Polysaccharide yield was 3.8 mg per ml of concentrated dyalizate, which corresponds to 6.16% of dried insoluble sediment after water extraction. The final quantity of polysaccharides that we managed to extract from reishi fruit bodies was 10.72% of the absolutely dry substance. On the whole, chemical composition of reishi from Altai is similar to that from other regions.