Fingerprint Characteristics Polyporus Umbellatus From Main Producing Areas In China And Exopolysaccharide Activities From The Mycelium

Polyporus umbellatus?Pers.?Fries is a medicinal fungus of Polyporaceae,riching in component of steroids and polysaccharides.Mountain areas such as Changbai Mountains,Yanshan Mountains,Taihang Mountains,Qinling Mountains and Yunnan-Guizhou Plateau are the main producing areas of P.umbellatus.The chemical composition of P.umbellatus in these producing areas varies greatly.In addition,there are also reports of indoor cultivation of P.umbellatus mycelium.At present,there is no systematic study on the difference of chemical composition of P.umbellatus in different producing areas and different growing periods,and the fermentation process and biological activity of exopolysaccharides from P.umbellatus mycelium are still unclear.In this paper,54 batches of P.umbellatus collected from 6producing areas were used as materials to determine their chemical compositions,including routine detection items,main active ingredients and mineral elements.LC/MS,GC/MS,UPLC-Q-TOF-MS/MS and other means were used to establish the chemical fingerprint of P.umbellatus from different producing areas.The similarity evaluation system software of traditional Chinese medicine chromatogram fingerprint?2004A edition?,principal component analysis?PCA?,Partial Least Squares Discriminant Analysis?PLS-DA?and XCMS online analysis platform were used to analyze the established fingerprints.Besides,response surface methodology was used to optimize the optimal fermentation conditions for exopolysaccharide from P.umbellatus mycelia.The exopolysaccharides of P.umbellatus mycelia were isolated and identified by means of fractional alcohol precipitation,Sephadex G-100 gel column chromatography,FT-IR spectrum,methylation and NMR spectrum,and the biological activities of which,such as in vitro antioxidant,phagocytosis of macrophages,inhibition of DNA lysis and delaying cell aging,were discussed.Scientific data for selection,cultivation,quality evaluation and active ingredient development of high-quality P.umbellatus were accumulated.The main research results are as follows:1. The factors such as mycelium source,initial p H,culture temperature,light,culture medium,culture material of original seed and culture material of cultivation seed were optimized and an efficient culture system of P.umbellatus sclerotium was established.The mycelium from Zhouzhi,Shaanxi Province has an excellent growth rate;the optimal culture medium for mycelium is bean cake powder medium,with the initial p H of 6–8 and suitable culture temperature of 23–28?.The optimal culture material for the original seed is birch sawdust medium.The optimal formula of culture material for cultivation seed is as follows:birch segment?80%?,bran?10%?,humus soil?10%?;well,the birch segment needs to be soaked in 0.25%NH₄NO₃ solution for a moment.Besides,the temperature of 22?and dark condition are good for the formation of sclerotium.By adopting the efficient culture system,the production cycle of P.umbellatus is shortened from 3 years to 3 months,and a new way is opened up for relieving the pressure of tight supply in the P.umbellatus market.2. Among the 37 samples of P.umbellatus from 6 producing areas,the ergosterol content of 29 samples was above 0.70 mg/g,and the total ash content of 34 samples was below 120.00mg/g,as well as the acid-insoluble ash content of 30 samples was below 50.00 mg/g.Among the 17 samples of P.umbellatus of different growth stages,the ergosterol content of 14samples was above 0.70 mg/g.The total ash content of 17 samples was below 120.00 mg/g,and the acid-insoluble ash content of 17 samples was below 50.00 mg/g.The results show that the collected P.umbellatus samples are basically in good condition and can be used for subsequent research.3. The contents of 5 active components of 37 batches of P.umbellatus from 6 producing areas and 17 batches of P.umbellatus of 5 different growth stages were different.The content of neutral polysaccharide ranged from 0.70 to 5.40 mg/g.The content of neutral polysaccharides in sample from Qinling Mountains was the highest,reaching 5.40 mg/g.The content of acidic polysaccharide ranged from 4.44 to 23.12 mg/g and sample from Guanzhong Plain was the highest,reaching 23.12 mg/g.The total content of free monosaccharide and oligosaccharide ranged from 0.58 to 4.45 mg/g and sample?S1?from Qinling Mountains was the highest,which was 4.45 mg/g.The total steroids content ranged from 1.54 to 3.25 mg/g and sample?S31?from Qinling Mountains was the highest,reaching 3.25 mg/g.The content of water-soluble protein ranged from 25.64 to 281.99 mg/g and sample?S9?produced in Taihang Mountain was the highest,reaching 281.99 mg/g.The sample in the stage of mycelial has the highest content of free monosaccharides and oligosaccharides,which was 6.25 mg/g.The content of water-soluble protein of sample in symbiosis period was the highest,reaching195.51 mg/g.The content of neutral polysaccharides,acidic polysaccharide,total steroids of fruiting bodies of P.umbellatus was the highest,with an average content of 11.03 mg/g,36.08mg/g,6.52 mg/g,respectively.This study showed that there were differences in the content of active components in different producing areas and growing periods of P.umbellatus,which provided a reliable basis for the internal quality control of P.umbellatus.This paper holds that the P.umbellatus produced in Qinling Mountains have a rich and relatively high content of active ingredients.The research results provide a theoretical basis for the application and development of P.umbellatus.4. The contents of 12 mineral elements of P.umbellatus samples from 6 producing areas were determined by atomic absorption spectrophotometry.The contents of Cu?5.39?g/g?,Zn?10.23?g/g?,Cr?1.04?g/g?are the highest in samples from Qinling Mountains,and the contents of Fe?603.47?g/g?,Mn?29.62?g/g?,Ni?10.70?g/g?,Pb?1.22?g/g?,As?0.85?g/g?,Cd?0.44?g/g?are the highest in those from Yanshan Mountains,and the content of Ca?24870.05?g/g?in samples from Taihang Mountains is the highest.In the symbiotic period,the accumulation of Cu?13.02?g/g??Mg?2268.15?g/g??Zn?1088.21?g/g??Fe?602.11?g/g??Mn?325.01?g/g??Ca?27850.12?g/g??Pb?0.71?g/g?is greatly promoted.Correlation analysis showed that the acid polysaccharide content was significantly and positively correlated with the contents of Fe,Mn and Ni.The total content of free monosaccharide and oligosaccharide is significantly and negatively correlated with the content of Pb,and the content of water-soluble protein is significantly and positively correlated with the content of Zn,while it is significantly and negatively correlated with the content of Fe and Mn.This paper holds that samples from Yanshan Mountains and the symbiotic P.umbellatus have a relatively high content of mineral elements,and the accumulation of active components in P.umbellatus can also be controlled through cultivation measures of exogenous application of mineral elements.5. LC/MS technology was used to determine the components of alcohol extract of P.umbellatus samples from 6 producing areas.Similarity analysis,cluster analysis and principal component analysis were carried out to realize the pre-classification of these samples.HPLC chromatographic peaks of the samples were determined,and 19 common peaks were manually calibrated for P.umbellatus samples from 6 production areas and 13 common peaks were calibrated for P.umbellatus samples in 5 different growth periods by using the similarity evaluation system software of chromatographic fingerprint of traditional Chinese medicine?2004A version?.Similarity analysis?SA?showed that the similarity of P.umbellatus samples from 6 production areas was 0.486–0.972,and samples in 5 different growth periods was0.201–0.922.Principal component analysis?PCA?showed that the cumulative variance contribution of the two principal components is higher than 70%,and the samples from 6production areas can be classified into 3 categories:the samples from Yanshan Mountains,Taihang Mountains,Changbai Mountains and Yunnan-Guizhou plateaus are in the same category,and the samples from Qinling Mountains and Guanzhong Plain are in the same category separately.The five different growth stages of P.umbellatus samples can also be classified into 3 categories:the white sclerotium period,black sclerotium period and symbiotic period samples are in one category,and the hypha period samples and fruiting body period samples are in one category,separately.The results showed that the components of alcohol extracts of P.umbellatus from 6 producing areas were obviously different,which could provide reference basis for quality evaluation and preclassification of P.umbellatus.6. Using UPLC-Q-TOF-MS/MS technology combined with Pairwise analysis method of XCMX Online analysis platform,the differential components of 15 batches of P.umbellatus from 3 production areas of Qinling Mountains,Taihang Mountains and Guanzhong Plain and15 batches of P.umbellatus in 3 different growth periods of hypha period,black sclerotium period and fruitingbody period were analyzed and identified.According to the interactive cloud picture,34 symbolic molecules,including 6 steroids,were screened from samples from different producing areas?p?0.005,fold change?10?.The analysis of peak area intensity of steroids shows that ergosterol,peroxygergosterol and ergosta-7,22-diene-3-one are key symbolic molecules to distinguish P.umbellatus from different producing areas,while peroxygergosterol and ergosta-7,22-diene-3-one are key symbolic molecules to distinguish P.umbellatus from Guanzhong Plain and other producing areas.Besides,ergosterone-7,22-diene-3,5,6-triol,ergosterone and polyporusterone F are key symbolic molecule to distinguish P.umbellatus in different growth stages.In combination with the cytotoxic activity of each key symbolic molecules,the cytotoxic activity of ergosterol and peroxyesterol in P.umbellatus from Qinling Mountains is stronger than that from Guanzhong Plain and Taihang Mountains,while ergosta-7,22-diene-3,5,6-triol and ergosterone in P.umbellatus of black sclerotium period is stronger than that in hypha phase and fruitingbody phase.This paper holds that the strong cytotoxic activity of P.umbellatus produced in Qinling Mountains and P.umbellatus in black sclerotium period is beneficial to ensure clinical efficacy,and may be one of the important reasons for the genuineness and harvest period formation of P.umbellatus.The research results provide new reference data for revealing genuineness and harvest period formation mechanism of P.umbellatus from the perspective of diversity of secondary metabolites.7. GC/MS was used to study the fingerprints of the volatile components of P.umbellatus from six producing areas and 85 common volatile components of P.umbellatus were identified,including 7 volatile components such as alkanes,alkenes,alcohols,esters,ethers,phenols and acids.The similarity of P.umbellatus from different producing areas ranged from 0.096 to0.999.The similarity of four samples is less than 0.511,and PCA can directly separate the four samples from other samples.Therefore,the similarity threshold value of 0.511 can be set to measure the quality of P.umbellatus.In addition,PLS-DA was used to select key differential components of P.umbellatus from three producing areas of Qinling Mountains,Taihang Mountains and Guanzhong Plain and the three growth periods of hypha period,black sclerotium period and fruitingbody period.As a result,a total of 34 potential symbolic molecules were screened out,comprising 7 active ingredients such as chrysophanol,and related to 7 metabolic pathways such as aspartic acid metabolic pathway.This paper holds that the different producing areas and growth periods are important driving force of the difference in volatile metabolites of P.umbellatus,and the diversity of metabolic pathways is the internal factor affecting the quality difference of P.umbellatus.8. Response surface methodology was used to optimize the optimal process conditions for exopolysaccharide?EPS?production from P.umbellatus mycelia.As a result,the 9#mycelia was selected for fermentation and the fermentation conditions were as follows:glucose 25 g/L,yeast extract 4.51 g/L,VB₁ 0.l g/L,KH₂PO₄ 1.5 g/L,Mg SO₄·7H₂O 1.05 g/L,initial p H=5.5,inoculum size 13.75%,27?,12 d,and 150 r/min.Under these conditions,the yield of exopolysaccharide was 1.23 g/L,which was 1.38 times bigger than before.The results showed that the optimized conditions could greatly increase the yield of EPS from P.umbellatus mycelia,which laid a foundation for the subsequent large-scale production of EPS from P.umbellatus mycelia.9. Three new uniform exopolysaccharides of PPS1,PPS2 and PPS3 were separated from P.umbellatus fermentation broth by fractional alcohol precipitation combined with Sephadex G-100 gel column chromatography technology.Their mean molecular weights were 6.9×10⁴Da,4.6×10⁴ Da and 3.7×10⁴ Da,respectively.Besides,they were mainly consisted of mannose,galactose and glucose,with the molar ratios of 43.6:2.5:1.0,17.7:3.1:1.0 and 4.6:2.6:1.0,respectively.In addition,FT-IR spectrum,methylation,GC/MS and NMR spectrum analysis showed that the three exopolysaccharides were neutral sugars with?-and?-glycosidic bond configurations,all of which were multi-branched polysaccharides.The branching degree of PPS1 is 25.49%,and the main chain is composed of?6)-?-D-Manp-?1?glycosidic bond connection,and some glycosidic bonds have branches at O-2 position.Branched chain consists of Man,Gal and Glc in the form of terminal residues,and part of Man is linked by?2?-?-D-Manp-?1?glycosidic bond.The branching degree of PPS2 is 34.39%and Man is linked by?6?-?-D-Manp-?1?glycosidic bond to form the main chain of polysaccharide PPS2,which has branches at O-2 position of some main chain residues;well,the branched chain consists of?2?-?-D-Manp-?1?,?2,6?-?-D-Galp-?1?,?6?-?-D-Glcp-?1?.PPS3 is a polysaccharide with a multi-branched structure with a branching degree of 38.87%.?6?-?-D-Manp-?1?and?6?-?-D-Galp-?1?form the main chains of PPS3,which existed branches at O-2 position of some main chain residues.The branched chain consists of?2?-?-D-Manp-?1?,?6?-?-D-Galp-?1?,?4?-?-D-Glcp-?1?,?6?-?-D-Glcp-(1?glycosidic bond.In addition,SEM was used to observe the branched structures of the three polysaccharides at nanometer scale.The results provide experimental basis for further study on the structure and structure-activity relationship of exopolysaccharide from P.umbellatus mycelium.The biological activities of exopolysaccharides of P.umbellatus mycelia were discussed from antioxidant,cell and molecular levels in vitro.The results showed that the scavenging ability of the three exopolysaccharides to different free radicals was ABTS⁺>DPPH>superoxide anion>hydroxyl,with a certain dose dependence.Besides,they have obvious activity of enhancing immunity of RAW264.7 macrophages in mice,and could up-regulate the secretion of NO and enhance the phagocytic ability of macrophages to neutral red.In addition,they also have obvious function of inhibiting DNA molecule cracking in vitro,and could obviously reduce the cracking rate of PUC-19 DNA under UV+H₂O₂?2%?.Furthermore,SA-?-Gal cell staining technique was used to confirm that the three exopolysaccharides have obvious anti-aging activity on RAW264.7 macrophages in mice.This paper holds that exopolysaccharides from P.umbellatus mycelium have obvious biological activities such as scavenging free radicals,improving cell immunity,inhibiting DNA lysis,delaying cell aging.The results provide theoretical basis for the development and utilization of biological activities of P.umbellatus fermentation broth.To sum up,this paper clarified the differences in chemical components and fingerprint characteristics of P.umbellatus from six main producing areas in China,and elaborated the structure characteristics and biological activities of exopolysaccharides from P.umbellatus mycelium.The results provide important scientific basis for the artificial cultivation,selection of producing areas&harvest period of high-quality P.umbellatus,as well as active ingredient development of P.umbellatus fermentation broth.