Vahide Payamnoor·Mohammad Reza Kavosi·Jamile Nazari

Abstract Macroscopic fungi on Caucasian alder wood(Alnus subcordata)were identified and tested as a source of betulin and betulinic acid(the most important metabolites of the Betulaceae family)to evaluate levels of phenols,flavonoids and antioxidant activity.Ganoderma applanatum,Lenzites betulina,Trichaptum biforme,Rigidoporus ulamrius, Fomes fomentarius, Schizophyllum commune,Auricularia mesenterica,and Trametes versicolor were among those identified,and they differed significantly in the level of betulin and betulinic acid and phenols,flavonoids and antioxidant properties in fungal tissues extracted with methanol and with ethanol(p ≤0.01).G.applanatum had the most betulin(3.642% )and S.commune the most betulinic acid(1.413% ).All tested fungi had high antioxidant activity,and L.betulina had the highest(97.775% ).The highest amounts of phenol(719.993 mg mL-1)and flavonoids(361.403 mg mL-1)were found in the ethanolic extract from G.applanatum.Considering the results of this study and the low cost and convenient access to these fungi,they should be good sources for producing different drugs.

Keywords Fungi·Alder·Betulin·Betulinic acid·Antioxidant

Introduction

Many fungi produce compounds that have medically useful properties such as antifungal,anti-inflammatory,anti-cancer,and antiviral(Ajith and Janardhanan 2007)and are widely used as antibiotics,exo-enzymes,mycotoxins,etc.in various industries(Saadatmand 2007).Fungi such as polypores that grow on different trees absorb and use metabolites from these trees(Faass 2012)and are a major source of pharmacologically active substances.Ganoderma lucidum for example,has high levels of antioxidant and biologically active compounds, consisting mainly of polysaccharides and terpenoids, that have anticancer properties, can modulate the immune system, and are therapeutic against HIV(Lin 2005;Paterson 2006;Kao et al.2013;Barbieri et al.2017).Aqueous extracts from this fungus also contain saponins,flavonoids,cardiac glycosides and steroids(Manasseh et al.2012).Schizophyllum commune(Agaricales)produces triterpenoid saponins(Wu et al.2012),antibacterial phthalic acid(Joel and Bhimba 2013)and antifungal compounds(Teoh and Don 2013).

Antiviral activity against type A influenza virus of birds and humans has also been reported for compounds from Daedaleopsis confragosa,Datronia mollis,Ischnoderma benzoinum, Laricifomes officinalis, Lenzites betulina,Trametes gibbosa and T.versicolor(Kabanov et al.2011;Teplyakova et al.2012).Abundant betulin was found in three parasitic Hymenochaetaceae fungi(Inonotus obliquus, Piptoporus betulinus and Daedalea confragosa growing on birch trees(Jasicka-Misiak et al.2010),and betulin,betulinic acid,phenol and flavonoids were found in Hyphodontia paradoxa and Stereum hirsutum associated with birch(Nazari et al.2016).

Betulin and betulinic acid,the product of botulin oxidation,are valuable therapeutic triterpenes,treating burns and eczema,and a wide range of different types of cancers,including cancers of the lung,intestine,uterus,prostate,breast,skin,blood and diseases such as AIDS,malaria and hepatitis(Kessler et al.2007;Feng et al.2013).Perhaps most important are the antitumor,antiviral and antibacterial properties of betulin(Krasutsky 2006).Betulinic acid is also antiviral against HIV and highly cytotoxic to different cancer cell lines(Cichewicz and Kouzi 2004).

Phenolic and flavonoids compounds are a group of aromatic secondary metabolites that are widely distributed throughout plants and fungi and have numerous biological effects such as antioxidant and antibacterial activity.These compounds are beneficial for treating many stress-related diseases such as cardiovascular disease, inflammatory bowel syndrome and Alzheimer’s disease.Some synthesized antioxidants have disadvantages,such as damaging the liver and causing cancer.Hence,many of these substances are extracted from plants,fungi and bark to provide antioxidants needed by the body but have fewer side effects and greater effectiveness(Rice-Evans 2004).Therefore,finding good natural sources to produce strong biologically synthesized antioxidants with greater efficacy but less health risk is a serious necessity.

In the present research, we therefore collected and identified fungal fruiting bodies on Caucasian aldertrees,and estimated levels of betulin and betulinic acid,phenols,flavonoids and antioxidant activity andy to assess their potential as a source of valuable medicinals.

Materials and methods

Sampling and crude extraction

Macroscopic fungi were collected and transferred to the Fungi Laboratory of Forest Sciences Faculty of Gorgan University of Agricultural Sciences and Natural Resources,Iran and identified using valid identification keys based on macro-and microscopic characteristics(cap shape and size,pore size and spore size)from Caucasian alder trees after forest monitoring in Golestan Province,Iran.Fungi were dried at room temperature, then ground with a wood chipper,and 10 g of powdered fungus in 100 mL of 95% HPLC-grade ethanol was placed on a magnetic shaker without heat for at least 48 h.Samples were then placed in an ultrasonic bath for 12 min.The extracts were then placed in a relatively dark environment,filtered through a 42×42 whatman filter(England)and were kept in the laboratory for HPLC analysis.

Measuring betulin and betulinic acid

Authentic betulin and betulinic acid were purchased from Sigma-Aldrich(St.Louis,MO);0.01 g in 10 mL methanol was used for liquid chromatography and as the standard solution for HPLC.Betulin(5-2000 mg L-1)and betulinic acid were prepared in concentrations of 0.05-1000 mg L-1for HPLC analysis and generating the calibration curve.The betulin and betulinic acid concentrations in each of the fungal samples were calculated using the area under the peak and the linear equation with high correlation coefficient obtained from the standard curve.The liquid chromatography system consisted of an L-7100 pump(Oven Hitachi,Japan),L-2450 software,O-ring chrome, C18 column,and reverse phase column of 4.6 mm×250 mm with 5 μm pore diameter.The mobile phase consisted of 84.16% acetonitrile in deionized water.Flow rate was 1.0 mL min-1),temperature was 25 C,and by UV detection at λ=210 nm(Zhao et al.2007).To estimate the amount of betulin and betulinic acid in the fungal samples,about 40 μL of the 0.42 μm-filtered ethanol extract was injected into the HPLC.The peaks in the chromatogram obtained for each sample were then compared to the standards(Fig.1).

Antioxidant properties measurements

The 2,2-diphenyl-1-picrylhydrazyl(DPPH)free radical scavenging method was used to measure antioxidant activity.DPPH was first prepared at 0.1 mM in methanolic and ethanolic solvent,and 1 mL was mixed with 1 mL of the extract in the desired solvent(methanol and ethanol 80% )and was shaken.The prepared solution was placed in the dark for 30 min, then absorbance at 517 nm was measured with the spectrophotometer(Biochrom Libra S12 UV/VIS,Germany).The blocking effect of DPPH free radical was then calculated as I=(Ablank-Asample)-×100,where Ablankis the absorption of DPPH alone at 517 nm and ASampleis the absorption of the sample at 517 nm.

Total phenol measurements

Total phenol compounds were determined using the Folin-Ciocalteu chelate(Merck,Germany).100 μL of Folin’s reagent was added to 20 μL of methanolic and ethanolic fungal extracts.After adding 1.16 mL of distilled water to the prepared solution,samples were placed in the dark for 5 to 8 min,then 300 mL of sodium carbonate(1 M)was added,and samples were placed in the dark in a steam bath(40°C)for 30 min.Absorption of samples was read at 760 nm with the spectrophotometer.Standard curves prepared using authentic gallic acid(0-200 ppm)(Merck,Germany) and plotted with excel software (Microsoft,Redmond,WA,USA)were used to estimate the amount of fungus phenolic compounds per gram of dried mass of fungi(Mashayekhi and Atashi 2014).

Fig.1 HPLC chromatogram for standards.a Betulin.b Betulinic acid.Units of X-axis is minutes,units of Y-axis is milliabsorbance units detected by the diode array detector

Total flavonoid measurements

Methanolic and ethanolic fungal extracts(0.5 mL)were mixed with 1.5 mL of 80% solvent,0.1 mL of aluminum chloride,0.1 mL of 1 M potassium acetate and 2.8 mL distilled water and held at room temperature for 30 min.Absorption was then measured at 415 nm with the spectrophotometer and compared with the standard curve for quercetin at 0-50 ppm generated by Excel. Flavonoid concentration was calculated and determined as milligrams quercetin equivalents per gram of fungal dry mass(Mashayekhi and Atashi 2014).

Fig.2 Fungi collected from the bark of Caucasian Alder(photos by authors).a Lenzites betulina(L.:Fr.)Fr.(Polyporales).b Auricularia mesenterica(Dicks.)Pers.(Auricularales).c Schizophyllum commune Fr.(Agaricales).d Trichaptum biforme(Fr.:Kl.)Ryv.(Polyporales).e Trametes versicolor(L.:Fr.)Pilat.(Polyporales).f Fomes fomentarius (L.: Fr.) Kickx. (Polyporales). g Ganoderma applanatum(Pers.:Wallr.)Pat.(Polyporales).h Rigidoporus ulmarius(Sowerby)lmazeki(Polyporales)

Statistical analyses

Significant differences among mean amounts or activities for the respective compounds produced by each fungus were compared using an one way ANOVA and two-way factorial test in MSTATC(Michigan State University,East Lansing,MI,USA)and SPSS version 16(SPSS,Chicago,IL, USA) software. Means were also compared using Duncan’s test.To analyze and determine the area under the curve for betulin and betulinic acid,EZChrom Elite 3.2.0(Japan)software was used,and the calibration curve was plotted.

Results

Fungi collected from Caucasian alder trees

Fungi collected from the bark of Caucasian Alder are shown in Fig.2.

Betulin and betulinic acid in fungal extracts

In the HPLC analysis,the betulin peak appeared after 20-22 min.According to the retention time of the betulin and betulinic acid standards, the peak for the active material in the samples was determined (Fig.3). The betulin content in each sample was then estimated by measuring the area of the peak and using this area in the linear equation for the standards as follows:y=3729.8×23,891 with a correlation coefficient of R2=0.9979 for betulin and y=3430.3×11,496.7 with a correlation coefficient R2=0.9953 for betulinic acid.

Based on the results of the variance analysis of betulin and betulinic acid from the fungal species on Caucasian alder presented in Table 1,the amount of betulin and betulinic acid differed among the fungi(p ≤0.01).

The results of the comparison of the mean percentage of betulin and betulinic acid in the fungi are presented in Fig.4(p ≤0.01).G.applanatum had the most betulin(3.642% ),and A.mesenterica and L.betulina had the least(very low and zero).The highest amount of betulinic acid was found in S.commune fungus(1.413% )and the lowest was obtained in L.betulina(0% ).

Antioxidant activity in fungal extracts

In the variance analysis of antioxidant activity of extracts from the fungi on the alder trees(Table 2),the antioxidant activity differed significantly among different species,and the interaction between fungal species and the solvents was significant(p ≤0.01).Based on the results,there is no significant difference between the two solvents in the extraction of materials with antioxidant activity.

The results of the comparison of mean antioxidant activity in extracts from the different species of fungi are presented in Fig.5.Although some species differed significantly in antioxidant activity,the activity for all species was high,with the highest in L.betulina(97.775% ).

In the comparison of the antioxidant activity in the extracts from two solvents,the highest activity was found in the methanolic extract from L.betulina.For the extracts of both solvents for all species,the antioxidant level was between 75 and 100% ,and the two solvents differed significantly in antioxidant activity of the extract only for L.betulina,S.commune,and T.biforme(Fig.6).

Total phenol content in fungal extracts

According to the equation obtained using the gallic acid standard curve (y=0.0059×0.0068, R2=0.9967), G.applanatum had the highest phenol level.Table 3 shows the results of variance analysis,and Fig.7 shows the average phenolic content in all species.The highest total phenol was in the G. applanatum fungus(659.186 mg mL-1),which is much higher than that of other macroscopic fungi.

The two highest total phenol content was found in the ethanolic extract(719.993 mg mL-1)and the methanolic extract of G.applanatum,followed by the methanolic extract of T.versicolor and F.fomentarius(approximately 100 mg mL-1).For all fungi except F.fomentarius,the two solvents differed significantly in the phenol content extracted.

Total flavonoid content in extracts from fungi

The quercetin standard curve fit the equation y=0.0057×0.0785 (R2=0.9927). The results of the variance analysis of the data showed a significant difference in the total flavonoid extracted with the two solvents among the fungal solvent and a significant effect of species solvent interaction on total flavonoid content(Table 4).Figure 9 shows the mean flavonoid content extracted from the various species of fungi from the alder trees.The highest total flavonoid (233.56 mg mL-1) content was found in G.applanatum.

The highest total phenol content was found in the ethanolic extract of G.applanatum(361.403 mg mL-1)(Fig.10).

Fig. 3 HPLC chromatograms of betulin and betulinic acid in macroscopic fungi of Caucasian alder.a Lenzites betulina.b Auricularia mesenterica. c Schizophyllum commune. d Trichaptum biforme.e Trametes versicolor.f Fomes fomentarius.g Ganoderma applanatum.h Rigidoporus ulmarius.Units of X-axis is minutes,units of Y-axis is milli-absorbance units detected by the diode array detector

Table 1 Variance analysis of betulin and betulinic acid macroscopic fungi of Caucasian alder trees

Fig.4 Mean amount of a betulin and b betulinic acid in fungi on Caucasian alder.A Lenzites betulina.Means with different lowercase letters differed significantly. B Auricularia mesenterica. C Schizophyllum commune.D Trichaptum biforme.E Trametes versicolor.F Fomes fomentarius.G Ganoderma applanatum.H Rigidoporus ulmarius

Table 2 Results of variance analysis of antioxidant activity in methanolic and ethanolic extracts from fungi of Caucasian alder trees

Evaluation and comparison of ethanol and methanol as extraction solvents

There was no significant difference between two solvents in the extraction of antioxidant properties(Table 2).The mean comparison of total phenols and total flavonoids from the species showed differences in the extraction efficacy between ethanol and methanol.Ethanol was better for solubilizing phenol and flavonoids of fungi extract and is thus preferred solvent(Fig.11).

Discussion

Basidiomycete fungi produce different types of secondary substances important for flavor,aroma,color and toxicity among others and used in medical sciences,agriculture and industry.In the present study,during summer and autumn forest monitoring,eight macroscopic fungi were collected and identified from the trunks of standing and fallen alder trees:G.applanatum,L.betulina,T.biforme,R.ulamrius,F. fomentarius, S. commune, A. mesenterica, and T.versicolor.

Fig. 5 Mean percentage antioxidant activity in different fungal species on Caucasian alder.A Lenzites betulina.Means with different lowercase letters differed significantly.B Auricularia mesenterica.C Schizophyllum commune.D Trichaptum biforme.E Trametes versicolor.F Fomes fomentarius.G Ganoderma applanatum.H Rigidoporus ulmarius

The amount of betulin in the studied fungal species from highest to lowest was G.applanatum(3.642% )＞S.hirsutum ＞S.commune ＞F.fomentarius ＞T.biforme ＞R.ulmarius ＞T.versicolor ＞A.mesenterica ＞L.betulina.I.obliquus,P.betulinus and D.confragosa associated withBetulaceae family(Betula pendula L.,Corylus avellana L.,Alnus glutinosa L.)were found by Jasicka-Misiak et al.(2010)to have 65.8% ,2.5% and 0.2% betulin,respectively.The amount of this active material in the natural bark of the tree species was 64.3% ,1.7% and 0% ,respectively.The high level of these metabolites in the fungi make them valuable sources of betulin(Jasicka-Misiak et al.2010).

Fig.6 Mean comparison of antioxidant activity in ethanolic(MeOH)and ethanolic(EtOH)extracts of different fungal species on Caucasian alder.Means with different lowercase letters differed significantly.A Lenzites betulina.B Auricularia mesenterica.C Schizophyllum commune.D Trichaptum biforme.E Trametes versicolor.F Fomes fomentarius.G Ganoderma applanatum.H Rigidoporus ulmarius

Table 3 Results of variance analysis of total phenol in methanolic and ethanolic extracts from fungi on Caucasian alder trees

Fig.7 Comparison of mean total phenol content in different species of macroscopic fungus of Caucasian Alder trees.Means with different lowercase letters differed significantly (p ≤0.01). A Lenzites betulina.B Auricularia mesenterica.C Schizophyllum commune.D Trichaptum biforme.E Trametes versicolor.F Fomes fomentarius.G Ganoderma applanatum.H Rigidoporus ulmarius

The amount of betulinic acid in the fungi in the present study from highest to lowest was S. commune(1.413% )＞A.mesenterica ＞G.applanatum ＞H.paradoxa ＞F.fomentarius ＞T.biforme ＞R. ulmarius ＞T.versicolor ＞L.betulina.Faass(2012)also confirmed the presence of betulin and betulinic acid in I. obliquus growing on birch trees.G.applanatum can thus be considered for producing betulin and S.commune and A.mesenterica for betulinic acid.In addition to their benefits as food and natural properties as therapeutics,these fungi can be cultured for a convenient,quick and cost-effective solution to produce these secondary metabolites,as done for inertol,a sterol produced by I.obliquus(Kahlos 1994).Betulin has also been extracted from I.obliquus using highintensity pulsed electric fields by Yin et al.(2007)and Bai et al.(2012)optimized betulin synthesis by mycelia of I.obliquus in suspension culture.

Table 4 Results of variance analysis of total flavonoid methanolic and ethanolic extracts from fungi on Caucasian alder trees

Fig.8 Comparison of total phenol content in methanolic(MeOH)and ethanolic (EtOH) extracts of different fungal species from Caucasian alder trees.Means with different lowercase letters differed significantly.A Lenzites betulina.B Auricularia mesenterica.C Schizophyllum commune.D Trichaptum biforme.E Trametes versicolor.F Fomes fomentarius.G Ganoderma applanatum.H Rigidoporus ulmarius

Fig.9 Mean total flavonoids in methanolic extracts of different fungal from Caucasian alder.Means with different lowercase letters differed significantly.A Lenzites betulina.B Auricularia mesenterica.C Schizophyllum commune. D Trichaptum biforme. E Trametes versicolor. F Fomes fomentarius. G Ganoderma applanatum. H Rigidoporus ulmarius

Our evaluation of the antioxidant activity in extracts of the studied fungal species showed that most of them had high antioxidant properties,especially L.betulina and T.versicolor(98% and 88% ,respectively in the methanolic extract).For similar extractions,Rajoriya et al.(2015)estimated antioxidant activity of 92% of G.applanatum and Tabari et al.(2013)determined activity at 20% for T.gibbosa.In our study,the highest total phenols among the extracts from the fungi was present in the ethanolic and methanolic extracts from G.applanatum.Because secondary metabolites in fungi vary in their solubility in extraction solvents (Jonathan and Fasidi 2003), not surprisingly,the amount of phenols in G.applanatum,F.fomentarius and T.versicolor varied in methanol(600,90 and 90 mg mL-1,respectively)and ethanol(720,90 and 50 mg mL-1,respectively).The levels that we obtained were higher than those found by Daniel and McElhenney(2013)in these three fungi in methanol solution(147.780,12.960 and 4.070 mg mL-1,respectively)and in ethanol(7,12 and 14 mg mL-1,respectively).In another study of different species of Ganoderma in India,Rajoriya et al.(2015)determined the total phenol content in a methanolic extract of G.applanatum was about 12 mg mL-1.These studies show the range in levels and highlight the need for selecting species and isolates that are the highest producers and optimizing the culture conditions.

Fig. 10 Mean flavonoids in methanolic (MeOH) and ethanolic(EtOH)extracts from fungal species on Caucasian alder trees.Means with different lowercase letters differed significantly.A Lenzites betulina.B Auricularia mesenterica.C Schizophyllum commune.D Trichaptum biforme.E Trametes versicolor.F Fomes fomentarius.G Ganoderma applanatum.H Rigidoporus ulmarius

Fig.11 Mean(a)total phenol and(b)flavonoid in ethanolic(MeOH)and ethanolic(EtOH)extracts from fungi on alder trees.Means with different lowercase letters differed significantly

The most flavonoids in the present study were found in the ethanolic extract of G.applanatum,in contrast to the very low level(1 mg mL-1)obtained from G.applanatum by Rajoriya et al.(2015).On the other hand,from G.applanatum,F.fomentarius and T.versicolor,Daniel and McElhenney(2013)extracted 139,38 and 9.5 mg mL-1,respectively,in methanol and 104,32 and 8 mg mL-1,respectively,in ethanol;thus,we obtained over 2.5 times more flavonoids from these fungi than did Daniel and McElhenney(2013).

Fungi are promising organisms for biotechnology research and production of important compounds for industry,medicine and agriculture,and those that can be used for food and drugs, are especially valuable and promising.Medicinal fungi have many properties to be explored and exploited for immunological and protective or toxic compounds against cancers, viruses, bacteria,parasites,hepatitis and diabetes and other serious diseases.The promising results from the present study provide the foundation for further research on cultivation and optimizing the use of these fungi.

Conclusions

As determined through 11 subcultures of Taxus cuspidata callus,the growth in FW and level of Taxol production were stable by the ninth generation.FW increased 2.77-fold,and Taxol production was 259.14 μg g-1DW.So the callus from the ninth generation could be used for selecting high Taxol-producing cell lines.After comparing normal plate culture, conditioned culture, and nursing culture,conditioned culture was used for single-cell cloning and an initial cell density 3×103cells mL-1was appropriate.After a stability analysis of 10 high-yielding cell lines,lines CL5,CL12,and CL21 were selected;Taxol contents were 0.0448% ,0.0477% ,and 0.0428% DW,respectively.The proliferation increase of CL5,CL12,and CL21 was 346.3% ,382.5% ,and 409.2% ,respectively.These three stable,high yielding cell lines are therefore candidate sources for the mass producing Taxol and promoting commercial production using cell culture.For obtaining a more stable,higher yield from such cells,selection and cultures methods will continue to be investigated in future research.This study on selection methods for high Taxolproducing cell lines of T.cuspidata lays a foundation for selection of other plant cells.Further research,such the optimizing the medium and culturing conditions,is now needed to move beyond the laboratory stage into full-scale production.