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Enhanced Anti-oxidant Activity as a Function ofSelenium hyperaccumulation in Agaricus bisporusCultivated on Se-rich Agri-residuesPoonam Bhatiaa, Satyendra Pandeyb, Ranjana Prakashb & Tejo Prakash Nagarajac

a Department of Biotechnology, Thapar Technology Campus, Patiala, Thapar University,Punjab, 147004, Indiab School of Chemistry and Biochemistry, Thapar Technology Campus, Thapar University,Patiala, Punjab 147004, Indiac School of Energy and Environment, Thapar Technology Campus, Patiala, Thapar University,Punjab 147004, IndiaPublished online: 19 Jan 2014.

To cite this article: Poonam Bhatia, Satyendra Pandey, Ranjana Prakash & Tejo Prakash Nagaraja (2014) Enhanced Anti-oxidant Activity as a Function of Selenium hyperaccumulation in Agaricus bisporus Cultivated on Se-rich Agri-residues, Journalof Biologically Active Products from Nature, 4:5-6, 354-364, DOI: 10.1080/22311866.2014.961103

To link to this article: http://dx.doi.org/10.1080/22311866.2014.961103

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Enhanced Anti-oxidant Activity as aFunction of Selenium hyperaccumulation in

Agaricus bisporus Cultivated on Se-rich Agri-residues

Poonam Bhatia 1, Satyendra Pandey 2, Ranjana Prakash 2, Tejo Prakash Nagaraja 3*

1 Department of Biotechnology, Thapar Technology Campus, Patiala,Thapar University, Punjab, 147004, India

2 School of Chemistry and Biochemistry, Thapar Technology Campus,Thapar University, Patiala, Punjab 147004, India

3 School of Energy and Environment, Thapar Technology Campus, Patiala, Thapar University, Punjab 147004, India

Abstract: Modulations in antioxidant properties of button mushroom, Agaricus bisporus, cultivatedon selenium-rich wheat straw from the seleniferous belt of Punjab (India) were examined in comparison to themushrooms cultivated on normal straw. Selenium (Se) uptake by Se-fortified mushrooms (122 ± 1.8 μg/g DW)was significantly higher than control (14 ± 0.7 μg/g DW). The antioxidant activity, as determined by variousassays viz., free radical scavenging, metal chelating activity, reduced lipid peroxidation; as well as inhibition ofcancer cell proliferation was higher in experimental mushrooms as compared to control. Further analysis of Serich extracts, using FTIR and MS indicated possible presence of selenoergothioneine-like moiety as an expressionof Se uptake by mushrooms. The present study demonstrates the increased anti-oxidant activity in mushroomscultivativated on Se hyperaccumulated agricultural residues.

Key words: Selenium, Agri-residues, Agaricus bisporus, Antioxidant activity, Selenoer-gothioneine.

IntroductionMushrooms have long been appreciated for

their nutritional value, as their fruiting bodies,on a dry weight basis, contain about 39.9 %carbohydrate, 17.5 % protein and 2.9 % fats, withthe rest being minerals 1. Medicinal mushroomsare source of good immunotherapeutic and anti-cancer agents, capable of interfering with parti-cular cellular signal transduction pathways 2.

Agaricus bisporus is the most widely producedmushroom in the world (4 million tons/year).These species are excellent source of Se,riboflavin, pantothenic acid and other important

nutrients, in addition to ergothioneine, animportant phytonutrient. Ergothioneine, is areadily bioaccessible compound and preventslipid proxidation by toxic compounds 3. Ourrecent report on the bioaccessibility andspeciation of Se in mushrooms, showed Se to bemainly present as selenomethioneine (73 %)indicating the rest of fraction (27 %) constitutingknown and un-known Se containing moieties 4.

Most of the studies reported till date has beenfocused on Se uptake by mushrooms throughexogenous supplementation of Se dominantly asselenite. However, similar studies on mushrooms

ISSN Print: 2231-1866ISSN Online: 2231-1874

*Corresponding author (Tejo Prakash Nagaraja)E-mail: < ntejoprakash@thapar.edu > © 2014, Har Krishan Bhalla & Sons

TBAP 4 (5 & 6) 2014 pp 354 - 364 354

Received 20 April 2014; accepted in revised form 11 July 2014

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cultivated on agricultural residues naturallyenriched with selenium have hitherto not beenreported to the best of our knowledge. Post-harvest agricultural residues such as thosegenerated in seleniferous region of Punjab, India,contain significantly high Se content in plant partssuch as grains and straw of wheat and othercereals 5.6.

Keeping this view, we examined the extent ofSe uptake by A.bisporus cultivated on wheatstraw harvested from seleniferous region and itscorresponding influence on anti-oxidant andcytotoxic activity expressed by these Se-richmushrooms.

Materials and methodsChemicals and reagents

Standards viz., BHT (tert-butylhydroquinone),and 2,2-diphenyl-1-picrylhydrazyl (DPPH),dithiothreitol (DTT), 2-mercapto-1-methyl-imidazole (MMI), betaine, sodium dodecylsulphate (SDS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Dulbecco’sModified Essential Medium (DMEM), Foetalbovine serum (FBS) were purchased from Sigma(St.Louis, USA). 2-tert-butyl-4-methoxyphenol(BHA), gallic acid (GA), 2,3-diaminonaphthalene(DAN) and Dimethyl sulphoxide (DMSO) werepurchased from Hi-Media (India). Methanol,ethanol , cyclohexane and hexane were obtainedfrom SDFine (India). Nitric (HNO3), hydrochloric(HCl) and perchloric (HCLO4) acids wereprocured from Merck (India). Rests of thereagents were obtained from Loba Chemie, India.

Mushroom cultivationThe strain of Agaricus bisporus (U-3) was

procured from Punjab Agricultural University(PAU, Ludhiana). This fungus was cultured onpotato dextrose agar (PDA) medium and wasstored at 4°C till use. The Se rich wheat strawwas collected from Jainpur (31°13’N, 76°21’E,Nawanshahr-Hoshiarpur region, Punjab), India.Similarly, straw from non-seleniferous region(30°34’N, 76°38’E Patiala, Punjab, India) wasused as control. The Mushrooms were cultivatedon compost prepared from agricultural residues,following method outlined by Punjab Agricultural

University, India 7. The compost prepared fromthe both varieties of straws was inoculated withthe fungal spawn. After inoculation, the compostwas filled in polypropylene bags and kept incultivation chamber (25 ± 4°C). At maturation(22-25 days after inoculation), fruiting bodieswere collected, dried at 40°C for near completedehydration and powdered using agate mortar.

Determination of Se concentrationThe Se content of powdered samples was

analysed using fluorescence spectrometry(Perkin-Elmer LS 45) 8. Briefly, this methodinvolved digestion with HNO3 and HClO4,reduction of Se from Se+6 to Se+4, complexing ofSe+4 with DAN and extraction of the piazselenolin cyclohexane. The emission spectrum ofpiazselenol complex formed during the reactionwas measured using fluorescence spectrometer(Perkin Elmer LS45) at excitation and emissionwavelength of 320 and 520 nm respectively. Sequantification in each sample was carried out byrelative method using emission spectrum of NISTcertified Se ICP standard solution (SRM1349).

Sample preparationFor various antioxidant properties

The samples (1g of dry powder) were extractedby stirring with 10 ml of 50 % methanol for 3 h,at room temperature using ultrasonicator bath, andfiltering through Whatman # 1 paper. The filtrateswere concentrated with a rotary vacuumevaporator at 40°C. The resultant extracts werestored at 4°C until use.

For extraction of ergothioneineOne gram of freeze dried and powdered fruiting

bodies were added to 20 ml of cold ethanolicextraction solution (10 mm DTT, 100 μm betaine,100 μm MMI in 70 % ethanol) and mixed byvortexing and subsequent sonication for 3 min.An ethanolic solution (4 ml) of sodium dodecylsulfate (SDS) was added and mixed by mildshaking. The mixture was centrifuged at 4000 rpmfor 15 min. 10 ml of the supernatant wasevaporated using rotary vacuum evaporator todryness and the residue was re-suspended in 10ml of distilled water (pH 7.3) followed by

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centrifugation at 4000 rpm for 15 min. Theresulting supernatant was further used for theanalysis.

For cytotoxicity assayOne gm of freeze dried mushrooms (both Se

and NSe) were extracted with different solventsin order of their increasing polarity (hexane,ethanol, methanol and water) in the ratio 1:10 (w/v). The extracts were vortexed and subsequentlysonicated for 3 min. The mixture was centrifugedat 6000 rpm for 10 min. The resultant supernatantwas evaporated to dryness at room temperature.The residue obtained was weighed and reconsti-tuted in respective solvents and were stored at -20°C till use. The extracts from various solventfractions were screened using MTT assayaccording to method outlined by Mossmann andMosmann 9 using human alveolar adeno-carcinoma (A549) cell line.

Determination of total phenol content and anti-oxidant activityTotal phenol content

Total phenolic compounds were measured usingLowry reagent 10 and expressed as gallic acid(GA) equivalents.

Lipid peroxidation activityLipid peroxides were extracted by grinding the

sample with 5 ml of 5 % (w/v) metaphosphoricacid and 100 μl of 2 % (w/v) butyl hydroxytoluene(in ethanol). Homogenates were filtered andcentrifuged at 15000 g for 20 min. The chromogenwas formed by mixing 50 μl of supernatant, 50μl of 0.2 % (w/v) butyl hydroxytoluene (BHT),250 μl of 1 % (w/v) thiobarbituric acid (TBA)(in 50 mm NaOH), and 250 μl of 2N (v/v) HCl,and by incubating the reaction mixtures at 95°Cfor 30 min 11. A blank for all samples was preparedby replacing the sample with extraction medium,and controls for each sample were prepared byreplacing TBA with 50 mm NaOH. The reactionwas stopped by cooling the samples in an ice bath.For determination of thiobarbituric acid reactivesubstances (TBARS), the chromogen formed wasextracted by adding 2 ml of 1-butanol, the tubeswere vigorously shaken, the organic (upper) phasewas separated by low speed centrifugation and

assayed fluorimetrically (Perkin-Elmer-LS45) atan excitation wavelength of 532 nm and emissionwavelength of 550 nm. Calibration curves weremade using Malondialdehyde (MDA;Sigma) inthe range of 0.5-5.0 μM.

Total antioxidant activity of methanolic extractof A. bisporus was measured using UV-visiblespectrometer (Hitachi U2900) according to themethod (phospho-molybdenum assay) outlined byImran et al. 12 with gallic acid (GA) (0.01 - 0.1mg/ml) as standard.

DPPH assayThe scavenging activity of the methanolic

extracts (0.05 to 1.0 mg/ml) from mushroom onDPPH radicals was measured spectrophoto-metrically following the method of Chu et al 13.The scavenging activity (% SA) of DPPH radicalswas calculated using equation [% SA= (1-Abs inthe presence of sample/Abs in the absence ofsample) × 100]. Butylated hydroxyl toluene(BHA) (0.1- 1 mg/ml) was used as standard.

Metal chelating activityThe chelating activity of the various concen-

trations of extracts (1-10 mg/ml) for ferrous ionswas measured spectrophotometrically followingthe ferrozine method 14. The metal chelatingactivity of the mushroom extracts was calculatedas: % chelating activity = [(Anegative – Asample) /Anegative] ×100, where A is absorbance. EDTA wasused as positive control while absence of extractof the mushroom was the negative control.

Determination of cytotoxicity (MTT assay)The human alveolar adenocarcinoma (A549)

cell line was grown in a humified atmosphere (5% CO2) at 37°C in Dulbecco’s modified essentialmedium (DMEM) containing 10 % FBS and 1 %Pen-Strep (pencillin-streptomycin).

The adherent cells were detached bytrypsinization from 25 cm flask on the first day.The viable cells were counted by using 0.5 %Trypan Blue dye exclusion method and cell countwas adjusted to 2 x 105 cells. 50 μl of variousextracts was added in triplicates in a 96 wellmicrotitre plate and air-dried overnight in asepticcondition to let the solvent evaporate completely.Wells with solvent alone were kept as controls

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and set for drying in similar way. The wells werethen seeded using a concentration of 2 x 105 cells/ml (100 μl/well). The microtitre plate wasincubated at 37°C, 5 % CO2 for 24h. Afterincubation, the medium of each well was removedby aspiration and replaced with experimentalbioactive residue (50 μl/well) and incubated fora period of 24 h. After this interval, 0.05 mg ofMTT was added to each well and the plate wasincubated for 4h in CO2 incubator. Subsequentlyafter 4h, the media was removed from each welland DMSO was added to all wells to dissolve theformazan crystals. Cell viability was determinedby measuring the absorbance at 570nm usingBiotek throughput reader, Power wave 34. Theinhibition ratio (I) was calculated according tothe following equation:

I (%) = [(Acontrol group – A AB treated group)/Acontrol group] X 100

Preliminary investigations on selenoergo-thioneine like moiety

FTIR analysis was carried out for the extracts(prepared for selenoergothioneine) whereininfrared spectra were recorded in the range of4000-500 cm on Magna FTIR-550 spectro-photometer using KBr pellets. These sampleswere analyzed by HPLC (Waters-Alliance 2795)equipped with X-terra C-18 column connected toWaters-Micromass Q-Tof mass spectrometer(MS). The HPLC conditions were: injectionvolume 20 μL with isocratic mobile phaseconsisting of 30 % acetonitrile and 1 % aceticacid in water at a flow rate of 0.5 ml/min. TheMS conditions were: electrons spray ionizationin positive ion mode, capillary voltage (3000-

3250 V), source temperature (120°C), and probetemperature (250°C).

Statistical analysisAll estimations were carried in triplicates and

were expressed as mean ± standard deviation (SD)values. The comparison between Se and non-Sesamples were drawn using student‘t’ test.

Results and DiscussionSe hyperaccumulation in fruiting bodies

The concentration of Se in straw fromexperimental (Se) and control (non-Se) sites, usedfor preparation of compost vis-à-vis the Se contentin fruiting bodies of mushrooms cultivating onthe straw varieties are presented in Table 1. Thefruiting bodies harvested from the compost of Se-rich straw containing a total Se concentration (indry weight) of 27.0 ± 0.2 μg/g were noted toaccumulate Se upto 122.0 ± 1.8 μg/g as comparedto control/ non-Se mushroom (14.2 ± 0.7 μg/g)cultivated on control straw (1.9 ± 0.8 μg/g).

Many investigations have been carried oncultivation of Se-enriched mushrooms usingsubstrates exogenously supplemented with Sewherein dominantly inorganic form of seleniumas selenite was used. The present study showedthat Se can be effectively mobilized bymushrooms such as A.bisporus cultivated onsubstrates naturally enriched with Se andcontaining bioaccessible organoseleniummoieties 4. The extent of Se removal fromsubstrate and subsequent accumulation in thefruiting bodies was comparable to concentrationreported in case of Agaricus sp. (ChampignonMushroom 110 μg/g) cultivated on compost

Table 1. Total selenium levels, yield of methanolic extracts, total phenolsand lipid peroxidation in Se-rich and control mushrooms. [n = 3]

Sample Se (μμμμμg g-1 dw) Yield of methanolic Yield of LipidStraw Mushroom extracts (%) total phenols peroxidation

(mg GA g-1 dw) (mg GA g-1 dw)

Se 27.0 ± 0.2 122.0 ± 1.8 36.1 ± 1.5 3.55 ± 0.085 10.7 ± 3.6Non-Se 1.9 ± 0.8 14.2 ± 0.7 31.3 ± 1.1 3.15 ± 0.094 32.0 ± 1.6

*** *** * ** ***

*: P<0.05; **: P<0.01; ***: P<0.001

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supplemented with 10 μg/g of sodium selenite 15

and Ganoderma lucidium (72 μg/g) cultivated oncompost supplemented with 100-250 μg/g ofsodium selenite 16. In comparison to the reportedlevels of Se uptake from supplemented substrates,our observations are important in demonstratingits mobilization by mushrooms cultivated onsubstrates that dominantly contain the organicforms of Se 4.

The influence of the Se hyperaccumulation inA. bisporus was further examined on theantioxidant properties of mushrooms usingvarious assay systems.

Bioactivity as function of Se hyperaccumu-lationYield of methanolic extracts and total phenolcontent

The yield of methanolic extracts frommushroom (Table 1) indicated that most of thesoluble components in mushrooms were high inpolarity. The yields of methanol extracts (% DW),were 36.1 ± 1.5 % in Se rich mushrooms whencompared to 31.3 ± 1.1 % dry weight detected incontrol mushrooms. The yield of methanolicextracts was higher (p<0.05) in Se-enrichedmushrooms as compared to those cultivated oncompost of non-Se (control) straw therebyindicating possible enhancement in biologicalefficacy of Se rich mushrooms. The amount ofphenolic compounds in the methanol extractsfrom the Se-enriched mushroom (3.55± 0.085 mgGA/g DW) was significantly higher (p<0.005)than control (3.15 ± 0.094 mg GA/g DW) (Table1).

With reference to the anti-oxidant profile of theSe accumulated mushrooms, the yields of themethanolic extracts obtained in this study aresimilar to that obtained in previous reports withyields varying from 15.9 % to 43.9 %.[17,18]Antioxidant properties of phenolic compoundsplay a vital role in the antioxidative defensemechanisms exhibited by the biological systems19. In addition, Se is well known for its associationwith antioxidant mechanisms in humans andanimals 20. Therefore, increase in the phenolcontent in Se-enriched mushrooms may be dueto the influence of Se in promoting the synthesis

and activity of antioxidant metabolites, a featurethat has been observed in the present study.

Profile of anti-oxidant activityMalondialdehyde (MDA) is the secondary

byproduct, which is released during the lipidperoxidation. A decrease in the production ofMDA in turn symbolizes the inhibition of lipidperoxidation. The potential of mushroom extractsin inhibiting lipid peroxidation was studied byTBARS assay (Table 1). The selenium richextracts (10.73 ± 3.6 nM/g) showed significantdecrease (p< 0.001) in MDA levels as comparedto control (32.0 ± 1.6 nM/g), indicating significantinhibition of lipid peroxidation by extracts fromSe-rich mushrooms. Similarly various researchershave defined selenium role in inducingantioxidant capacity in terms of decrease in lipidperoxidation 21,22.

The total antioxidant activity of the methanolextracts of Se-enriched mushrooms and controlincreased with increasing concentrations (Table2). Se-enriched mushrooms at 10 mg/ml ofmethanolic extract showed significantly (p <0.001) higher activity (97.0 ± 0.95 μg GA/10 mgextract) than (86.1± 0.71 μg GA/10 mg extract)mushrooms cultivated on control straw, whereasthe difference in the levels of activity was atp<0.05 to p<0.01 at extract concentration rangingfrom 1.0 mg/ml to 5.0 mg/ml.

The free radical scavenging potential ofmushroom extracts were studied by examiningthe effect of methanolic extract on scavenging ofDPPH. The scavenging effect of methanolicextract of Se-enriched and control mushroomsincreased with increasing concentration of extract(Table 2). At 1.0 mg/ml of Se-enriched extract,the scavenging effect was 91.4 ± 1.5 % whencompared to control (84.9 ± 0.5 %). In case offree radical scavenging ability of the methanolicextracts, the levels observed in controlmushrooms were relatively higher than thosereported by Huang et al. 23 wherein scavengingability of 97.1 % was reported at 2.5 mg/ml ofmethanolic extract from A. blazei. The results inthe present study revealed that methanolic extractsof Se-enriched mushroom acted as free radicalscavengers, acting possibly as primary

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Tabl

e 2.

DPP

H s

cave

ngin

g po

tent

ial;

met

al c

hela

ting

effic

acy;

and

tota

l ant

ioxi

dant

activ

ity o

f met

hano

lic e

xtra

cts f

rom

Se

enri

ched

and

con

trol

A. b

ispo

rus

(n =

3)

Sam

ple

Scav

engi

ng e

ffec

t (%

) of m

ushr

oom

spe

cies

on

DPP

HE

xtra

ct (m

g m

l-1)

0.05

0.1

0.2

0.3

0.4

0.5

1.0

Se48

± 0

.09

64.1

± 1

.368

.2 ±

0.7

872

.9 ±

2.3

76 ±

1.6

78 ±

1.7

91.4

± 1

.4N

on-S

e40

± 1

.059

.8 ±

2.2

62.8

± 1

.266

.9 ±

1.2

70.5

± 1

.474

.7 ±

1.0

84.9

± 0

.5*

***

**

***

BH

A@

96.7

± 0

.5M

etal

che

latin

g ac

tivity

(%)

Ext

ract

(mg

ml-1

)0.

41.

02.

03.

04.

05.

010

.0

Se-

10.8

± 0

.92

26.5

± 0

.98

39.0

± 0

.96

46.4

± 2

.83

53.4

± 0

.93

93.7

± 0

.79

Non

-Se

-1.

0 ±

0.73

4.4

± 1.

2216

.6 ±

1.7

828

.7 ±

1.7

933

.9 ±

0.8

378

.2 ±

1.7

4**

***

***

***

***

***

*ED

TA@

85.4

± 0

.7To

tal a

ntio

xida

nt a

ctiv

ity (μ

g G

A m

g-1

extr

act)

Ext

ract

(mg

ml-1

)1.

02.

03.

04.

05.

010

.0

Se11

.7 ±

0.6

13.9

± 0

.323

.7 ±

0.7

31.8

± 0

.538

.2 ±

1.0

97.0

± 0

.95

Non

-Se

9.4

± 0.

1711

.8 ±

0.6

20.6

± 0

.928

.4 ±

0.3

34.2

± 0

.986

.1±

0.71

****

***

***

***

@ :

posi

tive

cont

rols

*: P

<0.0

5**

: P<0

.01

***:

P<0

.001

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antioxidants.Chelating effect of methanolic extracts from

both mushrooms on ferrous ions increased withthe increase in concentration of extracts andsignificantly varied (p<0.001) between Se-richand control mushrooms (Table 2). Extracts fromSe-enriched and control reached their maximumchelating ability of 93.7 ± 0.79 % and 78.2 ± 1.79% respectively at 10 mg/ml. Metal ion chelatingcapacity plays a significant role in antioxidantmechanism since it reduces the concentration ofthe catalyzing transition metal in lipidperoxidation process. Since, ferrous ions are themost effective prooxidants in food systems, theenhanced chelating effects of methanolic extractsfrom Se-enriched mushrooms would be moredesirable over those obtained in controlmushrooms. Coordination of sulfur and Secompounds with metal ions has been proposedas an additional antioxidant mechanis 24. Evidencethat metal-antioxidant coordination leads toantioxidant activity are supported by in vitrostudies investigating metal-mediated oxidativestress and disease. Iron-mediated DNA damageinhibition is seen for variety of organoseleniumcompounds such as methyl-selenocysteine,selenocystamine, 3,3-diselenobispropionic acid,and 3,3-selenobispropionic acid 25. The high metalchelating activity of Se-enriched mushroom istherefore assumed to be due to specific metal-selenium compound interactions that greatlyaffect antioxidant activity based on the type ofmetal and the specific features of the Secontaining compounds.

Profile of cytotoxicityTo examine the effect of various fractions on

proliferation of cancer cells (A549), the crudeextracts of mushrooms (Se and NSe) from fourdifferent solvents viz. hexane, ethanol, methanoland water were tested. Out of the four solventfractions, hexane fraction showed significantinhibition of cell proliferation followed byethanol, methanol and water. The Se-rich fractioninhibited the proliferation by 89.6 %, 87.9 %, 64.9% and 47.5 % as compared to 83.2 %, 42.3 %,4.4 % and 9.4 % inhibition by Non-Se mushroomsin hexane, ethanol, methanol and water fractionsrespectively (Table 3).

The extracts obtained from Se-rich mushroomsindicated significant suppression of cellproliferation when compared to control which isbeing attributed to possible presence of varietyof known and unknown bioactive species in Sehyperaccumulating mushrooms 4. The presenceof such Se rich moieties might be directlyaffecting intracellular transduction pathways,triggering specific signaling reactions that mayadd to cancer inhibition. The present study issupported by the observations of Spolar et al. 26

where A.bisporus mushrooms enriched withexogenously supplemented selenium, were foundto suppress 7,12-dimethlybenz[a]anthracenebioactivation in mammary tissue. The majorbioactive metabolites found in mushroomspossessing anticancer properties can be highmolecular weight moieties such as Lentinan fromLentinula edodes 27, Schizophyllan (Sonifilan,Sizofiran, or SPG), from Schizophyllum commune28, or low molecular weight metabolites such asergosterol, caffeic acid phenethyl ester,cordycepin, panepoxydone and cycloepoxydonthat show specific cytotoxicity against tumor cells29. However, expression of such compounds and

Table 3. Percent inhibition of cancer cell proliferation against A549 cells by differentsolvent fractions from Se enriched and control Agaricus bisporus (n=3)

Sample % Inhibition of cell proliferationHexane fraction Ethanol fraction Methanol fraction Aqueous fraction

Se 89.6 ± 0.55 87.9 ± 2.2 64.9 ± 8.0 47.5 ± 3.2Non-Se 83.2 ± 3.70 42.3 ± 2.9 4.4 ± 3.9 9.4 ± 3.5

* *** *** ***

*: P<0.05; **: P<0.01; ***: P<0.001

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synthesis of other Se rich bioactive moieties asfunction of Se uptake is yet to be carried outextensively and therefore furthur characterizationof various chemical moieties in Se-rich mushroomis still required.

As an attempt to identify the presence of suchSe rich moieties, we looked at possible presenceof selenium containing analogue of ergothioneine,an important sulfur-rich antioxidant compound inmushrooms.

Preliminary observations on selenoergo-thioneine-like moiety

The FTIR spectra of extracts showed absorptionbands at 3424, 2925, 2516, 1630, 1409, 1045 and931 cm-1 (Fig. 1). A broad band centered at 3424cm-1 was assigned to hydrogen-bonded hydroxyland amine groups. The band at around 2925 cm-1

was assigned to a C-H stretching vibration. Thepeak at 2516 cm-1 was assigned to an O-Hstretching angle. The absorptions at 1630 cm-1

were assigned to the stretching vibrations of theCHO and C=O bonds. The broad absorptionbands with strong intensities at 1409 cm-1 couldbe assigned to bending vibrations of O-H bond.The peak at 931 cm-1 indicated the existence of

C=Se in the structure. These FTIR observationswere closely supported by the reports of AlMudhaffar and Hasan 30, where characteristicstrong bands due to ‹ C=Se were observed in therange between 864-987cm”1.

Extending further on the preliminary studies,mass spectral analysis showed the presence of amolecular ion [M+H]+ peak at m/z 278.1 (Fig. 2)which corresponds to the molecular formulaC9H15N3O2Se and calculated mass 277.1. Thiscompound with the molecular formulaC9H15N3O2Se appears to be Se analogue ofergothioneine (C9H15N3O2S) where sulphur isreplaced with Se (when accumulated at substantialrates). However, the FTIR and MS observationsare only suggestive and need detailed extractionand examination of various fractions.

Our earlier observations on mushrooms hyper-accumulated with selenium from agri-residuesindicated presence of about 23 % of unknownselenium moieties in addition to dominantpresence of selenomethioneine 4. In addition,studies carried out by Beelman and co-workerson mushrooms cultivated on substratesexogenously supplemented with Se as inorganicselenite indicated the synthesis of

Figure 1. FTIR spectrum of extract with Se-containing moiety

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selenoergothioneine with Se effectively replacingS in the ergothioneine moiety along with otherorganoselenium compounds 3,15. Pioneering workby this group envisaged enhanced potential ofselenoergothioneine in mushrooms due to com-bined benefits of two very strong antioxidantmoieties, viz., Se and ergothioneine.

The observations in the present study, thus,show enhanced anti-oxidant properties of Se-hyperaccumulated mushrooms during cultivationon agricultural post-harvest residues naturallyenriched with selenium. The increasing anti-oxidant activity in these mushrooms maypresumably be due to the formation of variety of

selenium containing moieties which otherwise arenot found in normally cultivated mushrooms.

AcknowledgementThe LCMS analysis by SAIF-Panjab University

(PU), Chandigarh and FTIR analyses carried outby SAIF-Indian Institute of Technology (IIT),Bombay are also duly acknowledged. The authorsare thankful to Punjab Agricultural University(PAU, Ludhiana) for providing the facility tocultivate mushrooms. Research grant under DBT-NDB to NTP and RGNF-UGC to PB are dulyacknowledged.

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