TAP CHl CONG THlflfNG
KHAO SAT HOAT TINH KHANG OXI HOA CUA CAO CHIET
METHANOL VO TRAI THOT NOT (BORASSUS FLABELLIFER L.)
• NGUYIN TRONG TUAN
TOM TAT:
Thot n6't (Borassus flabellifer L.) hay Th& iSt dutOc trfing nhieu d vftng dong bing s6ng CSu Long dac bidt la cac tinh nhiT An Giang, KiSn Giang v^ Dong Thap. Nghidn cii^ nay nham muc dich khSo sat hoat tinh khang oxi hoa cua v6 trdi Th^t n^t, mpt phia pham difdc viJt bo tCt hoat dpng kinh doanh thit qua Thot not thSng qua s^ dung cac phiTdng phdp thu: nghidm hoat tinh khdng oxi h6a khdc nhau nhif: DPPH, ATBS"*, reducing power (RP), khuT sdt (FRAP) va phosphomolyhdeum.
Kd't qud nghidn cii^ cho thay rHg, cao chiet methanol c6a v6 trdi Thd't nd't the hidn hoat tinh khdng oxi h6a t^t frong phifdng phap ATBS^, FI?AP, Phosphomolyhdeum v6i gia tri EC50 (hoac ODQ g) thap hdn hoac bang 100 ng/mL. Ket qud nay cQng phu hdp vdi vide xdc dinh hdm lifdng tdng flavonoid trong cao chid't methanol cua v6 la khd cao. Ket qua nay md ra tri^n vong trong viec s^ dung v6 trai Thd't not nhif san pham co hoat tinh khdng oxi hoa.
Tiir Idioa: Borassus flabellifer L. Flavonoid, khang oxi hoa, phu ph^m, polyphenol.
1. Mot diu
Ngay nay, tinh trang stress oxi hoa dang la mdt Irong nhffng van d^ difdc quan tdm nhieu cua cdc nha khoa hpc bdi vi tdc hai nguy hiem cua n6. Ttnh trang stress oxf hoa la sif mS't cdn bdng giffa vide san xuat cac goc tif do va sif tham gia hoat dpng cua cac chd't chpng oxi hpa. Stress oxi hoa cung la nguyen nhdn ciia cdc benh nhif tim mach, ung thff, suy giam than kinh va gay lao hoa sdm [1], Nhieu nghien cffu hidn nay dang tap trung vao vide tim kid'm cac hdp cha't chong oxi hoa tff nhidn tff cdc loai thffc vat vl cac hdp chai tff tff nhien nay dffpe xem nhff an toan cho con
ngffdi. Thot not (Borassus flabellifer L.) hay Thot 16't dffdc trong nhieu d vilng dong bing song Cii:u Long ddc bidt id cdc tinh nhff An Giang, Kidn Giang, va Dong Thap. Bo phan thffdng sff dung 14
• cuong cum hoa, re, dich cay, thdn cay va phan thit qua [2-41. Thot not dffpe sff dung trong y hoc dan gian nhff mot chat kich thich. chong co thdt ipi ti^u, chd'ng viem. Qui c6 tdc dung an thSn', nhuan trang. kho tien, day hpi, benh ngoai da, xua't huyd't, sd't va suy nhffpc. Re vi dich qui cd khi nang khang vidm. Re non la thuoc irti tieu va chd'ng giun. thud'c sdc cua re dffPc ding cho mdt so' bdnh ve dffdng hd hap. Tro cua hua cd khi
338 So 2 - Thdng 2/2020
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nang dieu tri tram cam, cac benh v l tim, \i lach va cam so't. Nhya cay cung co the dtfdc si> dung nhtf thuoc nhuSn trang va dieu tri loet va cac vd'n de vegan [5,6].
Cic nghien aSa ve hoat tinh khang oxi hoa cua cay Th6't n^t da diTdc thrfc hien trdn cac b6 phan khic nhau c6a cay. Theo Madhankumar el ai (2019), la cua cay Tho't not the hi$n hoat tmh khang oxi h6a manh vdi hieu qua gan tu'dng difdng vdi chat chu^n d cac phiTdng phap SOD (Superoxide dismutase, 26,4 ± 0,25 units/mg protem), CAT (Catalase, 44,58 ± 2,36 |imol HjOj tieu thu/phiit/rag proteins), GPx (Glutathione peroxidase, 231,4 ± 1,46 |ig glutathione bi oxi h6a/phijt/mg protein) v^ GST (Glutathione-s- transferase, 153,1 ± 1,43 |imol CDNB-GSH difdc Hnh th^nh/phiit/mg protein) [7]. Nghien ciJu cua Pramod el al. (2013) cho tha'y hoat tfnh khang oxi h(5a cfia thit qud Thot not trong phifdng phdp I ^ P H vi ATBS'+ CO su" tifdng quan vdi ham lifdng polyphenol vk flavonoid tdng co chu'a trong cao chiet [8], Cao chiet c6n c^a hoa Th6't nd't theo nghiSn ciJu cua Kavatagimath et al. (2016) cung co hoat tinh khdng oxi hoa tu'dng difdng cha't chuan BHT d phifdng phap DPPH va con co kha nang ha difdng huy6't rS't td't [9]. Ngo^i ra, vdi nguon gia tri dinh difdng cao, Thot not difdc ngifdi dan khai thic v^ kinh doanh nhieu, ddc biet la ph^n thit qua vdi him lifdng chd't dinh difdng den 1398kJ/100g va chiJa kha nhieu carbohydrate, protein, cha't xd va cdc chd^t khodng nhu" kali, canxi, ma-gi6 [10]. Tuy nhien, khi khai thac suf dung phIn thil qua, phan phu pham ttfc 1^ phan v6 se bi bo di. Theo tim hieu cua chiing toi tai mot so' cd sd kinh doanh Thot no't d DBSCL, phIn v6 phu phd'm nay chifa co each xtf ly, chi phdi kho diing lam nguyen lieu dot. Chinh vi the', nghien ctfu nay lu'a chpn phan v6 qua Thot n6't ldm doi ttfdng nghidn ctfu hoat tinh khdng oxi hoa, nham muc dich lang them gid tri stf dung cua phu pham v6 trai thdt n6't.
2. Thi^c nghidm 2.1. Nguyin lifu
Trdi Thot not dtfdc thu mua tai cac cd sd kinh doanh cac san pham didt ndi tai huydn Tjnh Bidn, tinh An Giang vao thdng 5/2019. Mdu trdi dtfdc xac
nhan bdi ThS. Phung Thi Hang va dtfdc Itfu trff tai Phong thi nghidm Hoa sinh hoc, bo mon Hoa hoc, khoa Khoa hpc Ttf nhien, Tnfdng Dai hoc Can Thd, vdi ma sd AG_Bo2019050002. Nguyen lieu sau khi mua ve dtfdc got lay phan v6 trai phia ngoai, rtfa sach, loai bd tap ban, cat nho. Mdu dtfdc say d 5CfC va xay thanh bpt.
2.2. Xdc dinh dg dm bgt nguyen lifu Dp am bdt nguyen heu dtfdc xdc dinh theo Dtfdc dien Viet Nam V, theo phtfdng phdp say [11].
2.3. Bieu che cao chiet
Bdt kho v6 trai Thd't nd't dtfdc chie't vdi dung mdi methanol bang phtfdng phdp ngam trong 24 gid (3 lan). Ta't ca dich chiet dtfdc gom lai va lpc de loai bo can, bui. Dich chiet sau khi lpc dem cd quay dudi dung mdi d nhiet dd 40-45"C thu dtfdc cao chid't methanol vd trai Thdt not. Cao chiet dtfdc dem can va tinh hieu suat chiet cao, sau do cao dtfdc bao quan trong tu lanh d nhiet dp 4'^C cho den khi thi nghiem.
2.4. Khao sdt hogt Hnh khdng oxi hda cua cao chie't
2.4.1. Khdo sdt hieu qud logi bd gdc tudo DPPH Kha nang loai bd goc ttf do DPPH cua cao chi^t dtfdc dtfdc thtfc hien theo Sharma et al. (2009) cd hidu chinh [12]. Hdn hdp phan tfng gdm 40 pL DPPH (1000 jJg/mL) va 960 pL cao chiet c6 nong do khac nhau ttf 30-150 pg/mL. Hdn hdp dtfdc ii trong tdi d nhiet dp 30°C trong thdi gian 30 phut, do dd ha'p thu quang phd d btfdc song 517 nm. Cha't chuan dtfdc stf dung la vitamin C. Ket qua hoai tinh khang oxy hoa cua cao chiet dtfdc bieu dien bang gia tri EC50 (Effective Concentfation of 50%) dtfdc tinh dtfa trdn phtfdng trinh tuyd'n tinh cua dich chiet khao sat. Phan tram loai bd gd'c ttf do dtfdc tinh theo cdng thtfc:
% Loai bd gd'c ttf do = ^ " ^ x 100%
Ac
Trong dd: A<; la gia tri ha'p thu cua dd'i chtfng.
As ha'p thu ciia mau.
2.4.2. Khdo sdl hieu qud logi bo gdc tu do ABTS'
Hoat dpng loai bo gdc Itf do ABTS * thtfc hidn theo Nenadis et al. (2004) cd hieu chinh [13].
Chuan bi dung dich ABTS *: 2 mL dung dich
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TAP CHJ CONG THI/(fN6
ABTS 7 mM va 2 mL dung dich K2S20g 2,45 mM, u trong bong tdi 16 gid, sau do pha loang bang ethanol (khoang 30 l^n), dilu chinh dp ha'p thu d btfdc sdng 734 nm dal 0,7 ± 0,05. Tien hanh cho 990 pL ABTS * vao 10 pL cao chie't d cdc ndng do khdc nhau ttf 20-100 pg/mL. Hdn hdp phan tfng dtfdc u trong thdi gian 6 phut. Sau do, do dp ha'p thu quang phd d btfdc sdng 734 nm. Chat chuan dtfdc stf dung la trolox. Ke't qua hoat tinh khdng oxy hoa cua cao chie't dtfdc bieu dien bang gid tri EC50 dtfdc tinh dtfa tren phtfdng tnnh tuye'n linh cua dich chie't khdo sat. Phan tram ldm sach gdc ttf do dtfdc tinh theo cdng thtfc:
% Loaibd gd'c tif do = ^~ ^ xlOO%
'V
Trong dd: A^ la gid tri hd'p thu cfla doi chtfng, Ag hap thu ciia mau.
2.4.3. Khdo sdt ndng luc khd: (Reducing Power-RP)
Ndng life khtf (RP) dtfdc thtfc hien theo phtfdng phdp Ferreira etal. (2007) cd hidu chinh [14]. Hdn hdp phan tfng gdm 0,5 mL cao chie't d ndng do khdc nhau ttf 100-700 pg/mL, 0,5 mL dung dich dem phosphate (0,2M, pH=6,6) va 0,5 mL K3Fe(CN)6 1%. Sau dd hdn hdp phan tfng dtfdc ii d 50»C trong 20 phut, them 0,5 mL CCI3COOH 10% rdi ly tdm 3.000 vong/phut trong 10 phut.
Nhe nhang nit 0,5 mL dich phia tren cho vao 0,5 mL ntfdc vd 0,1 mL FeClj 0,1%, l^c ddu. Do dd hd'p thu quang phd cua hdn hdp phan tfng d btfdc sdng 700 nm. Cha't dd'i chtfng dtfdng stf dung la trolox. Hieu qua khang oxy hda ciia cao chiel d cac ndng dp khac nhau dtfdc so sdnh vdi chd't chuan b^ng each stf dung ndng do ma tai dd chat chuan hay cao chid't (pg/mL) co gid tri OD=0,5 (ODo,5) cung nhtf ham Itfdng ttfdng dtfdng cha't khdng oxy hda pg/mL cha't chuan.
2.4.4. Khdo sdt khd ndng khd sdl (FRAP) Kha nang khtf sdt FRAP dtfdc thtfc hien theo Benzie era/. (1996) cd hieu chinh [15]. Tien hanh cho 10 pL cao chie't (cd ndng dp ttf 10-50 pg/niL) vao 990 pL dung dich FRAP. Cdc hdn hdp Iren dtfdc li d 37"C trong 10 phut, sau do lien hanh do gid tri dd ha'p thu quang phd d btfdc song 593 nm.
Cha'l chuan stf dung la irolox. Hieu qua khang oxy
hda cua cao chie'l d cac ndng do khac nhau dtfdc so sanh vdi chd't chu^n bdng cdch stf dung ndng dp ma tai dd cha't chuan hay cao chie'l (pg/niL) co gid tri OD=0,5 (OD0.5).
2.4.5. Khdo sdt hogi tinh khdng oxi hoa tdng (Phuang phdp phosphomolybdenum)
Phtfdng phdp dtfa trdn stf khtf cua Mo(VI) ve Mo(V) the hien qua stf tao phtfc Mo(V)-Phosphate mau xanh la d pH acid theo Prieto et al. (1999) [16]. Tie'n hanh cho 100 pL cao chie't ndng dp khdc nhau ttf 45-315 pg/mL vao dung dich phosphomolybdenum. Cac hdn hdp tren dtfdc li d 95°C trong 90 phut, sau do ti^n hanh do gid tn dp hap thu quang phd d btfdc song 695 nm. Chat chuan stf dung la vitamin C. Hidu qua khdng oxy hda cua cao chie't d cdc ndng dp khac nhau dtfdc so sdnh vdi chat chu^n bang cdch stf dung ndng dd ma tai do chd't chuan hay cao chie't (pg/mL) co gid tri OD=0,5 (OD0.5).
2.5. Dfnh li^g polyphenol tdng vd flavonoid tdng trong cao chiet
Ham Itfdng polyphenol tdng dtfdc xdc dinh bkng thud'c thtf Folin-Ciocalteu theo md td cua Rebaya et al. (2014) co hieu chinh [17]. Ham Itfdng flavonoid tdng trong cao chi^t dtfdc xac dinh theo phtfdng phdp so mau AICI3 ciia Bag et (j/. (2015) [18].
3. Ke't qua va thao lu^n
3.1. Dg dm b0t nguyen lieu vd hi$u sudt chiet cao
Dp ^m bdi nguyen lieu vd trai Thdt nd't dat 4,918 ± 0,028%, vdi gia tri dp am nay la khd tha'p va phii hdp vdi tidu chuan bdt dtfdc lidu cOa Dtfdc dien Viet Nam (dp am bdt nguyen lieu <:9%),
Hieu suat chiel cao dal 7,122%. Cao thu dtfdc d dang sanh ddc va co mau ndu da't.
3.2. Hogt tinh khdng oxi hda
Hoat tinh khang oxi hda cua cao chiet v6 trdi Thdt nd't dtfdc linh loan thanh cac gid tri EC50 hoacOD0,5 trinh bay d Bang 1.
Kd't qua d Bang 1 cho iha'y vd trdi Thdt nd't cd tidm ndng khdng oxi hda t^t d phtfong phdp ATBS-, FRAP. Phosphomolyhdeum vd, gid tri EC50 (hodc ODoO thap hdn hoac hjng 100 pg/mL. Tuy nhien. so vdi chat chuJn stf H.mo
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Bang 1. Ket q u a k h a o sat h o a t tinii k h a n g oxi h o a c u a c a o chiet v 6 trai Thot n6t TT
1 2 3 4 5
Phffdng phap DPPH(EC5j) ATBS-(ECs„) RP(OD„,s) FRAP(0D„,5) Phosphomolybdeum (ODQ g)
Chai chuan Qig/mL) 3,905±0,026 7,718±0,042 34,740±0,118 2,240±0,008 13,319±0,359
Caochl«(|ig/mL) 120,174+2,182 76,860±0,219 479,250±1,887 47,003±0,346 101,05710,574 Gki chii: Chat chudn sd dung I, 5: vitamin C; 2,3,4: trolox
trong ttfng phtfdng phdp thi hieu qua chd'ng oxi hda ciia cao chie't thap hdn chat chu^n khd nhieu, cu the h-ong phtfdng phap DPPH vdi EC50 dat 120,174 ± 2,182 pg/mL (cao hdn chd't chu^n hdn 30Ik), ATBS* la 76,860 ± 0,219 pg/mL (cao hdn ch^t chuan gin 10 lan), vdi gid tri ODQS la 47,003
± 0,346 pg/mL (cao hdn chat chuan 21 lan) vd 101,057 ± 0,574 pg/mL (cao hdn chd't chuan 7,5 lan) d ca 2 phtfdng phdp FRAP va phosphomolybdeum.
Ham Itfdng chd't khdng oxi hda ttfdng dtfdng cha't chuan (mg chat chuan/g cao chie't) cung da dtfdc tie'n hanh tinh loan d moi phtfdng phdp va ke't qua dtfdc trinh bay d Bang 2.
Bdng 2. H a m IUdng c h d t k h a n g oxi h o a t i f d n g d i i d n g c h d t c h u d n c u a c a o v d trai Thot not
TT
1 2 3 4
'
Phuong phap
DPPH ATBS- RP FRAP Phosphomolybdeum
Ham liidng chat khang oxihoati/dngdiiong chatchuan{rngchat chuan/g cao chiet)
32,868±5,660 109,046±13,375
66,533±13,158 47,105±1,813 127,398±8,703 Ghi chu: Chdt chudn sddung 1.5:
vitamin C: 2,3.4: trolox
Ke't qud dat dtfdc d Bang 2 cho thay, ham Itfdng chat khdng oxi hoa ttfdng dtfdng cha'l chuan ciia cao chie'l vd trdi Thd't not dtfdc xdc dinh cd ham Itfdng kha cao trong phtfdng phdp ATBS"* va phosphomlybdeum vdi gia tri lan Itfdt la 109,046 ± 13,375 (ttfdng dtfdng rag vitamin C/g cao chie't) va 127,398 ± 8,703 (ttfdng dtfdng mg trolox/g cao chid't). O cdc phtfdng phap thtf nghidm cdn lai, ham Itfdng nay dtfdc xdc dinh thd'p hdn lan Itfdt Id 66,533 + 13,158 va 47,105 ± 1,813 (ttfdng dtfdng mg trolox/g cao chie'l), 32,868 ± 5,660 (ttfdng dtfdng mg vitamin C/g cao chie't) trong phtfdng phap RP va FRAP, DPPH itfong tfng. Kd't qua nay chtfng minh dtfdc hieu qua khdng oxi hda td't d phtfdng phdp ATBS"^ va phosphomlybdeum cua cao chiet vd trdi Thd't nd't.
Nghien ctfu cua Pramodet al.(2013) tai An Dp thi hoat tinh khang oxi hda cua qua trdi Thdt not kem hdn rat nhieu so vdi nghien ctfu cua chung ldi d ca 2 phtfdng phap DPPH va ATBS* khi gid tri EC50 cua chung la 471,492 pg/mL (cao hdn 3,92 lan) va 567,905 pg/mL (cao hdn 7,39 lan) [8]. Tuy nhien, so vdi nghien ctfu trdn Id cay Thdt not cua Jamkhande et al. (2016) thi Id cho hieu qua to't hdn so vdi vd trai cua chiing tdi trong phtfdng phap DPPH vl gia tri EC50 la 40.19 pg/mL thd'p hdn 3 lan so vdi gia tri EC50 cua cao chie't methanol vd qua [19]. Nghien ctfu tren thil qua Thd't nd't ciia Arunachalam et al. (2011) cho tha'y gid tri EC50 d phtfdng phdp DPPH Id 515.46 pg/mL cao hdn 5 lan so vdi vd qua cua chiing tdi [10]. Ham Itfdng chat khdng oxi hda Itfdng dtfdng vitamin C d phtfdng phdp DPPH Iheo nghidn ctfu
So 2 - Thang 2/2020 341
TAPCHJCONfiTHirOfNG
cua Kunan et al. (2017) d Sn Lanka tren thit trai Thd't not d vung KJlinochchi va Batticalo cd ham Itfdng ttfdng dtfdng vdi nghien ctfu phan vd qua cua chiing tdi (32,858 ± 1,667 va 31,134 ± 0,470 mg vitamin C/g cao chid't) [20]. Nghien ctfu ciia Sastry et al. (2012) kha nang khang oxi hda cda mang vd bao phu cdm Thd't not cho tha'y cdc gia tri EC50 thu dtfdc d cdc phtfdng phdp DPPH va ATBS* ra't tdt, vdi ke't qua ttfdng dtfdng vdi cha't chuan vitamin C, d phtfdng phdp DPPH gia tri EC50 thu dtfdc la 7,90 pg/mL (cao hdn cha'l chuan chi 1,87 lan); d phtfdng phdp ATES"* gid tri EC^Q dat2,58 pg/mL (tha'p hdn chd't chuan 1,02 lan), vd ca 2 gid tri EC50 nay deu tha'p hdn gia tri EC50 trong thtf nghidm cua chiing tdi lln Itfdt la 15,21 lan va 29,79 lan [21].
3.3. Dinh lU^g polyphenol tong vd flavonoid tdng
Ham Itfdng polyphenol tdng va flavonoid tdng dtfdc xac dinh theo ham Itfdng ttfdng dtfdng acid gallic va quercetin dtfdc trinh bay d Bdng 3.
Polyphenol va flavonoid la nhffng hoat chat ttf nhidn cd nhilu tac dung nhtf chdng 0x1 hda, khdng vidm, khdng khuan, chdng di tfng, chd'ng lao hda va bdnh tat cho con ngtfdi. Kd't qua dinh Itfdng cho thay ham Itfdng cOa chiing cd trong vd trai Thd't nd't khd cao, ddc bidt la ham Itfdng flavonoid long vdi gid tti 115,522 ± 2,985 mg QE/g cao chie't, ket qua
Bong 3. Ham ladng polyphenol tong va flavonoid tdng cua cao chief methanol
v6 trdi Thdt ndt
Cao chiet vd trai
HanlUdng polyphenol t ^ TPC (mgGAE/g cao chiet)
27,813±0,469
Ham liidng flavonoid t ^ T F C (mg QE/g cao chieQ
115,522±2,985
nay gdp phan chtfng minh cho hoat linh khdng oxy hda manh ciia cao chie't tren.
Ham Itfdng polyphenol long trdn rS Thdt ndt theo nghien ctfu cda Sahni et al. (2014) cao hdn 3,57 lan so vdi vd trdi Thd't nd't khi co gid tri Id 99,34 mg GAE/g [5]. Trong nghidn ctfu cua Wijewardana et al. (2016), ham Itfdng polyphenol tdng cung cd stf chenh lech khi cd gid tri la 86,72 ± 0,02 (cao hdn 3,1 lian) [22].
4. Kd't lu$n
Khdo sdt boat tinh khdng oxi hda cao chiet methanol cua vd Irdi thd't not cho thay cao vd trdi cho hieu qua khang oxi hda tdt trong cac phtfdng phdp ATBS^, FRAP, Phosphomolybdeum. Ke't qud cung khd phu hdp vdi ham Itfdng flavonoid trong cao chiet dat dtfdc khd cao •
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So 2-Thang 2/2020 343
TAP CHi CONG THIf0KG
Ngay nhdn bai: 3/1/2020
Ngay phan bi^n danh gia va suTa chffa: 13/1/2020 Ngay chap nhdn dang bai: 23/1/2020
Thong tin tdc gid:
N G U Y I N TRQNG TUAN
Khoa Khoa hpc Ttf nliidn, TrtfoTng Dai hoc Can Thd
ESVESTIGATION OF THE ANTIOXIDANT ACTIVITY OF METHANOL EXTRACT OF BORASSUS FLABELLIFER L. HUSK
• NGUYEN TRONG TUAN College of Natural Sciences,
Can Tho University
ABSTRACT:
Borassus flabellifer L. is grown in the Mekong Delta, especially in An Giang, Kien Giang, and Dong Thap provinces. This study is to investigate the antioxidant activity of Borassus flabellifer L. husk, a by-product that is thrown away from the palmyra fruit through the use of various antioxidant activity tests as DPPH, ABTS *, reducing power (RP), FRAP and phosphomolybdeum methods. The tested results show that the methanol extract of the pahnyra fruit husk showed good antioxidant activity in ATBS •*, FRAP, Phosphomolybdeum methods with EC50 (or OD0.5) lower or equal lo 100 pg/mL. This result is also consistent with the determination of highly total flavonoid content in methanol extract oi Borassus flabellifer L. husk. This study opens up prospects in the use of Borassus flabellifer L. husk as the antioxidanl product.
Keywords: Antioxidanl, Borassus flabellifer L., by-product, flavonoid, polyphenol.
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