Nghien ciru tac dung uc che enzym tyrosinase / cua cac phan doan tach chiet tir y di ^
Bui Thanh Tung, Vu Dif c Loi, Le Thi Thu Hwong Nguyin Thj Thanh Binh, Nguyen Thanh Hai*
Khoa Y Dugc, Dgi hgc Quoc gia Ha Ngi ' E-mail: haipharma@yahoo. cam Summary
in view that melanin plays an important role in reduction of free radicals and protection of skin from ionizing radiations and ultraviolet, but abnormal increase in melanin leads to skin disorders such as freckles, melasma and even malignant melanoma, and noteworthy that tyrosinase Is one of the key enzymes involved in the biosynthesis of melanin, ...to prove the folk medicine use of Coix iaeryma- jobi for skin protection and treatment of melanin-related skin disorders, various fractions from Coix
iacryma-jobl were evaluated in vitro for tyrosinase-inhibitory activity. All the studied fractions exerted in vitro evident tyrosinase inhibition, n-hexane fractions were the best with IC^ value of 367 ± 1.4 pg/mL.
In conclusion, possessing tyrosinase inhibitory activity, these fractions of Coix lacryma-jobi, especially n-hexane one would be promising skin-whitening agents for antimelanogenesls formulations.
Keywords: tyrosinase, melanin, extraction, Coix lacryma-jobi
Dat van de
Melanin la chit quan trpng giup bao ve lan da ching lal cae tia birc xa, nhung su tieh tu bit thudng ciia melanin se gay ra cae rli loan sac t l , ching han nhu nam, tan nhang, v i t tan huang.
Qua trinh ting hgp melanin dupc tlln hanh trong t l bao melanonin, n i m a Idp day ciia thugng bi va duge kilm soat bd-i tyrosinase. Tyrosinase (EC 1.14.18.1), cdn dugc gpi la enzym polyphenol oxidase, la mpt enzym monooxygenase cd chira ding tham gia vao qua trinh tong hpp melanin.
Enzym nay duge phan b l rdng rai trong cac loai nim, thuc vat va ddng vat. Tyrosinase tham gia kiem soat qua trinh san xuit melanin. Enzym nay tham gia chii y l u vao hai phan irng khac nhau eiia su ting hgp melanin; mpt la, hydroxyl hda monophenol va hai la, viec chuyln ddi mdt o-diphenol d l tao thanh o-qulnon. o-Quinon trai qua mpt s l phan u-ng d l culi eung tao thanh melanin ro. Cae nghien eiru v l tyrosinase, eae ca chit va cac chit irc ehl dupc nhilu nha nghien ciru quan tam d l lam re han hoat ddng sinh hpc cua enzym nay va kilm spat nd P'. Gin day, eae hgp chit irc c h i tyrosinase cd nguin g l c tu nhien hoac mpt phin hay tong hgp hoan toan da dugc thif nghiem PI. Cae ehit irc ehl tyrosinase nhu arbutin, acid kojic va hydroqulnones da dugc
sir dung nhu chit lam trang, ehdng loan sac t l nhd kha nang ngan can san xuit melanin d da. Tuy nhien, arbutin va acid kojic khdng cho thiy hieu qua irc ehl sac t l d t l bao melanocyt nguyen ven hoac tren thii' nghiem lam sang va cac hydroquinon cd kha nang gay dpc t l bao vdi cac t l bao melanocyt va cae gay dot blln vdl t l bao thudng 6' dpng vat cd vu. Do dd, rit ein thilt phai nghien ciru d l tim kilm cac chit irc ehl tyrosinase mdi ma khdng cd tac dung phu.
y dT (ten khoa hpc: Coix lacryma-jobi) la mdt loai thuc vat nhiet ddi d l liy hat trong hp Hda thao (Poaceae), cd ngudn gde Ddng A va ban dao Malaysia nhung dugc gieo trdng a nhilu nai trong rupng, vudn nhu la mdt loai eay mdt nam. Nd dugc trdng rdng rai d- Viet Nam, Dai Loan, Trung Quic va Nhat Ban. V dT cd vi ngpt, khdng mui va dugc su- dung rdng rai nhu mdt loai thule truyin thing ciia Trung Quic qua hang ngan nam trong dllu trj cac benh nhu ung thu, dl can, tang huylt ap, viem khdp, hen suyin va cac rli loan miln djeh '" ^i. Ngoai ra, y dT ttf lau da duge su- dung trong cac san pham lam tring da va dep da, dae biet la d Nhat Ban. Tuy nhien, kha nang u-c ehl tyrosinase cua y dT v i n ehua dugc nghien ciru.
Tren ca so- dd chung tdi tiln hanh "Nghien eu-u tac dung irc ehl enzym tyrosinase ciia cac phan doan tach ehilt tu- y dT", vd-i muc tieu danh gia tac dung
TAP CHI DU'OC HOC -1/2016 (SO 477 NAM 56) 31
Nghien - Ky thuat
ire ehl tyrosinase ciia eae phan doan tach chiet tu' y dT (bao g i m n-hexan, ethyl acetat, n-butanol, nude va ethanol) va tinh gia trie ICg^ciia cac phan doan dd.
Nguyen lieu va phu'ang phap nghien ci^u
Hoa chit: L-dihydroxyphenylalanln (L-DOPA), acid phosphoric, enzym tyrosinase nim, acid benzoic.
Nguyen lieu: Y dT dugc thu hai 6' Mu Cang Chai, Lao Cai vao thang 10-2014 va duge giam djnh thuc vat hpc bd'i Bp mdn Duge lieu & Y hpc CP truyln-Khpa Y Dugc; M i u tieu ban (YD-001) dugc luu giu" tai Khoa Y Dugc, Dai hpc QuIc gia Ha Ndi.
Phu'O'ng phap nghien cu'u Chiet xuat cac phan doan tiry di
Chilt xuit cac phan doan tu' y dT theo sa d l d" hinh 1
Thu cao EtOH
Dich n-hexan Thu hoi n-hexan
Cao n-hexan (132 g)
Thu hoi EtOAc
Y dinhan (1kg)
Dich EtOH tong
Cao EtOH (478 g)
Dich nudc
Dich EtOAc
Cao EtOAc (112 g)
EtOH 96% 3 Lx 3 lan
Thu hoi EtOH
Tliem nudc
n-hexan 500 ml x 3 lan
ZI
Dich nudcEtOAc 500 ml X 3 i n
BuOH 500 ml X 3 Ian
Dich nudc
Dich BuOH
Thu hdi BuOH *-
Dich nudc
Cao BuOH (97 g)
Co
Cao nudc (65 g) Hinh 1: Sa dd chiit cac phan doan cao khd tuydf
32 T^P CHI DUOC HOC - 1/2016 (SO 477 NAM 56)
Phwang phap danh gia kha nang irc che enzym tyrosinase
Kha nang irc ehl enzym tyrosinase dugc thuc hien bing each sii' dung eg chit la L-DOPA, dugc md ta theo phuang phap eua Kubo '^i vdi nhO'ng thay doi nhd. Chuin bj cac dung djch 1 mg/mL tyrosinase (Sigma Chemical), 0,4 mM L-dlhydroxyphenylalanin (L-DOPA), 0,8 M acid benzoic va 1 mg/ml cae phan doan djch chilt.
• Miu thii': dung dich cac phan doan chilt tu' y dT hca tan trpng MeOH.
• M i u chirng duang: dung djch acid benzcic.
• Mlu ddi chirng: nude eit.
Tlln hanh ii bao gdm dung djch dem 1/15 M acid phosphoric (pH 6,8,1 mL), 10 pL miu cin thil' nghiem va 10 pL tyrosinase nim trong Ing nghiem.
Hdn hgp dugc u d- 25°C trong vdng 10 phiit. Sau dd, them 1 mL dung djch 1,5 mM L-DOPA d l tiln hanh phan irng va do dp hip thu quang d 475 nm trong vdng 5 phiit Su gia tang dp hap thu dl kem vdi qua trinh oxy hda ciia ca ehit (L-DOPA).
Ning dp euli eung cua dopaqulnon dupc tinh dua tren he s l hip thu moi 3.700 M'cm^ Mot dan vi hoat tinh enzym dupc djnh nghTa la lugng enzym chuyln hoa hoan toan 1 pmol ciia L-DOPA trong mil phut. Ty le irc ehl hoat tinh tyrosinase dugc tinh toan theo edng thire:
Cdng thuc tinh kit qua:
Ac-At
% Lfc ehl = x 100 Ac-Ao
Trong dd: A^: do hip thu cua mSu chimg A,; do hip thu cua mau thir A : do hip thu cua mau trang
Trpng dd A^ la su khac biet v l dp hip thu ciia miu chu'ng trong khoang tho'i gian ii la 0,5 va 1,0 phut, vaA, la su khae biet trong hip thu ciia mau thir trong khoang thd-i gian ii la 0,5 va 1,0 phiit M i u tring: dung djch dem acid phosphoric va do dp hap thu anh sang tai budc sdng 475 nm. T i t ea cac xet nghiem dugc lap lal 3 l i n .
K i t qua
Thyc nghiem va kit qua v l chilt xuit Hat y dT, edn vd (1 kg) sau khi sg ehl, loai bd vd cirng (thu dugc y dT nhan) dugc ngam chiet ky bing dung mdi ethanol 3 lin su- dung thilt bj ehilt sieu am 6' 40°C. Cae djch chilt ethanpl thu dwgc dugc Ipc qua giiy loe, gom lal va eit loai dung mdi dudi ap suit giam eho cao chilt ting ethanol (478 g). Hda tan can ehilt ethanol nay trong nudc c i t va chilt phan b l bing n-hexan, ethyl acetat va n-butanol (3 lin). Cac phan doan n-hexan, ethyl acetat, n-butanol dupe eit loal dung mdi dudi ap suit giam de thu dugc phan doan n-hexan (132 g), ethyl acetat (112 g), n-butanol (97 g) va H^O (65 g) tuang u'ng theo sa d l phan doan hinh 1.
Kha nang irc chi enzym tyrosinase tii' cac phan doan djch chilt y dT
Kit qua cac phan doan djch chilt y dT irc ehl enzym tyrosinase dupc trinh bay trcng bang 1 va hinh 2. IC^^ cua cao EtOH, cao n-hexan, cao EtOAc, cao BuOH va cao H^O lin lupt la 453, 367, 438, 427 va 489 pg/mL. Thep kit qua tren, phan doan n-hexan cd kha nang irc ehl cao nhit so vdi cac phan doan khae. Phan doan djch ehilt HjO la kha nang ire ehl thip n h i t
Bang 1: Phin tram uc chi enzym tyrosinase cua cac phan doan dich chiit y di Ndng do phan doan djch chiet
(pg/mi) 100 150 200 300 350 400 450 500 IC^
Cao EtOH
6,5 ±1,5 12,4 ±2,7 21,5 ±2,7 29,2 + 1,9 35,5 ± 3,2 45,3 + 41 49,9 + 2,5 65,6 + 2,8 453±2,7pg/mL
Caon-hexan
10,4 ±2,6 15,3 ±3,5 25,6 ±2,8 36,8 + 2,2 48,5 ± 2,7 56,8 ± 5,3 65,4 ±4,5 78,4 ± 4,2 367±1,4pg/mL
Cao EtOAc
7,8+1,3 12,3 ±3,6 18,5 + 17 28,4 + 2,9 35,2 ± 3,5 45,3 ± 2 7
53,2+1,2 67,5± 2,4 438±3,1 pg/mL
Cao BuOH
87 + 2,5 11,4 ±3,2 197 ±31 31,5 ±37 39,7 ± 3,9 47,6 ± 2,3 557 ±2,9 68,1 ±1,8 427±2,3pg/mL
Cao Hfi 7,2 ±2,3 13,1 ±2,8 19,5 ±1,6 25,9 ±1,5 327 ±1,9 39,3 ± 2,7 45,8 ± 2,4 53,4 ± 4 1 489±1,6pg/mL
TAP CHI DlTOC HOC - 1/2016 (SO 477 NAM 56) 33
Nghien - Ky thuat
S 20
0 100 200 300 400 500 600 Ndngddcacphan doan djch chiet (pg/mL)
Hinh 2: Dd thi kha nang uc chi enzym tynDsinase cua cac phan doan dich chiit y df Ban luan
Hat y dT chu-a mdt lugng Id-n cae chit dinh dud'ng, vi du nhu protein, acid amin bac cao va carbohydrat Ngoai ra mdt s l thanh phin quan trpng khae ed hoat tinh sinh hpe da dugc xae djnh trong hat y dT vdi nhilu chirc nang cd Igi cho SU'C khde, dae biet la eoixenolid, triglycerid, acid beo, va cac triterpen ^K
Y dT cd kha nang kich thich chu'c nang cua la lach, da day va phoi, giai phdng mu d cae v i t thuang thoat mii va gay Igi tilu; ttr lau da dugc sir dung d Trung Quic d l dllu trj mun, da nu-t ne, thip khdp, dau day thin kinh. Nghien ciru gin day cho thiy, hat y dT cd kha nang ching tang sinh ciia cac t l bao ung thu, ching khii u va diiu hda miln djch, chdng viem va ching dj u-ng.
Diu hat y dT cd the ngan ngira hoae giam bdt eae can CO rut ciia ca van va kich thich hoat dpng tir cung. Dau ma cung cd the lam giam nong dp ealei mau va glucose mau trong huylt thanh. Diu hat y dT cdn cd tac dung cd igi tren benh ung thu theo nhilu ca c h i khac nhau nhu da dugc chu-ng minh tren cac thii- nghiem lam sang. Y dT lam giam glucose mau va tac ddng ching oxy hda va dugc sir dung nhu la mpt thanh phin b l sung trong san phim thuc pham chu-c nang gliip cham sdc SU'C khde va cac loai thulc de phdng ngu'a cac benh man tinh (dac biet viem khgp va benh ddng mach vanh) i=' ^i. Trong khi cac thanh phin hoat tinh ciia y dT nhu eoixenolid, kieh thieh qua trinh mien djch t l bao va miln djch djch t h i ; coixan A, B va C lam giam dang ke mu'c dp glucpse trpng mau ''°- "J. Han nO'a, djch ehilt tir y dT cd t h i lam tang hoat ddng ciia t l bSo T g^y ddc t l bao va t l bao diet tu nhien (NK). Djch ehilt methanol eiia y dT U'C c h i su san xuit cua NO va O^- bing each
kich hoat cac dai thuc bao'^i. Nghien cu'u nay da chu'ng minh dugc tac dung u'c c h i enzym tyrosinase ciia cac phan doan chilt tu' y dT, trong dd phan doan n-hexan cho tac dung cao n h i t Dllu nay tao ca sa khoa hpc cho viec sii' dung y dT trong dan gian dung lam tring da va diiu trj cae roi loan lien quan d i n melanin.
Acid benzoic la mpt chit u-e c h i tyrosinase da dugc bilt ttr lau. Nghien cu-u nay sii' dung acid benzoic la ehit chuan de so sanh tac dung u'c ehl tyrosinase cua cac phan doan chilt xuit ttf y dT, kit qua cho thiy phan tram irc c h i cua acid benzoic la IC^^ bing 0,6 mM. Chung ta cd the thiy la mac du cac djch chilt cd ICj^eao han so vdi ehit chirng duang nhung do la cac phan doan ttr dugc lieu nen cd chira nhilu hdn hgp cae chit ndng dp rit nhd. Do dd, cac phan doan dich chilt vdi cac gia trj IC^^ U'c ehl enzym tyrosinase thi chiing la cac phan doan tilm nang cd kha nang U'C ehl tyrosinase. Cin tlln hanh cac nghiSn ciru tach chilt cac hgp chat tu' phan doan n-hexan d l phan lap dugc cae chat tinh khilt trong phan doan cd gia trj irc ehl enzym tyrosinase eao nhit trong cac phan doan da nghien cu'u d l tim ra chit iec c h i tyrosinase vgi gia trj IC^^ cao va cd kha nang sii' dung thanh thulc. Cg e h l irc ehl enzym tyrosinase ctia cac phan doan y dT cd the giai thich la cae chit trong y dT chira nhilu nhdm phenol, cd cac nhdm hydroxyl (OH) trong c i u true, tuang tu vdi ciu true ciia L-DOPA, ca chit ciia enzym tyrosinase. Do dd, cac ehit trong y dT canh tranh vdi L-DOPA va u'c ehl tae dung cua tyrosinase.
Cac benh lien quan d i n da cd nhilu v i n d l c i n quan tam, nhu qua nhilu hay suy giam sic t l d i u anh huang d i n su'c khde. Hien nay cd nhilu san pham lam trang da tren thj trudng, nhung kit qua thudng mang lai khdng kha quan.
Viec chu'ng minh cac phan doan chilt xuit tu' y dT cd kha nang u'c e h l enzym tyrosinase mang lai ca hdi cho cac san phim cd the u'ng dung trong cac benh lien quan d i n s i c t l , giup phdng va ngtfa cae benh lien quan d i n da.
K§t luan
Da danh gia dugc cac phan doan ehilt xuit ttf y dT ed kha nang u'c e h l enzym tyrosinase, trong dd phan doan n-hexan ed kha nang ire e h l cao nhit so vgi cac phan doan khac. Dllu nay ed t h i gial thieh dc phan doan n-hexan chu'a nhieu hpat chit chinh nhu la cpixenplid, triglycerid, acid beo va triterpenes. Nhdm tac gia de xuit tiln hanh
34 TAP CHi DUOC HOC - 1/2016 (SO 477 NAM 56)
nghien cifu tae dung ire e h l enzym tyrosinase tren md hinh in vivo, bao e h l dang my pham n h u kem bdi ngoai da tu' y dT va t l l n hanh danh gia tae dung ciia e h l p h i m .
LOT cam om: D l tai nay d u g c tai trp bgi d l tai thudc C h u a n g trinh Tay B i c , ma s l d l tai:
KHCN-TB.05C/13-18.
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{Ngay nh$n bar 06/11/2015 - Ngay duyet dang: 06/01/2016)
T o n g h o p v a thu* . . . (TUp theo trang 30)
chpn Ipc tren ddng KB. Hgp c h i t Bd cd hoat tinh manh n h i t tren ca 4 ddng t l bao vgi gia tri \C^
la 14,12-56 pg/ml. Nhdm t h i cdng k i n h d vj tri 3' tren vdng phenyl la y l u t l thuan Igi cho hoat tinh khang t l bao ung t h u ciia cac c h i t tong hgp d u g c .
L f f i c a m c n : Kinh phi thuc hien dwgc tai trg bai quy Phat triin Khoa hgc va Cdng nghe Qudc gia (NAFOSTED) cho di tai NCCB ma sd
^04.99-20^156.
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{Ngay nhan bai: 01/12/2015 - Ngay duyet dang: 06/01/2016)
T/^ CHi DUpc HOC - 1/2016 (SO 477 NAM 56) 35
