TAP CHI PHAT TRIk KUM TAP 17, 30 T3 - 2014 Nghien ci:ru hoat tinh khang khugn, khang oxi hoa, Crc the tyrosinase cua cao ethanol chiet xuat ti' cay Hujmh Anh (Allamanda neriifolia) • Trin Pham Tue Hung • Nguyen Thi M9 Hanh • Quach NO Diem Phirang Tru&ng Dai hoc Khoa hoc To, nhien, DHQG-HCM nkin n,gify 29 (hang 07 kin; 2014, n125)2 clang 'Tay 16 dicing 01 friar)? 2015) TOM TAT ce vai cac del ttrimg cOn lai Dinh tinh Cao ethanol cac 60 phen re, than trot, mat cac hop chit thir cap cao ethanol than leo, la dm cay Huynh anh (Allamanda ter than leo cho they su' hien dien caa neriifolia) ductc khdo sat kha nang khang alkaloid, steroid, triterpenoid, phenol, tannin the tyrosinase khuan, khang oxy haa, CPC va flavonoid Phan tich clang flavonoid cho Trong nhang be phen nay, cao ethanol ter they meu c6 the c6 flavan hoac chalcone than leo the hien cac hopt tinh cao Met so Tel' kh6a: Cfc the tyrosinase, Huynh Anh (Allamanda neriifolia), khang oxi h6a MD D AU Nam da, sam den la cac dau hieu benh lY sir bieu hien vuat mire melanin a da De khic phuc cac trieu chi:mg nay, nguiri to thuang sir dung cac hoa chat có kha nang irc the tyrosinase— enzyme tham gia sinh tong hop melanin.Tuy nhien, viec sir dung cac hoa chat co the gay tac dung phu cho nguai ding; s6 do, mot s6 'oat da bi cam sir dung dr nhieu nuac Thay vao do, nhiing hoat chat chiet xuAt tir dime lieu co kha rang irc the tyrosinase dirge sir dung bar hieu qua lam tring da va tinh an loan [11] Cay Huynh Anh co ngu6n gOc Trung va Nam My Ben canh gia tri lam canh, nhieu loci da- ductc chi nhu A carthartica, A blanchetti chUng minh co nhieu die tinh dune hoc khac eing nhir tic the tyrosinase Nghien ciru cua Omonhinmin A va ding sir nim 2011 di chimg minh kha Jiang cita A cathartina chita trt sot ret in vivo bing ca the kith thick hoat dOng cUa cac he thOng chOng oxi h6a cua co the Dich chiet re, la va hoa da chrac thing minh co kha nang khang su hinh khOi u in vitro tren to boo K562 leukemia [3-5,7,8] Melanin la nhirng hop chat mau, dang polyphenol, ton tai a da, long, t6c, mAt nguai, N6 gia vai tro quan trong can hang the oxi h6a cua da bai kha nang bit g6c tir Melanin duac sinh tong hap a melanocyte, bit dau tir ca chat L-tyrosin vai su tham gia cda enzyme tyrosinase Do vay, irc the enzyme tyrosinase, lam glom luong melanin tao [6] VAT LIEU VA PHOING PHAP Nguyen lieu Cay Huvnh Anh dirge thu hal tai Quan Thu Dire, Thanh phi-% HO Chi Minh va cluac dinh danh tai BO mon Sinh thai va Sinh hoc tien hoa, Truing Dai hoc Khoa hoc Tu nhien, DHQGTrang 61 Science & Technology Development, Vol 17, No.T3- 2014 HCM Ben be phan ctia cay: re-, than true, than leo va la duqc thu hal, say kho, xay nho Phtron phip Dietz the cao ethanol Cao ethanol da cac be phin dune, dieu the bang phtrang phap ngam 24 gi6 Sau ngam va chiet lin, cac mau duqc co quay thu cao tit& Khao sat hogt tinh khang khudn bang during kinh yang vo khudn Cac chimg khuan sir dung: Staphylococcus aureus K, Staphylococcus aureus, Bacillus subtilis, Shigella flexneri, Escherichia coil, Salmonella spp, Pseudomonas spp dtr6c hoot h6a va trai tren mai trung LB Cao ethanol be phin cita cay duqc pha loang ethanol tuyet dOi va dat len &a giay 5mm vai nOng de 500 gg/cria Kanamycin ding de 30 µg/dia lam chimg dirong, ethanol µl/dia lam chimg am Khcio sat Ming hit khzi theo Yen va Duh (1993) Cao ethanol be phan cita cay va vitamin E (cluing dtrcmg) &Km pha bang ethanol tuyet ClOi voi nang de 500.tg/ml Ethanol tuyet d6i la cluing am Hut Ian lust ml cac dung dich ten vao Ong nghiem nang biet, b6 sung 2,5m1 dung dich dem Na2(PO4)30,2M - pH 6,6, IL deu, them 2,5 ml K3(Fe(CN)6) 1% va u a 50 °C, 20 phin Them 2,5 ml TCA 10% va ly tam 7000 Ong phirt.Thu ml dich nOi, them 2m1 ntr6c cat, 0,5 ml dung dich FeCI3 1% San pham thu (lucre de hap thu a 700 nm Khao sat kha nang bdt gdc to DPPH Cao ethanol be phin cua cay duos pha loang ethanol tuyet d6i day nOng khac Vitamin E la chimg ducmg, ethanol tuyet dei la chting am Hut Ian loot 0,5m1 cac dung dich tren vao tirng Ong nghiem, them 3m1 ethanol tuyet d6i, 0,5 ml dung dich DPPH 0,6 mM duqc pha ethanol tuyet dOi Lac deu hem imp, u tai 36 phut a nhiet de ph6ng va de Trang 62 hap thu a bloc song 517 nm Phan tram bat gee ti DPPH duqc 'the djnh bang ding thire: 0/0 [(A thong am - A blank) - (A thu nghiem - A blank)] x 100 IC chung am - A blank) (1) Lutz ch9n dung mai pha cao va khao sat kha nang tic chef tyrosinase 70 gl dung dich cao ethanol than leo pha loang ethanol tuyet ai, DMSO 1%, DMSO 100% day nOng de khac duqc nap len dia 96 gieng Mau thing ant khong chira chit thir nghiem Them 30 pi dung dich tyrosinase nOng de 333 U/m1 va a nhiet de phong 10 phut Them 110 gl L-tyrosine mM va tiep tic 30 Olin Tien hanh de hap thu a btrac song 490 nm Kojic acid ducfc diing lam chimg during Cac miu di kern vai mk blank kh8ng chira tyrosinase Phan tram irc the tyrosinase duqc xtic djnh bang cling thirc (1) Tien hanh so sanh kha nang uc the tyrosinase cua cao ethanol be phin cda cay ming a 550 µg/ml bang each lap lai thi nghiem nhu ten Dinh tinh cac h9p chdt thti cap tren matt cao ethanol than leo Cao than leo pha bang ethanol tuyet del nOng a mg/ml va djnh tinh sir hien dien cac hop chat thir cap bang thuOc thin khac nhau: alkaloid (Wagner), triterpenoid - steroid (Lieberman Burchard, Salkowski), saponin (HCI 0,1N, NaOH 0,IN), phenol (FeC13 5%), tannin (gelatin man), flavonoid (chi acetat, H2SO4 dam dac, NaOH 1%, Shinoda) Cac mau thin di kern v6i Mau chimg am chi c6 dung mei va thu6c thu [I] Phurmg phap phan tick va tie li so lieu Cac se lieu dtruc xir 19 bang phan mem SAS 9.1.3 Service Park Si khac biet c6 nghia ye mat th6ng ke a mac P (p-value) < 0,05 Cac sO lieu dupe With bay dm% dang trung binh ± de , lech chuin TAP CHI PHAT TRIA KH&CN, TAP 17, Si T3 - 2014 Be KET QUA VA THAO LUAN Cao ethanol cac be phan re, than true, than leo, la dm cay Huynh anh c6 hien suet chiet cao len lust 10,23; 4,63; 4,47; 34,12 (%) tinh tren luting kho So voi cac be phan lot; la co chira nhieu hqp chat hem tan ethanol nhat Khoo eat hoot tinh khang khuin cua cao chat O Bang ket qua khang khuan caa kanamycin chi:mg mink thi nghiem hoot Ong Veit vi chimg am ethanol chimp mirth hoot tinh khang khuan cea cac loot cao hoan toan cac hcrp chit co cay ming dij 500 1.1g/dia, cao than leo the hien hoot tinh khang khan monh dei vied S aureus — vi khan gay cac berth viem da, co the troy nhan 16 Op pan quan trong phan my phi= &Ong da Cao than trvc c6 hoot tinh khang monh dei vol Samonella spp, S aureus Cao re kiting monh voi E colt, Samonella spp, S aureus So voi nghien tiro cna Mohammad Rashedul Islam (2010), cac phan don khac tircao methanol la caa loci day hujrnh khic (Allamanda carthartica) deu the hien kha nang khang khan thap cho chtIng S aureus va E colt Trong nghien ciru cua Swjanja Pulipati va tong sir (2012) en v&i hoa cua cay Allamanda carthartica, ly trich bang ethanol, voi ming de 500 µg/die cho hoot tinh khang khan trung birth yeti S aureus va E colt [10] Bing Ket qua Idiao sat hoot tinh khang khuan tir cao ethanol be phan cea cay (don vi tinh: mm) Chung vi khan La Thin leo Than tine Re Kanamycin Ethanol S aureus K 6,00:1-1,00c 7,67±1.15c 7,33=0,58, 12.33±2.08b 19,67*1,53a 6,67±0,58c S aureus 13.67=1,53b 18,33±2,08° 17:67+1,51° 16,31+1436 17,33±3,051 6.67±1,15c Shigella flemeri 5,67±0.58c 7,33±0.58, 5,67±0,58c 12,00=1,006 18,67±2,894 6,33*0,5Ss B subrilis 7,00:4,00 6,00±1,006 5,67=0.58' 7,33±I, l 5b 21.00±1,00' 6,67*l,53b 13,33±.2,086 14,33±0,586 14,67±0.586 18,6711,15° 19,67±0,581 6,67±1,53c 8,33±2,08c 8,67±-1.53, 13,67±4.516 14.67=1.53' 21.67±1.53, 6,67±0,58c 11,33=2,08, 10,67=2.08C 19,670.58" 16,33±3,21' 21.33=1.53' 6,33=0.58d khdo sat ' E coil Pseudomonas spp Samonella spp (a, b, c thir phan hang) Paulo sat nang lire khir cast cao chat Bang Ket qua khao sat nang lirc khir caa cac cao chiet OD700 Vitamin E Re Than trvc Than leo Là Ethanol 0,72+0,02' 0,234,02' 0,17±0,00d 0,52±0,056 0,19+0,00'd 0,10+0,02' Trang 63 Science & Technology Development, Vol 17, No.T3- 2014 Hinh Ket qua khao sat Ming luc kb./ cna cac cao chiet (Y1- vitamin E, Y2- re, Y3- than true, Y4- than leo, Y5- la, Y6 chUng am) Dua vao Bang 2, a loai cao ethanol cila cac bd phan re, than true, than leo, lab nOng dd 500 lig/m1 deu the hien nang luc sit Tit a cac thi nghiem deu co gia tri dd hap thu xep hang cao hcrn mau chirng am ethanol Cao than leo co nang lut khir math nhat, gat bing 3/4 men chirng duong vitamin E Khao sit kha nang bit gric tty DPPH cua die cao chiet Tir &rang cong the hien tuang quan tuyen tinh gifra ndng dd meu thi.1 va gia tri % bit cac g6c to DPPH (Hinh 2) not suy duot gia tri ICso irc the DPPH ciia cac men cao thi nghiem 100,00 100,00 x so,00 80,00 Op 60 00 OS 60,00 ' 40,00 40,00 20,00 if 0,00 20,00 0,00 7,81 15 63 31,25 62,50 125,00250,00 CP ,,pro 4"' — Dl Wing da uglml CPCP D3 en Ning ughnl —D5 Huth Phin trim bit g6c to DPPH dm vitamin E va cac cao ethanol tit bd phan cay Huynli anh cac ding dd khac (Dl- vitamin E, D2- re, D3- than true, D4- than leo, D5- la) Bang Gia tri 1050 irc the DPPH cua cac cao chiet to bd phan cay Hujmh Anh IC50 Vitamin E R6 Thin Ulm Thin leo La 62,04±17,970 713,44±188,896 1397,24± 263,800 100,65±2,830 936,8633,696 Dua vao Bang 3, cao than leo co gia tri IC50 thAp that so viii bd phan lai, dot 100,65 µg/ml So WU gia trj 1050 ctla vitamin E la 62,04 ttg/ml, cao than leo the hien kha nang bit g6c to rat match Ket qua cang Wang &tong v&i nghien ccru dtroc trinh bay boo cao oda Amjad Hameed va tong su nom 2014 mla loci A cathartica [21 Trang 64 Lira chon dung mai pha cao va khao sit kha nang tic ch6 tyrosinase Sit dung ethanol tuyet ddi va DMSO 100%, gia trj % itc the tyrosinase ctla mitt khOng tuong quan ding dd chat thit, nghiem Ngtroc lai, sir dung DMSO 1% pha loang cao, gia tri % itc the tang theo ding dd chAt thit nghiem (Bang 4) ' TAP CHI PHAT TRIEN DUN, TAP 17, S0 T3 - 2014 Bang Gia trt irc the tyrosinase cue cac cao than leo pha cac dung m8i khac 'Mau % tic the dm kojic acid Nang (10 kojic acid Nang da mazu TN (jig/m1) % tic the ink TN pha DMSO 100% % trc the mL TN pha ethanol 100% % tic the Mitt TN pha DMSO 1% 500 97,08 2000 -344,53 -1,46 87,07 250 96,35 1000 -102,19 18,25 74,14 125 96,35 500 -32,12 29,93 50,13 62,50 86,13 250 18,25 18,25 35,09 31,25 75,18 125 57,66 9,49 27,70 15,63 54,74 62,50 64,23 5,84 19,79 7,81 4,38 31,25 65,69 13,87 17,68 3,91 1,46 15,63 62,77 21,90 14,78 Nguyen nhan c6 the DMSO va ethanol c6 kha nang lam mat tinh en dinh cau trim protein, Anh huang den hoot tinh ctYa tyrosinase thi nghiem Theo Tjemberg va cong su (2009), ming DMSO > 5% c6 kha nAng anh hirang den enzyme vii st 'Nem cac tap chat DMSO tang nhu ethanol cam den tai he qua tucmg tu Nai suy tit Wang quan tuyen tinh gifta nOng de, mei' thtY va gia tri % irc the tyrosinase (Bing 4) cdo &rem gia tri IC50 irc the tyrosinase cite mL cao than leo (Ma tan DMSO 1%) la 537,63 µg/ml va mau chimg duong kojic acid dat 9,86 nOng 65 550 gen bang gia tri IC50 cda cao than leo, cac mit' than true, re, la dew c6 gia tri % irc the thap han men cao than leo So yeti cite nghien cim khac, ding chi Allarnanda thi meu than leo dtrac thtt nghiem (Allarnanda neriifolia) c6 hoot tinh chap han Allamanda carthartica ninmg cao han Allamanda blanchetti Cao than leo ming the hien hoot tinh irc the tyrosinase cao han cil nghe Curcuma longa vai IC50 785,10 lig/m1 ducir clan gian sir dung him ma WO, tri them den [4, 5, 9] Bing Phan tram irc the tyrosinase cda cao chiet tir ba phan cay Huynh Anh d nOng dO 550µg/ml % Tic the Re Than true Than leo La 27,671:0,441' 40,60±1,85'b 59,61+7,08' 15,40±4,25` (a,b,c thee hr pilau hang) Binh tinh cac hqp chit thfr cap tren mau cao thin leo cay Huynh Anh • Bing Ket qua dinh tinh cac nhom chat hoa tux tren matt cao than leo Alkaloid Triterpenoid Steroid Saponin Phenol Tannin -+ + + - + + Flavonoid + Trang 65 Science & Technology Development, Vol 17, No.T1 2014 (") 111% Triterpenoid iabot Flavonoid Hinh Ket qua djnh tinh cac nhom chat him hoc tren mau cao than leo Ket qua trinh bay Bang va Hinh ding Mang Bong voti nghien dru cda Elita Scio va cOng str, sang loc cac 'Thom chile tren mAu la cao Allamanda cathartica Bang Bang phan tich phAn flavonoid bang cac phan irng mau Thi nghiem Nhom chat dir doin Hien timing Flavane Chi acetate Tda vang + Shinoda Vang nhat + H2SO4 Cam, NaOH Cam, Trang 66 Flavanone Flavone Flavonol Flavanonol Chalcone + + + + + + + + + + TAP CHI PHAT TRIbl Ith&CH, TAP 17, 30 T3 - 2014 ITInh Ket qua cac thi nghiem phamtich phan flavonoid Bang cac phial img vOi cac thutic tit* co the dir (loan dugs clang flavonoid chinh cao than leo Khi phan ung voi chi acetate, hau het cac nhOm deu co kha ning tao phirc Ma Trong phan Ong Shinoda, mau c6 mau yang nhat (am tinh) chimg to cac flavonoid clang flavane hoac chalcone Khi phin fmg yeti H2SO4, NaOH, sin pham tau cam, chimg to thin co flavanone, flavonol va chalcone Tuy nhiert, nen tit* c6 flavanon, flavonol thi phan img Shinoda phai c6 mau dO, cam Vay nen khong co flavanone, flavonol Mat khac flavan 1:4 bien tinh tao mau cam, kin tic dung veri acid hay kiem manh Vi vay, flavonoid cao thin leo th the la chalcone va flavane [I] KET LU4Isi Cao thin leo Huy_ rth Anh co hoot tinh khang khan (S aureus), khang oxy h6a, frc the tyrosinase cao nhit Trong cao thin leo co: alkaloid, triterpenoid, steroid, phenol, tannin, flavonoid Flavonoid c6 the clang flavane hoac chalcone Trang 67 Science & Technology Development, Vol 17, No.T3- 2014 Studying antibacterial, antioxidantand tyrosinase inhibition activities of golden trumpet (Allamanda neriifolia) • Tran Pham Tue Hung • Nguyen Thi My Hanh • Quach Ngo Diem Phuong University of Science, VNU-HCM ABSTRACT Golden Trumpet (Allamanda neriifolia) Phytochemical identification of buds'extract was investigated through antibacterial, indicated the presence of alkaloids, steroids, antioxidant, anti-tyrosinase activities Ethanol triterpenoids, phenols, tannins, flavonoids extracts of plant'parts: leaves, stems, roots, Flavonoids possibly are flavanes or buds were used, which resulted in the most chalcones highlighted qualification of buds Key words: Tyrosinase inhibition, Golden Trumpet (Allamanda neriifolia),antioxidant TAI LIEU TRAM KHAO [1] N.K.P Phung, Phuong phi) co lap hap chat hau ca, Nha zit& ban Dal hoc qujc gia TP IM Chi Minh, 80-147 (2007) [2] A Hameed, G Nawazand T Gulzar, Short inhibitor and antioxidant agent, Journal of Biological Sciences, 10(2), 138-144 (2010) Isolation, identification and tyrosinase inhibitory activities of the extractis from Allamanda cathartica, Natural Resources, 2(3), 167-172 (2011) [5] I Batubara, L.K Darusman, T Mitsunaga, G Prota, Progress in the chemistry of melanins and related metabolites, Med Res Rev, 8(4), 525-556 (1988) D.F.N Schmidt, R.A Yunes, E H Schaab, A Malheiros and V C Filho et al., Evaluation of the anti-proliferative effect the extracts of Allamanda blanchetti and A schottii on the growth of leukemic and endothelial cells, Pharm Pharmaceut Sci, 9(2), 200-208 (2006) [8] R Singh, P Kharb, K Rani, Rapid micropropagation and callus induction of Catharanthus roseus in vitro using different explants, World Journal of Agricultural Sciences, 7(6), 699-704 (2011) [9] P.M Souza , S.T Elias , L.A Simeoni , J.E D Paula , S.M Gomes , E.N Guerra , Y.M M Rahminiwati, E Djahhari, Potancy of indonesian medicinal plants as tyrosinase Fonseca , E.C Silva, D Silveira , P.O Magalhaes , Plants from brazilian cerrado communication: Chemical composition, antioxidant activities and protein profiling ofdifferent parts of Allamanda cathartica, Natural Product Research (2014) [3] O.A Conrad., I.P Dike, U Agbara., In vivo antioxidant assessment of two antimalarial plants - Allamamda cathartica and Bixa orellana, Asian Pac J Trop Biomed, 3(5), 16 (2012) [4] K Yamauchi, T Mitsunaga and I Batubara, Trang 68 T413 CHI PHAT TRIEN ITH&CM, TAP 17, 30 T3 - 2014 with potent tyrosinase inhibitor• activity, Plos one, 7(11), 48589 (2012) [10] S Pulipati, V Siddartha, Evaluation of antibacterial activity of flower extracts of Allamanda cathartics, L., IJPWR, 3(2), 12 (2012) [1 ] Xiaoying, C.W.J.M Ronald, H.R.C.S.S Hongbo, Z.D.Q.H.I Maibach., Human in vivo and in vitro hydroquinone topical bioavailability, metabolism, and disposition, Toxicol Environ Health A, 54, 301-317 (1998) rang 6') ... hanh so sanh kha nang uc the tyrosinase cua cao ethanol be phin cda cay ming a 550 µg/ml bang each lap lai thi nghiem nhu ten Dinh tinh cac h9p chdt thti cap tren matt cao ethanol than leo Cao. .. dtrac thtt nghiem (Allarnanda neriifolia) c6 hoot tinh chap han Allamanda carthartica ninmg cao han Allamanda blanchetti Cao than leo ming the hien hoot tinh irc the tyrosinase cao han cil nghe... thire: 0/0 [(A thong am - A blank) - (A thu nghiem - A blank)] x 100 IC chung am - A blank) (1 ) Lutz ch9n dung mai pha cao va khao sat kha nang tic chef tyrosinase 70 gl dung dich cao ethanol than