Điều chế và kiểm tra chất lượng thuốc phóng xạ 90YDOTAnimotuzumab dùng trong điều trị ung thư đầu cổ

• Nghien cu'u - 1(9 thuOt Dieu cht va kiem tra chAt luvng thulk phong xa 90Y-DOTA-nimotuzumab dung dieu tri ung thtr dau co Nguyen Thi Thu'*, Vo Thi Cam Hoa', Nguyen Thi Khanh Giang' Nguy& Thi Ng9c', Mai Trong Khoa2, TrOn Dinh Ha' Nguy'Zn Linh Toa', HO Anh San' hot Muhl (Da Lot) Nah vier Bach Mai 'Hoc ri(M Quoit Y *E-mail: ngthithu2006(myahoo.cam Summary With regard that the radioactive isotope Yttrium-90 ( 90Y) with the physical half-life of 64.14 hrs and highly energetic beta-ray emission well serves as an ideal therapeutic radioisotope in clinical use, 90Y-DOTA-nimotuzumab radiopharmaceutical was prepared by conjugation of monoclonal antibody with the bifunctional chelating agent p-NCS-Benzyl-DOTA The chelating was covalently bonded to an antibody using isothiocyanate (NCS) chemically chelated to the radioisotope Ytrium-90 by attachment of carboxylate anions The coupling of p-NCS-Benzyl-DOTA to nimotuzumab at molar ratio 25:1 accomplished at 37°C and pH 8.5, achieved the efficiency of about 53.4 % after 12 hours The coupling labeled with 90Y isotope at 42°C and pH 6.0 attained a radiolabeling efficacy of 97.8±0.3 % after 60 minutes The obtained Radiopharmaceutical 90Y-DOTA-nimotuzumab proved to satisfy the in vitro stability and meet the quality requirements for use in the treatments of head and neck cancer Keywords: 90Y-DOTA-nimotuzumab, Radiopharmaceuticals, head and neck cancer therapy Mee clAU Trong nhOng nom gen day, thuoc phOng xa khang the dun d6ng danh deu &rig vi phOng xa da duvc nghien cOu dieu the va Ong dung chAn doan va dieu tri lam sang Khang the don d6ng nimotuzumab duvc danh d5u vai ding vi ph6ng xa 90Y to thuoc ph6ng xa 90Y-DOTAnimotuzumab de nghien cipu dieu tri ung thu, d'au co [1] Nimotuzumab la khang the nhan h6a, thuOc loai IgG1, thirc hien chipc nang nh5m dich len thu the cac you to tang true:mg bleu bi EGFR (Epidermal Growth Factor Receptor) tren cac to bao ung thu' bieu mo Ben c5nh ch(rc nang tieu diet to bao ung thu' theo cac ca the sinh hoc, 90 Y-DOTA-nimotuzumab diet to bao ung thu' theo co' the bi:Pc xa ion h6a [21 V6i thei gian ban 64,14 gio,, nang luvng tia beta 2.280 KeV, quang chay m6 4-11mm, 9°Y la &rig vi ph6ng )(ply tuerng dOng dieu tri benh gen vitei phan tiv khang the 131 D6 danh d5u vitei (long vi ph6ng xa TAP CHI DUQC HOC - 9/2015 (SO 473 NAM 55) kim loai co hoa tri Ill nhu, 9°Y, khang the door gen vai ph6,c trung gian la p-SCN-benzyl-DOTA to p-SCN-benzyl-DOTA-nimotuzumab Ph5n t6, p-SCN-benzyl-DOTA la ph& till, de t?o ph0,c ben vOi nhieu nguyen to co h6a tri Ill nhu' 90Y3*, vay, 90 Y &roc gan vao theo co' the phan irng the nucleophin SN tpo phirc hap 90Y-DOTAnimotuzumab [41 Vi thuoc ph6ng xa co china khang the va deng vi ph6ng xa 90Y co thOi gian ban r5 ngen, nen kh6ng the mua tO, nu,dec ngoai vi kh6 bao quen van chuyen, chu,a ke den la gia ce rat det Vi the, can thiet dieu the nu,dec de ch0 citing nguOn nguyen lieu cung cep cho y te Bai bao trinh bay nghien cu'u danh dAu dieu the thu6c phOng xa 90Y-DOTA-nimotuzumab de dieu tri ung thu' dhu co ten lam sang Phu'oing phap nghien CCPU Nguyen lieu, hoe chat, thiet bi Deng vi ph6ng xa 9°Y clang 90Yttriumacetat (CH3COO)3 90Y, nOng d(f) ph6ng xa 23 • Nghien ciru - Kji thuOt 592 mCi/ml Khang the nimotuzumab 50 mg/10 ml, h5ng CIMAB, Cu Ba C6ng thac phan tir C6566F110082N174602056S40 Cac hea chAt 2-ethylhexyl 2-ethylhexyl phosphoric acid C16H3503P (PC88A), Daihatchi; p-NCS-BenzylDOTA S-2-(4-lsothiocyanatobenzy1)-1,4,7,10 tetraazacyclododecan tetra acetic acid, C24H33N508.2,5 HCI.2,5 H2O, Macrocyclic; Sephadex G25 PD10, Hang GE Healthcare; Mang th5m tich Slide A lyzer 10 K (dialysis cassettes), MWCO 66453 Hang PIERCE; Te bao ung thu, HEp-2 (Human Epithelial Type 2), CCL-23, American Type Culture Collection (ATCC); Thiet bj phOng xa to, chop Cyclone B431200, Perkin Elmer, ph6 Ice gamma ORTEC, may beta GM, may beta nhAp nhay long Aloka 6110 May PTS 100, Hang Charles River Laboratory Cac thi nghiem duvc tien hanh tai Vien Nghien hAt nhan, DA Lat Khao sat ti le mol, nhiet do, thai gian va pH dieu the DOTA-nimotuzumab Thi nghiem thy'c hien vai n = cho tat ca cac diem khao sat va ket qual5y gia tri trung binh Cho vao cac chai phan Ong 50 ol bicarbonat 0,05 M, met luong p-NCS-benzyl-DOTA (pha bang DMSO den nong dd 10 mg/ml) va khang the nimotuzumab theo cac ti le mol IA 5:1, 10:1, 25:1, 50:1 100:1 va 200:1 (dung 0,66 nmol khang the m6i phan ang khao sat), dieu chinh pH 8,5 bAng pi dung dich NaOH 0,1 M, ten nhe dung dich h6n hap va tai cac nhiet de 4°C, 24°C, 37°C Khao sat thai gian phan Ong duvc thtyc hien tai ti le mol 25:1, nhiet de 37°C, pH 8,5, thai gian 10, 40, 120 , 180, 360, 720, 1080 va 1440 phOt Khao sat pH tao phU'c thac hien tai cac pH 3, pH 4, pH 5, pH 6, pH 7, pH 8, pH va pH 10, dieu chinh bAng dung dich NaOH M va HCI M Nhiet ph& O'ng la 37°C, ti le mol 25:1, thai gian 720 phOt Dieu the NY-DOTA-nimotuzumab va kiem tra chat luvng DOTA va khang the nimotuzumab theo ti le mol IA 25:1 (230 og DOTA va 2000 og khang the), dieu chinh pH 8,5 b5ng 6µl dung dich NaOH M, ten nhe dung dich h6n hop va tai nhiet 37°C thai gian 12 gia Hen hcyp duvc loc qua cot gel Sephadex G25, dung ch5t raa giai IA dung dich NaCl 0,9%, van toc rira giAi IA 30 cm3/gia, thu DOTA-nimotuzumab vai the tich IA ml tai phan doAn 4-6 ml Phirc hop &roc th5m tich yang 24 gi6 dem phosphat 0,1 M dung mang thArn tich dialysis cassettes Slide A lyzer 10.000 Da, sau ly tam va xac djnh ruing khang the tai baac song 280 nm Dieu the NY-DOTA-nimotuzumab M D6 di6U the 30Y-DOTA-nimotuzumab co hoat rieng 0,37 GBq/mg, danh dAu 592 MBq 30Y (1000 ml) tai thai diem dieu the vai 1000 og (750 ol) khang the DOTA-nimotuzumab dem acetat 0,5 M, pH U hen hap 60 phOt a nhiet de 42°C, sau de, phan tich lAng phaang phap sac k9 lap mong TLC dung m6i amoniacetat 10% va methanol theo ti le the tich 1:1, kiem tra hieu suet danh d5u bAng phiyang phap loc gel Sephadex, dung chAt raa giai IA dung dich NaCI 0,9%, vai toc dd raa giai IA 30 cm3/gity Kiem tra chat ng: Phac hap 30Y-DOTAnimotuzumab &roc kiem tra de tinh khiet h6a phOng xa bAng phaang phap sec ly lap mong, sec k9 loc gel (3.7] , Kiem tra de tinh khiet hat nh5n bang phaang phap chiet ten giey Extraction Paper Chromatography (EPC) 181, chAm PC88A va mAu vao vi tri g6c, phat trien dung dich NaCI 0,9%; Kiem tra sach gamma bAng phe Ice gamma 171; Kiem tra de on djnh b5ng phirang phap thuec huyet ngt.rai va cac dieu kien bao guar' nhir dem phosphat PBS 0,01 M, NaCI 0,9%, phan tich, theo del den ngay131 The, ye khu5n va tha not do, c to vi khu5n theo TCCSDPPX-16/15-2010 191 Kiem tra hoat tinh mien dich cue thuec ph6ng xa 30Y-DOTA-nimotuzumab, gen in vitro tren to bao ung thy bieu me HEp-2, s6 luvng 0,5 x 106 to bao/gieng gen, phaang phap Ap dung IA saturation binding assay (101 Dieu the phirc khang the clan dong vai chit tao pherc hai ch&c hang p-NCS-benzylDOTA-nimotuzumab: Thtyc hien phaang phap gen khang the vai ch5t tao phac trung gian p-NCS-benzyl-DOTA claa tren cac nghien cau (55 ceng be [5 ' 6] Cho vao chai phan O'ng 500 ol bicarbonat 0,05 M, met luang p-NCS-benzyl- Xtv ly s6 Cac ket qua nghien eau duvc xa 19 theo cac thu5t toan thong ke y hoc Tinh gia tri trung binh va de lech chu5n bAng Excel, tinh hoat tinh mien dich bAng phAn mem GraphPad Prism 6.0 24 TAP CHi DUOC HQC - 9/2015 (SO 473 NAM 55) • Nghien cu'u - K9 thulat Cac ket qua khao sat hieu suet gen theo ti le mol, nhiet dO, thai gian va pH gen DOTA vai khang the dem bicarbonat 0,05M tom tet bang nhu, sau: Ket qua va ban luan Ket qua dieu che phirc hap DOTAnimotuzumab Bang 1: Ket qua khao sat cac lieu kin tao phut hop DOTA-nimotuzumab Tile mot DOTA: Khang the (nmol:nmol) NhiOt dO 4°C Hiiu suit gin (%) Nhiet de, 24°C Hiiu suit gin (%) NhiOt c10 37°C HiOu suit gin (%) Then gian phan (mg (phtit) Hi#u suit gin (%) pH too phav Hieu suit gin (%) 5:1 10:1 25:1 50:1 100:1 200:1 85,0 63,3 54,6 46,3 12,5 12,3 80,7 56,0 53,9 41,0 19,1 12,6 72,0 60,1 55,0 49,6 48,5 46,8 10 0,9 6,0 40 4,4 9,7 Ket qua er bang cho they tai cac if le mol lb 5:1, 10:1, 25:1, 50:1 100:1 lib 200:1, hoet tinh gen DOTA vai khang the dat ter 12 den 85% Tai ti le mol 5:1 ket qua gen det khoang 72-85%, kr tinh duvc so nhem DOTA trung binh gen ten mei phgn ter khang the gen lb 3,9 (Hnatowich va Ong sky) twang if le mol 25:1 so nhOm DOTA trung binh gen ten mei phan ter khang the 13,5 Val ti le mol 50:1 b.& len, se nhern gen Ian hen 20,0 cac nhiet dO Wet qua khao sat cho they phU'c tao thbnh co hieu suet gen khac nhau, phan tey p-NCS-benzyl-DOTA tham gia cang nhieu thi hieu suet gen cua phOc vai 9°Y cang it, ljt co so canh tranh cua met luvng this DOTA chiem ch6 met so !Leong c6 hen cua 'long vi ph6ng xa 90Y Cac khdo sat nhiet de cho they nhiet de 4°C, 24°C va 37°C a gen DOTA vai nimotuzumab khOng lam thay dei Ian ye hieu suet gen Thai gian phan Ong gen cua DOTA va nimotuzumab 10 phut phirc hcyp DOTA-nimotuzumab teo lb 0,9% Thbi gian 360 phiit, phirc hap DOTAnimotuzumab tao 47,3% Thbi gian phan Ong 720 phut, phOc hap tao lb 60,1% va thai gian phan Ong 1440 phut, phirc hop O =N C + II 120 25,6 54,5 180 40,1 59,8 360 47,3 63,9 S 1080 53,2 77,0 1440 52,1 10 60,4 DOTA-nimotuzumab tao lb 52,1% Nho vay, thai gian cang tang, hieu suet gen Gang tang, deng hoc dm Olen Ong tao phOc hop p-SCN-BnDOTA-nimotuzumab tang theo thdi gian Anh hoeng dm pH len qua trinh gen cho they, mien pH acid, hieu suet gen cua p-SCN-BnDOTA va nimotuzumab de tao phOc hap DOTAnimotuzumab dat 10,0 - 50,0% Trong mien pH kiem, hieu suet gen dat 64,7-77,0% Co the ph& Ong gen p-SCN-Bn-DOTA len khang the nimotuzumab lb phan Ong acid-base theo thuyet Lewis, de, khang the dOng vai tro base p-SCN-Bn-DOTA clang vai tr6 lb acid Phan Ong gia,a nimotuzumab va p-SCN-Bn-DOTA lb phan Ong giu,a acid yet, va base yeu nen moi truing cang acid hay Gang base thi san phem se de bj ph8n hay Tern lei phOc hap p-NCS-benzyl-DOTA-nimotuzumab doec dieu che tai ti le mol la 25:1, nhiet de 37°C thai gian phan Ong 720 phut va pH 8-9 Giai thich ca che, DOTA lb tic nhen tao phOc hai chipc nang, lien ket Ong hoe tri nhOm isothiocyanat vai nhom amin cua lysin ten phan tot, khang the qua phan Ong the nucleofin SN nhu, sau: C Protein-NH: 720 60,1 64,7 O -N - C - NH-Protein II 11 S H Theo phan Ong tren, qua trinh tao phOc xay nguyen ter nits cua lysin co chrya del dien ter to va phan tir p-NCS-Bn-DOTA la chat nen chOa carbon cation, vai hieu Ong Ong twang am (-M) bad \tong benzen lam cho carbon mang dien tich &rang, Er lien ket isothiocyanat va amin tao TAP CHI DUQC HQC - 9/2015 (S6 473 NAM 55) 25 ▪ • Nghien aru - K9 thuOt Phirc tao va danh dau ph6ng xa vai 9°Y dupp bieu dien nhu' hinh sau: HOOC r—v COON 9try S )" NH—C—NH CH N (N soy = - N )— 011 Pal—C —NH Ss" N HOOC or/ COON O Hinh 1: Phirc hop p-NCS-Benzyl-DOTA-nimotuzumab va 90Y-DOTA-nimotuzumab Philp hgp p-NCS-Benzyl-DOTA-nimotuzumab truirc tinh sach phirc qua cot, hiOu suet gen vai 90Y dat duvc 53,4%, so phan tir DOTA ten m6i phan tir khang the la 13,4 va 3,9 Tai ti la mol 5:1, hieu suet gen vai 90Y det duvc 85%, de dieu the phirc p-NCS-benzyl-DOTA-90Y-nimotuzumab dung nghien ciru dOng hoc va dieu tri, dkra vao s6 nh6m gen can, ti le mol thay del cho phu hap SO nhOm gen DOTA ten phan tir khang the can khoang 4-10 nh6m va hoat dO rieng la 0,37 GBq/mg Phirc DOTA-nimotuzumab tao c6 nong d9 khang the nimotuzumab &pop xac dinh la 1066 mg/ml Mang them tich MWCO 10K se lopi cac phan tir nho va cac manh dirt gay cue khang the c6 krong phan tir nho han 10.000 Da khoi mang theo ca the khuyech tan chon 19c qua mang Bao quan DOTA-nimotuzumab nhiet d9 -20°C de danh dau dieu the 90Y-DOTAnimotuzumab Ket qua dieu the 90Y-DOTA-nimotuzumab va kiem tra chat luvng Higu suet danh clAu: Philp hap 9°Y-DOTAnimotuzumab duvc dieu the vai hiau suet danh dau 97,8 ± 0,3% (n = 6), (hinh 2a, 2b): Hinh 2: Hieu suet danh dAu 90Y-DOTA-nimotuzumab (a): 97,5%; (b): 99,1%, TLC, radioautograph Hieu suet danh dau cua phirc hop mien dich vai deng vi ph6ng xa dat tir 97,5% den 99,1% Cac dO thi 2a, 2b cho they DOTA-nimotuzumab sau tinh sach, danh dau vai 90Y dat hreu suet cao, hoat dO Hong 9°Y-DOTA-nimotuzumab dat 370 MBq/mg 41lb DO tinh khiet hem phong xa (RCP) va tinh On dinh: RCP dat han 99,3 ± 0,4% (n = 10) (hinh 3a) va phirc On dinh huyet ngtrol den ngay, On dinh dem PBS 0,01 M va NaCI 0,9% cho den bao quan 6' 4°C (hinh 3b): 11.0 0300 t 90 ea I80 "Y-Nhnotezumab 99,9% a 70 60 E- 50 * §a 40 • 30 al 20 raa 10 /Ma I • "Y - (b) On Sink Iron, huyil Mani, ngun31 —II— BOO 009n Iron° PBS 0,011A —16— Bin clan MCI UV Thing= (NOY) Hinh 3: 06 tinh khiet h6a ph6ng Xp (a) va On dinh 9°Y-DOTA-nimotuzumab (b) 26 i TAP CHI DUOC HOC - 9/2015 (SO 473 NAM 55) • Nghien C01.1 - Ky mot Dc) tinh khiet hcia phOng xa cua cltygc chat phOng xa 90Y-DOTA-nimotuzumab dat duvc la 99,3% Tren thi cho thay vi tri cua 90Y-DOTAnimotuzumab Ityu tai diem gOc cua b5ng sac k9 TCC, Rf = 0,0 - 0,1 va R cua 90Y la 0,9 - 1,0 90Y-DOTA-nimotuzumab On dinh dem PBS 0,01% hoac NaCI 0,9% cho thay co the bao quart de sir dung va on dinh human serum cho thay tinh ben cua thu& &pa vao ca the xa: DO tinh khiet DO tinh khiet hat nhan hat nhan ph6ng xa duvc kiem tra bang phtyang phap sac k9 chiet tren gray EPC, (hinh 4a): a INDIC no Emmy, (.04 FNE$1 Ina 181007 lm, Tow MOO U15 Aepuew 03.13/1014 17 70 75 e Or ChM 00 I WV0H10,10POOHOGYlop clop 0vPx,6 ilanW 07 P.C 40 Hinh 4: Ket qua ki6m tra dO tinh khiet hat nhan 90Y-DOTA-nimotuzumab, autoradiography (a) va sach gamma cua 90Y-DOTA-nimotuzumab, bang ph6 ka gamma ORTEC (b) Ket qua tinh khiet hat nhan cua 90Y-DOTAnimotuzumab dat han 99,998% Tai vi tri gOc, chat chiet dac hieu PC88A 90Y-DOTAnimotuzumab, 90Sr san phem 0,002% di chuyen ye tuyen ten cua dung mOi Phirc 90Y-DOTA-nimotuzumab dpt kiem tra dO sach gamma (hinh 4b), bang phO ice gamma PM cho thay khong co dinh gamma nao dung dich san phern Thu& dpt chat luting ye tinh khiet hat nhan phOng xa K& qua kiem tra vo khuan va not di)c to vi khuAn: Thu& phong xa 90Y-DOTA-nimotuzumab dat vo khuan, not dOc to vi khuan theo TCCSDPPX-16-2010, TCCS-DPPX-15-2010, IS017025 Wet qua kiem tra hopt tinh mien dich: Kat qua 90Y-DOTA-nimotuzumab gen ten to bao ung thu, dirac phan tich bang phan mem Graphpad Prism DO thi dpt den gia tri cu'c dpi bao h6a, hinh 5: Saturation binding assay 3.5 1.0 0.5 0.0 260 560 750 10130125014017.5020.002250 "Y-DOTA-nimotuzumab (nM/L) Hinh 5: DO thi gan bac, hoa 9°Y-DOTAnimotuzumab vat t6 bao ung thu, HEp-2 12;11' CHI DUVC HQC - 9/2015 (SO 473 NAM 55) Tren tryc hoanh la nOng dO tang den cua Y-DOTA-nimotuzumab, phan gen dac hieu fmol/ cell duvc ye trail tryc tung la kat qua so phirc On ten mOi to bao Gia tri Kd la 3,2 nmol/L Gia tri 13m„ ten phan mem tinh duvc la 2,9 x 105 Kat qua cho thay hopt tinh mien dich cua khang the dam bac) sau clanh dau vOl chat ph6ng xa Cac ket qua kiem tra chat luting cho thay, thu& phOng xa 90Y-DOTA-nimotuzumab dpt cac Weil chi ye kiem tra chat luting thu& phong On dinh in vitro, On dc hieu ten to bao ung thu, bleu mo, co the sir dung cac nghien ciru tien lam sang va nghien ciru lam sang 90 Ket luen Thu& phOng xa 90Y-DOTA-nimotuzumab duvc nghien cu,u dieu the dy'a ten nguOn nguyen lieu la khang the dan d6ng nimotuzumab va clang A ph6ng xa 90Y Phan Ong danh dau phOng xa dieu che 9°Y-DOTA-nimotuzumab duvc thy,c hien dam acetat 0,5 M, pH 6,0, dat hieu suat cao 97,8 ± 0,3% Thu& phOng xa thu duvc dpt tinh khiet hoa phOng xa han 99%, dO tinh khiet hat nhan han 99%, On dinh in vitro va apt cac chi tieu ve dc) vo khuan, nOi dOc to vi khuan, dam bao hopt tinh mien dich gen ten to bao ung thu, bieu ma c6 thy the EGFR Duvc chat ph6ng xa 90Y-DOTA-nimotuzumab dat tieu chuan chat luting thu& phOng xa dung lam sang dieu tri ung thu, du co 27 • Nghien ciru - K9 thuOt Tai lieu tham khao Denis Rolando Beckford Vera, Sebastian Eigner, Marco Chinol, et al (2011), "Preclinical evaluation of 177Lu-nimotuzumab: a potential tool for radioimmunotherapy of epidermal growth factor receptor over expressing tumors", Cancer Biotherapy and Radiopharmaceuticals, vol 26 No Barta P1, Laznickova A, Laznicek M, Vera DR, Beran M (2013) "Preclinical evaluation of radiolabelled nimotuzumab, a promising monoclonal antibody targeting the epidermal growth factor receptor", Pub Med NCB! J Labelled Comp Radioph, 56(5), 280-288 Gopal B Saha (2012), Fundamentals of Nuclear Pharmacy, Sixth Edition, Springer Brechbiel M W (2008), "Bifunctional chelates for metal nuclides", Q J Nucl Med Mo Imaging, 52(2), p.166-173 D J Hnatowich, R L Childs, et al (1983), "The preparation of DTPA- Coupled antibodies radiolabeled with metallic radionuclides: an improved method", JIM 02859 Elsevier Science Publishers B.V Sosabowski J K and S J Mather, (2006), "Conjugation of DOTA-like chelating agents to peptides and radiolabeling with trivalent metalic isotopes", Nat Protoc., 1(2), p 972-6 I.A.E.A (2009) "Therapeutic radionuclide Generators: 9°Sr/9°Y and 188W/188Re Generators", Tecchnical Series Number 470 Usha Pandey, Prem S Dhami, Meera Venkatesh, and M R A Pillai (2008), "Extraction paper chromatography technique for the radionuclidic purity evaluation of 90Y for Clinical Use", Anal Chem., 80 (3), pp 801-807 Vin Nghien ciru hat nhan, (2010), Tieu chua'n ca sO, DPPX-15/16-2010 ISO 17025 10 Lindmo, T., Boven, E., and Cuttita, E (1984), "Determination of the immunoreactive fraction of radiolabelled monoclonal antibody by linear extrapolation to binding at infinite antigen excess", J Immunol Methods, 27,77-89 (Ngay nhan Oaf: 29/07/2015 - Ngay duyat clang: 04/09/2015) Nghien cfru tong hcrp (S)-clopidogrel bisulfat tur clopidogrel racemic Nguyen Van Tai*, Phan Thi Trang, Nguyen Thi Thu Trang Nguyen Thi Ha, WI Dtic Nam Khoa H&J Time vat, Pen Dwaz lieu * E-mail: nguyenvantail 111@gmaiLcom Summary (S)-(+)-clopidogrel bisulfate was synthesized from clopidogrel racemic in high purity 99 %) The process proved simple (S)-clopidogrel was separated from clopidogrel racemic, then (S)-clopidogrel bisulfate was synthesized in form I and form II The form I exhibited better solubility and higher bioavailability than form II This suggested the form Ito be more suitable for medicinal use Keywords: (S)-(+)-Clopidogrel bisulfate, synthesis, chiral, form DOA yen de Tim mach dang la mdt nhOng can benh gay chet ngutri hang dhu Van the giai cung nhu, tai Viet Nam hien (S)-Clopidogrel bisulfat (ten Net du,gc Plavix), la thu6c chOng ngu'ng tap tieu cAu, ngan ngiva cuc mau dong (S)-Clopidogrel bisulfat thip6ng sir dung de du' ph6ng nguyen phat, du' ph6ng tho, phat va kiem soat cac roi loan mach huyet khdi, xci vim Ong mach 28 nhu' nhOi mau co' tim, ddt quy va benh ddng much vanh ngoai bran Thu& co kha nang ngan ngiva cuc mau thing manh hurl aspirin nhumg khong gay tac dung phu Von da day giOng nhu' aspirin Clopidogrel (C16F116CINO2S), ton tai dang clang phan enantiomer la (S)-(+)clopidogrel va (R)-(-)-clopidogrel Clopidogrel co clang dau, kem ben, kh6 tinh the va kh6 bao the hom clang muiii dm no la clopidogrel bisulfat TAP CHI DUOC HQC - 9/2015 (SO 473 NAM 55) • Nghien an' - Ky thuOt Tac dung sinh hoc lai chi co 6' &rig phan (S)Vi vey, hoat chat (+)-clopidogrel bisulfat cac clang bao che chli yeu la (S)-(+)-clopidogrel bisulfat, cho hieu qua chCra benh cao va tranh tac dung phu Tiou chuen cac Duvc dien M9, Ch5u Au deu quy dinh t9.1e (long phan (R)-(-)clopidogrel bisulfat phai nho han hoc bang 1% Trong bai bao nay, chi:mg tei trinh bay phyang phap tong hop (S)-(+)-clopidogrel bisulfat a clang Bang I va clang II tiv clopidogrel racemic thOng qua giai doan tao muOi vai acid (0-H-camphor10-sulfonic (10-L-CSA) de tach ley dOng phan (S)-clopidogrel Nguyen vat lieu va phwang phap nghien civu Nguyen vet lieu va thiet bi HOa chat duvc cung cep Nil cac hang Merck, CI Fisher, AK Scientific, hoc P.A Trung Qu6c, duvc sir dung kh6ng qua tinh che Sac k9 lap me:mg thiyc hien ten ban mong silica gel 60 GF25, (Merck) Ph6 Ong huang tip hat nhan tren may Bruker Avance 500 MHz vai TMS lam chat not chuen, ph6 hong ngoai tren may Impact 410, ph6 khoi luting tren may Auto Spec Premier- Vien I-16a hoc, Vien Khoa hoc Viet Nam Phipang phap phan tich HPLC ten may HPLC Agilent 1200-Tnyang Dai hoc Duvc Ha N6i, cot sec ky Chiral Ultrom ES-OVM + Guard cua Hang Shinwa - Nhet Ban Phirang phap xac dinh nhiet d6 n6ng chay (DSC), kiem nghiem san pham theo tieu chuan USP 34 duvc thus hien tai Vien Kiem nghiem thu6c Trung 'yang Phtrang phap nghien cOu (S)-Clopidogrel bisulfat &roc dieu che tip clopidogrel racemic theo so' dO nhu, sau: COOCH3 Nti\ 10-L-CSA Dung mei 10-L-CSA (I) Clopidogrel racemic (III) K61 Unit Baia Dung mei CI COOCH3 CI H2SO4 Dung mei (1) COOCH3 H2SO4 \ (S) I (IV) (+)-Clopidogrel bisulfate So, di) 1: Dieu che (S)-clopidogrel bisulfat ter h6n hop clopidogrel racemic (S)-Clopidogrel bisulfat la san pham ciia qua trinh tach dOng phan (S)-clopidogrel tir h6n hap clopidogrel racemic, sau teo muOi vai acid sulfuric Clopidogrel racemic phan Ong vai acid (L)-(-)-camphor-10-sulfonic dung m6i thich hap tao h6n hap hai mu6i la hai dOng phan: (S)-clopidogrel (L)-Camphor-10-sulfonic va (R)clopidogrel (L)-Camphor-10-sulfonic Day la hai dOng phanl5p the khong doi quang nen CO cac tinh chat vet 19 khac nhu, kha nang tao muOi, d6 tan dung m6i ; d6, muoi (S)-clopidogrel va (L)-camphor-10-sulfonic de &pop tao han, CO dO tan kern han muOi cua (R)-clopidogrel va (L)camphor-10-sulfonic nen de clang tach kh6i Won hop phan (Trig dirai clang tinh the ket tinh Trong qua trinh tao va ket tua muOi, main tinh the rat quan trong, giitp cho qua trinh xay nhanh va chon loc han Neu khong có mem tinh the qua trinh rat kh6 xay hoc can thai gian dai a nhiet d6 thap, san pham thu &roc co dO tinh khiet thap Loc, rira kat ttia thu &roc (S)-clopidogrel (L)-camphor-10sulfonic, sau du,cyc chuyen (S)-clopidogrel bang cach trung hoa vai dung dich NaHCO3 5% 6' 0-5°C dr, pH Y qua trinh CO the xay TAP CHI DU VC HOC - 9/2015 (SO 473 NAM 55) 29 Thu'c nghiem, ket qua va ban luen TOng hop S-clopidogel tip clopidogel racemic (IV) Khao sat qua trinh tach dong phan S-clopidogel tip clopidogel racemic • Nghien - Ky thulat hien tuvng racemic h6a (S)-clopidogrel nOng de base cao hoac nhiat do, phan Ong cao (S)Clopidogrel tao muoi vai acid H2SO4 de tao san pham (S)-clopidogrel bisulfat San pham duvc kiem tra nhiat de nOng chay theo phoang phap DSC de xac dinh la dang I hay clang II lai giam Khi sir dung dung moi co the tich ghp Ian khOi luvng clopidogrel racemic ban du thi tinh khiet cua san pham dat tren 99% va hiau suht dat 29% Vai aux tiou thu duvc (S)-(+)-clopidogrel co de tinh khiet quang hoc dat 99% chiing t6i tien hanh nghien ciru cac yet, to anh hverng den qua trinh ket tinh mu6i (S)-clopidogrel (L)-camphor10-sulfonic [1-4] Qua trinh ket tinh duvc kiem soat bang hiau suht, de tinh khiet (tinh theo % dian tich pic tren HPLC) cua (S)-clopidogrel Trong binh chu L day ton co lap vai may khuay tir gia nhiat, 96 g clopidogrel racemic (1 du'ang luvng) duvc him tan dung mai 768 ml aceton (1:8) (w/v), sau dO 34,6 g 10-L-CSA (0,5 &rang luvng) duvc them vao H6n hop phan Ong duvc dun h6i gi0, sau d6 lam lanh ve nhiat ph6ng Cho mhm (S)-clopidogrel (L)-camphor-10-sulfonic vao va tiep tuc khuay nhiat d0 phong 12 gitr LQC va rira kat tila bang aceton thu &pc (S)-clopidogrel (L)-camphor-10sulfonic clang bet mau Mu6i (S)-clopidogrel camphor sulfonat duvc h6a tan 300 ml H2O, them 300 ml DCM, trung hoa dung dich bang NaHCO3 5% den pH 7-8 Chiet dung dich vai dichloromethan (3 Ihn x 300 ml), pha hiru ca duvc rira vai NaCI bao hoa, lam khan bang Na2SO4, cat loai dung moi thu duvc 27,84 g (S)-clopidogrel Hiau suht toan quy trinh dat 29% va de tinh khiet quang hoc dat 99,08% Khao sat anh hu,deng cua logi dung moi den hiau sub' t va d5 tinh khiet cua (S)-clopidogrel: Sir dung cac dung mei ethyl acetat, aceton, toluen vai tj/ la dung moi/clopidogrel racemic =10/1 (ml/g), tj/ le 10-L-CSA/clopidogrel =1/1 (mol/ mol) Ket qua cho thay dung moi ethyl acetat cho hiau suht kha cao dat 44% nhu'ng de tinh khiet cua san pham (S)-clopidogrel chi dat 65,68%, dung moi la aceton va toluen hiau suht thu ducyc khong cao (37,5% va 37,0%), nhong tinh khiet cua san pham lai dat cao han (96,12% va 96,25%) Tuy nhien, so sanh ye mat dec hai va gia thi aceton van dogc u'u the han toluen Khdo sat anh hitting dm ti 15 acid (L)-camphor10-sulfonic tai hi6u suAt va dq tinh khiet caa (S)clopidogrel: Tien hanh phan u'ng dung mai aceton vai cac tj/ le 10-L-CSA/clopidogrel 0,5 va Ket qua thu duvc nhu, sau: Khi ti la acid 10-L-CSA la &rang luvng thi hiau suht phan Ong dat 37,5%, nhien de tinh khiet cua (S)clopidogrel lai chi dat 96,12%, nguyen nhan co the Itivng acid 10-L-CSA cao met phan ding phan (R)-clopidogrel cung tham gia tao mu6i Nib cling ket tinh vai mu6i cua (S)clopidogrel Khi ti la acid Om xuOng 0,5 doc/ng locyng mac d0 cho hiau suht thhp: dat 27,5%, nhong de tinh khiet quang hoc dat 99,35% Ring hop S-clopidogel tir clopidogel racemic (S)-clopidogrel (L) camphor-10-sulfonic IR (cm-I, KBr): 3064 (C-H khOng no); 2965,04 (C-H no); 1756,13 (-C=O-), 1507,45 (-C=C), 752,45 (the ortho) ESI-MS: [M+H]* = 322,8 (clopidogrel M = 321,82), [M-H]- = 231,23 (10-L-CSA, M = 232,3) 'H-NMR (500 MHz, CDC/3): (ppm): 1,42 (m, H); 1,58 (m, H); 1,86 (d, H); 2,04 (m, H); 2,34 (m, H); 2,70 (m, H); 2,77 (d, H); 3,75 (m, H); 3,85 (s, H, COOCH3); 4,83 (s, H); 6,81 (d, H);7,38 (d, H); 7,52 (d, H); 7,59 (m, H); 7,64 (d, H); 7,67 (d, H) ppm "C-NMR (125 MHz, CDCI3): 6(ppm): 20,14; 20,53, 23,18, 25,77, 27,79, 43,63; 44,13; 48,49; 54,70; 59,65; 67,38; 126,11; 126,85; 128,15; 128,28; 129,85; 131,40; 132,31; 132,90; 134,04; 136,71; 168,67; 218,32 (S)-clopidogrel Khao sat anh huang cua luceng dung moi den hiau sugt va tinh khiet cua (S)-clopidogrel: Chon tj/ la acid 10-L-CSA/clopidogrel racemic la 0,5; t9 la the tich aceton/khOi luting clopidogrel racemic la 5, 8,10 (ml/g) Kat qua cho thay tang luring dung moi tir Ian len 8, 10 Ian thi de tinh khiet cua san pham tang nhong hiau suht IR (cm-1, KBr) 3064,96 (C-H khong no), 2965,04 (C-H no); 1740,65 (-C=0); 1570,12 (-C=C-); 755,05 (the ortho yang benzen) ESI-MS: [M+2H].= 323,99 (M = 321,82); 'H-NMR (500 MHz, CDC/3): (ppm): 2,89-2,93 (m, 4H); 3,68 (d, H); 3,75 (s, H, -COOCH3); 30 TAP CHI DUQC HQC - 9/2015 (SO 473 NAM 55) • Nghien - Ky thuot 3,80 (d, H); 4,95 (s, H); 6,68 (d, H), 7,08 (d, H), 7,27-7,33 (m, H); 7,44 (d, H); 7,74 (d, H); "C-NMR (125 MHz, CDC/3); (ppm): 25,62; 48,38; 50,76; 52,20; 67,97; 122,82; 125,31; 127,23; 129,50; 129,88; 130,07; 133,35; 133,42; 133,97; 134,79; 171,40 Tong hop S-clopidogel bisulfat clang I ye dang II Kith() sat qua trinh tong hop S-clopidogel bisulfat cl?ng I tea dang II Cac tai lieu d5 cong be cho thAy cong met dung mei (nhu' aceton) nhu'ng a cac dieu kien CI COOCH3 )N khac c6 the cho cac clang tinh the khac cua (S)-clopidogrel bisulfat 151 Sty hinh tinh the c5u trim clang I duvc kiem soat bai qua trinh dOng hoc (kinetic control) dang II bol qua trinh nhiet dOng hoc (thermodynamic control), nen de c6 duvc cac ket qua On dinh CO dO lap lei cao can phai tim duvc cac dieu kien ye dung mei, nhiet rat chinh xac, neu kh6ng se thu duet dang I thay vi dang II hoac ngtrac lei, hoac hon hap cua ca clang: clang I va dang II Qua tham khao cac cong trinh d5 duvc ding b6[4-71 chOng tOi ch9n quy trinh tong hap (S)-clopidogrel bisulfat dang I va dang II duvc m6 to sa dO va sa CI COOCH3 H2SO4 ) 0-5°C Aceton H2SO4 (S) - Clopidogrel bisulfat dang I So' dO 2: T6ng hop (S)-clopidogrel bisulfat clang I (S)-clopidogrel bisulfat dang I duvc tong hap dung mei aceton a 0-5°C, nho tir tir acid sulfuric, khu5y b nhiet de thaang 12 gia Phan irng cho hieu suet tren 70%, san phArn CI COOCH3 thu duvc la tinh the mau CO nhiet de n6ng chay 184 ± 3°C (DSC), san ph5m da &vac kiem tra On dinh cho ket qua thei gian sir dung tren nem CI COOCH3 H2SO4 0-5°C MTBE )L\ H2SO4 (S) - Clopidogrel bisulfat clang II So' 3: T6ng hop (S)-clopidogrel bisulfat dang II (S)-clopidogrel bisulfat dang II thu duvc kha kh6 khan Chung tOi da thin rat nhieu dung m6i (isopropanol, ethylacetat, methanol, aceton, methyl tert butyl ether ), phaang phap khac nhu'ng van chi thu duvc dang I hoac h6n hap dm clang I va clang II (nhiet de n6ng chay cua san phArn nem khoang 176°C-184°C Sau met thai gian dai nghien ciru, tier' hanh ket tinh dung mei methyl tert butyl ether (the tich dung m6i gap 10 len khOi luvng (S)clopidogrel ban d5u) lam lanh a 0-5 °C tuen, they xuet hien ket tua, loc, rira ket tua veri aceton duet (S)-clopidogrel bisulfat dang II CO nhiet de nong chay 177 ± °C WA cac thi nghiem sau, thvc hien phan Ong co mem tinh the dang II thi san phArn tao de dang hen, sau Khdo sat anh htreyng ceia dung m6i den hieu suet va dO tinh khiet caa (S)-clopidogrel bisulfat: Tien hanh cac phan Ong vai cong met luang ch5t (feu, sir dung cac dung mai khac Wet qua TAP CHI DOC HQC - 9/2015 (SO 473 NAM 55) 31 lam lanh ir 0-5° thu &vac san ph5m (S)clopidogrel bisulfat clang II nhang hieu suet chi dat 33% Hieu suet tao (S)-clopidogrel bisulfat dang I cao hen clang II Mat khac dang I lei c6 sinh kha dung cao hen va ducyc san xuet cung nha sir dung (Ong rai han, nen chOng tOi da ch9n Iva nghien CCPU qua trinh ket tinh (S)-clopidogrel bisulfat dang I kr (S)-clopidogrel de thu duvc hieu suet cao nhet va net-1g cep len quy me 50 g/me, dinh hu&ng phat hien Crng dung san xuet cong nghiep • Nghien ciru - K9 thu4t cho they hai dung mOi aceton va isopropanol deu cho san phem co tinh khiet ten 99% (dat yeu cau USP 34) Tuy nhien, aceton la dung mai th6ng dung, de thu hoi, gia hp oda duvc Iva chon de ket tinh (S)-clopidogrel bisulfat clang I Bang 1: Anh huxing cua 100 dung mai HiOu suet (%) Dijtinh khiet (%) Ethyl acetat 72,3 98,33 Ethyl acetat - methanol (1:1) 68,3 97,42 Aceton 65,5 99,03 Isopropanol 60,8 99,47 STT Dung mai Khao sat anh hirang cua ti le acid H2S0, dac den hieu suet, tinh khiet cua (S)-clopidogrel bisulfat: Tien hanh phan Ong vai cac ti le so mol acid H2S0, dac/(S)-clopidogrel khac dung mOi aceton (Vaceton = 20 moopidogrei) Ket qua cho they, ti 16 acid H2SO4 dac/(S)clopidogrel la thi phan Ong cho hieu suet cao nhat 65,50% va de tinh khiet cao nhat la 99,35% Neu tang den ti le acid H2SO4 dac thi hieu suet giarn den va de tinh khiet Om den va co phan Ong bi oxy h6a manh kh6ng thu &pp san phem Bang 2: Anh Mrerng cern ti le acid H2SO4 dac H SO dOc/ STT (S)-clopidogrel Hieu suet (%) DQ tinh khiet(%) 65,50 99,35 1 1,2 42,40 98,61 1,5 Kh6ng thu &roc san phArn Khao sat anh huifmg cua luring dung mdi den hieu suet va dO tinh khiet cua (S)-clopidogrel bisulfat: Tien hanh phan u'ng vai the tich aceton Op 10, 15, 20 len khei luting (S)-clopidogrel (ml/g) Trong qua trinh thu'c nghiem they dung luting dung mai it, neng acid sulfuric dung dich cao, lam cho cac chat de bi oxi hoa va co hien twang chuyen sang mau den den den de tinh khiet cua san phern thap, hieu suet dm phan Ong cung thap Tuy nhien, tang dung mai qua cao lai lam Om hieu suet cua phan Ong vi qua trinh ket tinh kh6 khan hen De san phem dat 90%) va hieu suet phan tinh khiet dam bao Ong cao the tich dung mai Op 15 len khoi luting chat ban deu la ph0 hap 32 Bang 3: Anh huifmg cua ti re dung mdi SIT Dung mai Hieu suet (%) Da tinh khiet (%) 10 74,8 97,8 15 77,8 99,18 20 65,5 99,25 Quy trinh king hap (S)-clopidogrel bisulfat Bang I &pp Chung t6i ap dung de tong hop 6, quy m6 tren 50 g san phem/me Qua trinh duvc lap !pi nhieu len cho hieu suet 77,6 - 78,5% San phern duvc kiem nghiem dat tieu chuen cua &pop dien M9 USP 34 Tong hop (S)- clopidogrel bisulfat Ong I Trong binh cau L CO lap he thong lam lanh, may khuay tO, china 72,50 g (S)- clopidogrel (1 &yang locyng) hea tan 1088 ml aceton, lam lanh h6n hap nhiet de 0-5°C, nho tin tO, 11,23 ml acid H2SO4 dac (1 throng luting) vao h6n hap phan Ong de nhiet de luon a 0-5°C Hon hap phan Ong sau de duvc khuay 6, nhiet de 25- 30°C thai gian 24 gia Ket th0c phan Ong, loc thu kat tua, riy'a tua vai aceton lanh (2 x 10 ml), say san phem to say char) kh6ng gia thu ducyc 73,80 g (S)clopidogrel bisulfat (H = 78%) Nhiet nOng chay: 184-186°C [a]oi20 = 55,5°(C = methanol) De tinh khiet quang hoc: 99,53% IR (cm-1, KBr): v = 3116,6 (CH khong no); 2996,46 (CH khong no); 1753,61 (CO ester ); 1624,33; 1477,83; 1434,74 (-C=C- vong therm); 755 (the para-di cua wing benzen) ESI-MS: [M1=322,1; 'H-NMR (500 MHz,CDC13) S(ppm): 3,20 (s, 2H); 3,60 (m, 5H); 4,10 (s, 1H); 4,30 (s, 1H); 5,70 (s, 1H); 6,60 (d, 1H); 7,20 (d, 1H); 7,37,5 (m, 3H); 7,5 ( s, 1H) "C-NMR (125 MHz; CDCI 3) o(ppm): 21,60; 50,70; 54,54; 65,95; 125,03; 125,91; 126,0; 126,5; 128,75; 130,79; 131,14; 131,48,; 133,15; 135,14; 167,96 T6ng hop (S)- clopidogrel bisulfat Ong II Trong binh cau 50 ml c6 chCra 1,3 g (S)clopidogrel (1 &yang luting) hoe tan 13 ml methyl tert butyl ether (MTBE), hen hop doge lam lanh ye 0-5 °C, 0,21 ml acid H2SO4 dac (1 &yang luting) duvc nho giot tO, tu, vao hen hap phan Ong Khuey hen hap phan Ong a nhiet dO pheng 24 gia, sau lam lanh h6n hap phan Ong a 5°C Ket thuc phan u'ng, lec ley ket tua, riya tua bang aceton lanh (2 x ml), say TAP CHI DUQC HQC - 9/2015 (SO 473 NAM 55) • Nghien civu - K9 thulat Nguyen lieu, del tuvng va phu'o'ng phap nghien art, Dung mei, hoa chat Chit chuan + Acid vaiproic (AVP) — chuen USP, SKS: VA/11/04, ham lu•gng 99,31%; d6 Am: 0,5% + Methylparaben (MPB) — Vien Kiem nghiem thu6c Trung uvng, SKS: 0213108.02, ham lu,o,ng 99,74%; de Am 0,05% Methylparaben duvc sir dung lam chat not chuen (IS) phtrang phap phan tich Dung moi, hoa chat:Dat trail chuan tinh khiet dung cho HPLC va LC/MS Huy& twang Wang khong có AVP: Vien Hu* hoc N/A truyen mau Trung LK:mg cung cap Thiet bi va dieu kien phan tich Tien hanh phan tich tren may sac ky long kh6i ph6 sieu hieu nang LCMS Waters de duvc hieu chuen theo qui dinh cue ISO/IEC va GLP vai cac dieu kien sac ky va khoi ph6 nhu sau: Dieu kien sac ky + Cot C18; 100 x 2,1 mm; 1,7 pm + Pha d6ng: Acetontril — Acid acetic 0,1%, tY le phO hap Toc de d6ng 0,3 mL/phut + The tich tient mau: 0,5 pL Dieu kien khOi phO + Kieu ph6 khoi: SIM el& voi acid vaiproic va MS/MS dei vdei methylparaben, nguon ion hOa: ESI (-); Chuan bi va xiv ljf cac mau huyet twang china aicd vaiproic - Chuan bi maw Cac mau huyAt twang (HT) tv tao chiya AVP duvc chuan bi bang cach h6a tan chuan AVP vao cac mau huyet twang trAng vai cac n6ng d6 khac tir den 100 pg/ml - Phwang phap xa Ijr mit,: Mau HT de re (long o' nhiet de ph6ng Lay 0,5 ml HT, them 50 ill dung dich IS sau tua protein bang acetontril Lac, ly tam 6000 vOng/phot philt Hut dung dich lap tren, tiern sac ky Them dinh phwang phap phan tich Tien hanh them dinh dO dac hieu-chon loc, giai han dinh luvng du•ai, d6 dung, d6 chinh xac, tj, le thu hoi hoat chat, On dinh cita phu'ang phap theo cac hu,Ong den them dinh phiyang phap dinh luvng thu6c dich sinh hoc dm US-FDA va EMEA 261 Ket qua va ban luen Do dac hieu — ch9n I9c cue phwang phap Phan tich cac mau HT N/A cac mau HT tv, tao china chuen AVP vai n6ng de khoang 1,0 pg/ml (bang khoang 1/30 Cma9 uong lieu 500 mg) CO china not chuen MPB theo phtyang phap da duvc xay dung Tren sec ky mau HT tang (hinh 1) tai cac thol diem 0,9 va 0,8 ph0t (trung vai thoi gian kyu cOa AVP va MPB mau chuen — hinh 2), khong xuAt hien cac pic co cac manh ph6 khoi m/z = 143 (AVP); m/z = 150,97 —■ 91,95 (MPB) 1.1110.46/ Ounnels FS 15.97 a 919511.b1M90are866) 303 0A + Cac th6ng so cua thiet bi khoi ph6 de phat hien AVP va MPB nhu, sau: ,y 045 057 193 Host chit Thong so Dian the mao quan Dien thA bie phan chOp non Acid vaiproic Methylparaben 4800 V 4800 V 32V 34V 400 °C 400 °C 020 040 060 100 140 160 4.411(6 143 112 014 Nhi6t dO h6a hai d6ng pha dOng 120 40 060 100 130 140 167 100 1.6/41(6 100 TOc de thei hoa hai d6ng pha ng 800 UH 800 UH roc de thOi qua be phan chop non 20 UH Nang Lyng va them Ion ban du Ion tao 20 UH 18 V 914 114114 040 ISO 947M09 Channels 66.143 143 61464 0.86,0141 31093 180 61111.1 617 channels ES TIC 22293 07 142 Trne 020 040 060 080 100 120 140 160 180 m/z= 143,0 m/z = 150,97 m/z = 91,95 TAP CHI DUQC HQC - 9/2015 (SO 473 NAM 55) Hinh 1: S6c ky mau huyet tuung tr6ng 57 • Nghien cu'u - K9 thuOt 11,001 • 00 Own ES tea,1.117.1 01 06-(1 0640 5.5 *1 020 11001 IVO 1.1.001 20 040 061 320 100 120 040 000 000 i6o lit 140 40 0m 000 100 120 140 Ito 00001012 Ow* EP, 143 a 143 Noe 4M 02703 Ket qua phan tich cCing cho they, dap Crng cua mau tang tai thOi diem vai theri gian kru cua AVP (0,9 phut) nho hurl 2% clap u'ng cua pic AVP & nong dQ 1,0 pg/ml va dap Cvng cua mau trang tai th&i diem vai thoi gian dm MPB (0,8 phut), khong vuvt qua 1% dap Crng cua MPB Do vay, phirang phap dac hieu va chon 1QC d61 vai AVP 11.10 Cnamels ESC 60 00 Tn Hinh 2: S6c ky d6 mau huyet tucrng ttr Igo chira chuan AVP (1,0 mg/m1) not chugn (MPB) Ter tren xu6ng du& len lucrt: Sac d6 chon loc ion cua n6i chu4n MPB (MRM, m/z = 150,97 ,91,95); Sac ky d6 chon loc ion cua AVP (MRM, m/z = 143 -> 143); Sac kk d6 tong ion (Total Ion Chromatograms - TIC) DuNtvng chuan va khoang tuyen tinh Tien hanh pha cac mau HT china chuan AVP co nong dO khoang tit, 1,0 pg/ml den 100 pg/ml Phan tich theo quy trinh d5 xay dung Xac dinh sp, twang quan giCra nong c10 AVP (x) co mau va ty le dien tich pic AVP/MPB (y) bang phuvng phap hOi quy tuyen tinh, sir dung he so ty (1/nOng c102 ) Ket qua xac dinh mOi tuvng quan tuyen tinh duvc trinh bay bang Bang 1: DO dung, chinh xac cua cac mau thugc cac duang chuan Mau Along dd (pg/mL) S1 S2 S3 S4 S5 S6 S7 S8 1,0 2,0 5,0 10,0 20,0 40,1 70,2 100,2 Pharmg trinh hot quy (y = Ax + B) Dd dung e/o) Duong chudn Dudng chuan Dutmg chuan Duong chudn Dubng chuan 97,9 104,6 98,4 95,4 114,2 98,7 96,8 94,1 98,1 102,6 102,1 97,3 112,4 97,5 95,9 94,1 99,7 97,6 105,0 105,0 99,7 105,2 92,7 95,1 98,8 101,0 100,4 106,2 100,9 104,6 92,4 95,8 94,1 109,6 103,4 106,8 99,0 92,0 102,7 92,4 A= 0,0155 B = 0,0057 A= 0,0158 B = 0,0050 r = 0,9978 A= 0,0158 B = 0,0056 r = 0,9985 A= 0,0161 B = 0,0052 r = 0,9987 A= 0,0164 B = 0,0049 r = 0,9970 r = 0,9972 Ket qua them dinh cho they khoang nong dO tin 1,0 pg/ml den 100 pg/ml có skr twang quan tuyOn tinh giCra nong AVP vai t9 le dien tich pic cua AVP/MPB vai he so twang quan xOp xi bang Wing dO AVP xac dinh tin duteng chuAn so vol gia tri 19 thuyet deu dat x'ap xi 100% va nem giai han cho phep theo qui dinh cua phuvng phap phan tich thucic dich sinh hoc Gi61 han dinh luvng duevi (LLOQ) cua phtrang phap Phan tich cac mau huyet twang trang va mau huyet twang chira AVP va MPB vai nong dO Ian luvt khoang 1,0 pg/ml (mau LLOQ) Xac dinh dien tich pic AVP va MPB cua cac mau LLOQ va xac 58 dinh nong dO AVP co cac mau tiv duVng chuan tien hanh lam song song ding dieu kien Ket qua thArn dinh giffyi han dinh luvng dual cua phuvng phap duvc trinh bay b bang Bang 2: Ket qua xac dinh gia tri LLOQ Stt Wang clO' 'tic dinh Di'dong.sovoi Gia tri Ty l¢ S/N hi throng chuan nong thyr tich pic cua pic (119/m0 r%) 0,023 0,023 0,020 0,021 0,023 0,022 1,1 1,1 0,8 0,9 1,1 1,0 107,3 107,5 84,7 90,8 105,4 98,0 Trung Binh (%) 99,0 CV(%) 9,6 >10 >10 >10 >10 >10 >10 TAP CHI DUQC HOC - 9/2015 (SO 473 NAM 55) • Nghien clixu - K9 thulat Tien hanh them dinh dO dung, dO lap lei tren 16 meu thin LLOQ, LQC, MQC va HQC chira AVP co Hong c10 twang ring la 1,0; 3,0; 50; 80 pg/ ml Xac dinh ham luvng AVP co meu bang phkrang phap &rang chuen va tY le % giCra nong dO xac dinh duvc tir &rang chuen so vai ming c10 ljt thuyet Ket qua xac dinh TO dung, d0 lap lei cua phiyang phap dirge trinh bay 6' bang Ket qua them dinh cho they gia tri S/N cua pic AVP cac meu LLOQ (n6ng dO khoang 1,0 pg/ml) deu > 10; tY la niing dO AVP xac dinh tir &rang chart so vai nOng dQ thkrc co mAu nem khoang tir 80 -120% (trung binh = 99,0% va CV = 9,6%) dap Crng you cau giai hen dinh ILK/rig dkrai cua phiyang phap phan tich thu6c dich sinh hQc Xac djnh 60 dim, 014 10 dm phmong phap Bang 3: Kat qua than., dinh cla thing, cla lap !pi va khac DO dung, Da lap tai Miu HQC (80 pg/m0 Miu MQC (50 pg/m0 Miu LQC (3,0 pg/m0 DO dung (%) DO hp lai (C VA) DO dung (%) DO lOp lei (CV%) DO dung (%) DO 10p Igi (CV%) DO dOng (%) D6 10p 10 (CV%) Trong (n = 6) Khac (n = 5) Miu LLOQ (1,0 pg/m0 105,7 5,1 98,3 7,6 94,0 2,0 92,1 6,7 103,1 7,4 100,2 5,2 97,4 4,9 93,1 4,5 Xac dinh ti) 14 thu hoi hog chAt Xac djnh tY I¢ thu hoi AVP va MPB bang cach so sanh dian tich pic AVP va MPB cac 16 mei., co qua chiet tech va khong qua chiet tech (rneu pha dung dich nen rn5u) Ket qua xac dinh tY Ig thu h6i cua AVP b ca ba khoang nOng 60 thap, trung binh va cao duvc trinh bay bang Ket qua them dinh cho they a cac khoang nOng 60 thap, trung binh va cao, phirang phap CO dQ dung ngay, khac xep xi 100%; 60 lap lei ngay, khac vai gia trj CV < 15,0%; dap Ong cac yeu chu ye dO dung, 60 lap lei cua phuvng phap phan tich thu6c dich sinh hbc theo twang den cua US-FDA va EMEA Bang 4: Kat qua khdo sat t)% la thu hal cua AVP Dian tich pic (3,011°10 HT N6n rdu HT IS HQC (80,0 pg/mL) !sign mgu HT MQC (50,0 pg/mL) Nen mhu HT N6n mau Trung binh 1313 (n = 6) 1318 (n = 6) 19125 (n = 6) 18813 (n = 6) 29769 (n = 6) 28394 (n = 6) 21173 (n = 6) 21060 (n=6) 2,3 1,9 1,5 7,3 1,2 2,1 1,2 6,5 Cv (%) 100,5% 104,8% 101,7% 99,6% TS, Ig thu h6i Ket qua khao sat cho they, phuvng phap xir ly meu cho hiOu suet chiet host chat cao (99,6% 104,8%) va On dinh (CV%: 1,2% - 7,3%; sai khac giCra cac nOng dQ kh6ng qua 6%) Xac dinh anh hutong cua nen mAu dich nen mAu twang Ong Song song char, bi cac meu chi& & ming 60 LQC va HQC pha dOng Danh gia stir anh hkrang cua nen rnAu qua bi so MFAvp/MFis dO MFAvp, MFis duvc xac dinh bang cach so sanh bran tich pic cua AVP va IS cua cac rneu pha nen meu so vai din tich pic cua cac rnk pha pha dOng Kat qua danh gia skr anh Wang cua nen meu &vac trinh bay bang Chan bi cac 16 huyet twang tang co nguOn goo khac nhau, tien hanh xir ly theo qui trinh thu dirac cac dung dich nen Au Chan bi cac mAu chuen a nong dO LQC va HQC cac dung Bang 5: Ket qua clanh gia sy' anh Wong cua nat., mit, STT 0,72 TB CV(%) 0,66 0,67 0,67 7,1 MFAvr/MFis MF,5 MFAvp LQC 0,68 0,70 0,58 HQC 0,47 0,50 0,51 0,51 0,50 0,54 0,51 4,7 LQC 0,63 0,62 0,60 0,59 0,61 0,49 0,59 8,6 TAP CHI DU QC HQC - 9/2015 (SO 473 NAM 55) HQC 0,58 0,55 0,48 0,57 0,61 0,50 0,55 8,8 LQC 1,07 1,13 0,96 1,12 1,18 1,37 1,14 11,9 HQC 0,80 0,91 1,06 0,90 0,83 1,07 0,93 12,4 59 • Nghien cCvu - Ky thuOt Ket qua khao sat cho they khOng c6 so, sai khac giCra cac nen mau khac (CV < 15,0%) Mao sat d6 On dinh cua phu'o'ng phap On dinh cua mitt huyet twang sau chu ky thing - Khao sat di) on dinh sau ba chu ky d6ng - r5 thu'c hien tren nOng LQC va HQC Bao quan mau b nhiet -35°C ± 5° C va de d6ng 6, nhiet do, ph6ng Sau chu ky dong - r5, tien hanh phan tich xac dinh ming de AVP CO mau So sanh vai ket qua xac dinh nong de AVP co cac mau tien hanh phan tich sau pha (n6ng dO ban dau) Nang AVP mau sau chu ky Tong- phai sai khac vai nong de ban (Mu kh6ng qua 15% va gia tri CV% giDa cac ket qua dinh luting 6, m6i nOng de phai nhO hurl hoac bang 15% Kat qua duvc trinh bay qua bang DO so sanh \raj nong dO mau duvc xir ly sau dong Nang de AVP mau duvc x0, Iy sau bao quan met th&i gian nhat dinh b nhiet dO phong phai sai khac vai nOng dO mau xi) 19 kh6ng qua 15% va gia trj CV% giCra cac ket qua dinh luting a m6i nOng de phai nho hurl hoac bhng 15% Kat qua duvc trinh bay qua bang Bang 7: Ke't qua nghien ciru d6 an dinh cua mau huye't twang a nhi6t ph6ng that gian ng6n Mau Sau gia Xur ly Sau gi& 2,822 2,600 61995 65,191 2,428 2,856 65,802 68,417 2,662 2,551 63,468 61,277 2,476 2,705 68,415 65,860 2,763 2,566 67,815 59,196 2,554 2,862 65,575 76,470 TB 2,618 2,690 65,512 66,069 CV (%) 6,0 5,3 3,8 9,2 Bang 6: Kat qua nghien ceru on dinh cua mitt huye't Luang sau chu ky dOng-ra Along clO (pg/ml ) Agu LQC Afiu Mau HQC Sau chu kY Ban chu ky Banclau du Saudong-ra dong-ra" Ming (pg/mL ) M3u HQC Mau LQC 2,348 2,782 61,844 67,698 DO lOch (%) 2,337 2,998 60,277 66,410 2,419 2,473 60,832 64,370 2,545 2,718 62,945 63,072 2,626 2,631 61,410 68,626 2,680 2,555 61,648 65,485 DO On dinh cua mau huyet turyng the,' gian dai Khoang thoi gian thi) nghiem de On dinh dai tOi thieu phai du de tien hanh Ihy mau mau va phan tich het so mau huyet twang cua ngutei tinh nguyen Bao quan mau huyet twang a ruing de LQC va HQC b nhiet de -35°C ± 5°C Phan tich mau tai thol diem ban dau va sau *rig khoang tho'i gian bao quan nhat dinh (11, 35 va 53 ngay) So sanh nong JO dm mau huyet twang sau bao quan veyi nong dO cua mau tai thol diem ban dau Nang AVP mau sau bao qua(' met theyi gian nhat dinh phai sai khac vbi nong de ban dau khong qua 15% va gia tri CV% giO,a cac kat qua dinh luting a m8i nong phai nho hurl hoac bang 15% Kat qua duvc trinh bay a bang TB 2,493 2,693 61,493 65,943 CV (oh) 5,9 DO *eh (%) 6,9 1,5 3,1 8,0 7,2 DO On dinh cua mau huyet twang ey nhiOt phong that gian ngan Phan tich mau huyet twang b nOng de LQC va HQC sau d5 r5 d6ng va de 6, nhiet de phong sau met tho'i gian nhat dinh (5 gia), 2,8 0,9 Bang 8: Ke't qua d6 On dinh dai cua mau huyet twang Wing (pg/mL ) Miu 60 LQC Ban dau 2,348 2,337 Sau 11 2,822 2,428 Sau 35 2,526 2,674 HQC Sau 53 2,536 2,737 Ban dau 61,844 60,277 Sau 11 Sau 35 Sau 53 61,995 60,213 64,575 65,802 57,190 64,820 TAP CHI DUOC HOC - 9/2015 (SO 473 NAM 55) • Nghien cii'u - Ky thulat 2,419 2,662 2,410 2,622 60,832 63,468 61,696 70,318 2,545 2,476 2,396 2,705 62,945 68,415 59,497 66,508 2,626 2,763 2,518 2,605 61,410 67,815 62,616 65,345 2,680 2,554 2,506 2,711 61,648 65,575 63,446 63,659 TB 2,493 2,618 2,505 2,653 61,493 65,512 60,776 65,871 CV (h) 5,9 6,0 4,0 2,9 1,5 3,8 3,8 3,6 5,0 0,5 6,4 6,5 -1,2 7,1 DO loch (%) DO On (Oh coa tau sau xt:r Ijr (trong autosampler) So sanh n6ng dO cua mAu a n6ng dO LQC va HQC bao qudn auto-sampler sau met thai gian nhat dinh (20 Oa) va nOng de cilia mdu tiem sau xep 19 N6ng de mdu sau bao quan met thai gian nhet dinh auto-sampler sai khac vai n6ng dQ rneu tiem kh6ng qua 15% va gia trj CV% giCra cac ket qud dinh luvng & m6i n6ng de phdi nho han hodc bang 15% Ket qua de On dinh auto-sampler duvc trinh bay bang Bang 9: Kat qua d5 On dinh ctia ma'u sau ly auto-sampler Mau Nang (10 (pg/mL ) Mbu LQC M3u HQC Tram hem sau Tiam Tem sau 20 gib/20°C 20 gib/20°C 3,173 3,388 71,186 78,624 3,025 3,406 77,449 83,480 2,956 3,362 74,121 79,818 2,971 3,373 73,259 79,728 2,924 3,457 75,253 2,811 3,358 70,530 82,113 78,057 TB 2,976 3,390 73,633 80,303 CV (%) 4,0 1,1 3,5 DO lOch (h) 13,9 2,6 9,1 Ket luen Nghien dru xay dung dirqc phuvng phap dinh luvng acid valproic huyet twang ngutei bang UPLC/MS-MS Vai gia trj giai han dinh luvng dual 1,0 pg/ml x6p xi bang 1/30 gia trj Cam, khoang tuyen tinh reng tir 1,0 den 100 pg/ml, de dung cao, de Idp Ipi vai gia tri CV% nho (92,1%-105,7%; 2,0% - 7,6%), phirang phap xi, ly mdu nhanh, dan gian va thai gian phan tich ngAn (2,0 phut cho m6i mk); phuvng phap phan tich co the Ong dung cac nghien COU sinh kha dung va tuvng &rang sinh hoc the phem thu6c chu'a acid valproic Tai lieu tham khao BO Y to (2012), Dupe thu' quOc gia ViOt Nam, Nha xuAt ban Y hoc - Ha NOi European Medicines Agency, Committee for Medicinal Products for Human Use (2014), Guideline on validation of bioanalytical methods Laurian Vlase, Daniela-Saveta Popa, Dana Muntean, Sorin Leucuta (2008), "A new highthroughput LC-MS/MS assay for therapeutic level monitoring of valproic acid in human plasma", Sci Pharm., 76, 663-671 Kareem M Younes, Ehab F El Kady (2013), "A new validate bio-analytical liquid chromatographic tandem mass spectrometric method for the quantification of valproic acid in human plasma", International Journal of Analytical and Bioanalytical Chemistry, 3(4), 160-168 Rukhadze MD, Gonashvili MV, Zirakishvili AN, Okudzhava NV (2002), "HPLC determination of valproic acid as bromophenacyl ester in blood plasma", Pharm Chem J., 36, 45-47 U.S Department of Health and Human Services, Food and drug Administration, Center for drug evaluation and reseach (2003), Guidance for Industry - Bioanalytical method validation Zhong Y., Jiao Z., Yu Y (2006), "Simultaneous determination of mycophenolic acid and valproic acid based on derivatization by highperformance liquid chromatography with fluorescence detection", Biomed Chromatogr., 20, 319-326 (Ngay nhOn bei: 07/05/2015 - Ngay duyat clang: 04/09/2015) TAP CHt DOC HQC - 9/2015 (SO 473 NAM 55) 61 • Nghien civu - Ky thu'al Khao sat hoat tinh khang vi sinh vat cua alcol patchoulil va dan xuat f3-patchoulen ttr tinh dau hoac hu'o'ng Long Thi Noe Ha', Nguyen Thi Thao Tran2, Le NgoeThach2 " 'BO mon floa hoc, Khoa Khoa hoc Ca ban, Dai hoc Y Dagc Tp HO Chi Minh 'BO mon Iloa hoc Hii-u ca, Khoa flea hoc, Dai hoc Khoa hoc Tic nhien, Dai hoc Qudc gin Tplij Chi Minh * E-mail: lenthach@yahoo.com Summary Patchoulol or patchoulil alcohol (AP) was isolated from essential oils of the patchouli native of Vietnam (Pogostemon cablin Blanco., Lamiaceae) by column chromatography with 93.2 % output and 97.8 % purity /3-patchoulene was synthesized from AP by Wagner-Merrwein rearrangement with sulfuric acid as the catalyst, at an AP:H2SO4:Ac20 molar ratio of 1.0:0.1:1.0, at room temperature, for The yield was 95.67 % Patchoulil alcohol and its derivative f3-patchoulene were tested by disc diffusion assays the potential antimicrobial activity against five bacterial species and one fungal species in comparison with those of patchouli oil from Pogostemon cablin DMSO was used as solvent for dilution Patchoulil alcohol showed strong inhibitory activity on the growth of E faecalis and B subtilis, while f3-patchoulene showed inhibitory effect on V cholera, E coli, and C albicans Keywords: Pogostemon cablin, patchoulil alcohol, 13-patchoulene, antimicrobial activity Dal yen de Ngay nay, vai sky phat then virgt b5c cua cac nganh khoa hoc va c6ng nghe k9 thu5t, nhu cau cuOc sting cila x5 hOi ngubi cung duvc nang cao, cac san phArn cao cap thuang duvc Vu tien chon Ikra va sir dung, dac biet la nganh cOng nghiep m9 phAm, san xuAt mrac hoa De san xuOt mrac hoa danh cho nam giai thi alcol patchoulil la mot chat dinh huvng quan Qua trinh tong hap alcol phirc tap 121 dan den gia cao Han !lira, viec sir dung cac chat co nguOn gOc thien nhien cang duvc quan tam vi tinh an toan cho ngirai sir dung va tinh than thien m6i triyang Tinh dau hoac hiyang va alcol patchoulil co hoat tinh khang vi sinh vat tren mot so loci Tinh dhu hoac Wang, co tac dung khang khuAn tot voi Staphylococcus aureus va Bacillus subtilis 11, patchoulil alcohol (AP) co hoat tinh tren virus A/PR/8/34 (H1N1), va khOng co tac dung ten N Guizhou/54/89 (H3N1) 141 Ben canh nhivng gia tri ten thi alcol patchoulil 62 la nguyen lieu de tong hgp nen p-patchoulen DO tai thrgc thcrc hien nham lam ro kha nang khang vi sinh vat cua alcol patchoulil va dAn xuOt p-patchoulen tinh dau hoac twang Nguyen lieu va phtvang phap nghien cU'u Nguyen lieu va hoe chat La hoAc twang duvc thu hai vao gia sang tai thi trOn Cai L5y, tinh Tian Giang Mau nghien ciru d5 duvc BO mon Thgc vat hoc, Khoa Duvc, Dab hoc Y Duvc Thanh ph6 HO Chi Minh xac dinh la cay hoac (pogostemon cablin (Blanco.), Benth.) Tinh dhu la ho5c huvng duvc tach tir hai loci la (la twang va la gia) da duvc Cl 96 gia Viec tach tinh dau duvc thkrc hien theo phirang phap chung cat hai rariac truyen thong gia - da &vac khao sat va mO to nghien ciru triracI5I Thanh phan h6a hoc cua tinh dau va cau phan AP &vac xac dinh va phan tich bai phuvng phap GC/FID, GC/MSD va NMR TAP CHI DUQC HQC - 9/2015 (SO 473 NAM 55) • Nghien cu'u - Ky thuot Phan 10p alcol patchouli! Cho 2,5 g tinh dau hoac hirang vao cot sec kjr (3 cm x 50 cm) vai chat hap phi) la silica gel 60 (Himedia, flash chromatography, 230-400 mesh), tien hanh giai ly AP vai he dung mei hexan:diclorometan va &vac theo dei bang TLC va GC/FID Qua trinh sac kjt cot &vac thvc hien hai In San pham AP clugc kiem tra tinh khiet bang GC/FID va can khei liyang tinh hieu suht Ban tong hop fl-patchoulen Cho 0,46 g (2,00 mmol) AP vao binh cau ml day tron cOng vai 0,21 g (2,00 mmol) Ac20 va 0,02 g (0,20 mmol) H2SO4 dam dac Tien hanh khuay tip h6n hgp phan 0,ng khoang thai gian phOt a nhiet de ph6ng H6n hap san pham sau phan irng dugc chiet vai diethyl ether, trung hoa acid bang dung dich NaHCO3 bao hea va rira vai nuac nhieu Ihn cho den pH = Dung dich diethyl ether china san pham ()Vac lam khan bang Na2SO4, loc, thu hal dung mei Can, ghi nhan khOi luting san pham thu &Tic va kiem tra de tinh khiet bang GC/FID, tinh hieu suht phan Ong Cau triic san pham dirgc xac dinh bai GC/ MSD, ph6 hong ngoai (Infrared Spectrometry, IR) va ph6 Gang hkreyng tir hat nhan (Nuclear Magnetic Resonance, NMR) GC/FID: Tinh dhu hoac Wang va cau phan AP phan lap tin tinh dhu cluvc ph5n tich bai he thong GC Agilent 6890N dirgc trang bi cot mao quail Phenomenex 7HG G010-11 Zebron ZB-5, 30,0 m x 0,32 mm x 0,25 pm va Oki ion h6a ngon lira (flame ionization detector, FID) Khi mang cluvc sir dung la nita host Ong a the de clang tip 9,32 psi Nhiet de to cluvc lap trinh vai nhiet dau la 60 °C, tang °C/philt den 240 °C Mau barn vao dtrac hea hai 'a 250 °C va kh6ng chia d6ng (splitless) Ty le phan tram twang dei cua cac cau tir tinh dau du,gc tinh toan tu' Ong dva tren dien tich pic sec kjt dó GC/MSD: Tinh dau hoac Wang va cau phan sir dung lam mang vai the de clang ap 10,604 psi Nhiet de bueng barn va dhu dircyc thiet lap a 250 °C Mau cluvc pha bang vai dung mei thich hgp The tich met, tram vao la 0,1 pl vai the de chia d6ng (split) 25:1 Chuang trinh nhiet &vac thiet lap vai nhiet de dau 60 °C, tang °C/phiit den 240 °C Qua trinh ion h6a dirgc thvc hien & 70 eV Viec dinh danh cac cau tir tinh dau &vac thy'c hien bang cach so sanh cac gia tri chi so RAJ tuyen tinh (linear retention index, LRI) hay goi la chi so sO hoc (arithmetic index, Al) hay chi so Ikru nhiet diyac lap trinh (programmed-temperature retention index, PTRI = IT ) va ph6 khoi luting cua chOng vai cac hap chat tham khao diyac Gang b6 bai Adams va he thong dip lieu MS tirng hap chat tin thu, vien phe MS Wiley 8'h ket hap NIST 2008 Cac gia tri chi so luu tuyhn tinh cua terng cau tip tinh dau dirac xac dinh qua day thing clang alkan C5-C28 va &vac tinh toan dkra trail phiyang trinh de nghi boi Van den Dool va Kratz [10I Viec phan lap va xac dinh cau triic du,gc thkrc hien tai Phong thi nghiem hCru ca va Phong may B14, Twang Dai hoc Khoa hoc tu' nhien TP He Chi Minh Thu' nghiem hopt tinh khang vi sinh vat Hoat tinh khang vi sinh vat du,gc thlyc hien tai Ph6ng Vi sinh Thkrc pham, Khoa Xet nghiem, Vien Pasteur, ph6 HO Chi Minh theo phirang phap khuech tan tren dia thach: trai vi sinh vat vai met sO luting nhat dinh va deu len trail mat thach Dat dia gray (d = mm) da tam tinh dau, theo cac nong dO tin nguyen chat den pha bang dan, dung mai la dimethylsulfoxyd (DMSO) Mau can thir nghiem se khuech tan vao thach va 0,c the su' tang tnyang cua vi sinh vat tao vOng vo khuan Kich thipac cua &rang kinh vOng vo khuan cho Net kha nang khang vi sinh vat cua m'au can thin nghiem Ket qua va thao luan Cat tinh dAu AP phan lap fir tinh dau dirgc phan tich dinh danh bai he thong GC Agilent 7890A dhu dO phe kb& luting (mass spectrometry detector, MSD) 5975C VL MSD Triple-Axis sir dung cot mao guar) Phenomenex 7HG G010-11 Zebron ZB5, 30,0 m x 0,32 mm x 0,25 pm Helium dirgc Vai phvang phap chung cat hai nkrac dun n6ng truyhn thong, 300 g la hoac Wang, sau 96 gia, dkrac tin hanh chung cat gia Tinh dau thu cluvc co mau yang va mui tharn nhien Ket qua cluvc ghi nhan bang TAP CHI DOC HQC - 9/2015 (SO 473 NAM 55) 63 • Nghien cCpu - Ky thuot Bang : So sanh ham Ivceng tinh clAu va AP giika la gia va la Mixing Nguyen lieu Ham /u9ng tinh daiu (%) AP (% GC) La gia 0,7 48,7 La truteng '51 0,4 45,7 Tix bang 1, cho thay rang la hok hu,ang gia khang chi cho ham krang tinh dhu cao hail so vai la twang (0,7% so vai 0,4%), ma ch(ra tjt le phan tram AP cao han (48,7% so vai 45,7%) Nhir vay, vai nguyen lieu la la gia va six dung phu'ang phap chu'ng cat hai mac, ham luvng tinh dau la hoac hu,ang CO the dpt 0,7%, d6 AP chiern khoang 48,7% Phan lap alcol patchouli! Tien hanh sac k9 cot In vai chAt hap phi) la silica gel, giai ly he bkg dung mai hexan:dicloromethan, ti le 9:1 Ket qua sac ky cot thu duvc AP tu, tinh dau la hok hirang dat hiOu su6t 93,2% vai di) tinh khiet 97,8% So sanh vai ket qua thu duVc cua tac gia Xian 18] (six dung phirang phap tam chiet, hien su6t dat 65,1%) va Li [9] sir dung thiet bi High performance centrifugal partition chromatography (HPCPC), gia dat &vac hieu suAt khoang 65% va tinh khiet cua ca hai khokg 98%, cho thay phirang phap sir dung phirang phap sac 1(9 cot mang I?' hiOu qua cao han rat nhieu vai cac dung cu dan glen phong thi nghiem Phan tich va xac dinh cau trOc AP bkg cac phu,ang phap khac thu duvc ket qua nhu, sau: IR: vcDc,3 3506, 2937, 2807, 1465, 1375, 1324, 1056, 1000 cm-1 MS (m/z): 222 (M*,100), 207, 189, 179, 161, 151, 147, 138, 125, 121, 109, 105, 98, 95, 83, 67, 55, 41 'H va "C-NMR: ket qua duvc ghi nhan bang Bang 2: Rio 'H, 73C-NMR cOa mitt phan 10p (PPm) tri carbon Wong Om 'H-MNR (500 MHz, WO) 1,19 (br, s) 13 C-NMR (125 MHz, CDCI3) 39,2 40,1 75,6 1,51 (dd, 6,0, 3,5) 1,71(dd, 12,5, 5,5) 32,7 1,31, 1,47 28,6 1,97 (m) 28,1 1,44 43,8 37,7 1,03, 1,85 28,9 10 1,27, 1,46 24,6 11 1,26, 1,87 24,3 12 0,80 (d, 6,5) 18,5 13 0,85 (s) 20,6 14 1,07 (s) 26,8 15 1,08 (s) 24,3 TO' ket qua ghi nhan duvc, kat ho'p va so sanh vai ket qua dm nhirng nghien cCru truac 111-151 chung tai de )(At thu trOc hop chat phan lap duvc có thu truc theo hinh ca ten la alcol patchouli' 64 TAP CHI DUQC HOC - 9/2015 (SO 473 NAM 55) • Nghien CCM - KS"( thulat 14 Ban tong hop P-patchoulen 15 CH, H,C Tien hanh khuay tit, AP dieu kien ti le mol AP:H2SO4:Ac20 = 1,0 :0,1 :1,0 & nhiet dO phong hal phut thu &vac 13-patchoulen vai hieu suAt dat 95,7% vai tinh khiet 100% OH //4, 12 Phan tich, xac Binh eau triic p-patchoulen 13 Tien hanh ghi ph6 GC/MSD vai sari pham (-)-(1R,3R,6S,7S,8S)-2,2,6,8Tetramethyltricyclo[5.3.1.0381undecan-3-ol Hinh 1: C6u frac alcol patchoulil viva tong hap a' ten, ket qua phan tich trinh bay bang Bang 3: Ph6 MS cua san phAm tong hop so sanh vai f3-patchoulen cua tai lieu tham khao Sit Miu 0-Patchoulen1"1 San phOrn tong hap 204, 189(100), 175, 161, 147, 133, 119, 105, 93, 91, 79, 69, 67, 55, 41 204, 189(100), 175, 161, 147, 133, 119, 105, 93, 91, 79, 69, 67, 55, 41 Vi khuan gram (-1-) Staphylococcus aureus (to chit yang): la vi khuan gay mun nhot, 613 xe, viem MCli, viem ph6i, ng0 dOc thgc pham Bacillus subtilis: la vi khu6n gay nhiem khan huyet, ng0 dOc thgc pham Vibrio cholerae: la vi khuan gay dich ta, kiet ly Enterococcus faecalis: la vi khu6n gay nhiem mau, nhiem trOng &rang tiet niau, viem mang nao mu Vi khugn gram (-) - Escherichia coil: la vi khuan gay rOi loan tieu h6a, tiou chay cap tinh (1R,5S)-1,5,11,11-Tetramethyltricyclo[6.2.1.02.61 Vi nhm undecan-2(6)-en Hinh 2: Ca cau f3-patchoulen Candida albicans: la mot loaf vi narn gay benh a am dao, &yang runt Thu' nghiem hoat tinh khang vi sinh vat Thtyc hien the, nghiem hoat tinh khang vi sinh Alcol patchouli! va 13-patchoulen deu ducyc vat vai tinh deu hoac hu'ang, alcol patchoulil va the, nghiem ten chung vi sinh vat: I3-patchoulen, ket qua ghi bang Bang 4: Hopt tinh khang vi sinh vat cua tinh dAu hok huong, alcol patchoulil va 13-patchoulen Tir ket qua GC/MSD dong thai so sanh sty phan manh cua san pham tong hap vai sty phan manh cua f3-patchoulen tai lieu tham khao [11], chUng t6i de mat san pham tong hap la hap chat 13-patchoulen CO cau truc theo hinh Dutimg kinh king vo khuin (mm) Vi sinh vOt Tinh da‘u hoac huong ni th& nghi4m C, C2 C3 Co C1 C2 C3 6 15 10 6 21 11 9 6 30 25 18 17 6 6 6 22 12 12 6 6 6 25 10 6 10 9 28 10 6 Co CI C2 C3 S aureus 16 10 E faecalis 6 B subtifis 18 10 V cholerae 21 11 E coli 6 C albicans 6 NOng dO (C) 3-Patchoulen Alcol patchouli! C, Veri AP; Co = aloof patchouli' nguyen ch.4t, C, = 10-1CO3 C2 = l0-' C,, C3 = 10-1C2 Veri B-patchoulen; Co = 23,93%, C, = TAP CHI DUQC HQC - 9/2015 (SO 473 NAM 55) 0, C2 = C,, C3 = 10'C2 Dung rrrai DMSO 65 • Nghien cixu - K9 thual Cac ket qua th6, nghiem kr bang tren cho they rang: Tinh dau hoac twang c6 hoat tinh dOi vai chung vi sinh vat nhu' Staphylococcus aureus, Bacillus subtilis, Vibrio cholerae, Enterococcus faecalis; tot nhat tren chOng Vibrio cholerae; nhu,ng hoan toan khOng c6 tac dung tren chung Escherichia coli, Candida albicans Khi phan lap alcol patchouli! tip tinh dau hoac huang, alcol patchouli! lei c6 tac dung tOt han tinh dau dOi vai hai chung Bacillus subtilis, Enterococcus faecalis, c6 the nOng dO alcol patchoulil pha bang khoang gap dOi nong dO alcol patchouli! CO tinh dau pha bang Nhu, vay tinh dau co tac dung doi vai chung vi sinh vat su' tac dung ch6 yeu cue alcol patchouli! Alcol patchouli! CO hoat tinh yeu tren chung Staphylococcus aureus va hoan toan kh6ng co tac dung tren chung Vibrio cholerae; nhung tinh dau lei tac dung tOt han, dieu dO chOng to rang hoat tinh ch6 yeu tren chung kh6ng phai alcol patchoulil ma cac cau phan lei ca tinh dau P-Patchoulen duvc tong hap tu' alcol patchoulil, a nOng dO bang (Co = 23,93%) cung da cO hoat tinh ten ca chung dem thin nghiem, va CO tac dung manh ten chung Vibrio cholerae alcol patchouli! hoar) Wan khOng c6 tac dung Dac biet a chung Escherichia coli, P-patchoulen c6 tac dung rat manh tinh dau va alcol patchouli! hoan toan khOng CO tac dung hoat tinh manh ten cac chung Bacillus subtilis, Enterococcus faecalis Nhu' vay I3-patchoulen la hop chat c6 hoat tinh sinh hoc vircyt tr6i han han so vai alcol patchouli! va tinh dau, n6 khong chi CO tac dung rat manh ma c6 ph6 khang vi sinh vat rOng troll ca vi khuan gram (+), gram (-) va vi nem Ket Wen Qua ket qua khao sat viec dieu the alcol patchouli!, p-patchoulen tir tinh dau la hoac Wang Pogostemom cablin va sau dO th6, nghiem hopt tinh khang vi sinh vat, chimg t6i rut ket luan: - Ve mat nguyen lieu: la gia khong nhung chi CO ham luvng alcol patchouli! cao han ma CO ham luting tinh dau gen gap dOi so vai la tru,ang - Viec phan lap AP, chi dung sac k9 cot dan gian, chi phi thap, AP phan lap duvc c6 d6 tinh khiet va hieu suet rat cao - Viec ban tong hap P-patchoulen, vai nguyen lieu h6a chat gia thap de tim va dieu kien phan Ong de thu'c hien - Hap chat alcol patchoulil va I3-patchoulen c6 hoat tinh sinh hoc rat manh ten mot so vi sinh vat thin nghiem, dac biet p-patchoulen c6 phan vu,at tr6i han han so vai AP va tinh dau Chung tOi by vong de tai se gop phan vao su' phat then nganh cong nghiep m9 pham va lam ca so' cho nhCrng nghien ciru ke tiep dOi vai hap chat 13-patchoulen flOi vai chung vi nem Candida albicans, p-patchoulen c6 tac dung rat manh, alcol patchouli! (97,8%) thi CO tac dung twang dOi yeu Nhu, vay kha nang hoat tinh sinh hoc khong phai cua PA ma la tac dung c6a p-patchoulen (1,4%) I5n alcol patchoulil 97,8% Nguvc !al b tinh dau thi lei hoan toan khang co tac dung ten loaf vi nem mac du cung CO ham luting P-patchoulen la 1,4% ISI, dieu cO the f3-patchoulen bi Cyc the hoat tinh Uri mot vai hap chat nao da CO tinh dAu Neu so sanh vai nghien ciyu tnyac thi tinh dau tip la hoac hu,ang b Cal Lay — Tien Giang c6 cung hoat tinh ten hal chung Bacillus subtilis, Staphylococcus aureus va alcol patchouli! CO Tai lieu tham khao 1.J Arpana, D J Rao Bagyaraj, E.V S Prakasa, T N Parameswaran, B Abdul Rahiman (2008), "Symbiotic response of patchouli (Pogostemon cablin (Blanco) Benth to different arbuscular Mycorrhizal fungi", Advances in Environmental Biology, 1, 20-24 A Srikrishna, G Satyanarayana (2005), "An enantiospecific total synthesis of (-)-patchouli alcohol", Tetrahedron: Asymmetry, 16, 3992-3997 N Kongkathip, P Sam-ang, B Kongkathip, Y Pankaew, M Tanasombat, P Udomkusonsri (2009), "Development of patchouli extraction with quality control and isolation of active compounds with antibacterial activity", Kasetsart Journal: Natural Science, 43, 519 - 525 H Kiyohara, C Ichino, Y Kawamura, T Nagai, N Sato, H Yamada (2012), "Patchouli alcohol: 66 TAP CHI DUQC HQC - 9/2015 (SO 473 NAM 55) • Nghien cd3u - K9 thuOt in vitro direct anti-influenza virus sesquiterpene in Pogostemon cablin Benth", Journal Natural Medicine, 66, 55-61 U6ng Thj Ngpc Ha, Le Ngpc Thach (2012), "Khao sat tinh deu tir cay hoar huvng Pogostemon cablin (Blanco) Benth thu hal tai thi ter, Cai Ley tinh Tien Giang", Tap chi Ducrc hoc, 438, 38-45 Nguyen Xuan Dung (1989), "Chemical composition of patchouli oil from Vietnam", Journal of Essential Oil Research, 1(2), 99-100 S Bunrathep, G B Lockwood, T Songsak, N Ruangrungsi (2009), "Chemical constituents from leaves and cell cultures of Pogostemon cablin and use of precursor feeding to improve patchouli alcohol level", Science Asia, 32, 293-296 (2009) Y F Xian, Y C Li, S P Ip, Z X Lin, X P Lai, Z R Su (2011), "Anti-inflammatory effect of patchouli alcohol isolated from Pogostemonis herba in LPSstimulated RAW264.7 macrophages", Experimental and Therapeutic Medicine, 2, 545-550 X C Li, Q W Zhang (2011), "Preparative separation of patchouli alcohol from patchouli oil using high performance centrifugal partition chromatography", Journal of Essential Oil Research, 23, 19-24 10.H V D Dool, P D Kratz (1963), "A generalization of the retention index system including linear temperature programmed gas-liquid partition chromatograghy", Journal of Chromatography, 11, 463-471 11.R P Adams (2007), Identification of Essential Oil Components by Gas Chromatography / Mass Spectrometry, th Edition, Allured Publishing, Illnois (2007) 12.J A Faraldos, S Wu, J Chappell, R M Coates (2010), "Doubly deuterium-labeled patchouli alcohol from cyclization of singly labeled [2-21-11] farnesyl diphosphate catalyzed by recombinant patchoulol synthase", Journal of American Chemical Society, 132(9), 2998-3008 13.K Nishiya, T Tsujiyama, T Kimura, K Takeya, H Itokawa, Y litaka (1994), "Serquiterpenoids from Valerian fautieP, Phytochemistry, 39(3) 713-714 14 D H R Barton, J.-C Beloeil, A Billion, J Boivin, J.-Y Lallemand, S Mergui (1987), "Oxidation of patchouli alcohol by the "Gif system": Isolation and organoleptic properties of three new ketonic derivatives", Helvetica Chemica Acta., 70, 273-280 15 A Srikrishna, G Satyanarayana (2005), "An enantiospecific total synthesis of (-)-patchouli alcohol", Tetrahedron: Asymmetry 16, 3992-3997 (Ngay nhOn bai: 27/04/2015 - Ngay duyOt dang: 04/09/2015) Chi& xuAt, phan 14p phAn chinh dtryc We'u dau tam (Morus alba L., Moraceae) Vo Dtirc Phtro'ng Thao, DOng Thj Ngcoc Digp, Hujrnh Ng9c Thuy: BO mon Thew lieu, Khoa afar, DO hoc Y Duvc TR HO Chi Minh huynhngocthuyth@yahoo.com Summary Three polyphenol derivatives - Moracin C; Moracin M; Oxy-trans-resveratrol and 3-0-B-D glucopyranosyl sitosterol - were isolated from the ethylacetate fractions obtained from a total methanol extract of silk mulberry sprigs (Ramulus Mod albae) collected 2013, Feb in Da Lat (Lam Dong Province) Their structures were elucidated by analysis of their recorded spectroscopic data (UV, ESI MS 'H NMR, 13C NMR, DEPT HSQC, COSY and HMBC) in reference to the literal data Keywords: Morus alba, Moracin C, Moracin M, oxy-trans-resveratrol suet sir dung cao cac bai thu6c, cb tac dung khang khan, khang viem, tieu d0c, trj dau nhirc, cheng one hod, trj tieu duang11.3-51 Hien ten thj truang c6 net nhieu the phArn sir dyng dau tem Tuy nhien, chuyen 1u5n Dirac dien Viet Nam IV chira c6 phirang phap djnh luvng C15'c hieu de kiern djnh chet luvng &vac lieu chfnh vi vay d5 gay nhieu kh6 khan cho cling tac kiern tra, danh gia chAt luvng caa dirgc lieu va cac the phAm c6 china dau tern Nhern thu duvc chet tinh khiet d5c hieu c6 ham luvng cao dau tern TAP ClIf DU 1C HQC - 9/2015 (S6 473 NAM 55) 67 DOt vett de Dau tern (Morus alba L.) la duvc lieu c6 ten • Nghien civu - Ky thuot dung lam chat doi chieu de xay dkrng phuvng phap dinh luvng, chung toi Wen hanh chiet xuat, phan lap phan chinh duvc lieu canh dau tam (Ramulus Mod albae) Qua khao sat sa be phan h6a thy,c vat va danh gia phan CO tac dung chOng oxi hoa ten sac k9 ban mong vai thuOc thin DPPH, canh cho cac vet duvng tinh re vai TT DPPH va so sanh ten ban mong cac vet kh6ng khac brat nhieu vai v6 re Canh china it tap chlorophyl so vai v6 canh va la Han nira, canh co ngulin nguyen lieu sari co cat tia hang nam, sir dung re khong CO Io'i ich kinh to vi gay hal cho cay Theo tai lieu tham khao, phan hoa hoc cila cac be phan cay cling khOng nhieu khac biet so vai Bai bao se trinh bay ket qua phan lap va xac dinh cau trim met so phan chinh du'oc lieu canh dau tam 06i tuvng va phu'o'ng phap nghien cu'u Nguyen lieu Dau tam dung chiet xuat va phan lap: canh dau tam (Ramulus Mod albae) thu hai thang 2/2013 tai Da Let, Lam DOng Cac mau duvc xac dinh ten khoa hoc la Morus alba L., Moraceae va lu'u giir tai BO mon Du'o'c lieu - Khoa Du'o'c Dai Hoc Y Duvc Tp HO Chi Minh Dung m6i boa chat Cac h6a chat dung chiet xuat nhu' ethanol, dung phan lap nhu' ethyl acetat, dung tinh khiet h6a cac chat nhu' chloroform, ethyl acetat va cac dung mei thu6c thin khac dat tiou chuan tinh khiet dung kiem nghiem duvc lieu Dyng cu, thiet bi PhO UV thu,c hien ten may quang ph6 Shimadzu UV-vis 1700 PhO MS thu'c hien tren may Quattro Micro API (WATERS) Pho Ong hiking tip hat nhan (NMR) tai phOng cau truc Vien Hoa hoc, Ha Nei va duvc thvc hien Wen may Bruker AV500 sir dung chat chuan not la tetramethylsilan (TMS) Mau du'ac h6a tan DMSO-d6 hoac aceton-d6 Pho proton ( 1H-NMR) duvc a 500 MHz, carbon ( 13C-NMR) duvc 6' 125 MHz DO dich chuyen hoa hoc duvc tinh theo thang ppm (TMS = 0) vai chuan la tin hieu cOa TMS Cac hang so ghep (J) tinh 68 bang Hertz (Hz) HPLC Waters 2695, dau dO PDA 2996, bu,ac song phat hien 250 nm Get sac k9 Restek Pinacle C16 (5 pm, 150 mm x 4,6 mm) Phwang phap nghien cu'u Nguyen lieu cay dau tam dung chiet xuat va phan lap cac chat sau thu hai duvc phol kh6, xay bet, ray qua ray cho met, duvc dOng nhat Kiem tra va xac dinh nguyen lieu tiro:5'c thu,c hien nghien cu'u chiet xuat phan lap Chiet xuat, phan lap TO 9,5 kg bet canh dau tam, chiet xuat bang phuvng phap nghm kiet vai ethanol 96% thu duvc 356 g cao toan phan Cao toan phan (cao dac) duvc ten vai da !pot, dun hOi lu'u 30 phut 6' 50 — 60 °C vai ethyl acetat len, gan 10c lay dich chiet Gap cac dich chiet, thu hOi dung m6i thu duvc cao ethyl acetat g9i la cao A (cao dac) Phan cao lai khong tan (cao B) duvc lay rieng Cao A &roc phan tach 10 phan doan bang sac k9 cot silicagel ca hat viva (Merck),dung moi rira giai la chloroform — ethyl aceat vai t9 le ethyl acetat tang dan Phan doan va 10 co tia, loc hong lay tua, tinh the va dat ten la MA-2 (31,5 mg) Phan dich tir phan doan 10 cho cac vet phat quang va tat quang Ian, thu hOi dung m6i duvc 5,54 g can (goi la A10) TO' phan doan A10 bang k9 thuat sac k9 cot va dung m6i rira giai tuvng tu, thu duvc phan doan nho han (10.1 den 10.7) Phan doan 10.2 co tua trang, ket tinh lai nhieu Ian chloroform thu duvc chat ket tinh trang, dat ten la MA-1 (285 mg) Phan doan 10.7 xuat hien chat ket tinh, goi la chat MA-4 Tinh the MA-4 vai ethyl acetat thu duvc khoang 1350 mg KM qua va ban luan Chat MA-1 MA-1 la nhirng tinh the hinh kim ngan, mau trang, khOng MA-1 c6 cu'c dal hap thu tai cac buck song UV217 nm, 315 nm, 328 nm MA-1 c6 de tinh khiet tren HPLC/PDA la 95,46 % MA-1 co [M+H]' bang 243,13, twang u•ng vai tong thipc C14H1004 lieu phe 1H-NMR va 13C lieu phO cua moracin M ciia MA-1 so sanh vai ding dieu kien nhu' sau (bang 1) TAP CHI DU QC HQC - 9/2015 (SO 473 NAM 55) - K9 thulat • Nghien Bang 1: DO' lieu ph6 13C-NMR va 'H-NMR cua MA-1 so vai moracin M theo TLTKI31 'H-NMR (DMSO-d6) "C-NMR (DMSO-d6) MA-1 Moracin M Moracin M MA-1 7,38 7,38 C3-H 7,08 7,08 C7-H 6,92 6,94 C-3'/5' 158,73 158,8 C4-H C-7 155,66 155,7 C-6 155,21 155,3 C-2 153,91 154,0 C5-H 6,73 6,74 C-1' 131,60 131,7 C2'-H 6,67 6,70 C-4 121,04 121,1 C6'-H 6,67 6,70 C-3 120,73 120,8 C4'-H 6,20 6,23 C-5 112,40 112,5 C-4' 102,60 102,7 C-2'/6' 102,26 102,3 C3 101,48 101,6 C-7 97,40 97,5 Moracin M Ph6 NMR cua MA-1 co cac chuyen dich hoer hoc dm 13C vi 'H ph0 hop vai ch5t moracin M PhO khoi ESI-MS oh MA-2 a ch4 dd ion am cho pic EM-HI- b m/z 309 twang irng vai kh6i d5 cong b6 tai lieui3i, to nhirng du, lieu tren, chAt MA-1 duvc dinh danh la moracin M Chit MA-2 MA-2 diroc phan lap dual clang tinh the hinh kim, khong mau, co cu'c dal h5p thu tai cac boac song UV 217 nm, 315 nm, 328 nm va co dung ph6 UV twang to MA-1 luvng phan tf.r la 310 Ph6 cong hiropg tir hat nhan carbon 13 cho thAy co tin hieu cua 19 carbon Cac kat qua ph6 tren cho th5y MA-2 CO cong thErc phan to C15H 1804 Vai cong thirc phan tir nay, co chat da duvc phan lap dau tam la moracin C va moracin N H H OH I H HO Moracin C OH H OH HO Moracin N OH Cau trot cua moracin C va moracin N So sanh su' khac biet cau trim lien quan tai su' khac biet ph6 CO the nhan biet va phan bet giCra chat do,a vao nhOng chi tiet nhu' cau trim cua moracin C, ngoai proton dm day nhanh isopentenyl clipng a vi tri ke can nhau, co them proton ke can (vi tri ortho) vai vong benzofuran, moracin N chi CO proton cua day nhanh isopentenyl dirng a vi tri ke can Vai dac diem cau trCic nhu, tren, co the thay ph6 COSY dm moracin C se co cap proton c6 peak giao vai (do proton cua nhan thorn va proton dm day nhanh isopentenyl) moracin N chi co pic giao cua proton tren day nhanh isopentenyl, dO CO proton xuet hien a Wing cao dac tru'ng cua proton gun tren carbon spa Iron ph6 COSY cua MA-2 co dinh giao rat ro dm cap proton (cap proton er 6H 7,38 va 6,91 ppm, dac tru'ng cho proton ten nhan therm vai hang so ghep otho dac tru'ng la Hz va cap proton er titi 5,18 va 3,19 ppm dm proton ten day nhanh vai hang so ghep 6,5 Hz) Cac ket qua ph6 chirng to MA-2 co cau trim cua moracin C Vi tri cua day nhanh disc ding co bai cac ket qua ph6 HMBC dm MA-2 Tren HMBC, proton H1 Gila day nhanh cho thay twang tac vai carbon duvc gun cho cac vi tri C4 ( dc 114,92 ppm), C5 (tic 156,21 ppm), C2, (tic 123,12 ppm) va C3, (Sc 129,58 ppm) Chuyen dich hoa hoc cua proton, carbon va cac tuvng tac khac cua proton va carbon cua MA-2 duvc xac dinh bai cac ket qua ph6 'H-NMR, 13C-NMR, DEPT, TAP CHI DOC HOC - 9/2015 (SO 473 NAM 55) 69 • Nghien cU'u - K9 thu'Ot HSQC, COSY va HMBC Cac ket qua duvc so sanh vai cac du, lieu da cong to6 tai lieu m va duvc trinh bay bang du,ai day (bang 2) Nhu, vay, cau trot dm MA-2 &roc xac dinh chac than la moracin C HO MA2 Kh6, luong phan tit = 310.95 ()Mg thuc phan tit =CIA.0, Cgu frac cua moracin C Bang 2: DO' lieu ph6 NMR 13C va NMR H cOa MA-2 so vai Moracin C theo TLTKFI Moracin C (500 MHz.CD,OD) C-5" 18 C-1" 23,4 C-4" 26,2 C-7 98,6 C-3 101,3 C-2',C-6' 103,9 C-5 113,2 C-4' 117 C-4 121,9 C-3a 123,4 C-2" 124,5 C-1' 130,4 C-3" 131,5 C-2 156,6 C-7a 156,8 C-6 157,3 C-5' 157,7 C-3' 157,7 NMR 13C MA-2(125 MHz, DMSO-dam) 17,65 22,03 25,46 97,37 100,49 102,25 112,3 114,92 120,82 120,92 123,12 127,97 129,58 154,16 155,12 155,5 156,21 156,21 Pho NMR dm MA-2 CO cac chuyen dich hoa hoc cOa 13C va 'H tuvng d6i phO hap vai chat Moracin C da ding b6 tai lieu Chat MA-2 du'o'c dinh danh la moracin C ChM MA-4 MA-4 la nhOng tinh the hinh kim to lai hinh chu, mau yang xanh nhat, kh6ng mOi, dO tinh khiet ten HPLC-PDA la 95,13 % va CO hap thu cu'c dai & buck song UV 328,1 nm va 216,8 nm Ph6 kh6i ESI - MS cOa MA-4 O: the dO ion am co pic [M-H] 243 m/z cho biet khoi lu,cyng phan tip la 244 Wong u'ng vai cong thirc phan tip C14 H1204 Tren ph6 13C-NMR cho thay 14 tin hieu C; ph6 DEPT cho thay CO C bac 4, khOng Moracin C (300MHz, CD30D) C4-H 7,32 C7-H 6,88 C3-H 6,82 C2'-H,C6'-H 6,78 C5-H 6,72 C2"-H 5,26 C1"-H 3,32 C5"-H 1,78 C4"-H 1,66 NMR 'H MA-2 (500MHz, DMSO.d,) 7,38 6,91 6,90 6,73 6,72 5,18 3,20 1,71 1,62 co C bac 2, va cO C bac 1; tin hieu H cua OH ro vOng -9,5 vai tin hieu 6, 9,1 ppm cao cho thay tai day co OR Qua khao sat ket qua UV, MS, NMR du' doan cong dm MA-4 la oxy-trans-resveratrol Ket qua ph6 chieu va chieu cua MA-4 duvc so sanh vai di) lieu oho' NMR cOa oxytrans-resveratrol tip tai lieu tham khao[8] va duvc trinh bay bang sau (bang 3) OH OH oxy-trans-resveratrol Bang 3: DC, li6u ph6 '3C-NMR va 'H-NMR cua MA-4 va oxytransresveratrol theo TLTK [8] "C- NMR Oxyresverattol 181 (aceton-d6, 400 MHz) 158,5 C5' hoc 3' 158,5 158,1 70 C3' ho0c 5' C4 MA4 (DMSO-d6, 100 MHz) 'H - NMR Oxyresveratrol n1 (aceton-d6, 400 MHz) MA4 (DMSO-d6, 100 MHz) 158,49 158,49 158,6 TAP CH1 DOC HQC - 9/2015 (SO 473 NAM 55) • Nghien ciru - Ky thulat 156,4 140,0 127,1 124,6 123,2 115,2 107,2 104, 104, 102,5 101,3 C2 CI' C6 C6 Ca C1 C5 C6' ho#c 2' C2' ho#c 6' C3 C4' 156,021 140,032 127,168 124,646 123,212 115,287 107,233 104,003 104,003 102,589 101,373 SS lieu NMR dm MA-4 khdrp vai du,' lieu oxy-trans-resveratrol cho ket luan MA-4 la oxy-trans-resveratrol 7,38 d, J=8,5 Hz 6,86; d, J=16,5 7,13; d, J=16,5 7,340 J= 6,77 d J=16,5 7,14 J=16,5 6,38 dd, J 8,5, 2,4 Hz 6,50 s, 6,5 s 6,40 d, J 2,4 Hz 6,22 s, 6,31 J=2,5 6,33 J=2,5 6,33 6,25 d; J=2,5 6,06 s Dinh tinh sce IN) chat MA-1 va MA-4 ten HPLC/PDA MA-1 Sabi) danh gia chat ten sec ky cló then khai HPLC/PDA Ket qua ten sec ky d6 HPLC cCia dich chiet dau tem c6 thay n5 pic cOa MA-1 (moracin M) cO butec song hap thu cyc dal la 313,8 nm va 215,6 nm va MA-4 (oxy-trans-resveratrol) CO hap thu cu,c dbi a btrac song 328,1 nm va 216,8 nm Nhu' vay c6 the chon chat c6 ham luvng cao nguyen lieu lb moracin M (MA-1, 285 mg) va oxy-trans-resveratrol (MA-4, 1350 mg) lam chat desi chieu de xay ding quy trinh djnh luvng d6ng thdri host chat duvc lieu dau tem Ket Wen Chiet met 9,5 kg canh duvc lieu dau tem thu duvc 356 g cao Phan lap duvc chat ket tinh, tinh khiet tir cao ethyl acetat (35 g) Cac chat duvc xac djnh cau trim bang 1D-NMR va 2D, xac djnh moracin M (285 mg), moracin C (31,5 mg), oxy-trans-resveratrol (1350 mg) Trong d6 CO chat CO ham luvng cao (moracin M va oxy-trans-resveratrol) se du'p'c chon sir dung cho cac nghien tiep theo Tai lieu tham khao VO Van Chi (2012), Tip din Cay thu6c ViOt Nam, NXB Y hoc, tap 1, tr.710-712 Dupe di4n ViOt Nam IV, trang 736-738 Chen S K et al (2012), "Moracin M from Morus alba L is a natural phosphodiesterase-4 inhibitor", Bioorganic & Medicinal Chemistry Letters, 22(9), pp 3261-3264 Deepa M et al (2012), "Purified mulberry leaf lectin (MLL) induces apoptosis and cell cycle arrest in human breast cancer and colon cancer cells", Chemico-Biological Interactions, 200(1), pp 38-44 Hansawasdi C and J Kawabata (2006), "Alphaglucosidase inhibitory effect of mulberry (Morus alba) leaves on Caco-2", Fitoterapia, 77(7-8), pp 568-573 Piao, S J., Qiu, F., Chen, L X., Pan, Y., & Dou, D Q (2009), "New stilbene, benzofuran and coumarin glycosides from Morus alba", Helvetica Chimica Acta, 92(3), pp 579-587 Won-Gon Kim, Yu Kin Kim, Mi-Jin Sohn (2012), " Chalcomoracin and moracin C, new inhibitors of Staphylococcus aureus enoyl-acyl carrier protein reductase from Morus alba", Chemical & Pharmaceutical Bulletin, 35(5), pp 791-795 Shin N H., Ryu S Y., Choi E J., Kang S H., Chang I M., Min K R et al (1998), "Oxyresveratrol as the potent inhibitor on dopa oxidase activity of mushroom tyrosinase", Biochemical and Biophysical Research Communications, 243(3), pp 801-803 (Ngdy nhOn bak 26/02/2015 - Ngay duyOt clang: 04/09/2015) TAP CHI DIJQC HQC - 9/2015 (SO 473 NAM 55) 71 [...]... bao ung this & ngu,o1 ung this phOi (Lu-1), ung this gan (HepG2), ung this tier' liOt tuyen (LNCap), ung this vu (MCF7), ung this buOng tru,ng (SW626), ung this dai trang (SW480) va ung this bleu me mieng (KB) m6i thi nghiem duvc 10p !pi 3 Ihn de dam bao tinh chinh xac cua thi nghiem va dip lieu 20 22,29 0,91 (s) 22,3 0,91 (s) Hinh 1: C6u tritc has hoc cOa 1 164,8 5,66 (s) 115,1 5,67 (s) 172,4 trong. .. Wang den hoat tinh khang to bao ung thu' Hep-G2 dm chat 2a va 4a - Kat qua thii, hoat tinh khang to bao ung thu, cua 4 chat (lb - 4b) tren 4 d6ng to bao ung thu' ngu'ai cho they chat lb co hoat tinh khang to bao ung thu, tren 3 d6ng: to bao ung thu' ph& A549, to bao ung thu' t6, cung Hela va to bao ung thu, buOng tru'ng OVCAR-8 Chat 2b chi co hoat tinh khang to bao ung thu' bueng tru,ng OVCAR-8 Hai... khang to bao ung thu, • Nghien ciru - Ky thuOt 4,93 va 3,94 pg/ml Bon ch6t (lb - 4b) da" du,ac thP, ho@t tinh khang to bao ung thu, trOn 4 dOng to bao Ket qua cho thy 1 b co ho@t tinh ten 3 clang to bao: ung thu, bung trimg OVCAR-8 (IC50 = 53,7 pg/ml), ung thu, t0, cung Hela (IC50 = 60,6 pg/ml) va ung thu' phi A459 (IC50 = 80,4 pg/ml) Ch6t 2b co hopt tinh ten d6ng to bao ung thu, buOng trOng OVCAR-8... tong hap h6a hoc va tac dung sinh hoc Cac cong trinh nghien ciru cua cac tac gia trong va ngoai nultec da cong bo cho thay cac an chat rhodanin co hoat tinh sinh hoc da Bang nhu' khang khuan, khang n6m, chang ung thiy, khang virus 11 '2'5] Trap tuc phat then htrang nghien ciru ve thiazolidin-2,4-dion, rhodanin trong bai bao nay chUng toi trinh bay kat qua "TOng h9p va tac dung sinh hoc coa mot so can... dm nhan rhodanin va thiazolidin-2,4-dion trong vung 3182 - 3100cm-1 PhO IR cua 4 chet (la, 2a, lb, 2b) deu co dai hep thy cua nhOm >C=0 cua nhan rhodanin trong vung 1734-1699cm-1, dai hep thy cua nhOm >C=S trong vung tin 1235-1200 cm-1 Ph6 IR cua 4 san phem N-benzyl h6a cua cac chet tir 1a - 4a deu xuet hien dao (Ring h6a tri cua nhOm methylen (-CH2-) trong vung tir 2953 - 2930 cm-1 Ph6 IR cua 4 chet... chat deu CO tac dung tren chung nem moc Fusarium oxysporum va chung nem men Candida albicans TCF ket qua CO the nhan they tac dung cua chat 2a va 4a tren chung nem mOc Fusarium oxysporum manh hop so vai chat la va 3a Nhu' vay, 2 nhom the do trong nhan benzen lam tang hoat tinh khang nem cua cac chat 4a co hoat tinh khang to bao ung thu, Hep-G2 vai mik de khac nhau Nhu' vay, 2 nh6m the clo trong nhan benzen... ph0 hap (trong 190 ul mei twang) va de chUng phat trien trong yang tir 3-5 ngay Met khay 96 gieng khac khong CO chat thin nhung co TBUT (180 ul) se duvc sir dung lam clei chirng ngay 0 Sau 1 gitr, dia dei chi:mg ngay 0 se direc co dinh to bao bang Trichloracetic acid — TCA Sau giai doan phat trien trong to em CO2, to bao duvc co dinh vao day gieng bang TCA trong 30 phut, duvc nhuem bang SRB trong 1... hoat tinh gay dOc to bao clang ke clOi vai 07 to bao ung thu, nguai Lu-1, FlepG2, LNCap, MCF7, SW626, SW480 va KB vai gia tri IC50 narn trong khoang 13,93-25,05 pg/mL Trong so cac dOng to bao ung thu, thin nghiem, chat nay co tac dung manh nhat clai vai citing to bao ung thu, bung tru,ng SW480 Tuy nhien, chat 1 da kh6ng the hien hoat tinh tren cac chung vi sinh vat kiem dinh thin nghiem Day la lAn dhu... phu'o'ng phap nghien art, Dung mei, hoa chat Chit chuan + Acid vaiproic (AVP) — chuen USP, SKS: VA/11/04, ham lu•gng 99,31%; d6 Am: 0,5% + Methylparaben (MPB) — Vien Kiem nghiem thu6c Trung uvng, SKS: 0213108.02, ham lu,o,ng 99,74%; de Am 0,05% Methylparaben duvc sir dung lam chat not chuen (IS) trong phtrang phap phan tich Dung moi, hoa chat:Dat trail chuan tinh khiet dung cho HPLC va LC/MS Huy& twang... dec trong cho dao dOng hoa tri cua nhOm >C=0 amid va nhOm >0=0 imid trong cac vung twang irng tir 1754 - 1729 cm-1 va 1708 1671 cm-1 Phe IR cua 8 chet deu co dai hep thy dec tru'ng cho dao dOng hoa tri dm lien ket trong vung 1594 - 1500cm-1 va 1467 - 1376cm-1 Ca 8 chet duvc ghi phO hOng ngoai deu cho phep nhan biet sy' CO met cua lien ket C.rorn-X vai dao Ong hoa tri dec trong cua lien ket Carom-F trong