• Nghien
CCPU- Ky thuat
Xay drnig phirong phap dinh lirong hypoxanthin trong dia long (Pheretima aspergillum) bang sac ky long hieu nang cao (HPLC)
Pham Van Hiln', Le Due Hung S Vu Binh Duong'*
'Hoc vien Qudny - Bo Quoc phong -'Vien Yhoc cd ti-uyin Qudn doi - Bo Quocplmng
'E-mail: [email protected] Summary
An HPLC method for quantification of hypoxanthine in Pheretima (Pheretima aspergillum) was established and validated. The chromatographic analysis was perfomed with: C18 column (250 x4.6 mm, 5 gm); Detector UV (254 nm); Mobile phase: f/leOH and 50 mhAphosphate bufferpH 4.5 (10:90, v/v). The method was validated for the HPLC system compatibility, linearity range, tiie specificity, repeatability and acurate; the recovery greater than 95.0%.
Keywords: Pheretima aspergillum, hypoxanthine, HPLC.
Dat van de
Dia long {Pheretima aspergillum) la vi thudc da dugc sir dung lau ddi trong y hpc cd truyin vdi nhieu tac dung nhu ha nhiet, an thin, binh suyen, trin kinh, thdng kinh boat lae, Igi nieu, binh can, tieu dpe, ha liuylt ap'''. In vitro va thir nghiem lam sang cting che thiy dia long giup cai thien tuan hoan mau, diiu tri ddt quy do triing pheng, ha set Igi tieu, khang virus, khang ung thu va boat tinh khang khuan w. Trong dd tac dung diiu trj ddt quy de trung phong da dugc chirng minh in vitro va thir nghiem lam sang vdi cac tac dung lien quan din lam tan cue mau ddng va chdng ddng mau '^'"'. Heat tinh nay chu y l u dga tren eae tae dung eiia eae phirc hgp protein va enzym cd heat tinh sinh hpc. Tuy nhien, hyppxanthin cung gdp phin quan trpng vdi cae tac dung khang histamin va lam giam ap Ige tuin hpan mau. Dugc diin Viet Nam IV da cd chuyen luan rieng cua vi dugc lieu nay, nhung eae chi tieu ehit lugng mdi dirng lai d miPC dinh tinh, chua ed chi tieu dinh lugng heat chit cu t h i cd heat tinh sinh hpe. Nghien cipu nay duge thge hien nham xay dgng phuong phap dinh lugng hyppxanthin trong dja long, d l gdp phin vao danh gia chit lugng dugc lieu nay.
Doi tu'O'ng va phu'O'ng phap nghien CLPU Doi tu>gng va nguyen vat lieu nghien ciru Dugc lieu dja long (Pheretima aspergillum) dugc cung cip bdi Cdng ty Dugc lieu Son Lam, Ha Ndi dat tieu chuin Duge diln Viet Nam IV.
Chuin hypoxanthin dugc eung cip bdi Hang Sigma Aldrich, (> 99,0%, Lot. SLBC2747V).
Methanol (MeOH), aeetonitril (AON), nudc cit dat tieu chuin dung mdi HPLC. Cae hda chiL dung mdi khae nhu ethanol (EtOH) NaH^PO,, Na^HPO^, HjPO^... dat tieu ehuan tinh khiit phan tich (PA).
Phu'O'ng phap nghien cim Chuan bj mau
Miu chuan: Can chinh xae khoang 20 mg chuin hypoxanthin vae binh djnh mire 20 ml. Bo sung t h i tieh vira dii bing MeOH 40% dugc dung dich chuin gdc cd ndng dp 1000 pg/ml. Pha loang dung djch chuin gde vdi MeOH 40% d l ed eae dung djeh chuin lam viee.
Miu tring: MeOH 40%.
Miu thir. Can ehinh xae khoang 0,15 g bdt dja long da nghien mjn vao binh ndn, them 15 ml EtOH 40% chiit sieu am 2 gid d nhiet dd 60°C, ly tam d l rieng djch ehiit. Ba duge chilt tilp 2 lin nhu tren.
Tap trung tean bd djch ehiit vae binh dinh mirc 100 ml. Bo sung vira dii the tieh, lac diu, Ipe qua mang Ipe 0,22 pm d l dugc dung djeh thir tiem sic ky.
Lira chgn dieu kien sac ky
Chay sic ky HPLC d cae dieu kien khac nhau.
Trcng dd, cdt sic ky la Gemini 018 (250 x 4,6 mm, 5 pm, Phenemenex, My), detector UV do d 254 nm.
Khae sat Iga chpn pha ddng gdm 2 leai pha dpng khac nhau: (1) H20:MeOH:tetrahydrpfuran = 100:0,5:0,5; (2) dem 50 mM phosphat pH 4,5 va (3) MeOH : dem 50 mM phosphat pH 4,5 (10:90).
The tieh bom miu la 10 pi. Tdc dp dung mdi pha ddng la 1 ml/phut.
Tham djnh phircmg phap phan tich Phuang phap phan tich duge thim djnh theo yeu ciu chung eiia ICH (International Conference
TAP CHi DlTOC HOC - 6/2016 (SO 482 NAM 56) 27
Nghien ciru - Ky thuat
en Harmonisation) v i tham djnh phuong phap phan tich ^ gdm: Tinh tuong thich he thdng, chpn lpc - dac hieu; Khoang ndng dp tuyin tinh; Dd chinh xac; Dp diing; Gidi ban phat hien (LOD) va gidi ban djnh lugng (LQQ).
Xir ly so lieu
So lieu duge phan tich thdng ke tren phin mim Mierpsoft Excel Office 2010 va Origin Pre 8.0.
Ket qua nghien ciru
Ket qua khao sat Iga chon diiu kien sac ky
Tip chuan gde, pha chuan lam viec cd ndng dp thge t l la 5,05 pg/ml. Dung djch chuan nay duge sir dung d l khao sat de Iga chgn diiu kien sac ky.
Vdi 3 he dung mdi da neu tren kit qua cho thiy rang: Khi sir dung cac he dung mdi pha ddng la (1) va (2) thi cd xuit hien pic cua hypoxanthin, tuy nhien pic khdng can ddi, cd hien tugng doang pic.
Vdi he dung mdi (3) che pic can ddi, sac net, he sd bit ddi xirng pic xap xi bang 1, thdi gian xuit hien pic la ngan (t^ = 4,686 phiit) (hinh 1).
(a)
(b)
(c)
Hinh 1: Sie ky dd eiia chuin hypoxanthin chay sie ky vai eae diiu kien khae nhau (a): He dung moi (1); (b): He dung mdi (2); (c): He dung mdi (3) Tip kit qua nay, Iga chpn dugc dllu kien phan
tich hyppxanthin trpng dja lpng gdm: Cdt Gemini C18 (250 X 4,6 mm, 5 pm, Phenemenex, My) heac tuong duong; detector UV do d 254 nm; dung mdi pha ddng la MeOH: dem 50 mM phosphat pH 4,5 (10:90) chay d tdc dp ddng 1,0 ml/phiit; t h i tich bom miu 10 pi.
Kit qua tham djnh phu'O'ng phap djnh lugng hypoxanthin trong dja long
Tinh tirong thich he thong, chgn Igc - dac hieu Phan tich s i c ky 5 lin lien tiip miu ehuan hyppxanthin thep dieu kien da Iga chpn duge d tren. Thdi gian luu va dien tich pic ciia hypoxanthin dugc xac djnh lin lugt la 4,72 ± 0,04 (X_+ SD, RSD = 0,92%) va 2380108,80 ± 30583,92 (X ± SD, RSD 1,28%). Kit qua nay che thiy rang RSD ciia thdi gian luu va dien tich pic hypoxanthin deu nhd ban 2%. Nhu vay, phuang phap phan tich nay la tuang thich vdi he thdng sac ky.
(a)
hvpoxanthin
(b)
(c)
hvpoxanthin
Hinh 2: SKD thi hien tinh chgn loc - dac hieu cua phuang phap (a): SKD mau bing; (b): SKD miu chuan; (c): SKB miu thir
28 TAP CHI Dl/OC HOC - 6/2016 (SO 482 NAM 56)
Nghien cCeu - Ky thuat
Phan tich ddng thai mau chuan hypoxanthin, m i u trang, va m i u thip djch c h i i t dja long trong ciing d i i u kien sae ky da lga chpn d u g c . K i t qua cho t h i y rang, tai vj tri x u i t hien pie eiia hypoxanthin tren sac ky dd m i u thip, ddng thdi xuat hien pic t u o n g ipng tren mau ehuan. Tai v i tri nay khdng x u i t hien pic t u o n g irng tren mau t r i n g . Can cip vae sac ky dd mau thir ta t h i y pic hypoxanthin tach hoan toan khdi pic tap. Pic can ddi, sac net, dp rdng chan pic nhd. Dc vay, phuang phap nay dam bao dp ehgn lpe va dac bleu (hinh 2).
Khoang nong do tuyen finh
Phan tich sac ky 3 day c h u i n hyppxanthin cd ndng dp tip 0,1-10,0 pg/ml theo d i i u kien da Iga chpn d u g c . K i t qua cho thay, trong khoang ndng dp khae sat, cd mdi lien he t u y i n tinh giti'a dien tich pic va ndng dp c h i t phan tich. D u d n g hdi quy la d u d n g t h i n g (y = 216017,77x + 4150,46) vdl he sd t u o n g quan R ^ g i n bang 1 (0,9999) (hinh 3).
Concentration ( ppm)
Hinh 3: Dd tiii md ta moi quan he tuyin tinh cua dien tich pic va ndng dd 2,4-D trong khoang khao sat
Dp chinh xac
Chuin bj cac miu chuin LQC (1,1 pg/ml), MQC (5,5 pg/ml) va HQC (11,0 pg/ml). Mel ndng dp chuin bj 3 miu. Phan tich sic ky theo diiu kien da lga chpn dugc trcng ciing mdt ngay va sau 3 ngay de xac djnh dp chinh xac trong ngay va khac ngay. Kit qua che thiy dp lech chuin tuong ddi d cac diem khae sat trong ngay va khac ngay diu
< 2% (bang 1). Nhu vay, dp chinh xac eiia phuang phap dugc dam bao.
Bang 1 : Kit qua danh gia do ehinh xae trorig ngay va khac ngay
Bqehinhxac LQC(1,1 pg/ml) mC(5,5pg/ml) HQC(11,0iig/ml) Tjongngay 1,11+0,01* 5,51 ±0,07* 10,99 + 0,14*
(XtSD. pg/ml) RSD = 0.80% RSD = 1,31% RSD = 1,28%
Khac ngay (XtSD, pg/ml)
1,11+0,29*
RSD = 0,01%
5,50 ±0,05* 10,98 ±0,15*
RSD = 1,18% RSD = 1,36%
*: La ndng dd ngoai suy tir ducmg chuan Bg dung
Dp dung ciia phuong phap dugc thge hien theo phuang phap them ehuan. Chilt kheang
0,15 g dja long, chia thanh nhiiu phin khac nhau.
Them cac lugng chuan lan lugt la 1,1 pg; 5,5 pg va 11 pg vae mdi phin sae cho ham lugng heat chit treng miu phan tich d i u nam trong khoang tuyen tinh. Phan tieh sae ky theo dieu kien da lga chpn dugc d tren. Can cir vao dudng hdi quy tuyin tinh, dien tieh pic ciia miu thir va miu thip them chuan d l ngoai suy lugng them vae ban d i u . Op thu hdi heat chit duge xac djnh dga vao ti le % lugng tim thiy theo dudng chuan so vdi lugng thge t i them vao. Kit qua eho thiy, d 3 dilm khae sat, dp thu hdi ciia phuang phap diu Idn ban 85% (bang 2).
De vay phuang phap djnh lugng dap irng yeu cau ve dp dung.
Bang 2: Kit qua xac dinh do diing ciia phuang phap Luong them vao ban dau 1,1 pg 5,5 pg 11,0 pg Ddthuh6i(±SD,%) 96,2 ±1,71 98,3 ±1,46 99,1 ±0,64 Gl&i han phat hien va gioi han djnh lirgng Tiem sac ky 3 lin miu trang, ghi lai dp nhieu dudng nen tai thdi gian luu tuang irng vdi thdi gian luu cua hyppxanthin. LOD va LOQ dugc tinh dga vap cdng thirc k x SD/b (k = 3,3 che LOD va 10 chp LOQ). Treng dd SD la dp lech chuan ciia miu trang, b la he sd gdc dudng hdi quy tuyin tinh. Kit qua ehe thiy rang gidi ban phat hien va gidi han djnh lugng dudi eiia hypoxanthin lin lugt la 0,001 va 0,003 pg/ml.
K i t qua xac djnh ham lu'gng hypoxanthin trong mpt so m l u dja long tren thj tru'O'ng
Ap dung quy trinh da thim djnh d l xae dinh ham lugng hypoxanthin treng mdt sd miu dia long tren thj trudng hien nay. Kit qua thge nghiem eho thiy ring ham lugng boat chit nay trong dja long dae ddng d mipc tuang ddi rdng (bang 3).
Bang 3: Kit qua dinh lugng hypoxanthin trong mdt so miu dia long tren thi trudng
Mau Ham luomg hypoxanthin (pg/g)
1 174,4 2
3 4 5 6 7 8 9 10 11
184,1 178,3 184,7 190,9 189,8 250,5 387,9 1012,7 1645,4 546,7
Ban luan
Nghien cu'u nay sir dung cac thiit bj co ban eiia phdng thi nghiem phan tich d l khao sat dugc
TAP CHI DtrOC HOC - 6/2016 (SO 482 NAM 56) 29
Nghien cCfu - Ky thuat
diiu kien sic ky, cho phep cd the trien khai nghien ciru nay mdt each d i dang. Vdi dieu kien sac ky da lga chpn, pie xuit hien sdm, tilt kiem dung mdi, thdi gian phan tich. Trong dja long, ngoai hypoxanthin cdn ed cac thanh phin khac nhu eae pretein, chit bee gay anh hudng din qua trinh chilt xuit va lan nhiiu tap trong miu chiit, cd kha nang lam anh hudng din hieu Igc cdt sic ky va tinh chpn Ipe dae hieu eiia phuang phap. Nghien cipu nay sir dung dung mdi chilt xuit la EtOH thip dp (40%) vira cd the chiit duge boat ehit ddng thdi ban ehl chiit xuit cae tap ke tren va dam bao tinh dae bleu ciia phuang phap djnh lugng.
Kit luan
Da xay dgng va thim djnh dugc phuang phap djnh lugng hypoxanthin trong dja long bang phuang phap sac ky Idng hieu nang cao. Phuang phap da xay dgng tuong thieh vdi he thdng sic ky, dam bao dp chpn Ipe dac hieu, dp diing (> 95%) va dp ehinh xac cao (RSD < 2,0%).
Tai lieu tham khao
1. Bd Y t i (2009), Dugc dien Viet Nam IV, Nha xuat ban Y hpe. Ha Ndi.
2. Dd Tit Lgi (2004), Nhung eay thuoc va vi thuoc Viet Nam, Nha xuit ban Y hge. Ha Ndi, tr. 978.
3. Pham Xuan Sinh, Phiing Hda Binh (2002), Dugc hoc C6 truyin, Nha xuit ban Y hpc. Ha Ndi, trang 199-200.
4. Mira Grdisa (2013), "Therapeutic properties ef Earthworm", Bioremediation, Biodiversity and Bioavailability, 7 (1), 1-5.
5. Liu Kai, Zhang Yuhuan Yao Lin (2010),
"Chemical components and pharmacological function of TCM lumbricus", Harbin Pharmaceutical J., (30)1,57-59.
6. Zhang, Liping (1990), "Pharmacological study and clinical application of earthworm", Fujian J. of Traditional Chinese Medicine, 2 (6), 52.
7. ICH (2005), Validation of analytical procedures: text and methodology, Q2R1.
{Ngay nhan bai: 18/03/2016 - Ngay duyet dang: 02/06/2016)
7 r /v r
Tong hop mot so dan chat semicarbazon va thiosemicarbazon cua benzaldehyd the
Truong Ngoc Tuygn'*, Pham Canh Em', Nguyin Thi Phuong Nhung^
'Khoa Duac- DH Y-Duac TP. H6 Chi Minh 'Dai hoc Ky thuat YDuac Dd Ndng 'E-mail: [email protected]
Summary
As the antiviral antibacterial, antifungal antimalarial, anticancer, antiieprotic and antirheumatic bioactivities of semiearbazones and thiosemicarbazones ... largely depend on their parent aldehyde or ketone moieties, a sen'es of semiearbazones and thiosemicarbazones were synthesized by treating semicari^azide or/and thiosemicartiazide with different substituted aromatic aldehydes. All the synthesized compounds were stmcturally confinned by IR, H-NMR, "C-NMR, MS and evaluated antibacterial, antifungal and anticancer property for use as intermediates in preparing heterocyclic oxadiazoles and thiadiazoles to create new candidates for further cytotoxicity tests on some cancer cell lines.
Keywords: Semicarbazone, thiosemicartjazone, semicarbazide, thiosemicarbazide.
Dat van de
Semicarbazon edn dugc gpi la din chit ure, la san phim tir phan irng cdng-tach ciia mdt aldehyd (hoae eeton) vdi semiearbazid. Din ehit semicarbazon ed nhieu irng dung treng duge hpe va duge chirng minh cd nhiiu heat tinh nhu: khang nim manh, khang khuin, khang khdi u, ehdng co giat va khang pxy hda P-^'. Phipc hgp kim Icai eua
semiearbazcn dugc nghien ciru, ehe thiy ed heat tinh ehdng benh eiim, dau mtia, viy nln, thip khdp, ciu triing, sdt ret, va mpt sd loal khdi u 1"='.
Thiosemicarbazon cdn dugc gpi la d i n chit thicure, la d i n chit ciia semicarbazon cd chira mdt nguyen tip S thay t h i mdt nguyen tip O. Trong nhtrng nam gin day, cac din chit thiosemicarbazon tip 2-fermylpyridin va cae aldehyd tuang tg dugc
30 TXD r'xjT r\Tinr^
