• Nghien
CIPU- Ky thuat
(a) (b) (c) Hinh 8: SSc ky dd dung dich miu thu tif chi phim vien nen STADA (S)-ibuprofen 200 mg (a), vien nen (S)-
ibuprofen 200 mg (b), vien nang mim (S)-ibuprofen 300 mg (c) Nhan xet: K i t qua djnh lugng ba m i u ehe
p h i m (S)-ibuprofen eho t h i y ham lugng hoat c h i t d i u n i m trong khoang tir 90-110% so vdi ham lugng ghi trgn nhan. Trong khi dd c h i pham vien nang m i m (S)-ibuprofen 300 mg ed lugng tap (Rj- ibuprpfen eao n h i t 3,46%.
Kit luan
Qui trlnh phan tieh d i n g phan quang hgc (Sj-ibuprofen b i n g phuang phap HPLC, s u dung ept pha tTnh b i t d i i da d u g e xay dyng va tham dinh. Qui trinh cd tinh dac hieu, gidi han phat hign t h i p , eho k i t qua chinh xae va lap lai. Qui trlnh da duge ap dung d l dinh lugng ('Sj-ibuprofen, xac djnh tap d i n g phan (Rj-ibuprofen trong mdt s l e h l p h i m chira ('Sj-ibuprofen. K i t qua thu d u g c gdp p h i n vao viec phan biet eao dang dong phan va cd t h i d u g c irng dung d l xae djnh tap d i n g phan trong eae e h l pham (S)-ibuprofen dang luu hanh trgn thj trudng.
Tai lieu tham khao
1. P.O. Carvaltio, Q.B.Cass, S.A. Calafatti, F.J.
Contesini, R. Bizaco (2006), "Review - alternatives for ttie separation of drug enantiomers: ibuprofen as a model compound", Brazilian J. of Chemical Engineering, 23, 291-300.
2. Fumio Toda (2004), Enantiomer Separation Fundamentals and Practical Methods, Kluwer Academic Publishers, The Netherlands, pp. 308, 312, 316.
3. Satinder Ahuja (2007), "A strategy for developing HPLC methods for Chiral Drugs", LC GC North America, 25, 1118-1128.
4. Xiaoming Chen, Chiyo Yamamoto, Yoshio Okamoto (2007), "Polysaccharide derivatives as useful chiral stationary phases in high-performance liquid chromatograptiy", PureAppl. Chem., 79(9), 1564-1566,
5. ICH Harmonised tripartite guideline (2005), Validation of analytical procedures: text and methodology, pp.1-13.
6. Quyit djnh cua Cue truo-ng Cue Quan ly Du-gc, Bg Y t l s6 07/QD-QLD ngay 11 thang 01 nam 2013 \ii viec ban tianh So tay himng din dang ky thuoc, Ptiu luc 8, tr 30-35.
{Ngay nhan bai: 10/10/2016 - Ngay phan bien: 14/11/2016 - Ngay duyet dang: 02/12/2016) r r r
I Cai tien phirong phap chiet xuat rotundin 1/ tir cu binh voi
Tran Trong Bien'*, Nguyen Quoc Huy^, Nguyen Van Han' 'Truang Dgi hoc Duo'c Hd Noi -Cue Khoa hoc Cong nghe vd Ddo tgo, Bo Y te 'E-mail: trantronghien@gmail. com Summary
Crude L-tetrahydropalmatine (rotundin) was extracted from the fresh tubers of Binh voi (Stephanie brachyandra Diels) by cold maceration and further refined conventionally with 96% ethanol and novelly with a mixture of aceton - water or ethyl acetate. Practically these novel extractions showed superior to the conventional one in simplicity, efficiency and rather ecological safety. The product obtained by ail these solvents was of rather high purity (rotundin content > 95%,). But the novel extractions remarkably raised the yield from 71 % (with 96% ethanol) to 96.6 %> (with mixture of aceton - water) and the highest 98.5 % (with ethyl acetate).
In practical tests, 5 kg of fresh material (rotundin content of 1.02 %,), by such extraction and purification yielded 48.13 g purified rotundin (rotundin content of 99.42 %,); the whole extraction efficiency of rotundin was 93.3 %.
Keywords: Extraction, L-tetrahydropalmatlne, rotundin.
26 T A P C H i D l / O C " " ' '
Nghien
CCFU- Ky thuat
Dat van de
Cu cua cae loai cd ten ehung la binh vdi {Stephania spp.) tir lau da duge su- dung lam nguygn lieu d l ehilt xuit L-tetrahydropalmatin (C2,H2j.O^N) hay rotundin mgt alcaloid cd tac dung an thin, gay ngu, giam dau '''. Tuy nhien ham lugng rotundin trong cae loai binh vdi rit khac nhau P'^'. Trong dd, loai Stephania brachyandra Diels thu hai d Sa Pa, Viet Nam dugc xae djnh cd ham lugng rotundin trong cu eao (ed t h i dat 3,69%), thich hgp d l ehilt xuit rotundin P'.
Cac phuang phap thdng thudng d l chilt xuit rotundin tir cu binh vdi gim: ehilt rotundin dang base bing dung mdi hu-u ca nhu d i u hdaPi. eloroformPi, ethanoli"', n-hexan i^i, dieloromethani^i,...; chilt rotundin dang muli bing nudc^', nudc acidPi,.... San phim rotundin thd (ham lugng khoang 10-30%) dugc tilp tue tinh c h i tao rotundin tinh khilL Phuang phap duge ap dung phi bign la ehuyin dang nhilu lin gitra dang base va dang muli eua rotundin^ ^i hoac kit tinh lai trong ethanol^', methanol^. Nhin ehung cac phuang phap tren diu eho san phim rotundin cd ham lugng cao (> 95%). Tuy nhien nhilu quy trlnh tiln hanh phirc tap, san phim kem I n djnh, hieu suit quy trinh thip hoac khdng duge edng b l cu thl''^'*'.
Do dd, muc dich cua nghign ciru nay la cai tiln phuang phap chilt x u i t tinh ehl rotundin tir cu binh vdi cho hieu suit eao va on djnh.
Nguyen vat lieu va phu'o-ng phap nghien CLPU
Nguyen vat lieu
Mau nghien ciru: Cu binh vdi thu hai d Sa Pa, Lao Cai, Viet Nam thang 11/2012. IVliu dugc cu nhan Nguygn Chilu, Vien Duge lieu giam dinh tgn khoa hgc la Stephania brachyandra Diels. Tieu ban miu khd duge luu tai Phdng Tigu ban - Bd mdn Thyc vat - Trudng Bai hgc Duge Ha Npi, ma s l tigu ban HNIP/15308/07.
Hoa chat, thiet bi
- Rotundin chuin (ham lugng 99,6%, ham i m 4,86%, SKS 0203141, Vien Kilm nghiem thule Trung uang).
- Can phan tich Satorius TE 214S (Dire), can ky thuat may vit ly tam dung tich 5L Prolabo (Phap), may sie ky Idng hieu nang eao Shimadzu SPD-MIO Avp (Nhat Ban), may do nhiet dp ndng chay Ez-IVIelt (My). Cac hda ehit, thilt bj khae.
Phu-o-ng phap nghien cicu Phirong phap djnh luong rotundin
Ham lugng rotundin trong dugc lieu tugi, rotundin thd va rotundin tinh khiit dugc xae djnh bing phuang phap sie ky long hieu nang eao (HPLC). Diiu kien sic ky, mau ehuin, mau thu- duge chuan bj theo chuygn luan Binh vdi, Dugc diln Vigt Nam IV fi vdi mgt s l thay doi:
- Miu dugc lieu tugi: Sit- dung 2,000 g miu da xay nhd. Xu iy theo DDVN IV.
- Miu rotundin thd, miu rotundin tinh ehl: Pha miu trong pha dpng d l Sitae dung dich thu ed ning do rotundin khoang 50 pg/ml. Lge qua mang 0,45 pm.
Phuong phap chiet xuat va tinh chi rotundin Chiit xuit: phuang phap ngam lanh. Cu binh vdi tuai (1,00 kg) 3u/ac xay thanh bdt nhao sau dd ngam vdi dung mdi H^SO^ 0,4% (24 gid/lin lin lugt vdi 1,7 va 1,5 L dung mdi). Djeh ehilt dugc kilm hda bing dung djeh Ca(OH)2 bao hda din pH = 9 xuit hign tua. Lpe, siy tua d 50°C/1 gid thu duge rotundin thd.
Tinh chi: Rotundin thd dugc tinh ehl bing mgt s l phuang phap: sir dung ethanol 96%, hdn hgp aceton - nudc hoac ethyl acetat.
Tua rotundin thd (5,00 g) dugc thgm 20 ml dung mdi (ethanol 96%, aceton hoae ethyl acetat), khuiy diu trong 5 phiit d l hda tan rotundin, Igc qua giiy Ige. Ba cdn lai duge ehilt them 1 lin bing 15 ml dung mdi tuang irng. Lge, gpp cac djeh Ipe.
Oli vdi dung mdi ethanol: Djch Igc duge dun each thuy (50°C) va tiy mau bing than hoat (0,50 g) trong 10 phut sau dd d l kit tinh trong 24 gid, Igc thu tinh thi, siy tinh thi d 50°C/1 gid.
- D i i vdi dung mdi aceton: Djch Igc dugc thgm 5 phin nudc, de kit tua trong 24 gid, Ipe liy tua, siy tua d 50°C/1 gid.
- D i i vdi dung mdi ethyl acetat: Djeh Ipe dugc lie vdi dung djeh H^SO^ 0,1% (5 lin « 30 ml), gan liy Idp nude acid. Trung hda Idp nudc bing dung djch NaOH 10%, d l kit tua 24 gid, Igc liy tua, siy tua d 50°C/1 gid.
Phuong phap danh gia chat luong san pham
San phim rotundin dugc danh gia qua cae tieu chi:
-Cam quan.
- Nhiet do ndng chay - Ham lugng rotundin.
-Dp tinh khiit HPLC.
Phuong phap xd ly so lieu
Mil thi nghiem dugc tiln hanh lap lai 3 lin, kit qua dugc bilu diln dudi dang gia trj trung binh ± dp lech chuin {\ ± SD).
TAP CHI [alQCJi^Xl^T^ftH''^^'" ^°° N AIVI ^/:^ 27
• Nghien CIPU - Ky thuat
Kit qua nghien CLFU
Ham lu'o-ng rotundin trong nguyen lieu Bing phugng phap sie ky long hieu nang cao xae djnh duge ham lugng rotundin trong miu
nguyen lieu tuai la 1,02%. Hinh anh sic ky do (hinh 1) eho thiy trong duge lieu, ngoai pic cua rotundin, eon cd 2 pic khae, chilm tl le lin lugt Id 6,30% va 0,84% sg vdi rotundin. Nhu vay trong duge lieu tuai, rotundin chilm ham lugng cao nhat
=—
i !
1 1 3 I ,
(a) te
Hinh 1: Sic ky dd ciia rotundin chuin (a) va miu duac lieu tuui (b) Chiet xuat, tinh che rotundin
Tir 1 kg duge lieu qua giai doan chilt xuit thu dugc 36 g rotundin thd (ham lugng 27,44%).
Tinh theo ham lugng rotundin trong nguygn ligu.
Bang 1: Bang so sanh hieu qua cac phuang phap tinh chi rotundin
hieu suit chilt dat 96,85%. Rotundin thd dugc tinh ehl theo 3 phygng phap su- dung ethanpi 96%, hdn hgp aceton - nudc hoae ethyl acetat. Kit qua dugc trlnh bay d bang 1 va hinh 2, 3.
Tieu chi Cam quan Ntiiet (Jo nong chay (°C) Ham lu-ong rotundin (%) Pic tap chat tren sac ki do
Hieu suat tinh che (%)
Sir dung ethanol 96%
Tinh the mau vang nhat 141 -145 98,16 ±0,06
06 71,0 + 1,6
Sir dung hon hop aceton • nitac Tinh the mau vang
139-142 98,04 ±0,03
Co 96,6 ±1,3
Su'dung ethyl acetat Tinh the mau trang
142-143 99,44 ±0,08
Khong 98,5 ±0,7
(a) (b) (c) Hinh 2: Sac ky dd san phim rotundin tinh chi bang cac phuung phap khac nhau
((a) ethanol 96%, (b) hdn hop aceton - nuvc va (c) ethyl acetat
Nhan xet San phim rotundin tinh ehl theo 3 phuang phap d i u cd ham lugng cao (> 95%). Tuy nhien cd sy khae biet kha ro v l chi tieu cam quan,
„, , ^, , . nhiet dd ndng chay, pic tap tren sic k y d l HPLC va
• Ham luiTng rotundin (%) . 3 _ J ' r . i- j
.H«„ su4,«ch^ (%) hieu suat tinh chg.
Elhanol AceioD-nuoc Etliyl acetat Dung moi
Hinh 3: Dd thi so sanh hieu suit va ham luang rotundin tinh chi theo cac phuang phap khac nhau
28 T A P C H I D i r O C ' " " '
• Nghien
CLPU- Ky thuat
Su dung ethyl acetat thu dugc san phim mau tring, khoang nhiet dp ndng chay hep (142-143''C) phil hgp theo tieu chuin DDVN IV, tren s i c ky do khong thiy pic la ngoai pie rotundin, hieu suit tinh c h i cao nhit (98,5%).
Su- dung ethanol 96% hoac hdn hgp aceton - nudc thu dugc san phim tuang ii-ng ed mau vang nhat va mau vang, khoang nhiet do ndng chay rgng han, tren sic ky d l miu san phim v i n cdn pic phu ngoai pic rotundin. hieu suit tinh che lin lugt dat 71,0% va 96,6%.
Tif nhu-ng kit qua tren, lya ehgn ethyl acetat lam dung moi tinh ehl rotundin.
Chiet xuat va tinh che rotundin me 5 kg nguyen lieu tuoi
Be chirng minh tinh I n djnh eua quy trinh chilt xuit va tinh c h i rotundin tu- cu binh vdi, tren eg sd cac thdng s l thu duge ttr me 1 kg, tiln hanh thye nghiem trgn me 5 kg nguyen lieu. Kit qua duge ghi d bang 2.
Bang 2: Kit qua chiit xuit va tinh chi rotundin me 5 kg nguyen lieu tuui Rotudin tho
Khoi luong (g) Ham luong (%) 186,53±1.1 26.26i0.04
Rotundin Unh khiet Khdi luong (g) Ham luong (%)
43.13±1.3 99,42±0,02
Chiit xuit 96.0 ±1,5
Hieu suit (%) Tmh che 97,7 ±0,6
Ca quatrinh 93,8 ±1,2 Nhan xet: Rotundin sau khi tinh ehl dat cac
ehl tieu chit lu-gng theo DDVN IV v l cam quan (bgt kit tinh mau tring), nhiet do ndng chay (141- 143°C), ham lugng rotundin dat 99,42%. Hieu suit ca qua trlnh chilt xuit va tinh ehl dat 93.8%. Kit qua cho thiy quy trinh chilt x u i t tinh ehl rotundin da xay dyng I n dinh khi tang cd me nghign ciru tir 1 kg lgn 5 kg nguyen lieu.
Ban luan
Rotundin ia alcaloid chinh trong cu eua nhilu loai binh vdi, rit it tan trong nudc, d l tan trong dung djch acid sulfuric loang, kem bin vdi nhiet va anh sang •- ^' do dd khi chilt xuit cin han c h i tae dgng cua 2 y l u t l nay. Phuang phap ngam lanh su- dung dung mdi chilt la acid sulfuric loang (0,4%). tinh ehl sa bd ban diu bing each dung Ca(OH)2 bao hda d l kilm hda tao tua rotundin thd cd nhilu uu dilm: Quy trinh thilt bj dan gian, d l thyc hien, dung mdi re tiln, d l kilm.
D l tinh c h i rotundin, phuang phap biiyin thing dang ap dung tai nhilu ca sd san xuit trong nudc la tiy mau va kit tinh trong etiianol '•' '\ Tuy nhign san phim kem on dinh, d l bi nga vang. Mdt s l nguyen nhan cd t h i k l din la chit lugng tiian hoat khdng kilm soat duge, nhilu loai tiian hoat bj lin cac kim loai nang la chit xuc tac cho phan irng oxy hda rotuntin tiianh palmatin ed mau vang.
Mat khac, rotundin rit kem bdn vdi nhiet, qua ti"inh tiy mau ti^ong dung mdi etiianol cin nhiet dp cao d l gay phan huy dugc c h i t Kit qua kilm ti'a sic ky d l HPLC cho thiy ngoai pic rotundin vin edn xuit hien pic cua tap chiL Ngoai ra, higu suit qua ti-lnh tinh ehl chu-a cao, chT dat 71,0% do qua ti-lnh kit tinh khdng triet d l . Xuit phat ti> nhQ-ng vin d l gap phai trong tiiyc t l san xuit rohjndin tir cu
binh vdi, chiing tdi da cai tien phuang phap tinh ehl rotundin tif san phim rotundin thd, tranh su- dung nhiet dp. Hai phuang phap mdi dya tren viee su- dung hin hgp aceten - nudc va ethyl acetat dugc khao saL
Rotundin tan t i t fa-png aceton va rit it tan trong nude, aceton lai la mgt dung mdi trdn lin duge vdi nude, ngn khi pha loang dung djch rotundin ti^ong aceton bing nudc, do tan cua rotundin giam manh va kit tua lai. Lfng dung sy thay doi do tan cua rotundin trong hdn hgp aceton - nudc, ehiing tdi xay dyng phuang phap tinh ehl rotundin tir tua alaloid tiid bing each kit tua trong hdn hgp aceton - nude. Kit qua cho thiy san phim rotundin dat cac chi tieu chit lugng theo IDDVN IV, hieu suit tinh ehl eao han hin phugng phap su- dung ethanol 96%, dat 96,6%. Tuy nhign qua hinh anh sic ky HPLC vin cdn xuit hien pic tap chit.
Theo ly tiiuylt ehung, cac dung mdi hO-u ca it phan eye tiiudng hda tan t i t eae alcaloid d dang base, it hda tan cae tap chit khac. Dang muli eua alcalcid d l tan trong nudc, kem tan trong dung mdi hu-u ca it phan eye. Do dd cd t h i su- dung dung mdi hiru ca it phan eye thich hgp d l chilt rotundin tir rotundin thd. Qua khao sat, rotundin tan t i t trong clorofonn, dicloromethan, ethyl acetat.
Tuy nhien, ethyl acetat la dung mdi kha an toan va tuang dii tiian thien vdi mdi tru-dng, dung mdi nay eung duge chip nhan bdi Cue quan ly Thye phim va Dugc phim Hoa Ky diing trong Duge phim nen duge lya chgn d l hda tan rotundin. Djch Igc etiiyl acetat sau dd dugc chilt lai bing dung djeh H^SO^ 0,1% va kit tua rotundin bing dung djch NaOH 10%. Bing each nay, rotundin tinh khiit ttiu dugc dat eae chi tieu ehit lugng theo DDVN IV v l cam quan, nhiet dp ndng chay, hieu suit
T.A.PCH1 nirrtr Hnr^iT/Tntfi («;n ASS N A M ^\ 29
Nghien
CIFU- Ky thuat
tinh c h i cao (98,5%), hinh anh sic ky HPLC khong ecn xuit hign pic cua tap ehit. Phuong phap nay khdng cin budc tiy mau bing than hoat d nhiet do eao, do dd loai bo duge tac dgng ciia nhiet dp din dp I n djnh cua rotundin, ngoai ra each tiln hanh don gian, hda chit thdng dung va tuong dii than thign vdi mdi trudng.
Kit luan
Da khao sat, so sanh 3 phuang phap tinh ehl rotundin tir tua rotundin thd gim phuang phap tiy mau va kit tinh trong ethanol 96%, phuong phap kit tua trong hin hgp aceton - nudc va phuang phap sir dung ethyl acetat, trong dd phuang phap su- dung ethyl acetat che hieu suit tit nhit. Trgn ea sd cae kit qua da edng b l trudc dd, nghien cu-u da cai tiln giai doan tinh ehl rotundin su- dung dung mdi ethyl acetaL Kit qua tir 5 kg nguyen lieu tuai (ham lugng rotundin 1,02%) thu duge 48,13 g rotundin tinh khiit (ham lugng 99,42%). Higu suit toan qua trinh dat 93,8% tinh theo ham lugng rotundin
cd trong nguyen ligu. Day la tiln d l cho nhd-ng nghien CLPU d quy md Idn han.
Tai lieu tham khao
1. Bp mon Du-gc lieu (2004), Bai giang Duac lieu tap II, Trudng Bai hpc Dugc Ha Npi, tr 94 - 100.
2. Nguyen Quic Huy (2010), Nghien ciru v l thirc vat, thanh phan hda hpc, mpt so tac dung sinh hpc cua mpt s6 loai thupc chi Stephania Lour 6- Viet Nam, Luan an Tien si Dime hoc, Trudng Dai hpc Du-gc Ha Noi, tr 6, 73-99.
3. Bp mon Cong Nghiep Du-gc (2016), Ky thuat chiit xuit Duuc lieu, Trud-ng Dai hpc Dugc Ha Npi, tr74.
4. Nguyen Van Dan, Ngo Ngoc Khuyin (1999), Hap chat thien nhien dung lam thudc, NXB Y hpc, tr 106.
5. Bartley, J. P, et al. (1994), "Alkaloids of Stephania bancroftii", Phytochemistry, 36(5), pp. 1327-1331.
6. Kin, F. K., et al. (1965), "A study of the alkaloids of plants of the genus Stephania II. The alkaloids of Stephania glabra", Khimiya Prirodnykh Soedinenii, 1(6), pp. 392-394.
7. Bp Y te (2015), Dup-c diin Viet Nam IV, [An xu§t ban thu 4, NXB Y hgc, tr. 551, 696-697.
{Ngay nhan bar 30/09/2016-Ngay phan bien: 25/10/2016 - Ngay duyet dang: 02/12/2016)
M
Nghien ciru doc tinh cua cao long phan tren mat dat cay tien hac thao
Nguyen Van Linh'*, Pham Thanh Ky^
Nguyin Hoang Ngan^ Vu Manh Hung' 'F?en Y hoc co truyen Qudn dgi
^ Truang Dgi hoc Duac Ha Noi
^Hgc vien Qudny ' E-mail: nguyenvanlinh063@yahoo. com Summary
Experimental medical safety of the extract CL1 (prepared from the aerial parts ofAgrimonia pilosa Ledeb var pilosa) was investigated in mice and rabits For acute toxicity at the possible highest daily oral dose (360 Ogdry herb/kg body weight), CL 1 exhibited no acute toxicity in mice. For the subacute toxicity, rabbits were administered the CL1 at the dose of 1 gAg/24 h and 5 g/kg/24 h for 42 successive days. After such a treatment course, the basic biophysical parameters demonstrated no significant changes in the body weight,
h J ( f "^'°'°£"'^^' 9"^ biochemical ones. The histology of the liver, spleen and kidney remained normal In short, the CL1 exhibited no toxicities on the life-important systems of the experimental animals.
Keywords: Tien hac thao, Agrimonia pilosa Ledeb var pilosa, acute toxicity subacute toxicity.
Dat v i n dh
Cay tign hae thao (edn gpi la long nha thao, qua huong thao, til- miu thao...) ed ten khoa hpc la Agrimonia pilosa Ledeb var pilosa, hp Hoa hing - Rosaeeae, mgc hoang nhilu tren cac viing nui cao phia Bic nudc ta... Trong Ddng y, tign hae thao
dugc x l p vao nhom "thu liem chi huylf, diing lam thulc eim mau, chu-a t h i huylL khai huylL kiltly, s i t ret, mun nhgt Id loet ngoai da P'. Mpt s l nghien ciru eho thiy tign hae thao cd tac dung ching vigm, diet t l bao ung thu, khang virus '°i... Ciing vdi nghien eiru v l thanh phin hda hpe, chiing tdi
30 TAP CHI DLTOC I '