Hp tc khoa hc cng ngh v s pht trin bn vng nng nghip Lm ng - Ty Nguyn 2014

187

TIEN BO CONG NGHE CHIET TACH TAXOL T CAY THONG O

TAXUS WALLICHIANA ZUCC.

L TH KIM PHNG1, VNG CH HNG2,
TRN CNG LUN3, BI TH VINH3

1. t vn
Theo T chc Y t th gii nm 2008, ton cu c khong 7,6 triu ngi cht v bnh ung th.
Hin nay, s ngi mc bnh ln n 25 triu ngi v d on vo nm 2020, c khong 30 triu
ngi mc bnh.
Ti Vit Nam, c khong 200 ngn ngi mc bnh ung th hng nm v khong 150 ngn ngi
cht (B Y t). Mi nm, phi tn hng triu USD nhp thuc iu tr ung th, trong taxol
(paclitaxel) l thuc ch lc trong cc phc iu tr ung th phi khng phi t bo nh, ung th
bung trng v ung th biu m v. Do vy vic pht hin cy Thng l di (vit tt Thng ) v
trng thnh cng Lm ng lm ngun nguyn liu chit tch tin cht 10-DAB v taxol l cn
thit cho vic ch ng nguyn liu lm thuc theo ng ch trng ca nh nc ti Quyt nh s
61/2007/Q-TTg ngy 07/5/2007, trong cng nu rmc tiuNghin cu khai thc v s dng c
hiu qu cc hot cht thin nhin chit tch, tng hp hoc bn tng hp c t cc ngun dc
liu v ti nguyn thin nhin qu bu l th mnh ca nc ta, phc v tt cng nghip bo ch mt
s loi thuc c th ca Vit Nam, p ng nhu cu cha bnh v xut khu.
Do , vic nghin cu quy trnh cng ngh chit tch hot cht trong thng bng nhiu k thut
khc nhau nh chit tch theo cng ngh truyn thng, chit siu m hoc k thut CO2 siu ti hn,
nhm ti u ha cng ngh mang li hiu qu cao l iu cn thit v cng l mc tiu hng n.
2. Vt liu v phng php nghin cu
2.1 Vt liu nghin cu
Nguyn liu l thng trng Trung tm Nghin cu trng v ch bin cy thuc Lt, c
sy nhit 50oC v nghin nh tng kh nng truyn khi gia dung mi v nguyn liu.
Ha cht: ethanol, methanol, n-hexan, chloroform, acetonitrile, CO2 lng, nc
Cc thuc th: Mayer, Dragendorff, Bouchadat, Fehling
Cht chun: Paclitacel 99% c lp t Taxus brevifolia (hng Sigma, St Louis, MO, USA) v 10-DAB
98% c lp t Taxus baccata (hng Sigma, St Louis, MO, USA).
Trang thit b
- T sy KC-65
- Cn phn tch Melteler Toledo AB-204
- Cn phn tch Precisa (5 s l)
- My c quay BUCHI (c)
i hc Bch khoa Tp. H Ch Minh
Trung tm Nghin cu Trng v Ch bin cy thuc Lt
(3)
Trung tm Sm v Dc liu Tp. H Ch Minh
(1)
(2)

Hi tho Quc t

188

- n soi UV-VIS DESAGA SARSTEDT GRUPPE

- T sy chn khng VWS/P
- My nh k thut s FinePix S20 Pro
- My o UV: Heios Thermo Spectronic (UNICAM-UK)
- My o IR: Shimadzu FTIR-8201 PC
- My o LC-MS: My sc k lng cao p ghp khi ph AGILENT 1100 LC-MSD
- My ghi ph NMR AVANCE 500 BRUKER
- Bnh ngm kit inox dung tch 140 lt
- Ct sc k inox kch thc 15x150 cm
- H thng HPLC Shimadzu:

+ Bm LC-20AD

+ Detector PDA SPD-M20A

+ Ct Supelco C18 (250 mm x 4,6 mm, 5m)

+ B phn tim mu bng tay

- Thit b trch ly siu ti hn dung tch 100 mg (xut x Hoa K) v dung tch 20 lt x 3 (i Loan),
c s dng trong thc nghim ny, bao gm cc b phn chnh: (1) Bnh hnh tr cha dung mi
CO2. (2) Bnh cha dung mi h tr (3). Bnh trch ly. (4) B iu chnh p sut t ng. (5) B phn
thu hi sn phm. (6) B trn dung mi. (7) Thit b trao i nhit. (8) Thit b lm lnh. (9) Bm CO2.
(10) Bm dung mi h tr
2.2 Phng php nghin cu
2.2.1 Phng php nghin cu chit-tch

Quy trnh chit: Ngm bt nguyn liu trong 24 gi, chit ngm kit bng metanol (t l 1:6). Thu
dch chit v c gim p n dng cao st, thu c cao metanol th ha tan vi nc, lc vi
dicloromethan thu c cao CH2Cl2. Np cao CH2Cl2 vo ct diaion HP-20 (15 x 110 cm), ra gii ln
lt vi cc dung mi: nc, metanol 10%-60%-80% v 100%.

Hp tc khoa hc cng ngh v s pht trin bn vng nng nghip Lm ng - Ty Nguyn 2014

189

Chit tch v phn lp cc hp cht theo s di y:

2.2.2. Phng php nghin cu bng CO2 siu ti hn

S dng thit b CO2 my chit xut TST c dung tch 20 l x 3 = 60 lt ti Nh my Dc liu - Cng
ty DOMESCO, cho 04 kg nguyn liu vo bnh trch ly 20 lt. Cc dch trch c c quay thu hi dung
mi v cn khi lng cao thu c. Cao c em x l v ha tan trong methanol ri lc qua mng
lc em i phn tch HPLC. iu kin trch ly kho st nh bng 1.
Th nghim 1: 4 kg nguyn liu c trch p sut 295 bar, nhit 60oC, tc dng
3,13 l/pht, tc dng dung mi b tr 0 l/pht trch ly trong thi gian 6 gi. Ly mu theo thi gian,
c mi gi ly mu 1 ln.
Th nghim 2: 4 kg nguyn liu c ngm tm t trc vi dung mi ethanol, sau trch p
sut 295 bar, nhit 60oC, tc dng 3,13 l/pht, tc dng dung mi b tr 0 l/pht trch ly trong
thi gian 4 gi. Ly mu theo thi gian, c mi gi ly mu 1 ln.
Th nghim 3: 4 kg nguyn liu c trch p sut 295 bar, nhit 60oC, tc dng
3,13 l/pht, tc dng dung mi b tr 0,25 l/pht trch ly trong thi gian 4 gi. Ly mu theo thi
gian, c mi gi ly mu 1 ln.

190

Hi tho Quc t

Bng 1. Cc iu kin trch ly quy m pilot

Thi
gian
(gi)

Tc
dng ng
dung mi
Ethanol
(l/pht)

Th nghim

p
sut
(bar)

Nhit
(oC)

Tc
dng
CO2
(l/pht)

RE430414

295

60

3,13

Khng x nguyn liu trc

RE510414

295

60

3,13

Nguyn liu c ngm trc trong

ethanol

RE520414

295

60

3,13

0,25

Khng x nguyn liu trc

RE530414

295

60

3,13

0,25

Nguyn liu c ngm trc trong

ethanol

RE560414

295

60

3,13

0,25

Nguyn liu c ngm trc trong

ethanol v ngm CO2 trong qu trch ly

Tin x l nguyn liu v trch ly

Th nghim 4: 4 kg nguyn liu c ngm tm t trc vi dung mi ethanol v sau c

trch p sut 295 bar, nhit 60oC, tc dng 3,13 l/pht, tc dng dung mi b tr 0,25 l/pht
trch ly trong thi gian 4 gi. Ly mu theo thi gian, c mi gi ly mu 1 ln.
Th nghim 5: 4 kg nguyn liu c ngm tm t trc vi dung mi ethanol v sau c
trch p sut 295 bar, nhit 60oC, tc dng 3,13 l/pht, tc dng dung mi b tr 0,25 l/pht
trch ly trong thi gian 4 gi, trong qu trnh trch nguyn liu c ngm trong CO2 120 pht. Ly mu
theo thi gian, c mi gi ly mu 1 ln.
3. Kt qu nghin cu
3.1 V chit xut v phn lp
Chit ngm kit 25 kg bt nguyn liu thng bng metanol (1:6), thu c khong 150 lt dch
chit. C gim p thu c khong 8 kg cao metanol. Ha 1 lt nc vo 1 kg cao metanol, lc phn
on vi khong 12 lt CH2Cl2, c gim p dch trch ly thu c 200 g cao CH2Cl2.
Np 200 g cao CH2Cl2 vo ct diaion HP-20, ra gii ln lt bng cc dung mi nc (30 lt),
metanol 10% (60 lt), metanol 60% (150 lt), metanol 80% (60 lt), metanol 100% (60 lt). Phn on
ct diaion HP-20 metanol 60% (42 g) thc hin sc k ct trn silica gel pha thng v pha o vi
nhiu h dung mi khc nhau, pht hin 5 cht, k hiu: cht T1 (500 mg; 0,012%); cht T2 (160 mg,
0,0038%); T3 (50 mg, 0,0012%); T4 (3 g, 0,072%); T5 (93 g, 0,0022%).
Phn on ct diaion HP-20 metanol 100% (45 g), thc hin sc k ct hp ph trn silica gel pha
thng thu c T6 (60 g, 0,0014%).
3.2 V xc nh cu trc ha hc cc cht thu c

Hp tc khoa hc cng ngh v s pht trin bn vng nng nghip Lm ng - Ty Nguyn 2014

191

Hi tho Quc t

192

T cc d liu ph trn, kt hp so snh, i chiu ti liu tham kho, xc nh c hp cht T1

l Taxuspine F.

Tng t, da trn kt qu NMR mt v hai chiu, xc nh cc cht T2; T3; T4; T5; T6 ln lt l
19-hydroxybaccatin III; baccatin III; 10-deacetyl baccatin III; Taxchin A; taxol.

Hp tc khoa hc cng ngh v s pht trin bn vng nng nghip Lm ng - Ty Nguyn 2014

193

3.3 Kt qu thc nghim quy trnh cng ngh tch chit 10-DAB v taxol bng CO2 siu ti
hn trn bnh chit 20 lt x 3
3.3.1 Hm lng 10-DAB III v taxol trong nguyn liu th
Kt qu kho st hm lng 10-DAB III v taxol theo thi gian trch ly c trnh by hnh 3.1,
hnh 3.2.

Hnh 3.1. Hm lng 10-DAB trong nguyn liu th

Hnh 3.2. Hm lng taxol trong nguyn liu th

Khi trch ly 10-DAB III v taxol bng phng php Soxhlet vi dung mi methanol, t ngy th 2
tr i, hm lng 10-DAB III v taxol khng thay i nhng khi trch vi ethanol th n ngy th 3,
hm lng 10-DAB III v taxol khng thay i. p dng phng php Soxhlet hu nh trch c
hm lng 10-DAB III v taxol thp hn so vi phng php ngm dm. i vi 10-DAB III, hm
lng t cao nht khi trch vi methanol; 1,56741 mg/g nguyn liu. i vi taxol, hm lng t
cao nht khi trch vi ethanol; 0,26317 mg/g nguyn liu.

194

Hi tho Quc t

Bng 3.1. Hm lng 10-DAB III khi kho st trn quy m pilot theo thi gian v phng php chun b nguyn liu
Tn
mu

Khi
lng
cao (g)

Th tch
bnh nh
mc (l)

H s
pha
long

Nng 10DAB III (ppm)

Hm
lng
(mg/g)

Hm
lng
(%)

chn lc
(%)

1 gi

15.36

0.1

424.54600

42.45460

0.01141

0.00114

0.27640

2 gi

8.38

0.5

10

346.28600

173.14300

0.04654

0.00465

0.72933

3 gi

6.69

0.5

10

143.66900

71.83450

0.01931

0.00193

0.23606

4 gi

1.54

5 gi

2.13

0.5

10

85.93600

42.96800

0.01155

0.00116

0.13440

0.5

10

12.13850

6.06925

0.00163

0.00016

0.01780

6 gi

3.77

0.5

10

17.54425

8.77213

0.00236

0.00024

0.02316

1 gi

82.27

20

1101.10800

1101.10800

0.29600

0.02960

1.33841

2 gi

109.96

20

1132.35600

1132.35600

0.30440

0.03044

0.58906

3 gi

13.05

0.5

20

212.43200

106.21600

0.02855

0.00286

0.05174

4 gi

18.27

0.5

20

78.16100

39.08050

0.01051

0.00105

0.01748

Khi lng
(mg)

SFE vi nguyn liu th

SFE vi nguyn liu c ngm trong cn

SFE + ng dung mi
1 gi

47.51

20

364.55600

364.55600

0.09800

0.00980

0.76732

2 gi

37.44

0.5

20

2735.83400

1367.91700

0.36772

0.03677

1.61026

3 gi

19.07

20

794.16200

794.16200

0.21348

0.02135

0.76347

4 gi

18.36

0.5

20

1820.59400

910.29700

0.24470

0.02447

0.74383

SFE + ng dung mi vi nguyn liu c ngm trong cn

1 gi

107.36

0.5

10

496.05675

2654.87000

0.71367

0.07137

2.47287

2 gi

45.4

20

1280.40400

1280.40400

0.34419

0.03442

0.83818

3 gi

18.32

0.5

20

1101.82400

550.91200

0.14809

0.01481

0.32202

4 gi

8.83

0.3

50

931.04500

279.31350

0.07508

0.00751

0.15525

1 gi

111.87

2 gi

SFE + ng dung mi vi nguyn liu c ngm trong cn v CO2

0.5

20

2265.76000

1132.88000

0.30454

0.03045

1.01268

63.52

0.5

20

1965.01400

982.50700

0.26411

0.02641

0.56018

3 gi

18.84

0.5

20

1833.16000

916.58000

0.24639

0.02464

0.47190

4 gi

11.2

0.5

20

878.35400

439.17700

0.11806

0.01181

0.21378

Hnh 3.4. Hm lng taxol kho st theo thi gian ly mu v phng php chun b nguyn liu quy m pilot

Hp tc khoa hc cng ngh v s pht trin bn vng nng nghip Lm ng - Ty Nguyn 2014

195

Bng 3.2. Hm lng taxol khi kho st trn quy m pilot theo thi gian v phng php chun b nguyn liu
Nng
Khi lng
thc
(mg)
(ppm)
SFE vi nguyn liu th

Hm
lng
(mg/g)

Hm lng
(%)

chn lc
(%)

0.1

82.44800

8.24480

0.00222

0.00022

0.05368

0.5

38.33000

19.16500

0.00515

0.00052

0.08073

0.5

18.86200

9.43100

0.00254

0.00025

0.03099

1.54

0.5

5.30275

2.65138

0.00071

0.00007

0.00829

5 gi

2.13

0.5

6.07775

3.03888

0.00082

0.00008

0.00891

6 gi

3.77

0.5

10.16950

5.08475

0.00137

0.00014

0.01343

Tn
mu

Khi lng
cao (g)

1 gi

15.36

2 gi

8.38

3 gi

6.69

4 gi

Th tch
bnh nh
mc (L)

SFE vi nguyn liu c ngm trong cn

1 gi

82.27

209.84000

209.84000

0.05641

0.00564

0.25506

2 gi

109.96

151.20800

151.20800

0.04065

0.00406

0.07866

3 gi

13.05

0.5

45.67700

22.83850

0.00614

0.00061

0.01113

4 gi

18.27

0.5

16.09800

8.04900

0.00216

0.00022

0.00360

SFE + ng dung mi
1 gi

47.51

52.59000

52.59000

0.01414

0.00141

0.11069

2 gi

37.44

0.5

229.76200

114.88100

0.03088

0.00309

0.13523

3 gi

19.07

30.01400

30.01400

0.00807

0.00081

0.02885

4 gi

18.36

0.5

139.06800

69.53400

0.01869

0.00187

0.05682

SFE + ng dung mi vi nguyn liu c ngm trong cn

1 gi

107.36

0.5

390.90950

195.45475

0.05254

0.00525

0.18206

2 gi

45.4

108.53800

108.53800

0.02918

0.00292

0.07105

3 gi

18.32

0.5

168.61400

84.30700

0.02266

0.00227

0.04928

4 gi

8.83

0.3

0.00000

0.00000

0.00000

0.00000

0.00000

SFE + ng dung mi vi nguyn liu c ngm trong cn v CO2

1 gi

111.87

0.5

293.64600

146.82300

0.03947

0.00395

0.13124

2 gi

63.52

0.5

248.88600

124.44300

0.03345

0.00335

0.07095

3 gi

18.84

0.5

84.56400

42.28200

0.01137

0.00114

0.02177

4 gi

11.2

0.5

67.39200

33.69600

0.00906

0.00091

0.01640

IV. Kt lun
T cc kt qu thc nghim cho thy:
Bng 3.3. Hm lng 10-DAB III theo thi gian trch ly v phng php chun b nguyn liu
Hm lng 10-DAB III
(mg/g nguyn liu)

Hm lng taxol
(mg/g nguyn liu)

SFE vi nguyn liu th

0,09281

0,01280

SFE vi nguyn liu c ngm trong cn

0,63945

0,10536

SFE + ng dung mi

0,92391

0,07178

SFE + ng dung mi vi nguyn liu c ngm trong cn

1,28105

0,10438

SFE + ng dung mi vi nguyn liu c ngm trong cn v CO2

0,93310

0,09335

Tn mu

4.1. Chit tch theo phng php ngm kit

- Thit b n gin, gi thanh r v d dng nng cp quy m cng nghip. Hiu sut thu nhn
cc taxoid kh cao: 10-DAB (0,072%), taxol t nhin (0,0014%) so vi hm lng c trong nguyn
liu t 0,1-0,2% (10-DAB) v 0,004% (taxol).
- S dng nguyn liu ha cht r tin, c th ti s dng v thn thin mi trng.
4.2. Chit tch theo phng php CO2 siu ti hn
- quy m PTN, hm lng 10-DAB III v taxol ln lt l 0,25109 mg/g v 0,09872 mg/g vi
iu kin: p sut 300 bar, tc dng 20 g/pht, nhit 60oC v khng c ng dung mi ethanol.
- quy m pilot, hm lng 10-DAB III v taxol ln lt l 0,09281 mg/g v 0,0128 mg/g vi iu
kin: p sut 295 bar, tc dng 3,13 l/pht, nhit 60oC v khng c ng dung mi ethanol.
Nhng khi nguyn liu c ngm tm trc vi ethanol hay b sung thm ng dung mi ethanol,

196

Hi tho Quc t

Hnh 3.5. Hm lng 10-DAB III v taxol quy m pilot

hm lng 10-DAB III v taxol tng ln r rt. C th, khi trch ly c b sung thm ng dung
mi ethanol vi nguyn liu c ngm trc trong ethanol, hm lng hot cht t cao nht;
1,28105 mg/g nguyn liu i vi 10-DAB III v 0,10536 mg/g nguyn liu i vi taxol.
- Cc iu kin trch ly t c hiu qu cao quy m pilot ln lt l: p sut 295 bar, tc
dng CO2 3,13 l/pht, tc dng ng dung mi ethanol 0,25 l/pht, nhit trch ly 60oC, 4 kg
nguyn liu c ngm trc trong ethanol v thi gian trch ly 4 gi.
TI LIU THAM KHO
Nguyn Hong Qunh Hng, Nguyn Ngc Song Trm, Bi Th Vinh, Trn Cng Lun, 2008 - Xy dng quy trnh
nh lng 10-DAB v taxol trong l Thng (Taxus wallichiana Zucc.). Tp ch Y hc Tp. HCM, tp 12, ph bn ca
s 4, tr. 105-111.
Trn Cng Lun, Vng Ch Hng, Ng Thin T Khanh, Phan vn (2008). Nghin cu c im vi hc v thnh
phn ha hc ca l thng (Taxus wallichiana Zucc.). Tp ch Y hc Tp.HCM.2008, 12 (4), tr 98-104.
Bi Th Vinh, Trn Cng Lun, Vng Ch Hng, Nguyn Tin Hng. Studies on the Dynamic Variation of 10DAB and Taxol Contents of Taxus wallichiana needles Cultivated in Lm ng province. Pharma Indochina VI. The
development of Indochina Pharmacy in the Context of Global Economic Recession. 15 - 18/12/2009, tr. 621-624.
Arturo B., Ezio B., Paolo D., Andrea G., Alessandro P.. A process for the preparation of paclitaxel. Patentscope,
WO/2002/04617, (2002).
Hideji Itokawa and Kuo-Hsiung Lee, (2003). Taxus, The genus Taxus. First published 2003 by Taylor & Francis, 11
New Fetter Lane, London EC4P 4EE, Simultaneously published in the USA and Canada.
Madhusudanan K.P., Chattopadhyay S.K., Tripathi V.K., Sashidhara K.V., Kumar S. (2002), MS/MS Profiling of
Taxoids from the Needles of Taxus wallichiana, Phytochemical analysis, 13, page 18-30.
Madhusudanan K.P., Chattopadhyay S.K., Tripathi V.K., Shashidhara K.V., Kukreja A.K., Jain S.P. (2002), LC-ESIMS analysis of taxoids from the bark of Taxus wallichiana, Biomedical chromatography, 16, page 343-355.
Mclaughlin J. L., Miller R. W., Powell R. G., Smith C. R.. 19-hydroxybaccatin III, 10-deacetylcephalomannine, and
10-deacetyltaxol: New antitumor taxanes from Taxus wallichiana. Journal of Natural Products, 44 (3), pp. 312-319, (1981).
Tomasz Mroczek, Kazimierz Glowniak. Solid-phase extraction and simplified high-performance liquid chromatographic
determination of 10-deacetylbaccatin III and related taxoids in yew species. Journal of Pharmaceutical and Biomedical
Analysis 26, 89-102, (2001).