TP CH KHOA HC V CNG NGH, I HC NNG - S 2(43).

2011

NGHIN CU CHIT TCH ALKALOID CA R CY DA CN HOA HNG TI BNH NH

A STUDY OF ALKALOID EXTRACTED CATHARANTHUS ROSEUS ROOTS OF ROSES IN BINH DINH PROVINCE o Hng Cng, L Xun Vn
Trng i hc S phm, i hc Nng
TM TT Alkaloid c chit tch ca r cy da cn hoa hng, bng phng php soxhlet vi 0 dung mi chloroform (CHCl3) thi gian khong 10h, nhit khong 65 C, dch chit sau khi o ph cho thy c cc cu t alkaloid chnh (vincristine, vinblastine, ajmalicine v alstonine), trong vinblastine c hm lng ln nht 53,29%. Trong cc cu t tm thy th 2 cu t vincristine v vinblastine, theo cc ti liu tham kho trn th gii, chng ng vai tr quan trng trong vic iu tr ung th, trong mu dch chit c pht hin cn c 2 cu t ajmalicine v serpentine, theo khoa hc chng c tc dng cha cao huyt p. ABSTRACT Alkaloid extracted from Catharanthus roseus roots of roses, by means of soxhlet with a solvent chloroform (CHCl3) in about 10 hours time, at the temperature around 65 degrees Centigrade showed that after spectrometry it contained the major alkaloid component (vincristine, vinblastine, ajmalicine and alstonine), of which vinblastine had the largest concentration of 53.29 percent. In these components, vincristine and vinblastine were found. According to some worlds reference materials, they have played an important role in the treatment of cancer. In addition, two components were also detected in the sample extract: ajmalicine and serpentine. According to scientists, they can be used to treat hypertension.

1. M u Cy da cn cn gi l trng xun, hoa hi ng, bng da. Tn khoa hc Catharanthus roseus L. G. Don, thuc h Trc o Apocynaceae. Cy nh, cao 0,4 0,8m, c b r rt pht trin, thn g pha gc, mm phn trn, mc thnh bi dy, cnh thng ng. L mc i, thun di, u l hi nhn, pha cung hp nhn, di 3 - 8cm, rng 1 2,5cm. Hoa trng hoc hng, mi thm, nm cc k l pha trn, di 2 - 4cm, rng 2 3mm. Da cn pht trin tt v c quanh nm Bnh nh [1].

Hnh 1. Cy da cn hoa hng 85

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Da cn l loi thc vt t lu c dn gian xem nh mt trong nhng thn dc bi nhng cng dng m n mang li trong vic cha bnh, cng nh nng cao sc khe ca con ngi nh: ty giun, cha st, c tnh cht sn da (astringent), lc mu (dpuratif), dng cha mt s bnh ngoi da Trong cy da cn ngi ta pht hin ra hai alkaloid ng vai tr quan trng trong iu tr ung th, l vincristine v vinblastine [2].

Vincristine (vcr)

Vinblastine (vlb)

lm r hn thnh phn ha hc ca cy da cn hoa hng, chng ti tin hnh nghin cu chit tch alkaloid c trong r loi cy ny ti Bnh nh. 2. Thc nghim v phng php 2.1. Nguyn liu R cy da cn hoa hng thu hoch ti cc x ven bin thuc huyn Ph Ct, tnh Bnh nh. R da cn rt pht trin, thng ch c mt r ci v chm r ph di t. R ci m thng xung t, c th t chiu di 35 - 40cm, r ph mc thnh chm tha ngn, pht trin theo chiu ngang, vng v r l ni tch ly ch yu cc alkaloid (hnh 2).

Hnh 2. R v bt r cy da cn hoa hng

2.2. Phng php * Chit tch Alakaloid: R cy da cn hoa hng sau khi c thu hoch, em ra sch, cht nh, phi kh t nhin n m thch hp (9,13%), cui cng em xay mn thnh bt. Dng khong 10,23 (g) bt r da cn em loi tp (sc t, nha, tecpen, chlorophyll), sau tm bt da cn va loi tp vi dung dch amoniac 25% trong thi gian t 1h 2h. Chit soxhlet bt da cn sau khi tm dung dch amoniac, vi dung mi chloroform nhit khong 60 - 700C, thi gian chit khong 10h. Thu c dch chit c mu vng nu lm khan bng Na2SO4 khan [3].
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* Phn tch thnh phn ha hc: xc nh thnh phn ha hc v nh danh cc cu t c trong dch chit r cy da cn, chng ti s dng phng php sc k lng cao p - khi ph lin hp (LC MS), da trn thit b phn tch He HPLC/MS (Finigan Thermoquest): Bm cao ap 4 dong P4000, bo tiem mau t ong AS3000, au do MS Ion trap LCQ cua Thermo Finnigan ti phng Phn tch sc k Trung tm dch v PTTN CASE, qun 1 - TP H Ch Minh. 3. Kt qu v tho lun 3.1 Thnh phn ha hc trong dch chit alkaloid ca r cy da cn hoa hng Dch chit thu c sau khi x l, chng ti tin hnh o ph sc k lng hiu nng cao (HPLC). Kt qu c th hin hnh 3.

Hnh 3. Ph HPLC ca r cy da cn hoa hng

Sau khi chy ph HPLC (hnh 3), chng ti kt hp vi khi ph lin hp (MS) xc nh thnh phn ha hc cc cht c trong dch chit ca r cy da cn hoa hng. Kt qu c th hin hnh 4 v hnh 5.

Hnh 4. Ph LC/MS ca r cy da cn hoa hng 87

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Hnh 5. Ph LC/MS ca r cy da cn hoa hng

Trn sc k hnh 4 v 5, sau khi qua h thng LC/MS, trong khong khi lng 1001000, vi thi gian lu (RT) t 925,01; xc nh c 2 cht (Vinblastine v Vincristine) vi thi gian lu l 10,62; 10,82 pht (hnh 4). Ngoi ra, ti 2 peak vi thi gian lu l 10,09 v 10,29 pht, mi peak ny c hai cu t ging nhau v cng thc phn t nhng khc nhau v cu to (hnh 5). Bng phn mm Chemsketch, chng ti phn tch m/z cho tng peak c th nh sau: * Kt qu phn tch ti thi gian lu 10,09 pht. Trn sc k hnh 5, tng ng vi peak c m/z = 353,4 cn c cc peak tng i ln c khi lng 321,5; 222,3; 144,3. Trong peak 353,4 chnh l ion phn t hay peak gc [M+] c nhn din l ca mt trong hai cht Rhazimol v Ajmalicine. Kt qu s phn mnh c th hin hnh 6 v 7.
(1) H 2C (2) N (2) N H Ajmalicine Formula Weight H
3C

H N O (1) N H
+

CH O

+ 2

(1) O CH
3

Formula Weight

[A]

HC = 321.3923914 O H N
2+

= 352.42686 HN CH
+ 2

(2)

+
++

Formula Weight

[A

= 222.2839628

N Formula Weight
[B]
+

H = 144.1925514

Hnh 6. S phn mnh ca Ajmalicine c m/z 352.4 c m/z 352.4

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HOH2C HOH2C (3) CO2CH3 (4) (3) N N N (4) (3) CH3 Rhazimol (deacetylakuammiline) Formula Weight = 352.42686 N N (4) N Formula Weight = 280.3642

CH2 Formula Weight = 238.32752

Hnh 7. S phn mnh ca Ajmalicine c m/z 352.4

Peak 354,4 nm bn cnh peak gc, ng thi c khi lng bng [M+ 1]+ chnh l ion ng v ca [M+]. Cc speak ng lu nht l 321,5 v 144,3, chnh l cc ion mnh ch yu do s bn ph ca ion phn t c my ghi li. Da vo cng thc cu to ca 2 cht cng vi s h tr ca phn mm Chemsketch, chng ti cho rng c ch phn mnh nh hnh 6 v 7. Vi ph khi hnh 5 v s phn mnh hnh 6 v 7, cho thy s phn mnh m/z 352,4 c th theo 4 hng: + Hng (1): [M]+ (352,42686) [A]+ (321,3923914) [A]+ (321,3923914) [A1]++ (222,2839628) + Hng (2): [M]+ (352,42686) [B]+ (144,1925514) + Hng (3) v (4), mc d cng c s phn mnh nhng cc ion phn mnh li khng nm trong cc peak ca ph LC/MS. T nhng lp lun trn cng nh kt qu nhn c trn ph , chng ti xc nh c s xut hin ca cc mnh ion c m/z 144,2 v 321,4 do s ph v [M]+, cn ion [M+1]+ m/z 354,4 l ng v ca [M]+ m/z 353,4. Nh vy hp cht ti thi gian lu 10,09 pht chnh l Ajmalicine [5]. * Kt qu phn tch ti thi gian lu 10,29 pht. Trn sc k hnh 5, tng ng vi peak c m/z = 349,5 xut hin cc peak tng i ln c khi lng 335,4; 317,4; 295,3; 289,3. Trong peak 349,5 chnh l ion phn t hay peak gc [M+] c nhn din l ca mt trong hai cht Serpentine v Alstonine. Kt qu s phn mnh c th hin hnh 8 v 9.

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H 3C CH3 O (7) C O H N (1) N O O (1) H 3C N
+

O O=C

HN

+ +

Formula Weight[A] CH3 CH O N

= 335.3759114

Alstonine + Formula Weight [M] = 349.4024914 O + (2) C H N CH3 O N H 2C

+ + +

O N

Formula Weight
+

[A1]+

= 317.3606314

Formula Weight [B]

= 295.3540142

O N H 2C Formula Weight = 289.3505314

+

[A2]

Hnh 8. S phn mnh ca Alstonine c m/z 349,4

H N O CH3 (3) (4) O (4) NH Serpentine Formula Weight (5) N N H Formula Weight[E]
+

(3)

+ +

Formula Weight [C] (4)

= 277.3398314
+

N N H Formula Weight [D]

(3) O H 3C
+

CH3 = 235.3031514

= 349.4024914

= 223.2924514

N H + Formula Weight [D 1 ] = 220.2686314

Hnh 9. S phn mnh ca Serpentine c m/z 349,4

Peak 350,4 nm bn cnh peak gc, ng thi c khi lng bng [M+ 1]+ chnh l ion ng v ca [M+]. Cc speak ng lu nht l 335,4; 317,4 v 289,3; chnh l cc ion mnh ch yu do s bn ph ca ion phn t c my ghi li. Da vo cng thc cu to ca 2 cht cng vi s h tr ca phn mm Chemsketch, chng ti cho rng c ch phn mnh nh hnh 8 v 9. Vi ph khi hnh 5 v s phn mnh hnh 8 v 9, cho thy s phn mnh m/z 349,5 ca Serpentine v Alstonine nh sau: + Hng (1): [M+] (349,4024914) [A]+ (335,3759114) [A]+ (335,3759114) [A1]+ (317,3606314)

[A1]+ (317,3606314) [A2]+ (289,3505314)

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+ Hng (2): [M+] (349,4024914)

[B]+ (295,3540142)

Cc hng (3), (4) v (5) mc d cng c s phn mnh nhng cc ion phn mnh li khng nm trong cc peak ca ph LC/MS. Da vo nhng lp lun cng nh kt qu nhn c trn ph , chng ti xc nh s xut hin ca cc mnh ion c m/z 335,4; 317,4; 289,3 l do s ph v [M]+, cn ion m/z 350,4 chnh l ng v [M+1]+ ca [M]+ m/z 349,5. Nh vy hp cht ti thi gian lu 10,29 pht chnh l Alstonine [5]. Nh vy, cc thi gian lu 10,09 v 10,29 pht xc nh c 2 alkaloid l Ajmalicine v Alstonine, iu ny ph hp vi cc ti liu tham kho [2] [5]. T nhng kt qu xc lp, chng ti xc nh c trong dch chit ca r cy da cn hoa hng c 4 alkaloid (vinbalstine, vincistine, ajmalicine v alstonine) [5], trong c hai alkaloid quan trng l vinbalstine v vincistine. Ta tnh c hm lng ca 2 alkaloid theo total ion: %vlb =
311703752 x100% 584909354

= 53,29% ; %vcr =

73396665 x100% 584909354

= 12,55%

Hm lng alkaloid ca vinblastine (vlb) v vincrictine (vcr) c trong r da cn hoa hng (so vi hm lng alkaloid trong r da cn hoa hng) l 65,84 %, hm lng ca 2 alkaloid ny chim t l ln nht, ph hp vi cc ti liu tham kho [4]. 4. Kt lun Nhm tc gi nghin cu thnh cng qui trnh chit tch alkaloid trong r cy da cn hoa hng ti Bnh nh vi dung mi chloroform (CHCl3) vi mt s kt qu sau: Bng phng php chit Soxhlet nhit khong 650C, thi gian chit khong 10h th t hiu sut cao nht vi hm lng alkaloid ca vlb v vcr l 65,84 %. Bng phng php sc k lng ghp khi ph (LC MS) xc nh c hai alkaloid quan trng l vinblastine v vincristine, ngoi ra da vo phn mm Chemsketch cng xc nh thm c 2 alkaloid na, l ajmalicine v alstonine. TI LIU THAM KHO [1] Tt Li (1991), Nhng cy thuc v v thuc Vit Nam, Nh xut bn Y hc H Ni, tr. 307-309. [2] GS. Nguyn Vn n, DS. Nguyn Vit Tu (1985), Phng php nghin cu ha hc cy thuc, Nh xut bn Y hc. [3] Nguyn Vn Hnh (2002), Gio trnh cao hc, Tch chit v c lp hp cht t nhin, Nh xut bn i hc Quc gia Tp. H Ch Minh.
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[4] Jean-Hugues Renault, Jean-Marc Nuzillard, Galle Le Crourour, Phillippe Thpenier, Monique Zches Hanrot and Louisette Le Men Oliveir (14 July 1999), Isolation of indole alkaloids from Catharanthus roseus by centrifugal partition chromatography in the pH-zone refining mode, Laboratoire de Pharmacognosie, Moulin de la House, 51097 Reims Cedex, France. [5] Irshad Ali (1985), Isolation and structural on the alkaloids of catharanthus roseus, thesis submitted for the fulfilment of the degree of philosophy, H.E.J. research institute of chemistry university of Karachi.

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