Page 1

IH

NG IH

M

TR N TH

NGHI N C U PH N L P CH T TINH KHI T TRONG D CH CHI T N HEXAN C A V C Y HOA S A TR N A B N TH NH PH N NG

N T T NGHI P C

C

N NG, 2019


IH

NG IH

IH

M

NG IH

M

N T T NGHI P

TR N TH

NGHI N C U PH N L P CH T TINH KHI T TRONG D CH CHI T N HEXAN C A V C Y HOA S A TR N A B N TH NH PH N NG

NGHI N C U PH N L P CH T TINH KHI T TRONG D CH CHI T N HEXAN C A V C Y HOA S A TR N A B N TH NH PH N NG

c hi n: Tr n Th c

N T T NGHI P C

C

MSSV L p

: 314033151102 : 15CHD1 ng d

ng

N NG, 2019 N NG, 2019


L IC n t t nghi C

ng

c p ki n th c

n

c cu i t

ih

c Th y

kinh nghi

i

, cung nt t

ng d

m.

tc

y d em trong b n n th

C

ng d

em trong su t th i gian th c hi

nghi p. Em th

C

t nhi

n t t nghi p c

Nguy n Th

c nh

, truy n th nh ng u th c hi

ii


M CL C M U ..................................................................................................................... 1 ............................................................................................................. 1 1. 2. M u ....................................................................................................... 1 3. Nhi m v u...................................................................................................... 1 u................................................................................. 1 .............................................................................................................1 ...................................................................2 ................................................................................2 ........................................................................2 ..............................................................................................2 u................................................................................................ 2 ............................................................................................2 ...................................................................................2 ............................................................................................................... 3 6. B c c lu NG QUAN ....................................................................................... 4 1.1. Gi i thi u v a............................................................................................ 4 .............................................................................................4 ...............................................................................................4 ..........................................................................5 u Vi gi i v .............................. 5 u Vi t Nam ....................................................................5 ....................................................................6 1.3. Ancaloit ....................................................................................................................... 11 ........................................................................................................11 .............................................................. 11 1.3.3 ...........................................................................11 1.3.4 ...........................................................................................12 U ................... 15 tb m .......................................................... 15 .....................................................................................................15 .......................................................................15 2.2 u .......................................................................................... 16 2.2 .......................................................16 .....................................................................18 ........................18 ...........................................................................21 ................................................................ 23

iii

.....................................27 u th c nghi m ......................................................................... 27 .................................................................27 ......................................................28 K T QU O LU N ........................................................... 29 3.1. nh m t s ch s v ................................................................................... 29 3.1.1. ..............................................................................................................29 .................................................................................................29 ........................................................................................30 nh th i gian chi t ............................................................................................ 31 p ch t trong v a ........................................................... 31 nh m t s c trong d ch chi t n - hexan b p GC - MS .................................................................................................................... 33 ng cao chi t so v i m ...................................................................... 36 nh c p ch c ASVH10 .................................................... 36 ............................... 36 ......................................................39 K T LU N NGH .................................................................................. 46 K ............................................................................................................................... 46 ............................................................................................................................ 46

U THAM KH O......................................................................................... 47 PH L C .................................................................................................................. 49

iv


p ch

ASVH10: 13

C-NMR: Ph c

c 13

ng t h

C - Carbon Nuclear Magnetic Resonance

DEPT: Distortionless Enhancement by Polarisation Transfer kh

n t

- Electron Spray Ionization Mass

Spectroscopy FT-IR: Ph h ng ngo i bi GC-MS: S 1

H-NMR: Ph c

i Fourier - Fourier Transform Infrared Spectroscopy i ph - Gas Chromatography - Mass Spectrometry 1

ng t h

H - Proton Nuclear Magnetic Resonance

Spectroscopy J

H ng s

Rf: Th

NG

S hi u

Spectroscopy

ESI-MS: Ph

DANH M

VI T T T

DANH M

d ch chuy

c (ppm)

ng

b ng 1.1.

d

2.1.

S

3.1.

K t qu kh

3.2.

K t qu kh

3.3.

3.5.

K t qu

3.6.

M ts

3.10. 3.11.

v

u

29

ng tro trong b

ng m t s kim lo i trong b K t qu kh

3.9.

26 mc ab

i gian chi t v

u

30

u

30

-hexan

p ch

31 a

c trong d ch chi t n-hexane ng

36

h ng ngo i (FT - IR) c a ch t ASVH10 D li u ph

1

D li u ph

13

37

H - NMR c a ch t ASVH10

37

C - NMR, DEPT c a ch t ASVH10

li u ph NMR c a h p ch t ASVH10 v

vi

31 33

t so v i m

3.8.

- Retention factor

15

n m ng V8

3.4.

3.7.

Trang

38 - amyrin

39


U

M 1. DANH M

Trong th i gian qua, nh ng h p ch t t

S hi u

c ng d ng r

Trang

l p t th c v t

t nhi

p,...Vi c ph

1.1.

a

5

2.1.

15

2.2.

M u g n silicagel

2.3.

C ts

2.4.

M u r n ASVH

26

2.5.

S

27

kh

m ts y ut u ch

3.1.

S

3.2.

Ch t ASVH10

3.5.

p ch t tinh khi t

28

GC-MS d ch chi t n-hexane c a v

a

Ph h ng ngo i (FT - IR) c a ch t ASVH1 Ph

1

H - NMR (CDCl3, 125 MHz) c a h p ch t ASVH10

Ph

1

H - NMR (CDCl3, 125 MHz) c a h p ch t ASVH10 0.7 - 1.7)

3.6.

Ph

3.7.

Ph

13

C - NMR (CDCl3, 125 MHz) c a h p ch t ASVH10

Ph

13

C - NMR (CDCl3, 125 MHz) c a h p ch t ASVH10

Ph

13

3.9.

t o ra ch t m ch a b

c cao

c bi

H - NMR (CDCl3, 125 MHz) c a h p ch t ASVH10

0 - 50) C - NMR (CDCl3, 125 MHz) c a h p ch t ASVH10 50 - 145)

3.10.

Ph DEPT c a h p ch t ASVH10

3.11.

Ph DEPT c a h p ch

3.12.

C

s

nh phong, b qu

a, kh

n, c n [18].

s d ng ch ng l i b n nay

nv

37

V i nh

t, kh

ng d ng c a h p ch t a

41

tinh khi t trong d ch chi t n - hexan c a v

a.

nh ch

nh

u

p ch t

a khoa h

tr s d ng c a v

hoa s a trong y h c.

41

2. M

42

-

u nh c

t s h p ch t ancaloit quan tr ng trong v

p ch t tinh khi t trong d ch chi t n - hexan c a v nh c

a h p ch t

3. Nhi m v

44

-

10 - 55 )

44

-

a h p ch t ASVH10

45

-V c

a.

c.

tv nh c

hexan. a m t s h p ch

cc av

p ch t tinh khi t trong d ch chi t n - hexan c a v

-

4

a.

u

-

u

4

vii

n 40 m. V

h y, ki t l

ph

43

c

t lo i thu c b , h s t, ch a

36

43

g mu

nh nan y [3].

c s d ng ch y u trong y h

33

41

u

t chi

27

1

3.8.

u

25

n m ng VH4

2.7.

3.4.

24 pm u

2.6.

3.3.

h c, ho t

ng at i

ih c

m

1

ng.

a. a.


- Chi t b ;

u

4.2 4.2

tv i

-

t, d ng c t: n - hexan, axit axetic, thu c th Mayer,

p ch

tk

i,

i ph

i, buret, c c th y

-

s

p ch t b

i ph GC s

y, mao qu n s ch, b n m

c a ch

ng c

tb -

Lu

.

H - NMR, 13C - NMR, DEPT.

m 48 trang, 13 b ng, 20

Ph n m

-VIS.

-

-

c

u (3 trang)

:N

.

-

- MS.

t chi u 1H - NMR, 13C - NMR, DEPT.

ng t h

u (14 Trang)

: K t qu K t lu T

4.3

o lu n (17 Trang)

n ngh (1 Trang) u tham kh o (2 Trang)

Ph l c (4 trang) -

ut p m t s h p ch

-

ng. u cao chi t t v

nh c

p ch

5.

a.

c.

u th c hi

d

u sau:

t

5.1. - Thu th p, t ng h ; - H i th 5.2.

i kinh nghi m v c nghi m

-

li u tham kh o

: T ng quan (11 Trang)

.

-

nh, 18

ph l c. V i:

.

-

-

c: k t h

6. B c c lu

.

-

p m ng;

h ng ngo i (FT - IR), ph kh i (MS), ph 1

4.2

MS;

tk th pv is

th y tinh c r

a

t b

c c t, NH3

(LC - MS), ph h ng ngo i IR D ng c :

ch chi t t v

mb

ng;

-

ng tro b

-

ng kim lo i b

u; h p th

2

;

3


T NG QUAN 1.1. Gi i thi u v

a

1.1.1. a)

i -

ng g i - C

-

, tinpet, popeal -

.

c - Alstonia scholaris L. R. Br (

i - Echites scholaris L.

Mant., Pala scholaris L. Roberty). b)

1. 1.1.3.

ng c a v

i khoa h c - Gi i:

a u tr s

Plantae;

(amin ngu n g c t

- B

: Gentianales (

m);

- H

: Apocynaceae (La b ma);

: V c v t t

echitamine. Nh ng ch khu

a

cs d

t lo i thu

u tr

- Chi : Alstonia (Hoa s a); -

-

Aspergillus niger.

: A. Scholaris.

c) am

c tr ng d

Hoa (n

ng ph

l

c. V .

t nh

ng ct

c sau:

Trung Qu c: Qu Ti u l

(nam);

a

:

; Nepal; Sri Lanka;

: Campuchia; Myanma;

; Vi t Nam, Indonesia;

Malaysia; Papua New Guinea; Philippines; : Queensland.

1.2.

1.1.2.

cv t

(hi m g

-

u

Vi u

gi i v

Vi t Nam 1

u r ng t

n 20 cm n 5 cm. M

vi

n 3 cm. Cu y u gi to, cao t

(H

n

picrinin (1),vallesamin (2

3

.

n 40m. V

uv tn tn .H

mao

g

n

,

ong khi

a m .

.1).

1. Picrinin 4

2. Vallesamin 5

3. Scholaricin


Nguy n Th Thanh Huy

ng s

c trong v nh ph

u chi

at

cc av

GC - MS,

m cetylpyridinium (4), ellipatamine (5), ditamine (6 ho

t [5] 12. Akuammicine N-oxit CH 3COOH 2C

COOCH 3

13. Nb-demethylalstogustin u c a Nilubon Jong-Anurakkun, Megh Raj

Bhandari, Jun Kawabata, Nh t B n, t

a

u

HO

c 3 h p ch t c ch a -

N N

H3C

quercetin 3 - O -

1 glycoside flavonoid:

- D - xylopyranosyl (1

2 )-

2 glucoside linan: (-) - lyoniresinol 3a - O H3C

4. Cetylpyridinium

5. Ellipatamine

(+) - lyoniresinol 3a - O -

6. Ditamine

- D - galactopyranoside (14) - D - glucopyranoside (15),

D - glucopyranoside (16

gi i N QLD4072, Australia , t d ch chi t v m i, akuammiginone (7 19 - O -

2 [13].

a The University of Queensland, Brisbane, p 1 alkaloid indole

alkaloid indole glycosidic m i, echitamidine - N - oxide

- D - glucopyranoside (8

i 5 alkaloid

t, axit echitaminic (9),

echitamidine N - oxit (10), Nb - demethylalstogustin N - oxit (11), akuammicine N - oxit (12

- demethylalstogustin (13),

p ch

14. Quercetin 3 - O -

t

- D - xylopyranosyl (1

-

- D - galactopyranoside

t t [7].

7. Akuammiginone

8. Echitamidine - N - oxide19 - O -

- D - glucopyranoside

15. (-) - lyoniresinol 3a - O -

- D - glucopyranoside

16. (+) - lyoniresinol 3a - O -

- D - glucopyranoside , Anil Kanaujia,

Dipankar Ghosh, Rajeev Duggar v 1 apioglucosides, formononetin 7 - O -

(17) - D - apiofuranosyl -

glucopyranoside (18); biochanin A 7 - O glucopyranoside (19) [15].

9. Axit echitaminic

6

7

- D - apiofuranosyl -

isoflavone -

- D -

-D-


17. Alstonoside

19. R = OH Tao Feng, Xiang - Hai Cai, Zhi - Zhi Dua

Xiao - Dong Luo i

(A - D1) (20 - 23

isoboonein (24), alyxialactone (25)

(26) [16].

Tao Feng, Xiang - Hai Cai, Pei - Ji Zhao, Zhi - Zhi Du, Wei - Qi Li, Xiao - Dong Luo 21 alkaloid indo monoterpenoid

27- 32 picrinine (1), leuconoxine (33),

5 - methoxylstrictamine (34), (Z) - 16 - formyl -

- methoxystrictamine (35),

cao chi t chloroform c a v

19 - epi - ajmalicine (36), akuammidine (37), picralinal (38), 19(E) - vallesamine (2), 20 - epi - 19 - oxodihydroakuammicine (39), echitamidine (40), 19 - epi - scholaricine (41), nareline (42), N(4) - demethylechitamine (43),

a,

Jaipur 3 - hydroxy adiantulanosterol (46) lupeol (48),

- amyrin acetate (49),

: lupeol acetate (47), - amyrin acetate (50) [8].

3 - epi - dihydrocorymine - 17-acetate (44), echitamine (45) [17].

46. 3-Hydroxy adiantulanosterol

8

u

As

9

47. Lupeol acetate


1.3. Ancaloit m

1.3.1.

Theo Polonopski: "Ancaloi n

ng h p ch t h

ng ki

ng g p trong th c v

ng v 49. -amyrin acetate 2017, t

48. Lupeol

m t s thu c th g i l

a,

1.3.2. M t s ancaloit trong v

cao chi t n - hexane c

u

Chao - Min Wang, Kuei - Lin Yeh, Shang - Jie Tsai, Yun Chang - Hung Chou

nt

- lup - 20 (29), - ene - triol (55), lupeol (48), - amyrin (57), poriferastero (58), epicampesterol (59

c th chung c

sterol

y

].

m

.

m

-sitosterol (60),

y

t ch t b

C

u.

Ditamine (hay Echitamine) (C22H28N2O4)

- hydroxy - 4 - stigmasten - 3 - one (61), ergosta - 7,22 - diene -

y

-triol (62) [9], [11].

cv i

t ch t b t

Ditaine (C16H19NO2

- amyrin (56),

n

a

Echitenine (C20H27NO4

c 8 triterpenoid

axit ursolic (51), axit oleanolic (52), axit betulinic (53), betulin (54),

6 -

cm

50. -amyrin acetate

t ch t b

206oC, d

Echitamidine (C20H26O3N2) l

ng, d k t

c, clorofom, etanol tb

ete. y

o

135 C. 1.3.3.

t chung c a ancaloit

a. - Tr th r n Axit ursolic, axit betulinic, betulin c ch ho tn

- lup - 20 (29) - ene - triol at

li

i NSCLC,

t o

nl

cc ut

nhi

Nh

ng

17H19NO3), codein (C18H21NO3

cc ut

ng

th l

(C8H17N), nicotin (C10H14N2). -

trong

ts

-

58), (59), (61), (62)

c: H u h

cay

m

60 -

tan: h u h t ancaloit

d C nh

Nh 3OH,

K ph

C2H5OH. M t s

tan m t

c ho c ki m.

b. .

-

u, do s ki m c

10

ng nhau do 11

t c a N ng l

sp3.


m d n theo th t amoni b c 4, amin b c 1, amin b c 2, amin b c 3. V axit m

uc

t

i trong dung d chuy

u v n t n t i trong dung d

N u chi

i.

4. D ch chi t clorofom, c t thu h

s pKb

ih n pKb c

ng v

ancaloit t

-

nh chi

ng.

- Ancaloit k t h p v i kim lo i n

ng axit c n thi

ch chi t CHCl3

o ra mu i ph c.

5

lo i t p (clorophyl, ch

v

1.3.4. Chi

A

l c dung d ch axit ancaloit u, CHCl3 m

n;

nh. iv t ancaloit di chuy

7. D ch chi t CHCl3

p

n t i d ng t h p v i tanin (nh

t

u tanin) t

u tanin, c n t m b

ancaloit

3

ng Na2SO4

ho

i

natri sunfat khan, b

t ch t ki m.

n c n.

ch m s

u ki

dung d ch CHCl3

d ng dung d

c hay h

c chi

m

c ancaloit.

: Chi t b ng dung d ch axit h

chuy n

c khi chi t. C

c b ng

n trong clorofom ho c metanol

t thu h

iv i

c li u b ng ki m m t th

u

nh chi t;

n cm

ng ete ho c

cho t t dung d ch NH3 10% ho c lo i ki

c y

m

axit ancaloit

6. Cho dung d

c l a ch

c li

chuy n ancaloit

c. N

s n m l i trong CHCl3;

n ng v i m t s thu c th chung.

a.

1/2 hay 1/3 th

c v i dung d ch axit h

h pc -

tl il n2t

l y ki t ancaloit;

id

c ng d ng trong vi

.

tb

y trong

ng axit y u m t s T

ng

c li

3. Ti

th pv

chi

i d ng mu

ng tphoric.

b. Chi ng lo i ancaloit c n chi

: Chi t b chi t ancaloit b t m dung d

u

ch tb

chuy n ancaloit mu

c li u ph

n. Ti p theo,

c li ki m

ch NH3, Ca(OH)2, Na2CO3, NaOH. ofom, ete, benzen, etyl clorua. 1. B

c li u t m dung d

Chi t b ng axit:

c

b

c li

c li

p.

hu d ch chi t

v i axit r i

n l i trong axit r i lo i t

d ch chi t

i chi t l i b

chi

3

: Thu d ch chi

- axit. R i cho b

p

.

cv

; 12

c li u

l c d ch chi

lo i t p. Ti p theo, ki m 13

u

a phenol.

Chi t b ng dung d m ki

i c nhi u t

ki

c (ho c dung d ch Na2CO3,

i

b ch m s

c ti

; 2. Cho b

a ch

m

dung d ch


c axit r i chi t b

h

. Cu

b

thu

cc

N

CH

U tb

2.1.

V

m t t t nh

ch chi t l n nhi u t p.

iv

thu t chi

u

-

ch chi

cho m m

m

n d ch chi

ng axit,

nhi

d ch chi t c

- 600

), r a s

cao, ancaloit d b

chi t v

- hexane.

c khi c t thu h i ancol. Dung d ch thu r i lo i t p.

Trong s n xu t hi

chi p nh

t t a dung d ch chi t b ng ki m. L c l y t a, ch axit, k t t a l i b ng ki m. L

l

ng th i chi t l y ancaloit.

k tt

ct

p l i nhi u

l

c hi n

o

nhi

C. K t t

a. V

s ti p t c k t t a trong th ki

at

c. Thu c th -

cl yt al ib

1.

c.

n ancaloit c th

M ts ng t o k t t a v i ancaloit :

+ Thu c th Dragendorff t o k t t + Thu c th Mayer t o k t t

ng ho

c th

ng t

i ancaloit :

+ Dung d ch H2SO4

c (D = 1,84 g/ml);

+ Dung d ch HNO3

c (D = 1,4 g/ml).

tb

u

ts d

th c hi

B v i ancaloit;

+ Thu c th Wagner (Bouchardat) t o k t t -

ch amoniac ho c

i ancaloit; i ancaloit.

M (g/mol ) tinh khi t tos ( 0C )

b ng 1.1.

t s d ng

Acetone CH3COCH3 58.04

Chloroform CHCl3 119.38

Ethyl acetate CH3COOC2H5 88.11

Methanol CH3OH 32.04

N-hexane C6H14 86.18

99.5%

99.0%

99.5%

99.5%

97.0%

56

61

77

65

69

Xu t x

Trung Qu c

d ng b n m

n silicagel Merck 60F254

0.2 mm. Silicagel c h t 197 - 400 mesh (0.040 - 0.063 mm). B chi t soxhlet, thi t b s ih

m-

ih

AAS100 Perkin Elmer (Trung t Th

h p th K

ng Th

C ts 14

c ph c t th y tinh (

Hu

K -

t h p kh i ph GC ph m, D

Qu c gia,

c Trung Trung B

Ki m nghi m thu c, M

-

p l c th y tinh frit) cao kho 15

ng ng).

Hu ).


trong 3cm, 4cm.

ngo

= 254 nm v

b n m

soi

m-

ih

ng. M t s thi t b

+ Ph kh i ESI Vi

ng t

500 MHz t i Vi

h

t:

u 4 - 6 ti

% tro = ng

500 - 550 C, trong th i gian t

n kh

n nhi

ng kh

ng m4.

m4 m1 100% m2

( 2.3)

5

Vi t Nam.

%tro 1

( 2.4)

5

nh ch m

m1: Kh m ti

y m u trong t s y

0

95 -110 C. Ti

nhi

mv i5m uv

trong kho ng

ch HNO3 nh m c b

cc

h p th

W (%)

ng kim lo i b ng

[1].

(2.2)

h p th

: khoa h

(g)

m2: Kh

ng v

ng tro b

t k t h p. M

cx

ch t h

hu hay th

c nung

2 O2 ,

500 - 550o

t do

ng t

KMnO4 ,

16

n gi i h n n

c ppb

h p th

tr

c a cx

ng h p th

nh lu t h p th

M C2 H 2 -

th

p, th c ph m,

thu t hi

ts

t c

ub

c, h n h p H2SO4 v i HNO3, HClO4

t,

(nanogam) v i sai s

u d

-

ch uh

ng nh

ng m u: qu

n ph

t li u...V i trang b n

Wtb

ng

t nhi

u sinh h c, y h cu

mc am im u

c

n. t s

u sau khi s y (g)

i m i

ng d ng r

thu t

a (g)

m3: Kh

b.

t k thu

n m nh m m1: Kh

ab

quang ph h p th

5

5

v ng m t s kim lo i trong v

c

1

u (g)

m4: Kh

(m m2 ) m3 W(%) = 1 100% ( 2.1) m2

WTB(%) =

(g)

m2: Kh

y k t qu

mc am im u

H2SO4

nhi

u

a. 0

o

c tro tr ng. L y m

Vi t Nam; i d

c, Vi

2.2

c

.S

im

c, Vi

2.2.

. Ti p t

c ghi b

h ng ngo i FT -

Shimadzu t i Vi

m

t

Vi t Nam;

+ Ph c

+ Ph

nh c

- MSD - Trap SL t i

h c, Vi

ng m3

s d

y Buchi melting Point B - 545 t ih

u (kh

- Beer.

n l a, v i h n h ng b

ng chu n. 17

an


- Pha thu c th

t m u th c v t

2.2.2.

d chi

t r n - l ng v

+ Thu c th

t

hi

ng:

Mayer

c c

2

c c t. Thu c th

m. dung d

hu t chi t soxhlet:

cc

m u d ch l c

b t v n - hexane, r i ti

t soxhlet

nhi t

th

m u d ch chi ng (m1, gam). S n

100 mL. Nh

d ch l c

n th

t thu c th ng ho

t.

2

c c

cc c

, ta

100 mL. Nh

alcaloid s xu t hi n k t t

+ Thu c th

i nh ng

Mayer g m h n h p hai

t thu c th

u

xu t hi n k t t

b. Flavonoid

c ch a d ch chi t (m2, gam) [6]. m6 = m5 - mc

V i m6

ng c a d ch chi t. D

kh

ng

a d ch chi t sau m i 2h chi

th i gian t

c nhi u ch t nh t v i m u t chi

ch

c

c

y d ch l c. Cho 1mL

d ch chi

ng nghi m r

dung d ch NaOH 10%; dung d ch H2SO4

t.

-

m:

p ch t t

ch chi t

c l c qua gi y l c, thu h ab

n khi chi t ki t m t s l p ch t trong v

2.2.3.

pd

;n

-

g m hai ph

c theo t l th

i trong 24 gi

th nh tho ng l c tr n. L

mL c l y ph n d ch. D ch l c

nhi

n c

nghi m v i ph n ng m

c c -

ng nghi m b ng nhau m i ng 1mL d ch chi t:

c H2SO4

, trong 1 gi . L c, l y d ch l

c

th nghi m

-

2 ng nghi

ngu ng dung d ch H2SO4

v i hai lo i thu c th Mayer, Wagner. 18

ng

ch NaOH 10%. ng

th nghi m v i hai lo i thu c th Mayer, Wagner. n (5

ng v i dung d ch FeCl3 5%: H p ch

,

m cho d tan. L c, l y d ch l

- Ph n 2: B t v

. N

c. Coumarin

h n h p g m chloroform:ethanol 95o:NH4OH

ph n c n trong dung d

t dung dich NaOH 1% n cam -

t xanh l

n (5

ng ph

ng v i dung d

a -

c th c

.

isoflavon, chalcon, isoflavanon, flavanon, leucoantocyanidin s

[6].

a. Alkaloid

- Ph n 1: B t v

c

ho c xanh

ng nghi m ch a d ch l c s

t. Ti p theo, ta

pt

t dung d ch H2SO4

c bi t; n

cm cv t

ng sau:

ch FeCl3 5%.

ng v i dung d ch H2SO4

nx px pb m tc ab

c

n

ng nghi m ch a d ch l c. N

ng th y tinh ho c b

nhi

v

19

ng. N u ng 1 c


d. Saponin

l. Polysaccairid ng nghi m l r il cl yd

-

tv

th nghi m.

0.1N -

c qua gi y l

ng nghi m b ng nhau m i ng 1 mL d ch chi t: 3 gi t d ch ch a m u th .

ng th 2

ng 1

tb

u

tb

5 gi t thu c th Lugol.

ng th

2 mL

2 mL d ch chi t. N u ng 1

c c t, l c cho

nh m

cc t

n v ch.

m. Iridoid Thu c th Trim - Hill: tr n 10mL CH3COOH v i 1mL dung d ch CuSO4

c ch

t t m s

dung d ch AgNO3 0.1N (1mL

p m ng, gi i ly. Phun x t

ch NH4OH 5N (5mL)

. N u b n m ng xu t hi n v

ng t

c.

n m ng r i ng kh .

tv 1 mL d ch l

g. Steroid

ch HCl 1% trong 5 gi . L c l y ng nghi m, nh 2-3 gi t thu c th Trim - Hill. Kh

xu t hi mg cao chi t trong 2mL cloroform dung d

H2SO4

c. N u

.

n. Polyphenol

m, xanh, xanh -

b

h. Axit h

, ng 3 gam b t v c ti

ng nghi

cc tr i

N u

50 mL

ngu i r i

N

ng, n

ng nghi m.

t dung d

a

h p th

y

yn

M ts

r i l c. Nh 1 gi t d ch chi ng n

tm

nh

xu t hi n k t t a

c l y d ch l

ch l t H2SO4

c l c nh .

n - hexane trong

ng nghi

y

c th hi n ng ch

c chi t ra

h p th t ngo i - kh ki n

h p th quang c a d ch chi t. Quang ph h p th t ngo i - kh ki

vi

h p th b c x

th y xu t hi n

c c a dung d

ngo i - kh ki ng nh

20

h p th ys d

(UV a trong 10 mL

UV - VIS u qu c

h p th

y

k. Carotene am b t v

y 2 mL d ch l dung d ch chuy

2.2.4.

tv

s y

cc

Na2CO3.

i. Ch

ether d u h

c

t dung d ch FeCl3 5%

ngu i r i l c qua gi y l c, gi l y ph n d ch.

ng nghi m nh kho ng 2 mL d ch l

thu l y c n. S

ng nghi m: ng th

i 1g I2

tan h t r i chuy

ng kh Cao chi

gi y l

5 gi t thu c th Lugol.

Thu c th Lugo

nhi u so v i ng 1 th

s y

2 mL d ch chi

c 2

ng nghi m. N u c t b t trong c 2 ng nghi m cao b

e.

ch chi

ng th cc

b

t c c th y tinh 100 mL, thu l y

ph n d ch l

5 mL NaOH

3 gi t d ch ch a m u th ;

L c m nh c 2 ng nghi m r i

c c t,

t, t

u

nh y

i gian so v

21

nh ng th


i m t dung d max nh b.

h p th nh ng tia

i m u,

h p th b c x h ng ngo

p ch t c n th , s d

- 400cm . Vi tc

p gi a ph ch t th v i ph chu n cho s n ph

Th

ng s

hai,

n (GS

n: Ch

S ch

ng (GLC).

c h p ph tr c ti

ng

t ch t l

c gi i h n v i ch

u ki n.

b

cho s n ph

i th

cc

t h p ch t

trong c

tr

ng.

ng nh

c a ch

ch h

a m i ch

y,

t th i gian

nh ch t tan b gi l i trong c t s

, ion m

kh

nh

ra kh i

tc am

s

c ghi l

), tr Ph kh

cc

c ghi l

ts cx

th

s m/e g

i ph

cao nh

nh cao nh

m

, t

.

i d ng ph v ch hay b

ng ph

t chu

.S kh i m

nh ng th

- Chu n b m

n ch t

i ph (MS)

t c a m

c

t. Tu thu

n.

ng

ng

d ch chuy

S ti

HPLC

cc a

nh

i: s

u

p gi a ph ch t th v i ph c a ch t chu c.

S

t trong c

nh

c: Th nh t,

c

m t ch t

t

s d ng ph t ch t. Vi

tr

nh

c x h ng ngo i -1

(Atlas) ho

p ch t

i 250o

ts

Ph h ng ngo

s

ng trong

h

nh.

h ng ngo i IR

cs

c bi

i kh

nh ion

c a h p ch t

kh -

u ki n s

t chu

nh th i ta l

i ph (GC - MS)

ng s c b ng th

a t ng ch t.

a m i ch t;

y ph kh

-

c a ch t is

th

i th

d.

- MS d

c

a m t ch i ph (GC

ng (MS). Nh

ph kh

n. N u m t ch t trong m

i v i t tc

nh ng h p ph n v i kh

n.

t ch t ch

Cao chi c s

t

p ch 22

c, k c tt

p ch t u

pb S

c

p ph

ng ch c ppm

2.2.5.

MS)

i

d ng ph

bi n trong

h

tk th pv is p. S

t g m s

y 23

p m ng v ng s

d ng silicagel


tinh khi t c

0.040 - 0.063 mm. Ki t b ng s

c 3: N p ch t h

p m ng v i h

c ti

u ch

p [7]. -

yc ts l a ch

;

-

,

yc ts

ng r t nh cao chi t n - hexan

-

n p ch t h

p ch t tinh khi t:

c 1: L a ch

-

t

. - hexane;

ng mao qu n ch m dung d ch m

i b n m ng v

c ch

t m 2/3 chi u cao c t s ng

t th nh

140g silicagel v

kho ng

c c t kho ng 45cm - 4cm; c t th ng 70g silicagel v

-

nm

c ch y v l 10/1, 15/1, 20/1, 25/1, 30/1),

l 5/1,10/1, 15/1, 20/1, 50/1);

- Ti p theo hi

y c

(c t 1: n - hexane, c t 2: n silicagel. S

n m ng b

c hi

,

c, t

ch

i ly ph

p cho c

u

a l c nh , s

cl

-

ng 1

h n h p s l

Ph n cao n- hexane (7

- hexane

c

c n p ch t ch

ng nh

t th

u c t (k t h p r

nm ur

nb

c t

pm u

d ng b

: g silicagel kho ng 2 - 3 cm,

th t ch

+

t; cho m t l

ng 3 - 6 mm t

b m t c a l p silicagel. m

du kho ng 1 -

pl i uc

t kho ng

y xu ng t i khi m n (kho ng 2 - 3 l u ch y c t (H

2. M u g n silicagel 3. C t s 24

nh [7].

t

t quay

+ Sau

2).

t) kho ng 3 l

ng nh t.

c g

C o l y ph n

t

n khi h t. Sau khi n p xong,

c 4: N p m

n d n 10.5 g silicagel Merck c h t 0.04 - 0.06 mm u sao cho h n h

nh ps b

l ng xu ng. Ti p t

c 2: Chu n b m u

uc

t.

v

n - hexane/acetone (100:0

n - hexane/acetone (20/1) cho c t 2.

ts

t, v

khu

ng

m

-

u, l

u

hexane/acetone = 20/1) g p kho ng 1.5 l

t

d

v i H2SO4 10%;

u

ng

c c t kho ng 45cm - 3cm;

-

n - hexane, n - hexane/ethyl acetate (v n - hexane/acetone (v

ng silicagel s nh i (kho ng

in pm u 25

i y. Cu

3) [7].

,


c 5: Ch y c t Ch y c t silicagel v i h

15 mL 1

a gi i gradient n - hexane:acetone

c 120 l

(100:0

u t

n 120, m

c ki

c ki

n kho ng

nm

cg

n l n: V1

= (1

V2

= (10-17)

9)

V7

= (53-62)

V8

= (63-79)

V3

= (18-22)

V9

= (80-90)

V4

= (23-30)

V10

= (91-102)

V5

= (31-38)

V11

= (103-110)

V6

= (39-52)

V12

= (111-120)

c ki

ch t r

n: VH5 = (66 - 75)

VH2 = (16 - 37)

VH6 = (76 - 82)

VH3 = (38 - 50)

VH7 = (83 - 90)

VH4 = (51 - 65)

VH8 = (91 - 105)

n VH4 ch m b n m ng v c th (H

5).

n m ng v i h ng 2.1).

i cho v ti p t c tinh ch .

c 105 l .

nm

VH1 = (1 - 15)

m tv

n - hexane/aetone = 20/1 cho th

n V8

t th nh

n-hexane/acetone = 20/1. Ti

f

u V8

t kho ng 300 mg (H

h n h p.

y,

5. S 2.2.6.

c

ASVH 4).

C ph hi

n m ng VH4

nh c

cc

p ch

nh b

t th

ph h ng ngo i FT - IR, ph kh i ESI - MS, ph t

UV - VIS, ph c 2.3.

ngo i

t chi u 1H - NMR, 13C - NMR, DEPT.

ng t h u th c nghi m u th c nghi

2.3

kh

c th c hi

i

m ts y ut

kh

m ts y ut

4. M u r n ASV B ng 2.1. S S

B

n m ng V8

n m ng

u p ch t

t

1

2

3

b = 10 cm

Chi t v

m

Rf = ai /b [7]

0.3

0.5

Tro

Kim lo i n ng

D ch chi t n- hexane C t lo

0.8

Cao n- hexane

a1 = 0.3 cm a2 = 0.5 cm a3 = 0.8 cm

GC-MS

nh danh cao n- hexan

Ti p t c ch y c t th hai v

n ASVH. M

300 mg m u v i 600 mg silicagel). H 26

cn pb

6.

kh

a gi 27

m ts y ut

-hexan


u ch

p ch t tinh khi t

u ch

. K T QU

p ch t tinh khi

.

3kg B

u

3.1.

nh m t s ch s v

3.1.1.

m

O LU N

u ki n ti

t v i n - hexane (5 x 24h)

kho ng 3g b t

L

D ch chi t

, s y trong

u

nhi

100 C. C sau 5 gi l i l

ng c a m

C t lo

th c (2.1

mc ab t

c

V2

V3

V4

V5

V6

V9

V8

V7

V10

V11

V12

ASVH 300mg SKC, silicagel, H/A 20/1, 105 l

.

VH1

VH2

VH3

VH5

VH4

VH6

VH7

u

c

mc ab

u

STT

m1 (g)

m2 (g)

m3 (g)

W (%)

1

31.330

3.005

34.034

9.850

2

29.850

3.004

32.548

10.186

3

30.211

3.000

32.894

10.567

VH8

ASVH10 75 mg

m1

: Kh

m2

: Kh

m3

: Kh

W (%)

C

B ng 3.1.

1. K t qu kh V1

y, trong 30 gi

.

K t qu : K t qu kh

SKC, silicagel, H:A 100:0 0:100, 120 l

n khi kh i

c. Ti mc a3

Cao n- hexane (7g)

uc am u

0

ng b

Wtb (%)

10.201

u u sau khi s y

mc am im u

Wtb (%) Nh

( IR, MS, 1H-NMR, 13C-NMR, DEPT)

:

ab

u kho ng 10,20%. V

c b o qu n trong su u ch

p ch t tinh khi t

n mm

c nghi

m trong kho

c li u v

-12%) [1]. 3.1.2.

ng tro u ki n ti 5000

ph

L y3m uv n khi thu

c c n tr ng kh

n theo C K t qu : K t qu kh

). ng tro c a b t

B ng 3.2.

28

l

29

u


2. K t qu kh

ng tro trong b

u

nh th i gian chi t

STT

m1 (g)

m2 (g)

m4 (g)

% tro

1

31.330

3.005

31.596

8.852

2

29.850

3.004

30.116

8.855

3

30.211

3.000

30.4469

8.600

K t qu kh

i gian chi t m u b

4. K t qu kh Th i gian Kh

8.769

i gian chi t v i

n-hexan

4h

6h

8h

10h

12h

mc (g)

41.78

41.78

41.78

41.78

41.78

ng

1

: kh

ng c c

m5 (g)

74.13

88.38

87.98

88.41

88.26

m2

: kh

ng m u

m6 (g)

32.35

46.60

46.20

46.63

46.48

m4

: kh

ng c

% tro Nh

ng tro trong b t v

a.

:V

c ab t g nv

u kho ng 8,8 %.

c li u v

3.1.3.

u ki n ti

: M u sau

nh m

n 200ml. L y dung d

ng m t s kim lo i t ng Th

ng (mg/L) TCVN (mg/L)

ng d ch chi t

K

ng Th

ch chi t.

p ch t trong v

u c gia ,

K

ch chi t

- hexane chi t trong 6h s

nh K t qu

a

p ch

5. K t qu

b ng 3.3. L p ch t

u

Zn

Cu

Pb

As

Cd

0.3972

0.0663

0.2738

0.1364

0.0234

40

30

2

1

0.1

p ch t t

Thu c th

Hi

n Vi t Nam cho v c a b y t s 02/2011/ TT-BYT) v s

As: 1 mg/L, Cd: 0.1 mg/

1. Alkaloid

nhi u so v

y

Mayer

t

Dung d t

Dung d ch

Dung d ch H2SO4

30

(+++) Xu t hi n k t t a

Dung d m

Dung d

c

Dung d ch

c ph m [2].

Dung d

NaOH 10% 2. Flavonoid

s d ng v

K t lu n

Wagner

sinh th c ph m

L, Zn: 40 mg/L, Cu: 30 mg /L,

ng t i

K t qu Xu t hi n k t t a

Thu c th

ng kim lo i n ng t

ng kim lo i n

a u

Thu c th Nh

ng ch t nhi

B ng 3.5.

ng.

ng m t s kim lo i trong b

Kim lo i

m6: kh

hi u qu nh t. V y th i gian chi t t

ng m t s kim lo i trong b t 3.

ng c

ng dung d ch nh m c

c Trung Trung B -

K t qu : H

ng c c

m5: kh

Nh

ng kim lo i

HNO3

mc: kh

V : th

i s d ng

(theo th

ng

B ng 3.4.

(+) Dung d

m Dung d

(+++) D ch chuy n qua

31


L p ch t

Thu c th

Hi

u

FeCl3 5%

Nh

K t lu n

Ethanol 90 /

ch

Dung d

ng kh

6. Steroid

nh m t s

7. Axit h 8. Ch

9. Carotene

Ethanol

Dung d ch

10. Polysaccarid 11. Iridoid

t

c trong d ch chi t n - hexan b ng

t (+) S

B nm

AgNO3,

Dung d

c t ng h p

a trong d ch chi t

B ng 3.6.

m (+++)

Dung d

Na2CO3tt

(++)

Ether d u

Gi y l

h a

t

V

tm

m

(++)

Dung d

n-hexane/

Dung d GC-MS d ch chi t n-hexane c a v

nh t (++)

c

Thu c th

Dung d ch trong

Lugol

STT

su t

Thu c th

Dung d

Trim-Hill

6. M t s

ng

Dung d ch xanh

1

ch t n-Decane (C10H22)

T l %

8.399

0.61

10.966

1.28

11.580

0.74

12.025

2.35

p-Cymene 2 (C10H14)

t

u xanh th m, l c

dung d ch

1,4-Dimethyl-2-

nh th

FeCl35%.

t nh

Polyphenol

3

xanh th m (++)

ethylbenzene (C10H14)

Xu t hi n k t t a Benzene, 1,2,3,4-tetramethyl

10%. 4

: Ph n ng r

(++) : Ph n 32

(C10H14)

y hoa s a

c trong d ch chi t n-hexane tR

nh t Dung d

12.

H cc av

Chuy

nh t

c

nh danh m t s

n - hexane

x m (+++)

Chloroform/

GC - MS c a d ch chi t n -

K t qu

Xu t hi n v

NH4OH

H2SO4

alkaloid, flavonoid,

GC - MS

90o/HCl,NaOH

H2SO4

a c 12 l p ch t

iridoid.

NaOH, H2SO4

4. Saponin

Trong v

ng kh , polyphenol, sterol, coumarin, saponin, polysaccarid, carotene,

m

o

3. Coumarin

K t qu

n ng 33

cc ut o


STT

ch t

tR

T l %

12.140

3.86

cc ut o

STT

ch t

tR

T l %

cc ut o

1,2,3,5-Tetramethylbenzene 5 (C10H14)

14

-Lupenol

22.746

4.79

15

Lupenol

22.928

6.12

16

-Amyrenol

23.336

4.96

17

Lupenyl acetate

23.736

16.83

o-Allyltoluene 6

12.741

0.58

13.033

0.79

13.160

1.86

(C10H12) 5-Methylindan 7 (C10H12) Prehnitol 8 (C10H14) Cyclopentacycloheptene 9

14.223

1.30

(C10H8) n-Dodecane 10

H3CCOO

15.183

2-Decenoic acid, 5-hydroxy18

n-Tetradecane 21.824

2.22

.delta.-lactone

23.776

2.14

(C10H16O2)

(C14H30)

OH

Butyl hydroxyl toluene 19 -Amyrin

22.404

24.868

2.34 CH3

3.28 Cyclododecane 20

27.210

14.15

28.712

32.92

(C12H24) 13

-Amyrin

22.584

11.87

Tetrahydroactinidiolide 21 (C11H18O2)

34

t-but

tub-t

(C14H24O) 12

H

2.70

(C12H26)

11

HH H

35


ch t

STT

tR

li u ph :

cc ut o

T l %

Ph FT-IR: C 3-(Cis- -hydroxy-

22

cyclohexyl)propanol(C9H18O2)

T l %

l di

30.438

nh danh so v i t ng di n -

Nh

c.

22 c u t trong d ch chi t n - hexane c a v nh ng h p ch

ct

-amyrin, -

3 B ng 3.8.

ASVH10

12.38

Peak c a ch

at tc

- IR (H

a.

Lupenol, Lupenyl acetate,

p ch

pt h ng ngo i (FT - IR) c a ch t ASVH1

s a [14], [17]. 3.8.

ng cao chi t so v i m c t 3 kg b

u

STT

t so v i m

M uv

ng (3 kg)

Cao chi t Kh i

n-hexane

ng (g)

-1 )

)

ASVH10

c th hi n qua B ng 3.7. 7.

h ng ngo i (FT - IR) c a ch t ASVH10 -1

D ki n u

c -OH (alcol)

1

3441

3600 - 3300

2

2945, 2870

2800 - 3000

C-H

3

1638, 1462

1400 - 1660

C=C

385

% so v i m

u

[M+H]+

Ph kh i: Ch t ASVH10

12.83

,

409.2 (m nh), 298.9, 259.0, 231.0, 203.0, 177.0, 120.9. 3.6. X

nh c

p ch

3.6.1. H ng s v

li u ph c a h p ch

H p ch t ASVH10 hoa s a

d ng r

Ph

c ASVH10

nhi

2a), tan t t trong n - hexane, clorofom,

i axit H2SO4 10% (115o y 189.9oC (H

6): T i

i

n n - hexane c a v ng (H

4

H-NMR (H

c

axeton, metanol; Rf = 0.4 (Silicagel, H/A 20/1), Rf = 0.5 (Silicagel, H/E 90/10); hi n v

1

9. D li u ph d ch chuy

cc

1

H - NMR c a ch t ASVH10 d ch chuy

V

ng ( (J,Hz), ppm) proton

a. M u r n

b. Nhi

y

cc ng ( (J,Hz), ppm)

V proton

0.73 (1H, m)

H-5

1.51 (1H, d, 4,0), 1.33 (1H, m)

H-7

0.79 (3H, s)

H - 23

1.52 (1H, m)

H - 19

0.83 (3H, s)

H - 28

1.54 (1H, m), 1.41 (1H, m)

H-7

t ASVH10 36

c ghi nh n. S li u ph

ong B ng 3.9.

ng 75mg;

2b).

3.20 (1H, dd, J = 11.0 , 4.5 Hz, H - 3);

1.00 -

H-

5,18

H

proton c 1

H

u c a 1 proton olefinic

37


d ch chuy

cc

d ch chuy

V

ng ( (J,Hz), ppm) proton

cc ng ( (J,Hz), ppm)

proton

0.87 (3H, s)

H - 29

1.55 (1H, m)

0.87 (3H, s)

H - 30

1.60 (1H, m), 1.55 (1H, m)

H-2

0.94 (3H, s)

H - 25

1.64 (1H, m), 1.60 (1H, m)

H-1

1.76 (1H, ddd, 4.0, 4.0, 4.0),

H - 26

1.00 (3H, s)

H - 24

1.90 (1H, d, 3,5), 1.86 (1H, m)

H - 11

1.14 (3H, s)

H - 27

1.93 (1H, dd, 3.5, 3.5)

H-9

3.20 (dd, 4.5, 11.0)

H-3

5.18 (1H, t, 3.5)

H - 12

3.0, 3.0)

[M+H]+

Ph

(H

C -

7

1): Ph

13

C - NMR ch ra

u c a 2 cacbon olefinic thu c v

ut i - 3). D li u ph

CH3

Peak m/z

m nh, cho th y s

2

13

C (ppm)

13

79.07

>CH OH, 7

c th hi n 10. D li u ph

C

Ph

1

H - NMR, 13C d ch chuy

c

13

V

C (ppm)

ph -NMR c a h p ch t AVSH10 tr c a nh ng peak c

- amyrin

V C

C

ng h - amyrin cho th y s

ng. D li

ct

38.6 27.3 79.1 38.8 55.2

>CH2 >CH2 >CH OH >C< >CH

16 17 18 19 20

27.0 32.5 47.7 46.9 31,1

>CH2 >C< >CH >CH2 >C<

6

18.4

>CH2

21

37.2

>CH2

7

32.7

>CH2

22

34.8

>CH2

8

39.8

>C<

23

28.1

CH3

9

47.3

>CH

24

15.5

CH3

10

37.0

>C<

25

15.6

CH3

11

23.6

>CH2

26

16.8

CH3

12

121.8

CH=

27

26.0

CH3

13

145.2

>C<

28

28.4

CH3

14

41.8

>C<

29

33.4

CH3

15

26.2

>CH2

30

23.7

CH3

pv B ng 3.11 [12].

li u ph NMR c a h p ch t ASVH10 v i ASVH10

(CDCl3, 125Hz)

- amyrin

V proton

1

38.7

38.6

1

2

27.2

27.3

2

3

79.3

79.1

3

4

38.5

38.8

4

5

55.1

55.2

5

6

18.6

18.4

6

7

32.4

32.7

7

8

39.8

39.8

8

9

47.6

47.3

9

10

36.9

37.0

10

11

23.6

23.6

11

c

1 2 3 4 5

38

cc

B ng 3.10.

C-NMR, DEPT c a ch t ASVH10

p ASVH10

c ancol b c 1 ho c 2:

C - DEPT cho th y ASVH10

,

carbon b

V

13

kh i m/z ng CTPT C30H50O.

ASVH10

11. 13

c

Ph kh i

H - 15

1.00 (1H, m)

H - 21

a h p ch

H - 18

0.97 (3H, s)

1.40 (1H, m), 1.22 (1H, dt,

nh c

3.6.2.

V

H

- amyrin

ASVH10

(J, Hz) (CDCl3, 500 Hz)

1.55 (1H)

1.64 (1H, m),

1.49 (1H)

1.60 (1H, m)

1.52 (1H)

1.60 (1H, m),

1.55 (1H)

1.55 (1H, m)

3.20 (dd, 4.4,

3.20 (dd,

11.5)

4.5, 11.0)

0.71

0.73 (1H, m)

1.53 (1H),

1.54 (1H, m),

1.30 (1H)

1.41 (1H, m) 1.51 (1H, d, 4.0), 1.33 (1H, m)

1.95

39

1.84

1.93 (1H, dd, 3.5, 3.5)

1.90 (1H, d, 3.5), 1.86 (1H, m)


- amyrin

V C

ASVH10

C (CDCl3, 125Hz)

- amyrin

V proton

12

121.7

121.8

12

13

145.2

145.2

13

14

41.7

41.8

14

ASVH10

H (J, Hz) (CDCl3, 500 Hz)

5.16 (t, 3.5)

5.18 (1H, t, 3.5)

1.76 (1H, ddd, 4.0, 15

26.2

26.2

15

4.0, 4.0), 1.00 (1H, m)

16

26.1

27.0

16

17

32.6

32.5

17

18

47.2

47.7

18

1.89

1.55 (1H, m)

19

46.8

46.9

19

1.59

1.52 (1H, m)

20

31.0

31.1

20

21

34.7

37.2

21

22

37.1

34.8

22

23

28.0

28.1

23

0.77s

0.79 (3H, s)

24

15.4

15.5

24

0.98s

1.00 (3H, s)

25

15.4

15.6

25

0.92s

0.94 (3H, s)

26

16.8

16.8

26

0.94s

0.97 (3H, s)

27

25.9

26.0

27

1.11s

1.14 (3H, s)

28

28.4

28.4

28

0.81s

0.83 (3H, s)

29

33.8

33.4

29

0.85s

0.87 (3H, s)

30

23.7

23.7

30

0.85s

0.87 (3H, s)

1.66

K t lu n: T nh ng s in

c th hi n

H

H-NMR (CDCl3, 125 MHz) c a h p ch t ASVH10

1

H-NMR (CDCl3, 125 MHz) c a h p ch t ASVH10

1.40 (1H, m), 1.22 (1H, dt, 3.0, 3.0)

ng t h p ch t ASVH10 s h u

m kh o [10], [11], h p ch

1

c kh cc ut o

i C - 12/C -

i chi u v i

- hydroxylolean

12 - ene

c a h p ch t

(

12.

40

0.7-1.7)

41


8. Ph 1

7. Ph

13

H-NMR (CDCl3, 125 MHz) c a h p ch t ASVH10

13

C-NMR (CDCl3, 125 MHz) c a h p ch t ASVH10

42

C-NMR (CDCl3, 125 MHz) c a h p ch t ASVH10 (

13

0-50)

C-NMR (CDCl3, 125 MHz) c a h p ch t ASVH10 ( 50-145) 43


.C

a h p ch t ASVH10 p ch t

3 - hydroxylolean - 12 - ene ( ch

khu n, iv i

i HL - 60 c h

ho

i

i NTUB1. ng h p

ho - amyrin

c xem

t ngu n c ho

t chuy c

th c v t [8], [14].

44

ng d n c

p ch t

glycyrrhizin ho c ginsenosides.

p ch t trong th c v t

DEPT c a h p ch t ASVH10 (

,

i - amyrin

i v i t

- amyrin

DEPT c a h p ch t ASVH10

i

10-55) 45

t o ra trong


K T LU

N NGH

U THAM KH O

K

Ti ng Vi t 1.

h v

mc ab

u

[1] [2]

10.20%;

kim lo i n ng Cu, Pb, Zn, Cd, As n m trong kho nh c a

n Vi t Nam v

B Y t (2009),

n IV

i.

B Y t (2011),

-BYT - Quy chu n k thu t qu c gia

gi i h

c, H

Nguy n H

n Th

i.

[3]

ng kim lo i n ng t

c li u. 2.

p ch

ng g p trong th c v t b ng ph n ng

t qu trong m u b t v 12 l p ch

[4]

a

alkaloid

3.

kh

thu

c

B n

[5]

ng cao n

-

t v

uc

ng kh , polyphenol,

sterol, coumarin, saponin, polysaccarid, carotene, ch hexane

tb

c trong v khoa h

ch chi t

a bao g m ankan, xicloankan, acid h

d,

[6]

c.

Nguy n Th Thanh Huy n (2011),

.

c 35 c u t

ng d ng m t s

ih

u chi a t

nh ng, Lu

ng.

Nguy n Kim Phi Ph ng (2007),

p h p ch t t

,

d n xu - stigmasterol, - amyrin, lupenol, lupenone, lupenyl acetate, ho t ch

ng ch

4.

- sitosterol,

- amyrin,

-

ng

Ti ng Anh [7]

: ph h ng ngo i FT - IR, ph c 13

C-

nh c

l

ng t h

1

[8]

Ashutosh

Sharma1,

Maheep

K

Chahar,

Mahesh

C

Sharma,

Pradeep Parashar International Journal of Recent Research

c. H p ch t ASVH10

3 -hydroxylolean-12-ene ( -amyrin). H p ch

and Review, Vol. V ,pp. 2277 - 8322.

c

r tt

American Chemical Society

and American Society of Pharmacognosy, 67, pp. 1591 - 1594.

kh i ESI -

a h p ch

New Indole

Alkaloids from the Bark of

.

c h p ch t ASVH10 tinh s ch. Vi c k t h chi u 1H - NMR ,

Angela A. Salim, Mary J. Garso

pt v

a

[9]

Chao - Min Wang, Kue - Lin Yeh , Shang - Jie Tsai , Yun - Lian Jhan and Chang -

Vi t Nam

- Proliferative Activity of Triterpenoids and

Sterols Isolated from Alstonia scholaris against Non - Small - Cell Lung 1. Ti p t th ho

i c a cao chi t n - he cc

.B

a

Vi

nghi m v ho

d ch chi t t r

c bi c

t. 46

Molecules, 22, pp. 1 - 13. The Griess assay:

suitable for a bio - guided fractionation of anti -

c. ph

Carcinoma Cells

[10] Dirsch, V.M., H. Stupp

c,

s d ng c 2.

ng th i,

Planta Med, 64(5), pp. 423 - 436. [11] Hasandeep Singh, Rohit Arora, Saroj Arora and Balbir Singh (2017), Ameliorative potential of Alstonia scholaris (Linn.) R.Br. against chronic 47


BMC Complementary

constriction injury and Alternative Medicine, 17:63, pp 1 - 9. [12]

PH L C

zon and Arturo Navarro ThePent

- amyrins

A Review of Sources and

Biological Activities, 23, pp. 487 - 497. [13] Nilubon Jong - Anurakkun, Megh Raj Bhandari, Jun Kawabata (2006), -Glucosidase inhibitors from Devil tree (Alstonia scholaris)

Food

Chemistry, 103, pp. 1319 1323. [14] Ogwuche CE, Amupitan JO, NdukweIG and Ayo RG (2014) - Amyrin from the Aerial Plant Parts of Medicinal chemistry, vol .4, pp. 729 - 733. [15] P Steve Thomas, Anil Kanaujia, Dipankar Ghosh, Rajeev Duggar & Chandra Kant Katiyar

Alstonoside, a secoiridoid glucoside from Alstonia

scholaris Indian Journal of Chemistry, 47B, pp. 1298 - 1302. [16] Tao Feng, Xiang - Hai Cai, Zhi - Zhi Du and Xiao - Dong Luo (2008), Iridoids from the Bark of Alstonia scholaris

Helvetica Chimica Acta, 91,

pp. 2247 - 2251. [17] Tao Feng, Xiang - Hai Cai, Pei - Ji Zhao, Zhi - Zhi Du, Wei -Qi Li, Xiao-Dong Luo (2009

Monoterpenoid Indole Alkaloids from the Bark of

Alstonia scholaris Planta Med, 75, pp. 1537 - 1541.

Website [18] https://indiabiodiversity.org/species/show/7455

48

Ph l c 1. Ph MS c a ASVH10


Ph l c 2. Ph HSQC c a ASVH10

Ph l c 3. Ph HSQC c a ASVH10 ( ph


Ph l c 4. Ph HSQC c a ASVH10 ( ph

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