B Y T

TRNG AI HC DC H NI

NGUYN TH NGN

NGHIN CU C IM THC VT

& THNH PHN HA HC

CA L XOI TRN YN CHU,

SN LA

KHA LUN TT NGHIP DC S

H NI-2013

B Y T TRNG AI HC DC H NI

NGUYN TH NGN

NGHIN CU C IM THC VT

& THNH PHN HA HC

CA L XOI TRN YN CHU,

SN LA

KHA LUN TT NGHIP DC S

Ngi hng dn:

TS. Quyn

Ni thchin:

B mn Dc Liu

H NI-2013

LI CM N

Trong qu trnh hon thin kha lun ny, ti nhn c s ng h v

to iu kin ca ban gim hiu Trng i hc dc H Ni v b mn

Dc liu cng rt nhiu b mn khc trong trng.

Du tin, ti xin by t lng knh trng v bit n su sc ca mnh n

TS. Quyn l ngi thy trc tip hng dn ti hon thnh kha lun

ny.

Cng nhn dp ny, ti xin gi li cm n chn thnh ti TS Nguyn

Quc Huy hng dn ti nghin cu v cc c im hnh thi cy xoi

trn Yn Chu, Sn La.

Ti cng xin by t lng bit n n cc thy c gio cng cc anh ch k

thut vin khc ti B mn Dc Liu, B mn Thc Vt v cc b mn khc

trong trng nhit tnh gip v to iu kin cho ti trong sut qa trnh

hc tp v nghin cu hon thnh kha lun tt nghip.

Mt ln na ti xin chn thnh cm n!

H Ni, thng 5 nm 2013

Sinh vin

Nguyn Th Ngn

MC LC

DANH MC K HIU V CH VIT TT

DANH MC CC BNG

DANH MC TH V HNH V

T VN .............................................................................................................. 1

Chng I: TNG QUAN ............................................................................................. 2

1.1. c im thc vt chi Mangifera L. ..................................................................... 2

1.1.1.V tr phn loi chi Mangifera L. ........................................................................ 2

1.1.2. c im hnh thi v phn b ca ca chi Mangifera L. ................................. 2

1.3.3. Kha phn loi ca chi Mangifera L. ................................................................ 2

1.3.4. Chi Mangifera L. Vit Nam ............................................................................ 5

1.2. Thnh phn ha hc ca chi Mangifera L. ........................................................... 6

1.2.1. Nhm cc hp cht ca phenol v acid benzoic ................................................ 6

1.2.2. Coumarin ............................................................................................................ 6

1.2.3. Flavonoid ............................................................................................................ 7

1.2.4. Xanthon .............................................................................................................. 7

1.2.5. Tinh du v cc hp cht thm .......................................................................... 8

1.2.6. Triterpen ............................................................................................................. 9

1.2.7. Steroid ............................................................................................................... 10

1.3. Cng dng v ng dng ca cc loi thuc chi Mangifera L. trong Y hc

c truyn v Y hc hin i ............................................................................. 11

1.3.1. Trong lnh vc Y hc c truyn ....................................................................... 11

1.3.2. Trong Y hc hin i ........................................................................................ 12

1.4. Cc nghin cu v chit xut v nh lng mangiferin .................................... 12

1.4.1. Tnh cht ca mangiferin ................................................................................. 12

1.4.2. Mt s qui trnh chit xut mangiferin ............................................................ 13

1.4.3. Mt s phng php nh lng Mangiferin ................................................... 14

Chng 2: I TNG & PHNG PHP NGHIN CU ............................... 17

2.1. i tng nghin cu .......................................................................................... 17

2.2. Nguyn vt liu, ha cht, thit b nghin cu ................................................... 17

2.2.1. Nguyn vt liu ................................................................................................ 17

2.2.2. Ha cht ............................................................................................................ 17

2.2.3. Thit b .............................................................................................................. 17

2.3. Ni dung nghin cu ........................................................................................... 18

2.3.1. Nghin cu v thc vt .................................................................................... 18

2.3.2. Nghin cu v thnh phn ha hc .................................................................. 18

2.4. Phng php nghin cu ..................................................................................... 18

2.4.1. Nghin cu thc vt ......................................................................................... 18

2.4.2. Nghin cu v ha hc ..................................................................................... 19

Chng 3: KT QU THC NGHIM ................................................................... 21

3.1. Kt qu nghin cu v thc vt ........................................................................... 21

3.1.1. c im hnh thi ca cy xoi trn huyn Yn Chu, tnh Sn La ............. 21

3.1.2. c im vi phu l v cung l xoi trn ...................................................... 24

3.1.3. c im bt l xoi trn ................................................................................. 27

3.2. Kt qu nghin cu v ha hc ........................................................................... 28

3.2.1. nh tnh cc nhm cht hu c bng phn ng ha hc ............................... 28

3.2.2. nh tnh dch chit l xoi trn bng sc k lp mng ................................... 32

3.2.3. Kho st kh nng chit xut mangiferin ca EtOH bng sc k lp

mng ................................................................................................................ 34

3.2.4. nh lng mangiferin trong l xoi trn bng phng php HPLC ............. 35

3.2.5. Chit xut mangiferin t l xoi trn ............................................................... 37

3.3. Bn lun ............................................................................................................... 41

Chng 4: KT LUN V XUT .................................................................... 44

4.1. Kt lun ................................................................................................................ 44

4.2. xut ................................................................................................................. 44

PH LC

Ph lc 1: Phiu gim nh tn khoa hc v phiu chng nhn m s tiu bn ca

cy xoi trn Yn Chu, Sn La.

Ph lc 2: Pic sc k ca v ph hp th t ngoi ca dung dch mangiferin chun

1ppm

Ph lc 3: Pic sc k ca cc dung dch M1, M2, M3, M4, M5.

Ph lc 4: Ph 1H-NMR v 13C-NMR ca cht M.

DANH MC K HIU V CH VIT TT

DM: dung mi.

DMPM: dung mi pha mu.

HPLC: High Performance Liquid Chromatography (Sc k lng hiu nng cao).

SKLM: Sc k lp mng.

TT: Thuc th.

DANH MC CC BNG

Bng 1.1 : Mt s loi thuc chi Mangifera L. v phn b ti Vit Nam.

Bng 3.1: Cc vt (Rf, mu sc) trn sc k ca dch chit l xoi trn khai trin

vi h dung mi EtOAc-HCOOH-H2O [10:1,5:1].

Bng 3.2: Din tch pic ca dy dung dch chun cc nng khc nhau.

Bng 3.3: Kt qu nh lng mangiferin trong l xoi trn khi chit bng dung mi

EtOH 90o, 70

o v 50

o.

Bng 3.4: Kt qu nh lng mangiferin trong cc mu M4, M5.

Bng 3.5: Gi tr Rf v mu sc cc vt trn sc k ca cht M v mangiferin

chun khai trin h dung mi cc iu kin khc nhau.

Bng 3.6: Kt qu nh tnh cht M bng phn ng ha hc.

Bng 3.7: Ph 1H, 13C-NMR ca M v mangiferin.

DANH MC TH V HNH V

Hnh 1.1: Acid Gallic v cc dn cht ca n phn lp t chi Mangifera L.

Hnh 1.2: Cng thc cu to ca acid ellagic (I) v mangcoumarin (II).

Hnh 1.3: Mt s hp cht nhm flavonoid phn lp t chi Mangifera L.

Hnh1.4: Cc hp cht nhm xanthon phn phn lp t chi Mangifera L.

Hnh 1.5: Mt s hp cht monoterpen c trong chi Mangifera L.

Hnh 1.6: Mt s hp cht sesquiterpen c trong Mangifera L.

Hnh 1.7: Cu trc mt s dn cht cycloartan phn lp t chi Mangifera L.

Hnh 1.8: Cc hp cht triterpenoid tetracyclic nhm damaran c phn lp t chi

Mangifera L.

Hnh 1.9: Khung pregnan, pregnenolon, progesterol.

Hnh 1.10: Khung stigmastan v mt s dn cht stigmastan trong xoi.

Hnh 1.11: Cng thc ca mangiferin.

Hnh 3.1: Hnh nh cy v hoa xoi trn Yn Chu-Sn La.

Hnh 3.2: nh chp cc b phn ca hoa, qu, l xoi trn.

Hnh 3.3: Vi phu gn chnh v phin l xoi trn.

Hnh 3.4: Mt gc ca vi phu gn chnh l xoi trn.

Hnh 3.5: Vi phu phin l xoi trn.

Hnh 3.6: Vi phu cung l xoi trn.

Hnh 3.7: Mt gc ca vi phu cung l xoi trn.

Hnh 3.8: Cc c im ca bt l xoi trn.

Hnh 3.9: Hnh nh sc k ca dch chit dc liu bng dung mi ethanol ba

nng 50o,70o, 90o, khai trin h dung mi EtOAc-HCOOH-H2O

[10:1,5:1].

Hnh 3.10: th ng chun mangiferin.

Hnh 3.11: Hnh nh sc k cht M so snh vi mangiferin chun, khai trin h

dung mi EtOAc-HCOOH-H2O [2:0,3:0,2].

Hnh 3.12: Tinh th cht M.

1

T VN

Ngy nay cng vi s pht trin ca khoa hc k thut, thuc phng v iu

tr bnh ngy cng phong ph, bao gm thuc c ngun gc t nhin v tng hp.

Trong ngun nguyn liu t nhin l ngun quan trng nghin cu pht trin

thuc mi v sn xut trong cng nghip dc i vi thuc c ngun gc tho

dc.

Vit Nam l mt nc nhit i gi ma, nng m, ma nhiu, a hnh a

dng rt thun li cho s sinh trng v pht trin nhiu loi cy thuc qu. Cng

vi kinh nghim dn gian s dng cy thuc phng v cha bnh, rt nhiu cy

thuc qu c s dng trong cng nghip dc lm thuc.

Mt trong nhng tnh min ni pha Bc nc ta c s a dng sinh hc cao

l tnh Sn La. Sn La l tnh min ni cao nm pha ty bc Vit Nam, c kh

hu nhit i gi ma, ma ng lnh kh, ma h nng m, ma nhiu. Do a

hnh b chia ct su v mnh nn hnh thnh nhiu tiu vng kh hu, cho php pht

trin mt nn sn xut nng - lm nghip phong ph. Vng cao nguyn Mc Chu

ph hp vi cy trng v vt nui vng n i. Vng dc sng ph hp vi cy

rng nhit i xanh quanh nm. Trong tng s din tch t sn xut nng nghip

(247.684 ha), din tch trng cy n qu chim gn 10% (24.016 ha). Trong , xoi

trn (Mangifera indica L.) l mt c sn ca tnh Sn La, ni ting trn c nc.

Tuy nhin, cho n nay mi ch khai thc qu xoi trn, cn cnh, l cy xoi trn

thng qua vic ta cnh hng nm u khng c s dng. c th lm tng gi

tr s dng ngun nguyn liu ln, l xoi trn (Mangifera indica L.) ca huyn

Yn Chu, tnh Sn La, gp phn xa i, gim ngho cc tnh vng su, vng xa,

chng ti thc hin ti Nghin cu c im thc vt v thnh phn ha hc

ca l Xoi trn Yn Chu, Sn La vi hai mc tiu chnh: mt l gim nh

c tn khoa hc ca cy Xoi trn Yn Chu da trn cc nghin cu v c

im hnh thi v vi hc; hai l xc nh c s c mt v hm lng ca

mangiferin trong l xoi.

2

Chng I: TNG QUAN

1.1. c im thc vt chi Mangifera L.

1.1.1.V tr phn loi chi Mangifera L.

Theo h thng phn loi ca Takhatajan (1987, 2009) [6], [46], h o ln

ht v chi Mangifera L. c v tr nh sau :

Ngnh Ngc lan (Mangiphylata)

Lp Ngc lan (Magnoliopsida)

Phn lp Hoa hng (Rosidae)

Lin b Cam (Rutanae)

B Cam (Rutales)

H o ln ht (Anacardiaceae Lindl. 1830)

Phn h Anacardioideae (Anacardioideae)

Chi Mangifera (Mangifera Linnaeus, 1753)

1.1.2. c im hnh thi v phn b ca ca chi Mangifera L.

c im hnh thi chung ca chi Mangifera L.: Cy g thng xanh, c

kch thc ln. L n mc so le, c cung, phin l nguyn, dai. Cm hoa hnh

chy ngn cnh. Hoa nh, mu 4 hay mu 5, tin khai hoa lp. Cnh hoa i khi

hn lin gc. Cnh hoa c 1-5 sng ni bt. 5 nh hoa ri hoc dnh lin vi a

mt, thng c 1 hoc 2 nh ln hn. a mt to thnh t 5 tuyn ring bit

ngoi nh. Bu nhn mt , 1 non, 1 vi nhy nh bu. Qu c ht cng, v qu

gia c x, v qu trong dy, cng, chc. Ht c 1 hoc a phi [28].

Theo Koster mans v Bompard 1993, chi Mangifera L. gm 69 loi phn b

ch yu cc vng nhit i ca chu nh: n , Min in, Sri Lanka, Thi

Lan, Lo, Vit Nam, Campuchia, Nam Trung Quc, Malaysia, Indonesia, Papua

New Guinea, Philippin, qun o Solomon, mt vi loi trong qun o Thi Bnh

Dng (Hawai) [37].

1.3.3. Kha phn loi ca chi Mangifera L.

Chi Mangifera Linnaeus, 1753 gm 69 loi. Hooker (1862) l ngi u tin

phn chia chi Mangifera L. thnh hai phn chi da trn c im v kch thc ca

3

a mt. Sau ng, rt nhiu nh khoa hc nghin cu v a ra h thng phn

loi cho chi Mangifera L. vi rt nhiu quan im khc nhau. Trong , h thng

ca Kostermans v Bompard 1993 l ph bin nht. Trong h thng ny tc gi

da trn quan im ca Hooker chia cc loi trong chi Mangifera L. thnh hai phn

chi: phn chi Lumus (Marchand) Kosterm (c a mt ging cung hoa hoc khng

c a mt) v phn chi Mangifera (a mt ln rng hn v nm pha ngoi bu

nhy). Sau ng da vo 14 c im khc phn chi tip thnh cc nhm nh

hn v cc loi. Phn chi Limus c gn 10 loi trong c 2 loi M. odorata. v M.

foetida tm thy Vit Nam. Phn chi Mangifera c phn chia tip thnh nhm :

Nhm Marcbandora Pierre: nhm ny c mt loi duy nht M. gedebe Miq.

Theo 2 tc gi ny y chnh l hai loi M. Camptosperma v M. reba

c Phm Hong H m t ti Vit Nam.

Nhm Euantberae Pierre: nhm ny c 3 loi trong c hai loi M.

cochinchinensis v M. caloneura (hay M. dupperreana Pierre) c Phm

Hong H m t c mt Vit Nam.

Nhm Rawa Kosterm: nhm ny c 9 loi trong c loi M. minutifolia c

Vit Nam.

Nhm Mangifera: nhm ny c phn chia tip thnh 3 phn nhm nh

hn:

Phn nhm hoa mu 4: c 15 loi

Phn nhm hoa mu 4-5: c 5 loi trong c hai loi M. indica v

M. dongnaiensis c mt Vit Nam.

Phn nhm hoa mu 5: c 15 loi trong c cc loi M. indica; M.

laurina (hay M. longipes Griff.) v M. Flava c Vit Nam.

Loi M. indica c mt c hai phn nhm hoa mu 4-5 v hoa mu 5. Trong

69 loi, ngoi cc loi c phn loi trn cn c 12 loi khng c v tr phn

loi chnh xc v khng c chng c.

Cc c im m Kostermans v Bompard (1993) da vo phn loi chi

Mangifera L. gm c:

4

(1). Hnh dng a hoa.

(2). S nh hoa hu th.

(3). Ht c phc tp khng.

(4). Hnh dng ca cc nhnh th cp trong cm hoa.

(5). Lng t trn chm hoa.

(6). Hnh dng, s lng v c im gn l.

(7). Hnh dng kch thc cnh hoa.

(8). Hoa mu 4 hay mu 5 (y khng phi l c im c nh, thng kt hp

c hai loi).

(9). Mng li gn c bit mt di ca l.

(10). Hnh dng l trng thnh.

(11). Tnh nht qun ca l.

(12). Cy rng l hay cy khng rng l.

(13). Mu sc hoa.

(14). Mu sc v mn ca qu.

(15). S lng kch thc cc si x ht.

Cc c im c xp xp theo theo trnh t mc quan trng trong vic

nh loi [37].

Theo Thc vt ch ng Dng, H. Lecomte phn loi chi Mangifera L.

gm 11 loi trong c 9 loi c v tr phn loi r rng v 2 loi M. reba; M.

camptosperma khng c v tr phn loi xc nh [62]. Sau 2 loi M. reba v M.

camptosperma c hai tc gi Koster mans v Bompard (1993) xp vo cng vi

loi M. gedebe, loi M. duperreana tng ng c gi di mt tn khc l M.

caloneura [50].

Theo thc vt ch ba nc Campuchia, Lo, Vit Nam (1962) lit k v

phn loi 12 loi ging nh cc loi m Phm Hong H m t, trong xy

dng kha phn loi cho 9 loi, mt loi cha c bit y l M. minutifolia v

2 loi khng tm thy l M. macrocarpa Bl., M. odorata Griff [49].

5

Do c trng v v tr a l v kh hu nn s lng cc loi thuc chi

Mangifera ti Trung Quc khng phong ph nh ti Thi Lan, Vit Nam v cc

nc nhit i, cn nhit i khc. Theo thc vt ch Trung Quc chi Mangifera L.

ch c 5 loi l: M. indica, M. siamensis, M. persiciforma, M. laurina v M.

sylvatica [28].

1.3.4. Chi Mangifera L. Vit Nam

Theo Phm Hong H, Vit Nam c 12 loi thuc chi Mangifera L. [8].

Tuy nhin hai loi M. camptosperma Pierre, v M. reba Pierre c Kosterman v

Bompard (1993) xp vo mt loi chung l M. gedebe Miq [37].

Theo V Vn Chi v Nguyn Tin Bn Vit Nam c 11 loi (bng 1.1)

gm cc loi nh Phm Hong H m t tr loi M. cassia [1], [6].

Bng 1.1 : Mt s loi thuc chi Mangifera L. v phn b ti Vit Nam.

STT Loi Tn thng gi Phn b

1 M. indica L. Xoi Trung v Nam b

2 M. odorata Griff. Xoi thm Min Nam

3 M. cochinchinensis Engel. Xoi nt Bnh Dng, ng Nai

4 M. camptosperma Pierre. Xoi bi Bnh phc, Bnh Dng,

Ty Ninh, Tp H Ch Minh

5 M. dongnaiense Pierre. Xoi ng Nai Lm ng, Lt, Bnh

Dng, ng Nai

6 M. duperreana Pierre. Quo

Lm ng, Ninh Thun,

Ty Ninh, Bnh Dng, Tp

H Ch Minh, Kin Giang

7 M. reba Pierre. Quo ng Nai

8 M. foetida Lour. Xoi hi, mum,

xoi c lm

Qung Tr v nhiu ni

khc

9 M. flava Evr. Xoi vng Qung Nam, Lm ng,

Ninh Thun

6

10 Ma. longipes Griff. Xoi ni, Xoi

cng di

H Nam, Ninh Bnh, Kon

Tum, Bnh Dng

11 M. minutifolia Evr. Xoi rng, xoi

l nh Khnh Ha

1.2. Thnh phn ha hc ca chi Mangifera L.

Cho n nay c rt nhiu nghin cu v thnh phn ha hc ca cc loi

thuc chi Mangifera L. Trong loi M. indica L. l loi ph bin v c nghin

cu nhiu nht. Thnh phn ha hc ng ch gm c: Cc hp cht phenol v

acid benzoic, coumarin, flavonoid, xanthonoid, tinh du (monoterpen,

sesquiterpen), steroid, triterpen v vitamin. c bit mangiferin, mt xanthon

glycoside c nghin cu nhiu nht v l thnh phn c nhiu hot tnh sinh hc.

1.2.1. Nhm cc hp cht ca phenol v acid benzoic

Cc hp cht thuc nhm ny gm c: acid gallic [40], [43], v cc dn cht

ca n : methyl galate, n-propylga late [43]. Chng ch yu c tm thy hoa, l

v v thn ca cc loi thuc chi Mangifera L.

Hnh 1.1: Acid gallic v cc dn cht ca n phn lp t chi Mangifera L.

Ngoi ra cn c cc dn cht acid benzoic v acid 3,4-dihydroxy benzoic,

propyl benzoate [43], 5-[2(Z)-heptadecyl] resorcinol [24], 5-(12Cis- heptadecencyl)

resorcinol v 5-pentadecyl resorcinol c tm thy trong v thn xoi [26].

1.2.2. Coumarin

Hai hp cht thuc nhm coumarin c tm thy v thn, v r ca loi

M. indica: acid ellagic (I) [40], mangcoumarin (II) [39].

7

(I) (II)

Hnh 1.2: Cng thc cu to ca acid ellagic (I) v mangcoumarin (II).

1.2.3. Flavonoid

Flavonoid l mt trong nhng thnh phn ha hc chnh ca chi Mangifera

L., chng c tm thy trong l, qu v ht ca cy. Cc flavonoid c tm thy

trong cc loi ca chi Mangifera L. ch yu thuc cc nhm: Anthocyanidin:]7-O-

methylcyanidin 3-O- -D-galactopyranoside, 7-O- methylcyanidin [25]; Flavan-3-

ol : catechin, epicatechin [43]; Flavonol c quercetin, [16], [30], quercetin 3-O-

diglycosid, quercetin 3-O- galactosid, quercetin 3-O- glucoside, quercetin 3-O-

xyloside, quercetin 3-O- arabinopyranoside, quercetin 3-O- arabinofuranosid,

quercetin 3-O- rhamnoside, kemferol-3- glucoside [16], myrcetin [30]; Biflavon c:

I-4, II-4, I-5, I-5, I-7, II-7-hexahydroxy-[I-3,II-8]-biflavon [34].

Flavan-3-ol Flavonol

Biflavon Anthocyanidin (paeonidin-3-galactoside)

Hnh 1.3: Mt s cht thuc nhm flavonoid phn lp t Mangifera L.

1.2.4. Xanthon

y l nhm hot cht chnh trong cc loi thuc chi Mangifera L.. Cu trc

8

ha hc chung ca nhm l mt b khung xanthon c gn nhiu nhm OH, nhm

OH c th c thay bng lin kt O-glycosid hoc C-glycosid vi 1 phn t

ng. Cc hp cht thuc nhm ny gm c: mangiferin [16], [30], [32], [33],

homomangiferin [32]; euxanthon; 1,3,5,6,7-pentamethoxyxanthon; 1,3,6,7,8-penta

hydroxyxanthon [30], mangiferin-6-O-galate [16]. Trong mangiferin l hot

cht c hm lng kh cao v cng l cht c nhiu tc dng sinh hc: khng virut,

i tho ng, chng vim, iu ha min dch ..

Cng thc chung ca cc hp cht xanthon Mangiferin

Hnh 1.4: Cc hp cht nhm xanthon phn lp t chi Mangifera L.

1.2.5. Tinh du v cc hp cht thm

Cc hp cht monoterpen v sesquitecpen chnh l cc thnh phn to nn

mi thm c trng chi Mangifera L.. Cc thnh phn ny ch yu c tm thy

trong hoa, v qu, mt s c tm thy l cy.

Monoterpen

Camphren 2-Caren 3-Caren -Pinen

-Pinen - Myrecen Carveol

-Cymene -Geranial Linalol

Hnh 1.5: Mt s hp cht monoterpen c trong chi Mangifera L.

Cc monoterpen tm thy trong cc loi ca chi ny ch yu thuc nhm cc

dn cht khng cha oxy nh: 2- v 3-caren, camphren, limonen, -myrecen, -

9

v -phellandren, - v -pinen, -ocimen, Cis-ocimen caryophylen, -terpinolen,

- v -terpinen, -thujen, Ngoi ra, cn c cc dn cht cha oxy gm c: p-

cymen, carveol, -linalol ,[14], [38], [39], [44], [47].

Theo mt nghin cu v tinh du chit xut t v qu xoi M. indica L.

Nigeria, cho thy hm lng cc monoterpen cao (83,2%) trong thnh phn ch

yu l: 3-caren (58,2%), -pinen (13,0%) [15].

Sesquiterpen

Cc hp cht sesquiterpen tm thy chi Mangifera L. u l cc hp cht

c cu to hai vng nh: -cadinen, -cubeben, -caryophyllen, -bulnesen, -

elemene, -gurjunen, -guainen, -humulen, -murolene, -selinen, valenecen,.

hoc ba vng nh -copaen [14], [38], [39], [47].

-Cadinen - Caryophyllen - Guainen

- Copaene -Bulnesen Cadalene

Hnh 1.6: Mt s hp cht sesquiterpen t chi Mangifera L.

Cc hp cht khc

Trong thnh phn ca tinh du xoi cn c cc hp cht khc nh:

acetaldehyde, 2-hexenal, nonanal, 2-nonenal, ethyl butanolate, hexenol, methyl

hexanooate, methyl beoate, ...[14, [39], [44], [47].

1.2.6. Triterpen

Cho n nay tm thy khong 60 hp cht triterpen t cc loi thuc chi

Mangifera L. bao gm cc triterpen pentacyclic v triterpen tetracyclic. Cc hp

cht triterpen pentacyclic gm c cc nhm nh: olean, ursan, [17], [20], [22], [36],

fridelan [17], [18], [22], [29], [30]; lupan [19], [35], [36]; hopan [31]; taraxastan

[20], [21], [22], [23] v cycloartan [17], [18], [19], [20], [21], [22], [27]. Trong

10

nhm cycloartan chim s lng ln nht. Cng thc chung ca nhm bao gm b

khung cycloartan vi mt ni i cc v tr khc nhau (v tr C s 23, 24, 25) trn

mch nhnh, cc nhm th gm c: mt nhm OH hoc O gn vo cc v tr C s 3,

mt nhm OH gn vo mt trong cc v tr 22, 24, 25; nguyn t carbon s 26, 27

c th b oxy ha thnh COOH hoc CHO, CH2OH. Cc hp cht ny ch yu c

tm thy v cy.

Acid hydroxy mangiferolic. 3,24,25-Triol-cycloartane.

Hnh 1.7: Cu trc mt s dn cht cycloartan phn lp t chi Mangifera L.

Anjaneyulu v cng s phn lp c hai hp cht teriterpen tetracyclic

nhm damaran l 3,20(S)-diol-dermar-24-en (III) [17] [19] v 3-oxo-dammar-24-

en-20S,26a-diol (IV) [20] t mt s th ca loi M. indica L. ca n .

Hnh 1.8: Cc hp cht triterpenoid tetracyclic nhm damaran c phn lp

t M. indica L.

1.2.7. Steroid

Cho n nay tm thy khong 9 steroid c trong cc loi M. indica, M.

persiciformis, M. sylvatica. Cc steroid c tm thy thuc hai nhm khung c

bn l pregnan v stigmastan.

Nhm Pregnan: nm 1992, Anjaneyulu v cng s phn lp c hai hp

cht pregnenolon v progesteron t hoa xoi [19].

R1 R2

(III) -OH CH3

(IV) =O CH2-OH

11

Khung Pregnan Pregnenolon Progesteron.

Hnh 1.9: Khung pregnan, pregnenolon, progesteron.

Nhm stigmastan gm c: -sitosterol [20], [23], [29], [32], stigmasterol,

stigmas-7en-3-ol [29], daucosterol, 6-hydroxy stigmast-4-en-3one [23],

mangdesisterol. Trong -sitosterol c tm thy trong hu ht cc b phn ca

cy Xoi. Cc hp cht cn li ch yu c tm thy trong hoa, qu v ht.

Stigmastan -Sitosterol Stigmasterol

Hnh 1.10: Khung stigmastan v mt s dn cht stigmastan trong xoi.

1.3. Cng dng v ng dng ca cc loi thuc chi Mangifera L. trong Y hc c

truyn v Y hc hin i

1.3.1. Trong lnh vc Y hc c truyn

Xoi thng c trng ly qu n, ng hp xut khu. Qu xoi ngon,

b dng, c tc dng nhun trng li tiu, ra m hi, gii nhit, tr bnh lon huyt

v lon c, tiu ha km [11]. Xoi thi ming mng ngm ru vang vi ng,

thm mt t qu thm hoc thi ming sy kh lm ngun vitamin C t nhin [13].

V qu xoi dng cm mu, chng xut huyt, rong kinh, bch i, khi

huyt, chy mu rut di vi liu 20-40g/ngy, sc ung hoc dng di dng cao

lng, 10g cao lng pha vo 120ml nc, cch 1-2h cho ung mt tha c ph [11],

[13].

Hch qu dng tr giun, kit l, tr, xut huyt vi liu 5-10g/ngy, sc

ung [1]. Nhn xoi sy kh tn bt c ngi dn n , Braxin dng lm thuc

ty giun vi liu 1,5-2g. Philipin dng nc sc nhn xoi iu tr tiu chy [11].

12

V thn xoi c th dng ti hoc dng kh. V ti em gi vt ly nc

dng nh v qu xoi. V kh dng di dng thuc sc. Campuchia dng v

thn xoi cha thp khp (p nng bn ngoi) hoc ra cha kh h, bch

i ph n.

Nha v cy xoi mu nu en, khng mi, v ng hc, tip xc khng kh

th c li, khi ha vo nc c th dng bi gh [11].

L xoi c dng tr cc bnh ng h hp trn nh ho, vim ph qun,

tiu chy kit l, vim nga ngoi da. Liu 20-30g/ngy, sc ung [13].

1.3.2. Trong Y hc hin i

V thn, v cnh, l xoi dng chit xut dch chit ton phn hoc chit

mangiferin tinh khit lm thuc. Trn th trng Cuba hin c ch phm Vimang

(dch chit ton phn t v thn, l xoi) c s dng nh mt thc phm chc

nng h tr iu tr mt s bnh min dch, d ng, chng oxy ha, h tr trong

iu tr HIV. Trn th trng Vit Nam hin nay cng c rt nhiu sn phm vi

thnh phn chnh l Mangiferin ch yu dng cha bnh do virus herpes nh:

Vin nang cng Mangoherpin 100mg, kem Mangoherpin 5%, v dit khun nh

dung dch Manginovim 60ml.

1.4. Cc nghin cu v chit xut v nh lng mangiferin

1.4.1. Tnh cht ca mangiferin

Mangiferin l mt xanthonoid c cng thc ha hc l: C19H18O11 (M=

442,35), c Wiechowski (1923) phn lp t v cy Mangifera indica L.. Iseda

(1957) xy dng cu trc mangiferin. Sau Ramanahan v Sechadi (1960)

nghin cu iu chnh li cu trc. Cu trc c tha nhn hin nay l mt

glycoside c phn aglycon c b khung xanthon vi 4 nhm hydroxyl v mt phn

t glucose nh vo C s 2 .

Hnh 1.11: Cng thc ca mangiferin

13

Mangiferin c dng tinh th hnh kim, phin hay vy mng, mu vng nht,

t tan trong nc, tan trong methanol, ethanol, aceton nng. D tan hn trong hn

hp cc dung mi trn vi nc. Nhit nng chy: 269 270oC [10], [12].

1.4.2. Mt s qui trnh chit xut mangiferin

Do c tnh ca mangiferin tan c trong methanol, ethanol nng nn cc

qui trnh chit xut ch yu s dng hai loi dung mi ny lm dung mi chit xut.

tinh ch mangiferin c kh nhiu phng php nhng phng php c s

dng nhiu nht l kt tinh li nhiu ln trong dung mi thch hp. Di y l mt

s qui trnh chit xut magiferin t v v l xoi.

Phm Xun sinh v Phm Gia Khi (1991) chit mangiferin t v v l

xoi theo qui trnh sau: v hoc l sau khi lm sch sy kh c chit ba ln vi

methanol, un si cch thy 1-1,5h. Lc nng. Gp dch chit li ngui n nhit

phng. Flavonoid th ta xung, lc ly ta. Cho ta vo mt lng methanol,

un nng, thm mt lng than hot, ha cho tan ht ta. Lc nng. ngui, lc

thu ly ta c mu vng m hn. Lm li vi ln s thu c mangiferin tinh khit

hn. Hiu sut chit ca v thn l 3%, ca l kh l 1,6% [9].

Shashi Kant Singh v cng s chit mangiferin t v thn ca M. Indica L.

bng ethanol sau tch v tinh ch mangiferin bng phng php sc k hp ph

theo qui trnh: dc liu sau khi loi cht bo bng cch chit kit vi ether du ha

trong bnh Soxhlet (60-80oC, 56h) c chit kit vi ethanol 95% (56h). Dch chit

thu c c di p xut gim thu c bt v nh hnh mu vng. Bt thu c

c hp ph ln ct silicagel (60-120mesh), nhi ct bng ether du ha, ra ri

bng hn hp dung mi CHCl3/MeOH (1:1) thu c mangiferin mu vng nht

dng v inh hnh. Kt tinh li bng methanol thu c tinh th mangiferin hnh

kim mu vng nht [45].

S. Muruganandan v cng s cng chit mangiferin bng ethanol theo qui

trnh tng t nhng tinh ch bng phng php kt tinh. Dch chit ethanol thu

c em c di p xut gim n cn, loi cht bo nhiu ln ri ha tan vo

ethanol. 2 tun nhit phng thu c bt mu trng dng v nh hnh.

14

Kt tinh li nhiu ln trong hn hp dung mi H2O-EtOAc thu c mangiferin tinh

th mu vng, hnh kim. tinh khit c xc nh bng HPLC t 95,56% [41].

nh gi kh nng chit xut ca mt s dung mi v hm lng

mangiferin t l ca ba th xoi thuc loi M. indica L., Aranya Jutiviboonsuk v

Chanchai Sardsaengjun kho st ba dung mi methanol, ethanol v acetone 70%

chit xut mangiferin. Ngm lnh 100g bt l mi th xoi vi mi dung mi

chit xut nhit phng, sau mt tun rt dch chit v thm dung mi mi, lm

3 ln. Dch chit em lc, bc hi chn khng 40oC n cn. Phn tn cn vo

50ml hn hp dung mi gm dung mi chit xut v nc (t l 1:1, tt/tt). Sau

chit vi 100ml dichlomethan x 4 ln. Gn ly lp dung mi-nc em thy phn

trong bnh hi lu vi acid sulfuric pH=3 trong 1h, khuy trn lin tc. Lm lnh

n nhit phng, lc vi 100ml ethylacetat x 3 ln. Gn ly lp ethylacetat em

bc hi 40oC trong thit b bc hi chn khng. Cn thu c c ha tan vo

methanol 70% v t lnh qua m (4-8oC). Lc, ra tinh th, sy kh. Kt qu

cho thy s dng methanol chit theo qui trnh trn cho khi lng mangiferin cao

nht, v s dng aceton cho khi lng mangiferin thp nht [33].

1.4.3. Mt s phng php nh lng Mangiferin

Da vo cc c tnh ca mangiferin ngi ta nghin cu xy dng nhiu

phng php nh lng hot cht ny nh: phng php cn, phng php tp

phc, phng php o ph t ngoi, phng php sc k

1.4.3.1. Phng php cn

Cch tin hnh: Cn chnh xc khong 10,00g dc liu l lm nh v xc

nh m. Chit bng chloroform trong bnh soxhlet ti khi dch chit khng mu.

Chit bng methanol n khi dch chit trong bnh soxhlet nh ln giy lc khng

cho mu vng khi h trn hi NH4OH. Bc hi cch thy ti cn. Ha tan cn bng

hn hp dung mi aceton : nc (1:1), lc qua giy lc. Ct thu hi dung mi, c

n cn. Ha cn vo methanol nng 24h, mangiferin kt tinh. Lc, ra tinh

th, sy 60o n khi lng khng i. Cn v tnh ra hm lng. y l mt

15

phng php n gin, d thc hin nhng chnh xc khng cao nn hin nay t

c s dng [12].

1.4.3.2. Phng php o hp th

Mangiferin c kh nng hp th nh sng t ngoi, cc i hp th bc

sng 369nm. Khong nng tuyn tnh: 0,001-0,01%. Trong khong hm lng

ny, hp th tun theo nh lut Lambert-beer t l thun vi nng . Do c

th s dng phng php o hp th t ngoi nh lng mangiferin.

Cn chnh xc 1,00g dc liu xc nh m. Chit vi chloroform

trong bnh Soxhlet n khi dch chit khng mu. Chit tip vi methanol cho ti

khi ht mangiferin. Cho dch chit methanol vo bnh nh mc 100ml, trng bnh

chit 3ln bng methanol. Thm methanol th tch, lc u. Ht chnh xc 5ml

cho vo bnh nh mc 100ml th 2, b sung th tch. Dung dch trong bnh nh

mc 2 em o quang bc sng 369nm, o 3 ln, ly kt qu trung bnh, mu

trng l methanol. Xy dng ng chun bng cch chun b dy dung dch c

nng : 0,0075%; 0,0025%; 0,002%; 0,001% trong methanol. o quang bc

sng 369nm vi mu trng l methanol.

o hp th l mt phng php kh n gin c th thc hin nhiu

phng th nghim v chnh xc cao nn c th dng kim nghim dc liu

[10].

1.4.3.3. Phng php sc k s dng sc k lng cao p (HPLC)

Tc gi Bi Th Hng nghin cu xy dng phng php sc k lng cao

p nh lng mangiferin. Dung dch mu th c chun b nh sau: cn 1,00g

dc liu xay nh xc nh hm m. Chit bng methanol cho n khi ht

mangiferin trong bnh Soxhlet. C cn dch chit n 80ml, chuyn sang bnh nh

mc 100ml, thm methanol n vch chun. Dung dch mangiferin chun c nng

0,02% trong methanol. iu kin sc k: pha tnh: Hitachi gel 3050, pha ng:

methanol, ct: 2,5 x 500mm, detector t ngoi bc sng 369nm, tc dng:

1ml/pht [7].

16

Jeping Quen xy dng qui trnh nh lng mangiferin nh sau: L xoi

sau khi lm nh sy kh, c chit vi methanol 40% trong bnh nn, cn v ghi

li khi lng ca bnh. Lc nh, ngm 30 pht, siu m 40 pht nhit phng.

Lm lnh v thm methanol khi lng ban u. Lc dch chit qua mng lc

0,45m - 5l, s dng tim mu sc k. iu kin sc k: ct C18, di 250mm x

4,6mm, ng knh ht nhi ct 5m. Pha ng: dung dch phosphoric acid 0,1%

(tt/tt) (dung mi A) v acetonitrile (dung mi B). Chng trnh dng mi: 0 pht:

10% B; 15 pht: 12% B; 20 pht: 15% B; 30 pht : 45% B; 35 pht: 10% B; thi

gian cn bng sau qu trnh ra ri l 10 pht. Tc dng 1.0 mL /pht. Detector

UV 258nm. Duy tr nhit 30oC. hi phc trung bnh ca phng php ny

ba nng l 98,7-101,5%, tuyn tnh cao (r = 0,9998) i vi c hai cht trn

mt khong rng nng [32].

1.4.3.4. Phng php in di mao qun

Sau khi nghin cu cc iu kin tin hnh ca phng php in di mao

qun, Chaozan Nong v cng s a ra cc thng s ca phng php ny nh

sau: hn hp dung mi gm: dung dch m borat 0,5M (pH = 7,4) v methanol (t

l 1:0,3 tnh theo tt/tt) in p s dng l 20kV, b phn pht hin detector UV-VIS

vi bc sng l 254nm. Phm vi tuyn tnh ca phng php ny l 5-1000g,

gii hn pht hin 1,5g. ng ca phng php ba nng nh lng (10,

500,1000g) u ln hn 99% (99,00; 99,38 v 99,14%). lch chun tng i

di 3%. in di mao qun l mt phng php n gin v ng tin cy c th s

dng pht hin v nh lng mangiferin trong nhiu lnh vc c lin quan [42].

17

Chng 2: I TNG & PHNG PHP NGHIN CU

2.1. i tng nghin cu

Cy xoi trn Yn Chu, Sn La.

a im thu mu: bn Poong, x Ching Pn, huyn Yn Chu, tnh Sn La.

Thi gian thu mu: thng 3 nm 2012 v thng 1 nm 2013.

Mu nghin cu gm c l gi, l bnh t v l non.

2.2. Nguyn vt liu, ha cht, thit b nghin cu

2.2.1. Nguyn vt liu

Mu nghin cu thc vt:

L ti sau khi thu hi c ngm trong cn 50o nghin cu c im vi

phu.

L sau khi thu hi c sy kh, tn thnh bt mn nghin cu c im

bt.

Cnh mang hoa ti v l sau khi thu hi mt phn c ngm vi EtOH 50o,

mt phn c p tiu bn kh nghin cu c im hnh thi.

Mu nghin cu ha hc: cc mu l cy xoi trn huyn Yn Chu, tnh Sn La

sau khi thu hi c sy kh trong t sy nhit 50oC, nghin thnh bt th, bo

qun trong ti nilon kn, ni kh ro.

2.2.2. Ha cht

Dung mi: Acetonitril, CH3COOH bng, CHCl3, EtOAc, EtOH, MeOH,

HCOOH, H2O, HCl c...

Cc thuc th nh tnh thng dng: Bouchardat, Baljet, Diazo,

Dragendorff, Fehling, FeCl3 5%, Gelatin 1%, Lugol, Mayer, Nynhidrin, KI 10%,

Cc ha cht: anhydride acid acetic, NH4OH, FeCl3, HCl, H2SO4 c,

NaOH, Na2CO3, Mg,

Bn mng trng sn Silicagel GF 254.

2.2.3. Thit b

Knh lp soi ni Leica E24, knh hin vi Leica; My nh k thut s Canon

A2300, Canon A3200; Bp hng ngoi, ni cch thy; T sy; n t ngoi Vibez

18

lourmat; Cn phn tch Presica, cn k thut Sartorius, Mettler Toledo; Ct sc k

BDS Hypersil C18 (250x4,6mm; 5m); My o ph cng hng t ht nhn 1H-

NMR v ph 13C-NMR BRUKER AVANCE AM500 FT-NMR - Vin Ha hc.

2.3. Ni dung nghin cu

2.3.1. Nghin cu v thc vt

M t c im hnh thi v xc nh tn khoa hc ca cy xoi trn thu hi

huyn Yn Chu, tnh Sn La.

M t c im vi phu cung l v gn l xoi trn Yn Chu, Sn La.

M t c im bt l xoi trn Yn Chu, Sn La.

2.3.2. Nghin cu v thnh phn ha hc

nh tnh cc nhm cht chnh trong l xoi trn Yn Chu, Sn La bng

phn ng ha hc v sc k lp mng.

Xy dng ng chun mangiferin bng phng php HPLC.

Kho st hm lng mangiferin trong l xoi trn bng phng php HPLC.

Chit xut, phn lp v nhn dng mangiferin t l xoi trn.

2.4. Phng php nghin cu

2.4.1. Nghin cu thc vt

- Nghin cu c im hnh thi: M t phn tch c im hnh thi theo

phng php m t phn tch cy thuc [4], [5]. Lm tiu bn kh v lu mu tiu

bn ti Phng tiu bn thc vt, b mn thc vt trng i hc Dc H Ni. Da

trn c s cc kt qu phn tch c im thc vt kt hp vi cc ti liu xc

nh tn khoa hc ca cy xoi trn Yn Chu, Sn La.

- Nghin cu c im vi phu l xoi trn: Tiu bn vi phu l v cung l

c lm theo phng php ct ty v nhum kp. Tiu bn c chp trn knh

hin vi. Cc c im gii phu l v cung l c phn tch theo nguyn tc

nghin cu tiu bn vi phu [5].

- Nghin cu c im bt l xoi trn: Dc liu sau khi lm sch, ct

nh, sy kh c em tn nh, lm tiu bn soi bt [4]. Cc c im c m t

theo phng php mt t bt dc liu [3].

19

2.4.2. Nghin cu v ha hc

- nh tnh cc nhm cht hu c bng phn ng ha hc: Cc phn ng

ha hc c tin hnh theo nguyn tc chit xut v nh tnh cc hp cht hu c

c trong dc liu bng phn ng ha hc [2], [3].

- nh tnh bng sc k lp mng

Bn mng GF254 hot ha 110oC trong 1h.

Dch chm sc k c chit bng dung mi MeOH.

H dung mi kho st: (1). CHCl3- EtOAc-HCOOH [4 : 8 : 1,5].

(2). EtOAc-HCOOH-H2O [10 : 1,5 : 1].

Phng php pht hin: Quan st di nh sng thng, UV 254, UV 365,

hin mu bng hi ammoniac di nh sng thng [48].

- Chit xut v tinh ch mangiferin t l xoi

Chit xut: S dng phng php chit hi lu vi dung mi EtOH 70o.

Tinh ch: Loi tp nhiu ln bng n-hexan v chloroform, sau s dng

phng php kt tinh trong EtOH.

- Nhn dng cht phn lp: c da trn tnh cht ha l v ph 1H-

NMR v ph 13C-NMR.

- Kho st hm lng mangiferin trong l xoi bng phng php

HPLC

Tin hnh: Cn chnh xc khong 1,00g bt l xoi xc nh m cho

vo bnh chit hi lu 2h vi 20ml ethanol 90o, lc nng thu dch chit ln 1. Thm

tip 20ml EtOH 90o, tip tc chit hi lu trong 2h, lc nng, thu dch chit ln 2.

Tin hnh tng t chit ln th 3. Gp dch chit 3 ln vo bnh nh mc 100ml

b sung bng dung mi chit xut cho th tch (dung dch 1). Sau ly 1ml

dch chit cho vo bnh nh mc dung tch 50ml b sung bng dung mi chit xut

cho th tch (dung dch 2). Ly 1ml dung dch 2 cho vo bnh nh mc dung

tch 10ml, thm pha ng n vch (dung dch 3). Dung dch 3 c phn tch bng

HPLC.

20

iu kin sc k: Ct BDS Hypersil C18 (250x4,6mm; 5m); Pha ng:

Acetonitril - Dung dch CH3COOH 3% (14:86); Lu lng dng 1,0ml/pht; B

phn pht hin: Detector UV- VIS. Bc sng: 257nm. Th tch tim mu 20l.

Phng php x l s liu: S liu thu c c x l bng phn mm

Microsoft Ofice Excel 2007:

Dng ng chun mangiferin.

Tnh hm lng mangiferin:

Nng mangiferin (C) trong dch chit c tnh theo ng chun

mangifeirn

Lng Mangiferin /1ml dung dch 1: mmangiferin = C . h2.h3 = 500.C

Vi: h2: pha long t dung dch 1 thnh dung dch 2

h3: pha long t dung dch 2 thnh dung dch 3.

Hm lng Mangiferin c tnh theo cng thc:

6.100.10.100

.(1 )

Mangiferin

Dl

m

m HA

M Dl: khi lng dc liu (g).

HA: hm m ca dc liu

21

Chng 3: KT QU THC NGHIM

3.1. Kt qu nghin cu v thc vt

3.1.1. c im hnh thi ca cy xoi trn huyn Yn Chu, tnh Sn La

Cy thn g, cao, sng lu nm.

L n, nguyn, mc so le, c cung. Cung l di 2,5-3,5cm c th di ti

5-6cm. Phin l nguyn hnh mi mc, di 24-27cm c th di ti 35cm, rng 5,5-

6,5cm. Gc l thun nhn. Ngn l nhn hot, phn cui ko di bp nhn to

thnh ui di khong 1-1,5cm. Gn l hnh lng chim, c khong 21-23 i gn

ph. Mt trn l mu xanh m, bng. Mt di mu xanh nht. Mng li gn dy,

nhn thy r c hai mt. Cnh non mp.

Hnh 3.1: Hnh nh cy v hoa xoi trn Yn Chu, Sn La.

Cm hoa dng chm kp 3-4 ln, hnh chy hoc hnh thp, di n 30-35

cm, ph lng mn, trc chnh c mu hng tm hoc hng pht. L bc con hnh tam

gic, ph lng mn, di khong 1,5-2mm, mu xanh, rng sm. Trn chm hoa c 2

loi hoa: hoa lng tnh v hoa c. Hoa nh, u, rt thm. Cung hoa c khp,

di khong 2-3mm, ph lng mn. Hoa mu 5 (c khi l 4 hoc 6), 5 (c khi 4 hoc

22

6) l i ri di khong 2 mm mu xanh nht, ph lng mn; 5 (4 hoc 6) cnh hoa

ri, thng nhn, di khong 3,5-4mm. Trn cnh c 5 sng ni r mu vng hoc

hoc hng tm, phn gc dnh lin, phn nh sng khng tch khi b mt cnh.

Cnh hoa mi n c mu vng nht sau chuyn sang mu hng tm, cong ngc v

pha sau. Tin khai hoa dng van. a mt ging ci m, do 4-5 b phn hp

thnh, cao 1-1,5mm, nm ngoi bao ly nh v bu nhy. B nh ch c 1 nh hu

th c kch thc ln hn, ch nh hu th di khong 2mm; c th c 1 nh bt th,

nh bt th nu c di khong 0,5- 0,7mm, thiu bao phn hoc bao phn lp. Bao

phn nh lng, 4 . Hoa lng tnh c b nhy do 1 l non hp thnh bu thng,

mu xanh nht, ng knh 1-1,5mm, bu 1 vi 1 non, nh non bn. Vi nhy

nh lch sang bn pha i din vi nh hu th, di 1,5-2mm. Hoa c c bu

nhy tiu bin hon ton hoc rt nh, vi nhy vn cn hoc tiu bin.

Cng thc hoa * K5 C5 A1 hoc 2 G(1) v * K5C5A1 hoc 2.

Qu c hch, hnh xoi, c mi lch, t. V gia c x, v qa trong dy

cng chc.

23

Hnh 3.2: nh chp cc b phn ca hoa, qu, l xoi trn

Ch thch: (1a), (1b): Hoa ci nguyn vn c hoc khng c nh bt th, (1c): Hoa c,

(2a)-(2b): Hoa ct dc, (3a)-(3b): L bc cp 1-cp 2, (4): i hoa, (5a)-(5b:) Cnh

hoa, (6:) a mt, (7a): Nh bt th, (7b): Nh hu th, (7c): Bao phn, (8a): Bu

nhy,(8b): Bu ct dc, (8c): Bu ct ngang, (9): Cc b phn ca hoa khi xp trn mt

mt phng, (10): L, (11): Qu xoi, (12a)-(12b): Lt ct dc v ct ngang qu xoi,

(13): Ht, (14): Ni nh..

(1a) (1c) (1b) (2a)

(3a)

(2b)

(3a) (4)

(7a)

(7b)

(5a)

(5a) (8a) (8a)

(8c)

(6)

(7c)

(9) (10)

(11)

(12b) (13) (14)

(12a

)

24

Da trn bn m t c im hnh thi cy xoi trn c thu hi huyn

Yn Chu, tnh Sn La; cn c vo cc ti liu phn loi v kha phn loi hin c

nh Danh lc cc loi thc vt Vit Nam, Thc vt ch Trung Quc, Thc vt ch

ng Dng, Thc vt ch Campuchia-Lo-Vit Nam, di s hng dn ca TS.

Nguyn Quc Huy, chng ti gim nh mu xoi trn Yn Chu, Sn La

(HNIP/1782/13) l loi: Mangifera indica L, h Anacardiaceae.

3.1.2. c im vi phu l v cung l xoi trn

Vi phu gn chnh v phin l

Gn chnh:

Gn chnh c lt ct ngang li c hai mt. Lp biu b gm mt hng cc t

bo hnh ch nht xp u n. Ph ngoi lp biu b l mt lp cutin mng. Di

lp biu b l lp m dy trn vi cc t bo hnh bu dc hoc hnh trn. Tinh th

calci oxalat nm ri rc trong cc t bo m dy v m mm. Xen k trong lp m

dy l cc t bo cng v si, l cng gi th s lng cng nhiu c khi to thnh

mt lp gn nh lin tc ngay di lp biu b. mt di, cc t bo cng v si

nm ngay st lp biu b to thnh mt vng lin tc ngay st lp biu b. Lp t

bo m mm vi t bo hnh trn hoc hnh bu dc, thnh mng. Cc b libe- g

cu to cp 2 c kch thc khng u xp thnh vng trn. nh cc b libe- g c

cc m t bo m cng m ly b libe, to thnh mt vnh bn ngoi lp libe.

Phn libe thng c hnh gn trn hoc bu dc, rng gia. Di lp g cp 2 l

m mm rut vi cc t bo hnh trn c kch thc khng u thnh mng.

Phin l:

Bn ngoi l lp biu b gm mt hng t bo hnh ch nht xp u n,

biu b mt trn dy hn mt di. Bn di lp biu b l cc t bo m giu hnh

ch nht di xp vung gc vi lp biu b. Tip l lp m mm hnh bu dc

hoc hnh a gic thnh mng. Tinh th calci oxalat nm ri rc trong cc t bo m

mm.

25

Hnh 3.3 : Vi phu gn chnh v phin l xoi trn.

Hnh 3.4: Mt gc ca vi phu Hnh 3.5: Vi phu phin l xoi trn.

gn l xoi trn

Ch thch: (1): Lp Cutin, (2): Biu b, (3): M dy, (4): T bo m cng v si., (5): M

mm, (6): M cng bao ngoi libe, (7): Libe cp 2, (8): G cp 2, (9:) M mm rut, (10):

M du, (11): Tinh th calci oxalat.

Vi phu cung l

Lt ct cung l c hnh gn ging hnh trn. Ngoi cng l lp cutin mng

bao ngoi, tip n l lp biu b gm mt hng t bo hnh ch nht xp u n.

Lp t bo m dy hnh trn, hnh bu dc, vch t bo dy u (m dy trn). Xen

4

1

1

1

1

1

1

6

7

1

1

1

1

1

1

10

3

5

8

9

10

1

11

2

4

26

gia lp m dy l cc t bo cng v si xp thnh vng gn nh lin tc, l cng

gi th cc t bo si cng nhiu, vng cng lin tc. Di lp m dy l 1-2 hng t

bo m mm hnh trn hoc bu dc thnh mng. C nhiu tinh th calci oxalat

nm ri rc trong cc t bo. Cc b libe-g kch thc khc nhau, xp thnh vng

trn. nh cc b libe-g cng c vng t bo cng m ly b libe nh gn chnh

ca l. Libe cp 2 hnh trn hoc bu dc rng gia. Trong cng l m mm rut.

Hnh 3.6: Vi phu cung l Xoi trn.

Hnh 3.7: Mt gc ca vi phu cung l xoi trn.

Ch thch: (1): Cutin, (2): Biu b, (3): M dy, (4): M cng v si, (5): M mm, (6):

Libe, (7): G, (8): M mm rut, (9): T bo cng m ly b libe-g, (10): Tinh th

calci oxalat.

2

4

5

6

3

9

7

8

10

1

2

27

3.1.3. c im bt l xoi trn

Bt mu xanh lc, mi thm nh, v cht. Soi di knh hin vi nhn thy:

Mnh m mm gm cc t bo hnh bu dc, hnh trn, thnh mng (1); mnh m

mm c mang theo tinh th calci oxalat (2). Mnh mch ch yu l mch im (3),

mch vch (4). Cc mnh mang mu nu nm ri rc (5). Si gm cc t bo di,

thnh dy khoang hp thng tp trung thnh tng b (6). Cc t bo cng thnh

dy, khoang hp (7). Rt nhiu tinh th calci oxalat hnh khi kch thc khc nhau

nm ri rc (8), hoc thnh tng m (9). Mnh biu b (10) v mnh biu b c

mang l kh (11), l kh (12).

Hnh 3.8: Cc c im ca bt l xoi trn.

Ch thch: (1): Mnh m mm, (2): Mnh m mm mang tinh th calci oxalat, (3): Mch

im, (4): Mch vch, (5): Mnh mang mu, (6): B si, (7): T bo cng, (8): Tinh th

calci oxalat hnh khi, (9): m tinh th calci oxalat, (10): Mnh biu b, (11): Mnh biu

b c mang l kh, (12): L kh.

0,1mm

0,1mm

(1) (2)

(10)

(11)

(3)

(4)

(6)

(5)

(7)

(9)

(8)

(12)

28

3.2. Kt qu nghin cu v ha hc

3.2.1. nh tnh cc nhm cht hu c bng phn ng ha hc

3.2.1.1. Flavonoid

Cn 0,5g dc liu cho vo ng nghim ln, thm 15ml cn 90o un trc tip

10 pht. Lc nng qua bng, dch lc thu c dng lm cc phn ng.

- Phn ng vi NH4OH

Nh mt 2 git dch chit cn ln 2 t giy lc, sy kh, em mt t giy lc

h trn ming l NH4OH c thy mu vng ca vt dch chit m v sng hn

hn so vi vt dch chit trn t giy lc cn li. (Phn ng dng tnh).

- Phn ng vi NaOH 10%:

Cho vo ng nghim nh 1ml dch chit, thm 3 git NaOH 10%, thy xut

hin nhiu ta vng. (Phn ng dng tnh).

- Phn ng Cyanidyn

Cho vo ng nghim khong 1ml dch chit, thm mt t bt Mg kim loi,

sau nh vi git HCl c, thy xut hin mu . (Phn ng dng tnh).

- Phn ng vi FeCl3

Cho vo ng nghim 1ml dch chit, nh 2 git FeCl3 5% thy xut hin mu

xanh en. (Phn ng dng tnh).

S b kt lun trong l xoi trn c flavonoid.

3.2.1.2. Coumarin

Cn 10g dc liu cho vo bnh nn 100ml, thm 50ml cn 90, un trc tip

10 pht, lc nng qua giy lc. Dch lc thu c dng lm phn ng.

- Phn ng m ng vng lacton

Cho vo hai ng nghim mi ng 1ml dch chit. Thm vo ng 1 0,5ml

dung dch NaOH 10%. ng 2 nguyn. Sau khi thm kim, ng 1 xut hin ta

vng.

un c hai ng nghim n si. ngui. ng 1 c ta vng. ng 2 trong

Thm vo c hai ng mi ng 2ml nc ct, lc u. ng 1 c ta c. ng

2 trong sut.

29

Acid ha ng 1 bng 5-6 git HCl c, ng 1 vn c ta c, ng 2 trong

sut. (Phn ng m tnh).

Phn ng Diazo ha:

Cho 1ml dch chit vo ng nghim, thm 2ml dung dch NaOH 10%. Nh 5

git thuc th Diazo mi pha, lc u, un nng cch thy, thy xut hin mu .

(Phn ng dng tnh).

Phn ng hunh quang:

Nh 1 git dch chit ln giy lc, sy kh, nh mt git NaOH 10% chng

ln trn, sy kh. Che mt phn vt bng mt ng xu kim loi, t di nh sng

UV 366nm. Phn khng b che pht quang mu vng xanh. Sau 1 pht, b ng xu

kim loi ra thy phn b che v phn b che pht quang ging nhau. (Phn ng m

tnh).

Vi thng hoa:

Cho mt t dc liu vo np nhm. t ln bp hng ngoi, cho bay hi ht

hi nc trong dc liu. t ln np nhm mt phin knh, trn phin knh mt

t bng tm nc lnh, sau 5pht ly phin knh ra ngui, nh thm mt git

thuc th KI 10% ln phin knh. Soi di knh hin vi khng thy c tinh th.

(Phn ng m tnh).

S b kt lun trong l xoi trn khng c coumarin.

3.2.1.3. Saponin

Hin tng to bt: Cn 10g dc liu cho vo bnh nn dung tch 100ml,

thm 30ml nc, un si trc tip 10 pht, lc nng qua bng. Cho 5ml dch lc

vo mt ng nghim ln, thm 5ml nc lc mnh trong 5pht, ct bt tn ti trong

khong 6 pht. (Phn ng m tnh).

S b kt lun trong l xoi trn khng c saponin.

3.2.1.4. Alcaloid

Cn 10g bt l xoi cho vo bnh nn 100ml, thm 30ml H2SO4 1N, un si

20 pht. ngui, lc dch chit vo bnh gn dung tch 100ml, kim ha bng

NH4OH 6N n pH kim, th bng giy qu. Chit bng 5ml CHCl3 x 3 ln, gn ly

30

lp CHCl3. Sau chit tip vi dung dch 5ml H2SO4 x 2 ln. Dich chit thu c

chia u vo 3 ng nghim nh. Nh vo tng ng nghim ln lt 3 git cc thuc

th sau:

ng 1: TT Mayer. Qan st khng thy ta trng. (Phn ng m tnh).

ng 2: TT Bouchardart. Quan st khng thy ta nu. (Phn ng m tnh).

ng 3: TT Dragendoff. Quan st khng thy ta vng. (Phn ng m tnh).

S b kt lun l xoi trn khng c alcaloid.

3.2.1.5. Anthranoid

Cn 5g dc liu cho vo bnh nn thm 25ml H2SO4 1N, un si trc tip 5

pht, lc nng qua bng vo bnh gn 50ml. ngui, thm 5ml ethyl ether, lc

nh, gn b lp nc, gi li lp ether, c v 1ml, thm 1ml dung dch NaOH 10%,

lc nh. Lp nc khng thy xut hin mu sim. (Phn ng m tnh).

S b kt lun trong l xoi trn khng c athranoid.

3.2.1.6. Tannin

Cho vo ng nghim ln 1g bt l xoi, thm 10ml nc ct, un si trc

tip trong 10pht. Lc nng qua bng. Dch lc thu c em lm cc phn ng:

- Phn ng vi genlatin 1%

Cho vo ng nghim nh 1ml dch lc, thm 3 git gelatin 1%. Thy xut

hin ta bng trng. (Phn ng dng tnh).

- Phn ng vi ch acetat 10%

Cho vo ng nghim nh 1ml dch lc, thm 3 git ch acetat thy xut hin

kt ta bng vng ti. (Phn ng dng tnh).

- Phn ng vi FeCl3

Cho vo ng nghim 1ml dch lc, thm 3 git FeCl3 thy xut hin ta en.

(Phn ng dng tnh).

S b kt lun l xoi trn c tannin.

3.2.1.7. Cht bo, carotenoid, sterol

Cn 10g bt l xoi cho vo bnh nn c nt mi dung tch 100ml, thm

40ml ether du ha, y kn, ngm trong 24h, lc qua giy lc, dch chit thu c

31

dng lm phn ng.

* nh tnh cht bo

Nh dch chit ln mt mnh giy lc, h n kh, khng thy c vt m

trn giy. (Phn ng m tnh)

S b kt lun l xoi trn khng c cht bo.

* nh tnh steroid-tritepenoid

Cho vo mt ng nghim nh 5ml dch chit, c cch thy n cn. Ha tan

cn bng 1ml anhydride acetic, nghing ng nghim 45o nh t t theo thnh ng

nghim 1ml H2SO4 c, thy xut hin vng nu tm. (Phn ng dng tnh).

S b kt lun l xoi trn c sterol-triterpenoid.

* nh tnh carotenoid

Cho vo mt ng nghim nh 3 git dch chit, c cch thy n cn, thm

khong 3 git H2SO4 c, lc nh, thy xut hin mu xanh ve. (Phn ng dng

tnh).

S b kt lun l xoi trn c carotenoid.

3.2.1.8. ng kh, acid amin, polysacharid

Cn 5g bt l xoi cho vo bnh nn, thm 20ml nc. un si trc tip 10

pht, lc nng qua bng. Dch lc thu c em tin hnh phn ng xc nh s c

mt ca ng kh, acid amin, polysacharid:

* Acid hu c

Cho vo ng nghim nh 1ml dch chit, thm vo ng nghim mt t tinh

th Na2CO3. Khng thy xut hin bt kh. (Phn ng m tnh).

S b kt lun trong l xoi trn khng c acid hu c.

* ng kh

Cho vo ng nghim nh 1ml dch chit, thm vo ng nghim 3 git thuc

th Fehling A v 3 git thuc th Fehling B, un cch thy 10 pht thy xut hin

ta mu gch. (Phn ng dng tnh).

S b kt lun trong l xoi trn c ng kh.

* Polysacharid

32

ng 1: cho vo ng nghim 2ml dch chit + 5 git thuc th Lugol.

ng 2: 2ml nc ct + 5 git thuc th Lugol.

ng 3: 2ml dch chit.

Mu ca ng 1 m hn mu ca ng 2 nhng khng m hn mu ca ng 3.

(Phn ng m tnh).

S b kt lun trong l xoi trn khng c polysacharid.

* Acid amin

Ly 2ml dch lc cho vo ng nghim nh, thm khong 5 git thuc th

Nynhidrin 3%. un cch thy 10 pht, khng thy xut hin mu xanh tm. (Phn

ng m tnh).

S b kt lun l xoi trn khng c acid amin.

Nhn xt: Cc kt qu nh tnh bng phn ng ha hc cho thy trong l xoi trn

c cc nhm hp cht: flavonoid, tanin, ng kh, steroid-triterpenoid, carotenoid.

3.2.2. nh tnh dch chit l xoi trn bng sc k lp mng

Chun b dch chm sc k

- Dung dch th (T): cn 0,5g bt l xoi cho vo ng nghim nh, thm 10ml

methanol, un si trc tip trong 10 pht. Lc nng vo bnh nh mc 10ml, b

xung methanol cho th tch. Dung dch thu c dng chm sc k.

- Dung dch chun (C): Cn 2,5mg mangiferin chun vo bnh nh mc10ml, ha

tan bng methanol nng, b sung th tch. Dung dch thu c dng lm dch

chm sc k.

iu kin sc k:

Bn mng silicagel GF 254 hot ha 110 oC trong 30 pht.

a ln bn mng 5L dung dch th v dung dch chun.

H dung mi khai trin: (1). CHCl3-EtOAc-HCOOH [4:8:1,5].

(2). EtOAc-HCOOH-H2O [10:1,5:1].

Bng 3.2 v hnh 3.9 trnh by kt qu SKLM khi trin khai bn SKLM vi

h dung mi EtOAc-HCOOH-H2O [10:1,5:1]:

33

Bng 3.2: Cc vt (Rf, mu sc) trn sc k ca dch chit l xoi trn khai

trin vi h dung mi EtOAc-HCOOH-H2O [10:1,5:1].

STT Rf Quan st bn sc k lp mng

nh sng thng UV 254 UV 365 TT NH4OH

1 0,34 (-) (+) Nu Nu

2 0,41 Vng nu nht (+) Vng Nu m

3 0,58 (-) (+) Vng nu Nu tm

4 0,63 (-) (+) Vng ti Nu tm

5 0,77 (-) (+) Tm Vng nu

6 0,82 (-) (+) Tm Tm

7 0,89 Xanh (+) Xanh

8 0,97 Xanh (+) Xanh

Hnh 3.9: Sc k dch chit l xoi khai trin h dung mi EtOAc-

HCOOH-H2O [10:1,5:1].

Ch thch: (a): nh sng thng, (b): UV 254, (c): UV 365, (d): Hi NH4OH.

34

Nhn xt: Trn sc k dch chit l xoi trn khai trin h dung mi EtOAc-

HCOOH-H2O [10:1,5:1] nhn thy c 8 vt trong c 1 vt c Rf v mu sc

tng ng vi vt mangiferin chun. Chng t trong l xoi trn c mangiferin.

3.2.3. Kho st kh nng chit xut mangiferin ca EtOH bng sc k lp

mng

Bng SKLM, chng ti s b xc nh c l xoi trn Yn Chu c

mangiferin. Trn sc k cho thy vt cht mangiferin trong l xoi trn ln hn

so vi vt mangiferin chun (1,25g). V dung mi methanol c c tnh vi thn

kinh, nn bc u kho st kh nng chit mangiferin t l xoi chng ti thay th

methanol bng dung mi ethanol c th s dng dung mi ny chit xut quy

m cng nghip.

Chng ti chun b dch chit l xoi bng dung mi ethanol vi 3 nng

EtOH 50o, 70

o, 90

o. Dch chit c chun b tng t iu kin SKLM trn.

Bn mng c khai trin vi h dung mi EtOAc-HCOOH-H2O [10:1,5:1].

Hnh 3.10: Hnh nh sc k ca dch chit dc liu bng dung mi EtOH

ba nng 50o,70o, 90o, khai trin h dung mi EtOAc-HCOOH- H2O

[10:1,5:1].

Ch thch: (a): nh sng thng, (b): UV 254, (c): UV 365, (d): Hi NH4OH.

35

Kt qu cho thy khi quan st sc k cc iu kin khc nhau vt cht

mangiferin t dung mi EtOH 70o to nht, m nht, so vi 2 dch chit EtOH 50 o

v 90 o.

3.2.4. nh lng mangiferin trong l xoi trn bng phng php HPLC

Xy dng ng chun mangiferin

Chun b dung mi pha mu (DMPM): EtOH 96%-dung dch HCl 2% (7:1).

Cch pha 1000ml dung dch HCl 2%: ly 48ml dung dch HCl c(36-38%)

cho vo bnh cha 500ml nc, lc u, thm nc va 100ml. Lc k.

Dung dch HCl 2M:

Cch pha 1000ml dung dch HCl 2M: Ly 170ml dung dch HCl c (36-

38%) cho vo bnh cha 500ml nc, lc u. Thm nc va 1000ml. Lc k.

Chun b dy dung dch chun:

Cn 25,28mg mangiferin chun ha tan nng trong 30ml DMPM, cho vo

bnh nh mc 50ml c nt mi, b sung th tch thu c dung dch 1 c nng

0,5056mg/ml. Ly 1ml dung dch 1 cho vo bnh nh mc dung tch 50ml, b

xung DMPM cho th tch thu c dung dch 2. Ly 1ml dung dch 2 cho vo

bnh nh mc 50ml, thm DMPM cho th tch thu c dung dch 3. Ly 1ml

dung dch 3 thm pha ng n vch, lc qua mng lc 0,45m, thu c dung dch

4 c nng 4,0448g/ml. Ly 25ml dung dch 4 pha long 4 ln bng pha ng

trong bnh nh mc 100ml, lc qua mng 0,45m thu c dung dch 5 c nng

1,0112g/ml. Ly 25ml dung dch 5 pha long bng pha ng trong bnh nh mc

50ml thu c dung dch 6 c nng 0,5056g/ml.

Dung dch 4, 5, 6 c phn tch bng HPLC xy dng ng chun.

iu kin sc k:

Ct BDS Hypersil C18 (250x4,6mm; 5m); Pha ng: Acetonitril-Dung

dch CH3COOH 3% (14:86); Lu lng dng 1,0ml/pht; B phn pht hin:

Detector UV- VIS. Bc sng: 257nm. Th tch tim mu 20l.

Kt qu: Din tch pic ca cc dung dch chun c th hin trong bng sau:

36

Bng 3. 3: Din tch pic (S) ca dy dung dch chun cc nng khc nhau.

STT Dung dch Nng (g/ml) Din tch pic (mAU)

1 1 4,0448 286,5

2 2 1,0112 66,6

3 3 0,5056 34,2

Phng trnh tuyn tnh: y = 0,0139 x+0,0538. H s tng quan r2= 0,9998.

Trong : x: din tch pic thu c (mAU), y: nng dung dch (C).

Hnh 3.11: th ng chun mangiferin.

nh lng mangiferin trong l xoi bng phng php HPLC

Chun b 3 mu dung dch M1, M2, M3 l cc mu dch chit l xoi gi vi

dung mi ethanol 3 nng 90o,70o, 50o theo qui trnh chit xut c m t

trong phn phng php mc 2.4.2. Cc mu thu c tin hnh phn tch HPLC

trong cng iu kin khi xy dng ng chun.

T din tch pic, s dng phng trnh tuyn tnh trn ta tnh c nng

v hm lng mangiferin :

Bng 3.4: Kt qu nh lng mangiferin trong l xoi trn khi chit bng

dung mi EtOH cc nng 90o, 70o v 50o.

y = 0.0139x + 0.0538 R = 0.9998

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 100 200 300 400

Nn

g

m

an

gif

erin

tro

ng d

nu

g d

ch

C (

g/m

l)

Din tch pic S (mAU)

37

STT Mu l

Khi lng

Dc liu

m (g)

S1

(mAU1)

S2

(mAU 2)

Nng TB

C (g/ml)

Hm lng

mangiferin

1 M1 1,0120 62,8 61,7 0,9216 5,32%

2 M2 1,0117 80,6 81,6 1,1845 6,47%

3 M3 1,0116 78,4 80,2 1,1594 6,33%

Nhn xt: T bng s liu nhn thy mu M2 c nng mangiferin cao

nht, mu M1 c nng mangifrein thp nht, chng t dung mi ethanol 70o l

dung mi chit tt nht trong cc nng ethanol kho st.

Kho st hm lng mangiferin trong cc mu l xoi

Chun b 2 dung dch M4 v M5 theo th t l cc mu dch chit l bnh t

v l xoi non vi dung mi EtOH 70o theo qui trnh chit xut c m t trong

phn phng php mc 2.4.2. Cc mu thu c em phn tch bng HPLC cng

iu kin. Kt qu thu c c nh sau:

Bng 3.5: Kt qu nh lng mangiferin trong cc mu M4, M5.

STT Mu

l

m

(%)

Khi lng

dc liu

m (g)

S1

(mAU1)

S2

(mAU2)

Nng

mangiferin

C (g/ml)

Hm lng

mangiferin

(%)

2 M4 8,95 1,0125 94,5 94,1 1,3686 7,39%

3 M5 10,51 1,0127 93,2 93,2 1,3532 6,78%

Nhn xt: Tt c cc mu l xoi u c hm lng mangiferin kh cao (6-

7%). Trong hm lnng mangiferin trong l bnh t cao nht, tip n l non, l

gi c hm lng mangiferin thp nht.

3.2.5. Chit xut mangiferin t l xoi trn

3.2.5.1. Chit xut v tinh ch

Kt qu nh lng mangiferin trong l xoi trn cho thy hm lng

mangiferin l bnh t l cao nht so vi l gi v l non. trnh nh hng n

38

qu trnh pht trin cng nh nng sut cho qu ca cy xoi, ng thi tn dng

ngun nguyn liu ph thi t nng nghip, chng ti s dng l xoi gi nghin

cu chit xut v phn lp mangiferin.

Chit xut: Cn 100g l xoi gi sy kh, nghin nh ( m 9,87%) cho

vo bnh chit hi lu vi 500ml ethanol 70o, trong 2h. Lc nng gi li b thu

c dch chit ln 1. Thm tip 300ml ethanol 70oC, chit hi lu trong 2h, lc

nng, thu dch chit ln 2. Tip tc lm nh vy vi ln th 3, thu dch chit ln 3.

Gp dch chit, c c v 500ml, nhit phng qua m cho mangiferin ta

xung. Lc ly ta th, thu c 2,4979g ta th.

Tinh ch: Ra ta nhiu ln bng n-hexan n khi dch n-hexan khng mu.

Cn thu c bay hi ht n-hexan nhit thng sau phn tn vo 50ml

ethanol 50o, lc vi 100ml chloroform x 4 ln, gan b lp chloroform. Pha ethanol-

nc em c cch thy 80oC n cn, thu c 1,6836g cn. Ha tan cn vo mt

lng ti thiu ethanol 70o, lc qua giy lc, lnh qua m 4-8oC

mangiferin kt tinh li, lc ly tinh th, ra bng cn lnh. Kt tinh li 2 ln s thu

c 0,9327g cht tinh khit gi l M.

Hiu sut ca ton b qu trnh t 15,99%.

3.2.5.2. Kim tra tinh khit v s b nhn dng M bng sc k lp mng

Cht M c ha tan trong methanol tuyt i chm SKLM so snh vi

dung dch mangiferin chun 0,25mg/ml trong methanol.

iu kin sc k:

Bn mng GF 254 hot ha 100oC trong 1h.

H dung mi: (1). Et2O-HCOOH-H2O [2:0,1:0,05]

(2). EtOAc-HCOOH-H2O [2:0,3:0,2]

(3). BuOH- HCOOH- H2O [2:0,2:0,1]

Thuc th hin mu: hi NH4OH.

Khi quan st di nh sng UV 254, UV 365 v sau khi hin mu bng hi

NH4OH u thy trn sc k ca cht M ch c 1 vt vi v tr v mu sc tng

39

ng vi v tr v mu sc vt ca mangiferin chun. C th s b kt lun cht M

tinh khit.

Bng 3.6: Gi tr Rf v mu sc cc vt trn sc k ca cht M v

mangiferin chun cc iu kin khc nhau

H DM Rf nh sng thng UV 254 UV 365 Hi NH4OH

(1) 0,13 Khng nhn thy

vt (+) Vng sng Vng nu (2) 0,48

(3) 0,59

(a) (b) (c) (d)

Hnh 3.11: Hnh nh sc k cht M so snh vi mangiferin chun, khai trin

h dung mi Et2 O- HCOOH-H2O [2:0,3:0,2].

Ch thch: (a): nh sng thng, (b): UV 254, (c): UV 365, (d): Hi NH4OH.

3.2.5.3. Nhn dng cht M

Cm quan: Tinh th hnh kim mu vng nht.

Hnh 3.12: Tinh th cht M.

40

Tnh cht vt l: Tan c trong hn hp cn- nc, methanol-nc, tan nh

trong methanol, ethanol.

Nhn dng bng ph 1H-NMR v 13C-NMR

Bng 3.7: Ph 1H, 13C-NMR ca M v mangiferin.

V tr

carbon

Ph NMR ca M

(DMSO-d6)

Ph NMR ca mangiferin

(MeOD)

c

(ppm) H (ppm) c (ppm) H (ppm)

1 161.7 13.74 (1-OH) 161.7 13.80 (1-OH)

2 107.6 -- 107.5 --

3 163.8 -- 163.8 --

4 93.3 6.37, s 93.3 6.40, s

4a 156.2 -- 156.2 --

4b 101.3 -- 101.2 --

5 102.6 6.86, s 102.4 6.88, s

6 150.8 -- 150.9 --

7 143.7 -- 143.9 --

8 108.0 7.38, s 107.8 7.41, s

8a 111.7 -- 111.4 --

8b 154.0 -- 154.6 --

CO 179.1 -- 179.0 --

1 73.1 4.59, d, 9.5 Hz 73.1 4.60, d, 8.3 Hz

2 70.2 4.04, t, 8.5 Hz 70.3 4.03, t, 9.5 Hz

3 78.9 3.16, m 79.0 3.16, m

4 70.6 3.16, m 70.6 3.16, m

5 81.5 3.16, m 81.5 3.16, m

6 61.4 3.40, m

3.68, d, 11.5 Hz 61.4

3.40, dd, 11; 2.1 Hz;

3.60, dd, 11; 4.6 Hz

41

Da vo cn c d liu ph cng hng t ht nhn 1H, 13C-NMR, so snh

vi cc d liu ph cng b cho php kt lun cht thu c l mangiferin.

3.3. Bn lun

V c im thc vt

Nghin cu ca chng ti m t c im hnh thi v xc nh tn khoa

hc cho cy xoi trn Yn Chu, Sn La l loi Mangifera indica, gp phn lm

phong ph hn c s d liu v cc loi xoi ti Vit Nam. Mc d hnh dng qu

xoi trn Yn Chu trn v ngn hn so vi m t trong ti liu Cy c Vit Nam

tuy nhin vic xc nh tn khoa hc ch yu c da trn cu to ca hoa xoi

[8].

V cc kt qu nghin cu v c im vi hc: Vi phu l v cung l xoi

trn c rt nhiu t bo cng v si nm xen k trong lp m dy u, b libe gn

thnh hnh trn hoc bu dc, rng gia, bao quanh libe l mt vnh t bo cng,

tinh th calci oxalat nm ri rc trong m mm v m dy. Bt l xoi trn c rt

nhiu tinh th calci oxalat hnh khi kch thc khng u nm ri rc hoc thnh

m, cc t bo cng thnh dy khoang hp. Nhng c im c trng ny l c s

cho vic xy dng tiu chun kim nghim dc liu sau ny.

V thnh phn ha hc

Cc kt qu v nh tnh ha hc cho thy trong l xoi trn Yn Chu, Sn

La c flavonoid, tanin, steroid-triterpen, carotenoid, ng kh. Khng c

coumarin, saponin, anthranoid, alkaloid, cht bo, polysarchrid, acid amin, acid hu

c. Tuy nhin, trn th gii ngi ta phn lp c cc hp cht saponin

(Indicosid A v B), coumarin (mangcoumarin), cht bo, acid amin t cc b phn

khc trn cy xoi (M. indica).

V sc k lp mng chng ti kho st v tm ra h dung mi: EtOAc-

HCOOH-H2O [10:1,5:1] c kh nng tch tt cc thnh phn trong dch chit

methanol ton phn ca l xoi. Kt qu nh tnh bng sc k lp mng cng mt

ln na khng nh s c mt ca mangiferin trong l xoi trn (Mangifera indica

L.). iu ny hon ton ph hp vi cc nghin cu trc y v loi M. indica.

42

Bn cnh , chng ti cng tin hnh kho st kh nng chit xut

mangiferin bng hn hp dung mi ethanol-nc cc nng khc nhau, kt qu

cho thy ethanol 70o l dung mi chit xut tt nht. y l mt ng gp mi, to

c s ban u cho vic nghin cu xy dng qui trnh chit xut trn qui m cng

nghip.

Sau mt thi gian nghin cu chng ti chit xut v tinh ch c

mangiferin tinh khit t l xoi trn. Qui trnh chit xut s dng dung mi ethanol

70o v phng php chit nng tng t cc nghin cu trc y. Qui trnh tinh

ch s dng phng php kt tinh n gin. Dung mi chit xut v tinh ch ch

yu l EtOH, thn thin vi mi trng do c th nghin cu v ng dng vo

chit xut trn qui m cng nghip. Hiu sut ca ton b qu trnh chit xut v

tinh ch t 15,99%, thp hn so vi cc nghin cu trc y [9], [33]. Mangiferin

l mt xanthonnoid c nhiu tc dng sinh hc ang c quan tm nh: chng vi

rt c bit l vi rt herpet (hin ti trn th trng dc phm th gii cng nh

Vit nam c rt nhiu sn phm phc v cho iu tr bnh do loi vi rt ny gy

nn), chng i tho ng, chng vim, iu ha min dch, khng khun chng

oxy ha D liu ph 1H-NMR v 13C-NMR thu c ng gp vo c s d liu

ph so snh nhn dng mangfierin sau ny.

Cc kt qu nh lng bng HPLC cho thy hm lng mangiferin trong l

xoi trn Yn Chu-Sn La kh cao (6-7%), cao hn so vi loi quo Sn La (4-

5%) trong mt nghin cu ca Hng Lan [10]. Chng ti nhn thy y l mt

dc liu qu c tim nng a vo chit xut mangiferin trn qui m cng nghip.

Mt khc, hng nm c mt s lng ln l xoi trn b i t sn xut nng nghip.

Nu ngun nguyn liu l xoi ny c tn dng chit xut mangiferin s lm

tng thm gi tr v hnh thnh chui gi tr lin hon cho cy xoi. Phng php

HPLC l mt phng php c thi gian phn tch ngn, ng , chnh xc v

nhy cao hn nhiu so vi phng php o ph hp th UV-VIS. Cc cht c

phn tch ra trong qu trnh nh lng nn trnh c nh hng ca cht mu

cng nh cc tp cht khc trong dch chit c liu. Cng vi s pht trin ca

43

khoa hc k thut, phng php ny ngy cng c ng dng rng ri trong cc

lnh vc kim nghim ni chung v kim nghim dc liu ni ring. Vic s dng

phng php ny trong kim nghim dc liu s gip nh gi tt hn cht lng

ca dc liu trc khi a vo s dng.

44

Chng 4: KT LUN V XUT

4.1. Kt lun

Sau thi gian lm thc nghim chng ti thu c nhng kt qu sau:

V thc vt

m t c im hnh thi v gim nh tn khoa hc cho cy xoi trn thu

hi ti huyn Yn Chu, Sn La l loi Mangifera indica L., h Anacardiaceae.

M t chi tit c im vi phu gn l, cung l v c im bt l ca cy

xoi trn Yn Chu, Sn La gp phn lm c s cho vic kim nghim v tiu

chun ha dc liu sau ny.

V ha hc

Bng phn ng ha hc xc nh c trong l xoi trn Yn Chu c cc

thnh phn: flavonoid, tanin, carotenoid, steroid-triterpen, ng kh.

Kt qu nh tnh bng sc k lp mng c so snh vi mangiferin chun,

khai trin h dung mi EtOAc-HCOOH-H2O [10:1,5:1] cho thy trong l xoi trn

Yn Chu, Sn La c mangiferin.

kho st kh nng chit xut mangiferin ca hn hp dung mi EtOH-

H2O bng SKLM v HPLC, nhn thy EtOH 70o l dung mi c kh nng chit

mangiferin tt nht.

Bng phng php HPLC, kho st hm lng mangiferin trong l xoi

trn Yn Chu, Sn La: l bnh t c hm lng mangiferin cao nht (7,39%), tip

n l l xoi non (6,78%) v l xoi gi (6,47%).

tin hnh chit xut, tinh ch v xc nh c cu trc ca mangiferin t

l xoi trn Yn Chu, Sn La.

4.2. xut

Do thi gian nghin cu c hn nn ti ca chng ti ch nghin cu c

nhng bc u v loi xoi trn Yn Chu, Sn La. ngh tip tc nghin cu

tm ra qui trnh chit xut v tinh ch mangiferin c hiu sut cao trn qui m cng

nghip

45

TI LIU THAM KHO

Ti liu ting Vit

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3. B mn Dc liu Trng ai hc Dc H Ni (2010), Thc tp dc liu,

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4. B mn Thc Vt Trng i hc Dc H Ni (2007), Thc vt hc, Nh xut

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6. V Vn Chi (2007), Sch tra cu tn cy c Vit Nam, NXB Gio Dc, H Ni.

7. Bi Th Hng (1991), nh lng mangiferin bng phng php sc k lng cao

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8. Phm Hong H (2000), Cy c Vit Nam, Quyn II, NXB Tr, Tp H Ch Minh.

9. Phm Gia Khi v Phm Xun Sinh (1991), Nghin cu chit xut v xc nh

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10. Hng Lan (2002), Nghin cu s tch ly v bin ng hm lng

Mangiferin trong qu trnh sinh trng v pht trin ca cy quo Sn La v tip

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11. Tt Li (2011), Nhng cy thuc v v thuc Vit Nam, NXB Thi i, H

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12. L Th Thy (2001), Gp phn nghin cu thnh phn ha hc trong cy Quo

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46

13. Vin Dc Liu (2003), Cy thuc v ng vt lm thuc Vit Nam, Tp II,

NXB Khoa hc k thut, H Ni.

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50

PH LC

Ph lc 1: Phiu gim nh tn khoa hc v phiu chng nhn m s tiu bn ca

cy xoi trn Yn Chu, Sn La.

Ph lc 2: Pic sc k v ph hp th t ngoi ca dung dch mangiferin chun 1ppm.

Ph lc 3: Pic sc k ca cc dung dch M1, M2, M3, M4, M5.

Ph lc 4: Ph 1H-NMR v 13C-NMR ca cht M.

51

Ph lc 1:

Phiu gim nh tn khoa hc v phiu chng nhn m s tiu

bn ca cy xoi trn Yn Chu, Sn La.

52

53

54

Ph lc 2:

Pic sc k v ph hp th t ngoi ca ca dung dch

mangiferinchun 1ppm.

55

56

57

Ph lc 3:

Pic sc k ca cc dung dch M1, M2, M3, M4, M5.

58

59

60

61

62

63

Ph lc 3:

Ph 1H-NMR v

13C-NMR ca cht M.

64

65

66

67