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B Y T

TRNG I HC DC H NI

BI HNG TI

NGHIN CU C IM THC VT,

THNH PHN HA HC V

TC DNG KHNG KHUN L CY

HNG XIM

KHA LUN TT NGHIP DC S

H NI 2014

B Y T

TRNG I HC DC H NI

BI HNG TI

NGHIN CU C IM THC VT,

THNH PHN HA HC V

TC DNG KHNG KHUN L CY

HNG XIM

KHA LUN TT NGHIP DC S

Ngi hng dn:

1. PGS. TS. V Vn in

2.ThS. Ch Th Thanh Huyn

Ni thc hin:

1. B mn Dc c truyn

2. B mn Vi sinh-Sinh hc

H NI 2014

LI CM N

hon thnh ti ny, ti xin chn thnh cm n PGS.TS. V Vn in

Ngi thy tn tnh hng dn, ng vin v gip ti trong sut qu trnh

thc hin ti.

Ti cng xin chn thnh cm n ThS. Ch Th Thanh Huyn tn tnh hng

dn ti thc hin kha lun.

ng thi ti xin gi li cm n su sc ti:

PGS.TS. Cao Vn Thu gip ti thc hin th tc dng khng khun.

TS. Nguyn Quc Huy gip ti xc nh tn khoa hc ca mu cy

nghin cu.

Cc thy c gio trong b mn Dc C Truyn, i hc Dc H Ni.

Cm n nh trng to iu kin gip ti hon thnh ti.

Cm n gia nh bn b ng vin gip ti trong sut qu trnh thc hin

ti ny.

Xin chn thnh cm n!

H Ni, thng 05 nm 2014

Sinh vin

Bi Hng Ti

MC LC

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

CHNG I. TNG QUAN......................................................................................... 2

1.1. V TR PHN LOI, C IM THC VT V PHN B .................... 2

1.1.1. V tr phn loi ca chi Manilkara .............................................................. 2

1.1.2. c im chung ca h Hng xim (Sapotaceae) ...................................... 2

1.1.3. c im chi Manilkara .............................................................................. 2

1.1.4. c im thc vt v phn b ca cy hng xim ..................................... 3

1.2. THNH PHN HA HC CY HNG XIM............................................. 4

1.3. TC DNG SINH HC ................................................................................... 5

1.4. TNH V, CNG NNG ................................................................................... 6

1.5. CNG DNG .................................................................................................... 7

CHNG 2. I TNG V PHNG PHP NGHIN CU .......................... 8

2.1. NGUYN LIU, PHNG TIN NGHIN CU ........................................ 8

2.1.1. Nguyn liu nghin cu .............................................................................. 8

2.1.2. Phng tin nghin cu ............................................................................... 8

2.2. NI DUNG NGHIN CU ............................................................................ 10

2.2.1. Nghin cu v c im thc vt .............................................................. 10

2.2.2. Nghin cu v ha hc .............................................................................. 10

2.2.3. Th tc dng khng khun ........................................................................ 10

2.3. PHNG PHP NGHIN CU ................................................................... 10

2.3.1. Nghin cu v c im thc vt .............................................................. 10

2.3.2. Nghin cu v ha hc .............................................................................. 10

2.3.3. Th tc dng khng khun ........................................................................ 11

CHNG 3. THC NGHIM V KT QA ........................................................ 12

3.1. NGHIN CU V THC VT..................................................................... 12

3.1.1. c im dc liu l cy hng xim ....................................................... 12

3.1.2. c im vi phu l ................................................................................... 12

3.1.3. c im bt l .......................................................................................... 13

3.2. NGHIN CU V HA HC ....................................................................... 14

3.2.1. nh tnh cc nhm cht c trong dc liu bng phn ng ha hc ...... 14

3.2.2. Chit xut ................................................................................................... 21

3.2.3. nh tnh cc phn on bng SKLM ....................................................... 25

3.2.4. Phn lp cht t cn phn on Ethyl acetat ............................................ 31

3.3. TH TC DNG KHNG KHUN ............................................................ 39

3.3.1. Chun b ..................................................................................................... 39

3.3.2. Tin hnh ................................................................................................... 40

3.4. BN LUN ..................................................................................................... 42

KT LUN V KIN NGH .................................................................................... 44

1

T VN

Cy hng xim c ngun gc t Nam M, ch yu Mhic. Hin nay c di

thc vo nhiu nc nhit i. chu , hng xim c trng nhiu nht ti Thi

Lan, Philippin, Malaysia, Indonesia, n , Campuchia, Vit Nam [14]. Vit

Nam, ch yu c trng min Nam, nhng min Bc cng c trng nhiu

ni. Cy trng ch yu ly qu. Nha chit t v v qu hng xim s dng

trong thc phm v dc phm, l cy hu nh khng c s dng [14], [11].

Hin nay, Vit Nam cc nghin cu v cy hng xim rt hn ch c bit

nghin cu v l cy hu nh cha c. Tuy nhin nc ngoi c kh nhiu nghin

cu v thnh phn ha hc cng nh hot tnh sinh hc ca l cy. Vi mc ch

nghin cu tm hiu kh nng khai thc v s dng l cy hng xim, chng ti

tin hnh nghin cu ti Nghin cu c im thc vt, thnh phn ha hc

v tc dng khng khun ca l cy hng xim c thc hin vi mt s mc

tiu sau:

1. Nghin cu c im hnh thi, vi phu v bt l cy hng xim.

2. Nghin cu cc nhm cht v phn lp cht t l cy hng xim.

3. Th hot tnh khng khun l cy hng xim.

thc hin nhng mc tiu ra, ti c tin hnh vi cc ni dung sau:

1. Nghin cu c im thc vt l cy hng xim thu hi Ngh An: M t

c im hnh thi, c im vi phu l, c im bt l.

2. Xc nh cc nhm cht c trong l cy v cc phn on dch chit bng

phn ng ha hc v sc k lp mng.

3. nh lng s b khi lng cc phn on.

4. nh hng phn lp cht sch t phn on ethyl acetat.

5. Th hot tnh khng khun dch chit ton phn.

2

CHNG I. TNG QUAN

1.1. V TR PHN LOI, C IM THC VT V PHN B

1.1.1. V tr phn loi ca chi Manilkara

Cy hng xim c tn khoa hc l Manilkara zapota (L.) P.Van Royen [5].

Tn ng ngha: Achras zapota L. [5].

Tn nc ngoi: Sapota, sapodilla plum (Anh), sapotier, sapotillier (Php) [14],[17].

Chi Manilkara, h Hng xim (Sapotaceae), b Hng xim (Sapotales), phn lp

S (Dilleniidae), lp Ngc lan (Magnolisida), ngnh Ngc lan (Magnoliophyta).

Ngnh Ngc lan (Magnoliophyta)

Lp Ngc lan (Magnolisida)

Phn lp S (Dilleniidae)

B Hng xim (Sapotales)

H Hng xim (Sapotaceae)

Chi Manilkara [5], [1].

1.1.2. c im chung ca h Hng xim (Sapotaceae)

H Hng xim hay cn gi l h Xabch [8], [1].

Cy g, l n, thng mc cch, khng c l km hay l km nh rng sm.

Cy thng c m trng, hoa lng tnh, cnh hoa thng c phn ph bn hay

lng, vng nh ngoi thng l nh lp. Hoa thnh b, b nh gm 2-3 vng, b

nhy gm 4-12 l non, qu mng, ht c ni nh du [1], [6].

Vit Nam c 16 chi, trn 40 loi [1].

1.1.3. c im chi Manilkara

Chi Manilkara gm xp x 79 loi phn b khp cc vng nhit i (30 loi

Nam v Trung M, 35 loi chu Phi v 14 ng Nam ) [15].

Cy g, l n, thng mc cch, khng c l km hay l km nh rng sm.

Cy thng c m trng, hoa lng tnh, cnh hoa c ph b lng, tiu nhy bng

s cnh hoa, c tiu nhy lp. Hoa c 6 l i, 6 cnh hoa, ht dp, di [8].

3

Chi Manilkara c cha triterpenes, saponin v flavonoid. Dch chit c hot tnh

khng sinh, dit k sinh trng, chng cn trng c hi, khng cholinesterase [18].

1.1.4. c im thc vt v phn b ca cy hng xim

1.1.4.1. c im thc vt

nh ton cy Cnh mang hoa

Qu Ht

Hnh 1.1. Hnh nh mt s b phn cy hng xim

Cy to, cao 10-15m, phn cnh nhiu [14], m trng, v xm nu, l b trn [5].

Cnh v l khi cn non c ph lng t. L mc gn nhau cht nhnh [5], mc

so le, dy v dai, hnh trng hoc tri xoan, gc thun, u t hoc hi nhn, mt

trn mu lc bng, mt di nht, gn ph nhiu, xp song song, u n [14], cch

nhau 4-6 mm [5].

Hoa mc n c k l, lng tnh mu vng nht, i 6 rng, xp thnh hai

hng, ph lng mu vng, trng 6 cnh, nh 6, nh lp nhiu, bu 10-12 [14], [5].

Qu mng hnh trng, v mng mu nu, rn ko di mu nu nht v nhp, ht dt,

mu en, nhn hai u [14], [11]. Qu c ng knh 4-8 cm, cha 2-5 ht [30].

Ma hoa: Thng 5-8, ma qu: Thng 9-11 [14].

4

1.1.4.2. Phn b

Cy c ngun gc Trung M v Nam M, ch yu Mhic [14], [19], [24],

[31]. Hin nay c di thc vo nhiu nc nhit i. chu , hng xim c

trng nhiu nht ti Thi Lan, Philippin, Malaysia, Indonesia, Srilanca, n ,

Campuchia v Vit Nam [14], [17]. Vit Nam, cy c trng hu ht cc tnh

t Bc vo Nam, tr vng ni cao trn 1000m. Hng xim l cy a kh hu nhit

i nng m, vi nhit thch hp t 24 n 30C. Cy trng cc tnh pha Bc

c th chu c nhit di 10C v ma ng. Cy khng mc c vng kh

hn [14].

1.2. THNH PHN HA HC CY HNG XIM

L v v thn cha terpenoid, flavonoid, glycosid [21], [28].

L cy hng xim cha steroid, triterpenoid, terpenoid, phenol, coumarin,

tanin, glycosid tim, saponin, anthraquinon [26], flavonoid (apigenin-7-O--L-

rhamnosid, myricetin-3-O--L-rhamnosid [24]), acid bo trong acid bo khng

no chim 32,32% (thnh phn chnh l: acid oleic 13,95%, acid linoleidic 10,18%

v acid linoleic 5,96%) [17], [24], acid oleanolic, lupeol acetat [17]), acid bo no

chim 59,95% (acid palmitic 48,01%, acid stearic 8,3%), hydrocarbon chim

89,303% cc hp cht khng x phng ha, thnh phn chnh n-Triacontan 49,50%

v n-octacosan 23,83%, 2 sterol (sitosterol 3,40%, stigmasterol 2,61%) [24].

V cy cha 11,8% cht tanin [14], [17], [20], saponin v mt lng nh

alkaloid kt tinh gi l sapotin [5] v cht nha m. Nha m khi c c, khi

lng gim cn 30-33%, c mu trng sau chuyn thnh mu hng. Nha m

cha 44,8% cht nha, 17,2% carbohydrat, 3,4% cht gm v 9% ng. Khi tinh

ch cht nha m bng cch ra nhiu ln vi kim mnh, sau trung ha v sy

kh, s thu c mt cht bt v nh hnh, khng tan trong nc [14].

Qu cha carbohydrat, protein, cht bo, canxi, phosphor, st, acid ascorbic,

fructose 4,47-7,13%, sucrose 1,48-8,75%, tng s ng 11,14-20,43%, tinh bt

2,98%, tanin 3,16-6,45% [17], [24], [29], polyphenols (methyl chlorogentat,

5

dihydromyricetin, quercitrin, myricitrin, catechin, epicatechin, gallocatechin, gallic

acid) [22].

Qa hng xim cn xanh v v thn cha cht dch sa, trong gm chim

20-25% v nhiu tanin [14], [17]. Qu chn cha 14% ng m thnh phn ch

yu l sacharose, dextrose v levulose [17].

Ht cha 1% saponin v 0,08% sapotinin [14], [17], du bo v acid

cyanhydric [14], alkaloid, phenol (Quercitol [20], [21]), flavonoid [27].

1.3. TC DNG SINH HC

Hot tnh khng khun:

V thn v l: Dch chit ethanol, methanol v nc c hot tnh khng

khun vi 4 gram (+) (Bacillus subtilis BTCC19, Bacillus megaterium BTCC18,

Bacillus cereus ATCC258 and Sarcina lutea ATCC27803), 5 gram (-) (Escherichia

coli ATCC25922, Shigella sonnei ATTC8992, Shigella shiga ATCC27853, Shigella

dysenteriae ATCC561 and Salmonella typhi ATCC14228 v 5 chng nm

(Aspergillus flavus ACCT10558, Aspergillus fumigatus ATTC10231, Candida

albicans ATTC25889, Vasianfactum sp ATTC235561, Fusarium sp ACCT56390)

[21], [25], [28].

Dch chit methanol, ethanol c hot tnh mnh hn dch chit nc [25], [28].

Ht: Dch chit aceton c hot tnh khng khun vi Vibrio cholerae MTCC

3906, Salmonella paratyphi A, Shigella flexneri MTCC 1457, Staphylococcus

epidermidis MTCC 435 [25], [28].

H ng huyt:

Dch chit nc v cn ca l cy hng xim gim ng k nng ng

trong mu so vi tc dng ca metformin. Tuy nhin dch chit khng thay i ng

k nng ng trong mu ca nhng con chut bnh thng. C ch hot ng

c th tng t ca biguanid (metformin) [24], [28].

H cholesterol:

Dch chit nc v dch chit cn l lm gim ng k mc cholesterol trong

mu chut gn vi atorvastatin [24].

6

Tc dng chng oxy ha:

Dch chit ethanol, methanol, aceton v nc t l c tc dng chng oxy

ha trn chut. Trong dch chit methanol tc dng yu, dch chit aceton c tc

dng chng oxy ha mnh [16].

Dch chit methanol, aceton, ethanol, ethyl acetat, nc ca ht hng xim

c tc dng chng oxy ha, trong dch chit cn c tc dng mnh hn cng

nng [19]. Tc dng chng oxy ha ca cy hng xim ch yu do polyphenolic

[16] (gallocatechin, catechin) [28].

Tc dng dit giun sn:

Dch chit ethanol v chloroform phi ht hng xim c tc dng lm t lit v

dit giun sn (sau 8 v 23 pht vi dch chit chloroform, sau 3,3 v 7 pht vi dch

chit ethanol (12,5 mg/ml) [24].

Tc dng gim au :

Dch chit methanol v ether du ha l cy hng xim vi liu 200mg/kg c tc

dng gim au 96,82% v 94,27% trn chut [28], c ngha thng k (p0,001) so

vi nhm chng [23].

c tnh

LD50 ca dch chit nc v cn l hng xim l 8g/kg [24].

Liu hiu qu ti thiu 8mg/kg cn nng theo ng tim di da v 16mg/kg

cn nng theo ng ung [24].

1.4. TNH V, CNG NNG

Qu hng xim chn c v ngt, tnh mt, c tc dng b, mt, sinh tn dch, gii

kht, nhun trng [14].

Qu xanh c v cht, tnh bnh, c tc dng lm sn [14].

7

1.5. CNG DNG

Hng xim hin nay ch yu c trng ly qu n. Nhng cng dng khc

hu nh cha c ch .

Qu hng xim chn tr to bn, mi ln n 3-5 qa. Qu xanh v v cy tr

tiu chy, kit l [14], [17] vi liu 15-20g, sc ung [14]. Ht l thuc li tiu [16],

h st, mi ln 6 ht nghin thnh bt, ung vi ru [14], [19], [25], cha mt ng

[17]. Liu cao gy c v kh i [14].

V cy c tc dng khng khun, lm se, gii nhit v cha tiu chy [25],

[11], [19], [28], [16], ngy ung 6 n 12g [11].

Nha chit t cy hng xim c tn l chicle c dng nhiu trong cng

nghip thc phm v dc phm. Nha ly t v cy c gi l chicle covent,

nha thu t qu gi l chicle blanco hay chicle vergen. Nha ny tiu th rt nhiu

M, Canada ch to cao su [14], [17], dng trong phu thut nha khoa [28].

Cht nha c dng lm ko bc h, ko hi [11] c khi c thm cht pepsin

vi tn chewing gum, dng lm thuc cha ho, gip tiu ha hoc lm thm ming

[11], [28].

L sc ung hng ngy gim huyt p [17], cha ho, cm lnh, tiu chy

[16].

8

CHNG 2. I TNG V PHNG PHP NGHIN CU

2.1. NGUYN LIU, PHNG TIN NGHIN CU

2.1.1. Nguyn liu nghin cu

Mu nghin cu

Mu ti: L cy ti ra sch, dng lm vi phu v nghin cu c im thc vt.

Mu kh: L cy hng xim sau khi lm sch, c sy kh nhit di 60C:

- Tn thnh bt mn dng soi bt.

- Xay th, bo qun trong ti nilon kn, ni kh ro dng nghin

cu ha hc v th tc dng khng khun.

Ni thu hi: Thanh Chng Ngh An.

Thi im thu hi: 11/2013.

Mu dc liu c TS. Nguyn Quc Huy B mn thc vt Trng H

Dc H Ni gim nh tn khoa hc l Manilkara zapota (L.) P. Royen, h

Hng xim (Sapotaceae).

2.1.2. Phng tin nghin cu

Cc thit b dng trong nghin cu

T sy dc liu SHELLAB.

My xc nh m SATORIUS.

Cn k thut Satorious.

Cn phn tch Precisa.

My o ph t ngoi UV-VIS Spectrophotometer carry.

Knh hin vi Labomed.

H thng my chm sc k: thit b bm mu t ng (CAMAG-

LIMONAT5), my nn kh, phn mm winCATS, videoSCAN.

My nh Sony 12.1.

My sy Blustone.

My ct quay thu hi dung mi BUCHI Rotavapor R-200.

9

Ha cht v dng c

Ha cht v thuc th s dng trong nghin cu c mua ti H Ni, t

tiu chun tinh khit phn tch theo Dc in Vit Nam IV [9].

Ha cht: Javen, acid acetic, xanh methylen, carmin

Dung mi hu c: Chloroform, methanol, ethanol, ethyl acetat, n-hexan,

toluen, acid formic

Thuc th: Cc thuc th dng trong phn ng nh tnh v sc k.

Bn mng trng sn silica gel GF254 ca Merck.

Dng c: Dng c thy tinh v cc b dng c khc dng trong phng th

nghim nh: cc c m, bnh nn, a thy tinh, chy, ci, lam knh, ng nghim

Mi trng dng th hot tnh khng khun

MT canh thang nui cy VSV kim nh: NaCl 0,5%, pepton 0,5%, cao tht

0,3%, nc v 100ml.

MT thch thng: NaCl 0,5%, pepton 0,5%, cao tht, thch 1,6%, nc v

100ml.

Chng vi sinh vt

10 chng vi sinh vt (5 gram (+), 5 gram (-)) t tiu chun c ly b mn Vi

sinh-Sinh hc trng i hc Dc H Ni.

Nhm gram (+):

- Staphylococcus aureus ATCC 1128 (Sta)

- Bacillus subtilis ACTT 6633 (Bs)

- Bacillus cereus ACTT 9341 (Bc)

- Bacillus pumilus ATCC 10241 (Bp)

- Sarcina lutea ATCC 9341 (SL)

Nhm gram (-):

- Escherichia coli ATCC 25922 (EC)

- Pseudomonas aeruginosa VM 201 (Pseu)

- Salmonella typhi DT 220 (Sal)

10

- Shighella flexneri DT 112 (Shi)

- Proteus mirabilis BV 108 (Pro)

2.2. NI DUNG NGHIN CU

2.2.1. Nghin cu v c im thc vt

Nghin cu c im bt v vi phu l hng xim.

M t c im dc liu l hng xim.

2.2.2. Nghin cu v ha hc

nh tnh cc nhm cht chnh trong l cy hng xim bng phn ng ha

hc.

Chit phn on, xc nh hm lng cc phn on v nh tnh cc phn

on dch chit bng sc k lp mng.

nh hng phn lp cht sch bng sc k ct.

2.2.3. Th tc dng khng khun

Th tc dng khng khun ca 3 loi cao lng cn 3 nng 1:1, 1:2, 1:4.

2.3. PHNG PHP NGHIN CU

2.3.1. Nghin cu v c im thc vt

Nghin cu c im hnh thi:

Quan st, chp nh, m t c im l.

Nghin cu c im vi hc:

- Nghin cu c im vi phu v bt l theo ti liu: [3], [4], [12].

- Quan st cu to vi phu l v bt l bng knh hin vi.

- Chp nh cc c im vi hc bng my nh.

2.3.2. Nghin cu v ha hc

- nh tnh cc nhm cht hu c chnh trong dc liu bng cc phn ng

ha hc v bng sc k lp mng theo ti liu: [2], [3], [7], [10].

- Phn lp cc cht bng sc k ct theo ti liu [3].

11

2.3.3. Th tc dng khng khun

Tin hnh th tc dng khng khun ca cao lng cn 1:1, 1:2, 1:4 vi cc

chng vi khun Gram (+), Gram (-) theo phng php khuch tn trong mi trng

thch (dng khoanh giy tm dd th) [13].

Nguyn tc: Mu th c tm vo khoanh giy lc v trng, t ln trn

lp thch dinh dng cy VSV kim nh, trong iu kin nui cy thch hp.

Hot cht t trong mu th khuch tn vo MT thch v c ch s pht trin

ca VSV kim nh, to thnh vng v khun xung quanh mu th. o

ng knh vng v khun (nu c) nh gi kt qu.

nh gi kt qu: Da trn ng knh vng v khun v c nh gi

theo cng thc :

=

SD=

Trong : : ng knh trung bnh vng v khun.

Di: ng knh vng v khun.

SD: lch thc nghim chun c hiu chnh.

n: S th nghim lm song song (n=3).

Hnh 2.1. S t khoanh giy tm mu th trn thch.

Ch thch: 1: Mu cao lng 1:1

2: Mu cao lng 1:2

3: Mu cao lng 1:4

C: Khng sinh chun

12

CHNG 3. THC NGHIM V KT QA

3.1. NGHIN CU V THC VT

3.1.1. c im dc liu l cy hng xim

Hnh 3.1. nh chp l cy hng xim

L hnh tri xoan, gc thun, u t hoc hi nhn, di t 5 cm n 13 cm. Phin

l nguyn, dy, mp nguyn. Mt trn mu lc bng, mt di nht. Cung l nhn,

di 4-15 mm. Gn ph nhiu, xp song song, u n, cch nhau 4-5 mm. Th cht

dai. Khng mi, v cht, hi ng.

3.1.2. c im vi phu l

- Phn gn l: Gn l hai mt li, mt trn li t, mt di li nhiu. Biu b trn

(11) v biu b di (1) gm 1 lp t bo hnh ch nht, xp u n, ha cutin. M

dy (2) gm 5-6 hng t bo hnh trng, thnh dy. M mm v (3) gm cc t bo

thnh mng, hnh gn trn, kch thc khng u, xp ln xn. Vng b si cha

cc tinh th canxi oxalat (4) hnh khi, bao quanh b libe-g. Libe-g to thnh b

hnh cung, libe (5) ngoi, g (6) trong, cc mch g xp thnh hng u n.

M mm rut (7) gm cc t bo thnh mng, khng u, xp ln xn bn trong b

libe-g (Hnh 3.2.b).

- Phn phin l: Biu b trn (11), biu b di (8) gm cc t bo nh xp u

n thnh 1-2 hng. M du (10) gm 1-2 hng t bo hnh ch nht xp vung

gc vi cc hng t bo biu b. M khuyt nm ri rc trong phin l (9) (Hnh

3.2.a).

13

3.1.3. c im bt l

Bt mu xanh lc, khng mi, v ng. Soi di knh hin vi (Hnh 3.3) thy c

cc c im:

Tinh th canxi oxalat hnh khi (1), mnh biu b (2), mnh mch dn (3), b si

(4), mnh m mm (5), mnh mang mu (6), mnh biu b mang l kh kiu song

bo (7), mnh mch dn cha tinh th canxi oxalat (8) .

Hnh 3.2. nh chp vi phu l cy hng xim di KHV

Ghi ch : a. Phin l: 8. Biu b di 10. M du

9. M khuyt 11. Biu b trn

b. Gn l: 1. Biu b di 5. Libe

2. M dy 6. G

3. M mm v 7. M mm rut

4. B si cha tinh th

14

Hnh 3.3. nh chp mt s c im bt l cy hng xim

Ghi ch: 1. Tinh th canxi oxalat hnh khi 5. Mnh m mm

2. Mnh biu b 6. Mnh mang mu

3. Mnh mch dn 7. Mnh biu b mang l kh

4. B si 8. B si cha tinh th canxi oxalat

3.2. NGHIN CU V HA HC

3.2.1. nh tnh cc nhm cht c trong dc liu bng phn ng ha hc

3.2.1.1. nh tnh flavonoid

Cn khong 10g bt dc liu cho vo bnh nn, thm 50ml ethanol 90. un

cch thy 10 pht, lc nng qua giy lc. C dch lc ti cn (1), ha tan cn vo

nc nng, lc nng. C dch nc ti cn (2), ha tan cn vo cn 90 ly dch lm

cc phn ng sau:

Phn ng Cyanidin

Cho vo ng nghim nh 1 ml dung dch th. Thm mt t bt magnesi kim loi

(khong 10mg). Nh tng git HCl c (3-5 git). yn mt vi pht. Quan st

thy dung dch chuyn t mu vng sang mu thm Phn ng dng tnh.

15

Phn ng vi kim long

Cho vo ng nghim nh 1 ml dung dch th. Thm vi git dung dch NaOH

10%. Quan st thy xut hin ta vng, thm 1ml nc ct, ta s tan v mu vng

ca dung dch s c tng thm Phn ng dng tnh.

Phn ng vi hi amoniac (NH3)

Nh mt git dch chit ln giy lc. H kh ri ln ming l amoniac lng

c m nt, quan st thy mu vng ca vt dch chit c tng ln. Nh mt

git khc lm chng Phn ng dng tnh.

Phn ng vi FeCl3 5%

Cho vo ng nghim nh 1ml dung dch th. Thm vi git dung dch FeCl3 5%,

quan st thy xut hin ta mu xanh en Phn ng dng tnh.

Phn ng diazo ha

Cho 1ml dung dch th vo ng nghim, kim ha bng dung dch NaOH 10%,

thm vi git thuc th diazo mi pha, lc u, un nng nh. Quan st thy xut

hin mu vng cam Phn ng dng tnh.

S b kt lun: Dc liu c cha flavonoid.

3.2.1.2. nh tnh coumarin

Vi thng hoa

Cho mt t bt dc liu vo np chai bng nhm. t ln bp in c li amian,

cho bay hi ht nc trong dc liu. t trn ming np nhm mt phin knh,

trn c t t bng tm nc lnh. un nh di np nhm, sau 5 pht ly phin

knh ra, ngui, t lam knh ln, soi di knh hin vi thy tinh th hnh kim

khng mu. Nh thm 1 git dd KI 10% ln phin knh, soi di knh hin vi thy

tinh th hnh kim mu tm Phn ng dng tnh.

Cho 10g bt dc liu vo bnh nn c dung tch 100ml, thm 50ml ethanol

90%. un cch thy 5 pht, lc nng qua giy lc, dch lc thu c dng tin

hnh cc phn ng sau:

Phn ng m ng vng lacton

Cho vo 2 ng nghim, mi ng 1-2 ml dch chit ethanol.

16

+ ng 1: Thm 0,5ml dung dch NaOH 10%.

+ ng 2: nguyn.

un c 2 ng nghim trn ni cch thy vi pht, quan st thy:

+ ng 1: Xut hin ta c.

+ ng 2: Trong sut.

Thm vo 2 ng nghim 2ml nc ct, lc u thy:

+ ng 1: Tr li trong sut.

+ ng 2: Thy xut hin ta c.

Thm vo ng 1 vi git HCl c thy ng 1 c tr li Phn ng dng tnh.

Phn ng chuyn dng ng phn cis-trans:

Nh 2 git dch chit ln t giy lc, nh tip 1 git dung dch NaOH 10%, sy

nh cho n kh. Che 1 na din tch bng mt mnh kim loi, soi di n t

ngoi trong vi pht, b mnh kim loi ra. Quan st thy phn khng b che sng

hn phn b che Phn ng dng tnh.

S b kt lun: Dc liu c cha coumarin.

3.2.1.3. nh tnh Glycosid tim

Cho 10g bt dc liu vo bnh nn c dung tch 100ml, thm 50ml ethanol 25%,

lc u, ngm qua m. Thm vo dch chit 3ml ch acetat 30%, khuy u, lc

qua giy lc gp np vo mt cc c m dung tch 100ml. Nh vi git dch lc u

tin vo ng nghim, thm mt git ch acetat. Nu xut hin ta th ngng lc,

thm 1ml ch acetat 30% vo dch chit, khuy u, lc li. Tip tc th n khi

dch chit khng cn ta vi ch acetat na. Chuyn ton b dch lc vo bnh gn

125 ml. Lc k 2 ln vi hn hp chloroform - ethanol (4:1), mi ln 8ml. Gn lp

chloroform vo mt cc c m dung tch 100ml c sy kh. Gp dch chit

chloroform li. Cho dch chit vo 3 ng nghim c sy kh, em c cch

thy n cn. Cn thu c lm cc phn ng:

17

Phn ng Liebermann Burchard

Thm vo ng nghim 1ml anhydrid acetic, lc k cho tan ht cn, ng

nghing 450, cho t t theo thnh ng 0,5ml H2SO4 c, dch trong ng nghim

chia thnh 2 lp.

Kt qu: mt tip xc gia hai lp cht lng xut hin vng mu tm , cht

lng pha di mu hng, pha trn mu xanh l Phn ng dng tnh.

Phn ng Baljet

Chun b thuc th Baljet: Cho vo ng nghim 1 phn dd acid picric 1% v 9

phn dd NaOH 10%, lc u.

Cho vo ng nghim cha cn 1ml ethanol 90%, lc u cho tan ht cn, nh

tng git TT Baljet mi pha.

Kt qu: Thy dung dch xut hin mu cam Phn ng dng tnh.

Phn ng Keller Kiliani

Cho vo ng nghim 0,5ml ethanol 90%, lc u cho tan ht cn, thm vi git

dd FeCl3 5% trong acid acetic, lc u, nghing ng 450, thm t t theo thnh ng

0,5ml H2SO4 c.

Kt qu: mt tip xc gia 2 lp cht lng c vng mu tm , lc nh, lp

cht lng trn c mu xanh l Phn ng dng tnh.

S b kt lun: Dc liu cha glycosid tim.

3.2.1.4. nh tnh Saponin

Quan st hin tng to bt: Cho 0,5g bt dc liu vo ng nghim to, thm

5ml nc, un si nh, lc nng. Dch lc cho vo ng nghim to, thm 5ml nc,

lc mnh trong 5 pht theo chiu dc ng nghim. yn 15 pht, quan st thy bt

bn vng sau hn 15 pht Phn ng dng tnh.

S b kt lun: Dc liu c cha saponin.

3.2.1.5. nh tnh Alcaloid

Cho khong 2g bt dc liu vo bnh nn dung tch 100ml, thm m dc liu

bng dd NH3 6M. y kn bnh trong vng 30 pht. Chit hi lu chloroform trong

30 pht, lc ly dch chit cho vo bnh gn. Lc dch chit 2 ln vi dd H2SO4 1N

18

(mi ln 5ml). phn lp, gn ly dch chit nc, cho vo 3 ng nghim nh,

mi ng 1ml, tin hnh cc phn ng sau:

Phn ng vi TT Mayer: Thm 2-3 git thuc th Mayer, khng thy xut

hin ta trng Phn ng m tnh.

Phn ng vi TT Dragendroff: Thm 2-3 git thuc th Dragendroff, khng

thy xut hin ta da cam Phn ng m tnh.

Phn ng vi TT Bouchardat: Thm 2-3 git thuc th Bouchardat, khng

thy xut hin ta nu Phn ng m tnh.

S b kt lun: Dc liu khng c alcaloid.

3.2.1.6. nh tnh tanin

Chun b mu th: Cho vo bnh nn dung tch 100ml khong 2g bt dc liu,

thm 20ml nc ct, un si trn bp qua li amian trong khong 10 pht. Lc

nng qua giy lc gp np. Ly dch lc lm cc phn ng.

Tin hnh phn ng: Ly 3 ng nghim, cho vo mi ng 2 ml dch lc.

+ ng 1: Thm 2 git FeCl3 5%, xut hin ta mu xanh en Phn ng dng

tnh.

+ ng 2: Thm 2 git ch acetat 10%, khng xut hin ta bng Phn ng m

tnh.

+ ng 3: Thm 5 git gelatin 1%, xut hin ta trng c Phn ng dng tnh.

S b kt lun: Dc liu c tanin.

3.2.1.7. nh tnh ng kh

Chun b mu th: Cho 2g bt dc liu vo ng nghim to, thm 10 ml nc

ct, un si trc tip 5 pht. Lc qua giy lc gp np. Ly dch lc tin hnh phn

ng.

Tin hnh: Cho vo ng nghim 1 ml dch lc, 3 ml dd Fehling A (TT) v 3 ml

dd Fehling B (TT), lc k, un cch thy vi pht thy xut hin kt ta gch

Phn ng dng tnh.

S b kt lun: Dc liu cha ng kh.

19

3.2.1.8. nh tnh anthranoid

Phn ng Borntrager:

Cho 3g bt dc liu vo bnh nn dung tch 100ml, thm 50ml dd H2SO4 10%.

un si cch thy trong 15 pht. Lc nng vo bnh gn. ngui, lc vi 5ml

chloroform, yn phn lp, gn ly lp chloroform tin hnh phn ng.

Ly 1ml dch chit cho vo ng nghim, thm 1ml NaOH 10% vo lc nh,

khng thy lp nc c mu hng Phn ng m tnh.

S b kt lun: Dc liu khng c anthranoid.

3.2.1.9. nh tnh acid hu c

Ly 1g bt dc liu cho vo ng nghim ln, thm 10ml nc ct. un si trc

tip 10 pht trn ngn la n cn, ngui, lc. Ly 2ml dch lc cho vo ng

nghim to. Thm mt t Na2CO3 vo dch lc, lc nh, khng c bt kh Phn

ng m tnh

S b kt lun: Dc liu khng cha acid hu c.

3.2.1.10. nh tnh polysaccharid

Ly 2g bt dc liu cho vo cc c m, thm 10ml nc ct, un si cch thy

trong 5 pht, lc nng ly dch lc. Cho vo 2 ng nghim:

ng 1: 2ml dch lc v 2 git TT Lugol.

ng 2: 2ml nc ct v 2 git TT Lugol.

Quan st thy hai ng c mu nh nhau Phn ng m tnh

S b kt lun: Dc liu khng c polysaccharid.

3.2.1.11. nh tnh acid amin

Ly 2g bt dc liu cho vo ng nghim ln, thm 10ml nc ct. un si trc

tip 10 pht trn ngn la n cn, ngui, lc. Cho vo ng nghim 2 ml dch lc

v 3 git thuc th Ninhydrin 3%, un cch thy 10 pht, xut hin mu tm

Phn ng dng tnh.

S b kt lun: Dc liu c acid amin.

20

3.2.1.12. nh tnh cht bo, caroten, sterol

Cho 5 g bt dc liu vo bnh nn dung tch 50ml, ngp ether du ha, un

cch thy 15 pht. Lc ly dch lc lm phn ng.

nh tnh cht bo

Nh mt git dch chit ln giy lc, h nng cho bay ht dung mi, thy li

vt m trn giy lc Phn ng dng tnh.

S b kt lun: Dc liu c cht bo.

nh tnh sterol

Cho vo ng nghim 1ml dch chit trn, bc hi dung mi n kh, thm vo

ng nghim 1ml anhydrid acetic. Lc k ri nghing ng nghim 450, thm 1 ml

H2SO4 c theo thnh ng nghim, mt phn cch c mu nu Phn ng

dng tnh.

S b kt lun: Dc liu c sterol.

nh tnh caroten:

Cho vo ng nghim to 2ml dch chit trn, bc hi trn ni cch thy n cn,

thm vi git H2SO4 c vo cn, dch lng c mu xanh Phn ng dng tnh.

S b kt lun: Dc liu c caroten.

Kt qu nh tnh cc nhm cht trong dch chit ton phn l cy hng xim c

trnh by bng 3.1.

Bng 3.1. Kt qu nh tnh cc nhm cht trong l cy hng xim.

STT Nhm cht Phn ng nh tnh Kt

qu

Kt lun

1 Flavonoid - Phn ng cyanidin

- Phn ng vi FeCl3 5%

- Phn ng vi NaOH 10%

- Phn ng vi hi NH3

- Phn ng diazo ha

+

+

+

+

+

C

2 Coumarin - Phn ng m ng vng lacton

- PU chuyn dng ng phn cis - trans

+

+

C

21

- Vi thng hoa

+

3 Glycosid tim

- Phn ng Baljet

- Phn ng Lieberman-Buchardt

- Phn ng Legal

+

+

+

C

4 Saponin - Hin tng to bt + C

5 Alcaloid - Phn ng vi TT Mayer

- Phn ng vi TT Dragendorff

- Phn ng vi TT Bouchardat

-

-

-

Khng c

6 Tanin - Phn ng vi FeCl3 5%

- Phn ng vi ch acetat

- Phn ng vi gelatin 1%

+

-

+

C

7 ng kh - Phn ng vi TT Fehling + C

8 Antharanoid - Phn ng orntraeger - Khng c

9 Acid hu c - Phn ng vi bt Na2CO3 - Khng c

10 Polysaccharid - Phn ng vi TT Lugol - Khng c

11 Acid amin - Phn ng vi TT Ninhydrin + C

12 Cht bo - Vt m trn giy lc + C

13 Sterol - Phn ng Liebermann + C

14 Caroten - Phn ng vi H2SO4 c + C

Ch thch:

(-): Phn ng m tnh

(+): Phn ng dng tnh

Kt lun: Qua nh tnh bng phng php ha hc s b kt lun l cy hng

xim c cha cc nhm cht sau: Flavonoid, saponin, tanin, glycosid tim, acid

amin, ng kh, coumarin, cht bo, sterol, caroten.

3.2.2. Chit xut

Chun b bt dc liu

L c sy kh 60C, tn thnh bt th ng thi xc nh m dc liu.

22

- Xc nh m ca dc liu:

Lm nh dc liu xc nh m. Bt my o m, iu chnh nhit

110C. khong 1g dc liu vo a cn, tri u dc liu khp a, y np a

cn v ch my t ng hin kt qu trn mn hnh. Tin hnh o 3 mu ly kt qu

trung bnh.

- m ca dc liu: 9,21%.

Phng php v dung mi chit

- Chit theo phng php ngm lnh, 24h rt dch chit mt ln.

- Dung mi chit: Ethanol 70.

Dng c chit

Bnh gn 500ml c kha kn, ra sch, sy kh.

Chit xut cao lng ton phn

Lp bnh gn ln gi, lt 1 lp bng mng y bnh.

Cn chnh xc khong 100,0 gam bt dc liu kh vo cc c m, lm m bng

dung mi, trong 30 pht.

Cho bt dc liu lm m vo bnh gn, dung mi ngp, m kha, rt ly

15ml dch chit, sau tr li bnh gn, thm dung mi ngp dc liu 3cm, y

nt kn, ngm lnh.

Sau 24h, rt dch chit thu c dch chit 1 ln. Chit nhiu ln n khi dch

chit khng cn mu, gp dch chit li, ct thu hi dung mi thu c cao lng

cn, thm nc ct thu c cao lng 1:1, un nng n khi tan ht, t lnh 24h,

lc b ta, ly tm c dch trong. Dng dch ny chit cc phn on.

Chit cc phn on

Chit ln lt vi cc dung mi n-hexan, chloroform, ethyl acetat c phn

cc tng dn.

Chit vi n-hexan:

Cho dch chit trn vo bnh gn, thm n-hexan vi lng xp x 2/3 dch chit,

lc k, phn lp gn ly lp n-hexan, thm tip n-hexan, chit nhiu ln n khi

lp n-hexan khng cn mu, gp dich chit n-hexan, thu hi dung mi n khi

23

lng khng i, thu c cn, sy kh cn 60C, cn cn, tnh hm lng so vi

dc liu kh.

Phn dch chit cn li dng chit tip cc phn on sau.

Chit phn on chloroform v ethyl acetat tin hnh tng t nh chit

phn on n-hexan, thu c cc cn tng ng.

Cc cn c k hiu l : - H: Cn chit t n-hexan

- C: Cn chit t chloroform

- E: Cn chit t ethyl acetat

Qu trnh chit c tm tt hnh 3.4.

Qu trnh chit c lp li 3 ln, ly kt qu trung bnh.

Hm lng cht trong cc phn on c tnh theo cng thc sau:

F= a.100/(m.(100-x)) .100%

Trong : a: Khi lng cn (g)

m: Khi lng dc liu ban u (g)

x: m ca dc liu(%)

F: Hm lng cht (%)

Hiu sut cn 3 phn on so vi bt l cy hng xim c trnh by bng 3.2.

Bng 3.2. Hm lng cn 3 phn on chit xut t l cy hng xim.

STT Phn on m(g) x(%) mcn(g) Hm lng(%)

1 n-Hexan 1,0546 1,16

2 Chloroform 100,1054 9,21 2,4937 2,75

3 Ethyl acetat 5,5075 6,07

24

Hnh 3.4. S chit xut cn cc phn on t l cy hng xim.

Dc liu

Ethanol 70, Lc

Dch chit ethanol 70

Cao lng nc

Dch chit n-Hexan Dch chit nc

n-Hexan thu hi

Cloroform thu hi

Dch chit nc Dch chit cloroform

Cloroform

Dch chit nc Dch chit ethyl acetat

Ethyl acetat

Cn C

Nc ct 60C

Ethanol thu hi

Ethyl acetat thu hi

Cn E

Dch chit nc

n-Hexan

Cn H

25

3.2.3. nh tnh cc phn on bng SKLM

Chun b dch chm sc k: Ha tan cn tng phn on trong methanol.

Chun b bn mng: Bn mng trng sn Silica gel GF254 (MERCK), hot ha

100-105C trong 1 gi. ngui v bo qun trong bnh ht m.

Chm v xc nh pic sc k: S dng my CAMAG Linomat 5, phn mm

winCATS, phn mm videoSCAN chm.

Chm dch chit methanol ln bn mng. Sy nh cho kh, t vo bnh sc k

bo ha dung mi. Sau khi trin khai ly bn mng ra khi bnh, sy nh 5 pht

cho bay hi ht dung mi. Pht hin vt di nh sng t ngoi. Sau nhng

thuc th hin mu, sy nhit phng.

Hin mu : - Dd vanillin/ H2SO4 10%.

- Soi di n t ngoi bc sng 254nm, 366nm.

nh tnh cn phn on n- Hexan bng SKLM

- H dung mi khai trin: Tin hnh thm d trn cc h dung mi:

H I: Chloroform : Methanol (9:1).

H II: Chloroform : Methanol: Nc (8:1:1).

H III: Toluen: Ethhyl acetat: Acid formic (8:3:1).

H IV: Chloroform : Aceton : Acid formic (70:25:5).

H V: Toluen: Ethyl acetat: Acid formic (5:6:1).

H VI: Ethyl acetat: chloroform: Acid formic (3 :3 :1).

H VII: Toluen: Ethyl acetat: Acid formic (50:45:10).

Sau nhiu ln trin khai thy h VII tch tt nht.

Kt qu nh tnh bng SKLM ca cn phn on n-hexan khai trin vi h dung

mi VII c trnh by hnh 3.5 v hnh 3.6.

26

Hnh 3.5. nh chp sc k phn on n-hexan khai trin vi h

dung mi VII soi di UV366.

27

Hnh 3.6. Kt qu nh tnh cn phn on n-hexan bng SKLM.

Nhn xt: Sc k : - AST c 3 vt.

- Soi UV254 c 6 vt.

- Soi UV366 c 15 vt, trong 2 vt s 11, 15 c Rf (0,516;

0,861) hp th UV mnh nht.

- Hin mu bng dd vanillin/ H2SO4 10% c 10 vt.

3.4.2. nh tnh cn phn on chloroform bng SKLM




H III: Toluen: Ethyl acetat: Acid formic (8:3:1).




H VII: Toluen: Ethyl acetat: Acid formic (50:45:10).

Sau nhiu ln trin khai thy h III tch tt nht.

Kt qu nh tnh bng SKLM ca cn phn on chloroform khai trin vi h dung

mi III c trnh by hnh 3.7 v hnh 3.8.

28

Hnh 3.7. nh chp sc k phn on chloroform khai trin vi h dung mi

III soi di UV366.

29

Hnh 3.8. Kt qu nh tnh cn phn on chloroform bng SKLM.


- Soi di UV254 c 12 vt.

- Soi di UV366 c 13 vt, trong 3 vt s 5,6,7 c Rf

(0,274; 0,317; 0,357) hp th UV mnh nht.

- Hin mu bng dd vanillin/ H2SO4 10% c 11 vt.

3.4.3. nh tnh cn phn on Ethyl acetat bng SKLM




H III: Toluen: Ethyl acetat: Acid formic (8:3:1).




H VII: Toluen: Etyl acetat: Acid formic (50:45:10).

Sau nhiu ln trin khai thy h V tch tt nht.

Kt qu nh tnh bng SKLM ca cn phn on ethyl acetat khai trin vi h dung

mi V c trnh by hnh 3.9 v hnh 3.10.

30

Hnh 3.9. nh chp sc k phn on ethyl acetat khai trin vi h dung mi V

soi di UV366.

31

Hnh 3.10. Kt qu nh tnh cn phn on Ethyl acetat bng SKLM.


- Soi di UV254 c 7 vt.

- Soi di UV366 c 12 vt, trong 2 s 6, 8 c Rf

(0,457; 0,662) hp th UV mnh nht.

3.2.4. Phn lp cht t cn phn on Ethyl acetat

Phn lp ln 1

S dng phng php sc k ct hp ph vi cht nhi ct l silica gel loi dng

cho sc k ct, c ht 0,063 - 0,2mm (Merck).

Chun b ct

- Ct sc k c kch thc 1,5 cm 30 cm c lp thng ng trn gi, trng

sch bng cn.

- Cht nhi ct silica gel c em hot ha 110C/1h trong t sy, ly ra

ngui trong bnh ht m, cn khong 30g silica gel.

- Nhi ct: Lt 1 lp bng mng y ct. Trn silica gel vi mt lng va

hn hp dung mi Chloroform - Aceton (7:2,5) thnh hn dch, khuy u, a ln

ct, ra thnh ct bng hn hp dung mi trn, ct n nh trong 2-3h. M kha

32

ct, cho ct chy khng ti vi tc 5s/git trong 20-30 pht. ng thi thm

dung mi m bo dung mi lun ngp silica gel.

- Sau khi chy khng ti, ng kha ct, y kn.

Chun b cn

- Cn khong 0,5g cn ethyl acetat, thm 1 lng ti thiu hn hp dung mi

Chloroform-Aceton (7:2,5), thm tip lng va silica gel, phn tn u, cho

hn hp thu c vo t sy 60C/1h to thnh bt kh, mn.

a cn ln ct

- M kha ct cho dung mi chy n khi cch b mt silica gel khong 2 cm,

kha ct.

- Dng pipet sch a t t hn hp cn ethyl acetat v silica gel ln ct.

- Cho t t theo thnh ct hn hp dung mi Chloroform-Aceton (7:2,5) ra

sch cn bm trn thnh ct.

- Khi lp dung mi xung gn st b mt silica gel, cho 1 lp silica gel dy

khong 0,5 cm ln trn, cho tip 1 ming giy lc ln trn b mt lp silica gel.

- Ra gii bng hn hp dung mi: Chloroform-Aceton (7:2,5).

- Tc ra gii: 5 giy/ git.

- Hng dch ra gii vo cc ng nghim, mi ng 2ml.

- ng bay hi t nhin, kim tra dch ra gii bng SKLM vi h dung mi:

Toluen: Etyl acetat: Acid formic (5:4:1).

- Hin mu vt: Hi NH3, vanillin/ H2SO4 c.

Chn cc ng c cng s vt, cng Rf, gp vo vi nhau c cc phn on.

Kt qu:

- T ng 1 n ng 29 trn sc k ging nhau gm 6 vt, gp cc ng li

c phn on 1.


c phn on 2.


c phn on 3.

33


c phn on 4.


c phn on 5.


c phn on 6.

Sau khi gp cc ng, cc phn on bay hi t nhin.

Phn lp ln 2

Tin hnh phn lp ln 2 vi phn on 2 s dng phng php sc k ct hp ph

vi cht nhi ct l silica gel tin hnh tng t ln 1.

- Ct sc k 1 cm 40 cm.

- Dich chit phn on c a ln ct: Ly ton b cn phn on 2 ha tan

trong mt th tch ti thiu dung mi ra gii a ln ct.

- Dung mi ra gii: Tolulen- Ethyl acetat (5:4).

- Tc ra gii: 5 giy/git.

- Hng dch ra gii vo cc ng nghim, mi ng 1ml.

- Kim tra dch ra gii bng SKLM vi h dung mi:

Toluen: Ethyl acetat: Acid formic (5:5:1).

- Thuc th hin mu: Hi NH3, vanillin/ H2SO4 c.

Kt qu:


c phn on 1.


c phn on 2.


c phn on 3.


c phn on 4.

34

Kim tra li cc phn on 1 v 3 bng SKLM

Phn on 1

Phn on 1 c kim tra bng SKLM vi 3 h dung mi:

1. Chloroform: Ethyl acetat: Acid formic (7:2,5:0,5).

2. Toluen: Ethyl acetat: Acid formic (8:3:1).

3. Toluen: Ethyl acetat: Acid formic (5:5:1).

Kt qu kim tra phn on 1 bng sc k lp mng c trnh by hnh 3.11 v

3.12.

Hnh 3.11. nh chp sc k phn on 1 khai trin vi h dung mi s 3,

soi di UV366.

35

1 2 3 4

Hnh 3.12. nh chup sc k phn on 1 khai trin vi h dung mi 1 v 2.

Ch thch: - nh 1, 2, 3: Khai trin vi h dung mi s 1.

- nh 4: Khai trin vi h dung mi s 2.

Nhn xt:

C 3 h u cho kt qu 2 vt trn sc k soi di UV366.

C 2 vt u: - Khng mu AST.

- Hp th UV366 .

- Khng hp th UV254.

2 vt c Rf cch xa nhau, trong h 2 cho Rf ca 2 vt cch xa nhau nht.

Trong 2 vt, vt c Rf = 0,711 hp th mnh UV366.

Phn on 3

Phn on 3 c kim tra vi 3 h dung mi tng t phn on 1.

Kt qu kim tra phn on 3 bng SKLM c trnh by hnh 3.13, 3.14, 3.15.

36


soi di UV366.


soi di UV254.

37

1 2 3 4

Hnh 3.15. nh phn on 3 trin khai vi h dung mi 1 v 2.

Ch thch: - nh 1, 2, 3: Khai trin vi h dung mi s 1.

- nh 4: Khai trin vi h dung mi s 2.

Nhn xt:

C 3 h u cho kt qu 2 vt trn SK soi di UV366.

2 vt c Rf cch xa nhau, trong h 2 cho Rf ca hai vt cch xa nhau nht.

Kt qu so snh 2 vt c trnh by bng 3.3.

Bng 3.3. So snh c im 2 vt trong phn on 3.

Quan st Vt 1 (Rf= 0,804) Vt 2 (Rf = 0,563)

AST Khng mu Vng xanh

UV254 Khng hp th Hp th

UV366 Hp th Khng hp th

S b nhn dng thnh phn 1 v 3

inh tnh bng phn ng ha hc

Cn phn on 1 v 3 c ha tan trong ethanol 90, tin hnh cc phn ng :

- Phn ng cyanidin.

- Phn ng vi dd kim.

Kt qu nh tnh cn phn on 1 v 3 c trnh by hnh 3.16.

38

Hnh 3.16. nh chp kt qu nh tnh phn on 1 v 3.

Ch thch: 1. Mu trng.

2. Phn ng vi kim long.

3. Phn ng cyanidin.

S b kt lun: Phn on 1 khng c flavonoid, phn on 3 c flavonoid.

Kt qu hin mu sc k phn on 3 bng thuc th FeCl3, NH3 c c

trnh by hnh 3.17.

Hnh 3.17. nh chp sc k sau hin mu ca phn on 3.

39

Ch thch: 1.AST.

2.Hin mu bng FeCl3.

3.Hin mu bng NH3 c.

3.3. TH TC DNG KHNG KHUN

3.3.1. Chun b

Chun b ch phm th:

Cao lng chit bng EtOH 70 th 3 nng khc nhau l cao 1:1, cao 1:2,

cao 1:4.

iu ch cao lng: Cn 10g dc liu kh, lm nh, ngm lnh trong cn

70/24h (3 ln), lc, c n cn. Thm 10ml nc ct vo cn, un nng cho tan ht

thu c cao lng 1:1, k hiu C1:1.T C1:1 tin hnh iu ch cao lng 1:2, k

hiu C1:2 v cao lng 1:4, k hiu C1:4 nh sau:

C1:2: Ly 5ml C1:1 vo ng nghim, thm 5ml nc ct, lc u. c

10ml C1:2.

C1:4: Ly 5ml C1:2 vo ng nghim, thm 5ml nc ct, lc u. c

10ml C1:4.

Tin hnh: Cc khoanh giy lc v trng c sy kh, c tm 3 ln vi

dd ch phm th, sau mi ln tm c sy nhit < 60C cho n khi kh ht

DM.

Chun b ging vi sinh vt:

Cy truyn chng t ng gc ln mt ng thch nghing, vo t m 18h

37C, dng que cy tit trng gt vi khun pht trin trn b mt mi trng

vo nc mui sinh l (107 - 108 t bo/ ml).

Chun b mi trng v cy VSV kim nh:

Mi trng thch thng v trng:

Mi trng thch thng c cng thc:

Cao tht 5g

Pepton 10g

NaCl 5g

40

Thch 20g

Nc ct v 1000ml

Pha 800ml mi trng, chia vo 4 bnh nn dung tch 500ml, mi bnh 200ml,

em hp tit trng 121C trong 20 pht. ngui n 45 - 50C ri ln lt cho

mi vi khun kim nh nui cy vo mi bnh nn ni trn vi t l

1ml/200ml. Lc trn VSV kim nh phn tn u trong MT thch thng, ri

vo a petri v trng vi th tch 20ml/a v cho thch ng li.

3.3.2. Tin hnh

t giy lc c tm cht th ln b mt MT thch thng cha VSV kim

nh theo s nh sn (theo chiu kim ng h).

cc a petri c mu th c t nh trn trong t m nhit 37C trong

18 24h, ri ly ra, o ng knh vng v khun (nu c) bng thc kp Panmer

chnh xc 0,02mm.

Hnh nh khng sinh ca dch chit l hng xim c trnh by hnh 3.18.

nh gi kt qu:

ng knh vng v khun c tnh theo cng thc:

=

SD=

Ch thch:

D: ng knh trung bnh vng v khun

Di: ng knh vng v khun

S: lch thc nghim chun c hiu chnh

n: S th nghim lm song song

Khng sinh chun:

- i vi cc Gr (+): S dng Benzathin penicillin 20 IU/ml.

- i vi cc Gr (-) : S dng Gentamycin 20 IU/ml.

Mi mu tin hnh 3 ln, ly kt qu trung bnh.

Kt qu th tc dng khng khun cao lng l hng xim c trnh by bng 3.4.

Bng 3.4. Kt qu th hot tnh khng khun cao lng l hng xim.

41

Mu D s Mu D s

SA 1 11,13 0,38 BS 1 9,63 0,26

2 8,93 0,09 2 8,61 0,49

3 12,07 0,41 3 9,57 0,31

Chun 23,05 0,05 Chun 23,05 0,05

Shi 1 9,91 0,29 Ecoli 1 9,21 0,23

2 8,74 0,44 2 7,43 0,19

3 10,24 0,33 3 9,57 0,42

Chun 23,80 0,30 Chun 10,69 0,49

Sar 1 0,00 0,00 Pro 1 10,43 0,13

2 0,00 0,00 2 8,55 0,77

3 0,00 0,00 1 9,58 0,36

Chun 9,90 0,10 Chun 17,55 0,45

BC 1 10,02 0,41 BP 1 10,94 0,33

2 8,03 0,05 2 10,07 0,10

3 9,95 0,17 3 10,23 0,51

Chun 21,15 0,15 Chun 11,80 0,20

Sal 1 10,70 0,54 Pseu 1 10,43 0,42

2 7,68 0,23 2 10,54 0,74

3 12,80 0,22 3 11,04 0,21

Chun 11,61 0,01 Chun 13,05 0,05

Ch thch:

D: ng knh trung bnh ca vng v khun (trung bnh 3 kt qu).

s: lch chun (sai s thc nghim).

1. Staphylococcus aureus ATCC 1128 (SA)

2. Bacillus subtilis ACTT 6633 (Bs)

3. Bacillus cereus ACTT 9341 (Bc)

4. Bacillus pumilus ATCC 10241 (Bp)

5. Sarcina lutea ATCC 9341 (Sar)

6. Escherichia coli ATCC 25922 (EC)

7. Pseudomonas aeruginosa VM 201 (Pseu)

8. Salmonella typhi DT 220 (Sal)

9. Shighella flexneri DT 112 (Shi)

10. Proteus mirabilis BV 108 (Pro)

42

Hnh 3.18. Hnh nh khng sinh 3 loi cao trn mt s loi vi khun.

Ch thch: 1: Cao lng 1:1

2: Cao lng 1:2

3: Cao lng 1:4

Nhn xt: - C 3 loi cao u c tc dng khng 9/10 chng vi khun th nghim

(tr Sarcina lutea ATCC 9341).

- Cao 1:1 v 1:4 c tc dng mnh hn cao 1:2 .

- ng ch cao lng 1:4 c tc dng khng Salmonella typhi DT 220

mnh hn khng sinh chun nng th nghim.

3.4. BN LUN

Cy hng xim l mt cy rt d trng, l nhiu v hu nh c th thu hi quanh

nm. Hin nay, Vit Nam, l cy hng xim hu nh cha c nghin cu. V

vy, chng ti thc hin ti ny tm kim kh nng khai thc v s dng l

hng xim. V ngoi vic ly qu n, hu nh cc b phn khc u b b i.

L cy c kh nhiu cng dng cha bnh nhng cha c kim chng, v

vy cn tin hnh nghin cu lm c s cho cc nghin cu tip theo v tc dng

sinh hc, khng nh gi tr cha bnh ca v thuc ny.

V thc vt:

- Cu to gii phu l cy hng xim, ngoi cc c im tng t nh cc loi

l khc, gn l c cha vng b si cha tinh th canxi oxalat hnh khi, bao quanh

b libe-g. y l mt im khc bit kim nh l cy hng xim.

- Bt l c nhiu mnh biu b mang l kh kiu song bo, rt d quan st.

43

V ha hc:

- nh tnh dch chit ton phn l cy hng xim Vit Nam c cc nhm cht:

Flavonoid, saponin, tanin, glycosid tim, acid amin, coumarin, ng kh, cht bo,

sterol, caroten. Theo thng tin thu thp c t ti liu [21], [28], [26], [24], [17]

khng cp ti s xut hin ca acid amin, ng kh trong l hng xim c

nghin cu trn th gii.

- Xc nh hm lng cn cc phn on n-hexan, chloroform, ethyl acetat vi

dc liu kh ln lt l: 1,16; 2,75; 6,07. Hm lng cn phn on ethyl acetat

cao hn hn 2 phn on cn li. Cho thy thnh phn c phn cc gn vi

phn cc ca ethyl acetat trong l cy c hm lng tng i ln.

- nh tnh cc phn on bng sc k lp mng u s dng h dung mi c

cng cc cu t Toluen: Ethyl acetat: Acid formic vi t l khc nhau, cho kt qu

tch tng i r trn sc k .

- V phn lp, do kinh ph v thi gian c hn, chng ti mi ch tch c cc

phn on cha 2 cht v kim tra s b thnh phn trong cc phn on. Trong

phn on 3 s b xc nh c cha flavonoid. Cc cht trong cc phn on c Rf

cch xa nhau, c bit khi trin khai vi h dung mi Toluen: Etyl acetat: Acid

formic (8:3:1). y l c s quan trng cho cc ti phn lp cht tinh khit tip

theo.

V tc dng khng khun:

Cao lng cn cc nng khc nhau c tc dng khng 9/10 chng vi khun

th nghim. Kt qu ny tng i ph hp vi cc nghin cu v tc dng khng

khun ca l cy hng xim trn th gii [21], [25], [28].

Kt qu trn cho thy trin vng, kh nng ng dng cao tc dng khng khun

ca l cy trong iu tr bnh. y l c s cho cc ti tip theo tip tc nghin

cu cc loi dch chit khc nh nc, aceton, methanol so snh la chn

dung mi chit cho tc dng khng khun cao nht. ng thi tin hnh th tc

dng khng khun ca cc phn on dch chit cho kt qu chn lc hn

44

KT LUN V KIN NGH

Kt lun

Sau mt thi gian thc nghim, chng ti thu c nhng kt qu sau:

V thc vt:

- m t v phn tch c im hnh thi l hng xim.

- xc nh c im vi phu l, c im bt l, gp phn chun ha v kim

nghim dc liu chng nhm ln khi s dng.

V ha hc:

- nh tnh trong l cy hng xim c cc nhm cht: Flavonoid, saponin, tanin,

glycosid tim, acid amin, coumarin, ng kh, cht bo, sterol, caroten.

- tin hnh nh tnh cc phn on bng SKLM cho kt qu:

- Xc nh c hm lng ca cn 3 phn on so vi bt l cy hng xim.

- Bc u xc nh c phng php phn lp c cc phn on ch cha 2

cht t phn on ethyl acetat v nh tnh s b thnh phn bng SKLM v phn

ng ha hc. To c s cho cc nghin cu phn lp cht tinh khit.

V tc dng khng khun:

- th tc dng khng khun trn 10 chng vi khun th nghim ca

3 loi cao lng 1/1, 1/2, 1/4 .

45

Kin ngh:

Do thi gian v iu kin nghin cu c hn, nhng nghin cu trn y ca

chng ti mi ch l bc u. V vy, khng nh gi tr s dng ca dc liu

ny, chng ti kin ngh cc ti sau tip tc thc hin:

Nghin cu phn lp cc hp cht tinh khit v xc nh cu trc cc hp

cht.

Nghin cu th tc dng sinh hc ca cc hp cht phn lp c.

46

TI LIU THAM KHO

I. TING VIT

1. Nguyn Tin Bn (1997), Cm nang tra cu v nhn bit cc h thc vt ht

kn Vit Nam, NXB nng nghip, trang 28.

2. B mn Dc liu (2004), Bi ging dc liu tp I, Trung tm thng tin- th

vin H Dc H Ni.

3. B mn dc liu (2012), Phng php nghin cu dc liu, Trng i

hc Y- Dc TP H Ch Minh.

4. B mn Thc Vt (2012), Thc tp thc vt v nhn bit cy thuc, trung tm

thng tin-th vin H Dc H Ni, H Ni.

5. V Vn Chi (1996), T in cy thuc Vit Nam, NXB Y hc, H Ni.

6. V Vn Chuyn (1976), Tm tt c im cc h cy thuc, NXB Y hc, H

Ni.

7. Nguyn Vn n, Nguyn Vit Tu (1985), Phng php nghin cu ha

hc cy thuc, NXB Y hc, H Ni.

8. Phm Hong H (1991), Cy c Vit Nam, Nh xut bn tr.

9. Hi ng dc in (2010), Dc in Vit Nam IV, NXB Y hc, H Ni.

10. Phm Thanh K, Nguyn Th Tm, Trn Vn Thanh (2007), Dc liu hc

tp 2, NXB Y hc, H Ni.

11. Tt Li (1999), Nhng cy thuc v v thuc Vit Nam, Nh xut bn Y

hc, H Ni, trang 170.

12. Nguyn Vit Thn (2010), Thc tp dc liu, Trung tm thng tin - th vin

H Dc H Ni.

13. Cao Vn Thu (2005), Thc tp vi sinh k sinh, Trung tm thng tin - th

vin H Dc H Ni.

14. Vin dc liu (2004), Cy thuc v ng vt lm thuc Vit Nam tp I,

NXB khoa hc v k thut, trang 1006-1008.

47

II. TING ANH

15. Kathleen Ellen Armstrong, James Richardson, Richard Milne, Graham Stone

(2011), Systematics and biogeography of the pantropical genus Manilkara

Adans. (Sapotaceae), The University of Edinburgh.

16. Chanda SV, Nagani KV (2010), Antioxidant capacity of Manilkara zapota

leaves extracts evaluated by four in vitro methods, Nature and science 8(10),

p.797-802.

17. Julia F. Morton, Miami, FL (1987), Fruits of warm climates, Creative

Resource Systems, p.393-398.

18. Eduardo Coriolano De Oliveira, Caio Pinho Fernandes, Eladio Flores

Sanchez, Leandro Rocha, Andr Lopes Fuly (2014), Inhibitory Effect of

Plant Manilkara subsericea against Biological Activities of Lachesis muta

Snake Venom, United States National Library of Medicine.

19. K.Shanmugapriya, P.S.Aravana, Harsha Paya, S.Peer Mohammed, Williams

Binnie (2011), Antioxidant activity total phenolic and flavonoid contents of

artocarpus heterophyllus and manilkara zapota seeds and its reduction

potential, International Journal of Pharmacy and Pharmaceutical Sciences,

3.

20. Kirtikar K.R, Basu B.D (1956), Indian Medicinal Plants, Allahabad India, II,

p.1486-1487.

21. M. Abu Osman, M. Abdul Aziz, M. Rowshanul Habib, M. Rezaul Karim

(2011), Antimicrobial Investigation on Manilkara zapota (L.) P. Royen,

International Journal of Drug Development & Research, p. 185-190.

22. Manirujjaman, Farhana Sultana, Md.Arifur Rahman Chowdhury, Md. Tanjir

Hossain, Md. Imran-Ul-Haque (2014), In Vivo Assay of Analgesic Activity

of Methanolic and Petroleum Ether Extracts of Manilkara zapota leaves,

British Journal of Pharmaceutical Research, 4(2), p.186-191.

23. Ma J, Luo XD, Protiva P, Yang H, Ma C, Basile MJ, Weinstein IB, Kennelly

EJ (2003), Bioactive novel polyphenols from the fruit of Manilkara zapota L.

(Sapodilla), J Nat Prod, p. 983-986.

48

24. Nesrin M. Fayek, Azza R. Abdel Monem, Mohamed Y. Mossa, Meselhy R.

Meselhy, Amani H. Shazly (2012), Chemical and biological study of

Manilkara zapota (L.) Van Royen leaves (Sapotaceae) cultivated in Egypt,

Pharmacognosy Research, 4(2), p. 85-91.

25. R Nair, Sumitra Chanda (2008), Antimicrobial activity of Terminalia

catappa, Manilkara zapota and Piper betel leaf extract, Indian Journal of

Pharmaceutical Sciences, 70(3), p. 390-393.

26. Sharmila S, Jeyanthi Rebecca L, Merina Paul Das, Md Saduzzaman, Shashi

Bala (2013), A Comparative Study on Phytochemical Analysis of Murraya

Koenigii and Manilkara zapot, Research Journal of Pharmaceutical,

Biological and Chemical, 4(2), p.1104.

27. Seshadri, Vijay Kothari and Sriram (2010), In vitro antibacterial activity in

seed extracts of Manilkara zapota, Anona squamosa, and Tamarindus indic,

Biological Research, 43, p. 165-168.

28. T.K.Lim (2012), Edible Medicinal And Non-Medicinal Plants, Springer of

Dordrecht Heidelberg New York London, 6, p.110-116.

29. U. N. Bhale, P. M. Wagh, J. N. Rajkonda (2013), Antagonistic confrontation

of Trichoderma spp against fruit rot pathogens on Sapodilla (Manilkara

zapota L.), Journal of Yeast and Fungal Research, 4, p. 4-11.

30. Yashwanth Kumar D.R, Vurivi Hema, Mayank Agrawal, Pramoditha Struthy

C, Vedamurthy (2012), Manikara zapota seek embryo extract: A potent

anthelminthic, Asian Journal of Pharmaceutical and Clinical Research, 5(3),

p.159.

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