Nghiên cứu thành phần hóa học của cây hương nhu tía (Ocimum Sanctum L).pdf

Embed Size (px)

DESCRIPTION

các bạn liên hệ e-mail: [email protected] hoặc sms via 0949 278 106 ( không nhận cuộc gọi ) để có thể có được file. Ngoài ra nhận tải mọi tài liệu ở trang http://125.235.10.97/opacdigital/ ( thư viện đại học dược hà nội)

Citation preview

  • B Y T

    TRNG I HC DC H NI

    NGUYN NGC HIU

    NGHIN CU THNH PHN

    HA HC CA CY HNG NHU TA

    (OCIMUM SANCTUM L.) KHA LUN TT NGHIP DC S

    H NI - 2013

  • B Y T

    TRNG I HC DC H NI

    NGUYN NGC HIU

    NGHIN CU THNH PHN

    HA HC CA CY HNG NHU TA

    (OCIMUM SANCTUM L.) KHA LUN TT NGHIP DC S

    Ngi hng dn :

    1. TS. Nguyn Vn Ti

    2. TS. Quyn

    Ni thc hin :

    1. Khoa Ha thc vt - Vin

    Dc liu

    2. B mn Dc liu - trng

    i hc Dc H Ni

    H NI - 2013

  • LI CM N

    Kha lun ny l kt qu ca qu trnh hc tp ti trng i hc Dc H

    Ni v qu trnh nghin cu ca em ti Vin Dc liu.

    Vi tnh cm chn thnh, em xin by t lng bit n n cc thy c ti khoa

    Ha Thc vt - Vin Dc liu v cc thy c b mn Dc liu, trng i hc

    Dc H Ni tn tnh gip , to iu kin cho em trong qu trnh hc tp,

    nghin cu hon thnh kha lun ny.

    c bit em xin by t lng bit n su sc v tri n n Tin s Nguyn Vn

    Ti v Tin s Quyn, d bn rt nhiu cng vic nhng tn tnh hng dn,

    gip , du dt em trong qu trnh nghin cu ti v hon chnh kha lun.

    Mc d bn thn rt c gng trong ln u tin nghin cu khoa hc nhng

    chc chn kha lun khng trnh khi nhng thiu st, em rt mong nhn c

    nhng kin ng gp qu gi ca cc thy c.

    H Ni, thng 5 nm 2013

    Sinh vin

    Nguyn Ngc Hiu

  • MC LC

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

    CHNG 1: TNG QUAN .......................................................................................... 2

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

    1.2. Thnh phn ha hc ca Hng nhu ta (Ocimum sanctum L.) ......................... 5

    1.3 Tng quan v acid ursolic ........................................................................................ 6

    CHNG 2 : I TNG V PHNG PHP NGHIN CU ....................... 12

    2.1 i tng nghin cu ............................................................................................. 12

    2.2 Phng php nghin cu ....................................................................................... 13

    CHNG 3 : THC NGHIM, KT QU V BN LUN ................................ 15

    3.1 nh tnh cc nhm cht hu c bng phn ng ha hc .................................. 15

    3.2 nh tnh acid ursolic trong cnh l Hng nhu ta (Ocimum sanctum L.)

    bng SKLM ................................................................................................................... 22

    3.3 Phn lp acid ursolic t cnh l Hng nhu ta (Ocimum sanctum L.) bng

    sc k ct ....................................................................................................................... 23

    3.4 Xy dng quy trnh chit tch acid ursolic t Hng nhu ta (Ocimum

    sanctum L.) .................................................................................................................... 29

    BN LUN ................................................................................................................... 37

    KT LUN V XUT ......................................................................................... 39

    TI LIU THAM KHO ........................................................................................... 40

    PH LC ...................................................................................................................... 43

  • DANH MC CC K HIU,

    CC CH VIT TT

    CTCT cng thc cu to

    DCM dichloromethan

    dd dung dch

    EtOAc ethyl acetat

    EtOH ethanol

    IR Ph hng ngoi

    kl khi lng

    MeOH methanol

    NMR Ph cng hng t ht nhn

    MS Ph khi

    Rf h s lu

    SKLM sc k lp mng

    tt th tch

    TT thuc th

  • DANH MC CC BNG

    Bng 1.1 : Hm lng acid ursolic trong l ca cc loi thuc chi Ocimum L. ....... 6

    Bng 3.1 : Kt qu nh tnh cc nhm cht hu c trong cnh, l Hng nhu

    ta (Ocimum sanctum L.) bng phn ng ha hc. ................................................... 21

    Bng 3.2 : Ph 1H, 13C-NMR ca D1 v acid ursolic. ............................................... 27

    Bng 3.3 : Hm lng acid ursolic th (%) khi chit bng cc dung mi khc nhau. .. 32

    Bng 3.4 : Hm lng acid ursolic th (%) khi chit cc nhit khc nhau. .......... 34

    Bng 3.5 : Hm lng acid ursolic th (%) khi chit vi t l dung mi/dc

    liu khc nhau. .............................................................................................................. 34

    Bng 3.6 : Hm lng acid ursolic th (%) vi s ln chit khc nhau. ................. 35

  • DANH MC CC HNH V, S

    Hnh 1.1 : CTCT ca acid ursolic. ................................................................................ 7

    Hnh 1.2 : CTCT ca acid corosolic. ............................................................................ 9

    Hnh 1.3 : S bn tng hp acid corosolic t acid ursolic. .................................... 9

    Hnh 1.4 : S chit xut v tinh ch acid ursolic t l Hng (Diospyros kaki). . 10

    Hnh 1.5 : S chit xut v tinh ch acid ursolic t v to (Malus domestica) .. 11

    Hnh 3.1 : Sc k ca cn MeOH ton phn vi acid ursolic i chng trn

    cc h dung mi khai trin. ......................................................................................... 22

    Hnh 3.2 : Tm tt quy trnh chit xut cc phn on t cnh, l Hng nhu

    ta (Ocimum sanctum L.) .............................................................................................. 24

    Hnh 3.3 : Sc k cc cn phn on vi acid ursolic i chng. ...................... 25

    Hnh 3.4 : Sc k acid ursolic th v sau khi kt tinh vi cht i chng ......... 31

    Hnh 3.5 : Sc k acid ursolic th ca cc m chit bng cc dung mi khc nhau ... 33

  • 1

    T VN

    Hng nhu ta t lu c s dng rng ri trong y hc c truyn lm

    thuc iu tr cm nng, cm hn, st nng s rt, nhc u, au bng ... Dc in

    Vit Nam IV c chuyn lun v Hng nhu, xem hai loi Hng nhu trng

    (Ocimum gratissimum L.) v Hng nhu ta (Ocimum sanctum L.) c s dng

    nh nhau trong iu tr. Thc t, cy Hng nhu trng l ngun nguyn liu chit

    xut tinh du Hng nhu v iu ch eugenol, cn cy Hng nhu ta ch yu dng

    trong Y hc c truyn.

    Trn th gii, Hng nhu ta (Ocimum sanctum L.) c dng nh mt ngun

    nguyn liu giu acid ursolic, do c cha hm lng acid ursolic cao [19, 22]. Acid

    ursolic l mt saponin thuc nhm ursan c chng minh c nhiu tc dng

    sinh hc ng ch nh chng vim, bo v gan, h lipid mu, chng khi u [10,

    15]. c bit acid ursolic cn l mt cht trung gian quan trng bn tng hp

    nhiu cht c hot tnh sinh hc cao nh acid corosolic (mt cht c ch glycogen

    phosphorylase c nghin cu nhiu trong iu tr i tho ng) [20, 24].

    Xut pht t thc t cy Hng nhu ta Vit Nam cha c nghin cu

    nhiu v thnh phn acid ursolic, chng ti thc hin ti : "Nghin cu

    thnh phn ha hc ca cy Hng nhu ta (Ocimum sanctum L.)"

    vi cc ni dung chnh nh sau :

    - nh tnh cc nhm cht hu c trong cnh, l Hng nhu ta (Ocimum sanctum L.).

    - Chit xut, phn lp acid ursolic t cnh l Hng nhu ta (Ocimum sanctum L.).

    - Xy dng quy trnh chit xut acid ursolic t Hng nhu ta (Ocimum sanctum L.).

    - Kho st mt s yu t nh hng n hm lng acid ursolic chit t dc liu.

  • 2

    CHNG 1: TNG QUAN 1.1 c im thc vt chi Ocimum L. 1.1.1. V tr phn loi chi Ocimum L.

    Theo h thng phn loi ca Takhtajan 1987, chi Ocimum L. thuc :

    Gii thc vt (Plantae)

    Ngnh Ngc lan (Magnoliophyta)

    Lp Ngc lan (Magnoliidae)

    Phn lp Bc h (Lamidae)

    B Bc h (Lamiales)

    H Bc h (Lamiaceae)

    Chi Ocimum L. [4]

    1.1.2 c im thc vt v phn b ca chi Ocimum L.

    Chi Ocimum L. thuc h Bc h (Lamiaceae), tn gi c th bt ngun t ozo,

    ozein ngha l tot mi thm, lin quan n mi thm phng pht ca cc cy thuc

    chi ny; hoc do ch okimon, okys ngha l nhanh, cp tc v cc cy ny mc rt

    nhanh. Tn khc ca chi : Hng nhu, Hng, .

    C hay cy bi nh, sng hng nm hay sng nhiu nm. Thn thng vung,

    nhn hay c lng. L mc i, mp nguyn hay x rng ca, c lng hay nhn,

    thng c cc im tuyn trn trn phin. Cm hoa dng chy hay hnh thp, gm

    cc xim b to thnh cc vng gi, mi vng thng c 6 hoa. L bc tn ti hay

    sm rng. i hnh chung, thng c lng v im tuyn pha ngoi, 2 mi; mi

    trn c mt thy ln t nhiu men xung ng; mi di 4 thy, vi 2 thy bn nhn

    ngn, 2 thy di nhn, di. Trng c ng th khi i, nhn hay c lng, 2 mi :

    mi trn 4 thy ngn; mi di mt thy di v ln hn mi trn, hi cong v lm

    hnh thuyn. Nh 4, hng xung mi di, ch nh t nhiu th khi ng trng, bao

    phn hnh trng hay hnh thn, 1 . Bu nhn, vi nhy x 2 thy nh. a mt

  • 3

    c thy trc ln. Qu hnh trng hay gn hnh cu, nhn, nm trong i ng

    trng.

    C khong 150 loi, phn b ch yu vng nhit i chu Phi v Nam M.

    Vit Nam c 4 loi [5].

    1.1.3 Chi Ocimum L. Vit Nam

    Vit Nam, chi Ocimum L. c 4 loi :

    Ocimum americanum L. - hoang, chu M. nc ta c gp ti H Ni

    v vng Cam Ranh tnh Khnh Ha.

    Ocimum basilicum L. - Hng qu, Hng gii, Hng ch, ta, qu.

    nc ta, c gp t H Giang, H Ni, Tha Thin Hu, Ninh Thun, thnh

    ph H Ch Minh v Long An. Cng c trng nhiu ni.

    Ocimum basilicum L. var. pilosum (Willd.) Benth. - Hng trng, trng, Tr

    tin. nc ta, th ny c gp t H Giang, Ha Bnh, H Ni vo Qung

    Nam, Kon Tum, c Lc, Khnh Ha, thnh ph H Ch Minh v Long An.

    Cng c trng nhiu ni.

    Ocimum gratissimum L. - Hng nhu trng, l ln. nc ta, c gp t

    H Giang, Sn La, Lng Sn, Ph Th, Vnh Phc, H Ni vo ti thnh ph

    H Ch Minh.

    Ocimum sanctum L. (tn khc O. tenuiflorum L.) - Hng nhu ta, ta,

    , rng. nc ta, c gp t Ha Bnh, Bc Giang, H Ni, Ninh Bnh

    vo Khnh Ha, Ninh Thun, thnh ph H Ch Minh cho n An Giang.

    Cng c trng nhiu ni [5].

    1.1.4 c im thc vt loi Hng nhu ta (Ocimum sanctum L.)

    1.1.4.1 Tn gi

    Tn khoa hc : Ocimum sanctum L. (tn khc : O. tenuiflorum) - h Bc h

    (Lamiaceae).

    Tn khc : , ta.

  • 4

    Tn nc ngoi : Monk's basil, sacred basil, holy basil, rough basil, tulsi,

    mosquito plant of South Africa (Anh), basilic saint, basilic sacr (Php) [7].

    1.1.4.2 c im thc vt

    Cy nh, sng hng nm hay sng dai, cao n gn 1m. Thn, cnh mu

    ta, c lng mn. L mc i, c cung di, hnh mc hoc thun, di 2 - 5 cm, rng

    1 - 3 cm, mp kha rng, hai mt mu tm ta, c lng mm.

    Cm hoa mc u cnh thnh chm xim phn nhnh; l bc nh; hoa mu

    trng hoc tm ta, xp thnh tng vng 5 - 6 ci trn cm hoa; i hoa di 3 - 5 mm,

    thy trn hnh mt chim, thy di hnh gii di hn, nhng thy bn rt ngn;

    trng hoa c cnh hi ln sng mp; nh 4, vt ra ngoi trng.

    Qu b t, gn hnh cu, hi dt, mu nu nht hoc c m en nh, nm

    trong i tn ti.

    Ma hoa qu : thng 5 - 7 [7].

    1.1.4.3 Phn b, sinh thi

    Hng nhu ta vn l cy c nhit i chu , c trng ri rc Trung

    Quc, Lo, Thi Lan lm thuc v lm rau gia v. Vit Nam, Hng nhu ta

    mi c trng hn ch trong vn cc gia nh hoc cc c s cha bnh theo y

    hc c truyn. Cy a kh hu nhit i nng v m; nhit trung bnh nm

    khong 25 - 300C; lng ma 1800 - 2600 mm/nm. cc vng ni cao c kh hu

    cn nhit i v hi lnh, khng thy trng.

    Hng nhu ta mc t ht vo khong cui ma xun, sinh trng nhanh trong

    ma h, n cui ma thu hay u ma ng th tn li. Cy ra hoa qu nhiu. Qu

    chn t m, ht ri xung t v ny mm sau 5 - 6 thng. Cy trng d dng bng

    ht [7].

  • 5

    1.2. Thnh phn ha hc ca Hng nhu ta (Ocimum sanctum L.) Tinh du : y l thnh phn ng ch v c gi tr cao trong cy Hng nhu

    ta, theo Dc in Vit Nam IV, dc liu phi cha t nht 0,5% tinh du (tnh

    theo dc liu kh tuyt i) [3].

    V thnh phn ha hc,tinh du hng nhu ta Vit Nam cha cc thnh phn

    chnh l eugenol (trn 70%), methyleugenol (trn 12%) v -caryophylen [7].

    Thnh phn ha hc khc trong cy :

    - Hng nhu ta cha cc hp cht thuc nhm flavonoid nh apigenin, luteolin,

    apigenin-7-glucuronid, luteolin-7-glucuronid, orientin v nhm polyphenol nh :

    acid galic, acid galic methylester, acid galic ethylester, acid protocatechic, acid

    rosmarinic. [7].

    - Acid ursolic l mt thnh phn quan trng v c hm lng cao trong Hng nhu

    ta.

    + Nm 2008, Silva MG v cng s tin hnh nghin cu hm lng acid

    ursolic trong l ca 8 loi thuc chi Ocimum L., l : O. americanum, O.

    basilicum, O. basilicum var purpurascens, O. basilicum var. minimum, O.

    gratissimum, O. micranthum, O. selloi v O. tenuiflorum bng phng php phn

    tch HPLC. Nghin cu ch ra Ocimum tenuiflorum l loi c hm lng acid

    ursolic trong l cao nht (ln n 2,02%) trong cc loi thuc chi Ocimum L. Kt

    qu ca nghin cu c trnh by bng 1.1 [19].

    + Nm 2013, Mangesh D v cng s kho st cc yu t trong qu trnh

    chit xut acid ursolic t l Ocimum sanctum L. bng sng siu m. Nghin cu

    xc nh iu kin chit xut ti u trn mt m 2 g dc liu l s dng dung mi

    MeOH, t l dung mi/dc liu l 1/30, nhit 45 0C, tn s sng siu m 25

    kHz v trong thi gian 12 pht, lng acid ursolic c chit ra iu kin ny l

    1,647% (s dng phng php nh lng HPLC) [22].

  • 6

    Bng 1.1 : Hm lng acid ursolic trong l ca cc loi thuc chi Ocimum L.

    STT Loi Hm lng (%)

    1 Ocimum americanum 1,03

    2 Ocimum basilicum 0,29

    3 Ocimum basilicum var. purpurascens 0,38

    4 Ocimum basilicum var. minimum 0,27

    5 Ocimum gratissimum 1,04

    6 Ocimum micranthum 1,05

    7 Ocimum selloi 0,45

    8 Ocimum tenuiflorum 2,02

    1.3 Tng quan v acid ursolic 1.3.1 Cng thc ha hc, tnh cht l ha ca acid ursolic

    Cng thc phn t : C30H48O3.

    Khi lng phn t : 456,7 (g/mol).

    Cng thc cu to (hnh 1.1)

    Tn khoa hc : 3-hydroxyurs-12-en-28-oic acid.

    Hnh dng tinh th : Kt tinh dng tinh th lng tr to t cn tuyt i, dng kim t

    cn long.

    Nhit nng chy : 2840C.

    tan : 1/88 (MeOH, kl/tt), 1/178 (EtOH, kl/tt), 1/35 (EtOH si, kl/tt), 1/140

    (ether, kl/tt), 1/388 (chloroform, kl/tt), 1/1675 (CS2, kl/tt), tng i tan trong

    aceton. Tan trong acid acetic bng nng v dung dch 2% NaOH trong EtOH.

    Khng tan trong ether du ha. tan trong nc : 1,02.10-4 mg/L 250C (d

    on) [26].

  • 7

    Hnh 1.1 : CTCT ca acid ursolic.

    1.3.2 Tc dng dc l ca acid ursolic

    1.3.2.1 Tc dng chng vim

    Acid ursolic t Pyrola rotundifolia c tc dng chng vim trong th nghim

    gy ph chn bng carageenan trn chut [14].

    Trong dch chit ca Rosmarinus officinalis, acid ursolic l mt trong cc

    thnh phn ngn nga ph tai chut trong th nghim gy ph tai chut bng 12-o-

    tetradecanoylphorbol-13-acetat [11, 12].

    Acid ursolic phn lp t Plantago major th hin tc dng chng vim trong

    th nghim in vitro sinh tng hp prostaglandin (c s xc tc ca COX-2). Acid

    ursolic th hin tc dng chng vim ng k trn COX-2 vi t l chn lc COX-

    2/COX-1 l 0,6 [13]. Trong th nghim in vivo, tc dng chng vim ca acid

    ursolic ln hn acid oleanolic v indomethacin, c th l gi tr ID50 ca acid

    ursolic, acid oleanolic v indomethacin ln lt l 0,14/0,36/0,26 mol/cm2, iu

    ny cho thy acid ursolic c tc dng chng vim mnh hn gn 2 ln so vi

    indomethacin v gn 3 ln so vi acid oleanolic [8].

    1.3.2.2 Tc dng bo v gan

    Acid ursolic c tc dng bo v gan trong cc tn thng gan do ha cht,

    cng nh bnh x ha gan cp tnh, x gan, acid ursolic c s dng n c hoc

    phi hp vi cc tc nhn bo v gan khc. Tc dng bo v gan ca acid ursolic c

    th do tc dng chng oxy ha v chng vim ca n mang li. Mt trong nhng c

  • 8

    ch bo v gan ca acid ursolic l lm tng lng metallothionein - mt protein giu

    cystein c chc nng ging glutathion trong vic bo v gan khi cc c t, loi b

    cc kim loi nng. Tuy nhin khng phi l c ch bo v gan duy nht ca acid

    ursolic [16].

    Bn cnh tc dng bo v gan trong cc th nghim gy tn thng gan bi

    CCl4 hoc D-galactosamin, acid ursolic cn c tc dng ngn nga tc nghn ng

    mt trong th nghim gy tc nghn ng mt bi acetaminophen [18].

    1.3.2.3 Tc dng chng khi u

    Acid ursolic t Eriobotrya japonica th hin tc dng c ch s t bin trong

    th nghim s dng aflatoxin B1 trn Salmonella typhimurium [25].

    Acid ursolic tch t Rosmarinus officinalis c hiu qu trong c ch s hnh

    thnh lin kt cng ha tr ca B[a]P vi DNA ca da v c ch s hnh thnh khi

    u do B[a]P v 7,12-dimethyl-benz[a]anthracene [12].

    1.3.2.4 Tc dng h lipid mu

    Acid ursolic gip ngn nga x va ng mch, gim cholesterol mu (44%)

    v mc -lipoprotein (50%) th v chut [21].

    Acid ursolic c ch hot ng ca enzym FAS (fatty acid synthase) vi IC50 =

    6,0 g/mL. Tc dng c ch l do s i khng ca acid ursolic vi acetyl-CoA v

    malonyl-CoA, nhng khng i khng vi NaDPH [17].

    1.3.3 Acid ursolic l nguyn liu ban u bn tng hp mt s hp cht c

    tc dng sinh hc quan trng

    T acid ursolic c th bn tng hp nn cc hp cht c tim nng c ch

    enzym glycogen phosphorylase iu tr i tho ng type 2 [23], c bit l

    acid corosolic (ch khc acid ursolic l c thm 1 nhm OH v tr s 2-alpha).

  • 9

    Hnh 1.2 : CTCT ca acid corosolic.

    X.Wen et al (2007) tng hp acid corosolic t acid ursolic theo s [24]

    Hnh 1.3 : S bn tng hp acid corosolic t acid ursolic.

    1.3.4 Mt s phng php phn lp acid ursolic

    1.3.4.1 Phn lp acid ursolic t l Hng (Diospyros kaki L.f.)

    Nguyn Th Ngc Lan (2012) chit tch acid ursolic t l Hng (Diospyros

    kaki L.f.) lm cht i chiu phc v Cng tc tiu chun ha v kim tra cht

    lng dc liu. T 2 kg dc liu, tc gi chit tch c 125 mg acid ursolic.

    Phng php chit tch c trnh by hnh 1.4 [6].

  • 10

    L Hng (2kg)

    Dch chit MeOH

    MeOH (7L x 3 ln)

    Cao MeOH (170g)

    - MeOH

    + 1,5L nc

    Dch nc

    + DCM (2L x 3 ln)

    Cn DCM (80g) Dch chit nc

    Phn on IV Phn on I-II Phn on III 8,5g

    Phn on III-1 Phn on III-2 Phn on III-3

    Acid ursolic (125 mg)

    Hnh 1.4 : S chit xut v tinh ch acid ursolic t l Hng (Diospyros kaki).

    SK ct pha o MeOH-H2O

    SK ct pha o MeOH-H2O

    SK ct pha o MeOH-H2O (4:1)

    20/1, 10/1 5/1 1/1

    5/1 9/1 1/1

  • 11

    1.3.4.2 Phn lp acid ursolic t v to (Malus domestica)

    Rosa T.S. Frighetto (2007) chit tch v tinh ch acid ursolic t v to

    (Malus domestica) bng phng php sc k phn b ngc dng tc cao

    (HSCCC - high speed counter-current chromatography) [9].

    V to

    + n-hexan loi b nht

    V to loi b nht

    + EtOH 96% trong 15h Ct thu hi dung mi

    Cn EtOH ton phn

    Sc k ct silicagel pha thun

    Ra gii bng CHCl3

    Gom cc phn on c acid ursolic

    Acid ursolic

    Tinh ch bng phng php HSCCC

    Hnh 1.5 : S chit xut v tinh ch acid ursolic t v to (Malus domestica).

  • 12

    CHNG 2 : I TNG V PHNG PHP

    NGHIN CU

    2.1 i tng nghin cu 2.1.1 Nguyn liu v ha cht

    Nguyn liu : cnh, l Hng nhu ta c thu hi ti huyn Ba V, H Ni.

    Mu ti c gim nh tn khoa hc b mn Thc vt trng i hc Dc H

    Ni vi m s tiu bn l HNIP/18039/13, tiu bn c lu ti Phng tiu bn cy

    thuc (HNIP) - trng i hc Dc H Ni (ph lc 1).

    Ha cht : Dung mi dng trong chit xut l dung mi cng nghip ct li.

    Cc ha cht, dung mi dng cho kt tinh, tinh ch l dung mi tinh khit ca Trung

    Quc. Acid ursolic i chng c cung cp bi Khoa Ha thc vt - Vin Dc

    liu, c s dng lm cht i chng cho cc th nghim sau ny.

    2.1.2 Trang thit b v dng c nghin cu

    - Cc dng c cn thit trong qu trnh thc nghim nh bnh nn, cc c m, ng

    nghim, pipet, ...

    - Thu hi dung mi bng my ct quay Rotavapor R-200 ca hng BUCHI - Vin

    Dc liu.

    - Xc nh m trn my PRECISA HA60 - Vin Dc liu.

    - Cn phn tch PRECISA - Vin Dc liu.

    - Bn mng silicagel GF254 (Merck) trng sn pha thun - Vin Dc liu.

    - Ph hng ngoi o trn my quang ph hng ngoi IMPACT 410 NICOLET -

    Vin Ha hc.

    - Ph cng hng t ht nhn 1H, 13C o trn my BRUKER AVANCE AM500 FT-

    NMR - Vin Ha hc.

  • 13

    - Ph khi o trn my sc k lng- khi ph SHIMADZU LC-MS 2010EV - Vin

    Ha hc.

    2.2 Phng php nghin cu 2.2.1. nh tnh cc nhm cht hu c c trong cy Hng nhu ta (Ocimum

    sanctum L.)

    nh tnh cc nhm cht chnh trong dc liu theo phng php ha hc ghi

    trong sch Thc tp Dc liu v Bi ging Dc liu tp 1 ca trng i hc

    Dc H Ni [1, 2].

    2.2.2 nh tnh acid ursolic trong cnh l Hng nhu ta (Ocimum sanctum L.)

    bng SKLM

    - Bn mng Silicagel GF254 pha thun (Merck) c hot ha trong vng 1 gi

    nhit 1100C.

    - Chun b dch chm sc k ca mu th v mu i chng :

    + Mu i chng : ly khong 1 mg acid ursolic i chng ha tan vo 1 mL

    MeOH.

    + Mu th : ly khong 5 mg cn MeOH ha tan vo 1 mL MeOH (cn c

    chun b nh mc 2.2.3).

    - Cc h dung mi khai trin :

    H 1 : CHCl3 : MeOH = 9 : 1 (tt/tt).

    H 2 : ether du ha : EtOAc = 4 : 6 (tt/tt).

    Dung mi khai trin c bo ha trong cc sc k, t bn mng chm

    dch chit vo cc, chy theo chiu t di ln trn. Sau khi khai trin xong, ly bn

    mng ra, kh t nhin.

    - Phng php pht hin : Quan st cc vt di nh sng t ngoi (UV) 2 bc sng

    254 nm v 365 nm, phun dung dch H2SO4 10% trong EtOH, h nng trn bp in.

  • 14

    2.2.3 Phn lp bng sc k ct

    - Chit xut : Chit hi lu cnh l Hng nhu ta (phi sy kh, xay nh) vi

    MeOH. Sau , gp dch chit, ct thu hi dung mi di p sut gim ti cn.

    - Chit cc phn on : Phn tn u cn MeOH ton phn vo mt lng ti thiu

    nc nng, sau chit ln lt vi cc dung mi theo th t tng dn phn cc

    l n-hexan, DCM, EtOAc. Thu cc phn dch chit, ri em ct thu hi dung mi

    di p sut gim ti cn.

    - Phn lp bng sc k ct :

    + La chn phn on DCM tin hnh sc k.

    + Cht hp ph : Silicagel 60 (c ht 0,04 - 0,063 mm) c hot ha 110 0C

    trong 1 gi. ngui bnh ht m.

    + Ct sc k : kch thc 5x25 cm.

    + H dung mi ra gii : S dng h dung mi DCM v MeOH tng dn phn

    cc (gradient lng MeOH t 0% n 10%).

    - tinh khit ca cht phn lp c kim tra s b bng SKLM, o im chy.

    Sau s dng cc phng php phn tch ph (ph hng ngoi IR, khi ph ESI-

    MS, ph cng hng t ht nhn 1H/13C-NMR), tham kho c s d liu ph ca

    cc ti liu cng b bin gii cu trc cht phn lp c.

    2.2.4 Phng php chit xut acid ursolic t Hng nhu ta (Ocimum sanctum L.)

    Chit cnh, l Hng nhu ta bng phng php hi lu vi h dung mi

    EtOH v EtOAc, dch chit c ct thu hi dung mi di p sut gim ti cn.

    Phn tn cn vo mt lng ti thiu nc nng, chit nhiu ln vi DCM, gp cc

    phn dch chit DCM. Ct thu hi bt dung mi, sau un hi lu lp DCM vi

    than hot than hot hp ph acid ursolic, ra gii acid ursolic ra khi than hot

    bng dung mi aceton, ct thu hi dung mi thu c acid ursolic th.

  • 15

    Kt tinh acid ursolic : s dng phng php kt tinh bng nhit , da trn s

    chnh lch tan ca acid ursolic trong EtOH nhit si (1/35, kl/tt) v trong

    EtOH nhit thng (1/178, kl/tt) [26].

    2.2.5 Kho st mt s thng s ca qu trnh chit xut

    Tin hnh kho st mt s thng s nh hng n hiu sut chit xut :

    Dung mi.

    Nhit .

    T l dung mi/dc liu.

    S ln chit xut.

    Hm lng acid ursolic th chit c so vi khi lng dc liu kh c tnh

    theo cng thc :

    A(%) = x 100

    Trong :

    A : hm lng phn trm (%) ca acid ursolic th.

    m : khi lng acid ursolic th thu c.

    b : hm m dc liu.

    M : khi lng dc liu.

    CHNG 3 : THC NGHIM, KT QU V

    BN LUN

    3.1 nh tnh cc nhm cht hu c bng phn ng ha hc nh tnh cc nhm cht trong cnh, l Hng nhu ta (Ocimum sanctum L.)

    bng phn ng ha hc.

    m

    M (1-b)

  • 16

    - Chun b dch chit cn : Cho 5 g dc liu vo bnh nn dung tch 100 mL, thm

    50 mL EtOH 900, un cch thy 10 pht, lc nng. Dng dch lc lm cc phn ng.

    - Chun b dch chit ether du ha : Cho 5 g dc liu vo bnh nn dung tch 50

    mL, ngp ether du ha, ngm qua m, lc. Dch lc dng lm cc phn ng.

    3.1.1 nh tnh alcaloid

    Ly 5 g dc liu cho vo bnh nn dung tch 50 mL, thm dung dch H2SO4

    2% cho ngp dc liu, un si vi pht. ngui, lc dch chit vo bnh gn,

    kim ha dch lc bng dung dch amoniac 6N n pH kim. Chit alcaloid bng

    cloroform (CHCl3) 3 ln, mi ln 5 mL. Dch chit cloroform c gp li v lc

    vi H2SO4 2%. Gn ly lp nc acid, cho vo 3 ng nghim, mi ng khong 1

    mL ri thm vo :

    - ng 1 : 2-3 git TT Dragendorff, dung dch trong sut, khng c ta vng cam.

    - ng 2 : 2-3 git TT Mayer, dung dch trong sut, khng c ta vng nht.

    - ng 3 : 2-3 git TT Bouchardat, dung dch trong sut, khng c ta nu.

    Kt lun : dc liu khng c alcaloid.

    3.1.2 nh tnh anthranoid

    - Phn ng Borntraeger: cho 5 g dc liu vo bnh nn dung tch 100 mL, thm 10

    mL dung dch H2SO4 25% cho ngp dc liu ri un si trong vi pht. Lc dch

    chit vo bnh gn, ngui ri lc vi 5 mL ether. Ly 1 mL dch ether cho vo

    ng nghim, thm 1mL KOH 10%, lc k, quan st thy lp KOH khng c mu.

    Kt lun : dc liu khng c anthranoid.

    3.1.3 nh tnh flavonoid

    - Phn ng Cyanidin : cho 2 mL dch chit cn vo mt ng nghim, thm mt t

    bt Mg kim loi, ri thm vi git HCl c. un nng trn bp cch thy sau vi

    pht thy xut hin mu tm .

  • 17

    - Phn ng vi dung dch FeCl3 5% : cho 2 mL dch chit cn vo mt ng nghim,

    thm 2-3 git dung dch FeCl3 5%, thy dung dch chuyn mu xanh sm.

    - Phn ng vi kim : Nh vi git dch chit cn ln mt mnh giy lc, h kh ri

    t mnh giy ln ming l amoniac c thy mu vng hin r, khi soi di n t

    ngoi thy c mu vng sng.

    Kt lun : dc liu c cha flavonoid.

    3.1.4 nh tnh saponin

    - Hin tng to bt : Ly 2 ng nghim c bng nhau, cho vo ng th nht 5 mL

    HCl 0,1 N v ng th 2 l 5 mL NaOH 0,1 N. Cho thm vo mi ng 2-3 git dch

    chit cn ri bt ng nghim, lc mnh c 2 ng trong 15 giy. yn, thy ct bt

    2 ng bn vng v bng nhau.

    - Phn ng Salkowski : Ly 10 mL dch chit cn cho vo bnh nn v thm 10 mL

    H2SO4 long. un si cch thy vi pht. ngui, chit vi cloroform. Ly

    khong 2 mL dch chit cloroform cho vo ng nghim. Thm t t 1 mL H2SO4

    c theo thnh ng nghim, quan st thy gia hai lp xut hin vng mu xanh l.

    - Phn ng Liebermann - Burchardt : Ly 1 mL dch chit cloroform trn cho vo

    1 ng nghim ri c ti cn. Cho vo cn 0,5 mL anhydrid acetic, lc u, t

    nghing ng 450 ri thm 0,5 mL acid sulfuric c theo thnh ng nghim dch

    lng trong ng chia thnh 2 lp, quan st thy mt tip xc gia 2 lp cht lng

    trong ng nghim c mu .

    Kt lun : dc liu c cha saponin, s b l nhm triterpenoid.

    3.1.5 nh tnh tanin

    - Phn ng vi dung dch gelatin 1% : ly 1g dc liu cho vo bnh nn dung tch

    50 mL. Thm nc ngp dc liu ri un si vi pht. Lc nng, ly 1 mL dch

    lc cho vo ng nghim, thm vi git dung dch gelatin 1%, khng quan st thy

    ta bng.

    Kt lun : dc liu khng c tanin.

  • 18

  • 19

    3.1.6 nh tnh coumarin

    - Phn ng m v ng vng lacton :

    + Cho vo 2 ng nghim mi ng 1 mL dch chit cn, ng 1 thm 0,5 mL dung

    dch NaOH 10%, ng 2 nguyn.

    + un c 2 ng nghim cch thy n si, ngui. Quan st thy ng 1 c mu ta

    c mu vng, ng 2 trong sut.

    + Thm vo c 2 ng nghim, mi ng 2 mL nc ct, lc u. Quan st thy ng 1

    trong hn, ng 2 c ta c, acid ha ng 1 bng vi git HCl c, ng 1 c.

    - Phn ng vi thuc th diazo : cho vo ng nghim 1 mL dch chit cn, thm

    vo 2 mL dung dch NaOH 10%. un cch thy ti si, ngui, thm vi git

    thuc th diazo (mi pha), thy xut hin ta mu gch.

    Kt lun : dc liu c coumarin.

    3.1.7 nh tnh acid amin

    - Phn ng vi ninhydrin : ly 3 mL dch chit cn cho vo ng nghim. Thm vi

    git thuc th Ninhydrin 0,5%, un si 2 pht, khng quan st thy dung dch

    chuyn mu tm.

    Kt lun : dc liu khng c acid amin.

    3.1.8 nh tnh acid hu c

    - Phn ng vi Na2CO3 : cho vo ng nghim 1 mL dch chit cn v c ti cn.

    Ha cn trong 1 mL nc v thm vi tinh th Na2CO3 thy c bt kh ni ln.

    Kt lun : dc liu c acid hu c.

    3.1.9 nh tnh ng kh

    - Phn ng vi TT Fehling : ly 1 mL dch chit cn cho vo ng nghim, thm 3

    git TT Fehling A v 3 git TT Fehling B, un cch thy trong 10 pht, khng quan

    st thy ta mu .

  • 20

    Kt lun : dc liu khng c ng kh.

    3.1.10 nh tnh cht bo

    Nh vi git dch chit ether du ha trn giy lc, h kh, khng quan st

    thy vt m trn giy.

    Kt lun : dc liu khng c cht bo.

    3.1.11 nh tnh carotenoid

    - Phn ng vi H2SO4 c : c 5 mL dch chit ether du ha ti cn, thm 1-2 git

    acid sulfuric c, khng thy xut hin mu xanh ve.

    Kt lun : dc liu khng c carotenoid.

    3.1.12 nh tnh phytosterol

    - Phn ng Liberman : Cho vo ng nghim 1 mL dch chit ether du ha. Bc hi

    dung mi n kh, cho vo ng nghim 1 mL anhydrid acetic, lc k, thm 1 mL

    H2SO4 c theo thnh ng nghim. Kt qu cho thy gia 2 lp cht lng xut hin

    mt vng tm .

    Kt lun : dc liu c phytosterol.

    3.1.13 nh tnh tinh du

    Nh vi git dch chit ether du ha ln giy lc, thy c mi thm.

    Kt lun : dc liu c tinh du.

    Kt qu nh tnh cc nhm cht hu c trong Hng nhu ta bng phn ng

    ha hc c tm tt trong bng 3.1.

  • 21

    Bng 3.1 : Kt qu nh tnh cc nhm cht hu c trong cnh, l Hng nhu

    ta (Ocimum sanctum L.) bng phn ng ha hc.

    STT Nhm cht Phn ng Kt qu Kt lun

    1 Alcaloid

    Phn ng vi TT Dragendorff -

    Khng Phn ng vi TT Mayer -

    Phn ng vi TT Bouchardat -

    2 Anthranoid Phn ng Borntraeger - Khng

    3 Flavonoid

    Phn ng Cyanidin +

    C Phn ng vi dd FeCl3 5% +

    Phn ng vi kim +

    4 Saponin

    Phn ng to bt ++ Saponin

    triterpenoid Phn ng Salkowski ++

    Phn ng Liebermann - Burchardt ++

    5 Tanin Phn ng vi dd gelatin 1% - Khng

    6 Coumarin Phn ng m v ng vng lacton +

    C Phn ng vi thuc th diazo +

    7 Acid amin Phn ng vi TT Ninhydrin 0,5% - Khng

    8 Acid hu c Phn ng vi Na2CO3 + C

    9 ng kh Phn ng vi TT Fehling - Khng

    10 Cht bo Vt m trn giy lc - Khng

    11 Carotenoid Phn ng vi H2SO4 c - Khng

    12 Phytosterol Phn ng Liberman + C

    13 Tinh du C mi thm ++ C

    Ghi ch : (-) m tnh, (+) dng tnh, (++) dng tnh r.

    Nhn xt : Kt qu nh tnh bng phn ng ha hc cho thy cnh, l Hng

    nhu ta c cha saponin triterpenoid, flavonoid, coumarin, acid hu c, phytosterol,

  • 22

    tinh du v bng phng php ny khng thy s c mt ca alcaloid, tanin,

    anthranoid, acid amin, ng kh, cht bo v carotenoid.

    Nh vy, theo kt qu nh tnh trn, trong thnh phn ha hc ca Hng

    nhu ta c nhm saponin triterpenoid. Tip theo chng ti s tin hnh nh tnh

    acid ursolic trong Hng nhu ta bng SKLM.

    3.2 nh tnh acid ursolic trong cnh l Hng nhu ta (Ocimum sanctum

    L.) bng SKLM Tin hnh chy sc k cn MeOH so snh vi acid ursolic i chng trn 2 h

    dung mi.

    Sc k ca 2 h dung mi hnh 3.1.

    Hnh 3.1 : Sc k ca cn MeOH ton phn vi acid ursolic i chng trn

    cc h dung mi khai trin.

    (a) H 1 : CHCl3 : MeOH = 9 : 1 (tt/tt).

    (b) H 2 : ether du ha : EtOAc = 4 : 6 (tt/tt).

    Phng php pht hin : soi UV bc sng 254 nm, 365 nm v thuc th H2SO4

    10%/EtOH, h nng trn bp in.

    (1) acid ursolic i chng ; (2) cn MeOH ton phn ; (1+2) chm chng acid

    ursolic v cn MeOH ton phn.

  • 23

    Nhn xt : Sc k cho thy acid ursolic khng hp th UV 2 bc sng

    254 nm v 365 nm, hin mu tm vi thuc th H2SO4 10%/EtOH khi h nng

    trn bp in. Trn 2 h dung mi khai trin khc nhau, SKLM cho thy trong dch

    chit MeOH ton phn c vt c mu sc (tm ) v c Rf tng ng vi acid

    ursolic i chng khi dng thuc th H2SO4 10% trong EtOH, do c th s b

    xc nh trong mu Hng nhu ta nghin cu c acid ursolic.

    3.3 Phn lp acid ursolic t cnh l Hng nhu ta (Ocimum sanctum L.)

    bng sc k ct Chit hi lu 1 kg dc liu xay nh ( m 15,63%) vi dung mi MeOH

    nhit 600C trn bp cch thy, chit 3 ln, mi ln vi 5 L dung mi trong 2

    gi. Gp cc dch chit, lc qua giy lc thu ly phn dch, ct thu hi dung mi

    di p sut gim ti khi ht dung mi, thu c 42,45 g cn MeOH ton phn.

    Phn tn cn MeOH ton phn trong mt lng ti thiu nc nng, sau

    chit ln lt vi cc dung mi : n-hexan, DCM, EtOAc (chit mi phn on 3

    ln), thu c dch chit cc phn on, ri ct thu hi dung mi di p sut gim

    cc dch chit ti cn. Cn khi lng cc cn.

    Qu trnh chit cc phn on t dc liu c tm tt hnh 3.2.

  • 24

    Hnh 3.2 : Tm tt quy trnh chit xut cc phn on t cnh, l Hng nhu

    ta (Ocimum sanctum L.)

    (*) : ct thu hi dung mi

    Cnh, l Hng nhu ta xay nh (1kg)

    Chit hi lu vi MeOH 5L x 3 ln, mi ln 2h

    Dch chit MeOH

    Cn MeOH (42,45 g)

    Nc nng

    (*)

    Dch chit nc Dch chit n-hexan

    n-hexan (*) Cn n-hexan

    (10,12 g)

    Dch chit nc

    Dch chit DCM

    DCM (*) Cn DCM

    (11,34 g)

    Dch chit nc Dch chit

    EtOAc EtOAc

    (*) Cn EtOAc (3,75 g)

    Dch chit nc

  • 25

    Tin hnh chm cc cn phn on trn SKLM vi acid ursolic i chng

    xc nh phn on tch cht.

    - Chun b mu th v mu i chng :

    + Mu i chng : 1 mg acid ursolic ha tan trong 1 mL MeOH.

    + Cc mu th c chun b nh sau : 5 mg cn ha tan vo 1 mL dung mi

    tng ng.

    - H dung mi khai trin : CHCl3 : MeOH = 95 : 5 (tt/tt).

    - Phng php hin mu : phun dung dch H2SO4 10% trong EtOH, h nng trn

    bp in.

    Hnh 3.3 : Sc k cc cn phn on vi acid ursolic i chng.

    (C) acid ursolic i chng ; (H) cn n-hexan ; (D) cn DCM ; (E) cn EtOAc.

    Thuc th hin mu l H2SO4 10% trong EtOH.

    Nhn xt : Da trn hnh nh sc k hnh 3.3, ta thy cht c Rf tng

    ng vi acid ursolic nm ch yu phn on DCM (vt m mu nht), do

    chng ti s tin hnh tch cht t phn on DCM.

  • 26

    Tin hnh phn lp cht t phn on DCM bng sc k ct :

    - Chun b cht hp ph

    ong 300 mL Silicagel 60 (c ht 0,04 - 0,063 mm) c hot ha 110 0C

    trong 1h. ngui bnh ht m.

    - Chun b ct

    Chn ct sc k kch thc 5x25 cm, lp thng ng trn gi. Vn kha ct.

    Lt 1 lp bng sch y ct. Thm DCM vo silicagel hot ha, khuy u to

    hn dch, ch loi b ht bt kh. Rt t t hn dch ln ct theo thnh ct. Dng

    pipet ra thnh ct bng DCM. Cho dung mi chy qua ct vi ln n nh ct.

    - Chun b mu a ln ct

    Ha tan hon ton cn (11,34 g) vo lng ti thiu dung mi DCM, ri cho

    20 mL silicagel vo khuy u cho cht hp ph ht ln silicagel. Tip tc khuy

    cho dung mi bay hi t nhin n khi kh ti.

    - a cht ln ct

    + M kha ct cho dung mi chy t t n khi lp dung mi trong ct gn b

    mt silicagel.

    + a t t silicagel hp ph cht ln ct, trnh xo trn ct. Ch phi

    a t t trnh silicagel vn cc v phn tn khng u.

    + B sung nh nhng dung mi DCM, trnh gy xo trn.

    - Ra gii

    + S dng h dung mi DCM v MeOH tng dn phn cc (gradient lng

    MeOH t 0% n 10%).

    + iu chnh kha ct sao cho tc ra gii khong 20 git/pht.

    + Dch ra gii c hng vo cc ng nghim nh s th t, mi ng

    hng khong 20 mL.

    + Kim tra dch ra gii cc ng nghim bng SKLM, chm i chng vi

    acid ursolic chun, h dung mi khai trin l CHCl3 : MeOH = 95 : 5 (tt/tt), thuc

    th hin mu l H2SO4 10% trong EtOH, h nng trn bp in.

  • 27

    + Thu ly cc ng cha cht c Rf tng ng vi acid ursolic i chng, k

    hiu l D1. Ct thu hi dung mi di p sut gim thu c 1,56 g cht rn.

    - Kt tinh D1

    Tin hnh kt tinh D1 nh sau : un hi lu D1 trong EtOH cho tan hon ton,

    sau tip tc khuy nhit thng, qua m t lnh, lc ht chn khng,

    ra mu bng DCM lnh, sy chn khng thu c 0,94 g tinh th mu trng.

    3.3.3 Nhn dng cht phn lp

    Tnh cht ca D1 : D1 c dng tinh th mu trng, c nhit nng chy l

    288-2900C, khng hin UV trn SKLM, hin mu tm m sau khi phun dung dch

    H2SO4 10% trong EtOH v h nng trn bp in.

    D1 sau khi tinh ch, c em o cc ph hng ngoi (IR), khi ph (ESI-

    MS), ph cng hng t ht nhn (1H-NMR v 13C-NMR) xc nh cu trc.

    Ph hng ngoi IR (KBr, cm-1) : 3475 (nhm hydroxyl), 1693 (nhm

    carbonyl) (ph lc 2).

    Ph khi ESI-MS : cho thy khi lng phn t ca D1 l 456,31 ng vi

    cng thc phn t C30H48O3 ca acid ursolic (ph lc 3).

    Ph cng hng t ht nhn NMR : d liu ph NMR ca cht c th hin

    bng 3.3 (ph lc 4,5).

    Bng 3.2 : Ph 1H, 13C-NMR ca D1 v acid ursolic.

    V tr

    carbon

    Ph NMR ca D1

    (CDCl3/MeOD)

    Ph NMR ca acid ursolic

    (DMSO-d6)[19]

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

    1 36,87 1,54 (2H,m) 38,22 1,56 (2H,m)

    2 26,79 1,37 (2H,m) 26,79 1,43 (2H,m)

    3 78,82 3,21(1H,t,J =8,5;7,5Hz) 76,82 3,01 (1H,dd,J=5,2;9,5Hz)

    4 38,62 38,35

    5 55,19 0,71 (1H,t) 54,77 0,66 (1H,s)

  • 28

    6 18,24 1,37 (2H,m) 17,97 1,47 (1H,m)

    1,29 (1H,m)

    7 30,60 1,26 (2H,m) 30,77 1,27 (2H,m)

    8 39,41 39,09

    9 48,66 1,59 (1H,t) 47,00 1,58 (1H,s)

    10 36,75 36,51

    11 24,14 1,92 (2H,dd) 23,80 1,92

    (2H,dd,J=13,7;3,5Hz)

    12 125,44 5,24(1H,t,J =3,5;3,5Hz) 125,54 5,14

    (1H,dd,J=13,7;3,5Hz)

    13 138,12 138,18

    14 42,01 41,62

    15 32,98 1,48 (2H,m) 32,69 1,01 (2H,m)

    16 23,20 1,52 (2H,m) 22,82 1,53 (2H,m)

    17 49,51 46,81

    18 52,76 2,18(1H,d,J =11Hz) 52,37 2,12 (1H,d,J=11,1Hz)

    19 38,83 1,31 (1H,m) 38,42 1,31 (1H,m)

    20 39,02 1,57 (2H,m) 38,48 1,52 (1H,m)

    21 27,95 1,29 (2H,m) 27,52 1,29 (2H,m)

    22 36,75 1,47 (2H,m) 36,29 1,54 (2H,m)

    23 27,95 0,92 (3H,s) 28,23 0,90 (3H,s)

    24 16,89 0,77 (3H,s) 16,90 0,68 (3H,s)

    25 16,79 0,98 (3H,s) 16,03 0,87 (3H,s)

    26 15,49 0,81 (3H,s) 15,18 0,69 (3H,s)

    27 23,42 1,09 (3H,s) 23,24 1,05 (2H,s)

    28 180,54 178.23

    29 15,31 0,85 (3H,d,J =6Hz) 16,97 0,82 (3H,d,J=5,9Hz)

    30 21,03 0,94(3H,d,J =6,5Hz) 21,10 0,92 (3H,d,J=6,8Hz)

  • 29

    Da vo cn c d liu ph hng ngoi IR, ph khi ESI-MS v 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 acid ursolic [19].

    3.4 Xy dng quy trnh chit tch acid ursolic t Hng nhu ta (Ocimum

    sanctum L.)

    Phng php chit tch acid ursolic t l Hng ca Nguyn Th Ngc Lan

    (hnh 1.4) s dng n sc k ct pha o nhiu ln, tn nhiu thi gian, h dung

    mi ra gii l MeOH - H2O c hi v t tin, hn na lng acid ursolic thu

    c thp (2 kg l hng sau qu trnh chit tch ch thu c 125 mg acid ursolic).

    Phng php ca Rosa T.S. Frighetto phn lp acid ursolic t v to (hnh 1.5)

    s dng sc k ct pha thun v sc k phn b ngc dng tc nhanh tch

    acid ursolic, phng php ny tn thi gian v kh p dng cho sn xut ln.

    Hai phng php chit tch acid ursolic trn s dng phng php sc k ct

    nn mt nhiu thi gian, ch hp vi quy m phng th nghim, khng th p dng

    vo quy m sn xut ln hn. Hn na cch chn i tng chit xut acid

    ursolic cha hp l cng nh lng acid ursolic thu c cha cao.

    Do , chng ti tin hnh nghin cu quy trnh chit xut acid ursolic t

    Hng nhu ta vi mc ch a ra quy trnh chit tch c hiu sut cao hn, n

    gin hn v c th p dng vo quy m ln hn.

    3.4.1 Phng php chit tch acid ursolic t cnh, l Hng nhu ta (Ocimum

    sanctum L.)

    Cnh l Hng nhu ta phi sy kh c xay nh, xc nh m. m

    ca dc liu l 14,67%.

    Cho 100 g dc liu xay nh vo bnh cu dung tch 2 L, chit hi lu dc

    liu bng 1 L h dung mi EtOH v EtOAc t l 1 : 1 (tt/tt) trn bp cch thy

    nhit 65 0C c lp sinh hn hi lu dung mi bay hi, trong thi gian 2h,

    thnh thong c khuy trn. Lc ht chn khng, thu dch lc, phn b tip tc c

  • 30

    chit tng t vi 0,8 L hn hp dung mi trn thm hai ln na. Gp tt c cc

    phn dch lc, thu c dch chit.

    Dch chit c ct thu hi dung mi di p sut gim ti cn, thu c

    4,40g cn. Phn tn u cn vo nc nng, chit vi DCM 3 ln, gp cc dch

    chit DCM, ct thu hi dung mi di p sut gim ti cn, thu c 2,46 g cn

    DCM. Ha tan cn vo 50 mL DCM, thm 1,2 g than hot (lng than hot bng

    khong 50% v khi lng so vi cn) un hi lu trong 15 pht cho than hot hp

    ph acid ursolic. Sau lc nng, ra tp mu trn than hot bng DCM, cui cng

    dng 100 mL aceton ra gii acid ursolic. Lp DCM sau khi lc c un hi lu

    vi than hot, x l tng t nh trn. Gom cc dch aceton, ct thu hi dung mi

    thu c 0,2358 g acid ursolic th. Hiu sut chit acid ursolic th ca phng php

    l 0,28% trn khi lng dc liu kh.

    Kt tinh acid ursolic c thc hin nh sau : lng acid ursolic th trn

    c a vo bnh cu. Thm 3,3 mL EtOH tuyt i, un hi lu cho tan hon

    ton. Sau ngui, tip tc khuy nhit phng, qua m nhit 5 0C.

    Tinh th c lc ht chn khng, ri ra tp mu bng DCM, sy chn khng, thu

    c 0,1558 g tinh th mu trng. Hiu sut qu trnh kt tinh l 66,07%.

    Sc k acid ursolic sau kt tinh vi cht i chng cho thy qu trnh kt

    tinh loi c nhiu tp (hnh 3.4)

  • 31

    Hnh 3.4 : Sc k acid ursolic th v sau khi kt tinh vi cht i chng.

    (C) : acid ursolic i chiu.

    (T) : acid ursolic th (cha kt tinh).

    (KT) : acid ursolic sau khi kt tinh.

    H dung mi khai trin : CHCl3 : MeOH = 95 : 5 (tt/tt).

    Thuc th hin mu : dd H2SO4 10% trong EtOH, h nng trn bp in.

    Chng ti tip tc kho st mt s yu t nh hng n quy trnh chit xut,

    t tng bc hon thin quy trnh t hiu sut cao hn.

    3.4.2 Kho st mt s yu t nh hng n qu trnh chit xut

    Cc yu t chng ti kho st bao gm : dung mi, nhit , t l dung

    mi/dc liu v s ln chit.

    3.4.1 Kho st h dung mi chit xut

    Chng ti tin hnh kho st dung mi chit xut bng cch so snh lng

    acid ursolic th thu c khi chit vi cc dung mi khc nhau. Tin hnh kho st

    vi khi lng dc liu 50 g, t l dung mi/dc liu gia cc ln ln lt l

    8/6/6 (tt/kl), nhit 65 0C trn bp cch thy, thi gian mi ln chit l 2h.

  • 32

    Cc dung mi kho st l :

    EtOH.

    MeOH.

    EtOH v EtOAc t l 1 : 1 (tt/tt).

    MeOH v EtOAc t l 1 : 1 (tt/tt).

    Kt qu kho st bng 3.3

    Bng 3.3 : Hm lng acid ursolic th (%) khi chit bng cc dung mi khc nhau.

    STT H dung mi

    chit xut

    Khi lng acid

    ursolic th (g)

    Hm lng acid

    ursolic th (%)

    1 EtOH 0,0945 0,22

    2 MeOH 0,0863 0,20

    3 EtOH : EtOAc = 1 : 1 0,1226 0,29

    4 MeOH : EtOAc = 1 : 1 0,1345 0,32

    T bng trn ta thy c s chnh lch gia hm lng acid ursolic th khi

    dng cc dung mi khc nhau. Khi dng mt dung mi ring l (EtOH hoc MeOH)

    chit xut, ta thu c lng acid ursolic th t hn khi chit bng h dung mi

    (EtOH/EtOAc v MeOH/EtOAc). Trong khi chit bng h MeOH/EtOAc t l

    1:1, ta thu c lng acid ursolic th cao nht.

    Do trong cc qu trnh tip theo, chng ti s kho st vi h dung mi

    MeOH/EtOAc t l 1:1.

    Sc k acid ursolic th ca cc m chit bng cc dung mi hnh 3.5.

  • 33

    Hnh 3.5 : Sc k acid ursolic th ca cc m chit bng cc dung mi khc nhau.

    (C) : acid ursolic i chiu.

    (M) : acid ursolic th ca m chit bng MeOH.

    (ME) : acid ursolic th ca m chit bng MeOH/EtOAc.

    (E) : acid ursolic th ca m chit bng EtOH.

    (EE) : acid ursolic th ca m chit bng EtOH/EtOAc.

    H dung mi khai trin : CHCl3 : MeOH = 95 : 5 (tt/tt).

    Thuc th hin mu : dd H2SO4 10% trong EtOH, h nng trn bp in.

    3.4.2 Kho st nhit chit xut

    kho st nh hng ca nhit n hm lng acid ursolic th chit t

    dc liu, chng ti tip tc tin hnh chit vi khi lng dc liu 50 g, dung

    mi l MeOH/EtOAc t l 1 : 1, t l dung mi/dc liu gia cc ln ln lt l

    8/6/6 (tt/kl), kho st 3 nhit trn bp cch thy : 45 0C, 55 0C v 65 0C (ch

    l 65 0C hn hp dung mi hi lu), thi gian mi ln chit l 2 h. Kt qu kho

    st bng 3.4.

  • 34

    Bng 3.4 : Hm lng acid ursolic th (%) khi chit cc nhit khc nhau.

    STT Nhit chit xut

    (0C)

    Khi lng acid

    ursolic th (g)

    Hm lng acid

    ursolic th (%)

    1 45 0,1164 0,27

    2 55 0,1255 0,29

    3 65 0,1345 0,32

    Chng ti ch kho st n nhit 65 0C v nhit ny l nhit hi lu

    ca hn hp dung mi. T bng trn ta thy c nhit c nh hng n lng

    acid ursolic th chit c. Khi tng nhit chit th lng acid ursolic th thu

    c cng tng ln.

    V l do trn, trong cc qu trnh tip theo, chng ti s kho st vi h dung

    mi MeOH/EtOAc t l 1:1 nhit 65 0C trn bp cch thy.

    3.4.3 Kho st t l dung mi/dc liu

    kho st nh hng ca t l dung mi/dc liu n hm lng acid

    ursolic th chit t Hng nhu ta, chng ti tip tc tin hnh chit vi khi lng

    dc liu 50 g, h dung mi chit xut l MeOH/EtOAc t l 1 : 1, nhit 65 0C

    trn bp cch thy, thi gian mi ln chit l 2h, kho st 2 t l dung mi/dc

    liu 3 ln chit ln lt l 8/6/6 v 10/8/8 (tt/kl). Kt qu kho st bng 3.5.

    Bng 3.5 : Hm lng acid ursolic th (%) khi chit vi t l dung mi/dc

    liu khc nhau.

    STT T l dung mi/

    dc liu

    Khi lng acid

    ursolic th (g)

    Hm lng acid

    ursolic th (%)

    1 8/6/6 0,1345 0,32

    2 10/8/8 0,1371 0,32

  • 35

    L do s dng lng dung mi nh hn cc ln chit tip theo l do sau ln

    chit u, dc liu thm dung mi nn ch cn lng dung mi nh hn. Tin

    hnh kho st bt u t t l 8/6/6 (tt/kl) v y l t l dung mi/dc liu nh

    nht ngp dc liu, m bo ton b dc liu c tip xc vi dung mi.

    T bng trn cho thy khi tng lng dung mi ln, lng acid ursolic thu

    c tng khng ng k, do chng ti s kho st yu t tip theo vi t l dung

    mi/dc liu l 8/6/6 (tt/kl).

    3.4.4 Kho st s ln chit xut

    kho st s ph thuc ca hm lng acid ursolic th vo s ln chit,

    chng ti so snh lng acid ursolic thu c khi chit 2 ln v 3 ln vi khi

    lng dc liu 50 g, dung mi chit l MeOH : EtOAc = 1:1, nhit 65 0C trn

    bp cch thy, thi gian mi ln chit l 2h, t l dung mi/dc liu cc ln chit

    ln lt l 8/6 (tt/kl) i vi chit 2 ln v 8/6/6 (tt/kl) i vi chit 3 ln. Kt qu

    kho st bng 3.6.

    Bng 3.6 : Hm lng acid ursolic th (%) vi s ln chit khc nhau.

    STT S ln chit xut Khi lng acid

    ursolic th (g)

    Hm lng acid

    ursolic th (%)

    1 2 ln (8/6) 0,1058 0,25

    2 3 ln (8/6/6) 0,1345 0,32

    Theo bng trn ta thy lng acid ursolic thu c sau 2 ln chit bng

    78,66% so vi khi chit 3 ln. Do chng ti s tin hnh chit 3 ln thu c

    lng acid ursolic cao nht.

    Cc kt qu trn cho thy iu kin chit xut cho hm lng acid ursolic

    th cao nht vi 50 g dc liu l s dng hn hp dung mi MeOH/EtOAc t l

    1:1; ln chit u vi 400 mL hn hp dung mi trn; hai ln sau, mi ln vi 300

    mL hn hp dung mi trn. Cc ln chit c tin hnh nhit 65 0C trn bp

    cch thy trong thi gian 2 gi.

  • 36

    Vi iu kin chit ny, t 50 g nguyn liu ban u thu c 0,1345 g acid

    ursolic th, tng ng vi hm lng acid ursolic th l 0,32% trn khi lng

    dc liu kh. S quy trnh chit xut acid ursolic hnh 3.6.

    Cnh v l Hng nhu ta (50 g)

    MeOH/EtOAc (1:1)

    Chit 3 ln/mi ln 2h

    65 0C/lng DM = 400/300/300 mL Lc

    Dch chit

    Cn

    Ct thu hi dung mi

    Dch DCM

    Phn tn trong nc chit vi DCM

    Thm than hot, un hi lu 15 pht, lc ly than hot

    Than hot hp ph acid ursolic

    Dch aceton cha acid ursolic th

    Ra gii bng aceton

    Acid ursolic th (0,1345 g)

    Ct thu hi dung mi

    Dch DCM sau khi lc

    Hnh 3.6 : S quy trnh chit xut acid ursolic t Hng nhu ta.

  • 37

    BN LUN

    V nh tnh cc nhm cht hu c trong Hng nhu ta

    - Kt qu nh tnh cho thy Hng nhu ta c nhm saponin triterpenoid.

    iu ny ph hp vi s c mt ca acid ursolic trong Hng nhu ta.

    V phn lp acid ursolic bng sc k ct

    - Chng ti kho st s c mt ca acid ursolic bng SKLM cc cn phn

    on n-hexan, DCM, EtOAC (hnh 3.3) v kt qu cho thy acid ursolic tp trung

    ch yu phn on DCM; phn on n-hexan v EtOAc ch cha lng nh,

    khng ng k. iu ny l do acid ursolic c phn cc trung bnh nn s tp

    trung phn on DCM.

    - Tuy cn n-hexan chim t l khi lng ln trong cn MeOH (23,84%) (t l

    ca cn DCM v EtOAc ln lt l 26,71% v 8,83%) nhng theo sc k hnh

    3.3, phn on n-hexan ch cha lng nh acid ursolic. Do , trc khi tin hnh

    phn lp cht, cn loi b phn on n-hexan tit kim ha cht, dung mi cng

    nh thi gian tin hnh sc k ct.

    - Qua vic phn lp thnh cng v thu c lng ln cht cho thy acid

    ursolic l mt thnh phn ha hc chnh trong Hng nhu ta (Ocimum sanctum L.),

    iu ny ph hp vi cc nghin cu trc nc ngoi [19, 22].

    V phng php chit xut acid ursolic t Hng nhu ta (Ocimum sanctum L.)

    - Kho st v dung mi : chng ti kho st 4 dung mi chit xut l EtOH,

    MeOH v hn hp ca chng vi EtOAc. Kt qu cho thy khi s dng h dung

    mi c EtOAc th thu c lng acid ursolic th cao hn dng ring l EtOH hoc

    MeOH. L do l acid ursolic l mt cht c phn cc trung bnh nn khi s dng

    EtOAc s lm gim phn cc ca h dung mi, do s ha tan acid ursolic

  • 38

    trong dc liu tt hn. Hn na, y u nhng dung mi thng dng, r tin, c

    th p dng sn xut ln. Khng s dng DCM chit v DCM l mt dung mi

    c hi, bay hi nhanh v dch chit thu c s c c phn khng phn cc (cha

    t acid ursolic) to ra nhiu tp, gy kh khn trong qu trnh chit tch.

    - Kho st v nhit chit : chng ti so snh lng acid ursolic th thu c

    khi tin hnh chit 3 nhit khc nhau 45, 55 v 65 0C tm ra nhit ti u

    cho qu trnh chit xut, va thu c nhiu acid ursolic, va tit kim nng lng.

    Kt qu cho thy nhit hi lu ca h dung mi (65 0C) cho lng cht ln nht.

    - Kho st v t l dung mi/dc liu v s ln chit xut vi mc ch gim

    lng dung mi s dng, rt ngn thi gian chit xut nhng vn m bo thu c

    hm lng acid ursolic cao. Kt qu cho thy cn chit 3 ln v t l dung mi/dc

    liu tt nht l 8/6/6.

    - Phng php chit xut ca chng ti s dng than hot hp ph acid

    ursolic, y l mt nguyn liu r tin, d kim. Sc k hnh 3.4 cho thy acid

    ursolic th c t tp, do d tinh ch. Thc t l chng ti kt tinh c acid

    ursolic vi hiu sut cao (66,07%). Da trn sc k vi thuc th H2SO4 10%

    trong EtOH (hnh 3.5), acid ursolic sau kt tinh c sch cao. V cm quan, acid

    ursolic th c mu vng chanh, sau qu trnh kt tinh v ra mu thu c tinh th

    c mu trng.

    - Chng ti khng s dng sc k ct nh hai phng php m t trn

    (hnh 1.4 v 1.5) phn lp cht, m dng cc phng php tch chit n gin v

    cc dung mi, ha cht thng dng, r tin nn c th p dng quy m ln.

    Nghin cu cn cho thy Hng nhu ta l mt ngun nguyn liu chit xut

    acid ursolic tim nng v cn c nghin cu nhiu hn.

  • 39

    KT LUN V XUT Kt lun

    Sau qu trnh thc hin kha lun, chng ti thu c mt s kt qu :

    - Xc nh c cnh l Hng nhu ta (Ocimum sanctum L.) c cc nhm

    cht : saponin triterpenoid, flavonoid, coumarin, acid hu c, phytosterol, tinh du

    v bng phng php ny khng thy s c mt ca alcaloid, tanin, anthranoid, acid

    amin, ng kh, cht bo v carotenoid.

    - Xc nh c s c mt ca acid ursolic trong Hng nhu ta (Ocimum

    sanctum L.) bng SKLM khi khai trin vi h dung mi :

    CHCl3 : MeOH = 9:1 (tt/tt).

    ether du ha : EtOAc = 4 : 6 (tt/tt).

    v hin mu bng thuc th hin mu : H2SO4 10%/EtOH, h nng trn bp in.

    - phn lp c acid ursolic bng sc k ct t phn on DCM.

    - nhn dng c cht phn lp l acid ursolic bng cc phng php ph,

    kt hp vi d liu ph ca acid ursolic cng b.

    - kho st mt s yu t v rt ra iu kin chit xut cho hiu sut chit

    acid ursolic th cao nht vi 50 g dc liu l s dng hn hp dung mi

    MeOH/EtOAc t l 1:1; t l dung mi/dc liu trong 3 ln chit ln lt l 8/6/6.

    Cc ln chit c tin hnh nhit 65 0C trn bp cch thy trong thi gian 2

    gi. Vi iu kin chit ny thu c lng acid ursolic th bng 0,32% trn khi

    lng dc liu kh.

    xut - Nng cp quy trnh ln quy m ln hn.

    - Xy dng phng php nh lng acid ursolic trong Hng nhu ta v sn

    phm th.

  • 40

    TI LIU THAM KHO

    TI LIU TING VIT

    1. B mn Dc liu (2004), Bi ging dc liu, trng i hc Dc H

    Ni, tp I.

    2. B mn Dc liu (2006), Thc tp dc liu, trng i hc Dc H Ni,

    phn ha hc.

    3. B Y t (2009), Dc in Vit Nam IV, NXB Y hc.

    4. B Y t (2007), Thc vt hc, NXB Y hc, tr. 321.

    5. V Vn Chi (2004), T in thc vt thng dng, tp II, NXB Khoa hc v

    k thut, tr. 1808 - 1810.

    6. Nguyn Th Ngc Lan (2012), Nghin cu phn lp mt s hot cht lm

    cht i chiu phc v cng tc tiu chun ha v kim tra cht lng dc

    liu, Vin Dc liu, tr.3-12.

    7. Vin Dc liu (2006), Cy thuc v ng vt lm thuc Vit Nam, tp 1,

    NXB Khoa hc v k thut, tr. 1027 - 1029.

    TI LIU TING ANH

    8. Baricevic D., Sosa S., Della Loggia R., Tubaro A., Simonovska B., Krasna

    A., Zupancic A. (2001), "Topical anti-inflammatory activity of Salvia

    officinalis L. leaves: the relevance of ursolic acid", J Ethnopharmacol, 75(2-

    3), pp. 125-32.

    9. Frighetto Rosa T. S., Welendorf Rodolfo M., Nigro Eduardo N., Frighetto

    Nlson, Siani Antonio C. (2008), "Isolation of ursolic acid from apple peels

    by high speed counter-current chromatography", Food Chemistry, 106(2),

    pp. 767-771.

    10. Gautam R., Jachak S. M. (2009), "Recent developments in anti-inflammatory

    natural products", Med Res Rev, 29(5), pp. 767-820.

  • 41

    11. Hirota M., Mori, T., Yoshio, M. and Iriye, R. (1990), "Suppression of tumor

    promoter-induced inflammation of mouse ear by ursolic acid and 4,4-

    dimethylcholestane derivatives.", Agricutural and Biological Chemistry, 54,

    pp. 1073-1075.

    12. Huang M. T., Ho C. T., Wang Z. Y., Ferraro T., Lou Y. R., Stauber K., Ma

    W., Georgiadis C., Laskin J. D., Conney A. H. (1994), "Inhibition of skin

    tumorigenesis by rosemary and its constituents carnosol and ursolic acid",

    Cancer Res, 54(3), pp. 701-8.

    13. Jachak S. M. (2006), "Cyclooxygenase inhibitory natural products: current

    status", Curr Med Chem, 13(6), pp. 659-78.

    14. Kosuge T., Yokota M., Sugiyama K., Mure T., Yamazawa H., Yamamoto T.

    (1985), "Studies on bioactive substances in crude drugs used for arthritic

    diseases in traditional Chinese medicine. III. Isolation and identification of

    anti-inflammatory and analgesic principles from the whole herb of Pyrola

    rotundifolia L", Chem Pharm Bull (Tokyo), 33(12), pp. 5355-7.

    15. Liu J. (1995), "Pharmacology of oleanolic acid and ursolic acid", J

    Ethnopharmacol, 49(2), pp. 57-68.

    16. Liu J., Liu Y., Mao Q., Klaassen C. D. (1994), "The effects of 10 triterpenoid

    compounds on experimental liver injury in mice", Fundam Appl Toxicol,

    22(1), pp. 34-40.

    17. Liu Y., Tian W., Ma X., Ding W. (2010), "Evaluation of inhibition of fatty

    acid synthase by ursolic acid: positive cooperation mechanism", Biochem

    Biophys Res Commun, 392(3), pp. 386-90.

    18. Shukla B., Viser, S., Patnaik, G.K., Tripathi, S.C., Srimal,, R.C. Day, S. and

    Dobhal, P.C. (1992), "Hepatoprotective activity in the rat of ursolic acid

    isolated from Eucalyptus hybrid", Phytotherapy Research, 6, pp. 6.

    19. Silva M. G., Vieira I. G., Mendes F. N., Albuquerque I. L., dos Santos R. N.,

    Silva F. O., Morais S. M. (2008), "Variation of ursolic acid content in eight

    Ocimum species from northeastern Brazil", Molecules, 13(10), pp. 2482-7.

  • 42

    20. Stohs S. J., Miller H., Kaats G. R. (2012), "A review of the efficacy and

    safety of banaba (Lagerstroemia speciosa L.) and corosolic acid", Phytother

    Res, 26(3), pp. 317-24.

    21. Vasilenko Iu K., Lisevitskaia L. I., Frolova L. M., Parfent'eva E. P., Skul'te I.

    V. (1982), "Hypolipidemic properties of triterpenoids", Farmakol Toksikol,

    45(5), pp. 66-70.

    22. Vetal Mangesh D., Lade Vikesh G., Rathod Virendra K. (2013), "Extraction

    of ursolic acid from Ocimum sanctum by ultrasound: Process intensification

    and kinetic studies", Chemical Engineering and Processing: Process

    Intensification.

    23. Wen X., Sun H., Liu J., Cheng K., Zhang P., Zhang L., Hao J., Zhang L., Ni

    P., Zographos S. E., Leonidas D. D., Alexacou K. M., Gimisis T., Hayes J.

    M., Oikonomakos N. G. (2008), "Naturally occurring pentacyclic triterpenes

    as inhibitors of glycogen phosphorylase: synthesis, structure-activity

    relationships, and X-ray crystallographic studies", J Med Chem, 51(12), pp.

    3540-54.

    24. Wen X., Xia J., Cheng K., Zhang L., Zhang P., Liu J., Zhang L., Ni P., Sun

    H. (2007), "Pentacyclic triterpenes. Part 5: synthesis and SAR study of

    corosolic acid derivatives as inhibitors of glycogen phosphorylases", Bioorg

    Med Chem Lett, 17(21), pp. 5777-82.

    25. Young H. S., Chung H. Y., Lee C. K., Park K. Y., Yokozawa T., Oura H.

    (1994), "Ursolic acid inhibits aflatoxin B1-induced mutagenicity in a

    Salmonella assay system", Biol Pharm Bull, 17(7), pp. 990-2.

    TRANG WEB

    26. http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=64945#x291

  • 43

    PH LC PH LC 1 : GIY CHNG NHN M S TIU

    BN

  • 44

    PH LC 2 : PH HNG NGOI (IR) CA

    D1

  • 45

    PH LC 3 : PH KHI (ESI-MS) CA D1

  • 46

    PH LC 4 : PH 13C-NMR CA D1

  • 47

    PH LC 4 : PH 13C-NMR CA D1 (TIP)

  • 48

    PH LC 4 : PH 13C-NMR CA D1 (TIP)

  • 49

    PH LC 5 : PH 1H-NMR CA D1

  • 50

    PH LC 5 : PH 1H-NMR CA D1 (TIP)

  • 51

  • 52

    PH LC 5 : PH 1H-NMR CA D1 (TIP)

  • 53

    T VN CHNG 1: TNG QUAN1.1 c im thc vt chi Ocimum L.1.2. Thnh phn ha hc ca Hng nhu ta (Ocimum sanctum L.)Bng 1.1 : Hm lng acid ursolic trong l ca cc loi thuc chi Ocimum L.

    1.3 Tng quan v acid ursolicHnh 1.1 : CTCT ca acid ursolic.Hnh 1.2 : CTCT ca acid corosolic.Hnh 1.3 : S bn tng hp acid corosolic t acid ursolic.

    Hnh 1.4 : S chit xut v tinh ch acid ursolic t l Hng (Diospyros kaki).Hnh 1.5 : S chit xut v tinh ch acid ursolic t v to (Malus domestica).CHNG 2 : I TNG V PHNG PHP NGHIN CU2.1 i tng nghin cu2.2 Phng php nghin cuCHNG 3 : THC NGHIM, KT QU V BN LUN3.1 nh tnh cc nhm cht hu c bng phn ng ha hcBng 3.1 : Kt qu nh tnh cc nhm cht hu c trong cnh, l Hng nhu ta (Ocimum sanctum L.) bng phn ng ha hc.

    3.2 nh tnh acid ursolic trong cnh l Hng nhu ta (Ocimum sanctum L.) bng SKLMHnh 3.1 : Sc k ca cn MeOH ton phn vi acid ursolic i chng trn cc h dung mi khai trin.

    3.3 Phn lp acid ursolic t cnh l Hng nhu ta (Ocimum sanctum L.) bng sc k ctHnh 3.2 : Tm tt quy trnh chit xut cc phn on t cnh, l Hng nhu ta (Ocimum sanctum L.)Hnh 3.3 : Sc k cc cn phn on vi acid ursolic i chng.Bng 3.2 : Ph 1H, 13C-NMR ca D1 v acid ursolic.

    3.4 Xy dng quy trnh chit tch acid ursolic t Hng nhu ta (Ocimum sanctum L.)Hnh 3.4 : Sc k acid ursolic th v sau khi kt tinh vi cht i chng.Bng 3.3 : Hm lng acid ursolic th (%) khi chit bng cc dung mi khc nhau.Hnh 3.5 : Sc k acid ursolic th ca cc m chit bng cc dung mi khc nhau.

    Bng 3.4 : Hm lng acid ursolic th (%) khi chit cc nhit khc nhau.Bng 3.5 : Hm lng acid ursolic th (%) khi chit vi t l dung mi/dc liu khc nhau.Bng 3.6 : Hm lng acid ursolic th (%) vi s ln chit khc nhau.

    BN LUNKT LUN V XUTTI LIU THAM KHOPH LC