Nghiên cứu thành phần hóa học của dịch trích bã hột cây bông Gossypium herbaceum bởi nước 5

Nghiên cứu thành phần hóa học của dịch trích bã hột cây bông Gossypium herbaceum bởi nước 5

Ph'an t(lng quan -Chl/(/flg II

ChuangII CAC PHUONG I)HAI) SU DfjNG DE TIM HIEU THANH PHAN

HOA HOC CUA DJCH TRICH LY BX H(rr BONG nal NUOC.III

MU(Jim(')t (II) phudng phap khac nhau d3: duQc ap d~ng de Om hieu thanhph&n hoa hQc cling nhu ho:;tt tinh IQi sua cua dich trich ly b3: hOt bong bdi nudc dd<;tngddng d) chao khdng

II.l Phli<ingpluip sicu IQcd~ tach chie't s(i bO mOt sa diu td hoa hQc cua djch

trich Iy bi! hOt bong bi~imid'c :

Trong th~p nien 60, nhil3u lac gia da d) I~p protein tu' ba h(')t hdng h~ng cachcha ke't tua cha't trich Iy vdi dung dich mu6i NaCI 10 % GOngphosphat 0, I M d

pH 7,4 [531, vdi dung dich phosphat 3 % [15] , vdi dung dich NaCl2 M [35] ho~cydi dung dich NaOH 0,015 N [8]

Cu6i th~p nien 70, vai lac gia da co l~p protein trong h3: h(')t bong h~ngphu(jng pilar sieu Iqc 1411, [621 f)e'n cu6i th~p nien 80, phu(ing pilar sieu IQc duQcpIlat tri0n them, dung mang I<,>cvdi 10 co duong kinh khac nhau de Iqc cac phan tac6trqnglu'(Jngphan ta khac nhau Cac lac gia da dung phuclngpilar nay de khan

, satcac polisacaridco trQngluQngphan ta > 10 KD va > 30 KD cua 8 loC;\i cay

vungTrung A, trong do co h(')t cay hdng [391

0.2 Phuong pilar djnh lu(jng mi~n djch phong x~ (R.I.A = RADIO IMMUNO

ASSAY) di thd nghi~m Hnh l(ji sua cua cac pilau do~n sieu IQc :

11.2.1.Cac yeu t6 co anh hu'dng den su'pilat trien tuye'n sua:

Vdi ph~ nIT, tC'hao cua mo tiel chi xua't hi~n trong Ilk mang thai, dudi st! taGdQng cua steroid bu6ng trang (oestrogen, progesteron, corticoid); hormon tuye'nyen ( hormon tang trudng va nha't la prolactin)

d cac loai dOng v~t co vu, Lyon L va GOng st! [46] chang minh rang steroidhu()ng trang (E2 17p progesteron) kh()ng th0 lam pilat trien tuye'n vu cua chu(')t,th6 cai, de cai Neu cac steroid huang trang khong hi~n di~n, nhung khi cho lacdyng mOt lu(Jng can hormon tuye'n yen ho~c ca'y tuye'n yen van chuOt , co the lamtang trudng tuye'n sua[49] Khi cho lac dyng cha't CB 154 ,mOt cha't ac che' st! t<;toprolactin d giai do<;tnd&u cua vi0c tiC'tsua, sua se khdng tie't ra duQC [33]

Chinh prolactin ,chd khong phai cac steroid bu6ng trang ,giu vai tro chu yC'utrong vi<;c tiC'tsua Progesteron khong phai la hormon tuyC'n sua chinh yC'u ma chike't hQp trong qua tdnh ban d'au vdi oestrogen lam tang trudng tuyen sua, lam cac hormon tuyC'n sua khac nhu prolactin, glucocorticoid, insulin, hormon tang trudngnh:;tycall h(jn Trong s6 nay prolactin dong vai tro quan trQng ,con khac hormonkia chi gap phtln lam tang kha nang ho:;ttdOng cua prolactin

~

Phl1n tdng quan -ChU(fng Il

-9-Ndl loai dOng v~t co vu sail khi sinh ,song song vdi hi~n tuQng liSt sua, ham luQngprolactin cfing tang theo mQt cach dang k€ trong mall

Do do, nSu dich trich ly cua cay thu6c c6 anh huang dSn vi~c kich thich slf

LiClra sua, no cfing kich thich SlrtiCt ra prolactin VI the ,vit;c dinh phan prolactinLronghuyCLLu<Jngcua dOng V~Ldu<Jc thli' nghi~m vdi chilLLrich cua cay se cho bit\'LLinhIQisua cua dich Lrich cay Lren

Chung Wi chc,mphuong phap cua lac gia MESSINGER H G [50] d€ do hamlu'Qngprolactin trong huyet tu<1ngcua truu va chuOt cai b~ng phuong phap dinhlu'(}ngmiCn dich phung x~ ,nh~m tIm hi0u ho~t Hnh tang sua trcn truu va chu(}tcuacae phan do~n sicu I<}ccua dich Lrich bi! h(}t b<1ngbdi nudc

Vi~c Lhinghi~m trcn cac sinh v~t s6ng kh6ng phai hao gi('Jcfing d';lt du(Jc kCt

qua kha quaIl VI m6i dQng v~t c6 dO nh';ly cam hormon khac nhau Ngoai ra c6nhi0u yCu t6 anh huang dSn stf tiel prolactin n<1icon truu

Ham lu'(Jng prolactin cua trtJ'u thay d<1itheo chu ky dOng dl;lc, thai gian ngayd0m, slf ding thiing th'an kinh khi hi lily mall fan d'au, nhi~t d(} (thco mua co iLhaynhi0u anh Hang m~t tn'Ji)[611

1I.2.2.Binh phan prolactin:

Tilt ca cac phU(1ng phap do hormon dell sli' dl;lng phuong phap dinh luQngchiiLmi~n dich phung x~ Phuongphap nay dlfa vao slf c';lnhtranh giua mQtkhang

nguyen kh6ng mang Linh phung X';lva mQt khang th€ co mang dang vi phung X~voi s6 lu<jng hiet tru'dc, ca 2 se gall vao m(}t khang the d~c bit;t, c6 s6 luQng biet

Lru'ocva st; lu'(,1ngnay sc iLh(1nho~c h~ng bing st; cac khang nguycn

Phan ling chung la :

*

Phu'c kSt tl;lgiuakhang nguyen va khang th€

h~ngmay do so'phung X';l,

Ph'an t6'ng quan - Chu(1ng II

-10-Ve'duong bi€u di~n chuffn bieu thi so' ph'an tram cua khang nguyen cophung X<;ln6i Ilen vdi khang the ,khi co s,! hi~n di~n tang d'an cua khang nguyenkh6ng ph6ng xC;l.Songsong, chuffn bi cac 6ng nghi~m trong d6 ph'an tram cua cackhang nguycn phung XC;ldIng du<jc xac dinh nhu tren, nhung cac khang nguyenkh6ng ph6ng x':ldu(jc thay the' b[ing mfiu c'an dinh pIlau

HINH 1 : Buong bieu di~n chuffn

" *

(}()ho':lt Hnh chung cua lAg -Acl

so' do dudc Mfiu c'an doN()ng dO khang nguycn /

Ndng de)Ag (nglml)Voi Ag* 2 Ac, ta co diing thlic :

: S61uQng khang nguyen ph6ng XC;lbi giin

Phu(jng trlnh dudc bieu di~n b[ing. mot hiperbon dang y = ~x + B

va trong phong thl nghi~m, cac du ki~n duQc xli' 19 bdi chuang trinh may tinh

1I.3.Bjnh phan ham hiqng dliong t6ng quat bdng phlidng phap so mau [74] :

II f)u(}ng trung Hnh thuong hi~n di~n trong thao mOc la aldohexoz nhuD-galactoz, D-glucoz, D-mannoz ho~c nhung aldopentoz nhu D-arabinoz,D-xiloz hay deoxihexoz nhu' L-ramnoz , L-fucoz ( Hlnh 2)

~

PhCin tt/ng quan - Chuang II

-11-Duui lac dl;lng cua acid VO C(j, pentoz va hexoz trd thanh aldehid nhuformaldchid, acctaldchid, crotonaldchid cung vui dftn xua't cua furfural nhu'5-hidroximctilfurfural ho~c 5-metilfurfural va cac san phftm giam ca'p kh6ng chu'ah~ Ihi)ng n6i (1(,ilien IH.ip

Tll~ tu(jng d6i giii'a dftn xuc1tcua furfural vui cac chc1tham s~c thay d0i tuytheo n6ng dO cua acid, nhi~t dO, thoi gian phan ling Phu(jng phap nay c6 sai s6trung blnh khoang 9 'Yo

Dtnh phan b~ng phuong pIlar so mall, cac lac cha't orcinol, antron, phenol,cistein duQCsa dl;lng va antron du(Jc dung nhi~u nha't

C(j che' phan u'ng: antron hay 9, IO-dihidro-9-oxoantracen trong moi trui'jngacid n()ng , k<3th(Jp vui san phftm giam cc1pcua duong, chc1tdftn xua't cua furfural

dC t(;lothanh cha't c6 mall xanh du(jng ll;lc 134J (hlnh 3).

Phan t1ng nay h! u'c ch<3hdi cac chc1tox id h6a va du(.ic ho(;lt h6a hdi cac chc11khu' hay chc1tkhang- oxid h6a Vai lac gia dC ngh! dung chc1tkhang- oxid h6a nhutioure d«36n dtnh dung djch antron

£-)0 hc1p tho clfc dC;licua cac chc1t mall t(;lo thanh ttr chc1t duong d khoang

II 620 11m,nhung dO dai s6ng du'<jcdo d 585 nm d€ tranh hi~n tu<jng giao thoa gay rahdi slf hi~n di~n cua cac protein cilng cho phan u'ng vdi lac chc1tantron

[-~._.n :", "

I r'~ " I J

,;.li II (~ , ~_.:~: , -'Iu () J

HINH 2: Cc1utruc cac duong trung tinh trong thao mOc

I)-galactof, I)-mannoz D-glucoz L-fucoz

Acid uronic cling cho mau y€u vdi antron, c6 dO hilp thu cl{c dC;\id 560 nm ;

d 585 nm, dO hilp thu cua acid uronic kh6ng dang k€ so vdi dO hilp thu cua cae chillduong Osamin, acid sialic kh<1ngt.ic dl;lng vdi antron

11.4 Dinh philo cae dlion~ drill trun~ tinh qua ddn xua't ace tat alditol biin~pllli<1n~phap sdc ky khi 191

Polisacarid thuy giiii cho monosacarid, sau do monosacarid acetiJ hoa thanhd~n xuitt ace tat bay h(1i, xac dinh bdi phuong phap s~c ky khi

f)i<3uki~n c'an thi€t d€ chuy€n hoa cae polisacarid thanh monomer thay d<1ituy then diu true va thanh ph'an ca'u tc;\o monomer cua cae polisacarid Voi polisacarid d(1ngian, s\!,thuy giiii ra't de dang va kh6 khan h(1n n€u polisacarid chuad(jn vi acid uronic hoi;tc osamin Cae nh6m amino d vi tri C-2 cua monomer cuahau h€t cae poliglicosamin bi proton hoa khi thuy giiii, do d6 6n dinh c'au n6i

. glicosic Mu6n be gay diu n6i nay, c'an di<3u kit?n mC;\nh me hon vdi HCl 4 N,

100 °c ,2 gi(J,

Ph'an r6ng Quan - Chztmzg II

-13-OH

OHNH2

NHCOCH3Glucosamin

(2-amino- 2-deoxi-p- J)-glucoz)

N-acctil glucosamin(2-ace tamido- 2-deoxi- p- 0- g lucoz)

Voi pobsacarid chua d(jn vi acid uronic, nghla Ia chua nh6m -COOH d C-6cua monosacarid, dIu n6i gbcosic se 6n dinh Slf thuy giai mOt cach dinh lu<;Jngthanh U(jnvi acid uronic chi xay ra vui HF , - 23 °c [54] Sau khi thuy giai va khu,cac dftn xuftt acid aldonic trd thanh diin xuftt trimetilsilil (TMS) do lac dl;!ng vdihcxametil disilazon ; d~ng ace tat cua acid aldonic khong bay he5i

Thong thuong sau giai do~n thuy giai, monosacarid bi khu bdi borohidrurnatrium NaBH4 ,d nhi~t dO phong; ngo~i tru glucoz, ramnoz hi khif ch~m hejn.Cac dftn xuftt cua bor se t~o phac vui alditoI, tam ngan can stf acetil h6a d

giaiuo~n ke tiep Cac chftt nay bi lo~i bdi mctanol duui d~ng borat trimctiJ

Thlfc hi~n sl;!'acetil h6a bdi anhidrid acetic vdi xuc lac piridin Trkh cacacetal alditol vita t~o thanh bdi ace tat ctil

Chftt dftn xuftt acetal alditol rftt ben, c6 Hnh bay hdi, va thuong dung d6 phantich uuong trung Hnh b~ng phu(jng phap s~c ky khl 169), ho~c s~c ky long can apqua dftn xuftt 2-aminopiridin 128J

U.S l)hlMng phap dinh philo acid uronic [74]

Co nhi'Cu Io~i acid uremic nhu acid D-gbcuronic, acid L-iduronic, acidl)-galacluronic, acid D-manuronic nhu'ng cho den nay, nguoi la chi tlm thfty acidI)-galacturonic va acid D-manuronic trong poliosid thlfc v~t

Philn Tling Quan - Chtll1ng II

-14-OH

HOAcid (J-D-glicuronic

Carhazol hay dihenzopirol, trong m6i tn()ng acid, n6ng , ke't hQp vdi aciduremiccho san ph~m co man till d~c hiQt 1171

Theo ROWNESS [101 , san khi so sanh ph6 ha'p tho to' ngo~i cua nhfeu dftnxuiit cua acid [uranic va cac san ph~m giam ca'p cua acid uronic bdi H2SO4, vdiSl;1'hiQn diQn ho~c khie'm diQn cua carhazol, chlnh acid 5-[ormil furoic la nguyennhan t~o ra man

Sl;I'ha'p tlm cua cae man thay d6i tily theo lo:;ti acid uronic Hexoz cfing chovdi lac cha't carhazol-sulfuric mQt man yang nh~t, nhung ha'p tho eve d~i d 410 om,Lrongkhi dO hap tho clfc d~i cua cac acid uf(mic la 530 om

Osamin, acid sialic kh6ng cho ph an ung vdi carbazol-sulfuric

Protein kh6ng cho hi~n tuQng giao thoa, ngo~i tru d nong dQ 200 Ilg/ml Ne'unong (lQ can hc$n200 Ilg/ml, slf giam cu'ong de?man xay fa

Cae mu6i natrium, kalium, amonium lam giam cuong de?cua philo ung.PhU(ingphap sa dl;1ngcarbazol ti~n IQi, toy v~y m6i fan dinh phan vftn phai

do I~i cae dung dich acid uronic chu~n d~ thie't l~p duong bi~u di€n chu~n

11.6.I'htMng phap dinh lliqng protein toan ph'an :

den nay, phuong phap nay vftn con hi~u qua ,vdi nhieu cai tie'n b~ng each sa dl;1ng

Lhu6c Lha Coomassie Brilliant Blue G.250 [65J

Khi ph~m man Coomassie nOi vdi protein trong m6i truong

chuyen dQhap tho tu 465 mm de'n 595 mill

acid, co slfvdi slf djch

Philn Tong Quan-ChLtl7ng II

Ph:1m mall xanh Coomassie Brilliant G-250

Trudc day, theo DIAMANT B [16] , ph:1m mall gall vdi protein ngang quanh6m sulfonic, nha llfc ha'p tllU tinh di~n G'an day, theo COMPTON S.J.[ 14]Coomassie hi~n di~n d 3 tr<;lngthai can bling : cation, trung tinh va anion vdi cacmlii ha'p thu quang ph6 l'an luQt la 595, 650 va 465 om D<;lnganion gall vdi proteindl{atren slf dich chuyen ha'p thu ta 465 mm de'n 595 mm S6luQng coomassie gallvao m6i phan tit protein tl l~ vdi di~n tich duOng cua protein :khoang tu 1,5 de'n 3philo ta cha't mall cho 1 dit; n tich [64]

Thu<1cthu' coomassie cua hang PIERCE ctfc nh<;lyvdi n6ng dQ protein tha'p,

(thi dl} vdi bovine serum albumin 1 flg/ml) ; kh6ng bi anh huang bai h'au he't cac

11.7.Hinh phan Amino Acid h~ng may siic ky Mng cao up .

Trong 10 nam g'an day, ngu()i ta thu(Jng dinh phan cae amino acid blingphuOng phap sac ky long cao ap, qua dftn xua't cua amino acid vdi fluoreniJ

me tiJchJorofor mat (FMOC-C 1), orthophtaldiaJdehid (OP A), phenilisotiocianat(PITC) 113J va 6-aminoquinoliI-N-hidroxisucinimidil carbamat (AQC)

III

DAnxutt cua PMOC

Phan U'ngxay ra nhanh d nhi~t d(>phong; chill dftn xuilt cua no b~n trongnhilsugio Dftn xufft FMOC bi anh hudng bdi muO'iva cac t~p chill C'an sa dl;!ngdungm6i hull C(jde trich san phftm truoc khi ap dl;!ngs~c ky lOngcaD ap

kh6ngmiluphan tich, lam bay hdi cae chill diln xuat cua amino acid, tinh che' chungh~ng s~c ky trao d6i ion, din thdi gian it nhat J05 phut de ph an rich m(>tmilu

V(li OPA (orto-phtaldialdchid) 1241

Phu(jng phap nay hi~n dang th6ng dl;!ng, VI OP A t~o thanh chill dftn xuatnhanh vdi amino acid Vi~c tach cae dftn xufft co the th1;1'chi~n t6t d nhi~t d(>phong,vdi uQ nh~y cao.Phuong phap it cho ph an U'ngphl;!, d€ thao lac t1;1'dQng tren may.Tuy nhien, OPA kh6ng phan lIng tr1;1'ctie'p voi amin nhi nhu prolin,hidroxiprolin Do do, din oxid hoa vang imino (acid) cua prolin bdi c1oramin-T,

I

, ~

-17-vdi sl;\' hi~n di~n cua NaBH4 , d~ t~o thanh 4-amino- J -butanol Ke' d6, chill trendu'ljcbien d<1ithanh chill dfin xuilt cua OPA

PluMng phap c6 au di~m ]a cae lac chill thira trc) thanh aminoquinolin(AMQ), vdi dQ hilp thu A ctfc d~i =520 nm khac h£n so vdi dQ hilp thu cua AQC

Cae phuong phap tren d-euc6 uu va khuye't di~m, tily thuQcdi-euki~n, su d~ng mQt

trongnhang phu<.jngphap neu tren d~ phan tich cae amino acid

H3N+-C-H

COO-H-t-OH

IHSerio (Ser)

COO-+ I

H3N -C-HCH

«00-CH2

/~HH3C CH3Leucin(Leu)

COO-+ IH3N -y-HCH2IC(jHsPhenilalanin (Phe)

Ph'anT6ng Quan -Chtldng II

-19-U.8 Phli<1ngphap siic ky IQc GEL

M(H trong nhung phuong phap dau Lien thuang duQC sa dl;(ng d€ tinh che cacd~i phan ta nhu protein, acid nucleic hay polisacarid Ia siic ky IQc gel(chromatographic par gel filtration) Nguyen tiic cua phuong phap la cac phan tatrong dung djch duQc tach do kich thudc khac nhau cua chung va tach theo phan takh6i giam daD Cac gel th6ng dl;(ng co ten thuong m~i nhu Sephadex, Sephacryl,Scpharoz chill gel la m()t lo~i polimer phan nhanh cua dextran va hiacrilamid,

, kh6ng tan nhu'ng riltl1g~m nu'(Je N6 c6 d~ng hinh Call tf()n, du<'1ngkinh trung hinh

Ii! 1001-1111.

Uim tntdng nd gel trong dung m6i se dung trong phep siic ky ; cho gel vaoC(lt; cho ti6p mau chill van d'au C()tgel Khi giai ly, cac phan ta co trQng luQngphan ta nho, phan b6 trong dung m6i d khoang tn')ng giua cac h~t gel, no cling cothe xuyen van va niim trong nhung Iwt gel; cac phan ta co trQng luQng phan tu'Idn,kh6ng th6 chui van cac h~t gel, niim trong dung djch , chiem kh6ng gian giuacac h<;ttgel Khi giai ly, phan ta ldn se di chuy€n nhanh , ra kh6i cQt gel trudc tien

HINH4 : SI1tach cac phan ta do kich thudc khac nhau biing phuong phap siic ky

Khuyct diem cua phu'(jngphap la kha Dang tach co gidi h~n.Thi dl;!: protein

la chiltda di~n phan ma he mi;itcua no mang nhung tam it, nhi'eu kY.nudc ; cac tam

cua no118].Vai lo~i gel thue}ngm~i cua hang PHARMACIA (Bang 3)

BANG 3: Vai lo~i gel thue}ng m~i cua hang PHARMACIA

11.9 1)lnl(ing phap sdc ky trao d6j ion:

Il.9.I.Khai niem t6nuuat ve thanh tt baD thuc vat:

Theo TREVOR ROBINSON 167] , vach cua thanh tt baD thl{c v*t gum baIo~i polisacarid :ccluloz, hemiccluloz, polisacarid pectic Nhom polisacarid pectic

g6m tfftca cac polisacarid trich ly tll'thanh tt baD bdi nudc nong Hemiceluloz du<jclrich bdi dung dich ki6m tu'<lngdoi m~nh va ph'an ba con I~i sau khi trich ki6m Iaceluloz

Polisacarid pectic la h6n h<jp phuc t~p polimer acid va trung Hnh [73] No

la thanh ph'an chu ytu trong trai, hQt rau.Chu6i day chanh gum cac acid galacturonic n6i vdi nhau b~ng a I -4 va day chanh n'ay xen ke bdi nhung don viL-ramnoz L-ramnoz noi vao acid uronic bdi C-I va C-2 cua no d~ t~o nen g6c I4ch

D 900 so vdi lrl}c chanh gQi la khuyu pectic Tren nhung carbon CD 2, CD 3, CD 4 cuaramnoz va acid galacturonic, d<1ikhi c6 g~n them nhung dudng khac nhu B-D-xiloz,I3-D-galactoz,L-arabinoz Do chuc acid, pectin d~ tan trong nudc

Ca'u truc Lieubi~u cua pectin [79] du<jc trlnh bay trong hlnh 5

Cha't pectic cua cay Hm tha'y nhieu trong tt baD nha'p ca'p (55 %) va lien tt

III baa, hi~n di~n nhi€u trong cay 2 la m'am he}nla cay 1 la m'am No gum h6n h<jparaban, galactan, metil eter cua galacturonan, chua xiJoz, ramnoz Ca phan ta lamQtkh0i di- polimer, c6 trQngIueJngphan Iii'IOngom 1000 de}nvi ho~c nhi'Cuhon

Ilondagcl 12-125 2000 - 50000 Dung cho may HPLC 20 - 50