Tl/CAC THl/C VAT KHAC NHAU TACH CHlfiT VA PHAN TICH HAM LUONG ANTHOCYANIN

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:)JW: /y-QD Tap ehi SINH HOC 12-2011

TACH CHlfiT VA PHAN TICH HAM LUONG ANTHOCYANIN Tl/CAC MAU THl/C VAT KHAC NHAU

PHAM THI THANH NHAN

Trudng dai hge Suphgm, Dai hge Thdi Nguyen NGUYfiN HOU CUdNG, ht TRAN BINH

Vien Cong nghe sinh hge Cac loai sde td d thue vdt ed vai trd rd't quan

trong trong qua trinh quang hgp. Chung bao gom: sde td lue (chlorophyll), sde td vang (carotenoid), sde td eua thue vdt bde thd'p (phycobilin) vd sde td djch bao (anthoeyanin).

Tren CO sd hdm lugng cde dang sde td trong Id, ngudi ta cd the danh gia kha ndng quang hgp ciia thuc vdt va xlp thuc vdt thude nhdm ua sang hay ua bdng, thue vdt C3 hay C4. Anthoeyanin dugc tim thd'y trong djch khdng bdo eua t l bdo bie'u bl, md mach ddn [I, 4, 8, 10]. Chung xud't hien trong rl, tru dudi la mdm, bao la mdm, than, cii, la va tao mau eho ea bl mat, viln sge, hay cac vet dd'm. Anthoeyanin la nhfing glucozit, thude hg flavonoid, do gd'e dudng glucose, glactose... kit hgp vdi gd'e aglueon cd mau (anthocyanidin). Aglueon eua chung cd cd'u tnic CO ban duge md ta trong hinh 1, Cac gd'c dudng thudng duge gdn vao vj tri 3 va 5, it gdn vao vj tri 7, Cac aglycon cua anthoeyanin khde nhau chinh Id do cde nhdm gdn vdo vj tri Rl va R2, thudng la H, OH bode OCH3 [2], '

Hinh 1. Cd'u true co ban cua aglycon cua anthoeyanin

Anthoeyanin la ehdt mau thien nhien dugc sir dung an toan trong thuc pham va dugc phdm v6i gia thdnh cao (khoang 1000 USD/lOOmg), Chiing tdn tai trong hdu he't eae thue vdt bde cao va CO nhilu trong rau, hoa, qua, hat cd mdu tfi do de'n tim nhu: qua nho, qua ddu, la tia td, gao

dd, hat ngd den.,,, Gdn ddy, ehfic ndng eua anthoeyanin duge nhilu nha khoa hge quan tdm nghien efiu [6, 7, 12, 14], Cac ehfic ndng cua anthoeyanin bao gdm: bao ve lue lap khdi tac ddng bdt Igi eua anh sdng, ban ehi bfie xa cua tia UV-B, hoat tinh chd'ng oxi hod va ehdng viem. Ngodi ra, chung cdn tao diiu kien cho su thu phdn, phdt tan hat nhd mdu sde sac sd tren canh hoa va qua. Sinh tdng hgp anthoeyanin d la dugc tang cudng dl dap fing vdi stress m6i trudng: ban, anh sang manh, UV-B, nhiet do eao, thilu nito va photpho, nhilm ndm va vi khudn, tdn thuong, edn trung, d nhilm [9, 15].

Vdi kha ndng chd'ng oxy hda eao, anthoeyanin duge sfi dung d l ehdng lao hda, hoae chd'ng oxy hda cde san phdm thuc pham, ban ehe su suy giam sfie dl khdng, Diiu nay md ra mdt trien vgng vl viee san xud't duge pham chfic ndng ehfia benh ed hieu qua.

O Viet Nam, anthoeyanin ed the duge tach chie't tfi eae nguyen lieu thuc vdt sdn ed. Bai bao nay trinh bdy mdt sd dung mdi tach ehilt anthoeyanin vd kit qua xae djnh ham lugng anthoeyanin trong mdt sd nguyen lieu tuoi bdng phuang phdp pH vi sai, ldm co sd eho viee lua ehgn dung mdi va nguyen lieu gidu anthoeyanin dl khai thae sfi dung.

I. PHU'ONG P H A P NGHIEN CtfU 1. Vdt lilu

Cac loai rau, cfi, qua dugc thu thdp tai Hd Ndi (thang 12 ndm 2009),

2. Hda chat

Chung tdi sfi dung cac hda chat nhu:

ethanol, acetone, chloroform, methanol, KCl

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(dung dieh dCm pH 1,0), K,S205, CHjCOONa (dung dieh dCm pH 4,5) ciia hang Fluka; HCI dae cua Trung Qude.

3. Phuong phdp

(/. Tdch I hict anthocvcinin

Anthoeyanin dugc tach ehilt theo quy trinh sau [5. 11]: NgiiyOn liCii tirai thu thdp vl duge rfia sach. dl rao hit nude a nhiCt dd phdng, mdi mdu thi nghiem cdn la'y 10 g (mdi loai nguyen liCu lay 3 mdu); nghiln nhd mdu thdnh bdt mjn trong nito Idng. bd sung 20 ml dung mdi ed HCI, dl mdu d 4"C trong 24 h; Igc chan khdng thu la'y djch (lap lai 3 Idn), bd sung chloroform (theo tl le lv:lv) va thu dieh mdu phia tren. Do phd hd'p thu vd mdt dd quang

b. Xde dinh ham lugng anthoeyanin theo phuang phdp pH vi sai 13}

Dua tren nguyCn tde: ehd't mdu anthoeyanin thay ddi theo pH. Tai pH 1,0 eae anthoeyanin tdn tai d dang oxonium bode flavium cd dd hd'p thu cue dai, edn d pH 4,5 thi chung lai b dang carbinol khdng mdu. Do mdt dd quang eua mdu tai pH 1,0 vd pH 4,5 tai bude sdng hd'p thu cue dai, so vdi dd hd'p thu tai bude sdng 700 nm.

Dua tren edng thfie eua djnh ludt Lambert- Beer:

l g ^ = £ x / x C

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Trong dd: Ig—. dae trung eho mfic dd anh sang ylu ddn khi di qua dung djch (mdt dd quang. A); I. Cudng dd dnh sdng sau khi di qua dung djch; IQ. Cudng dd dnh sang chilu vdo dung djch; C. Ndng dd ehd't nghien eihi, mol/l; 1.

Chilu day eua Idp dung djch md dnh sdng di qua; e. He sd hd'p thu phdn tfi, moi' cm"'

Ham lugng sde td anthoeyanin don tu theo cdng thfic:

AxMxHSPLxV^ ^2) a = -

exl

Trong dd: A. Mdt dd quang, A = (Ax^ax. pH=i.o

" A700nm. pH=l.o) " (AXmax. pH=4.5 Ayoon^, pn=4.5); Axmax»

Ayoonm- Dd hd'p thu tai budc sdng cue dai vd 700 nm d pH i,0 va pH 4,5; a. Lugng anthoeyanin (mg/I); M. Khdi lugng phdn tfi cua anthoeyanin (g/mol); HSPL, He sd pha loang;

V, Thi tich dieh ehilt (I); 1. Chilu day cuvet (cm); e = 26900,

Tfi dd tinh dugc phdn trdm ham lugng anthoeyanin todn phdn:

%anthocyanin =

mx(100-w)xlO' • X l 0 0 % (3)

Trong dd: a, Lugng anthoeyanin tinh dugc theo cdng thfie (2); m. Khd'i lugng nguyen lieu ban ddu (g); w. Dd dm nguyen lieu (%.).

Djch chi^t anthoeyanin duge xu ly bdng nude cd't thay cho eae dem pH d tren, dem do d cac bude sdng tuang ii:ng va xac dinh mdt dd sac mdu theo edng thfic [11]:

M =[{A 420nm '700nm ) + (A ...-'4,<K,.J]xHSPL(4) Djch chii"! anthoeyanin cdn lai dugc xu ly bdng dung djch K2S2O5 20% thay eho dem pH, dem do d edc budc sdng tuong irng vd tinh ti 16 phdn trdm mdu da tfi theo cdng thfic [II]:

D=[(A,,,„„-A^^„J + (A D

w-^7oo«J]xHSPL

% Mdu da tfi = — X100%

M ⁽⁵⁾

e. Phuang phdp tinh todn sdlieu

Sfi dung toan thdng ke dl xae djnh cac trj sd thd'ng ke, mdi mdu nghien cihi dugc nhde lai ba Idn. Cac sd lieu dugc xfi ly tren may vi tinh bdng chuang trinh Excel theo Chu Hodng Mdu (2008) [13].

II. K^T QUA VA T H A O LUAN

1. Phd hd'p thu ciia anthoeyanin a mot sd rau, cij, qua

Nguyen lieu tuoi thu thdp vl duge xfi ly theo quy trinh b mue 3.a vd sfi dung 4 loai dung m6i chie't anthoeyanin khae nhau: ethanol: dung djch HCI 1%= 1:1 (v/v), acetone cd 0.01% HCI (v/v), methanol cd 0,1% HCI (v/v), methanol co 0,01% HCI (v/v) [2, 5, 11], Sau dd chfing tdi Id'y 0,5 ml dieh chie't pha loang vdi 2 ml dung djch dem pH 1,0, quit phd hd'p thu budc sdng tfi 250 nm de'n 750 nm tren may quang phd. Kit qua dugc minh hga d hinh 2,

Ke't qua trin cho thd'y, phd hd'p thu eua djch chie't tfi cac mdu nghien cihi nam trong vung phd hd'p thu cua cac anthoeyanin (510-540 nm), bude sdng hd'p thu cue dai eua djch ehilt anthoeyanin tfi la tia td la khoang 520 nm- 524

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nm, qua ddu ta la 513-526 nm, vd qua nho la nm, vd nho Id 523,5 nm [2], Diiu nay da chfing 519-523,5 nm. Trong khi k i t qua cdng bd trude td dd tin edy cua phuang phap thfi nghiem va day d la tia td Id 524 nm, qua ddu Id 513,5 dung mdi tdch ehilt la kha eao.

3,449

4,360

43

<

2,200

0,0411—

Hinh 2. Phd hd'p thu eua djch chie't anthoeyanin tfi mdt sd nguyen lieu tuoi d pH 1,0 A. than ng6 non; B. rau din; C. la tia t6; D. Ld mo; E. Phd anthoeyanin chudn d pH 1,0 va pH 4,5

(theo M. Monica Giusti va Ronald E. Wrolstad, 2001); 1. dung m6i ethanol: dung djch HCI 1%

(Iv: Iv); 2. dung m6i acetone cd 0,01% HCI (v/v); 3. dung ni6i methanol cd 0,1% HCI (v/v);

4. dung m6i methanol co 0,01% HCI (v/v).

Bdng I Gia trj mat do quang cua cde dieh chiet anthoeyanin tiir bdn dung mdi nghien efiu S

TT 1 2

Mdu Than eua lodi ngd (Zea mays L.) La ehua, bup dd'm

X„,ax(nm) 512 521

Gid tri mdt c Ethanol

HCI J%

0,23 1,21

Acetone

0,01% HCI

0,29 1,27

d quang A (Abs.) Methanol

0,1% HCI

0,27 ' 1,26

Methanol

0,01% HCI

0,25 1,25

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3 4

5 6 7 8 9 10

11 12

{Hibiscus sabdarijja L.) Hoa cua loai chudi tieu {Miisa paradisiucci L.) La cua lodi hoa sdi {Chloranthus spieatus (Thunb.) Makino) l,a ciia loai huylt du ((.'orilyti/u'fi-uticosa (L.) Gocpp.)

Qua ciia lodi ddu tam {Moras alba L.) La cua loai tia td {Perilla fiutescens (L.) Britt.)

Cii cua loai khoai lang tim {Ipomoea batatas (L.) Poir.) Vd qua cua loai nho

(\ /7/.V vinifera L.) La ciia loai mo leo

{Paederia scandens (Lour.) Merr.)

Gil eiia loai sdm dai hanh {Eleutherine bulbosa (Mill.) Urban)

La cua loai rau din tia {Amaranthus tricolor L.)

516 522

519 513-526 520-524

520 519- 523,5

522

511 540

0,10 1,08

1,57 2,43 1,81 2,08 2,06 0,59

1,49 1,04

0,12 1,18

1,65 2,55 2,32 2,12 2,10 0,62

1,86 1,06

0,10 1,16

1,59 2,43 2,21 2,02 2,04 0,51

1,75 1,02

0,10 1,06

1,50 2,34 2,02 2,02 2,02 0,44

1,55 1,00 So sanh phd hd'p thu vd gid trj mdt dd quang

eua djch ehilt anthoeyanin thu duge tfi bd'n loai dung mdi cho thd'y, sfi dung dung mdi acetone ed 0,01% HCI (v/v) thu duge lugng anthoeyanin td'i uu ban ea, vd phd hd'p thu gid'ng vdi phd chudn [11]. Kit qua nay tuong tu d cde dd'i tugng cdn lai (bang 1).

2. Ham lugng anthoeyanin d mot sd rau, cii, qua

Do hd'p thu cua anthoeyanin lien quan mdt

thie't de'n mdu sde, ndng dd cua chung va phu thudc vdo pH cua dung djch (thudng pH thudc vfing acid manh cd dd hd'p thu Idn).

Sau khi tach ehilt anthoeyanin bdng dung m6i acetone, chfing tdi tiin hanh do mdt dd quang cua cde mdu nghien cuu tai bude sdng hd'p thu cue dai, 420 nm vd 700 nm, 6 pH 1,0 va pH 4,5, tfi dd dp dung cdng thfic (2), (3), (4) vd (5) tinh duge ham lugng anthoeyanin trong cac loai nguyen lieu tren. Kit qua duge thi hien trong biing 2.

Bdng 2 Ham lugng anthoeyanin trong cac mdu nghiln cihi

s

TT 1 2 3

Mau Thdn cua loai ngd (Zea mays L.) La chua, bup dd'm

(Hibiscus sabdarijfa L.) Hoa cua loai chudi tieu (Musa paradisiaca L.)

Ham lugng toan phdn (%)

0,59 1,49 0,34

Sde td anthoeyanin don tur (mg/I)

19,57 84,63 7,88

Mau da tijf(%)

31,42 23,30 35,21

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4 5 6 7 8 9 10 11 12

La cua lodi hoa sdi

(Cliloranthus spieatus (Thunb.) Makino)

La cua loai huylt du

(Cordylinefiutieosa (L.) Goepp.) Qua cua loai dau tdm

(Moras alba L.) La cua loai tia td

{Perilla fi-utescens (L.) Britt.) Cu cua loai khoai lang tim {Ipomoea batatas (L.) Poir.) Vd qua cua lodi nho

{Vitis vinifera L.) La cua loai mO leo

{Paederia scandens (Lour.) Merr.) Gil cua lodi sam dai hanh

{Eleutherine bulbosa (Mill.) Urban) La cua loai rau din tia

(Amaranthus tricolor L.)

0,56 1,28 1.75 1,72 0,46 1,27 1,05 2,04 1,74

78,89 110,28 170,26 154,77 141,67 140,20 41,28 124,37

70,54

23,66 22,12 17,73 19,97 21,95 22,06 25,88 21,87 24,02 Kit qua bang 2 eho thay, ham lugng

anthoeyanin toan phdn cua cac mdu nghien efiu dao dgng trong khoang tfi 0,34 % din 2,04%.

Dang chu y la ham lugng anthoeyanin cua la tia to la 1,72%, qua dau ta 1,75%, vd nho 1,27%, cao hon kit qua cua tac gia Huynh Thj Kim Cue (2007) (0,397%, 1,188%, vd 0,564%) [2], Su khac nhau nay cd thi do mdu dugc lay d dja

180 160 140 120 100 80 60 40 20 0 1

(mg/I)

) 1 1 / ' \

r t

M - / - r \ - -! - -

/ * \ ' /

1 \ ^ 1 1

V

1 ^

f-i- -

1 i i

I t t I /

I

- r -

- t- -

1 1

4'--.

/ 1 1

1

S ^

- t -

1

t t,.:

^^^

- - 1 ^

I

1 1

1

— 1 ~ -

1 1

1 I

1'

r

1

" T - T

1

r

I

i 1

1 1 1 ' 1 Bv

iy/T ii

• ^ * ^ ( i

1 t

diim vd thdi gian khac nhau, nhung khi so sanh ham lugng anthoeyanin tach dugc tfi dung mdi acetone vdn cho kit qua eao ban cac dung mdi khae, dae biet dd'i vdi than cay ngd.

Khi phan tieh sd lieu vl sde td anthoeyanin dan tfi va mau da tfi cho thay, gia trj cua hai dai lugng nay ti le nghjch vdi nhau. Kit qua dugc minh hga trong hinh 3.

(%) 40 35 30

25 20 15 10 5 0

-Sac 16' anthoeyanin doti tu (mg/1) -Mku da tu (%)

8 9 10 11 12 Nguyen lieu thuc vat

Hinh 3. Do thj tuong quan gifia lugng sac td anthoeyanin don tfi va ti le phdn tram mdu da tfi Ghi chii: 1. Than cua loai ng6; 2. La chua, bup da'm; 3. Hoa cua loai chudi tieu; 4. La ciia loai hoa sdi;

5. U cua loai huylt du; 6. Qua ciia loai dau tam; 7. La ciia loai tia t6; 8. Cu cua loai khoai lang tim; 9. Vd qua cua loai nho; 10. La ciia loai mo leo; 11. Cii cua loai sam dai hanh; 12. Ld ciia loai rau din tia.

m . KET LUAN

Djch chie't anthoeyanin duge tach tfi dung moi acetone ed 0,01% HCI (v/v) ed phd hdp thu

vd ham lugng td'i uu ban so vdi 3 dung mdi edn lai (ethanol: dung djch HCI 1% (Iv: Iv), methanol cd 0,1% HCI (v/v), methanol ed 0,01% HCI (v/v)).

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Djch anthoeyanin tfi cac nguyCn lieu khae nhau cd phd hap thu cue dai tai budc sdng tfi 511-54{) nm. Gia trj hd'p thu ciia sde td anthoeyanin dan tfi va ti le phdn tram mdu da tfi cd mdi tuang quan nghjch.

Ham lugng anthoeyanin todn phdn d thdn ca\ n\io nun Id 0,59'/,. la chua 1.497,. hoa ehud'i 0,347, la cay hoa sdi 0.567 . la huylt du 1,287 . qua ddu ta 1,757 , la tia td 1,727 . cu khoai lang tim 0,467. vd qua nho l,277r, la mo l,()57r, cii sdm dai hanh 2,047 la rau din tia 1,747.

Loi cam on: Cdng trinh hoan thanh duge sir hd tra tfi kinh phi dl tai nghien eihi khoa hge cd'p bd B2009- TN04- 25, phdng Thi nghiem trgng diim cong nghe gen va phdng thi nghiCm Cdng nghe tl bao thuc vdt, Vien Cdng nghe sinh bgc, ViCn Khoa bgc vd Cdng nghe Viet Nam,

TAI LIEU THAM KIIAO

1. Chalker Scott L.. 1999: Environmental significance of anthoeyanins in plant stress responses. Photobiol, 70: 1-9.

2. Huynh Thj Kim Ciic, Nguyin Thj Lan, Chau The Lieu Trang, 2005: Td'i uu hda diiu kien ehilt tach ehd't mau anthoeyanin tfi bdp cai tim trong mdi trudng trung tinh. Tap chi Khoa bgc va Cdng nghe, trudng dai hge Da Ndng, 4(12): 44-50.

3. Huynh Thi Kim Cue, 2007: Nghien efiu thu nhdn va fing dung anthoeyanin edng nghe thuc phdm. Ludn an Tiin sT, trudng dai bgc Da Ndng.

4. David R., Kristen Bell, Gochenaur, 2006:

Direct vasoactive and vasoproteetive properties of anthoeyanin-rich extracts, Apllied Physiology, 00626(4): 1164-1170, 5. Fulcki T., Francis F. J., 1968: Quantitative

Methods for Anthoeyanins, 2. Determination of total anthoeyanin and

degradation Index for Cranberry Juice.

J. Food Science, 33: 78-83.

6. Gould K., 2004: Nature's Swiss amy knife.

The diverse protective roles of anthoeyanins in leaves. J. Biomed Biotech, 5: 314-320, 7. Gould K. S., Lister C , 2006: Flavonoid

functions in plants. Flavonoids: Chemistry, Biochemistry, and Applications. CRC Press, Boca Raton: 397-441.

8. Hooijmaijers C. A. M., Gould K. S., 2007:

Photoproteetive pigments in red and green gametophytes of two New Zealand liverworts. J. Bot, 45: 451-461.

9. Kevin Gould, Kevin Davies, Chris Winefield, 2009: Anthoeyanins:

Biosynthesis, Functions, and Applications.

Qc Springer Science+Business Media, LLC, New York, NY 10013, USA.

10. Lee D. W.. Collins T. M., 2001:

Phylogenetic and ontogenetic influences on the distribution of anthoeyanins and betaeyanins in leaves of tropical plants. J.

Plant Sci., 162: 1141-1153.

12. Manetas Y., 2006: Why some leaves are anthoeyanie, and why most anthoeyanic leaves are red. Flora, 201: 163-177,

13. Chu Hodng Mdu, 2008: Phuang phap phan tich di truyin hien dai trong ehgn gid'ng cay trdng, Nxb, Dai hge Thai Nguyen,

14. Stintzing F. C , Carle R., 2004: Functional properties of anthoeyanins and betalains in plants, food, and in human nutrition. Trends Food Sci, Teehnol, 15(1): 19-38.

15. Winkel-Shirley B., 2002: Biosynthesis of flavonoids and effects of stress, Curr Opin Plant Biol,, 5: 218-223.

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EXTRACTING AND ANALYSISING ANTHOCYANIN CONTENT FROM DIFFERENT PLANTS

PHAM THI THANH NHAN, NGUYEN HUU CUONG, LE TRAN BINH SUMMARY

Anthoeyanins are natural pigments used safely in foods and functional food. They play the important role in photoprotection, antioxidant activity, biological defense and also in symbiotic functions between microbes and plant cells. In addition, they arc attractants for pollinating via flower color and dispersing seeds via brightly colored fruit. This article presents some anthoeyanin extraction buffers and the result of determination of anthoeyanin content in different plants by pH-differential method.

Using the buffer containing acetone and 0.019, HCI lo extract anthoeyanin from different materials was more effective than three other types of buffers (ethanol: distilled water with HCI 17 (Iv: Iv), methanol with 0.1% HCI (v/v), methanol with 0.017 HCI (v/v)) and the absorption spectrum of anthoeyanin diluted extract was similar to that of standard anthoeyanin.

Maximum absorption spectrum of the different diluted extracts was ranged from 511nm to 540nm. In addition, the nonomeric anthoeyanin pigment is in inverse proportion to the percent of polymeric color.

The content of anthoc>anin was 0.597 in young stem of Zea mays L., 1.49% in leaf of Hibiscus sahdariffa L.. 0.347f in flower of Musa paradisiaca L., 0.56% in leaf of Chloranthus spieatus (Thunb.) Makin, 1.28% in leaf of Cordyline j'ruticosa (L.) Goepp., 1.757f in fruits of Morns alba L., 1.72% in leaf of Perilla fiutescens (L.) BriU., 0.467r in tuber of Ipomoea batatas (L.) Poir., 1.27% in fruit skin of Vitis vinifera L., 1.05% in Ic'df of Paederia scandens (Lour.) Merr., 2.047f in bulbus of Eleutherine bulbosa (Mill.) Urban and 1.74% in leaf of Amaranthus tricolor L..

Key word: Acetone, Anthoeyanin, maize, absorption spectrum, pH-differential method.

Ngdy n/ign bdi: 23-6-2011