HDA HOC-CONG NGHE THIfC PHAM
HOAT TINH KHANG OXY HOA CUA CAO CHIET ETHANOL TRAC BA DIEP
{THUJA ORIENTALIS L.)
• N G U Y I N TRONG TUAN - vo VAN LUAN
TOM TAT:
Nghidn cii'u nay nham muc dich khao sat hoat tinh khang oxy hda cua dich chid't ethanol cua cSy Trac ba didp (la va canh) siJ dung cac phtfdng ph^p khang oxy hda khac nhau nhtf phtfdng phap DPPH, ABTS"", nang Itfc khtf (reducing power), kh^ sat (Ferric ions Reducing Ability Power) va phosphomolybdenum (khang oxy hda tdng). Ke't qua cho tha'y, dich chid't ethanol cua cay Trac ba diep the' hien hoat linh khang oxy hda td't Irong cac phtfdng phap thu"
nghiem Kd't qua nay cung phii hop vdi stf hien dien cac thanh phan cd hoat tinh sinh hoc nhtf polyphenol va flavonoid trong cao chid't. Nghidn ctfu nay md ra htfdng mdi trong viec sv( dung thtfc vSt nay trong linh vtfc Y Dtfdc.
Tiif khoa: Khang oxy hda, cay Trac ba didp (Thuja orientalis), flavonoid, polyphenol.
1. Md d^u
Stress oxy hda va viec san xuat qua nhilu gd'c ttf do trong cd th^ la nguyen nhan gay ra nhieu benh nguy hi^m nhtf ung thtf, dm mach, thoai hda than kinh, xd vtfa ddng mach, tieu dtfdng [1]. Cac hdp chat chd'ng oxy hoa nhtf polyphenol va flavonoid cd tac dung loai bo gd'c ttf do va do do tfc che' cd chd' oxy hda dEn dd'n cac benh thoai hda [2]. Trong nhffng nam gan dSy, cd nhieu nghidn ctfu dtfdc den hanh nhSm tim ra cac hdp cha't ttf ttf nhien va tong hdp cd tac dung khang oxy hda, ngan chan cac bdnh do qua trinh oxy hda gay ra dang dtfdc quan tam va diy manh. Tuy nhidn, viec stf dung cac chat tuf tdng hdp cung gSy ra nhieu tac dung phu. Do do, vide srf dung cac hdp cha'l chie'l xuS't ttf ttf nhidn cd
kha nang khang oxy hda khdng g§y lac dung phu cho stfc khoe la Itfa chgn hang dau trong vide nang cao chat Itfdng thtfc pham, my pham va dtfdc ph£m
Trac ba diep (Thuja orientalis) la loai cay trong lam canh va lam thud'c trdn nhieu vdng d Vidt Nam. Trac ba diep cd vi chat, dang, hdi han cd tic dung cam mau, chtfa thd huye't, mau cam, ly ra mau. Ba tu" nhan vi ngot, linh binh, co tac dung bd tam, ty dinh than dung chtfa hdi hdp, mat ngu, hay qudn, ngtfdi ye'u ra nhieu md hdi, tao bdn [3].
Cac nghien ctfu tren the' gidi cho tha'y rang, thtfc vat nay the hien nhieu boat tinh sinh hoc nhtf thdc dly stf tang Irtfdng cua tdc, khang virus, khang di tfng, chdng ddng kmh, khang khuan va nam, khang vidm [4]. Tmh dau la Trac ba diep tfc che' stf phat
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tnen mdt so' vi khuS'n va na'm [5,6]. Cao chie't ttf vd cay va qua cd tac dung tfc che'enzyme lipase [7].
Hdp cha't tCf tinh dau cua la, a-pinene, cd kha nang cai thidn tri nhd, phong ngtfa va dieu Iri cac bdnh do thoai hda than kinh nhtf chtfng mat tri nhd hay rdi loan nhan thtfc [8], a-cedrol cd tac dung khang na'm Altemaria alternate [6], cac hop cha't flavonoids phan lap ttf cao chie't methanol qua cd hoat tinh khang oxy hda va hoat tinh tfc chd' neuUopil elastase d ngtfdi [9].
Mot so' nghien ctfu ve thanh phan hda hgc ngoai ntfdc ihtfc hidn trdn tinh dau la TrSc ba diep cho tha'y tinh dau chtfa cac hop cha't terpenoids nhtf monoterpen, sesquiterpen... [6,10]. Cao methanol trai chtfa cac flavonoids nhtf cupressuflavone, amenloflavone, robuslaflavone, myricitrin, (+)-catechin, quercitrin, hypolaedn, isoquercitrin 7-O-^-xylopyranoside va afzelm [9].
Trong ntfdc, nghien ctfu cua Dinh Nggc Thtfc va cdng stf cho tha'y tinh dau cua la Trac ba didp chtfa cac thanh phan terpenoids va the hidn boat tinh khang vi khuan [II]. Nguyen Thi Thao Ly va cong stf cung phan lap dtfdc mot so' hdp chat ttf cao hexan cua la nhtf acid ferulic, 3-oxo-a-ionol, acid pinusolidic, acid 3j6-hydroxyisopimaric, 7- hydroxy-5, 6, 4'-lrimethoxyflavon, «-docosyl frulat [12]. Theo tim hieu cua chiing ldi, cho de'n hidn nay, cac nghidn ctfu ve boat tinh sinh hgc cua cay trac ba diep trong ntfdc van cdn ban chd'. Vi vay, nghien ctfu nay dtfOc thtfc hien nham danh gia hoat tinh khang oxy cua cay trSc ba diep vdi muc dich dm kid'm thdm ngudn thao dtfdc trong thidn nhidn phuc vu cho bao vd stfc khde con ngtfdi.
2. Thu'c nghiem 2.1. Nguyen lieu
Cay Trac ba didp (phan la va canh cay ttfcti) dtfdc thu hai lai huydn Chd Lach, tinh Be'n Tre vao thang 1/2018 dtfdc dinh danh bdi Thac s7 Phung Thi Hang, Bo mdn stf pham Sinh hoc, Trtfdng Dai hgc Can Thd Mau thtfc val (BT_Thu 2018010001) dtfdc Itfu gitf tai Phdng thi nghiem Hda sinh, Khoa Khoa hoc Ttf nhidn, Trtfdng Dai hgc Can Thd. Mau thtfc vat diing lam thi nghiem dtfdc ru'a sach, phdi khd va nghien thanh bOt. Bdt dtfdc trtf trong tu ddng -20"C cho dd'n khi thi nghidm.
Bdt kho (lOOg) dtfOc cho vao tui vai, cot kin, ngam trong IL dung mdi ethanol 96" moi lan ngam trong 24 gid (ngam 3 lan). Sau do dich chid't dtfdc Igc qua gia'y Igc roi tid'n hanh co quay dtfdi ap sua't tha'p d nhidl do khoang 55"C, dudi he't dung moi thu dtfdc cao chie't ethanol trac ba diep.
Cao thd dang set dtfdc bao quan d nhidt 4"C diing lam thi nghidm.
2.2. Binh tinh thdnh phdn hoa hoc cdc cao chief
Thanh phan hda hoc cua cac cao chid't nhtf:
alkaloid, flavonoid, steroid va Iriterpene, glycoside, hdp cha't phenolic va tannins dtfdc dinh dnh bang cac thudc thiJ dac trtfng [13]
2.3. Dinh lUang polyphenol tong vd flavonoid tong trong cao chie't
Ham [tfdng polyphenol dtfdc xac dinh bSng thud'c tbiJ FoHn-Ciocalteu theo md ta cua Rebaya et al., 2014 [14], Ham Itfdng flavonoid loan phan trong cac cao chie't dtfdc xac dinh theo phtfdng phap so mau AICI-, cua Bag et al., 2015 [15].
2.4. Khao sdt hoat tinh khdng oxy hoa ciia cao chie't
Khdo sdt hieu qud loai bo gdc tudo DPPH Kha nang loai bd gd'c ttf do DPPH cua cac cao chie't Trac ba didp dtfdc dtfdc thtfc hien theo Sharma et al, 2009 [16]. Hon hdp phan tfng gdm 40 pL DPPH (1000 pg/mL) va 960 pL cao ethanol (d cac nong do ttf 0 - 350 pg/mL) u trong td'i d nhiet do phdng trong thdi gian 30 phdt, do dd ha'p thu quang phd d btfdc sdng 517 nm. Do'i chtfng dtfdng siJ dung la vitamin C. Ke't qua hoat tinh khang oxy hda cua cac cao chie't dtfdc bieu dien bang gia tri EC^Q (Effective Concentration of 50%) dtfdc dnh dtfa trdn phtfdng trinh tuye'n tinh cua dich chie't khao sat.
Khdo sdt hieu qud loai bo gdc tu do ABTS*
Hoat ddng loai bd gd'c itf do ABTS+ thtfc hien theo Nenadis et at., 2004 [17]. Chuan bi dung dich ABTS +: 2 mL dung dich ABTS^ 7 mM va 2 mL dung dich K7S2OS 2,45 mM, u trong bdng td'i 16 gid, sau do pha loang bang ethanol (khoang 30 lan), dieu chinh do ha'p thu d btfdc sdng 734 nm dd'n 0,7±0,05. Tie'n hanh cho 990 pL ABTS + vao 10 pL cao ethanol (d cac ndng do ttf 0 - 70 pg/mL). Hdn hdp phan tfng dtfdc u trong ihdi gian 6 phdt. Sau do, do do ha'p thu quang phd d btfdc sdng 734 nm. Dd'i
HdA HOC-CONG NGHE THIfC PHAM
chtfng dtfdng dtfdc siJf dung la trolox. Kd't qua boat tinh khang oxy hda cua cac cao chid't dtfdc bieu didn bang gia tri ECgo (Effective Concentradon of 50%) dtfdc tinh dtfa trdn phtfdng irinh tuyen dnh cua dich chid't khao sat.
Khdo sdt ndng luc klid (Reducing Power - RP) Nang Itfc khu- sat (RP) dtfdc thtfc hidn theo phtfdng phap Ferreira et al., 2007[I8]. Hdn hdp phan tfng gdm 0,5 mL cao ethanol (d cac ndng dp ttf 0 - 500 pg/mL), 0.5 mL dem phosphate (0,2 M, pH = 6,6) va 0,5 mL K3Fe(CN)(, 1%. Sau dd hdn hdp phan tfng dtfdc ii d 50"C trong 20 phiit, them 0,5 mL CCI3COOH 10% rdi ly tam 3000 vdng/
phiit trong 10 phiil. Nhe nhang riit 0,5 mL cho vao 0,5 mL ntfdc va 0,1 mL FeC^ 0,1%, lac deu. Do dp ha'p thu quang phd ctia hdn hdp phan tfng d btfdc sdng 700 nm. Cha't dd'i chtfng dtfdng sit dung la butylated hydroxianisole (BHA). Hidu qua khang oxy hda cua cac cao chid't d cac ndng dd khac nhau dtfdc so sanh vdi cha't chuan bang each siJ dung ndng do ma tai do cha'l chuan hay cao chie't (|Jg/mL) cd gia tri OD = 0,5 (OD0,5).
Khdo sal khd ndng khd sdt (FRAP)
Kha nang khu" sat FRAP dtfdc thtfc hidn theo Benzie and Strain, 1996 [ 19]. Tid'n hanh cho 10 pL cao ethanol (d ciic ndng do ttf 0 - 45 pg/mL) vao 990 pL dung dich FRAP. Cac hdn hdp tren dtfdc u d 37"C trong 10 phut, sau dd tie'n hanh do gia tri do ha'p thu quang phd d btfdc sdng 593 nm. Dd'i chu'ng dtfdng s^ dung la irolox. Hidu qua khang oxy hda cua cac cao chie't d cac ndng dp khac nhau dtfdc so sanh vdi cha't chuan bang each stir dung ndng dd ma tai dd cha't chuan hay cao chie't (pg/mL) cd gia tri 0D = 0,5(000,5).
Khdo .sdt hogt tinh khdng oxj hda td'ng (Pliuang phdp phosphomolybdenum)
Phtfdng phap dtfa tren stf khu' cua Mo(VI) ve Mo(V) the hidn qua stf tao phtfc Mo(V) Phosphate mau xanh la d pH acid (Prieto et al., 1999 [20]). Tid'n hanh cho 100 pL cao ethanol (d ndng do ttf 0 - 120 pg/mL) vao 900 pL dung dich phosphomolybdenum. Cac hdn hdp trdn dtfdc u d 95<'C trong 90 phut, sau dd tie'n hanh do gia tri dp ha'p thu quang phd d btfdc sdng 695 nm. Dd'i chtfng dtfdng Sli dung la vitamin C. Hieu qua khang oxy hda cua cac cao chie't d cac ndng dp khac nhau dtfdc so sanh vdi cha'l chuin bang each si^ dung
ndng dp ma tai dd chat chuan hay cao chidt (pg/mL) cd gia tri OD = 0,5 (OD0,5).
3. Kd't qua va thao luan
3.1. Dinh tinh thdnh phdn hoa hoc cao chie't Kd't qua dinh dnh thanh phan hda hgc cao chie't ethanol cho tha'y stf cd mat cua flavonoid, alkaloid, hdp cha'l phenolic va tannin, steroid, glycoside. Ke't qua nay cung kha phii hdp vdi nghien ctfu cua Jasujal et al. 2013 [21]. Nhdm cha'l flavonoid va polyphenol da dtfdc bie't de'n la nhtfng nhdm hdp chat cd hoat dnh khang oxy hda td't. Ttf viec dinh tinh cho tha'y trong la va canh cay Trac ba diep chtfa nhieu cac nhdm hdp chS't cd tidm nang sinh hpc.
3.2. Hdm liiting polyphenol tong vd flavonoid tong
Ham Itfdng polyphenol tdng va flavonoid long trong cac cao chid't dtfdc xac dinh ttfdng dtfdng ham Itfdng gallic acid, quercedn vdi phtfdng trinh dtfdng chuan y = 0,1052x + 0,083 (R^ = 0,9999) va y = 0,0053x + 0, 0068 (R^ = 0, 9923) ttfdng tfng.
(Bang 1).
Bang 1. Ham ltfdng polyphenol tong va flavonoid tong cua cao chie't
IPC (mg GAE/g cao chiet) TFC (mg QE/g cao chiet)
Cao ethanol
81,82±0,69 93,86+0,81
3.3. Hoat tinh khdng oxy hoa
Kd't qua Bang 2 cho tha'y, cao chid't ethanol cua Trac ba diep cho hidu qua khang oxy lot trong cac phtfdng phap thtf nghiem. Hidu qua lot trong cac phtfdng phap ABTS*, FRAP va Phosphomolybden vdi gia tri EC50 tha'p hdn nhieu (< lOOpg/mL) so vdi phtfdng phap DPPH va RP. Tuy nhidn, hieu qua nay vln cdn tha'p hdn so vdi cac do'i chtfng dtfdng.
Tiiy theo phtfdng phap va dieu kidn phan tfng lie'n hanh khac nhau ma cac hoat cha't ttf cao chid't sd cho kha nang phan tfng khac nhau. Kd't qua nay cung phii hdp vdi ke'l qua dinh tinh, dinh Itfdng thanh phan hda hpc cao chid't ethanol cho tha'y cd chtfa polyphenol va flavonoid. Theo stf hieu bie't cua chung toi, cac nghidn ctfu trong va ngoai ntfdc ve hoat tinh khang oxi hda cua cao chid't la Trac ba
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Bdng 2. Gia trj EC50 (ODQ 5) cua cao chiet ethanol vdi cac phUdng phap khdng oxy hoa
PhUdng phap
DPPH ABTS*
Nang lire khil(RP) Khd sat (FRAP) Phosphomolybdenum
Glaln(ECs„hayOD„,5) Chat chuan
3,9 ^g/mL 7,1^g/mL 34,56 ng/mL 2,07 ixglmL 10,13 |jg/mL
Cao ethanol 236,97 pg/mL 49,91 pg/mL 496,44 ng/mL
34,06 pg/mL 62,03 pg/mL Ghi chii • Chat chuan trong col theo thd tu vitamin C, trolox,
BHA. trolox, vitamin C
didp van cdn han che'. Mot sd^ lac gia chi sii' dung rieng re cac phtfdng phap nhtf DPPH, ABTS^ RP, hoac ke'l hdp 2 phtfdng phap khi khao sal boat tinh khang oxi hda. Dieu nay cung con nhieu ban chd' khi xac nhan boat dnh khang oxy, bdi vi stf hidn dien hdn hdp thanh phan cac hdp cha't cd boat dnh trong cao chie't cd the hieu qua do'i vdi phtfdng phap thtf nghidm nay nhtfng khdng hieu qua ddi vdi phtfdng phap khac. Nghidn ctfu nay the hidn tfu diem la stf ke'l hdp cua 5 phtfdng phap khac nhau de danh gia hieu qua khang oxy hda cua trac ba diep.
Nghien ctfu cua Efsharkawy el al. (2017) cho tha'y, dich chid't Trac ba didp cho hidu qua khang oxy hda (phtfdng phap DPPH) 73,35 % d ndng do 300pg/mL (EC50 = 202, 46pg/mL) [22], Nazir et al. (2016)
bao cao dich chiet methanol cua cay ciing chi Thuja occidentalis cho hoat tinh khang oxy hda bang phtfdng phap DPPH d ham Itfdng 490pg/mL va ABTS" la 450pg/mL [23]. Nizam et al.
(2017) bao cao dich chid't ethanol ciia la cho hidu qua khang oxy hda bang phtfdng phap DPPH va nang life khij kha cao (>500 pg/mL) so vdi nghien ctfu nay [24). Tuy nhien, nghien ctfu cua Jasujal et ai, (2016) cung cho tha'y hoat dnh khang oxy hda cua cao chid't methanol 70% bang phtfdng phap DPPH cd hieu qua tfc chd' 85,25% d ndng do lOOpg/mL [21]. Kd'tqua td't hdn so vdi kd't qua trong nghidn ctfu nay bdi dung mdi methanol- ntfdc se hoa tan lot cac hdp cha't flavonoid va polyphenol hdn so vdi dung moi ethanol. Diiu nay cung phu hdp vdi ham Itfdng polyphenol va flavonoid irong cao chid't cua Jasujal el al., (2016) Idn hdn trong nghidn ctfu nay (95 va 100 mg/g).
4. Kd't luan
Nghien ctfu boat tinh khang oxy hda cda cao chie't la va canh cay Trac ba didp cho tha'y cay cho boat tinh khang oxy hda td't. Kd't qua nay cung phu hdp vdi stf hidn dien cua cac thanh phan polyphenol va flavonoid, ciing nhtf ham Itfdng xac dinh cua nd cd trong cao chid't •
TAI LIEU THAM KHAO:
/. Lee. J., Koo, N, Miii. D B., 2004. Reactive oxygen .species, aging, ond anlioxidative nutraceuticals Comprehensive reviews in food science and food .safety 3: 21-33.
2. Wu. Y.Y., Li. W.,Xu, Y.. Jin, E.H.. Tu. Y.Y.. 2011. Evaluation of the antioxidant effects of four main theaflavin derivatives through chemiluminesceiice and DNA damage analyses Journal ofZheJiang University Science B 12:
744-751
3. Do Tdt Lai, 2004. NliUng cdy thudc vd vi thudc Viet Nam. Nhd xudi bdn Y hoc
4. Jain, N. and Etbanobouiny, M. S., 2017. Phylochemical and Pharmacological Aspects of Thuja onenlalis: A Review International Journal of Pure and Appied Bioscience 5 (4): 73-83.
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5. Jain, R. K. and Garg, S. C. 1997. Antimicrobial Activity of The Essential Oil Of Thuja Orientalis L Ancient science of Life 16(3): 186-189.
6. Sanjay, G., Ashok, K, andAshok, K. T., 2007. Chemical Composition and Fungitoxic Activity of Essential Oil of Thuja orientalis L. Grown in the North-Western Himalaya. Z Naturforsch63c: 211 -214.
7. Kim, H. Y., and Kang, M H., 2005 Screening of Korean Medicinal Plants for Lipase Inhibitory Acdvilv Phytotherapy research 19 (4) 359-361.
8. Lee, G-Y, Lee, C, Park, G. H., and Jang, J-H., 2017 Amelioration of_Scopolamine-lnduced Learning and Memoiy Impairment by d-Pinene in C57BL/6 Mice. Evidence-Based Complementary and Alternative Medicine 2017: Article !D4926815,9pages https://doi.0rg/JO.l 155/2017/4926815.
9. Xu, C-H.. Ryoo, IJ., Kim, Y-H., Choo, S-J.. and Yoo, I-D., 2009. Free Radical Scavenging and Antielaslase Activities of Flavonoids from the Fruits of Thuja orientalis. Archives of Pharmacol Research 32(2). 275-282.
10. Suresh, S. K, and Ramavtar. S., 2016. Essential oil constituents and antimicrobial potency of Thuja orientalis grown in Rajasthan. Suresh Cyan Vihar University International Journal of Environment, Science and Technology 2(2): 1-7.
11 Dinh Ngoc Thiic. Nguyen Thi Hd. Mai Thi Tho vd Le Nguyen Thdnh. 2017. Nghien cdu thdnh phdn hoa hoc vd hoat tinh khdng sinh cua tinh ddu cdy trac bach diep (Plalycladus orientalis (L) Franco). Tap chi Duac hoc 57 (5).
12. Nguyen Thi Thao Ly. Nguyen DiSu LiSn Hoa va Nguyen Thi LS Thu, 2016. Khdo sdt thdnh phdn hoa hoc cao hexan cua Id cdy trac bdch diep (Biota Orientalis). Tap chi Phdn tich Hoa, Ly vd Sinh hoc 20.
13. Nguyen Kim Phi Phung, 2007. Phuang phdp co lap hop chd't hUu ca. NXB Dai hpc Quoc gia TP. Ho Chi Mmh. Trang SO-138.
14 Rebaya, A, Belghith S.I., Baghdikian B.. Leddet V. M., Mabrouki F., Olivier E., Cherif J K, Ayadi, M.T..
2014. Total phenolic, total flavonoid, tannin content, and antioxidant capacity of Halimium halimifolium (Cistaceae). Journal of Applied Pharmaceutical Science 5( 1): 52-57.
15. Bag, G.C., DeviP.G, Bhalgyabad Th., 2015 Assessment of Total Flavonoid Content and Antioxidant Activity of Methanolic Rhizome Extract of Three Hedychiuin Species of Manipur Valley. Internationai Journal of Pharmaceutical Sciences Review and Research 30( I): 154-159.
16 Sharma, O. P., andBhat, T. K, 2009. DPPH antioxidant assay revisited. Food chemistly 113(4): 1202-1205.
17. Nenadis. N, Wang, L-F., Tsimidou. M., and Zhang. HY., 2004. Estimation of Scavenging Activity of Phenolic Compounds Using the ABTS + Assay. Journal of Agricultural and Food Chemistry 52:4669-4674.
18. Ferreira. I C. F. R, Baptista, P., Vilas-Boas M. and Barros L, 2007. Free-radical scavenging capacity and reducing power of wild edible mushrooms from northeast Portugal: Individual cap and stipe activity. Food chemistry 100(4): J5JJ-I516.
19. Benzie, 1. F., and Strain, J. J.. 1996. The ferric reducing ability of plasma (FRAP) as a measure of'andoxldant power": the FRAP assay. Analytical Biochemistry 239(!): 70-76.
20. Prieto, P., Pineda, M, Aguilar, M., 1999. Spectrophotometrlc quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: Specific application to the determination of vitamin E Analytical Biochemistry 269(2): 337-341.
21. Jasujal, N. D., Sharma, S. K., Saxena. R.. Choudlwry. J. Sharma. R.. and Joshi. S. C. 2013. Antibacterial, antioxidant and phylochemical investigation of Thuja orientalis leaves. Journal of Medicinal Plants Research 7(25): 1886-1893.
22. Elsharkawy. E,R., Aljohar. rt, and Donia, A E. R. M. 2017. Comparative Study of Antio.xidani and Anticancer Aclivin- of Thuja orientalis growing In Egypt and Saudi Arabia. British Journal of Pharmaceutical Research 15(5): 1-9.
So 19-Thang 10/2019 339
