Nguyin Thi Thiiy vd Dig Tap chi KHOA HOC & CONG NGHB 169(09)'33-37
M O T S O PHU'CfNG PHAP XAC DINH THANH PHAN HOA HQC, HOAT TINH SINH HOC CUA DAU HAT THlTC VAT
Nguyen Thi Thuy", Hira Thi Toan Trudng Dgi hgc NdngLdm —DH Thai Nguyen
TOMTAT
Vi^t Nam n3m trong khu vuc nhipt ddi gid miia, c6 diam thuc v|t da dgng va phong phu ca ve so lupng cung nhu chat lugng. Trong s6 10.400 loai thuc v | t bac cao da duoc phat hipn c6 khoans 564 loai la cac cay cd dlu [2]. Chiing cd mOt vai tro quan trpng trong toan canh biic tranh tai nguyen thyc vat nude ta va cuoc song cOng dong, khong chi la nguyen heu thiet yeu cho cong nghiep che bien (son, xa phong, thuc pham, my pham . ) va xuk khau ma nhieu dau hat thirc vat con duoc sur dyng trong dan gian nhu cac bai thu6c co tmy^n de dieu tn cac benh viem nhiem, chong lao hda va mot s6 can benh hiem ngheo Trong khi m$t s6 loai dau hat thuc vgt chii luc nhu dau dau nanh, dau lac, dau vimg, dau trau . da dupc nha nude dau tu ap dung nhihig cdng nghe tien tien d l san xuat thi nhihig nghien ciiu chuyen sau khoa hpc va co he thong ve lipit va cac axit bdo tir nguon goc thien nhien d nude ta gan nhu con bd ngo, Vay de c6 ket qua chinh xac chiing ta can cd nhOng phucmg phap nghien cihi khoa hoc, phii hgp vdi tirng doi tugng nghien ciiu Trong bii b4o nay chimg toi da dua ra mot so phuong phap xac dinh thiinh phan hoa hoc va hoat tinh sinh hoc ciia dau hat thuc vat Vipt Nam.
Tli khoa; Lipid, thdnh phdn hoa hpc, hogt tinh sinh hgc, sdc ki, oxi hoa
D A T V A N D E
Hipn nay, cac nguon tai nguyen dang ngay cang khan hiem, d o do nhiing kien thiic ve tiem nang di truyen cua hgt t h y c vat ngay cang trd nen quan trong hon. Cac loai hat cd thanh phan dinh d u o n g chinh l a carbohydrate, protein va chat b e o , dong thai chiia mpt phan Idn cac hop chat cd hogt tinh sinh hpc khac nhau c h u a d u g c s u dyng, day dugc coi la nguyen lipu hap dan cho viec san xuat cac thyc pham bo dudng va an toan cung nhu la nguon tai nguyen sii dyng trong cdng nghiep y, dugc. Dp lam dugc dieu nay, chiing tdi cung cap mgt so phuong phap chinh xac, khoa hoc de xac dinh thanh phan hda hpc va hogt tinh sinh hpc cua dau hat thuc vgt d Viet Nam.
K E T QUA N G H I E N C U U T o n g q u a n ve lipid
Vao dau nhirng nam 1950, ti-ong linh v y c Hoa hpc cac H p p chat thien nhien tren the gidi da xuat hipn nganh nghien cuu h o a h o c cac chat beo (lipid). Lipid (tieng H y lap co - lipid nghTa la m d hay la chat beo) la nhiing hpp
' Tel: 0977 008553. Email. Thuy.hoa.nK^gniail.com
c h i t hiiu c o t y nhien rat pho bien trong te bao cac CO the song, trong dpng vat, thyc vgt va vi sinh vgt. Lipid la hgp phan cau tao quan trpng cua cac mang sinh h p c , la ngudn cung cap nang lugng (376,106 J/kg), nguon cung cap cac vitamin A, D, E, F, K va F cho co the song, gdp phan tao ra ket cau ciing n h u vi dgc trung ciia rat nhieu thyc pham. Trong t u nhien nhiing hgp chat thuoc ve Idp chat lipid hieu theo nghTa rgng rat da dang, chiing cd the ton tgi dudi cac dang: cac hydrocarbon b a c cao, sterol, alcol, aldehyde hydrate, axit beo v a cac san pham thii cap ciia chiing n h u glyceride, sap, phospholipid, glycolipid, sulfoHpid, Trong s6 do cac dan xuat tocopherol va axit beo cd hogt tinh sinh hpc duoc iing dyng rpng rai trong cong nghiep t h u c pham, ray pham va d u g c pham.
Hipn nay, cac nguon tai nguyen dang ngay cang khan hiem, do dd nhiing kien thuc ve tiem nang di truyen ciia hgt thyc vat hoang da ngay cang trd nen quan trpng hon Do do chiing tdi lua chpn mdt so p h u o n g phap phii hgp va chinh xac de xac dinh thanh phan hda hgc va hoat tinh sinh hpc cua dau hat thyc vgt n h u dudi day.
Nguyin Thi Thliy vd£)/g Tap chi KHOA HQC & C 6 N G NGHB 169(09) 33-37 Phuong phap xac dinh th^nh phSn hoa hoc
ciia lipid
Chiit lipid theo phuang phdp ISO 659/2009 Dau beo tir mau hat dupc chiet tach va xac dinh ham lugng lipid theo phuong phap ISO/DIS 659'1988 Mau hat duoc Iya chpn theo tieu chuan, dem xay nhd trong may xay roi dupc chiet bang dung mdi hihi co n-hexan va dupc ho trp bdi sdng sieu am trong 6 gid Dich chiet thu duoc dem cat logi kiet dung moi tren may quay cat dung mdi chan khdng, Xdc dinh thdnh phdn ciia cdc axit beo theo phirang phdp ISO/FDIS 5509/1998 LB DH-c.
Phdn tich trin mdy sdc ky khiHP-6890, ghep noi vdi Mass Selective Detector Agilent 59 73 - Thanh phan va ham lugng axit beo xac dinh theo phuang phap ISO/FDIS 5509:1998 LB Diic' lay lOOmg dau beo hoa tan trong Iml heptan sau dd chuyen hda thanh dang methyl ester de phan tich tren may sac ky khi May sac ky khi hang Hewlett Packard instrument Model 5890 Series H, CP-Sil 88 (cot mao quan chuy6n dyng CP-Sil 88, I00mm/0,25mm/0,25p,m vdi hp chat chuan C16:0, Ci8:0) [3] Chuang tiinh nhipt dp:
nhipt dp 155-220''C(1.5''C/min), toe do: IO°C/
phiit, 260^/5 phiit; Split:150; injector 250°C, detector 250''C, khi mang 36cm/s hydrogen, detector gas 30ml/mm hydrogen, 300ml/min khdng khi va 30ml/min nitrogen, bam mau ty ddng vdi the tich 0,9pl. Sii dyng pho dii lieu chat chuan thu vipn Nhan dang cac axit beo bang phan mem chuyen dung tinh toan chuyen doi qua gia tri thai gian luu tuong duong ELC (Equivalent Chain- lengths of methyl ester denvaties of fatty acids) cho cpt mao quan chuyen dung CP-Sil 88, cd sii dung he chat chuan CI6:0, C18:0 tren may C- R3A theo cong thiic sau:
2{LaIiT^ - Z-gfiTifao) ECL = 16 + -
LgRT^z ~ igRT^^
Lay lOmg dich chiet hexan ciia mau hat dugc metyl hda bdi tac nhan CH30Na/MeOH (1%). Sau dd metyl ciia lipit tong dugc phan tich tren may GC
34
Xdc dinh thanh phan cua cdc tocopherol theo phirang phdp ISO 9936/2016 - Hda tan phan mau thii Irong n-heptan va dung sic ky ldng hieu nang cao de tach tirng logi tocol) Dung he so hieu chuan xac dinh dugc tir cac dung dich hipu chuan de tinh ham lugng cua tirng logi tocol.
- Thanh phan va ham luong tocopherol xac dinh theo phuong phap ISO/9936:20i6- hoa tan 70mg-100mg dau beo trong lOOpl heptan sau dd lay 20pl ra va dem phan tich tren thiet bi sic ky ldng cao ap HPLC hang Merck- Hitachi F-1000 Fluorescence Spectrophotometer, 295/330nm, D-2500, Mau dugc bom ty dpng trong buong bom mau tir dong Merck 655-A40, cpt 25cm x 4 6mm ID, toe do: 1.3ml/phut, he mobilephase sii dyng heptan/tert.butyl metylete (99+l,v/v), Chromato integrator, hp dung moi chgy heptan/tert,butyl metylete (99+I.v/v), NghiSn cihi xdc dfnh thdnh phhn vd hdm lir^tg ciia cdc triacylglycerol, phytosterol se dir^c xdc dinh bdi:
Sdc ki lap mong (TLC)
sic ki Idp mdng dupc thyc hien tren ban mdng trang san DC-Alufolien 60 F254 (Marck 1,05715), RPig F254S (Merck), Phat hipn bing den tii ngoai d hai budc sdng 254 nm va 368 nm hoac diing thuoc thii la dung dich HjSO^lOyo dugc phun deu tren ban mdng, say khd roi ha ndng tren bep dien tir tir tdi khi xuat hipn mau,
Sdc kl Idp mong diiu che
Sac ki lap mdng dieu che dupc thyc hien tren ban mdng trang san sihca gel 60G F254 (Merck, ki hipu la 105875), phat hien chat bang den tii ngoai d hai budc sdng 254 nm va 368 nm, hogc cat ria ban mdng de phun thuoc thii la dung dich la H^SO, 10%, ho ndng de phat hipn vet chat, ghep lgi ban mdng nhu cii de xac dinh vimg chat, sau do ego lop silica gel cd chat, giai hap phy bang dung moi thich hgp, 5dc ki cdt (CC)
Sac ki cpt dupc tien hanh vdi chat hap phy la silica gel pha thudng va pha dao. Silica gel pha
Nguyin Thi Thliy vd Dig Tgp chi KHOA HQC & CONG NGHE 169(09) 33-37 thudng cd kich Ihudc hat la 0,040-0,063 mm
(240430 mesh). Silica gel pha dao ODS hogp YMC (30-50^m, Fujisilisa Chemical Ltd.), Xdc dinh thdnh phhn cua phospholipid bdng phuang phdp: LC-DAD, LC-MS, GC-MS
• Nghien ciiu xac dinh thanh phan va ham luong cac lop chat trong lipit tong - Nghien cuu tiianh phan va ham lugng ciia phospholipid Thanh phan va ham lugng cac axit beo trong cac lap chat phospholipit TL, PL, NL Su dyng sic ki cot, sic ki ban mdng, sac ki ldng hipu nang cao HPLC de phan Igp cac Idp chat phospholipit' PE, PC, PI, LPE, PA, PG va 01 lop chat SQDG
Cic phuong phap xac dinh hoat tinh ch6ng oxy hda
Phirang phdp Trolo Equivalent Antioxidant Activity (TEAC)
TEAC la phuang phap xac dinh hoat tinh chong oxy hda so sanh vdi kha nang chong oxy hda ciia Trolox (Demirel va cpng sy, 2009) [3]. Cation ABTS* [2,2'-azinobis(3- ethylbenzothiazoIme-6-suIfonate)(ABTS)] la mpt goc ty do ben.
Phirang phdp FRAP (ferric reducing- antioxidant power)
Nguyen tic xac djnh hogt tinh chong oxy hda ciia phuong phap nay la dya tren kh^ nang cua cac chat chong oxy hoa trong vipe khii phiic Fe^*-TPTZ [2,4,6-tiipyridyI-s-triazine (TPTZ)] (mau tia) tiianh phirc Fe=^-TPTZ (mau xanh) trong mdi trudng acid Khi dd, do tang cudng do mau xanh ty le vai ham lupng chat chong oxy hoa cd trong nguyen lipu.
Mlic do tang cudng do mau nay dugc do d budc sdng 593 nm trong su so sanh vdi chat chuan la dung dich FeS04 hay BHT (Butylated Hydroxy Toluene) (Strain va Bpnzie, 1996) Khi cho phirc Fe^*-TPTZ vao moi trudng chiia chat chong oxy hda, cac chat chong oxy hda se nhudng dipn tii cho phirc nay va sinh ra Fe^*-TPTZ Ket qua tinh toan la mmol Fe^V g chat kho. Do dd, khi ket qua tinh toan ra Idn thi chiing ta cd the suy doan
i ^ g trong mdi trudng phan img do, so lugng cac phan tii cd the nhudng dipn tii la cao. Tuy nhien, dieu nay khdng hoan toan diing vi mot phan tli chat chong oxy hda cd the khii nhieu phiic Fe^*-TPTZ cirng Iuc. Day la mgt hgn che ciia phuong phap FRAP.
Phirang phdp DPPH
Nguyen tic cua phuong phap: I,I-diphenyI-2- picrylhydrazyl (DPPH) cd kha nang tgo ra cac goc ty do ben trong dung dich ethanol bao hoa. Khi cho cac chat thii nghiem vao hon hgp nay, neu chat cd kha nang lam trung hoa hogc bao vay cac goc tu do se lam giam cudng dp hap phy anh sang ciia cac goc tu do DPPH Hoat tinh chong oxi hoa duac danh gia thong qua gia tn hap phy anh sang ciia dich thi nghiem so vdi doi chiing khi dpc tren may EHsa d budc sdng 515 nm.
Phirang phdp ORAC (oxygen radical absorbance capacity)
Phuong phap ORAC xac dinh kha nang hap thu goc ty do chiia oxy hogt ddng [3]. Phuang phap nay do mirc do phan hiiy do bi oxy hda ciia fluorescein khi cd su hipn dien cua goc peroxy Phan iing trong dieu kipn nay dupc so sanh vdi 16 phan iing trong su hien dien cua chat chuan Trolox (hay vitamin E) va trong hipn dipn ciia mau chiia chat chong oxy hda can xac dinh hoat tinh Khi fluorescein bi oxy hda, cudng do phat huyiih quang se giam di.
Tien hanh do dp giam cudng do phat quang nay lien tye trong 35 phiit sau khi them chat oxy hda vao. Khi cd mat chat chong oxy hda, su phan ra fluorescein se chgm han. Xay dyng dudng cong bieu dien sy phy thupe do giam phat huynh quang theo thdi gian va viing dudi dudng cong diing de tinh toan Ket qua tinh toan la mmol Trolox/g mau.
Phirang phdp TRAP (total radical-trapping antioxidant potential)
Phuong phap TRAP sii dyng goc peroxyl duoc tao thanh tu 2,2'-azobis(2- amidinopropane) dihydrochloride (AAPH), Khi cho AAPH vao mdi trudng plasma, cac chat khu se 17 bi oxy hda. Qua trinh oxy hda 35
Nguyen Th| Thuy vd Dig Tap chl KHOA HOC & CONG NOHE 169(09): 33
f ^
nay dugc do dgt thong qua ham lugng oxy tieu thy bang mot dien cue. Khi cd mat chat chong oxy hda trong mdi trudng plasma, qua trinh oxy hda se xay ra cham hon. Gia tri TRAP ciia mau thi nghiem dugc tinh toan dua vao dp dai pha lag cua mau so vdi do dai pha lag cua mau trang va do dai pha lag ciia chat chuan la dung dich Trolox. Ket qua tinh toan la mmol Trolox/kg mau rin hogc mmol Trolox/I mau long
Phirang phdp Lipid assay
Phuang phap nay danh gia su khac biet trong toe dp oxy hda acid linoleic bdi goc ABA?
trong moi quan he vdi chat chuan la tocopherol [3] Trudc va trong suot qua trinh xdy ra phan iing, nhiet dp cua hSn hpp {[70 p,I ciia linoleic acid (2,3 mmol/l)]; 100 ml dpm phosphate 0,05 M (Natri phosphate hda tan trong nude; 2 88 g SDS, pH 7.4)} dugc dieu chinh va duy tri d 4''C. Lay 2 pJ c6a dung dich nay cho vao 0,01 ml ABAP (0,04 M) Sau 2-5 phiit phan iing, lay 0,02 ml djch chiet cho vao hon hgp nay. Do dp hap thu ciia hon hgp d 236 nm,
Phuang phdp FTC (ferric thiocyanat) Myc dich ciia phuong phap nay la khao sat kha nang lam giam sy peroTode hda lipid ciia chat khang oxy hda. Thong thudng, cac goc oxy ty do se peroxide hda lipid, tao thanh peroxide. Peroxide se oxy hda Fe^ thanh Fe^*, phiic ciia Fe^* vdi SCN cd gia tn hap thu toi da tgi 500 nm Gia tri hap thu cao dong nghia vdi sy peroxide hda lipid cao trong suot qua trinh u dung dich. Mau dung dich
cd chiia chat khang oxy hda se cho gia tri dp hap thu thap hon mau tring khong c6 chat khang oxy hda
Phifongph^ tdng nang luc khu (redudngpower) Nguyen tic xac dinh hoat tinh chong oxy hoa ciia phuang phap nay la dya tren kha nang cua cac chat chong oxy hoa trong vipc khii phiic K3[Fe(CN)6] tiianh phirc K4[Fe(CN)6], phiic nay tac dung vdi FeCU thanh KFe[Fe(CN)6] (xanh Pniss) [3], Khi dd, dp tang cudng do mau xanh ty Ie vdi ham lupng chat chong oxy hda cd trong nguyen li^u, Mlic do tang cudng dp mau nay duoc do d budc sdng 690 nm trong su so sanh vdi chat chuan la dung dich acid ascorbic, KET LUAN
Chiing toi da dua ra duoc ket qua tong quan ve mpt so phuang phap xac dinh thanh phan hda hpc va phuang phap xac dinh hogt tinh chong oxi hda ciia dau hgt thyc vat ia tai lipu tham khao can thiet cho cac co sd san xuat lua chpn, ap dyng trong danh gia chat lugng san pham cua co sd.
TAI LIEU THAM KHAO 1, Pham Quoc Long, Chau Van Minh (2005), Lipit vd cdc axil beo hogt linh sinh hgc eo nguon goc thien nhien, Nha xu4t ban khoa hgc va kJ thugt 2, Pham Van Nguyen (1981), Nhung cdy co ddu beo a Viet Nam, Nha xual ban khoa hgc ki thugt 3 Ulleberg, E.K.; Comi, I , Holm. H„ Henid, E.P,; Jacobsen, M , Vegarud, G E . (2011),
"Human gastrointestinal juices mtended for use in m vitro digestion models". Food Digestion 2, pp 52-61
Nguyin Thi Thuy vd Dig Tgp chi KHOA HOC & CONG NGHE 169(09). 33 - 37
S U M M A R Y
T H E M E T H O D S T O D E F I N E C H E M I C A L C O M P O S I T I O N S , B I O L O G I C A L A C T I V I T I E S OF V E G E T A B L E S E E D OIL OF V I E T N A M
Nguyen Thi Thuy', Hua Thi Toan University of Agriculture and Forestry - TNU Vietnam locates in monsoon tropical area with abundant and diversified vegetation m quality as well as quantity, Of the 10 400 tracheophyta species were discoversed, there were about 564 species of oil plants,These plant species play an important role in the whole of plant resource picture and community life of our country, not only are they be essential ingredients for the processing industry (paint, soap, food, cosmetics . ) and export but many kinds of vegetable seed oil also were used in the folk such as traditional remedies for treatment of ageing resistant, inflamed diseases, and other serious diseases While some mam vegetable seed oils such as soybean, peanut, sesame, tung oil, etc, have been invested and applied the advanced technologies to produce by the state, the scientific deeply and systematic researches about fat acids and lipids from natural sources in our country have been still almost opened Therefore, to obtain accurate results we need to have suitable scientific research methods for each studied object.In this paper, we suggested several methods to determine Oie chemical compositions and the biological activities of vegetable oil of some plants in Vietnam
Keywords: Lipid, chemical composition, biological activity, chromatography, oxidation
Vgdy nh^n bdi:l 7/4/2017; Ngdy phdn biSn: 21/6/2017; Ngdy duyet d&ng: 30/9/2017 Tel- 0977 008553. Email: Thuy.hoa [email protected]
