TAP CHi KHOA HOC DAI HQC VAN LANG Mi Bich Nhu
DIEU KIEN TACH CHIET VA TAC DUNG HA DlTonVG HUYET CHONG OXY H 6 A CUA DICH CHIET HUNG QUE
THE STUDY OF EXTRACTION CONDITIONS AND THE EFFECT OF REDUCING BLODD SUGER AND ANTIOXIDANT ACTIVITIES OF GLUE EXTRACTED FROM BASIL
MA BICH NHlf('>
TOM TAT: Hung qui (Ocimum basilicum var thyrsiflora) duge sie dyng rpng rai nhu mpt logi thdo duge. Khd ndng khang, khd ndng ire chi enzyme thuy phdn linh bpt eiia cao chiit hdng qui eung duge xde dinh. Kit qud cho thdy, khi thay doi diiu kien trich ly se thu dupe hdm lugng phenolic tdng khde nhau. Diiu kien tdi uu (ty le mdu dung mdi la l/IOO vd khudy & nhiet dp 55''c) trong thai gian 35 phiit v&i hdm lugng phenolic long thu duac la 43.6 ±0.03 mg GAE/g chdt khd.
Khd ndng ire chi hopt dpng cua gdc tudo DPP Hid 38.2+1.1 %. Dich chit hdng qui cd khd nang ire chi doi vai a-amylase vd a-glueosidase tuang duang 45.8 ± 0.82 %, 22.7 ± 1.1%. Tir kit qud nay, ed the thdy dieh ehit thu duge tu hdng que cd chua cdc chdt phenolic co khd ndng khdng oxy hda eao khi su dung diiu kiin chiit thich hgp.
TU khoa: hung qui; phenolic tdng; DPPH; a-amylase. a-glueosidase.
ABSTRACT: Basil (Ocimum basilicum) is widely used as an herb. Ability of resistance, inhibiting the starch hydrolytie enzyme of the glue extracted from basil had been determined. The results showed that, when changing extraction conditions, different total phenolic content would be obtained. Optimal conditions (with 1/100 sample solvent ratio, the duration of 35 min at the temperature of55°c), obtaining the highest phenolic content is 43.6+0.03 mg GAE / g dry matter.
Ability of inhibiting the activity of free radical DPPH is 38.2 +1.1%. Extracted liquid from basil has ability of inhibiting a-amylase and a-glueosidase equivalent to 45.8 ± 0.82%, 22.7 ± 1.1%.
From this result, it ean be seen that the liquid extracted from basil contains high antioxidant phenolic substances when appropriate extraction conditions are used.
Key words: Basil; total phenolic content; DPPH; a-amylase; a-glueosidase.
I. D^T VAN DE amylase va a-glucosidase, bdi vi hai enzyme Dii thio dudng li bdnh man tinh khdng nay ed vai tid quan trpng trong he thdng tidu lay Ian do roi logn ehuydn hda, dgc trung bdi hda lidn quan den su thiiy phan tinh bpt thanh ndng dp glucose l i nguydn nhan dan ddn cac dudng [2, y.247-252]. Vide ire ehd a-amylase bidn ehiing vi mgch nhu b^nh tim mach, dot ya a-glucosidase cd thi lam giam ding kl su quy, mil v i bpnh thgn [1, tr.547-553]. Mgt gia tang dudng huylt sau i n do chi cd thi hap phuong phip dilu tri thye t l dl kilm soat thu monosacarit qua nidm mge mdt, do dd lam b?nh tilu dudng la hgn chd tang dudng huydt g[^un dudng huylt d bdnh nhan tilu dudng [3].
sau in. Didu niy cd thd dgt dugc bang tie chd j^i^t vai ylu to khae ddng vai trd tiong enzyme thuy phan carbohydrate nhu a- sinh bdnh hgc dii thao dudng nhu lipid raau
<•>ThS. Tradng Dai hpc Vin Lang mabichnhu@vanlanguni eduvn, Masd: TCKH22-13-2020
^ ^ W W { O A » O C | i.C DAI HQC VAN LANG ting v i stress oxy hda. Dac biet, stress oxy hda la nguyen nhan hinh thinh eie gdc tu do trong CO thd va cung la nguydn nhan chinh din din khang insulin, rdi logn lipid mau, roi loan ehiic ning te bio, giam dung ngp glucose va cudi cimg din den bdnh dii thao dudng type 2 [4, y.670-674]. Bang each bd sung eie chat oxy hda tu nhidn ed trong thue vat se cd tic dyng ngan ehgn sy tidn yidn eua bdnh dii thio dudng bdi vi cac chit chdng oxy hda nay ed kha nang lim sgch cic gdc tu do cd hai cho ca thi [4].
De didu tij bdnh dii thio dudng, nhidu logi thio dugc cd nguon gdc ty nhidn da dugc ua chuong vi chiing an toin vi it ddc hon so vdi thude duge tdng hgp [5, ti.1354-1360]. Hon 400 loii thyc vat ed hogt dgng hg dudng huydt di dugc edng bd, tuy nhidn vide tim kidm cic logi thuc vit mdi vin ludn la su quan tam ciia cic nhi khoa hpc. Ngoii y i xanh, khd qua, giao ed Iam, thd phye Iinh, diy thia eanh, hiing qud... cd tie dyng tdt yong didu tri dudng huydt v i chdng oxy hda. Tgi Viet Nam, hiing que duge su dyng rdng rii tiong chi biln thiie an va y hpe ed tmydn. Mye tidu eua bii vidt nay l i tim ra didu kidn tdi uu nhit dd trich ly cic hgp chit cd hogt tinh sinh hpe tir hiing qud. Vide sii dyng eao chilt ciia logi thio dugc niy dl thii boat tinh uc chi a-amylase v i a-glucosidase nhim tgo ra cac thude dgc hipu hon dd chdng bdnh tilu dudng tir ngudn dugc lieu nay.
2. N Q I DUNG 2.1. Nguyen lieu
Cac hda chat dupc sir dung de phan tich tdng him lugng phenolic vi khi nang chdng O?Q' hda bao gdm: methanol, thude thu Folin Cioeaheu, natri carbonate, axit gallic, rutin, 2 enzyme a-amylase va a-glucosidase, ddmcitiate 0.01, pH4, DNSA(3, 5-dinitrosaIicyiie acid), p-nitrophenyl-a-D- glueopyranoside (jM^G) deu dugc mua tu Meik hogc Sigma-AIdrich, Hoa Ky. Hiing que tuai dut;*:
mua t^ mpt cd sd d Thanh phd Hd Chi Minh vio
khoang thing 9-10 Sau dd, miu dugc ehin qua nude sdi trong thdi gian 10 giay. Li dugc siy khd d 5(fC.
2.2. Phuong p h i p nghien cirn 2.2.1. Chiit xudt mau thuc vgt
Li khd duge nghiin bing may xay IKA Al 1 v i duge bio quin yong tiii sgch trong binh hut im eho din khi phin tieh. Viec chilt xuit la hiing qui theo phuong phip [6, 325-331]. Bpt khd tir miu thyc vit dupc chiet vdi 75%
methanol va dupe yich ly them 2 lin n&a de chilt xuit hoin toan cic hgp chit cd hogt tinh sinh hgc.
2.2.2. Cdc yeu to dnh hu&ng den qud trinh chief xuat
Anh hudng nhidt do ddn hgp chit cd hoat tinh sinh hpc: 35,45, 55, 65°C;
Anh hudng thdi gian lie din hgp chit cd hogt tinh sinh hpc: 10, 35, 60 vi 75 phut;
Anh hudng ty le mau va dung radi trich ly din hgp chit cd hoat tinh sinh hgc: 1:20, 1:50, 1:100, 1: 150, 1: 200 g/mL. Mdi thi nghiem duge lgp lai 3 lin.
2.2.3. Nghiin cuv kba sang ire cbe enzyme a-glucosidase
Kha nang lic chd enzyme a-glucosidase bdi cao chilt hiing qui theo phuang phap [7].
Tmdc tidn, hdn hgp phin iing bao gdm 0.05 mL p-niyophenyl a-D-glucoside (pNPG) (10 mg tiong 2 mL phosphate buffer) vi 0.01 mL eao hiing que vdi dem phosphate 2.8 mL (pH 6,8). Sau dd, 0.02 mL ciia arayloglueosidase duge them vao. Tilp theo, hdn hgp dugc u d nhidt dg 37°C yong 60 phiit, phan iing duge kit tiiiic ii d nhidt dg 90°C, yong 10 phut. Hdn hgp phin ling dugc do d bude sdng 405 nra. MSu ddi chirng buffer dupe tiidm vio thay eho eao chilt thye vgt. Kha ning ire chi enzyme a- glucosidase dugc tinh bang cdng thiic;
phan tr%m <xc chS (%) =
'5 t^u Ml chirng
TAP CHl KHOA HQC DAI HQC VAN LANG Ma Bfch Nhu^
Z2A.l^bieBCttuldianai^dct:beat^mec6-amyIase Phan irng lie ehe sy thdy phin tinh bdt ciia enzyme a-amylase bdi cae eao chidt dugc thyc hien theo phucmg phap ciia Bhandari va cdng sy [3]. 0.5 mL cao yich hdn hgp phin iing trong 0.02 M dung djch dem natri phosphate pH 6.9. Hdn hgp duge ii d 25''C, yong 10 phut, them vao 1% tinh bgt. Cudi cimg, thdm 1 mL ciia 96 mM dinitrosalicylic acid, dung dich dugc u yong bd nude 95''C yong I phiit. Hdn hgp phin ung duge do bing miy do quang phd vdi biie sdng 540 nm. Tat e i cic phep do ddu dugc thye hidn trong ba lan. Mau ddi chiing dugc tiiuc hipn bang thuoc Acarbose. Khi nang uc ehd enzyme a-amylase dugc tuih bing edng thiie;
phan tram uc che {%) =
"SiO m l u dSi ching
2.2.5. Xdc dinh phenolics tong
Tong ham lugng phenolic trong dieh chiet dugc xac dinh bing thudc thii Folin-Ciocalteu [8]. 300 ^iL miu eao dugc pha loang trdn vdi thude thii 300 jiL Folin-Ciocalteu. Sau 2 phiit, 2,4 ml dung djch natri cacbonat 5% da duge them vao. Miu sau dd duge li 1 gid d 25°C yong bdng tdi. Dp hap thy dugc do d bude sdng 760 nm. Axit gallic dugc sii dyng lam dudng ehuan.
2.2.6. Xdc dinh tong hdm luffng flavonoid Tong hira Iupng flavonoid trong miu chiet duge xie dinh bing phuong phap so mau
nhdm cloma ciia Chang et al [9] vdi ragt sd sua ddi nhd. 0,5 ml djch chilt duge thdra vao 1,5 ml ethanol 95%, 0,1 ml nhdm cloma 10%, 0,1 ml kali axetat va 2,8 ml nude cit. Hdn hgp nay sau dd dugc ii d nhiet do mdi tmdng trong 30 phut. Cudi cimg, do hip tiiy ciia hdn hgp phin ling duge do bang miy do quang pho d bude sdng 415 nm. Hidu chuin duge thue hidn bing each su dung dung dich rutin vdi ndng dp 20, 40,60, 80 v a i 00 g/mL
2.2.7. Xdc dinh khd ndng logi gdc tu do Hogt tinh chdng oxy hda duge xac dinh tiieo md ta bdi Huang et al [8, y.669-675]. Diu tidn, 0,1 ml djch chilt dugc them vio 3,9 ml DPPH. Sau do, hdn hgp duge giir yong tdi d nhidt do phdng. Sau 30 phiit, hdn hgp duge do bing may quang pho d 515nm. Phan yim quet gdc tu do dupe tinh tiieo phuang trinh sau:
Phan tram quet goc ty do (%)= (•^n-lnj-'^niluVlOO
Trong dd: Anjag: la do hip thu ciia miu trang; Amiu; li dd hip thu cua hdn hgp phan iing cd miu thir. Thi nghidm dupe lgp lgi ba lin, tinh kdt qua tmng binh.
3. KET QUA VA THAO L U ^
3.1. Cac yeu to anh hirdng den qua trinh chiet xuat
3.1.1. Anh hu&ng nhiet do din hgp chdt cd hopt tinh sink hgc
Hinh 1. Anh huong cua nhift dd din ham luong phenolic tdng (TPC)
Ghi chu: Cac chu cai khac nhau (a, b, cvdd) chi ra su khac biet coy nghTa thong ke (p <0,05)
TAP CHI KHOA HQC DAI HQC VAN LANG Nhidt dp khac nhau anh hudng ding kl ddn tdng ham lugng phenolic tdng (TPC) cua djeh chilt nhu trong FTinh I. Chilt xuit d 35X^ cho TPC tiiip hon so vdi 55''C (21,88 mg GAE/g trpng lugng khd vi 24,59 rag GAE/g trpng lugng khd, tuong img). Bdi vi nhidt dp cang eao, polyphenol v i dung mdi dd hda tan vio nhau, iupng polyphenol se trich ly ra nhieu hon, vide tang nhidt dp thieh hgp ed thd lim giam dd nhdt va dan din sy gia ting cae he sd khuech tin cua cic chit hda tan. Mat khac, nhidt giup eho qua trinh trich ly dd ding bang each phi huy mang td bao bdi vide lam bidn tinh mang t l bio vi sy hinh
tiiinh cic bpt khi, nd lim tang kha nang hda tan cua bdt hiing qui cin trich ly. Nhiet dp trich ly qui tiiip, hidu xuit trich ly khdng cao vi ham lugng polyphenol trong ba cdn nhidu. Do do, nhidt dp tic ddng phiic tgp ddn phenolic tdng, khi tang nhidt dd ciing tang hidu suit tiich ly. Tuy nhidn, nhiet eao nhu 65°C tid ldn se gay tac ddng nguge lai bdi vide lim bidn tinh sin phira tiich ly [10, y.159-169]. Vi vgy, yong khao sit niy chiing tdi chgn nhidt do dd trich ly polyphenol tir hiing qui la 55°C dk thye hien eac thi nghidm tiep theo.
3.1.2. Anh hu&ng thdi gian Idc din hep chdt cd hoat tinh sinh hoc
Hinh 2. Anh hudng cua thdi gian lie din him iupng phenolic tdng (TPC)
Ghi chu: Cdc chSr cai Uiac nhau (a, bvac) chi ra su Ichdc bi^t co y nghTa thong ke (p <0,05) Cd djnh nhi^t dg SS^C, anh hudng eua thdi
gian lic ldn ting him lugng phenol dugc thi hidn tiong hinh 2. TPC chat chilt xuit cd thdi gian lie khic nhau cd su khic biet ding kd.
TPC cao nhit ciia djch chilt thu duge sau 35 phiit lie (24,6 mg GAE/g chit khd). Luc diu, TPC eua djch chidt thap ban so vdi cae miu khac vi qui yinh ehilt cin nhieu thdi gian lie hon dl thu dugc nhidu hgp chit phenol hda tan yong dung mdi. Do dd, sy keo dai ciia thdi gian keo theo sy gia tang hieu suit yich ly, nhung khdng nen keo dii vi didu nay se lim giam hipu suit trich ly, bdi vi polyphenol yong ba ngiy cing giim. Thdi gian toi uu la 35 phiiL 3.1.3. Anh hu&ng ty If mdu vd dung moi trich ly din hgp chdt co hogt tinh sinh hpc
Khi tang ty Id nguydn lipu (dung mdi), TPC ciing tang din. Kht tang ty Ip nguydn lieu (dung mdi tir 1:20 Idn 1:150), TPC ting 14 mg GAE/g chit khd (tang 1.58 lin). Vdi ty Id dung mdi 1:100 v i 1:150 se cho him lugng cao nhat TPC. Kit qua nay cd thi duge giii thich bing nguydn tie ehuydn khdi giira chit rin va dung mdi, sy thay doi ndng do giiJa te bao tiiuc vgt bdn tiong vadung radi [10, ti. 159-169]. Nghien ciiu ti^dc diy cung cho bidt ty Ip l/iOO Ii toi uu dl chilt xuit tdng phenolics va flavonoid tir hung qui bing dung mdi nude [11, y.864-870].
Trong nghien cim nay, ty Ip mau/dung mdi 1:100 hoic 1:150 la dilu kipn tot nhit d l cd dugc TPC cao.
TAP CHi KHOA HQC DAI HQC VAN LANG Ma Bfch Nhu
Hinh 3. Anh hudng ciia ty le mlu va dung mdi den ham lugng phenolic tdng (TPC) Ghi chu: Cdc chd cai khac nhau (a, b vac) chi rosy khde billed y nghia thing l^(p<0.05) 3.2. Xac dinh tong ham lirong flavonoid,
phenolic tong v^ kha nang loai goc tu- do tren hiing que trich ly dieu kien toi iru Bing 1. T6ng ham lirpng flavonoid, phenolic tong va kha nang lo^i glc ty do tren hiing qui trich ly di^u ki|n tli iru
Hn> chiit c6 hof t tinh sinh hfc
TPC TFC DPPH
Gii trj 43.6 ± 0.03 mg GAE/g chit Itho 10.2 ± 0,05 ingRE/gchhldlS 3 8 . 2 ± l . i %
Gii yj TPC, TFC v i DPPH cua dieh chidt tir la hung qud d dieu kien tdi uu dugc the hidn tiong bing 1. Gii tij TPC la 43,6 ± 0,03 mg GAE/g chit khd, eao hon gii tii dugc bao cio [12, ti.85-91] tiich ly TPC t\s 15 loii hiing qui khie nhau. Kdt qua niy cd thi do khac bidt Ioai Ocimum vi dilu kien chiet xuat. Kdt qua eung chi ra rang gia tij TFC ciia dich chiet la 10,2 ± 0,05 mg RE/g ehit khd, thip ban gia tij dugc bio cao. Ngoai ra, khi nang logi gdc tu do DPPH ciia djch chilt la 38,2 ± 1,1%. Mgt nghidn ciiu tmdc diy eung eho bilt kha nang logi goc ty do DPPH eua Ocimum basilicum dao dpng tCr 19,23 din 89,46% [11, y.864-870].
3.3. Kha nang ire che enzyme oc-amylase va a-glucosidase
Bitng 2. Ty le phan tram (%) kha n5ng uc che hai lo9i enzyme tu cao chiet hiing qui Kh£ nang irc che enzyme
cc-amylasc a-glucosidase
Gii tri 45.8 ± 0 . 8 2 % 2 2 . 7 ± l . l % Khi nang uc chd ciia 2 enzyme a-amylase va a-glucosidase tir cao hung que dugc thi hipn tiong bang 2. Khi ning ire ehi eac enzyme thiiy phin tinh bdt dugc giii thieh la do cic hgp chat phenolic phan iing vdi protem tao thanh phiie ehit khdng hda tan, din din sy biln dgng cua enzyme, do dd ngan cin kha ning lien ket vdi chit nln dd giai phdng dudng hip fliu [12, tr.85-91]. So sinh vdi nghidn ciiu eua Lam va Pham [13, ti.439-445], ham lugng flavonoid ciia mudp ding, die bidt la him lugng charantin, ed thi lam giam lugng dudng tiong mau, tuong tu chiic ning eua insulin. Khi nang iie chi e n ^ m e thiiy phan tinh bpt tir cao hiing qui kha eao.
4. KET LUAN
Dilu kidn chilt toi uu cie hgp chat cd hogt tinh sinh hpc tii hung qui li: ty Id dung mdi raiu (g/niL) la 1/100, tiidi gian lie 35 phut va nhipt dp 55''C. Cao chilt tir hung qui d dilu kipn tdi uu cd khi ning khang 2 logi enzyme a- amylase va a-glucosidase.
TAP CHI KHOA HOC DAI HQC VAN LANG TAI LIEU THAM KHAO
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[7] Pistia-Bmeggeman, G. and R.I. HoUingsworth {20QI), A preparation and screening strategy for glycosidase inhibitors, Tetiahedron.
[8] Thaipong, K., et al (2006), Comparison of ABTS, DPPH. FRAP, and ORAC assays for estimating antioxidant activity from guava fruit extracts. Journal of food composition and analysis.
[9] Chang, C.-C, et al (2002), Estimation of total flavonoid content in propolis by two complementary eolorimetrie methods. Journal of food and drug analysis.
[10] Nguyen, N.Q. and T.Q. Dang (2017), Optimization of Ultrasound-Assisted Extraction of Natural Pigments from Annatto Seeds (Bixa Orellana) Using Response Surface Methodology, EC Nutiition.
[II] Izadiyan, P. and B. Heramateenejad (2016), Multi-response optimization of factors affecting ultrasonic assisted extraction from Iranian basil using central composite design. Food chemistry.
[12] Hossain, M., et al (2010), Effect of drying method on the. antioxidant capacity of six Lamiaeeae herbs. Food Chemistry.
[13] Lam, Q. T and Pham, V. H (2016), Extraction and determination of inhibitory capacity against stareh-hydrolyzing enzymes and antioxidant capacity of crude extract of Momordica Charantia, Joumal of Biotechnology.
Ngiy nhin bii: 17-01-2020. Ngiy bien tip xong: 13-7-2020. Duyet ding: 24-7-2020