Dong Quang Huy va Dtg Tgp chi KHQA HQC & CONG NGHE 177(01): 61-66
NGHIEN CUtI T O I UtJ HOA QUY TRINH CHIET XUAT BERBERIN T f T H A N , R E C A Y VANG D A N G B A N G D U N G D J C H K I E M
DSng Quang Huy'*, Nguyin Qudc Thjnh', Le Thu HoaP ' Trudng Dai hgc Y Dugc - DH Thai Nguyen. ^Trudng Cao ddng Y le Thai Nguyen TOM TAT
Myc tigu: Xay dyng va tdi uu hda quy Ulnh chilt xuat berberin tCr than, re cay vang ding d quy md phdng thi nghigm.
Phirffng phap: Ap dung cac phin mem thdng minh trong nghign ciiu quy trinh chiet xuat gdm: Thiet ke mo hinh thyc nghigm D-Optimal bing phin mim Modde, phan tich cac yeu td anh hudng bing phin mim FormRules va toi uu hda thdng sd bdi phin mem INForm va thyc nghiem kiem chiing.
Ket qua: Dit' ligu phan tich cho thay khdi lupng va ham lupng berberin bj anh hudng bdi 3 yeu td khao sat la ndng dd nude vdi, t;^ Ig dupc ligu/dung mdi va thdi gian chiet
Ket lugn: Cac thdng sd tdi uu ciia quy trinh da duoc xac d^ih bao gdm: Ndng dp nude vdi (4,2%), tj lg dupc ligu/dung moi (1/8) va thdi gian chiet (11,6 gid),
Tii' khda; chiilxudl, berberin, vdngdang, Modde, FormRules, INForm.
D A T V A N DE
Viet Nam chiing ta la mpt nude giau tiem nang cay thudc. Trong nhirng nam gan day xu hudng trgn the giai dimg thuoc tir dugc ligu ngay cang tang len, vigc nghien ciiu va phat trien thuoc tii dupc lieu tao thuan Igi de nganh cdng nghiep dugc nude ta phat trien theo hudng hien dai hda cac thuoc y hpc co truyen, thuoc cd nguon goc dupc ligu va tan dyng ngudn tai nguyen dupc lieu.
Vang ding la mdt cfiy thu6c quy, moc hoang chii ylu d cac nude vimg nhigt ddi. d nude ta, cay mpc phd bien d mien Ddng nam bp. Nam trung bp, Tay nguyen. Trong dan gian, vang ding thudng dugc diing de chii'a li, dau mat, viem rupt [6]. NhO'ng nghien ciru gin day da chi ra ring berberin cd tac dyng chong ung thu, lam giam lugng cholesterol, khang nam, khang virus,,, D§c biet han ca, berberin cd tac dyng lam giam duang huylt va giam khang insulin d6i vdi bgnh tieu dudng tuyp II [7]. Vi vay hign nay, nhu ciu sii dung cac chl phim cd berberin ngay cang Idn, vnk cdng nghigp tong hgp hda dugc chua san xuit dugc. 100%
berberin trgn thj trudng hign nay dupc san xuit bing each chiet xuat tir dugc lieu.
Email: HuyDongQuangh@gmail com
Da cd mpt so nghien cuu vl phuang phap chiet xuat berberin nhu phuang phap chiet berberin tir vang dang bang dung djch acid sulfuric 0,4% [5], hay phuang phap chiet berberin bang ethanol 96" [3]. Hign nay chii yeu dung dung dich acid sulfuric loang dg chiet xuat, tuy nhien quy trinh chiet van cdn mpt so nhugc dilm nhu: Keo dai thdi gian, tdn nhieu dung m6i, djch chilt lan nhigu tgp khd tinh chl, hieu suit chilt thip, ddi hdi thiet bj chiet chiu dugc acid...
Vdi mong muon n^ng cao hieu suit chiet xuit chiing tdi thyc hipn de tai: "Nghign ciiu toi uu hda quy trinh chilt xuit berberin tir than, re cay vang dang bang dung djch kigm". Myc tieu de tai nham xay dyng dugc quy trinh toi uu chilt xuit berberin tii vang dang bang dung djch kiem vdi su h6 trg ciia cac phan mem thdng minh [1], sao cho khoi lugng va ham lugng hogt chat danh gia dua vao berberin cao nhat.
VAT LIEU, PHUONG PHAP NGHIEN CUU Nguyen lieu
Vang ding thu mua tgi huygn Dak N6ng, tinh Dak Lik. Xii li dupc lieu: ThSn vang ding nia sach, thai thanh lat m6ng, say khd d 50''C, xay thanh bgt thd.
61
Dong Quang Huy va Dig Tgp chi KHOA HQC & CONG NGHE 177(01): 61 -06 Chat chuin doi chilu: Berberin clorid do Vign
kilm nghiem thudc Trung uang cung cap, ham lupng 99,72% C20H18NO4CI, hien trang nude 0,23%, SKS: 0103168
Cac phan mem chuyen dung Modde (2002): Thiit kl md hinh thuc nghiem.
FormRules v3.3 (2007) Intelligensys:
Nghign Cliu nhan qua.
INForm v3.7 (2008) - Intelligensys: Tdi uu hda quy trinh.
Chilt xuat
Can bgt dupc ligu, Lap ludi va lot bdng vao binh chilt. Cho b6t dugc lieu vao binh chiet.
Them dung mdi chiet ngap mgt dugc lieu, khuay deu, ngam trong thdi gian thich hgp.
Cir 1 gid khuiy lai mdt lin. Riit djch chilt.
Tien hanh chiet tuang ty de thu dugc djch chilt lan n. Them acid hydrocloric loang den pH=3 trudc khi them NaCI. Khuay mgnh cho tan hit natri clorid rdi de 24 gid cho berberin ket tiia. Gan lay phin tiia. Lpc va nia tua bang
dung djch acid hydrocloric 0,4% dg loai tgp chit. Sau do nia tiia bing nude cho het phan ling acid. Thu dupc berberin clorid thd [2].
Djnh luvng:
Nguyin tdc: Theo phuong phap sac ky long higu nang cao (Phy lyc 5.3 - Dugc diln Vigt Nam IV)
Tien hdnh:
Dung dich chudn: Can chinh xac 25,0 mg berberin clorid chuan (m^), hda tan hoan toan vao binh dinh miic 250 ml trong pha dpng thu dupe dung djch cd nong dp 0,1 mg/ml (Cc).
Dung dich thir:
Mlu thii la berberin clorid th6 hogc berberin clorid tinh chl: Can chinh xac mdt lupng mau (m,) tuong duong vai 25 mg berberin clorid cho vao binh dinh miic 250 ml. Them 150 ml pha dpng, lac cho den khi tan het, bo sung pha dpng den vach thu dupc dung dich cd n6ng do C], Lpc qua mang lpc 0,45 \x.m, bd 20 ml dich lgc diu thu dupc dung djch thd.
Tbia, Kn^og-diaf
IMsim bsi, ^iH, 5ax 50 X.
'&ht!B^'}d>as.^&s.
Maafli te* vav BMHG yM. te^MY. mm
a s B sJd aCl 5%. pH 3-t; ^ o a MaCi. lasi nia SBft Bjstxaa <d<u:id ilio
Ethanol 96%. ihan Jua^j, BeittePn .d»Eid Hnh ciie
;Eiflfe.li
Hamlui:nist>ertwoa
Sa dd quy trinh chiet xudt vd tinh cite berberin clorid fir du-gc lifu vdng ddng bdng nu &c vdi
Ddng Quang Huy va Dtg Tap cht KHOA HQC & C6NG NGHE 177(01): 61-66 MSu thu la bpt dugc lieu: Can chinh xac
khoang IOO mg (m,) bdt dugc lieu (qua ray 710) vao binh dmh muc IOO ml, thdm 80 ml pha ddng, lac sieu am trong 40 phiit, de ngudi, thgm pha dpng din vach, lie deu, lgc qua mang lpc 0,45 nm thu dugc dung djch thii.
Pha dgng: Hda tan 1,0 gam natri laurylsulfat trong hdn hgp acetonitril - acid phosphoric 0,1% (1 : I) va pha loang thanh 1000 ml vdi cimg dung m6i.
Dieu kien sdc ky:
Cdt thep khdng gi (15 cm x 4,6 mm) dupc nhSi pha tmh Cis ( 3 ^im)
Detector quang pho tu ngogi dgt d budc sdng 345 nm, nhigt dp cdt: 30°C, t6c dp ddng: 1,2 ml/phiit
The tich tiem: 5 ^il
Cdch tien hdnh: Lan lugt tiem 5 ^1 dung dich chuin va dung dich thir vao he thong sic ky, tien hanh sac ky theo dieu kien da md ta.
KET QUA NGHIEN ClTU THAO LUAN X&c djnh ham Iircmg berberin trong dircrc iif u vang dang
Xac dinh ham lupng berberin trong than cay vang dang dg danh gia chat lupng nguyen lieu va ti'nh dugc higu suat ciia qua trinh chigt.
Thi nghiem dugc lgp lgi 3 lan.
Bang I. Hdm lugng berberin trong dugc liiu nghiin cuu
„™,™, Ham lutmg berberin (C2oH,gN04)
_!I! m
1 2,46 2 2,52 3 2,49 TB 2,49 ± 0,035
Ham lupng berberin trung binh ciia nguyen lipu dem khao sat la 2,49% ± 0,035% nhu vgy dugc lipu dem khao sat dgt tieu chuan chat lugng theo DDVN FV (Ham lupng berberin (C20H18NO4) khdng it hon 1,5% tinh theo dugc lieu kh6 kiet).
Hlnh \.Sdc ky do mdu chudn berberin clorid
Hlnh 2. Sdc ky do HPLC mdu duac lieu Thiet lie mo hinh chiet xuat
Quy trinh chiet xuat berberin dupc thiit ke bdi phan mem Modde gom 32 thi nghigm, berberin clorid tuong iing vdi cac thi nghigm theo thigt ke dugc kiem nghigm va ket qua dupc tdm tit trong bang 2, Ba th6ng so dau vao quan trpng ciia qua trinh chiet xuat can nghien ciiu gdm:
thdi gian chilt (X|), ty le duoc ligu - dung m6i (X2), nong dp nude vdi (X3), dugc ma hda thanh cac biin (X'l), (X'2), (X's). Cac thi nghigm dugc thiet ke theo tryc tam quay.
PHAN TICH LIEN QUAN NHAN QUA Dii ligu trong Bang 2 dupc diing lam ylu t6 dau \ko cho phan mem thong minh FormRules.
Bang 3. Xu huang vd mice do hen quan nhdn qud W luygn
100 100
Dong Quang Huy va Dtg
STT 1 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
Bang Thfri gian chiet (X'l)
-1
-1 -1
0 0 0 Ghi chii: Xi: That
>
Y,: Khoi Cac bien
Tap chl KHOA HOC & CONG NGHS 2. DQ lieu thuc nghiem quy
Dugrc ii|ii-dung mSI (X'2) 1/3 1/5 1/5 1/5 1/5 1/6 1/6 1/6 1/6 1/7 1/7 1/7 1/7 1/7 1/7 1/7 1/7 1/8 1/8 1/8 1/8 1/9 1/9 1/9 1/9 1/10 1/10 1/10 1/10 1/10 1/10 1/10
Nong at) nu-dt v6i (X'3)
-1
-1
-1
-1
0 0 0
177(01): 61-66 trinh chiet xudt berberin tic vdng ddng bdng nu&c vdi T h i i
gian chilt (XI) 4 24 4 24 14 4 24 4 24 4 24 4 24 4 24 14 14 4 24 4 24 4 24 4 24 4 24 4 24 14 14 14
Duvc lifu-dung
moi (X2) 1/5 1/5 1/5 1/5 1/5 1/6 1/6 1/6 1/6 1/7 1/7 1/7 1/7 1/7 1/7 1/7 1/7 1/8 1/8 1/8 1/8 1/9 1/9 1/9 1/9 1/10 1/10 1/10 1/10 1/10 1/10 1/10 gian chiet (gid). X2: Ty le duac lieu/dung mdi, Xi luang berberin clorid tho (gam). Yi ma hoa (X'l)
^.m^a~0
,(X':),(X'i),0, = micc
Nong d9 nir^c vdi (X3)
1 1 10 10 5,5 1 1 10 10 I 1 10 10 5,5 5,5 1 10 1 1 10 10 1 1 10 10 1 1 10 10 5,5 5,5 5,5
Kiloi lug'ng berberin
(VI) 1,14 1,92 1,55 2,41 1,61 1,9 1,98 1,56 2,47 1,19 1,93 1,57 2,49 1,57 2,18 1,62 2,09 1,24 1,97 1,59 2,51 1,29 1,98 1,59 2,56 1,3 2,0!
1,58 2,59 2,08 2,13 2,11 Ndng do nuac voi (%m/v) Ham luang berberin clorid (%) g6c, X = khoang bien thISn
Him lu'gng berberin
(V2) 83,7 84,9 87,1 85,2 85,7 83,2 83,5 87,6 85,3 84,3 83,5 86,9 85,8 86,7 85,1 84,6 87,1 84,7 85,1 86,7 85,6 84,4 84,6 86,9 85,2 83,6 84,3 86,7 85,6 86,7 86,6 86,9
Khoi lugng va ham luong berberin clorid cd lien quan trirc tiip vdi tit ca 3 ylu t6 khao sat la thdi gian chilt, ty le dugc ligu/dung mdi va nong dp nude vdi. Trong do ylu to thai gian anh hudng idn tdi hai bien dau ra, ylu to ty le dugc lipu - dung mdi it anh hudng.
Ddng Quang Huy vd Dtg Tgp chi KHOA HQC & CONG NGH$ 177(01): 6 1 - 6 6
Hinh 3. Mmh hpa moi lien quan giifa khdi luang berberin vdi thai gian chiit vd ndng d0 nu&c voi Nhgn xet; Thdi gian chiet cang dai, nong dp nude v6i cang cao thi khoi lugng berberin clorid th6 thu d u p c cang nhieu
Hinh 4. Minh hga moi lien quan giUa hdm lugng berberin vai thdi gian chiet vd nong d^ nuac voi Nhgn xet: Thdi gian chiet dai ham lugng berberin kh6ng tang them, qua trinh chiet x u i t dat tdi can bang sau 12 gid chiet.
Hinh 5. Minh hga moi lien quan giita khoi lugng vd hdm lugng berberin v&i thdi gian chiet Nhgn xet; Thdi gian chiet cang dai tgp chat
cang khuech tan, hda tan vao dich chiet nhieu hon vi vay khdi lugng san pham th6 tang trong khi ham lupng hogt chat chinh la berberin khong tang them.
Toi iru hda t h o n g so q u y t r i n h Bang 4. Xu hic&ng vd muc dg tiin quan nhdn qud
GU trj R^
Nhdm luy^n Nh6m thj
98,2%
95,0%
96,6%
87.7%
D I I li?u trong Bang 2 d u p c diing lam dau vao cho p h i n m i m INForm v3.7 (2008). Vdi nhdm thir gom 2 mau 14 va 16 va thugt toan RPROP, cac md hinh lien quan nhan qua dugc thiit lap va danh gia v l mgt thong ke theo Bang 4.
M 6 hinh YI c6 tinh tuang thich tot (R^ = 98,2%) va kha nang dy doan chinh x i c (R'^ = 95,0%). M6 hinh Y2 cd tinh tuong thich tot (R'^ = 96,6%) va kha nang du doan chinh xac ( R 2 = 87,7%). Ca hai m6 hinh deu c6 thg ap dyng trong giai doan t6i uu hda thdng so.
Biiu kien toi tru hoa X]! Thdi gian chiet = so duong
X2: Ty lg dugc lipu/dung m6i = s6 nguyen ducmg, X3: N o n g dp nude v6i = s6 duong.
Y I : Hi?u suat = max = Up.
Y2: Ham lugng berberin = max = Up.
Thong so toi tru
X I : Thdi gian c h i l t = 1 1 , 6 gid, X 2 : Ty Ip d u g c ligu/dung mdi = 1/8, X 3 : N o n g dp n u d e vdi = 4 , 2 % ,
65
Dong Quang Huy vd Dtg Tgp chi KHOA HOC & CONG NGHE 177(01):6I -66 Tinh chat da- dodn
Y I : Kh6i lupng = 2,43 gam, Y 2 : Ham lugng berberin = 87,2%.
Kiem chirng thuc nghiem
Dugc lieu vang dang dugc chiet xuat hai lan vdi cung dieu kign va quy trinh. San pham dugc xac djnh khoi lugng va ham lucmg berberin, cac gia tri kiem nghiem dupc so sanh vdi gia tri <i^ ^oan cho bdi phan mem INForm (Bang 5).
Bang 5. Kel qud thuc nghiem vd gid tri du dodn Tfnh chat
san pham Khoi lupng (gam) H&m lirgng berberin (%)
Thu'c n g h i f m L i n 1 Lan 2 T B
2,43 2,41 2,42 86,8 87,2 87,0
Dir doait 2,43 87,2
Ket qua phan tich phuang sai hai yeu to khdng lap doi vdi dQ lieu thuc nghiem: Quy trinh cd tinh lap lai (p < 0,05) va cac gia tri d y doan bdi phan mem INForm so vdi gia trj thuc nghigm (trung binh) cd y nghia thdng ke (P < 0,05).
KET L U A N
Ket qua tdi uu hda cho bdi phan mem INForm: Thai gian chiet 11,6 gid; ty le dugc ligu/dung moi 1/8 va ndng dp nude voi 4,2%.
Vigc ap dung phuong phap truygn thong vdi sy hd trg ciia phan mem thdng minh trong
quy trinh chiet xuat giiip giam chi phi, cong sue va riit n g i n thdi gian nghien ciiii. Diic biet da hoan thign quy trinh djnh lupng berberin trong vang d i n g b i n g p h u o n g phap HPLC giup nang cao c h i t lugng cac san pham co ngudn goc tii d u g c llpu.
TAI LIEU T H A M K H A O 1. D$ng van Giap (2002), Thiit ki vd tdi icu hoa cong thicc vd quy trinh, Nxb Y hpc, TP. HCM, tr.
1-97.
2. Ding Quang Huy (2014), Nghien cuu chiitxudi berbein lie vdng ddng bdng dung dich kiim, Luan van thgc si Dupc hpc, Trudng Dai hpc Duoc Hi Ndi.
3. Nguyin LiSm (1980), "Chilt xuit Berberin bing i^ luc ndng", Tgp chi Dugc hgc, so (3), tr. 10.
4. Phgm Vilc Trang, Nguyen Liem (2000), "Gop phan ve nghign cuu thyc vat va hda hpc ciia cay viing dang", Tgp chi Dicgc liiu, tap 5, sl (5), tr. 131.
5. Ho Die Trinh (1983), "Chilt berberin clorid trong vang dang bang dung djch acid sulfuric loang". Tap chl Dugc hgc, so 3, tr.l9, 6. Kulkami S. K. (2008), "On the mechanism of antidepressant-like action of berberin clorid", European Journal of Pharmacology, 589, pp.163-172, 7. Patil J. B, et al (2010), "Berberine induces apoptosis in breast cancer cells (MCF-7) through mitochondrial-dependent pathway", European Journal of Pharmacology, 645, pp. 70-77.
S U M M A R Y
O P T I M I Z A T I O N O F T H E E X T R A C T I O N B E R B E R I N FOR C O S C I N I U M F E N E S T R A T U M C O L E B R
Dong Quang Huy'*, Nguyen Quoc Thinh', Le Thu Hoai' 'TNU- University ofmMedical and Pharmacy, ^Thai Nguyen College of Medical Objective: Development and optimization ofthe extraction procedure for Coscinium fenesffatum Colebr. at laboratory
Methods: Intelligent softwares were applied in the exyaction procedure. This process was developed through steps: experimental design using Modde software, cause-effect study using FormRules software and optimization using INForm intelligent software and experimental observation.
Results: Data analysis showed that the amount and concentration of berberin were affected by three factors including caIci hydroxyd concentration, material-solvent ratio and extraction times.
Conclusions: The optimized extraction parameters were determined as calci hydroxyd concentration (4,2%), material- solvent ratio 1/8 and extraction times (11,6 hours).
Keywords: extraction, berberine, Coscinium feneslratum Colebr., Modde, FormRules, INForm Ngdy nlidn bdi: 13/11/2017; Ngdy phdn biin: 07/12/2017; Ngdy duyit ddng: 30/01/2018
Email NuyDangQuangh@gmail ci 66
