Nghien ciru - Ky thuat
\
Xay dung phirong phap dinh tinh, dinh Itfong cynarosid va scolymosid trong actiso
(Cynara scolymus L., Asteraceae)
Nguyin Thi Anh Nguyet', TrSn Thj Hoang ChP, Pham Dong Phirong^*
' Bo mon Dugc lieu - Khoa Dugc - DH YDugc Tp. Ho Chi Minh
• Vien Kiem nghiem thuoc Tp. Ho Chi Minh
^Triing tdm Ddo tao vd Nghien ciru phdt trien thuoc nguon goc tir nhien, DH YDugc Tp. Ho Chi Minh
*E-mail: phamdongphuongl 954(a),yahoo. com Summary
An assay (HPLC) was developed for the simultaneous determination of cynaroside and scolymoside in the leaves and extracts of Artichoke. The recoveries of cynarsoide and scolymoside in the leaves were 98.3 - 101.2% and 98.1 -100.9%, respectively against 101.5 -103.9% and 99.68 -102.9% for those in the extracts The precisions were 0.79% and 0.76%, respectively, in the leaves and 0.29% and 0.27% in the extracts. The calibration curve was found linear in the concentration range of 3- 45 pg/ml with the correlation coefficient of 0.9992 (R^) for both cynaroside and scolymoside. LOD of scolymoside and cynaroside were 0.5 and 0.4 pg/
mi, respectively
Keywords: Cynara scolymus L, cynaroside, scolymoside
Dat van de
Actiso da duge sir dung lam thudc va thge phim ehire nang d treng nudc cung nhu d nhiiu nude tren t h i gidi tip rit lau. Thanh phin ehinh ciia aetisd la cac polyphenol (cynarin, acid elorogenie, flavonoid). Cac chit nay ed nhtrng edng dung bao ve t i bao gan, dieu trj thieu nang gan... tang tilt mat, thdng mat, Igi tieu, giai dpe, chdng dj irng, giiip an ngon mieng, ehdng tao bdn '^l Mdt sd thir nghiem tren lam sang da chirng td aetisd cd tae dung chu'a chirng khd tieu, diy hoi, lam giam dang k i hdi chirng rupt kich thieh, ha lipid huyit, dai thac dudng typ 2 va diiu tri eao huyit ap. Cae tac dung tren la do thanh phin polyphenol cd d trong cay actiso. Ngoai ra, flavonoid (luteolin, scolymosid va cynarosid) cdn cd tae dung phdng ehdng ung thu, giam md mau, giam cholesterol giiip ngan ngira xo vu-a ddng mach va tai biin mach mau nae P"*'. Flavonoid cdn ed tac dung khang viem, chdng oxy hda va lam tang qua trinh sae chep eua cac gen tdng hgp nitric-exid (nitric oxide synthase: eNOS) lam tang ham lugng nitric oxid va vi vay cd tac dung lam gian mach, ha huylt ap, giiip ich cho viee dllu trj eae benh v i tim maeh '=1
Hien nay tren thi trudng thude Viet Nam ed rit nhilu c b l pham dugc san xuit cd chua eao
aetisd dang dugc luu hanh rdng rai. Cac ccng ty dugc phim trong nudc da nghien ciru va san xuit dudi nhieu dang bao c h i nhu dang cao dac, vien bae dudng, vien nang cirng, vien nang mim, dang dung djch udng va phd biin ban do la tra till Ipc, tra hda tan. Hai hgp ehit eynaresid va scolymosid da dugc phan lap va bao cae treng bai bao trudc day w. Tuy nhien, cho din nay van chua cd bao eao nao v i dinh lugng flavonoid chinh trong aetisd. Do dd, viee xay dgng phuong phap djnh lugng hai flavonoid nay nham gdp phin nang cao tieu chuin chit lugng duge lieu la mdt nhu c i u ed y nghTa thiit thue.
Doi tu'O'ng va phu'O'ng phap nghien cu'u Nguyen lieu
- La aetisd tuoi duge thu hai tai phudng 12, Tp. Oa Lat vae thang 9/2014 (miu 1) va thang 3 nam 2015 (miu 2). Sau khi thu hai, rge bd gan chinh, s i y khd, xay, ray qua ray 0,710 mm va 0,355 mm nhim loal bd phin Idng che ehd, liy phin bdt dugc lieu di qua ray 0,71 mm va khdng qua ray 0,355 mm.
- La khd aetisd (miu 3) dugc mua d cira hang Thinh Phat, sd 62 Mai Thj Lgu, Phudng Da Kae, Q. I, Tp. Hd Chi Minh va cung dugc loai bd gan chinh va tiin hanh nhu tren.
22 TAP CHI DlTgfC HQC - 6/2016 (SO 482 NAM 56)
Nghien CLPU - Ky thuat
Cao khd aetisd d u g c c h i i t x u i t d u d i dang bdt phun s u a n g va ddng gdi trong tui nhdm.
- Mdt sd c h i pham tren thi t r u d n g cd chira cao aetisd d u g e ma hoa la dang vien hay mau tra (xem bang 6).
Hoa c h i t , t h i i t bj
C h i t c h u i n ddi c h i l u : Cynarosid Taure Biotech (Trung Qudc), sd Id 1320331, ham l u g n g 99,25% tinh tren nguyen trang bang HPLC;
Scolymosid cd ham l u g n g 95,38 % tinh tren nguyen trang bang HPLC do Trung tam Dae tae va Nghien ciru phat t r i l n thudc ngudn gde t g nhien - D H . Y D u g e Tp. Hd Chi Minh cung c i p . May HPLC Shimadzu UFLC 20A, d i u dd PDA SPD M20A (Nhat). Cdt sac ky: cdt HPLC C18 (250 X 4,6 m m ; 5 pm), t i i n cdt C18 (4,6 mm x 5 pm). Dung mdi: acid formic (99,6%), methanol (99,9%), aeetonitril (99,9 % ) , ethanol (99,9 %) ciia Hang J. T. Baker - My dat tieu c h u i n dinh l u g n g .
Phu'O'ng p h a p n g h i e n c i r u
Khao sat dieu kien HPLC: Lua chpn b u d c sdng phat hien ehe cynarosid va scelympsid, pha ddng, e h l dp rira giai, nhiet dp cdt, va tdc dp ddng tren 2 ddi t u g n g nghien ciru (la va cap aetisd)
Khao sat phu'O'ng phap chuan bi miu tren la va cao actiso: Tham dd dung mdi e h i i t (nude, cdn 70 %, cdn 96 %, methanol 60 %, methanol), nhiet dp c h i i t (70 °C, 80 °C, 90 "C va 100 °C) va sd l i n c h i i t de dam bae e h i l t kiet 2 hgp c h i t tren.
Tham djnh qui trinh djnh lirgng: Qui trinh dinh l u g n g d u g e t h i m dinh theo h u d n g d i n ciia ICH ^ bao gdm: tinh t u y i n tinh, tinh phii hgp he thdng, gidi han phat hien, gidi han dinh l u g n g , tinh chpn Ipe, dp diing va dp chinh xac.
ij'ng dung quy trinh dinh lugng da xay dgng d l danh gia ham l u g n g cynarosid va scelymesid trong la, cao va mdt sd e h i p h i m dang luu hanh tren thi t r u d n g ed chira cae actiso:
- C h i p h i m la tra tui Ipe d u g e t i i n hanh xip ly m i u n h u quy trinh danh che la.
Nguyen lieu ban thanh p h i m n h u cap dae hay eae khd se d u g e t l l n hanh n h u quy trinh c h u i n bi m i u danh che cao c h i l t .
- C h i p h i m dang vien (vien nen va vien bao d u d n g . . . ) d u g c t i i n hanh xir ly m i u n h u quy trinh dinh l u g n g danh che cao c h i i t .
- Ham l u g n g scolymosid va cynarosid trong c h i p h i m d u g e tinh theo edng thirc:
St ^ k X = 7 ^ X Cc X -TTT
Sc m (1-h) x p x 100 T r o n g d d :
X: Ham luong ciia chat djnh lugng co trong dugc lieu (%) Sc: Dien tich dinh cua mau chuan (pV" s)
St: Dien tich dinh ciia miu thir (pV" s) Cc: Nong do ciia mau chuan (mg/ml) k: Bo pha loang ciia mau do (mi)
m: Khdi luong can cua cao hoac dugc lieu (mg) h: dd im ciia dugc lieu
p: dd tinh khiet ciia chat chuan (%) K i t q u a v a b a n l u a n
D i i u kien HPLC da d u g c khae sat d i djnh tinh, dmh l u g n g ddng thdi scolymosid va cynarosid trpng dich chiet la, cao aetisd va c h i pham ed eao aetisd
- Cdt Gemini C18 (250 mm x 4,6 mm; 5 pm) - B u d c sdng phat hien: 348 nm
- Toe dp ddng: 0,7 ml/phiit - Nhiet dp cdt: 30 °C - The tieh tiem: 20 pi - Thdi gian phan tieh: 30 phut
- Pha ddng: acid formic 0,1 % (A) - aeetonitril (B). C h u a n g trinh rira giai tiem t i i n : 20 % B (10 phiit); 20-40 % B (2 phiit); 40 % B (6 phiit);
40 - 20 % B (4 phiit); 20 % B (8 phut).
s i c ky dd d u g c trinh bay d hinh 1.
K h a o sat p h u o n g p h a p c h u a n bj m a u thCr (la va cao k h d actiso)
Quy trinh chief xuat tren la da dirge khao sat: Can ehinh xae kheang 0,5 g bdt la, cho vao binh ndn 250 ml, them khoang 50 ml ethanol 70%, dun hdi luu 30 phut d 100°C, d l ngudi, Ipc qua g i i y Ipc vao binh dinh mipc 100 ml, t i i n hanh them 2 l i n ntpa, mdi l i n vdi 20 ml ethanol 70
%, them VLPa dii ethanol 7 0 % tdi vach, lac d i u , Ipc qua mang Ipc 0,45 pm. T i i n hanh phan tich HPLC thee d i i u kien da Iga chpn.
Quy trinh chiit xuat tren cao da dirge khao sat: Can chinh xae kheang 200 mg eao vae binh dinh mirc 100 ml, them khoang 50 ml ethanol 70
%, sieu am d nhiet dp 40 °C, trpng 15 phiit, l i e , de ngupi 15 phiit, them ethanol 70 %, vipa dii tdi vach, l i e d i u , Ipc qua mang Ipe 0,45 pm. T i i n hanh phan tieh HPLC n h u d i i u kien da Iga chpn.
TAP CHI DirgfC HOC - 6/2016 (SO 482 NAM 56) 23
Nghien CCFU - Ky thuat
0 - 1
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10, 2 0 3CSKD eiia hdn hgp c h u i n (eynaresid va celymosid)
10 20
SKD ciia dich c h i i t cao SKD ciia djch e h i l t la Hinh 1 : Sic ky dd ciia dieh ehiit eao va la actiso
Quy trinh chief xuat cho thanh pham da dugc khao sat: C h i p h i m ed dang bao c h i la vien bao dudng, bao phim cd chira cae aetisd thi d u g e nghiin mjn va can khdi lugng t u a n g irng vdi 200 mg eao aetisd, sau dd t l l n hanh xip ly mau nhu quy trinh chiit x u i t danh eho cao c h i i t da xay dgng.
Tham djnh quy trinh djnh j y g n g theo ICH Khao sat khoang tuyen tinh: Pha dung djch c h u i n gdc scolymosid va cynarosid ed ndng dp 0,1 mg/ml b i n g dung mdi ehilt x u i t da khao s a t Tip dung djch ehuan gdc, pha b a n g d i d u g c hdn hgp ehuan l i n lugt ed ndng dp n h u sau:
t!^ 1 2 '3 4 ~5 6 T (mg/ml)
Cynarosid 0,003 0,005 0,008 0,010 0,018 0,028 0,045 Scolymosid 0,003 0,005 0,010 0,017 0,022 0,033 0,045 T l l n hanh phan tieh tren may HPLC-PDA mdi dung dich c h u i n 2 l i n vdi d i i u kien sac ki da
khao sat. Xac djnh mdi t u a n g quan giti'a ndng dp va dien tich dinh, xay d g n g p h u o n g trinh hdi quy t u y i n tinh t u o n g irng v d i mdi e h i t phan tieh.
Tinh phil hgp he thong: RSD ciia cae thdng sd dien tich dinh (S), thdi gian luu {{^) d i u nhd hon 2 %, sd dTa ly thuylt (N > 2.000); cac thdng sd khae d i u dat yeu c i u [He sd dung lugng (k" > 1); He s i b i t ddi (0,8 < As < 1,5); Dp phan giai (Rs > 1,5)].
Dp dac hieu: Quy trinh dat dp dae hieu do thdi gian luu, phd UV ciia pic scolymosid va cynarosid trong cac dung djch de la tuong duong nhau (Xem hinh 2). Mau trang khdng cd tin hieu ciia 2 pic nay Pic ciia scelymesid va cynarosid khdng ed thanh phin nae khac khi sir dung ehire nang kiim tra dp tinh khiit.
Op lap lai va do dung: dat yeu eau eho mpt quy trinh dinh lugng vdi ham lugng heat c h i t tip 0 , 0 0 1 % - 0 , 0 1 % ty le phuc hdi trung binh cho phep t u 90,0 % - 1 0 7 , 0 % f'^i (Xem bang 1-5).
Hinh 2: Khao sat tinh dae hieu ciia phuang phap tren djch chiit ciia cao va ia aetisd (1. Miu thir + chuan; 2. Thir; 3. Hdn hop chuin; 4. Miu tring)
24 TAP CHI DlTgfC HQC - 6/2016 (SO 482 NAM 56)
Nghien CIPU - Ky thuat
Bang 1 : Khoang tuyin tinh, LOD va LOQ cua cynarosid va scolymosid
u „ ,,', Phu^gtrinh „, ' ^ ° ^ " p . LOD LOQ Hop chat , ' , • , . / f nong do , , ,. , , „
tuyen tmh ^^^^^ (pg/ml) (pg/ml) Cynarosid y = 83031429 x 0,9992 3 - 4 5 0,4 1,0 Scolymosid y = 57079201 x 0,9992 3 - 4 5 0,5 1,25
Bang 2; S o chinh xac ciia phuang phap dinh lugng tren la aetisd
u „', X + S D ( n = 6) , " ^ ' " RSD% fiSD% f^'^' Hap chat - J i ' i " "; /(,(,,,„ , , j^ng
(mg/0,5glaAcUsd) fi/^j (Ngay 1) (Ngay 2) ^^.^ = Cynarosid 0,808 + 0,005 0,161 0,39 1,19 0,79 Scolymosid 1,664 ±0,02 0,332 0,95 0,57 0,76 Bang 3.' D p chlnh xac eiia phuang phap djnh lugng
tren eao khd aetisd
X ' + SD(n = 6) Ham j ^ j ^ j , , ^ g ^ j , . RSD%
H^ehit (m,0,g^o iu^g ^,^,^,, ^,^,^,^ Tning
Bang 4: Do dung eua phuang phap dinh lugng tren la aetisd
Lirong Hap chat sin co
(mg) Cynarosid 0,808
Scolymosid 1,664 Tile them vao
(%)
80 100 120 80 100 120
Lirgng them vao
(mg) 0,646 0,808 0,970 1,331 1,664 1,997
Lirong tim thay thue te
(mg) 1,429 1,614 1,799 2,983 3,265 3,694
Tile phue holTB
(%)
98,3 99,9 101,2 99,6 98,1 100.9 Bang 5: Do diipg ciia phuang phap dinh lugng
tren cao khd aetisd
Lumig Tile Lirong Luvngtim Tile phue Hgp chit san ed them vao them vao thay thuc te hoiTB
(mg) (%) (mg) (mg) (ii)
Cynarosid 0,757
Scolymosid 1,649 80 100 120 80 100 120
0,606 0,757 0,908 1,319 1,649 1,979
1,386 1,537 1,730 3,021 3,394 3,516
101,7 101,5 103,9 101,8 102,9 99,68 Chu thich: TB (tmng binh, n = 3)
Lfng dung quy tnnh djnh lu-gng da xay dirng va tham djnh de djnh lugng cynarosid va scolymosid trong la, cao va mot s6 che pham Bang 6: Ham lugng scolymosid va cynarosid trong cac dang chi pham
Cynarosid 0,757 + 0,003 0,377 0,27 0,30 0,29 Scolymosid 1,649 + 0,008 0,821 0,29 0,25 0,27
Stt Doi tirang djnh /irong 1 Miu la 1
2 Miu la 2 3 Miu la 3 4 Cao kho actiso 5 Dang vien 1' 6 Dang vien 2"
7 Dang vien 3=
8 Dang vien 4' 9 Miu trat'' 10 Miu tra 2'
Ham luong scolymosid (%)
0,61 0,44 0,34 0,84 N.D 0,12 0,04 0,04 N.Q N.D
Ham luong cynarosid (%)
0,40 0,21 0,17 0,40 0,29 0,03 0,05 0.01 N.Q 0,01
Stt
11 12 13 14 15 16 17 18 19 20
Do/ hrong djnh luong Mau tra 3'
Miu tra 4' Miu tra 5"
Miu tra 6"
Miu tra T Miu tra 8=
Miu tra 9=
Miu tra 10' Miu tra 11'^
Miu tra 12=
Ham luong scolymosid (%)
n.d n.d n.d n.d 0,37 n.d n.d n.d n.d 0,01
Ham lirong cynarosid (%)
n.d n.d n.d n.d 0,21 n.d n.d n.d n.d 0,01 Ghi chii:
- ND: khong phat hien du'p'c (ham Iwng thip hon LOD) - NQ: khong dinh lu-gng du'ge (ham lu'gng thip hgn LOQ) - Miu la 1 va 2: la tu-gi lam kho
- Miu la 3: mua g thj Inrgng Tp. Ho Chi Minh
Nhin chung, la khd, cao c h i l t va cac e h l pham dang vien san x u i t tip cao aetisd d i u cd s g hien dien cua ca scolymosid va cynarosid; tuy nhien ham l u g n g hai e h i t nay trong m i l e h i p h i m la khae nhau, ngoai trip met e h i p h i m dang vien (dang vien 1=) khdng cd scolymosid. H i u h i t cac c h i p h i m dang tra d i u khdng phat hien d u g c pic ciia hai flavonoid ndi tren heac ham l u g n g qua t h i p khdng t h i djnh l u g n g (xem hinh 3 va
'vien bao dTOng;
'vien bao phim:
' tra tui loc.
bang 6), ngeai trip 2 c h i p h i m cd ham l u g n g r i t t h i p va met c h i p h i m ( m i u tra 7"=) ed ham l u g n g kha eae. Nguyen nhan chii y i u la do hau h i t cac dang tra dang luu hanh tren thj t r u d n g d u g c san x u i t chii y i u tir p h i n than, re treng khi ehi ed la eiia aetisd mdi cd chira boat chat nhdm polyphenol n h u cynarin, acid elorogenie, cynarosid va scolymosid.
TAP CHI DirpC HOC - 6/2016 (SO 482 NAM 56) 25
Nghien CIFU - Ky thuat
30000-1
25000-
0-
J Ul 1
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1 1
5 8
10
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t j
20
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30 40 30000-
20000-
10000-
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20
1
DV1
30 40
25000-
0-
j
s
6
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10 20
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MT2
30 4(
Hinh 3: Sic ky dd dinh lugng cynarosid va scolymosid trong mdt vai che pham Chti thich: DV: Dang vien MT: Mau tra
Ngoai ra, k i t qua nghien ciru cung cho t h i y ham lugng 2 flavonoid trong la kha cao nhung ham lugng trong eao c h i i t lal khdng t u o n g xirng.
Ly do ehu yeu la chi diing dung mdi n u d c hay hon hgp edn - n u d c ed ndng dp cdn qua t h i p trong qua trinh ehilt x u i t va vi vay khdng t h i ehilt x u i t duge 2 flavonoid ndi tren.
K e t l u a n
Tip cae k i t qua nghien ciru eho thiy, quy trinh dinh lugng ddng thdi cynarosid va scelympsid trpng la va cao ehilt aetisd ed tinh t u a n g thieh he thdng, dat tinh dae hieu, ehe dp dung va dp chinh xac kha cap. Quy trinh da d u g c ap dung de danh gia ham lugng cynarosid, scelymesid trong la, cae va cae e h i pham (tra till Ipc, vien bao phim, bao dudng), trong dd met sd c h i pham cd chira n h i i u leai cao chiit tip cae d u g c lieu khac nhau ngeai aetisd. Quy trinh dinh lugng flavonoid trong aetisd l i n d i u tien d u g e bao eao trong nude.
T a i lieu t h a m k h a o
1. Bo T i t Lgi (2000), Nhu-ng cay thudc va vi thudc Viet Nam, NXB. Y hpc, tr. 221-222.
2. Jeremy P. E. Spencer (2009), "Flavonoids and {Ngay nhan bai: 01/03/2016 -
brain health: multiple effects underpinned by common mechanisms", Genes & Nutrition, 4(4), pp. 243-250.
3. Gabriel K. Harris, Yong Qian, Stephen S. Leonard, Deborah C. Sbarra, Xianglin Shi (2006), luteolin and chrysin differentially inhibit cyclooxygenase-2 expression and scavenge reactive oxygen species but similarly inhibit prostaglandin-E2 Formation in RAW 264.7 Cells", American Society for Nutrition J. Nutr, 136 (6), pp. 1517- 1521.
4. Rate Bundy Ann R Walker, Richard W. MIddleton, Carol Wallis, Hugh C. R. Simpson (2008), "Artichoke leaf extract {Cynara scolymus) reduces plasma cholesterol In othenwise healthy hypercholesterolemic adults: A randominzed, double blind placebo controlled tria", Phytomedicine, (15), pp. 668-675.
5. Robbins W. W., M. K. Bellue, W. S. Ball. (1970), Weeds of California, State of California, Dept. of Agriculture, pp. 547.
6. NguySn Th| Anh Nguyet, Pham Bong Phuong (2013), "Nghien ciru phan lap mpt so flavonoid trong la actiso", Tap chi Dugc hoc, 8, tr. 25-30.
7. ICH Harmonised Tripartite Guideline (CPMP/
ICH/281/95), Validation of analytical procedures:
Methodology, The European Agency for the Evaluation of Medicinal Products, pp. 3-9.
8. L. Huber, Method validation (2005), Validation of analytical methods, pp 1-15.
Ngay duyet dang: 02/06/2016)
26 TAP CHi DLTOC HOC - 6/2016 (SO 482 NAM 56)
