Nghien CIFU - Ky thuat
Xay dmig quy trinh dinh tinh, dinh lirotig acid ganoderic A bSng phuong phap HPLC trong nam linh chi - Ganoderma lucidum (Leyss ex. Fr.) Karst
Nguyin Ngoc Vinh'*, Le Thi Thu Ciic', Phan Nhu- TriicS Trin Viet Hiing^
'Vien Kiim nghiem thuoc TP Ho Chi Minh,
- Khoa Dugc, Bai hpc YDuac TP Ho Chi Minh,
•' Vien Kiem nghiem thuoc Trung uang
*E-mail: nguyenngocvinhvn@yahoo. com
Summary
An HPLC method was developed for analysis of the triterpenoid ganoderic A acid in the commercial Ganodenria lucidum (Leyss ex.Fr.) Karst): Column - Synergi Fusion - RP 18 (100 x 4,6 mm; 4 pm); isocratic mobile phase -1 % acetic acid and acetonitrile (70:30); Detector - Photodlode auay (254 nm). The calibration curve was linear over the concentration range of 15- 300 pg/mL, and the recovery range was 98 -102 %. The proposed HPLC method showed quite time-saving, precise and accurate. By this method, ganoderic A acid, as the main triterpenoid constituent of Ganoderma lucidum, was for the first time separated.
Keywords: Ganodenva, ganoderic A acid.
Dat van de
Nim linh ehi - Ganodenria lucidum (Leyss ex.
Fr.) Karst - la leal thae dugc quy duge trdng va san xuit dai tra d Nhat, Han Qude, Trung Qudc va Viet Nam do nd ed tae dung dugc ly kha pheng phu:
tae dung chdng oxi hda, chdng ung thu, bae ve gan, chdng dj irng, giam cholesterol. Cac nghien ciru gin dayPi da chimg minh nam linh chi (NLC) chira din 120 ehit, bao gdm eae hgp chit hu'u ca, cac nguyen td vi lugng, cac vitamin. Treng NLC, nhdm heat ehit chinh la cac pelysaccharid va cac triperpeniod. Acid ganoderic A la thanh phin chinh quan trpng nhit trpng cac hgp chit triterpencid eiia NLC. Chit lugng NLC lien quan din ham lugng pelysaccharid va triterpenoid trong dd cd acid ganoderic A. D l xae djnh ham lugng acid ganoderic A trong NLC, phan Idn eae cdng trinh nghien cuu diu sir dung phuang phap HPLC pha dao vdi chuang trinh rira giai gradient i^*'.
Bai bao nay gidi thieu phuang phap djnh lugng acid ganoderic A treng mdt sd miu nim linh chi ed tren thj trudng bang phuang phap HPLC - PAD pha dao rira giai isocratic cd thdi gian phan tich dugc rut ngin se vdi phuang phap HPLC rira giai gradient da duge cdng bd trudc day.
Nguyen lieu va phu-cng phap
Chat do! chieu, trang thiet bj, dung moi va hoa chat
Chit doi chieu
Acid ganoderic A (sd Id: Gan01/0-8/14) do Vien Kiim nghiem thudc TP Hd Chi IVIinh cung cip, ham lugng 98,92%.
Trang thief bi
May HPLC Shimadzu UFLC 20A vdi diu do PAD, can phan tich Mettler Toledo PX26, b l sieu am Hwashin.
Dung moi va hoa chat
Acid acetic (leal P. A), aeetonitril (leal HPLC) va methanol (leal HPLC).
Ddi tu'ong nghien ciru
Sau miu nim linh chi: Viet Nam, Nhat Ban, Han Qudc, A1 va /\2 do Cdng ty Dugc phim A cung cip va nim lim xanh (leal nim linh chi mpc tren cay lim xanh) duge thu hai tir Quang Nam da phai siy khd va xay mjn.
Phu'O'ng phap nghien ciru va thge nghiem Xay dung quy frinh djnh tinh, dinh lirgng acid ganoderic A frong nam linh chi bang phircmg phap HPLC
Chuin bi miu
+ Miu thip: Can chinh xac kheang 2 g bdt nam linh chi cho vae binh ndn nut mai dung tich 250 ml, them 40 ml dung djch NaHCOj 5% ngam trong kheang 2 gid, sau dd ehilt liy djch kiem. Tilp tuc them 40 ml dung djch NaHCQj 5% vao can ngam trong kheang 30 phut va chilt djch kiem. Tilp tue
48 TAP c m DlTOC'
Nghien CCFU - Ky thuat
ngam can vdi 20 ml dung djch NaHCO, 5% trong 30 p h u t Tap trung djeh c h i i t k i l m , acid hda bang dung djch acid hydrocloric 10% d i n pH <3. Djeh sau khi acid hda d u g e c h i l t bang cloroform 3 lan (40 x 30 x 30 ml). Tap trung dich cloroform va bay hoi den c i n . Hda can treng 10 ml methanol. Lpc qua g i i y Ipe Milllpore 0,45 pm.
+ Dung djeh ddi e h i i u goe: can chinh xac khoang 6 mg acid ganoderic A ehe vae binh djnh mu'c 20 ml, hda tan trong kheang 15 ml methanol, sau dd them methanol vira dii the tich, l i e d i u (Dung dieh cd ndng dp khoang 300 pg/ml). Tu"
dung djch gde pha thanh day dung djeh ddi e h i i u ed ndng dp acid ganoderic A khoang tip 15 pg/ml - 300 pg/ml.
Diiu kien phan tich HPLC
+ May HPLC Shimadzu UFLC 20A - d i u dd PDA.
+ Cdt Synergi Fusipn - RP 18 (100 x 4,6 mm;
4 pm).
+ Pha ddng: aeetonitril - acid acetic 1 % (30:70).
+ B u d c sdng phat hien: 254 nm.
+ Nhiet dp ept: 30 °C.
+ Tde dp ddng: 1,5 ml/phut.
+ T h i tich tiem: 20 pi.
Tham djnh quy frinh
Tinh t u a n g thieh he t h i n g , tinh dae hieu, tinh t u y i n tinh, dd lap lai, dd chinh xac trung gian, dp diing, gidi ban phat hien, gidi ban djnh lugng i^l
Ket qua va ban luan
Tfnh tu'ong thich he thdngK i t qua t u a n g thich he thdng d u g e trinh bay d bang 1.
Bang 1: Kit qua tuong thich he thing So dia ly He so doi
thuyet ximg _x,.. ^.. Thai gian luu Dien tich
So lan tiem , ,.,,
(phut) pie 1
2 3 4 5 6
9,705 9,704 9,714 9,708 9,703 9,686
890754 887825 895482 891383 889766 888420
150607 150283 150364 150983 151800 151141
1,16 1,17 1,17 1,17 1,16 1,16 Taing binh 9,703 890602 150863 1,17
RSD (%) 0,10 0,308
Ket qua thdng ke che thay, sau 6 lan tiem dien tich pie va thdi gian luu cd RSD < 2%; He sd ddi xipng 0,8 < A^ < 1,2. Vay p h u a n g phap dat tinh t u o n g thich he t h i n g .
Tinh dac hieu
T i i n hanh s i c ky m i u tring, m i u ddi ehiiu va m i u thir. K i t qua cho t h i y m i u tring khdng cd pic triing vdi thdi gian luu ciia pie acid ganoderic A. S i c ky dd m i u thir cd pic cd thdi gian luu tnJng vdi thdi gian luu eiia pie acid ganoderic Atrong m i u ddi chilu.
Khi them e h i t ddi ehiiu acid ganoderic A vao m i u thip thi dien tieh eiia pie acid ganoderic A tang.
Phd tip ngeai eiia pic acid ganoderic A treng mau thir gidng phd tir ngeai ciia mau ddi e h i i u acid ganoderic A. Dp tinh khiit eua cac pic Idn ban 99%. Vay phuang phap cd tinh dae hieu.
(A) (B) (C) Hinh 1 : Sie ky dd miu tring (A), acid ganoderic A chuin (B) va miu thir NLC (C)
thuan Aaileanodenc A SuedramOonatifln H:nt
'N-^ v.
0 M 250 22Q 2» 2^0 2J0 M ' ?!|j BO ' 13 a 30] 21c ys : y j c
: i . / -.1 : 3 m •™n .50 m ; . : 220 iV} loi :&! :30 : : : •;; sa •-: 35: a: 370
(A) (B) Hinh 2: P/76 UV - VIS eua pie acid ganoderic A tren sie ky dd dung dieh chuin (A) va dung dich thir (B)
TAP CHI DtTOC HOC - 6/2016 (SO 482 NAM 56)
49
Nghien CIFU - Ky thuat
Tinh tuyen tinh
Mdi tuong quan giO'a dien tich pic va ndng dp (pg/ml) acid ganodricAduge xay dgng tren 8 ndng dp khac nhau trcng kheang tu" 15 - 300 pg/ml.
Kit qua khao sat tuyin tinh dugc trinh bay d bang 2.
Bang 2: Kit qua khao sat tuyin tinh Nong do (ig/ml) Dien tich pic
15 30 60 75 105 150 225 300
84976 176289 360792 452332 633514 905849 1361851 1812373
Kit qua cho thay cd sg tuang quan tuyen tinh giO'a ndng dp acid ganoderic Ava dien tich pic.
- Gidi ban phat hien: khoang 0,5 pg/ml.
- Gidi ban djnh lugng: khoang 1,5 pg/ml.
Khao sat do lap lai va dp chinh xac trung gian
Kit qua dp lap lai va dp chinh xac trung gian duge trinh bay d bang 3.
Bang 3: Kit qua khao sat do lap lai va dp chinh xac trung gian
Ugayl
Ngay 2Mau
Ndng dd
(mg/100gNLC)'
Ndng do
• (mg/100gkc)
12 3 4 5 6
1072713 1086184 1085811 1090898 1084304 1072418
86,87 89,59 89,56 89,04 89,43 88,06
1091042 1099488 1100272 1099134 1099425 1099801
89,90 90,14 90,34 88,40 87,44 88,09 Trung binh (mg/IOOg NLC)
RSD(%)(n = 6) RSD(%)(n = 12)
88,76 1,23
1,26
89,04 89,04
Nhan xet: quy trinh dat dp chinh xac (RSD <
2%).
Hinh 3: Dd thj bieu dien mdi tuung quan ndng dd acid ganoderic A va dien tich pic
Bang 4: Kit qua khao sat dp diing
Khao sat do dungKit qua khao sat dp dung dugc trinh bay d bang 4.
Ty le them
vio{%)
80100
120
Lirong acid ganoderic A them vao (mg/2gNLC)
1,76 1,74 1,75 1,79 1,78 1,78 1,77 1,78 1,77
Luong acid ganoderic A cd sin (mg/2gNLC)
1,40 1,40 1,40 1,76 1,76 1,76 2,10 2,10 2,10
Nong do (pg/ml)
Trung binh 158,0 157,0 157,5 177,5 177,0 177,0 193,5 194,0 193,5
Luong acid ganoderic A tim lal (mg/2gNLC)
1,75 1,73 1,75 1,78 1,77 1,76 1,80 1,82 1,78
Tyle phuc hdi
(%)
99,40 99,70 100,06
99,76 99,71 99,23 101,57 102,15 100,54
Trung binh
(%)
99,72
99,57
101,42
100,24
Nhan xet: Quy trinh dat dd diing (quy dinh 95 -105%).
LCng dung
Lfng dung quy trinh da tham djnh d l xac djnh ham lugng acid ganoderic A trong mdt sd miu nim linh chi tren thj trudng (bang 5).
Bang 5; Ket qua khao sat ham lugng acid ganoderic A trong mot sd mau nim linh ehi
Loai nam Viet Nam Ntiat Ban Han Quoc Lim xanh
Al
/^
Dien tieh pie 247712 1008361 543612 567319 19276 18093
Ham luong acid ganoderic A (mg/IOOg NLC)
20,29 82,95 47,19 49,13 1,93 1,83
50
TAP CHI DlTOC'
Nghien CCFU - Ky thuat
Hinh 6: Sic ky dd mau nim Viet Nam Hinh 7: Sic ky dd mau nim Nhat Ban
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Hinh 8: Sac ky dd miu nam Han Qudc Hinh 9: Sic ky dd miu nim lim xanh
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^'^^^-MuvA,
Hinh 10: Sae ky dd miu nam Al
K i t luan
P h u a n g phap djnh l u g n g a d d ganoderic A treng n i m Ilnh chi b i n g HPLC -PAD v d i edt Synergi Fusion - RP 18 (100 =< 4,6 mm; 4 pm) va pha dpng gdm hon hgp acid acetic 1 % va aeetonitril ed tinh dac hieu, dp dung va dp chinh xae cao. D a y la l i n d i u tien acid ganoderic A trong n i m linh ehi d u g e djnh lugng b i n g HPLC b i n g rira giai isocratic vdi thdi gian d u g c rut n g i n thdi gian phan tich so vdi rira giai gradient trong eae edng trinh da edng bd ^^-^K
Tai lieu tham khao
1. Huber, Ludwig (2007), Validation and qualification in analytical laboratories, CRC Press, New/ York, pp. 146-148.
2. Jiang-Jing Gao (2004), "Quantitative determination of bitter principles in specimens of Ganoderma lucidum using high- peri'ormance liquid chromatography and its application to the evaluation of Ganoderma products",
Hinh 11: Sac ky dd miu nam A2
Chem. Pharm. Bull., 52(6), pp. 688-695.
3. Krystyna Skalicka- Wozniak et al (2010),
"Evaluation of polysaccharides content in fruit bodies and their antimicrobial activity of four Ganoderma lucium (W Curt: Fr) R Karst. Strains cultivated on different wood type substrates", Acta Societatis Botanicorum Poioniae, 81 (1), pp. 17-21
4. Sissi Wachtel- Galo et at (2011), "Ganodenria lucidum (Lingzhi or Reishi)", HeriDai Medicine:
Biomolecular and Clinical Aspect, 1 (2), chapter 9.
5. W. Tang et al (2006), "Separation of targeted ganoderic acids from Ganoderma lucidum by reversed phase liquid chromatography with ultraviolet and mass spectrometry detections", J. Biochemical Engineering, 32(1), pp. 205-210.
6. Ying Qi, Lingling Zhao, Hao. H Sun (2012),
"Development of a rapid and confirmatory method to identify ganoderic acids in Ganodenva mushrooms", J.
List Front Pharmacol., 3, 85.
{Ngay nhan bai: 11/05/2016 - Ngay duyet dang: 02/06/2016)