• Nghien ciru - Ky thuat
^Phan lap va xac dinh cau true quercitrin va vitexin tir vo hat dau xanh
(Vigna radiata (L.) R. Wilczek var. radiata)
Nguyen Thu Hang
B6 mdn Duac li^u - Trucmg Bai hoc Duac Ha NoiE- mail hangminhanh@gmail com
Summary
Two flavonoids, quercitrin (DX3) and vitexin (DX5). were isolated from ethyl acetate fractions of the seed coats of'mung bean" (Vigna radiata (L.)R. Wilczek var radiata) by chromatographic technichques.
Their structures were confirmed by MS and NMR in reference to the literature data. Quercitrin was reported for the first time from the seed coats of Vigna radiata (L.) R. Wilczek van radiata.
Keywords: Vigna radiata (L.) R. Wilczek var. radiata, mung bean, seed coat, quercitrin, vitexin, flavonoid.
Dat van de
Dau xanh la eay luong thye d u g c trong pho bien d n u d c ta. Ngoai gia trj ve dinh dudng, dau xanh cdn d u g e s i j dung theo kinh nghiem dan gian d dang toan hat hoae vo hat de lam thuoe ehUa benh. Hat dau xanh dh ehij-a sdt ndng, phi^n khat, phu thung, ta ly, mun nhgt sung t i y , loet mieng ludi, cac trudng hgp ngd ddc '*'®'. Vd hat dau xanh cd tac dung giai nhiet, tieu ddc tdt hon so vdi hai la m§m'^i. Flavonoid la hogt chat chinh cija vd hat dau xanh vdi nhieu tae dung quan trgng n h u bao vi co the ehdng phdng xa P', ehdng stress P', chong u thye nghiem •'!. Muc tieu cua nghien euu nay la phan lap va xae djnh cJiu tnjc cua mgt so flavonoid t u vd hat dgu xanh de ed them thdng tin khoa hgc ve thanh p h i n flavonoid cua d u g c lieu nay.
Nguyen iieu va pliu'cng phap
nghien CLPUNguyen lieu
Cay dau xanh cd hoa d u g c thu hai tai huyen Hoai S u e , Ha Ngi vao thang 4 nam 2013. Dya theo khoa phan loai eua Thi.Fc vat chi Trung quoc '^1, eay dau xanh nghiSn cuu da d u o c xac djnh tin khoa hgc ta Vigna radiata (L.) R. Wilczek var.
radiata, hg fl^u (Fabaceae). Hat dgu xanh duge thu tai Hoai Ode, Ha N6i vao thang 6 nam 2013
va d u g c tach rieng lay vd hat. Vd hgt dau xanh dwgc phoi khd rdi tan thanh bdt thd b i n g thuyen tan, sau do bao quan trong tui nilon kin d noi khd rao, thoang mat de nghien cuu.
Phuo'ng phap nghien c u u Chiet xuat
Vd hgt d | u xanh (2 kg) d u o c chiet hdl luu 3 lan vdi ethanol 80%, mdi \in trong 1 gid. Ggp cac dich chidt ethanol, eat thu hoi dung mdi d u d i ap s u i t giam thu duge hdn dich. Phan tan deu hdn djch trong n u d c cat rdi l i n luot ehiet vdi eae dung mdi eo do phan eye tang d i n nhu n-hexan, chloroform, ethyl acetat thu d u g c cac phan doan djeh chiet tuong ung, cac phan doan djch chiet d u g e cat thu hdi dung mdi d u d i ap s u i t giam rdi cd each thuy den khd thu d u g c eae can t u o n g ung. C i n phan doan ethyl acetat d u g c s u dung de phan lap eae ehat tinh khilt.
Phan lap cac chat tip phan doan ethyl acetat
Phan lap cac ehat t d phan doan ethyl acetat bang phuong phap s l e ky cdt hap phu vdi chat nhdi cdt la silica gel pha thudng (MERCK, c d hgt 0,040 - 0,063 mm), he dung mol rua g\i\
diehloromethan - methanol gradient 100:1, 2 0 : 1 , 15:1, 10:1, 4:1 va 2:1 thu d u g c 6 phan doan ky hieu t u o n g ung t u PD, d^n PD^. Kiem tra cae phan doan b i n g s i c ky ldp mdng vdi he dung mdi
TA P CHI DirOC HOC - 5/2014 (SO 457 NAM 54)
Nghien CIFU - Ky thuat
ethyl acetat - aeid formic - n u d c (8:1:1), hien v4t b i n g AICI3 3%/ethanol cho t h i y eae phan doan PB3 va PO5 eho mdt v i t tren s l e ky do. Phan doan PD3 duge e l t thu hdi dung mdi den can, rua e i n b i n g aceton thu duge e h i t vd djnh hinh mau vang ky hieu la D X , (15 mg). Phan doan PDg dieq/c eat thu hoi dung mdi d i n can, rua c i n bang aceton rdi t i l p tuc tmh e h l b i n g sac ky ldp mong ehe hda vdi hg dung mdi ethyl acetat - aeid formic - n u d c (8:1:1) thu d u g c ehat vd djnh hinh mau vang ky hieu la DX^ (22 mg).
Xac djnh cau true hoa hgc cua cac chat phan lap dwgv
Xac dinh c l u true cua cac c h i t phan l | p duge d y a tren so lieu pho khdi (MS) va pho egng hudng t u hat nhan (NMR). Phd khoi d u g c do tren may AGILENT 1100 LC-MSD, pho cdng hudng t u hat nhan d u g c do tren may BRUKER AVANCE AM500 FT-NMR tai Vien Hda hgc - Vign H^n lam Khoa hgc va Cdng nghe Viet Nam.
Ket qua va ban iuan
Chat 0X3
MS m/z : 448,9 [M+H]* t u o n g ung vdi cdng thue phan t u C j i H j o O , ,
'H-NMR (500 MHz, CD3OD, 6): 6,21 (1H, d, J
= 1,5 Hz, H-6); 6,37 (1H, d, J = 1,5 Hz, H-8); 7,35 (1H, d, J = 2 , 0 H z , H-2');6,93(1H, d, J = 8 , 5 H z , H-5'); 7,32 (1H, dd, J , = 2,0 Hz, J^ = 8,5 Hz, H-6');
5,37 (1H, d, J = 1,0 Hz, H-1"); 0,96 - 5,37 (6H, m, eae proton cua dudng)
'^C-NMR (125MHz, CD3OD, 5): 158,5 (C-2);
136,2 (C-3); 179,6 (C-4); 163,1 (C-5); 99,8 (C- 6); 165,8 (C-7); 94,7 (C-8); 159,3 (C-9); 105,9 (C-10); 122,7 (C-r); 116,4 (C-2'); 146,4 (C-3');
149,7 (C-4'); 116,9 (C-5'); 122,9 (C-6'); 103,5 (C- 1"); 72,1 (C-2"); 72,0 (C-3"); 73,3 (C-4"); 71,9 (C- 5"); 17,6 (C-6").
Phd 'H-NMR cua DX^ x u i t hien cac tin hieu nam proton cua vdng thom, trong dd cd hai proton n i m d vj tri meta tai 8^ 6,21 (1H, d, J = 1,5 Hz, H-6) va 6,37 (1H, d, J = 1,5 Hz, H-8), ba proton thudc vdng B eua khung flavon tai 8^ 7,35 (1H, d, J = 2,0 Hz, H-2'), 6,93 (1H, d, J = 8,5 Hz, H-5') va 7,32 (1H, dd, J , = 2,0 Hz, J^ = 8,5 Hz, H-6'). Ngoai ra cdn cd eac tin hieu cua mdt phan t u d u d n g rhamnose gom: tin hieu eiia proton anomer tai 6^
5,37 (1H, d, J = 1,0 Hz, H-1") vdi hang so t u o n g
58
t^e be (J = 1,0 Hz) ehung td d u d n g ed h u d n g a, tin hieu cua proton methyl eua rhamnose tai 5^^
0,96 (H-e"), tin hieu cac proton khac cua dudng ed do djch ehuyen hda hge nam trong vung 3,33 - 4,87 ppm.
Phd "C-NMR eua DX^ x u i t hien tin hieu cua 21 nguyen t u carbon, trong do cd 15 tin hieu cua khung flavon va 6 tin hidu cua d u d n g rhamnose.
Cac tin hieu cua phan aglycon eho t h i y nhdm earbonyl x u i t hien tai 5^ 179,6 (C-4), hai tin hieu CH dien hinh l u o n g ung vdi cac vj tri C-6 va C-8 eua vdng A tai 5^, 99.8 (C-6) va 94,7 (C-8), tin hieu eua carbon anomer tgi B^ 103,5 (C-1"), tin hieu eua nhdm methyl tai S^ 17,6 (C-6") va bon tin hieu CH noi vdi oxy edn lai cua phan tip d u d n g rhamnose r i t gan nhau tai S^, 72,1 (C-2");
72,0 (C-3"); 73,3 (C-4"); 71,9 (C-5").
Dya vao phd 'H-NMR v^ pho '^C-NMR ed the d y doan DX^ IS quercetin-3-O-o-L- rhamnopyranosid hay quercitrin ed e i u true hda hge d u g e trinh bay d hinh 1.
OH
Hinh 1: Ciu true hda hoc cua DX^ - quercitrin C a e s d lieu pho ^H-NMRvi pho " C - N M R cua chat 0X3 d u g c so sanh vol so lieu pho tuong ung cua quercitrin cdng bd trong tai lieu ''^ cho ket qua phu hgp.
Can c u vao so lieu pho khoi [MS) va pho cdng h u d n g t u hat nhan {NMR), ehat DX^ d u g c nhgn dang la quercitrin.
Chat OXg
MS m/z : 433,0 [M+H]* t u o n g ung vdi cong thue phan t u C^^H^oO,;,.
T^P CHI Dl/gfC HQC - 5/2014 (S6 457 NAM 54)
• Nghien ciJu - Ky thuat
'H-NMR (500 MHz, DMSO, 5): 13,16 (1H, =, 5-OH); 8,02 (2H. J = 8,5 Hz, H-2',6'); 6.82 (2H, J
= 8,5 Hz, H-3',5'); 6,27 (1H, s, H-3); 6,77 (1H, s, H-6);4,69(1H, d, J = 10,0 Hz, H-1"), 3,24 - 4,69 (6H, m, eae proton eua dudng).
"C-NM/? (125MHz, DMSO, 5): 164,8 (C-2);
102,4 (C-3); 182,1 (C-4); 160,4 (C-5); 98,2 (C-6);
162,7 (C-7); 104,0 (C-8); 156,0 (C-9); 104,6 (C- 10); 121,6 (C-1'); 128,5 (C-2',6'); 115,8 (C-3', 5');
161,1 (C-4'); 73,4 (C-1"); 70,8 (C-2"); 78,7 (C-3");
70,6 (C-4"); 81,8 (C-5"); 61,3 (C-6").
Ph6 'H-NMR eua DX^ eho cac tin hieu proton thom cua khung flavon bao gom: mdt tin hieu singlet cua proton trong vdng A tgi 8^ 6,77 ( I N , s, H-6); hai tin hieu doublet cija hai cap proton vdng B d v| tri orto tgi 6^ 8,02 (2H, d, J = 8,5 Hz, H-2',6') va 6,82 (2H, d, J = 8,5 Hz, H-3',5'). Ngoai ra cd tin hieu singlet cCia mgt proton methin tai 6^^
6,27 (1H, s, H-3). (y vOng trudng thap cd lin hieu eCia nhdm 5-OH tai 6^^ 13,16. Tren phd 'H-NMR cdn cho tin hieu eua proton anomer d 6^ 4,69 (1H, d, J = 10,0 Hz, H-1") vdi h i n g sd t u o n g tae Idn (J = 10,0 Hz) Chung td dudng ed hudng p.
Tin higu eac proton khae eua d u d n g ed do djch chuyen hda hge nam trong vung 3,24 - 3,84 ppm.
Tren pho '^C-NMR cua DXj ed 6 tin hieu dae tning eua d u d n g glucose tai 6^ 73,4 (C-1"); 70,8 (C-2''); 78,7 (C-3"); 70,6 (C-4"); 81,8 (C-5"); 61,3 (C-6"). Lien ket cua nhdm glucopyranosyl vao vj tri C-8 cua vdng A gay ra s y ehuyen djch S,, d cae vi tri C-7 (A5f. -1,6), C-8 (A5^. +10,0) va C-9 (AS^.
-1,4). Do dd, £>Xj cd d u d n g la glucose lien ket vdi aglycon d C-8 cua vdng A.
T d s y phan tich tren cd the d y doan DX^ la vitexin (apigenin 8-C-/3-D-glucopyranosid) cd cau true hoa hge d u g c trinh bay d hinh 2.
DH
Hinh 2 : Cau tnJc hda hoc cOa DX^ - vitexin
Cic so lieu pho 'H-NMR va pho "C-NMR cua c h i t DXj d u g c so sanh vdi so lieu phd tuong ung cua vitexin cdng bd trong tai ligu I'^i cho ket qua phu hgp.
Can e u vao sd iidu pho khdi [MS) va pho edng h u d n g t u hat nhan (NMR), c h i t DX^ duge nhgn dang la vitexin.
Ban iuan
V6 hgt dau xanh ed thanh phan ehinh la flavonoid '^l Flavonoid la nhdm hgp c h i t l y nhien ed hoat tinh sinh hge manh vdi nhieu tac dung quan trpng nhu ehdng oxy hda, bao ve gan, khang khuan, khang virus, ehdng ung thu, ehdng viem, ehdng di ung, ehdng l o e f ^ . G i n day, cac hgp chat flavonoid edn duge dac biet quan tam bdi Chung u c c h l hoat dgng eua nhieu enzym n h u cyclooxygenase, Ca^'-ATPase, xanthln oxidase, acethyl eholinesterase, phosphodiesterase, lipooxygenase, aldose reductase va tyrosin kinase la nhung enzym ed lien quan d i n eo ehe benh sinh cua nhieu loai benh tat '^•^•''''. Hdn hgp flavonoid ehilt tip vd hat dau xanh da d u g e edng bo ed eac tae dung bao ve c o the chong phdng xa '^1, ehdng stress '^', ehdng u thye nghiem ' ' l Trong mdt nghien eiiu trudc day ''^', chung tdi da phan lap d u g c mgt flavonoid la isovitexin (apigenin 6-C-^-D-glucopyranosid) t u vd hgt dgu xanh. Trong bai bao nay, ehung tdi t i l p tuc phan lap d u g c hai flavonoid t u vd hat dau xanh la quercitrin va vitexin. Quercitnn ed tae dung khang khuan, khang n l m i ' ° ' , chong oxy hda ''^i, uc c h l enzym acethyl eholinesterase i'", chong ung t h u i'^'. Vitexin la ddng phan eua isovitexin. Hai hgp c h i t nay ed dgc diem cau true C-glycosid, p h i n dudng lien k i t tryc tiep vdi aglycon qua lien k i t C-C. Vitexin va isovitexin ed tae dung ehdng oxy hda va dc c h l enzym phosphodiesterase '^"l Cac k i t qua nghien euu da cho I h l y vd hat dau xanh la mgt d u g e lieu tiem nang giau flavonoid hua hen nhieu hoat tinh sinh hge cd the ung dung d l phdng va chda benh.
K i t iuan
T u phan dogn djch ehilt ethyl acetat eua vd hat d|iu xanh [Vigna radiata (L.) R. Wilczek var. radiata) da phan lap d u g c hai Havonoid tinh
TAP CHi Dl/gfC H p c - 5/2014 (SO 457 NAM 54)
Nghien ciru - Ky thuat
k h i l t ky hieu la DX^ va DX^. Dya trSn cac sd lieu pho khoi (MS) va pho cdng hudng t u hgt nhan (NMR), 0X3 da d u g c nhan dang la quercitnn, 0X5 la vitexin. Day la lan dau tien quercitrin d u g c phan lap t u vd hat dau xanh.
Tai iieu tham l^hao 1. Tran Luu Van Hien, Van Quoc Hoa, Nguyin Thu Hien, Tran Thanh Loan (2002), "Tac dung ch6ng u thye nghiem tren chupt nhat trang cCia flavonoid vitexin", Tap chi Sinh ly hgc. 6(1), tr. 20-23.
2. Tran Luu Van Hien, Nguyin Thj V§n Thai (2000),
"Tham do tac dung antistress cua flavonoid vitexin doi vdi chugt nhit tring", Tap chi Sinh lyhoc. 4(2), tr 32-36
3. Nguyen Boi Huong, Tran Van Hien, Vu Tan Trao (2002), "Ham lugng MDA huyet thanh va dap ung chuyen dang lympho bao d benh nhan ung thu vu dilu trj tia xa ket hop vdi ehe pham antioxidant-vitexin", Y hoc thuc hanh. 12, tr. 7-8.
4 B 3 Tat Lgi (1999), NhOng ciy thuoc va vi thudc Viet Nam, NXB Y hoc, tr. 932-933.
5. Le Khanh Trai (1994), "Dau xanh mpt thuc an thong dung, mot vj thuoc thanh nhiet giai dpc", Tap chi Dupc/Jpc, 3, tr. 12-14.
6. Vien Dugc lieu (2004), Cay thuoc vi d^ng vat lam thuoc & Viet Nam, tap I, NXB Khoa hpc va ky thuat, tr. 771-774.
7. Harleen Kau Sandbar, Bimlesh Kumar et al.
(2011), "A review of phytochemistry and pharmacology of flavonoids", Intemational Pharmaceutica Sciencia, 1 (1), pp. 25-41.
8 J B. Harborne(1993), T/7eflavono/ds-Advances in research since 1986. Chapman and Hall, London.
9. Jiang Dou Sou, Wu Delin, Mats Thulin (2010), Flora of China 10, pp, 2005- 2009.
10. K. Hema, R. Knshnavenl (2014), "Antibacterial and antifungal activities ofAllamanda cathartica Linn.", Int J. Pharm. Bio. Sci., 5(1), pp. 588-593,
11. Mankil Jung, Moonsoo Park (2007),
"Acetylcholinesterase inhibition by flavonoids from Agrimonia pilosa", Molecules, 12, pp. 2130-2139.
12. Myoung-Chong Song, Hye-Joung Yang et al.
(2008), "Bioactive flavonoids from Trapapseudo/nc/sa", J. Korean Soc Appl. Biol. Chem., 51(4), pp. 334-337 13. Nguyen Thu Hang, Nguyen Thi Huong (2013),
"Pharmacognostic study of mung bean [Vigna radiata (L.) R. Wilczek) seed coats". Proceeding of the Eighth Indochina conference on Pharmaceutical sciences.
Medical Publishing House, pp. 958-961.
14. Oyvind M. Andersen, Kenneth R. Markham (2006). Flavonoids - Chemistry, Biochemistry and Applications, ORG Press, pp. 902
15 Zhang Jing, Wang Ying et al. (2009),
"Chemical constituents from the leaves of Lophatherum gracile", Chinese Journal of Natural Medicines. 7(6), pp. 428-431.
{Ngiy nhin bii: OB/03/2014 - Ngiy duyet ding: 12/05/2014)
Phan lap mot so hop chat coumarin
trong re xao tam phan (Paramignya trimera (Oliv.) Burkill)
Tran Thi Thiiy Quynh', Le Thi Kim Thoa^, Pham Ddng Phirong^' ' BM Duac hoc CO truyen-Khoa Y hoc co truyin. DH Y Duac TP. H6 Chi Minh
^Bo man Duac lieu, Khoa Duac, Dgi hoc YDuac TP. H6 Chi Minh
^ Trung tdm Dao tao vd Nghien cieu phdt trien thudc ngudn gdc tu nhien - Dgi hpc YDuac TP Ho Chi Minh
* E-mail: phuongpd56@yahoo com.vn
Summary
Dried root powders of Xao tam phan (Paramignya trimera (Oliv.) BurktII) grown in Khanh Hoa province (4.5 kg) were extracted with ethanol 96%, evaporated in vacuum to condensed extracts. The obtained extracts were successively partitioned with n-hexan, chloroform and ethyl acetate. From the