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PHN LP HAI HP CHT TINH KHIT T V TRI MNG CT (GARCINIA MANGOSTANA L.) V TH HOT TNH CA CHNG
Thanh Xun1, Trn Vn Quc1, Nguyn Ngc Hnh2 v Phng Vn Trung2

ABSTRACT
From the alcohol extract of Garcinia mangostana L. pericarp, -mangostin (VMC1) and -mangostin (VMC2) were isolated. Their structures were elucidated by modern spectrometric methods as 1H- NMR, 13C- NMR, DEPT, HSQC, HMBC and compared with reported data. Besides, antioxidant and antibacterial activities of VMC1 and VMC2 were also surveyed. Keywords: Garcinia mangostana L., xanthone, antioxidant, antibacterial Title: Isolation two pure compounds from pericarp of Garcinia mangostana L. and their bioactivities assay

TM TT
T dch chit cn ca v tri Mng ct, hai hp cht -mangostin (VMC1) v -mangostin (VMC2) c c lp. Cu trc ca cc cht c nhn danh bng cc phng php ph hin i nh 1H, 13C- NMR, DEPT, HSQC, HMBC v so snh vi ti liu cng b. Ngoi ra, hot tnh khng oxi ha v khng khun ca VMC1 v VMC2 cng c kho st. T kha: Mng ct, Garcinia mangostana L., mangosteen, antioxidant, antibacterial

1 M U Cy Mng ct c tn khoa hc l Garcinia mangostana L., thuc h Ba (Clusiaceae), l loi cy n tri nhit i rt quen thuc ng Nam . Cy Mng ct cn c tn khc l Ging Chu hay cy Mng, ngi Tu gi l Sn Trc T, ngi phng Ty gi tri Mng ct l n hong ca cc loi tri cy (Queen of fruits). Do thch hp vi kh hu nng m nn Vit Nam cy Mng ct c trng nhiu cc tnh min ng Nam b v ng bng sng Cu Long, mt s t c trng min Trung, khng thy trng min Bc. ( Huy Bch v cc tc gi, 2004) V tc dng dc l, t lu tri Mng ct ngoi hng v thm ngon cng hin nhiu bi thuc qu. Ngi Vit Nam, n , Thi Lan, Indonesia, Phillipines, dng v tri Mng ct tr tiu chy, au bng, bnh vng da, chng vim, c ch d ng, khng vi khun, vi sinh vt, lm gin ph qun trong iu tr hen suyn, (Hyun-Ah Jung, Bao-Ning Su, William J Keller, Rajendra G.Mehta, A.Douglas Kinghorn, 2006; Y.Sakagami, M.Iinuma, K.G.N.Pijasena, H.R.W._ Dharmaratne, 2005). Nhng nghin cu mi nht cho bit v tri Mng ct cn tr c ung th v khng HIV, hot cht cha trong v tri Mng ct gy c rt
1 2

Trng i hc Cn Th Vin Cng ngh Ha hc, Vin KH&CN Vit Nam

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mnh cho dng t bo ung th gan, ung th v SKBR3, c ch t bo ung th mu HL60 ca ngi. (Yukihiro Akao, Yoshihito Nakagawa, Munekaju Iinuma and Yoshinori Nozawa, 2008) Thnh phn ha hc ch yu trong v tri Mng ct c nghin cu v cng b c cha mt lot cc cht thuc nhm xanthone (Wilawan Mahabusarakam, Pichaet Wiriyachitra, Walter C.Taylor, 1987; T.R.Govindachari, P.S. Kalyanara_ man, N.Muthukumaraswamy and B.R.Pai, 1971). Vit Nam cho n nay ch c mt s t tc gi phn lp v kho st hot tnh sinh hc ca mt vi xanthone t v tri Mng ct (Nguyn Diu Lin Hoa, Phm nh Hng, Nguyn Vn Vy khoa Ha trng i hc Khoa hc T nhin vi cht -mangostin v 3-O-metil_ normangostin t v tri Mng ct Garcinia mangostana). Trong bi bo ny chng ti cng b kt qu phn lp, xc nh cu trc v th hot tnh sinh hc ca hai xanthone l -mangostin v -mangostin. 2 THC NGHIM 2.1 Nguyn liu Dch chit cn ca v tri Mng ct do Trung tm Nghin cu v Sn xut Dc liu min Trung cung cp ngy 14 thng 07 nm 2007. 2.2 Chit xut v c lp T dch chit cn ca v tri Mng ct, ly phn rn tch ra t dch chit c mu nu . T 200 g cao rn ny tin hnh ly trch nng vi petroleum ether (60C90C) (PE), sau khi lc qua giy lc v loi dung mi di p sut km thu c 47 g cao GE c mu vng ti. T 7 g cao GE tin hnh sc k ct vi h dung mi gii ly c phn cc tng dn: PE, PECHCl3, CHCl3 thu c ba phn on: GM 1, GM 2, GM 3. phn on GM 2 vi h dung mi gii li (PE:CHCl3 = 85:15), thu c cht rn c mu vng sau khi loi dung mi, sc k lp mng, hin mu bng hi iod cho mt vt trn mu vng c Rf = 0.537 (CHCl3:MeOH = 9:1), Rf = 0.67 (EP:EtOAc = 2:8), cho tinh th hnh kim mu vng (PE:MeOH = 95:5), c im tan chy mp = 178C v c k hiu l VMC1 (2.37 g). phn on GM 3 vi h dung mi gii li (PE:CHCl3 = 70:30) thu c cht rn c mu vng nht sau khi loi dung mi, sc k lp mng vi h (CHCl3:MeOH:CH3COOH = 90:10:2), hin mu bng hi iod cho mt vt trn mu cam c Rf = 0.375; Rf = 0.23 trong h PE:EtOAc = 1:1; cho tinh th hnh vy mu vng nht (PE:MeOH = 95:5), c im tan chy mp = 200C v c k hiu l VMC2 (523 mg). 2.3 Phng php xc nh cu trc Ph hng ngoi c o trn my VECTOR 22, dng vin nn KBr. Ph UV-Vis c o trn my UV-2450.

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Ph cng hng t ht nhn: 1H-NMR, 13C-NMR, DEPT, HSQC, HMBC c ghi trn my Bruker Avance 500 MHz dch chuyn ha hc c tnh theo (ppm), hng s tng tc (J) tnh bng Hz. im nng chy c o trn my Electrothemal 9100 (UK) dng mao qun khng hiu chnh. Sc k lp mng s dng bn nhm silicagel Merck 60F254 trng sn dy 0.2 mm. Sc k ct dng silicagel 60; 0.040.06 mm Scharlau GE 0048. 3 KT QU V THO LUN 3.1 Xc nh cu trc VMC1 VMC1 kt tinh trong (PE:MeOH = 95:5) cho tinh th c hnh kim mu vng sm, im tan chy mp = 178C, sc k lp mng (CHCl3:MeOH = 9:1), hin mu bng dung dch H2SO4 10% trong cn cho vt trn mu vng sm, khi hin mu bng hi iod cho vt trn c mu vng ti, c Rf = 0.537. Ph 1H-NMR (MeOD, ppm) cho cc tn hiu hp thu ca cc proton (Bng 1): nhm mi trong khong 1.67; 1.68 l tn hiu proton ca hai nhm CH3 ng vi C14 v C20. Hai mi n 1.78; 1.82 ng vi proton ca hai nhm CH3 v tr C15 v C19. Nhm methylen C11 cho tn hiu i v tr 3.26 v 3.33 (d, 2H, J = 7.5 Hz) v C16 cho mi i 4.03 v 4.05 (d, 2H, J = 6.5 Hz). Mt mi n c cng mnh v tr 3.75 l tn hiu ca proton OCH3 gn C7 ca khung xanthone. Mi a v tr 5.23 v 5.24 l proton ca hai nhm methyl C12 v C17. Hai mi n 6.20 v 6.65 l tn hiu proton ca C4 v C5 trn khung xanthone khng b thay th bi nhm OH v dy prenyl. Ph 13C-NMR (MeOD, ppm) v ph DEPT (Bng 1) cho bit trong phn t VMC1 c tt c 24 C, trong c 4 C loi CH= C4, C5, C12, C17 ( ppm bng 93.2; 102.7; 123.9; 125.2), 2 C loi CH2 cc v tr C11 v C16 ( ppm bng 22.2; 27.1), 5 C methyl cc v tr C14, C15, C19, C20 v OCH3 (25.9; 17.9; 18.3; 25.9; 61.3), 1 C carbonyl = 183.1 ppm v 12 C t cp. Ph HSQC cho bit s tng quan gia C v H tng v tr. Ph HMBC cho thy proton ca nhm OCH3 tng tc vi C7, proton nhm olefin H11 tng tc vi C1, C2, C3, C12, C13 v H16 tng tc vi C7, C8, C8a, C17, C18, proton H4 tng tc vi C2, C3, C4a, C9, C9a, proton H5 tng tc vi C6, C7, C8a, C9, C10a. T cc ph 1H-NMR (MeOD, ppm), ph 13C-NMR (MeOD, ppm) kt hp vi ph DEPT, HSQC, HMBC v so snh vi ti liu tham kho chng ti nhn danh VMC1 l -mangostin v c cng thc cu to nh hnh 1.

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Bng 1: D liu ph 1H-NMR, 13C-NMR, DEPT v HMBC ca VMC1

V tr

13

C-NMR ( ppm)

DEPT

H-NMR ( ppm)

HMBC 1H13C

1 2 3 4 4a 5 6 7 8 8a 9 9a 10a 11 12 13 14 15 16

161.5 111.4 163.5 93.2 156.1 102.7 157.7 144.7 138.4 112.2 183.1 103.8 156.6 22.2 123.9 131.6 25.1 17.9 27.1

>C= >C= >C= CH= >C= CH= >C= >C= >C= >C= >C=O
>C=

6.21 (s, 1H)

H4C2, C3, C4a, C9, C9a H5C6, C7, C8a, C9, C10a

6.65 (s, 1H)

>C= CH2 CH= >C= CH3 CH3 CH2 1.67 (s, 3H) 1.78 (s, 3H) 4.03; 4.05 (d, 2H, J = 6.5 Hz) H14C12, C13, C15 H15C12, C13, C14 H16C7, C8, C8a, C17, C18 H17C8, C19, C20 H19C17, C18, C20 H20C17, C18, C19 H1'C7 3.26; 3.33 (d, 2H, J = 7.5 Hz) 5.23 (m, 1H) H12C2, C14, C15 H11C1, C2, C3, C12, C13

17 18 19 20 1'

125.2 131.7 18.3 25.9 61.3

CH= >C= CH3

CH3

5.24 (m, 1H)

1.82 (s, 3H)

1.68 (s, 3H)

OCH3

3.75 (s, 3H)

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3.2 Nhn danh cu trc VMC2 Tinh th VMC2 hnh vy mu vng nht kt tinh trong trong h dung mi PE:MeOH = 95:5, im tan chy mp = 200C, sc k lp mng trong h dung mi (CHCl3:MeOH:CH3COOH = 90:10:0.2), hin mu bng dung dch H2SO4 10% trong cn cho vt trn mu vng nu, cho vt trn mu cam khi hin mu bng hi iod, c Rf = 0.375. Ph 1H-NMR (CDCl3-MeOD, ppm) cho cc tn hiu gn tng t VMC1 (Bng 2), nh l cc mi v tr 1.68 c cng mnh l mi ca hai nhm proton CH3 mi i khong 4.15; 4.16 (d, 2H, J = 6.5 Hz) l mi ca proton olefin C16, mi a khong 5.28; 5.29 l proton CH= ca C12 v C17. Khc vi VMC1, khng tm thy tn hiu proton ca OCH3 trong ph 1H-NMR ca VMC2. Ph 13C-NMR (CDCl3-MeOD, ppm) v ph DEPT ca VMC2 (Bng 2) cho thy VMC2 c tt c 23 C, ch t hn VMC1 mt carbon, cc tn hiu cn li tng t vi VMC1, nh tn hiu 182.2 ca nhm carbonyl, ppm = 17.4; 17.7 l tn hiu ca carbon nhm CH3,... iu ny chng t VMC2 v VMC1 c cng cu to l khung xanthone ch khc l nhm OCH3 gn C7 trong VMC1 th c thay bng nhm OH i vi VMC2. Ph HSQC cho bit tng quan gia C v H tng v tr. Ph HMBC c ghi trong bng 2. T cc ph 1H-NMR, 13C-NMR (CDCl3- MeOD, ppm) kt hp vi cc ph DEPT, HSQC, HMBC v so snh vi ti liu tham kho chng ti kt lun cht VMC2 l -mangostin c cu to nh hnh 2.
19 17 18 20 15 11 2 4a 4 3 12 13 14
19 18 17 20 15 11 2 3 12 13 14

16 8 8a

H 3CO
7

1'

O
9 9a

OH
1

16 8 8a

O
9 9a

OH
1

HO HO

7 6 5

HO

10 a

OH

10 a

O 4a

OH

Hnh 1: -Mangostin

Hnh 2: -Mangostin

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Bng 2: D liu ph 1H-NMR, 13C-NMR, DEPT, HMBC ca VMC2

V tr

13

C-NMR ( ppm)

DEPT

H-NMR ( ppm)

HMBC H13C

1 2 3 4 4 5 6 7 8 8 9 9 11 12 13 14 15 16 17 18 19 20

160.4 109.7 161.4 92.0 150.6 99.9 154.8 140.0 127.9 111.3 182.2 103.1 21.1 122.3 131.6 25.4 17.4 25.5 122.8 131.7 17.7 25.4

>C= >C= >C= CH= >C= CH= >C= >C= >C=

>C=

6.25 (s, 1H)

H20C1, C2, C3, C9a, C10 H5C7, C8a, C10, C10a

6.68 (s, 1H)

>C=O >C= >C= CH2 CH= >C= CH3 CH3

CH2

10a 152.7

3.36 (m, 2H) 5.28 (m, 1H)

H11C1, C2, C3, C12, C13 H12C8, C15 H14C12, C13, C15 H15C12, C13, C14 H16C7, C8, C8a, C17, C18 H17C8, C15 H19C17, C18, C20 H20C17, C18, C19

1.68 (s, 3H) 1.81 (s, 3H)

4.15; 4.16 (d, 2H, J = 6.5 Hz)

CH= >C= CH3 CH3

5.29 (m, 1H)

1.86 (s, 3H) 1.69 (s, 3H)

3.3 Kt qu th hot tnh khng oxi ho trn cao chit v cht sch 1,1-Diphenyl-2-picrylhydrazyl (DPPH) ha tan trong ethanol nng 250 M. Mu c ha tan trong Dimethyl sulfoxid (DMSO) nng 500 g/ml. Cho 2 ml dung dch mu vo 8 ml dung dch DPPH, hn hp c lc u, phn ng

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c thc hin iu kin nhit phng v trnh nh sng c cng mnh trong khong 30 pht, sau tin hnh o hp thu bc sng 517 nm. Kt qu l cao petroleum ether (GE), cao tng (GT) v cao nc (GW) ln lt c phn trm c ch l 64.61%, 72.5% v 81.27%. Trong khi vi cht tinh khit VMC1 khng c hot tnh (0.05%) v VMC2 c hot tnh mnh nht (92.63%). Kt qu kho st cho thy VMC2 c hot tnh khng oxi ha rt mnh nn chng ti tin hnh xy dng ng chun v tm gi tr IC50 nh gi kh nng oxi ha ca n, gi tr IC50 cng thp kh nng khng oxi ho s cng cao. Kt qu l:

th ng chun ca VMC2

Phng trnh ng chun ca VMC2 nh sau: y = 2.10-9x6 6.10-7x5 +7.10-5x4 2.8.10-3x3 1.58.10-2x2 + 4.4556x 0.5799 R2 = 0.9965 Da vo phng trnh ng chun, gi tr IC50 ca VMC2 tnh c bng 12.9 g/ml. 3.4 Kt qu th hot tnh khng khun Phi hp vi Khoa Thy sn trng i hc Nng Lm Thnh ph H Ch Minh, hot tnh khng khun c thc hin trn cc loi vi khun gy bnh trong thy sn: Vi khun Aeromonas hydrophyla (A. hydrophyla) c phn lp t ch. Vi khun Edwardsiella tarda (E. tarda) c phn lp t c tra. Vi khun Streptococcus.sp c phn lp t c r phi. Hot tnh khng khun c thc hin trn cao tng GT cc nng : X1 : 3 g/15 l (0.2 g/l) X2 : 30 g/15 l (2 g/l) X3 : 300 g/15 l (20 g/l) X4 : 3000 g/15 l (200 g/l)

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Kt qu th khng khun ca cao tng trn ba dng vi khun

Vi khun trung bnh (mm) Streptococcus.sp X2 0 X3 0 X4 0 0 Edwardsiella tarda X1 0 X2 0 X3 0 X4 A.hydrophyla X1 X2 0 X3 0 X4 0 Vng khng khun X1

10.4 0

Nhn xt: Cao tng ch c tc dng c ch s pht trin ca vi khun E. tarda nn ch tin hnh th hot tnh khng khun ca VMC1 trn loi vi khun ny. Kt qu th khng khun ca cht sch VMC1 trn vi khun E. tarda nng 3 g/15 l (0.2 g/l)
S a 1 2 3 4 5 6 7 8 9 10 KVVK trung bnh (mm) ng knh vng v 10 9 khun (KVVK) 10 10 9 10 10 10 9 10 9.7

Nhn xt: VMC1 c hot tnh khng khun cao trn dng vi khun E. tarda vi ng knh vng v khun trung bnh 9.7 mm nng 3 g/15 l (0.2 g/l), t hn 1000 ln so vi cao tng. 4 KT LUN T v tri Mng ct chng ti c lp c hai cht tinh khit. Theo cc s liu ca cc phng php ph hin i v so snh vi cc ti liu cng b chng ti xc nh c hai cht trn l -mangostin v -mangostin. Ngoi ra chng ti cng th hot tnh khng oxi ha v khng khun ca cao chit v cc cht tinh khit. TI LIU THAM KHO
Huy Bch v cc tc gi, 2004. Cy thuc v ng vt lm thuc Vit Nam. NXB Khoa hc v K thut H Ni, 3p. Hyun-Ah Jung, Bao-Ning Su, William J Keller, Rajendra G.Mehta, A.Douglas Kinghorn. 2006. Antioxidant Xanthones from pericarp of Garcinia mangostana (Mangosteen). in: J. Agric. Food. Chem. 54: 20772082. T.R.Govindachari, P.S.Kalyanaraman, N. Muthukumaraswamy and B.R.Pai. 1971. Xanthones of Garcinia Mangostana Linn. in: Tetrahedron. 27: 39193926. Wilawan Mahabusarakam, Pichaet Wiriyachitra, Walter C. Taylor. 1987. Chemical constituents of Garcinia mangostana. in: J. Nat. Prod. 50: 474478. Y.Sakagami, M. Iinuma, K.G.N.Piyasena, H.R.W.Dharmaratne. 2005. Antibacterial activity of -mangostin against vancomycin resistant Enterococci (VRE) and synergism with antibiotics. in: Phytomedicine. 12: 203-208. Yukihiro Akao, Yoshihito Nakagawa, Munekaju Iinuma and Yoshinori Nozawa. 2008. Anticancer effects of xanthones from pericarps of mangosteen .in: Int.J.Mol.Sci. 9: 355-370

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