TP CH KHOA HC V CNG NGH, I HC NNG - S 3(44).

2011

PHN LP CC HP CHT STEROL, FLAVONOID, COUMARIN T CY LC VNG TNH QUNG NAM

ISOLATION OF STEROLS, FLAVONOIDS AND COUMARINS FROM THE LUOCVANG PLANT IN QUANGNAM PROVINCE Phm Vn Hai, Trn Th nh Hng
i hc Nng
TM TT Bi bo ny trnh by v kt qu chit tch v phn lp cc hp cht chnh c trong thn l ca cy Lc vng Tnh Qung Nam. Qua thc nghim bng phng php chit soxhlet vi cc dung mi c phn cc khc nhau nh n-hexan, ethyl axetat, methanol v 13 1 bng cc phng php sc k, cc phng php ph hin i MS, C-NMR, H-NMR phn lp v xc nh c cu trc cc hp cht c trong thn l cy Lc vng l: hp cht HLVE_1 c tn 3-O-(-D-glucopyranosyl)--sitosterol, hp cht HLVH_1 c tn (24Rstigmasta-5-en-3--ol) cn gi l -sitosterol, hp cht HLVE_2 l (2S, 3S, 4R)-2-(2Rhydroxyhexacosanoyl)-amino-hexadeca-1,3,4-triol hay l Calliceramide v hp cht HLVH_2 l stigmasterol . SUMMARY This paper presents a study on the extraction and determination of the chemical composition of the Luocvang plant from Quangnam Province. We first used the soxhlet extraction method with different polarization solvents such as n-hexan, ethyl acetate, methanol 13 and then with Chromatography methods and the model spectro methods such as IR, MS, C1 NMR, H-NMR to isolate and determine its chemical structures. Our experimental results show that there are four extracted chemical compounds such as 3-O-(-D-glucopyranosyl)-sitosterol, 24R-stigmasta-5-en-3--ol or -sitosterol, Calliceramide, and stigmasterol. This is the first report on the successful isolation of the compound of (2S, 3S, 4R)-2-(2Rhydroxyhexacosanoyl)-amino-hexadeca-1,3,4-triol from the Luocvang plant in Quangnam Province.

1. t vn Cy Lc vng c tn khoa hc l Callisia fragrans, h Thi Li (Commelinaceace). Gn y, trn cc thng tin i chng, cy Lc vng c xem nh mt thn dc tr bch bnh. Thc vt Callisia fragrans l mt cy cnh di thc t Lin bang Nga sang Vit Nam, ban u n c trng Thanh Ha, gi y cy Lc vng nhanh chng pht trin n cc tnh, thnh ph khc nh H Ni, Hi Dng, Bc Giang, Ninh Bnh, Qung Nam,[2,3] Cy Lc vng c s dng theo kinh nghim dn gian iu tr rt nhiu bnh t nhng bnh thng thng nh mn nht, d ng, au hng, au rng, n nhng bnh nan y nh ung th, tiu ng [2,4]... Trong khi , cho n nay trn th gii v Vit Nam, cha c cng trnh no cng b y v thnh phn ha hc cng nh tc dng sinh hc ca loi cy ny.
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Nhm mc ch cung cp thm mt s thng tin c ngha khoa hc v loi thc vt m dn gian s dng lm thuc cha bnh by lu nay, chng ti tin hnh nghin cu chit tch, phn lp v xc nh cu trc ha hc mt s hp cht chnh c trong thn l ca cy Lc vng Tnh Qung Nam. 2. Thc nghim 2.1. Nguyn liu Mu cy ti thu hi v c thi thnh ming nh, a i sy ngay nhit 110 C khong 10 pht dit men. Sau phi v sy kh 50-60 0C cho ti khi m 10%. Mu kh c nghin nh v c ngm chit vi cc dung mi c phn cc khc nhau. Mu cy c TS Hu Th Vin Sinh thi v Ti nguyn Sinh hc Trung tm Khoa hc T nhin v Cng ngh Quc gia gim nh tn khoa hc l Callisia fragrans [2].
0

2.2. Thit b v ha cht 2.2.1 Thit b 1. Sc k ct, s dng silicagel Merck 60, c ht 100-400 mesh (0,040 n 0,063mm hoc 0,1 n 0,16mm) v nhm xit (Al2O3, Aluminiumoxid 150-PF254+366Typ T , Art. 1065 - Merck) vi h dung mi ra gii l n- hexan:etylaxetat c phn cc tng dn. 2. Sc k bn mng: Cc tm sc k lp mng nhm DC-Alufolien Kieselgel 60F254 Art.5554 v nha (polyme) DC-Plastikfolien Kieselgel 60F254 Art.5735 trng sn, dy 0,2mm cng c s dng xc nh mt s thnh phn c trong cc dch chit, cc phn on chy ct v kim tra s b sch ca sn phm thu c. Cc tm SKLM sau khi sy kh c soi di n t ngoi (UV-BIOBLOCK) bc sng =254nm (365nm) ri c phun thuc th Vanilin 1% trong metanol H2SO4 10% v sy trn 100 0C, a vo bung I2 xc nh thnh phn cu t. 3. Cc dng c trong phng th nghim (cc loi bnh cu chu nhit thy tinh, thit b ct quay chn khng, cc thit b nghin cu thch hp khc). 2.2.2 Ha cht - Dung mi: n-hexan, ethyl axetat, methanol, ... dng tinh khit (P) v phn tch (PA); - Thuc th : Dragendoff, Mayer, Wagner, LiebermannBurchard v cc ha cht: dung dch H2SO4 c, dung dch H2SO4 5%, dung dch NaOH 10%, dung dch CH3COOH 5%, dung dch HCl c, dung dch FeCl3,; - Thuc hin hnh: Vanilin trong H2SO4 10%; - Cc h dung mi trin khai bn mng. 2.3. Phn tch thnh phn ha hc thn l Lc vng Tnh Qung Nam Thnh phn ha hc thn l Lc vng bng phng php ngm chit soxhlet
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trong dung mi n- hexan c phn tch bng cc thit b ph khi kt hp sc k kh (GC-MS). 3. Kt qu v tho lun 3.1 Phn lp cc hp cht ha hc trong thn l cy Lc vng Tnh Qung Nam 3.1.1. Hp cht HLVH_1 (-sitosterol) Thn l cy Lc vng c ngm chit trong dung mi methanol v chuyn ln sc k ct ra gii ct bng h dung mi n-hexan: ethyl axetat (30:1), thu c cht rn v nh hnh v kt tinh li trong n-hexan thu c nhng tinh th hnh kim, khng mu, nng chy 136-138 0C. Trong ph EI-MS cht HLVH_1 cho mnh ion phn t (m/z) M+ = 414, nh vy khi lng phn t bng 414 vc ng vi cng thc phn t C29H50O. Trong ph FT-IR cho bit c dao ng ca lin kt nhm OH vng 3430cm-1, c trng dao ng ho tr ca cc lin kt i (C=C) vng 1640cm-1 v dao ng vng 3010cm -1. Trong ph 1H-NMR cho bit: Tn hiu H 3,53 ppm c trng cho proton trong nhm CH carbinol , tn hiu H 5,36 ppm ca H trong lin kt olefin (C=C) vi C ln lt l 140,8 ppm v 121,7 ppm. Trong cc ph 1 H-NMR, 13 C-NMR v kt hp vi ph DEPT cho bit trong phn t ca cht HLVH_1 c 29 nguyn t cacbon trong c 3 nguyn t cacbon bc 4, c 9 nhm metin (CH), 11 nhm metylen ( CH2) v 6 nhm metyl (CH3). T c th quy kt hp cht HLVH_1 c mt ni i gia v tr C-5 v C-6. Ngoi ra, trong ph 13 C-NMR cn nhn thy C 71,8 ppm c trng cho cacbon lin kt trc tip vi oxy ca nhm hydroxyl. So snh ph 1 H-NMR v 13 C-NMR kt hp vi ph DEPT ca cht HLVH_1 vi ph -sitosterol thy hon ton tng thch sai s cho php. T cc kt qu phn tch thnh phn ho hc, tnh cht vt l, tnh cht quang ph v da trn c s so snh cc s liu ph ca cht HLVH_1 vi ph ca cht -sitosterol chun, kt hp vi vic xc nh im chy ca hn hp ca HLVH_1 vi -sitosterol khng i, c th cho php quy kt cht HLVH_1 phn lp c t l cy Lc vng l 24R-stigmasta-5-en-3--ol hay -sitosterol, v cu trc ca HLVH_1 nh sau:

-sitosterol
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3.1.2 Hp cht HLVH_2 (Stigmasterol) Ra gii ct bng h dung mi n-hexan: ethyl axetat 5:1 t cao hexan thu c cht rn mu trng hnh kim, tan trong clorofom v methanol. Nhit d nng chy ca cht HLVH3 155-157 0C. Ph 1 H-NMR c cc tn hiu c trng H 5. 35 ppm (1H, d, J = 5Hz v 2Hz) l tn hiu ca proton trong lin kt olefin (C=C) trong vng. Hai tn hiu H 5.14 (1H, dd, J = 5 v 15 Hz) v H 5.03 (1H, dd, J = 15 Hz v 5Hz) c trng cho hai proton lin kt vi hai cacbon lin kt i ngoi vng, iu ny chng t lin kt C=C ngoi vng c cu hnh trans (E). Tn hiu H 3,49 (1H, m) thuc proton H-3 nhm carbinol trong vng. Ph 13C-NMR c cc tn hiu c trng C 138.32, 129.31, 140.71, v 121.67 ppm l cc tn hiu cacbon trong cc lin kt i C=C, ti C 71.8 ppm l tn hiu ph ca C-3 lin kt vi nhm OH. Ngoi ra, c cc tn hiu ph kh dy c trong vng trng mnh nm trong vng t 12.2 - 60 ppm c trng cho tn hiu ca cc nhm metyl (CH3), metylen (CH2) v metin (CH) ca HLVH_2. Trong cc ph 1 H-NMR, 13 C-NMR v ph DEPT cho bit thm trong phn t HLVH_2 c 29 nguyn t cacbon, trong c 3 nguyn t cc bon bc 4, c 11 nhm nhm metin (CH), 9 nhm metylen (CH2) v 6 nhm CH3. T cc kt qu phn tch thnh phn ho hc, tnh cht vt l, tnh cht quang ph nht l da trn c s so snh cc s liu ph ca cht HLVH_2 vi ph ca cht stigmasterol hon ton c th cho php quy kt cht HLVH_2 phn lp c t l cy Lc vng l stigmasterol v cu trc ca HLVH_2 l:

Stigmasterol 3.1.3. Hp cht HLVE_2 (Calliceramide) HLVE_2 l cht rn mu trng, v nh hnh thu c t dch chit ethyl axetat ca cy Lc vng, nng chy 116-118 0C, khng tan trong nc, tan rt tt trong h dung mi CHCl3/MeOH (t l 1:1). Ph HR-MS ca n (Hnh 1) cho pic ion gi phn t vi (m/z) 684,6713 [M+H]+, (tnh theo cng thc: C42H86NO5), do vy khi lng phn
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Hnh 1. Ph HR-MS ca HLVE 2

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t lm trn s ca HLVE_2 s l M = 683, iu ny chng t cht HLVE_2 c cha nit v cng thc nguyn s l C40H85NO5. Phn tch cc s liu ph 1 H, 13C-NMR, DEPT-135, DEPT-90, HMQC v HMBC ca n cho thy, cht HLVE_2 l mt amit, c gc alkyl mch di no khng phn nhnh (c trng cho cc cerebrozit). Cc Hnh 2. Ph 1H-NMR ca HLVE_2 ph DEPT-90 v DEPT-135 cho bit, phn t HLVE_2 c 4 nhm metin (CH), vi cc dch chuyn ha hc : C 51,3(C2); 70,9(C-4); 71,0(C-2) v 74,5ppm (C-3). Ph HMQC cho bit, pha trng yu nht c 3 nguyn t C lin kt trc tip vi xi, nh vy trong phn t ca n c 3 nhm hydroxyl thuc 3 nguyn t C bc 3, nguyn t C cn li C-2 51,3 lin kt trc tip vi nguyn t nit. Ph 1H-NMR (hnh 2) ca n cho thy H 7,3ppm l mt dublet (J=8,9Hz), y l tn hiu ca nguyn t H thuc nhm amit; H 3,95ppm (m) l tn hiu ca nguyn t H-2, cc nguyn t H-2 v H-4 c cng dch chuyn ha hc 3,42ppm (br, s), cn cc nguyn t H-1 c dch chuyn ha hc 3,6 v 3,7 ppm (2H, dd, J=6,1 v 12,5Hz, H-1a v H-1b), H-3 c dch chuyn ha hc 3,85ppm (1H, dd, J=4,2 v 4,6Hz, H-3). Cc nguyn t H1 v H1' c dch chuyn ha hc khong 0,9ppm (t) v dch chuyn ha hc ca cc nguyn t C1, C1' l 13,2ppm. Cc nguyn t H cn li trong phn t thuc cc nhm CH2 c dch chuyn ha hc trong khong t 1,2 - 2,0 ppm i vi cc nguyn t H v khong 22-35ppm i vi cc nguyn t C. Cc ph 13C-NMR, DEPT -135, DEPT- 90 (hnh 3), v HMQC (hnh 4) cn cho bit thm trong phn t HLVE_2 c mt nhm CO (cacbonyl) vi C-1 =173,2ppm (s), c mt nhm metylen (CH2) lin kt trc tip vi xi c dch chuyn ha hc 60,4ppm.

Hnh 3. Ph 13C-NMR v DEPT ca HLVE_2

Hnh 4. Ph HSQC ca HLVE_2

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Do vy c th thy trong HLVE_2 c tng cng 4 nhm hydroxyl (OH). Ph HMBC (hnh 5) c th quan st thy mt s tng tc quan trng C-1 vi H-2; C-2 vi H-1, H(NH) v H-3; C-3 vi H-2, H-3; C-4 vi H-3, H-5; C-1' vi H(NH), H-2'; C-2' vi H-3' v.v. Kt qu phn tch cc ph 1H, 13C-NMR, 1D v 2D vi cc k thut khc nhau c trnh by Hnh 5. Ph HMBC ca HLVE 2 trong bng 3.5. xc nh phn axt v phn aminoancol, phn t HLVE_2 c acyl ha nh nhng vi anhydrit axtic trong piridin nhit phng qua m, thu c peraxetat ca HLVE_2. Ph 1H-NMR ca Peracetyl HLVE_2 (hnh 6), cho thy 4 tn hiu H 2.05, 2.08, 2.16 v 2.18 thuc v 4 nhm metoxy (CH3CO), chng t 4 nhm hydroxyl (OH) c acyl ha hon ton. Cc s liu khc hu nh khng thay i so vi ca HLVE_2. Sn phm thy phn axt trong metanol ca HLVE_2 l metyleste ca 2-hydroxyaxt bo (HLVE_2.1) Hnh 6. Ph 1H-NMR ca Peraxetyl HLVE_2 v mt aminoancol mch di HLVE_2.2 (khng thu c c th l do khi lng thy phn t, v sn phm to thnh b phn hu trong qu trnh phn ng v cha nhiu nhm hydroxy). Ph EIMS ca HLVE_2.1 cho pic ion phn t m/z: [M]+= 426 tng ng vi cng thc phn t: C27H54O3 (hnh 7). Ph 1H-NMR ca HLVE_2.1 (hnh 8) cho bit, c mt nhm metoxy (MeO) vi H 3,8 ppm (s) v c mt nhm hydroxyl lin kt trc tip vi C-2, v H-2 c H 4,2 ppm (t). T s liu ph EI-MS v 1H-NMR ca HLVE_2.1 c th

Hinh 7. Ph EI-MS ca HLVE_2.1 138

Hnh 8. Ph 1H-NMR ca HLVE_2.1

TP CH KHOA HC V CNG NGH, I HC NNG - S 3(44).2011

khng nh HLVE_2.1 l axt 2-hydroxyhexacosanoic, cng thc phn t: C26H52O3 khi lng phn t M=412. T thy rng 3 nhm hydroxyl cn li trong HLVE_2 thuc phn aminoancol v phn t lng ca n s l: M= 683 - 395 + 1 = 289 tng ng vi cng thc phn t: C16H35NO3. Nh vy, qua vic phn tch cc s liu ph ca HLVE_2, cc sn phm acyl ha v thy phn axt trong methanol ca n, kt hp so snh cc s liu ph 1H v 13CNMR ca cc cerebrozit tng t [2], chng t rng cht HLVE_2 l mt cerebrosid c cng thc phn t: C42H85NO5 (M=683) c hnh thnh bi axit 2hydroxyhexacosanoic v 2-amino-hexadeca-1,3,4-triol vi cu trc ha hc (2S, 3S, 4R)-2-(2R-hydroxyhexacosanoyl)-amino-hexadeca-1,3,4-triol v chng ti t tn l Calliceramide, vi cng thc cu to nh hnh 9.

Hnh 9. Calliceramide [(2S, 3S, 4R)-2-(2R-hydroxyhexacosanoyl)-amino-hexadeca-1,3,4-triol]

Sn phm acyl ha Peracetyl HLVE_2 to thnh theo phn ng di y:

Peracetyl HLVE_2 Cn cc sn phm HLVE_2.1 v HLVE_2.2 c hnh thnh theo phn ng thy phn axt trong methanol theo phng trnh phn ng sau:

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3.1.4. Hp cht HLVE_1 (3-O--D-glucopyranosyl--sitosterol) Ra gii ct bng h dung mi clorofrom: methanol 7:1 t cn ethyl axetat thu c cht rn v nh (28.6 mg) hnh mu trng, khng tan trong clorofrom, d tan trong h dung mi clorofrom v metanol. Nhit d nng chy ca cht HLVE_1 273275 0C. Quan st trn ph 13 C-NMR v DEPT thy c 35 tn hiu ca nguyn t cacbon, trong c 7 nguyn t cacbon gn vi oxi c trng cho phn ng (nm trong vng 62.1 ppm n 100.5 ppm). C 2 tn hiu 140.7 v 122.4 ppm thuc v mt lin kt olefin C=C. Trong ph 1H-NMR, quan st thy proton ng xut hin dng doublet ti H 4.4ppm, c J = 7.8Hz ca H-1 phn ng tng ng vi C-1 100.5ppm. S liu cc ph IR, MS, 1H-NMR v 13 C-NMR thu c cho php xc nh HLVE_1 c cu trc ca mt hp cht glucosid vi cng thc phn t C35H60O6. Nhng im trnh by trn kt hp vi vic o im nng chy ca hn hp HLVE_1 vi -sitosterol-glucosid chun c nhit nng chy khng i cho php khng nh HLVE_1 l 3-O--D-glucopyranosyl --sitosterol. Do vy, cht HLVE_1 phn lp c t cy Lc vng l 3-O-(-Dglucopyranosyl)--sitosterol vi cu trc ha hc nh sau:

3-O-(-D-glucopyranosyl)--sitosterol
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4. Kt lun Ln u tin cy Lc vng trng Qung Nam c thu hi, xc nh tn khoa hc l Callisia fragrans. xc lp c qui trnh ngm chit thch hp cho vic phn lp cc hp cht trong cy Lc vng Tnh Qung Nam v phn tch nh tnh cc lp cht c trong cy Lc vng l: cc sterol, coumarin, flavonoid. phn lp, o cc hng s vt l, cc ph (IR, MS, 1D v 2D-NMR) cho 6 cht v xc nh c cu trc ha hc ca 4 hp cht c trong thn l cy Lc vng Tnh Qung Nam, gm: -sitosterol (HLVH_1), stigmasterol (HLVH_2), 3-O--Dglucopyranosyl--sitosterol (HLVE_1), calliceramide (2S, 3S, 4R)-2-(2Rhydroxyhexacosanoyl)-amino-hexadeca-1,3,4-triol (HLVE_2). Ln u tin phn lp c mt hp cht ha hc calliceramide (2S, 3S, 4R)-2(2R-hydroxyhexacosanoyl)-amino-hexadeca-1,3,4-triol (HLVE_2) t cy Lc vng Tnh Qung Nam v c th ni y l mt cht mi c cng b. TI LIU THAM KHO [1] Nguyn Tin Bn v cc cng s (2003), Danh mc cc loi thc vt Vit Nam, NXB Nng nghip, tp III. [2] Trnh Th ip, Th Phng, Phng Kim Nguyn, Nguyn Minh Khi (2008), Bc u nghin cu v thnh phn ha hc v tc dng sinh hc ca cy Lc Vng Callisia fragrans ( Lindl.) Woods, Tp ch Dc liu, tp 13, tr.276 279. [3] Trn Thu Hng, L Huyn Trm, Trn Thng Qung, Trn Th Minh, Nguyn Tun Anh, H c Cng (2009), Ginsenoside RG1 v L-Tryptophan t cy Lc vng (Callisia fragrans), Tp ch KH v CN cc trng i hc K thut, S 74, tr.107-112. [4] Trnh T Long, (2008), Lc vng - cy thuc ca mi nh, Tp ch Vietnam Time, Tp 14 (9). (BBT nhn bi: 14/04/2011, phn bin xong: 12/05/2011)

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