全 文 ：波 谱 学 杂 志第 24卷第 1期
2007年 3月　 Chinese Journal of M agnetic Resonance
Vo l. 24 No. 1
　Mar. 2007
Article:1000-4556(2007)01-0077-07
Two Neolignans Isolated from Hydnocarpus Annamensis
Studied by NMR Spectroscopy
S HI Hai-ming 1 , 2 , L IU Y i 3 , J IANGYong1 , 2 , SUN Zhe 3 ,
TU Peng-f ei 1 , 2* , LING X iao-mei 3
(1. Department of Natural Medicines , S chool of Pharm aceut ical Sciences , Pekin g Universi ty H ealth S cience
Center , Beijing 100083 , China;2. Modern Research Center for Tradi tional Chinese Medicine ,
Peking University H ealth Science C enter , Bei jing 100083 , China;3. Department of Pharmaceutical Analy sis ,
Sch ool of Pharmaceutical S ciences , Peking University Heal th S cien ce Cen ter , Beijing 100083 , C hina)
Abstract:Two neolignans w ere isolated from the bark o f H ydnocarpus annamensis. Their
structures w ere elucidated using high-reso lution ESI mass spect rometry and NM R spectrosco-
py. The pro ton and carbon chemical shifts of the two compounds was assigned using 2D NM R
experiments , including HSQC , HMBC , DQF-COS Y and NOESY . The results o f capillary e-
lectropho resis(CE) analy sis demonstra ted that the two compounds are racemates.
Key words:NM R, comple te assignment , 2D NMR , neo lignans , H ydnocar pus annamensis ,
capilla ry electrophor esis
CLC number:O641　　Document code:A
Introduction
Hydnocarpus annamensis (Gagnep. ) M. Lesco t et Sleum. (Flacourt iaceae) is an
eve rg reen tree , mainly dist ributed in Guangxi province o f P. R. China , and used in folk
medicine for t reatment rheumatoid arthrit is and syphilis
[ 1]
. Chemical consti tuents and
biolo gical act ivities of this plant have no t been studied. As part o f our research on bio ac-
tive compounds f rom Flacourt iaceae plants , we have invest igated the barks of H. anna-
mensis and iso lated tw o neolignans , 1 and 2. The tw o neolignans have been isolated
Received date:26 May 2006;Revised date:17 Jul. 2006
Biography:Shi H ai-ming(1976-), male , postdoctor , phytochemist ry;Corresponding author:Tu Pen g-f ei , Tel:010-
82802750 , E-mail:pengfeitu@bjmu . edu. cn.
f rom Picea excelsa
[ 2] , Larix lep tolepis[ 3] and Zantedeschia aethiopica [ 4] and their NMR
results are rather old and incomplete. Therefo re , it is essential to report a detailed NMR
data of these tw o compounds based on modern 2D NMR measurements. In addi tion , we
analysed 1 and 2 by capilla ry elect ropho resis and found that 1 and 2 occurred as race-
mates. Al though this is a w ell-known phenomenon among the plant lignans[ 5] , the oc-
currence of tw o racem ic natural products in the same plant is rare.
1　Experimental
1. 1　Plant material
The barks o f Hydnocarpus annamensis (Gagnep. ) M. Lescot et Sleum. w ere co l-
lected f rom Pingxiang , Guangxi province , P. R. China , in December 2004 , and w ere i-
dent ified by M r. Chao-Liang Zhang . A voucher specimen (No. 041212)was depo si ted in
the herbarium of Modern Research Center for TCM , Peking Unive rsity , Beijing , P. R.
China.
1. 2　General experimental procedures
High-reso lution mass spect rometry w as pe rfo rmed on an AccuToFCS JM S T100CS
spect rometer. Semi-preparative HPLC:Waters 600 contro ller , Water s co lumn (Prep
Nova-Pak HR C18 7. 8×300 mm), Waters 2487 dual λabsorbance detector , detection
w avelength 210 , 254 nm. Chiral HPLC co lumn:Shiseido chiral CD-Ph (4. 6×250 mm ,
5μm). Capi llary electrophoresis:Beckman P /ACETM MDQ system (Beckman Coulter ,
Inc. , Fullerton , CA , USA) equipped wi th a pho todiode array detecto r as w ell as the 32
KaratTM sof tw are version 5. 0 (Beckman). A capillary tube (Yongnian Optical Fibre
Co rp. , Hebei , P. R. China) with an internal diame ter o f 50μm was used.
1. 3　Extraction and isolation
The dried barks (21. 4 kg) were mil led and ex tracted three times wi th 95% EtOH
fo r 2h each time , w ith the so lvent removed under reduced pre ssure. The 95% ethano lic
e xt ract w as suspended in w ater , then w as partit ioned w ith CHCl3 and n-BuOH succes-
sively . The n-BuOH-soluble fraction (198. 2 g)was concentrated and subjected to silica
gel column eluting w i th a CHCl3-CH 3OH (100∶1 , 100∶2 , 100∶4 , 100∶8 , 100∶16 ,
100∶32 , 100∶50 , 100∶100) g radient system to yield f rs. 1-8. Fr. 6 (16 g)was chro-
matographed on a silica gel co lumn eluting w ith CHCl3-CH 3OH (10∶1) to afford f rs.
6-1 , 6-2 , 6-3 and 6-4. Fr. 6-3 (2. 8 g)was purified by semi-preparative HPLC (CH 3OH
∶H 2O=12∶88) to afford 1 (5. 1 mg , t R =20. 1 m in), and 2 (4. 2 mg , t R =30. 5 min).
1. 4　CZE separation of 1 and 2
Fo r the separation of 1 and 2 , the running buffer w as 25 mmo l /L Bo rax-NaOH
buf fer (pH 10. 0) in the presence of 120 mmol /L HP-β-CD , the temperatures o f the car-
t ridge and sample room were kept a t 15 ℃ and 4 ℃, respectively. The applied vol tage
w as 30 kV. The samples w ere injected using the pressure injection mode a t 0. 5 p. s. i.
fo r 20 s (1 p. s. i. =6 894. 76 Pa). The capillary w as w ashed betw een runs w ith the run-
78 波　　谱　　学　　杂　　志　　　　　　　　　第 24卷　
ning buffer for 5 min at 20 p. s. i. Each concentration w as run in duplicate. Af ter every
tw o runs , the capillaries w ere rinsed w ith running buf fer fo r 5 min.
1. 5　NMR spectra
The NMR spect ra w ere recorded on Varian INOVA-500 and Bruker Avance DRX-
600 NMR spect rome ters equipped wi th 5 mm probes , respectiv ely . 5. 1 mg of compound
1 and 4. 2 mg of compound 2 were disso lved in 0. 5 mL solvent and transferred into a 5
mm NMR tube , respectively , which we re measured at 298 K. The pulse condi tions in
Varian INOVA-500 NMR spect rometer w ere as fo llow s:fo r the 1H NMR spectrum , ob-
servation f requency =499. 90 MHz , acquisi tion time (AQ)=1. 892 s , relaxat ion delay
(RD)=4. 0 s , spect ral w idth (SW)=7 998. 4 Hz , Fourier t ransfo rm (FT) size =
65 536;for the 1 3C NMR spect rum , obse rv ation f requency =125. 70 MHz , AQ=0. 7 s ,
RD=1. 0 s , SW=31 421. 8 Hz , line broadening =3. 0 Hz , FT size =65 536;fo r the
DQF-COSY spect rum , AQ=0. 228 s , RD =1. 0 s , SW =4 499. 4 (1H) and 4 499. 4
(13C)Hz , FT size =4 096×4 096;fo r the HSQC spectrum , AQ=0. 228 s , RD=1. 0 s ,
SW=4 499. 4 (1H) and 21 367. 5 (13 C) Hz , FT size =2 048×2 048;fo r the HMBC
spect rum , AQ=0. 228 s , RD=1. 0 s , SW=4 499. 4 (1H) and 30 165. 9 (13 C) Hz , FT
size =2 048×8 192;for the NOESY spect rum , AQ =0. 228 s , RD =1. 0 s , SW =
4 497. 9 Hz , FT size =4 096 ×4 096 , mixing time =0. 6 s. The pulse condit ions in
Bruker Avance DRX-600 NMR spect romete r w ere as fo llow s:for the 1H NMR spec-
t rum , observ ation f requency =600. 13 MHz , AQ=4. 955 s , RD=4. 0 s , SW=7 998. 4
Hz , FT size =65 536;fo r the 13 C NMR spect rum , obse rvation f requency =150. 92
MHz , AQ=0. 416 s , RD =1. 0 s , SW =31 421. 8 Hz , line bro adening =3. 0 Hz , FT
size =65 536;for the HSQC spectrum , AQ=0. 071 s , RD =1. 0 s , SW=4 499. 4(1H)
and 21367. 5 (13C)Hz , FT size =2 048×2 048;fo r the HMBC spect rum , AQ=0. 071
s , RD=1. 0 s , SW=4 499. 4 (1H) and 30 165. 9 (13 C) Hz , FT size =2 048×8 192.
2　Results and Discussion
Compound 1 was obtained as an amo rphous light-yellow ish powder wi th a molecular
fo rmula of C20H26O 9 , as dete rmined by da ta f rom positive-ion HR-ESI-MS , show ing an
[ M+Na] + ion at m /z 433. 1461 (calcd fo r C20H 26O9Na:433. 1475). T he 1H NMR of
compound 1 (Table 1)exhibited characteristic proton signals(δ6. 71 ～ 7. 01) o f tw o me-
ta and para-disubst ituted phenyl ring s. Addit ional ly , there are tw o sets o f signals be-
tw een δ3. 34 ～ 4. 81 , which a re typical g lycery l substituents commonly present in neoli-
gnans. Fur thermo re , tw o methoxyl g roups w ere observed at δ3. 80. The 13 C NMR
spect rum showed 20 signals , of w hich 12 we re aromat ic carbons and 8 w ere oxygen-
bearing carbons. The above spect ral data showed tha t compound 1 includes tw o pheny l-
propanoid f ragments.
79
　第 1期　SHI Hai-ming et al:Tw o Neo lignans Isolated f rom H ydnocar pus Annamensis
Studied by NMR Spec troscopy
Table 1　NMR spectral data at 500 /125 MHz in CD3OD for compound 1
No. δC δH HMBC NOESY
1 134. 2 /
2 111. 8 7. 01 (d , J=2. 0) C-1 , 4 , 6 , 7 H-7 , 8 , 3-OCH3
3 148. 7 /
4 147. 0 /
5 115. 6 6. 71 (d , J=8. 0) C-1 , 3 , 4 H-6
6 121. 0 6. 82 (dd , J=2. 0 , 8. 0) C-2 , 4 , 7 H-5 , 7
7 74. 1 4. 81 (d , J=6. 0)C-1 , 2 , 6 , 8, 9 H-2 , 6 , 8
8 86. 2 4. 34 (dt , J =6. 0 , 4. 0) C-1 , 7 , 9 , 4′ H-2 , 6 , 7 , 9 , 5′
9 62. 2 3. 84 (dd , J=6. 0 , 12. 0) C-7 , 8 H-9
3. 76 (dd , J=4. 0 , 12. 0)
1′ 137. 7 /
2′ 112. 3 6. 99 (d , J=2. 0) C-1′, 3′, 4′, 6′, 7′ H-7′, 3′-OCH3
3′ 151. 7 /
4′ 148. 6 /
5′ 118. 6 6. 87 (d , J=8. 0) C-1′, 3′, 4′, 6′ H-8 , 6′
6′ 120. 5 6. 82 (dd , J=2. 0 , 8. 0) C-2′, 7′ H-5′, 7′
7′ 75. 1 4. 53 (d , J=6. 0) C-1′, 2′, 6′, 8′, 9′ H-2′, 6′, 8′
8′ 77. 4 3. 64 (dt , J =6. 5 , 4. 0) C-1′, 7′, 9′ H-2′, 6′, 7′, 9′
9′ 64. 2 3. 47 (dd , J=4. 0 , 11. 0) C-7′, 8′ H-9′
3. 34 (dd , J=6. 5 , 11. 0)
3-OCH 3 56. 5 3. 80 (s) C-3 H-2
3′-OCH3 56. 3 3. 80 (s) C-3′ H-2′
O n the assignment of the proton and carbon signals , i t is easiest to start from the
gly cerol group by DQF-COSY and HSQC. HMBC co rrelations betw een H-7(δ4. 81)
and C-1 (δ134. 2), C-2 (δ111. 8) and C-6 (δ115. 6) established linkage betw een the
gly cerol and the pheny l ring. Then , H-2 (δ7. 01) and H-6 (δ6. 71) can be assigned by
HSQC (Table 1). The rest of the phenyl ring atoms can be ident ified by the combina-
tion o f DQF-COSY , HSQC and HMBC spect ra. As the first phenyl ring is completely
assigned , it is easy to ident ify the signals o f the second one. Furthermo re , a cross-peak
in the HMBC spect rum be tw een H-8 (δ4. 34) and C-4′(δ148. 6) indicated that tw o
pheny lpropanoids connect wi th an (8→4′) linkage. Complete 1H and 13C assignments of
compound 1 are g iven in Table 1. From the above spectral data , the st ructure of com-
pound 1 was established as 1-(4-hydro xy-3-methoxy)-pheny l-2-[ 4-(1 , 2 , 3-t rihydro xy-
propy l)-2-methoxy]-phenoxy-1 , 3-propandiol.
80 波　　谱　　学　　杂　　志　　　　　　　　　第 24卷　
　　　Fig. 1　S tru ctures of compounds 1 and 2
Compound 2 was obtained as an
amorphous light-yellow ish powder w ith a
molecular formula of C20 H26 O 9 , as deter-
mined by data from posit ive-ion HR-ESI-MS ,
show ing an [M +Na] + ion at m /z 433. 146 0
(calcd fo r C20H 26O 9Na:433. 147 5). The 1H
and 13C NMR spect ra of compound 2 (Table
2) show ed much simi lari ties to compound 1
indicated that these tw o compounds possess
the same planar skeleton and functionali ties.
Consequent ly , compound 2 was an isome r of
compound 1 and also assigned as 1-(4-hy-
droxy-3-methoxy)-phenyl-2-[ 4-(1 , 2 , 3-t ri-
hydro xy-propy l)-2-methoxy]-phenoxy-1 , 3-propandio l.
Table 2　NMR spectral data at 500 /125 MHz in CD3OD for compound 2
No. δC δH HMBC NOESY
1 133. 8 /
2 111. 7 7. 02 (d , J =1. 5) C-1 , 3 , 4 , 6 , 7 H-7 , 3-OCH 3
3 148. 8 /
4 147. 2 /
5 115. 8 6. 74 (d , J =8. 5) C-1 , 3 , 4 H-6
6 120. 7 6. 85 (dd , J =8. 5 , 1. 5) C-2 , 4 , 7 H-5 , 7
7 74. 0 4. 87 (d , J =5. 5) C-1 , 2 , 6 , 8 , 9 H-2 , 6 , 8
8 87. 2 4. 27 (dt , J =6. 0 , 5. 5) C-1 , 7 , 9 , 4′ H-2 , 6 , 7 , 5′
9 61. 9 3. 72 (dd , J =6. 0 , 12. 0) C-7 , 8 H-9
3. 47 (dd , J =5. 5 , 12. 0)
1′ 137. 9 /
2′ 112. 2 7. 06 (d , J =1. 5) C-1′, 3′, 4′, 6′, 7′ H-7′, 3′-OCH3
3′ 151. 5 /
4′ 148. 9 /
5′ 118. 6 7. 01 (d , J =8. 5) C-1′, 3′, 4′, 6′ H-8 , 6′
6′ 120. 6 6. 86 (dd , J =8. 5 , 1. 5) C-2′, 4′, 5′, 7′ H-5′, 7′
7′ 75. 1 4. 56 (d , J =5. 5) C-1′, 2′, 6′, 8′, 9′ H-2′, 6′
8′ 77. 4 3. 66 (dt , J =6. 5 , 4. 0) C-1′, 7′, 9′ H-2′, 7′
9′ 64. 2 3. 50 (dd , J =4. 0 , 11. 5) C-7′, 8′ H-9′
3. 36 (dd , J =6. 5 , 11. 5)
3-OCH 3 56. 5 3. 87 (s) C-3 H-2
3′-OCH3 56. 3 3. 81 (s) C-3′ H-2′
81
　第 1期　SHI Hai-ming et al:Tw o Neo lignans Isolated f rom H ydnocar pus Annamensis
Studied by NMR Spec troscopy
　　The optical ro tat ion values o f 1 and 2 were found to be zero , which suggested that
they are racemates. This was confirmed by the lack of Co tton ef fects in the ci rcular di-
chroism (CD) spect rum o f 1 and 2. A ll our a ttempts to separate them including using a
chiral HPLC co lumn , how ever , were to no avail. To determine w hethe r compounds 1
and 2 are racemates , they w ere analy zed by CE. Tw o peaks w ere observed in 1 (tm =16.
48 and 16. 81 min) and 2 (tm =15. 20 and 15. 52 min), respectively , which suggested
that both of them are racemates. Compounds 1 and 2 (1-(4-hydro xy-3-methoxy)-phen-
y l-2-[ 4-(1 , 2 , 3-t rihydroxy-propyl)-2-methoxy]-phenoxy-1 , 3-propandiol)were isola-
ted f rom Picea excelsa in 1967[ 2] and their complete NMR da ta hadn’ t been repo rted un-
til 1998 by G reca et al[ 4] . Previous studies only included 1H NMR in CD3OD and 13 C
NMR in acetone-d6 . M oreover , these results did no t match w ell wi th our new er ones ,
which stimulated us to assign the 1H and 13C NMR spect ra of 1 and 2 in CD3OD (Tables
1 and 2). The question remained is that the relat ive stereochemistry of C-7 , C-8 and C-
7′, C-8′in compounds 1 and 2. NOE co rrelations w ere observed be tw een H-7 /H-8 , H-
2 /H-8 , and H-6 /H-8. Furthermo re , the coupling constant of H-7 w as 6. 0Hz. A ll these
evidences indicated the threo form s o f the g ly cery l chain (betw een C-7 and C-8) in com-
pound 1
[ 6-9]
. M eanwhi le , the NO E correlations we re observed between H-7′/H-8′, H-
2′/H-8′, and H-6′/H-8′. The relatively high coupling of H-7′(6. 0Hz) and the chemi-
cal shif t at C-7′(δ75. 1) and C-8′(δ77. 4)10 further suppo rted the threo fo rms of the
gly ceryl chain (betw een C-7′and C-8′) in compound 1. By the same w ay , the g lyce ry l
chains in compound 2 we re also indicated as threo fo rms.
From the biosynthetic view , compounds 1 and 2 a re presumably formed from the
dimerization of threo-1′, 2′-guaiacy l gly cerol , the majo r pheny lpropanoid of the barks of
H. annamensis
[ 11]
. Bo th compounds 1 and 2 occurred as racemate s , suggest ing that
they are fo rmed in the plant cells wi thout the par ticipation o f enzymes.
References:
[ 1] 　Zhonghuab encao Edi to rial Board. Zhongh uab encao[ M] . S hanghai:Sh angh ai Scient ifi c and Technological Pres s ,
1999 , 5:4 479 - 4 480.
[ 2] 　Nimz H. Is olat ion of guaiacylg lycerol and it s dimericβ-aryl eth er from spru ce lignin [ J] . C hem Ber-Recl , 1967 ,
100(1):181 - 186.
[ 3] 　Miki K , Sasaya T , Sakakibara A. Lignans f rom heartw ood of La rix lepto lepis gord [ J] . M okuzai Gakk aishi ,
1980 , 26(9):633 - 636.
[ 4] 　Greca M D , Ferrara M , Fiorentino A , et al. Ant ialgal comp ounds f rom Zan ted eschia aeth iop ica [ J] . Phyto-
chemist ry , 1998 , 49(5):1 299 - 1 304.
[ 5] 　Suzuki S , Umezaw a T , Shimada M . Stereochemical diversity in lignan biosynthesis of Arctium la ppa L[ J] . Bio-
s ci Biotech Bioch , 2002 , 66(6):1 262 - 1 269.
[ 6] 　Matsuda N , Kikuchi M . Studies on the con sti tu ents of Lonicera S pecies. X . Neolign an g lycodides f rom the leav-
es of Lonicera g raci l ip es var. g landulosa M axim [ J] . Chem Pharm Bul l , 1996 , 44(9):1 676 - 1 679.
82 波　　谱　　学　　杂　　志　　　　　　　　　第 24卷　
[ 7] 　Shen X H , Van Heiningen A. Synthesis of β-O-4 lignin model dimmers and thei r chlorinated derivat ives [ J] . Can
J C hem , 1992 , 70:1 754- 1 761.
[ 8] 　Yuan Z(原忠), Zh ou BY(周碧野), Li X(李铣). NM R study of cit rusin A (橙皮素 A的 NM R研究)[ J] . Chi-
nese J Magn Reson(波谱学杂志), 2002 , 19(3):309- 313.
[ 9] 　Yuan Z(原忠), Li X(李铣). NM R meth ods for determining the configu rat ion of 8-O-4′neolignan s(8-O-4′型异
木脂素立体构型的测定方法)[ J] . Chinese J Magn Reson(波谱学杂志), 2003 , 20(3):307 - 314.
[ 10] 　Is hikawa T , Fujim atu E , Kitajima J . Water-solub le const ituents of anise:new glucosides of anethole glycol and
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[ 11] 　Shi H M , Wen J , Jia C Q , et a l . T wo new phenolic g lycosides f rom the barks of h ydnocarp usannamen sis and
their an ti-inf lammatory and ant i-oxidat ion activi ties [ J] . Plan ta M ed , 2006 , 72(10):948- 950.
广西大风子中 2个新木脂素的 NMR研究
史海明1 , 2 , 刘　一3 , 姜　勇1 , 2 , 孙　哲3 , 屠鹏飞1 , 2* , 凌笑梅3
(1.北京大学 药学院天然药物学系 , 北京 100083;2.北京大学中医药现代研究中心 , 北京 100083;
3.北京大学药学院药物分析教研室 , 北京 100083)
摘　要:从广西大风子的皮中分离得到两个新木脂素 1和 2 , 并通过高分辨质谱和核磁
共振确定了其结构. 利用多种二维核磁共振技术 , 包括 HSQC , HMBC , DQF-COSY 和
NOESY谱 , 对其碳氢信号进行了全归属. 毛细管电泳分析显示 1和 2均为外消旋体.
关键词:核磁共振;全归属;二维核磁共振;新木脂素;广西大风子;毛细管电泳
　*通讯联系人:屠鹏飞 , 电话:010-82802750 , E-mail:pengfeitu@bjmu . edu. cn.
83
　第 1期　SHI Hai-ming et al:Tw o Neo lignans Isolated f rom H ydnocar pus Annamensis
Studied by NMR Spec troscopy