Megastigmane Glycosides from Polygala hongkongensis Hemsl

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CHEM. RES. CHINESE U. 2007, 23 ( 5 ) , 530-532 Article ID 1005-9040( 2007) -05-53043

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Megastigmane Glycosides from Polygala hongkongensis Hemsl WU Jian-feng'.2.3s4,CHEN S i - b a ~,~GAO * ~ Jing-chuns , WU Li-jun' , CHEN S h i - l i t ~* ~and . ~ TU Peng-fei"2* 1. School of Traditional Chinese Materia Medica , Shenyang Pharmaceutical University, Shenyang 110016, P. R. China; 2. School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100083 , P. R. China ; 3. State Key Laboratory of Chinese Medicine and Molecular Pharmacology, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Horn, Shewhen 518057, P. R. China; 4. College of Medicine, Foshan University, Foshan 528000, P. R. China ; 5. Institute of Medicinal Plant Development, Chinese Academy of Medical Science, Peking Union Medical College, Beijing 100094, P. R. China Received Feb. 5 , 2007 Two new megastigmane glycosides, 72-roseoside, and 72-trifostigmanoside I , as well as, four known compounds, trifostigmanoside fl , epimedin C , 3', 7-dihydroxy4'-methoxyisoflavone-7-D-glucopyranoside,and formenonetin, were isolated from Polygnln hongkongensis Henisl. The structures of the isolated compounds were established on the basis of U V , IR, NMR, and MS spectral data. Megastigmane glycosides( 1-3) were isolated from the family Polygalaceae for the first time. Keywords Polygala hongkongensis ; Polygalaceae ; Megastigmane glycosides ; 7Z-Roseoside ; 7Z-Trifostigmanoside I

Introduction In recent times, there has been an increasing interest in genus Polygala with a broad spectrum of biological activities and it is possible to be used as a novel source for unique natural products. Several chemical constituents have been isolated from Polygala plants that mainly contained s a p ~ n i n s [ ~,- ~anthones'~"' ~~ , and oligosaccharide esters[741. However, there has been no chemical report on P. hongkongensis yet. This plant is a widely spread herb that grows in the south of China used in Chinese folk medicine"" for heat-clearing, detoxication , promotion of blood flow, and expelling phlegm to arrest coughs. In the search for biologically active materials, it was found that the n-butanol extract of the herb P. hongkongensis showed antioxidant activity. Subsequent chromatographic separation and purification led to the isolation of two new megastigmane glycosides, ( 6S, 7 2 , 9R ) -6, 9-dihydroxyme-gastigma4, 7-dien-3 -one-9-0-P-D-glucopyrano-side ( 1 ) and ( 6 S , 7 2 , 9R ) -6, 9-dihydroxymegastigrna-4, 7-dien-3-one-9-0-~-D-apiofuranosyl-( 1 -+ 2 ) -P-D-glucopy-ranoside( 2 ) , which were named as 7Z-roseoside and 7Z-trifostigmanoside I ( Fig. 1 ) , respectively. In addition, four known compounds, trifostigmanoside I I ( 3 ) [ " ] , epimedin C ( 4 ) [ I 2 ] , 3 ' , 7 -

*

dihydroxy4 '-methoxyisoflavone-7 -D-glucopyranoside ( 5 ) ' I 3 ] , and formenonetin(6) 'I4] were also obtained. Megastigmane glycosides( 1-3 ) were isolated from the family Polygalaceae for the first time. This study deals with the isolation and the characterization of the two new megastigmane glycosides.

1

2

Fig. 1 Structures of compounds 1 and 2

Results and Discussion The molecular formula of compound 1 is C,,H,,O, as established by HR-ESI-MS ( [ M + Na] + at m/z 409. 1833; calcd. for 409. 1 8 4 7 ) . Its IR spectrum shows the presence of hydroxyl groups (3401 c m - ' ) and a hydrogen-bopded ketone ( 1654 cm-' ). The I3C and ' H NMR spectra of compound 1 showed the presence of a P-D-glucopyranosyl moiety from the sign a l s a t 6 , 102.7 a n d 6 , 4 . 2 5 ( 1 H , d , J = 7 . 6 Hz). Compared with the known compounds, aglycone shows great similarity to megastigmane"" . In the known compounds, the coupling constant of the 7,8-olefinic protons was 15.7 Hz, which was a result of trans confor-

To whom correspondence should be addressed. E-mail: pengfeitu@ bjmu. edu. c n ; bcslchen@ inet. polyu. edu. nk

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WU Jian-feng et al.

mation; however, in compound 1, the coupling constant of the 7,8-olefinic protons was 10. 0 Hz, which indicates a cis conformation of the double bond. Furthermore, the conformation was confirmed by the Stereochemical correlation of NOESY experiments, in which cross peaks were found between an olefinic proton signal of H7 ( 6 5 . 7 8 ) , two tertiary methyls of 1 I -CH, ( 6 0. 9 4 ) , 12-CH3( 6 0. 93 ) , and olefinic proton signal of H8 ( 6 5. 77 ) ( Fig. 2 ) . The cyclohexenone skeleton was deduced to be in half-chair conformation from the above-observed correlation , which also coincided with the known compounds. On the other hand, in the NOESY spectrum of compound 3 , cross peaks were found between an olefinic proton signal of H7 ( 6 6. 6 6 ) and two proton 2-H, ( 6 1 . 9 4 ) and 9-H ( 6 4 . 2 0 ) ; an olefinic proton signal of H8 ( 6 6. 6 4 ) had a correlation with the 6 H ( 6 2 . 5 9 ) signals( Fig. 3 ) . All the abovementioned evidence indicates that the 7 ,8-olefinic protons of compound 1 were in cis conformation. The new compound 1 was identified as ( 6S, 7 Z , 9 R ) -6,9-dihydroxyme gastigma-4 ,7-dien-3 -one-9-0-/3-D-glucopyra-noside and named as 72-roseoside.

531

at m/z 409[ M - Api] + , indicating that the apiose was the terminal sugar. Comparing the l 3 C and ' H NMR spectral data of compound 2 ( Table 1 ) with those of compound 1 reveals that compound 2 was an apiofuranosyl derivative of 7Z-roseoside ( compound 1 ) as indicated from the signals at 6 , 110.8 ( Cl") together with 6, 5 . 2 7 ( l H , d , J = 3 . 2 Hz, Hl") of the p-Dapiofuranosyl moiety and the signals at 6, 101. 3 ( C 1 ' ) with 6,4. 3 1 ( 1 H , d , . I = 7 . 6 , Hl") of the p-D-glucopyranosyl unit. Their attachment together by a ( 1-2) inter glycosidic linkage was deduced from the downfield shift of C2" of glucose to 6, 79. 3 and upfield shift of C1" to 6, 101. 3 when compared with the corresponding shifts of compound 1 at S,75. 3 and 102.7, respectively. Thus, the new compound 2 was identified as ( 6 S , 7 Z , 9 R ) -6,9-dihydroxyme gastigma4,7-dien-3-one-90-p-D-apiofuranosyl- ( 1 +2 ) -P-D-glucopyranoside and named as 72-trifostigmanoside I . Table 1 ' H NMR(400 MHz) , I3C NMR( 100 MHz) data of compounds 1 and 2 * Compound 1

Posiiion 614

1

2

2.42a(lH,d,J= 16.8 Hz), 2.04b (lH, d , J=16.8 Hz)

3 4

5.76(18, s )

5 6 7

Fig. 2

Stereochemical correlation of compound 1 obtained from NOE experiment n

'I

W l2

Me 11

Stereochemical correlation of compound 3 obtained from NOE experiment The molecular formula of compound 2 is C,,H,,O,,

Fig. 3

as established by HR-ESI-MS [ M + N a ]

at m/z 541.2256 ( calcd. for 541. 2269). Acid hydrolysis of +

compound 2 with 1 moVL HCl afforded D-glucose and D-apiose identified by direct comparison with authentic samples. The FAB-mass spectral data of compound 2 support the presence of an extra apiosyl moiety as compared with those of compound 1 by showing a quasi-molecular ion at m/z 541 [ M t Na] + and a fragment peak

5.78(1H,d,l= 10.0 Hz)

8

Sc 42. 5

Compound 2 St1

50.7 2 . 4 3 a ( l H , d , J = 16.8 Hz), 2.06b

SC 42.5

50.7

(lH, d,J=16.8Hz) 201.3 201.4 127.2 5.75(1H, s ) 127.2 167.3 167.4 80. 1 80.0 131.6 5 . 7 7 ( 1 H , d , J = 131.5 9.2 Hz)

5.77(1H, dd, J = 10.0, 1.6 Hz) 9 4.32(18, dd, J = 6 . 4 , 1.6 Ha) IO-CH3 1.19(3H, d , / = 6.4 Hz)

135.3 5.76(1H, dd, J = 9 . 2 , 2.0 Hz)

135.2

77~.4 4.31(1H, dd, J = 6 . 4 , 2.0 Hz) 21.3 1. 18(3H, d , J = 6 . 4 Hz)

76.9

11-CH3 12-CH3 13-CH3 Glc-1

23.5 24.8 19.7 102.7

0.94(3H, s ) o . 9 3 ( 3 ~ ,s ) 1. 83(3H, s ) 4.25(IH, d , J = 7 . 6 Hz)

Glc-2 Glc-3 Glc4 Clc-5 Glc-6

75. 3 78.0 71.6 78. 1 62. 8

Api-1

23.4 24. 8 19.6 101.3 79. 3 11.9 71. 7 78.0 62. 8

5.27(1H, d , J = 3.2 Hz)

Api-2 Api-3 Api4

110.8 78. 6 80.7 66. 1 75.4

Ari -5

*

0.94138, s) 0.91(3H, s ) 1.82(3H, s ) 4.31(1H, d , J = 7.6 Hz)

21.2

Measured in MeOD-d,

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CHEM. RES. CHINESE U.

Experimental 1 Apparatuses Optical rotations were performed on a Perkin-Elmer digital polarimeter. Melting points were measured on a Fisher-Johns apparatus and were uncorrected. IR spectra were recorded on a Shimadzu FTIR 8400 infrared spectrometer with KBr disc technique. UV spectra were measured on a Perkin-Elmer Lambda 35 UV/Vis spectrometer. NMR spectra were measured on a Bruker AV 400 spectrometer with TMS as the internal standard. ESI-MS data were obtained with a Micromass ZabSpec high-resolution mass spectrometer. Silica gel 60H ( 400-500 mesh ) and silica gel GF,,, sheets ( 0 . 2 0 4 . 2 5 mm ) ( both from Qingdao Haiyang Chemical Group Co. , Shandong Province, People's Republic of China) were used for column chromatography and TLC, respectively. DlOl resin was obtained from Tianjin Chemical Company. 2 Plant Material Polygulu hongkongensis Hemsl. ( Polygalaceae ) was collected at Guangzhou ( China) , in May 2005, and identified by Prof. TU Peng-fei, from the School of Pharmaceutical Sciences, Peking University ( China). A voucher specimen( PH200504) was deposited in the Herbarium of Peking University Modem Research Center for TCM. 3 Extraction and Isolation The air-dried herb P. hongkongensis ( 10.0 kg) was pulverized and extracted with 70% MeOH under reflux ( 1. 5 h x 3 ) . The extracts were combined and evaporated in vacuum to yield 4. 1 kg of residue, 2 kg of which was suspended in water and successively partitioned with EtOAc and n-BuOH. Part of the n-BuOH extract( 297.5 g) was subjected to a macroporous resin DlOl column(ll.Ocmx80.Ocm). Theadsorbed material was eluted successively with H,O, then 30% , 60%, and 95% EtOH, respectively. The 30% EtOH eluate( 76.5 g) was subjected to silica gel chromatography using a gradient system of CHC1,-MeOH( 19: 1 , 9: 1 , 6: 1 , 4: 1 , 3: 1 , 1: 1 , 0: 1 , volume ratio) , yielding 10 pooled fractions: P1-P10. P2( 3. 6 g) was subjected to silica gel column, eluted with CHC1,-MeOH( 85: Com15; volume ratio) to produce fractions 1-16. pound 6 ( 13.2 mg) was obtained from fractions 7-1 1 by Sephadex LH-20 after repeated silica gel chromatography. P3 ( 4. 8 g ) was re-crystallized from MeOH thrice to give compound 5 ( 20.0 mg) . P5( 4 . 5 g) was

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purified by Sephadex LH-20 column to give compound 4 ( 77 mg) . P8 ( 16. 2 g) was initially subjected to reverse phase C,, column, then purified by semi-preparative HPLC with MeOH-H,O ( 4 : 6, volume ratio) as mobile phase to give compounds 1 (68. 1 mg) , 2 ( 88 mg) , and 3 (44.8 mg) . 7Z-roseoside ( compound 1) : an amorphous powder, m. p. : 132-133 "I:, [ a]: = + 77.2' ( c 0. 1 , MeOH) ; IR ( KBr) , it,,,,/crn-l : 3402, 1654, 1074, 1036; UV ( MeOH) : A, ( lge) = 235 ( 4 . 2 4 ) nm; HR-ESI-MS [ M + Na ] + , m/z: 409. 1833 (calcd. 409. 1847 for C,, H,, 0, ) . ESI-MS, m / z : 387 [ M + l ] + , 225[M+l-glc]'. ' H a n d "C NMR(Tab1e 1 ) . 7Z-trifostigmanoside I ( compound 2 ) : an amorphous powder, m. p. : 167-168 9: , [ a ] = + 35.6' ( c 0. 1 , MeOH) ; IR( KBr) , Y,,,,,/cm-I : 3414, 1653, 1071; U V ( M e 0 H ) : A,(Ig&) = 2 3 6 ( 4 . 2 1 ) nm; HR-ESI-MS [ M + Na ] + , m/z: 541.2256 ( calcd. 541.2269 for C,H380,2). ESI-MS, m / z : 519[ M + l ] ' , 3 8 7 [ M + l - A p i ] + , 2 2 5 [ M + l -Api-Glc]. H and I3C NMR( Table 1 ) .

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