Substituent Chemical Shift of Rhamnosides from the Stems of Cinnamomum osmophleum

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1 The Chi nese Phar ma ceu ti cal Jour nal, 2004, 56, Substituent Chemical Shift of Rhamnosides from the Stems of Cinnamomum osmophleum Chung-Yi Chen a ( ), Shu-Ling Hsieh b ( ), Ming-Mu Hsieh c ( ), Sung-Fei Hsieh a ( ) and Tian-Jye Hsieh a * ( ) a Ba sic Med i cal Sci ence Ed u ca tion Cen ter, Fooyin Uni ver sity, Kaohsiung County 831, Tai wan, R.O.C. b De part ment of Nu tri tion and Health Sci ence, Fooyin Uni ver sity, Kaohsiung County 831, Tai wan, R.O.C. c De part ment of Ap plied Chem is try, Fooyin Uni ver sity, Kaohsiung Hsien 831, Tai wan, R.O.C. (Re ceived March 1, 2005; Ac cepted May 4, 2005) ABSTRACT The chem i cal con stit u ents in the stems of Cinnamomum osmophloeum were sep a rated by col - umn chro ma tog ra phy. Six flavonoids, in clud ing kaempferol (1), kaempferol 3-O-α-L-rhamno - pyrano side (2), kaempferol 7-O-α-L-rhamnopyranoside (3), kaempferol 3-O-α-L-rhamno pyrano - side- 7-O-α-L-rhamnopyranoside (4), kaempferol 3-O-α-L-rhamnopyranosyl-(1 2)-α-L-rhamno - pyranoside (5), and dihydrokaempferol as well as (+)-yangambin, (+)-sesamin, fumaric acid, p-hydroxybenzadehyde, p-hydroxybenzoic acid, cinnamic acid, coumarin, p-dihydrocoumaric acid, and trans-cinnamaldehyde, were iso lated from this plant. The com plete elu ci da tion of the 1 H NMR spin sys tems and the chart ing of substituent chem i cal shifts pro vides great po ten tial for the struc ture elu ci da tion of glycosides. Key words: Cinnamomum osmophleum; Flavonoids; Kaempferol; Rhamnosides. INTRODUCTION Cinnamomum osmophloeum is an en demic tree that grows in Tai wan s nat u ral hard wood for - ests at el e va tions be tween 400 and 1500 m. Hu and co-workers an a lyzed the com po si tion of the es sen - tial oil of C. osmophloeum leaves col lected from 21 prov e nances in cen tral, south ern, and east ern Tai - wan. 1 On the ba sis of the chem i cal com po si tion of dif fer ent leaf es sen tial oils, C. osmophloeum was clas si fied into nine types: cassia type, cinnamal - dehyde type, coumarin type, linalool type, eugenol type, cam phor type, 4-terpinenol type, linalool - terpinenol type, and mixed type. 2 Cin na mon oil is com monly used in the food in dus try be cause of its spe cial aroma. In ad di tion, its antimicrobial and antifungal prop er ties have also drawn great at ten - tion from many re search ers. 3-6 Chang and coworkers 7 found that the leaf es sen tial oils of C. osmophloeum have an ex cel lent in hib i tory ef fect against bac te ria. Re cently, we re in ves ti gated the con stit u ents in the stems of C. osmophloeum. The MeOH ex tract of its stems was sub jected to sol vent par ti tion ing and chro mato graphic sep a ra tion to af - ford fif teen pure sub stances. In ad di tion to transcinnamaldehyde, kaempferol 7-O-α-L- rhamno py -

2 142 Chin. Pharm. J. Vol. 56, No. 3, 2004 Chen et al. rano side, kaempferol 3-O-α-L-rhamno pyrano side - 7-O-α-L-rhamnopyranoside, and fumaric acid, oth - ers were found for the first time from this plant. 8,9 The pres ent pa per deals with the iso la tion and char - ac ter iza tion of the glycosides. RESULTS AND DISCUSSION The CHCl 3 - and n-buoh-soluble layer of the methanolic ex tract of the stems of C. osmophloeum was sub ject to SiO 2 chro ma tog ra phy to yield fif teen com pounds, in clud ing six flavonoids, kaempferol (1), kaempferol 3-O-α-L-rhamnopyranoside (2), kaempferol 7-O-α-L-rhamnopyranoside (3), kaemp - ferol 3-O-α-L-rhamnopyranoside-7-O-α-L-rhamno - pyranoside (4), kaempferol 3-O-α-L- rhamno - pyrano syl-(1 2)-α-L-rhamnopyranoside (5), and dihydrokaempferol as well as (+)- yangambin, (+)- sesamin, fumaric acid, p-hydroxybenzadehyde, p- hydroxybenzoic acid, cinnamic acid, coumarin, p-dihydrocoumaric acid, and trans- cinnamal de - hyde. The ca pac ity of de tailed 1 H NMR anal y sis to help in struc ture de ter mi na tion can best be dem on - strated by look ing at closely re lated com pounds. There fore, bisdesmosidic kaempferol glycosides con tain ing α-l-rhamnose units, which were re - cently iso lated from C. osmophloeum, were cho sen as ex am ples. Fully as signed sets of pro ton and car - R'O OH 10 O O 2 3 OR OH 4' R R' 1 H H 2 Rha H 3 H Rha 4 Rha Rha 5 Rha 2 1 Rha H 1' Table C NMR chemical shift data of the flavonoids 1-5 (in CD 3 OD) Position bon res o nances were thus ob tained and the 13 C NMR re sults are sum ma rized in Ta ble 1. In prin - ci ple, the rel a tive stereochemistry of the hexo - pyranoses can be de duced from the cou pling pat tern of nu clei us ing first-order as sump tions as fol lows: H-1 (d, J = 1.9 Hz), H-2 (dd, J = 1.9, 3.5 Hz), H-3 (dd, J = 3.5, 9.5 Hz), H-4 (t, J = 9.5 Hz), H-5 (dq, J = 5.9, 9.5 Hz), and 3H-6 (d, J = 5.9 Hz) for rhamnose. 10 How ever, as will be shown be low, this straight for ward la bel ing be comes im per fect in many cases due to the pres ence of higher or der sit u - a tions caused by sig nif i cant substituent chem i cal shifts. It must also be em pha sized that the choice of sol vent has sub stan tial im pact on the com plex ity of

3 Rhamnosides from Cinnamomum osmophleum Chin. Pharm. J. Vol. 56, No. 3, the 1 H NMR spec trum. While we have ob served the most fa vor able sig nal dis per sion with CD 3 OD so lu - tions of glycosides and other po lar, nonglycosidic nat u ral prod ucts, most phe no lic glycosides and es - pe cially flavonoids are re ported in DMSO-d 6 so lu - tion. On the ba sis of our ex pe ri ence it is pref er a ble to use CD 3 OD as the main sol vent. While sig nal dis - per sion and, there fore, spec tral in for ma tion are fa - vor able in meth a nol so lu tion, the main dis ad van - tage of los ing the hydroxyl pro ton res o nances can still be over come by us ing CD 3 OD. De pending on the site of link age, the sug ars are sub jected to dif fer ent in flu ences by the fla - vonoid nu cleus in terms of ar o matic substi tuentinduced shifts aris ing from the ring cur rent ef fect. There fore, the res o nances of rhamnose show re - mark able dif fer ences con cern ing chem i cal shift, sig nal mul ti plic ity and cou pling be hav ior. The rhamnose moi ety dis plays re mark ably dif fer ent sig - nals when com par ing the 3-O (2) and 7-O (3) link - ages. The prom i nent up field shift of all of the ter mi - nal rhamnose pro tons trans lates into a con for ma - tion where the ter mi nal sugar elec tron i cally in ter - acts with the shield ing cone of the B-ring. Be cause the ar o matic and the sugar res i due elec tron i cally in ter act with each other, the ef fects can be ob served on both sides. While the oc cur - rence of higher or der spin sys tems in the sugar por - tion is caused by the ar o matic moi ety, the lat ter is in flu enced by the chiral na ture of the sugar res i - dues, which also leads to higher or der sit u a tions. 10 An ex am ple of asym met ric in duc tion caused by chiral sugar res i dues is the diastereotopism of the p-oh ar o matic B-ring pro tons of the flavonoid nu - cleus of kaempferol and an a logues. Even in the very re cent lit er a ture, the AA XX 1 H sig nals cen - ter ing around 8.0 and 6.9 ppm are er ro ne ously re - ported as straight dou blets with one J value given. A de tailed eval u a tion is nec es sary to es tab lish the cou plings, the shift val ues, and the sig nal mul ti plic - ity. 10 The flavonols 1~5 were char ac ter ized from the ap pear ance of an AA XX pat tern for H-2, H-3, H-5 and H-6, and an AX pat tern for H-6 and H-8 in the 1 H NMR spec tra. The flavonols quercetin, quercetin 3-O-α-L-rhamnopyranoside, and quercetin 3-O-α-L-rhamnopyranosyl- (1 2)-α-L- rhamno - pyranoside, were char ac ter ized from the ap pear - ance of an ABX pat tern for H-2, H-5, and H-6, and an AX pat tern for H-6 and H-8 in the 1 H NMR spec tra. The lo ca tion of sugar sub stitu ents at C-3 was in di cated by the glycosidation shift to C-2. For in stance, the sig nal of C-2 shifted from δ in 1 and quercetin to δ in 2 and quercetin 3-O-α - L- rhamnopyranoside due to rhamnosidation at 3-OH. Al though the diglycoside 5 was re ported from Cassia hir sute, NMR anal y sis of the par ent com - pound is lack ing. 11 Most sugar pro ton sig nals over - lap be tween 4.00 and 3.30 ppm. They were clar i fied and as signed in the COSY, NOESY, and HMBC spec tra. With sig nals of two anomeric pro tons as mark ers (δ 5.49 and 5.03) in the COSY spectrum, the sig nals at δ 4.32 and 3.98 were iden ti fied to be H-2. From the COSY spec trum, the pro ton sig nals of each sugar unit were elu ci dated. The split of the sig nal of the anomeric pro ton of the rhamnose moi - ety (δ 5.49 and 5.03) showed the re layed pro ton sig - nals at δ 4.32 and 3.98 (dd, H-2 and H-2 ), 3.86 and 3.61 (dd, H-3 and H-3 ), 3.36 and 3.33 (t, H-4 and H-4 ), 3.40 and 3.63 (m, H-5 and H-5 ) and 0.94 and 1.23 (d, H-6 and H-6 ). These as - sign ments were con firmed with NOESY data. The H-2 res o nance of one sugar was un usu ally down - field (δ 4.32), be cause of the 1 2 interglycosidic link age of the sugar moi ety. Anal y sis of the 13 C NMR spec trum con firmed the iden tity of the aglycon as 1 and the two sugar units as α-l-rhamnose. Due to typ i cal shifts ex pe ri enced by the in ner rhamnose (sig nals were dis placed downfield by 6.9 ppm for

4 144 Chin. Pharm. J. Vol. 56, No. 3, 2004 Chen et al. C-2 and up field by 1.2 and 0.1 ppm for C-1 and C-3, re spec tively, from those of 2), it was pos si ble to con firm the 1 2 interglycosidic link age. The val ues are also in good agree ment with those from anal o gous com pounds. Place ment of the disac - charidic moi ety at C-3 was de ter mined on the ba sis of the typ i cal glycosylation shifts that oc curred with re spect to the aglycon 1: downfield shifts of C-2 and C-4. Finally, acid hy dro ly sis of 5 lib er ated rhamnose and 1, which was iden ti fied by TLC with an au then tic sam ple. Ac cord ing to the 1 H NMR data, the com plete 13 C NMR as sign ment of 5 was made with a HMBC com bined with NOESY ex per i ment. The HMBC spec trum dis tin guished the sig nals of C-2 (δ 159.0) and C-9 (δ 158.2) by re veal ing the long range cou - pling to H-2 & 6 (δ 7.72) (C-2) and H-8 (δ 6.31) (C-9). EXPERIMENT SEC TION General Methods UV spec tra were ob tained on a Hitachi spectrophotometer in MeOH. IR spec tra were mea - sured on a Hitachi spectrophotometer. 1 H NMR (400 and 200 MHz), 13 C, NOESY, and COSY spec tra were ob tained on a Varian NMR spec trom e - ter. Low-resolution EIMS were re corded on a JEOL JMS-SX/SX 102A mass spec trom e ter or Quattro GC-MS spec trom e ter hav ing a di rect in let sys tem. High-resolution EIMS were mea sured on a JEOL JMS-HX 110 mass spec trom e ter. Sil ica gel 60 (Merck, mesh) was used for col umn chro - ma tog ra phy, precoated sil ica gel plates (Merck, Kieselgel 60 F-254, 0.20 mm) were used for an a lyt - i cal TLC and precoated sil ica gel plates (Merck, Kieselgel 60 F-254, 0.50 mm) were used for pre - para tive TLC. Spots were de tected by spray ing with 50% H 2 SO 4 and then heat ing on a hot plate. Plant Material The spec i men of C. osmophloeum was col - lected in Pingtung county, Tai wan in March A voucher spec i men was iden ti fied by Dr. Pei-Fang Lee (Grad u ate In sti tute of Bio tech nol ogy, Fooyin Uni ver sity, Kaohsiung County) and was de pos ited in the Ba sic Med i cal Sci ence Ed u ca tion Cen ter, Fooyin Uni ver sity, Kaohsiung County, Tai wan. Ex trac tion and Sep a ra tion The stems (7.0 kg) of C. osmophloeum were ex tracted re peat edly with MeOH (10 L 6) at room tem per a ture for hrs. The com bined MeOH ex tracts were evap o rated and par ti tioned to yield CHCl 3 and aque ous ex tracts. The aque ous ex tract was evap o rated and ex tracted with n-buoh to give n-buoh and aque ous lay ers. The CHCl 3 ex tract was dried and evap o rated to leave a vis cous res i due (112.4 g). The res i due was placed on a sil ica gel col - umn and eluted with n-hex ane grad u ally en riched with EtOAc to af ford ten frac tions. Each frac tion was rechromatographed over sil ica gel with a gra di - ent of n-hex ane/etoac to ob tain (+)-yangambin (1.4 mg), (+)-sesamin (21 mg), fumaric acid (31 mg), p-hydroxybenzadehyde (24 mg), p-hydroxy - benzoic acid (23 mg), cinnamic acid (1.2 g), coumarin (2.1 g), p-dihydrocoumaric acid (11 mg) and trans -cinnamaldehyde (45 mg), re spec tively. The n-buoh layer (123.1 g) was con cen trated and chromatographed over a sil ica gel col umn (50 10 cm) us ing MeOH/EtOAc as eluent to pro duce ten frac tions. Each frac tion was rechromatographed over sil ica gel with a gra di ent of EtOAc/MeOH to ob tain kaempferol (1) (67 mg), kaempferol 3-Oα-L-rhamnopyranoside (2) (23 mg), kaempferol 7-O-α-L-rhamnopyranoside (3) (45 mg), kaemp - ferol 3-O-α-L-rhamnopyranoside-7-O-α-L- rhamno - pyranoside (4) (27 mg), kaempferol 3-O-α-Lrhamnopyranosyl-(1 2)-α-L-rhamnopyranoside

5 Rhamnosides from Cinnamomum osmophleum Chin. Pharm. J. Vol. 56, No. 3, (5) (30 mg) and dihydrokaempferol (31 mg), re - spectively. Kaempferol (1) Yel low nee dles (MeOH); mp C; UV λ max nm (MeOH): 220, 265, 365; IR (KBr) ν max cm -1 : 3500 (OH), 1680, 1625, 1560; EIMS m/z (rel. int.): 286 ([M] +, 90), 258 (15), 230 (10), 153 (20), 121 (65); 1 H NMR (CD 3 OD): δ 6.25 (1H, d, J = 2.0 Hz, H-6), 6.56 (1H, d, J = 2.0 Hz, H-8), 6.73 (2H, d, J = 8.8 Hz, H-3 and H-5 ), 7.37 (2H, d, J = 8.8 Hz, H-2 and H-6 ); 13 C NMR (CD 3 OD): see Ta ble 1. Kaempferol 3-O-α-L-rhamnopyranoside (2) Yel low nee dles (MeOH); mp C; 24 D (c 0.65, MeOH); UV λ max nm (MeOH): 255, 290, 375; IR (KBr) ν max cm -1 : 3400 (OH), 1670, 1600; EIMS m/z (rel. int.): 286 ([M- rhamnosyl] +, 100), 285 (50), 258 (23), 229 (40); 1 H NMR (CD 3 OD): δ 0.92 (3H, d, J = 5.6 Hz, H-6 ), 3.32 (1H, m, H-5 ), 3.35 (1H, t, J = 9.2 Hz, H-4 ), 3.70 (1H, dd, J = 9.2, 3.6 Hz, H-3 ), 4.22 (1H, dd, J = 3.6, 1.6 Hz, H-2 ), 5.37 (1H, d, J = 1.6 Hz, H-1 ), 6.19 (1H, d, J = 2.0 Hz, H-6), 6.35 (1H, d, J = 2.0 Hz, H-8), 6.93 (2H, d, J = 8.8 Hz, H-3 and H-5 ), 7.76 (2H, d, J = 8.8 Hz, H-2 and H-6 ); 13 C NMR (CD 3 OD): see Ta ble 1. Kaempferol 7-O-α-L-rhamnopyranoside (3) Yel low nee dles (MeOH); mp C; 24 D (c 0.63, MeOH); UV λ max nm (MeOH): 255, 270, 370; IR (KBr) ν max cm -1 : 3400 (OH), 1650, 1600; EIMS m/z (rel. int.): 286 ([M- rhamnosyl] +, 100), 285 (45), 258 (32), 229 (35); 1 H NMR (CD 3 OD): δ 1.26 (3H, d, J = 5.6 Hz, H-6 ), 3.47 (1H, t, J = 9.0 Hz, H-4 ), 3.58 (1H, m, H-5 ), 3.82 (1H, dd, J = 9.0, 3.5 Hz, H-3 ), 4.03 (1H, dd, J = 3.5, 1.6 Hz, H-2 ), 5.58 (1H, d, J = 1.6 Hz, H-1 ), 6.45 (1H, d, J = 2.0 Hz, H-6), 6.75 (1H, d, J = 2.0 Hz, H-8), 6.75 (2H, d, J = 8.8 Hz, H-3 and H-5 ), 7.52 (2H, d, J = 8.8 Hz, H-2 and H-6 ); 13 C NMR (CD 3 OD): see Ta ble 1. Kaempferol 3-O-α-L-rhamnopyranoside-7- O- α-l-rhamnopyranoside (4) Yel low nee dles (MeOH); mp C; 24 D (c 0.65, MeOH); UV λ max nm (MeOH): 240, 260, 345; IR (KBr) ν max cm -1 : 3400 (OH), 1660, 1600; FABMS m/z (rel. int.): 577 ([M-H] +, 20), 431 ([M-rhamnosyl] +, 14), 285 ([M- rhamnosylrhamnose] +, 35); 1 H NMR (CD 3 OD): δ 0.94 (3H, d, J = 6.0 Hz, H-6 ), 1.26 (3H, d, J = 6.0 Hz, H-6 ), 3.33 (1H, m, H-5 ), 3.33 (1H, t, J = 9.2 Hz, H-4 ), 3.48 (1H, t, J = 9.5 Hz, H-4 ), 3.61 (1H, m, H-5 ), 3.70 (1H, dd, J = 9.2, 3.2 Hz, H-3 ), 3.83 (1H, dd, J = 9.5, 3.2 Hz, H-3 ), 4.04 (1H, dd, J = 3.2, 2.0 Hz, H-2 ), 4.22 (1H, dd, J = 3.2, 2.0 Hz, H-2 ), 5.41 (1H, d, J = 2.0 Hz, H-1 ), 5.57 (1H, d, J = 2.0 Hz, H-1 ), 6.42 (1H, d, J = 4.0 Hz, H-6), 6.64 (1H, d, J = 4.0 Hz, H-8), 6.92 (2H, d, J = 10.0 Hz, H-3 and H-5 ), 7.74 (2H, d, J = 10.0 Hz, H-2 and H-6 ); 13 C NMR (CD 3 OD): see Ta ble 1. Kaempferol 3-O-α-L-rhamnopyranosyl- (1 2)- α-l-rhamnopyranoside (5) Yel low nee dles (MeOH); mp C; 24 D (c 0.65, MeOH); UV λ max nm (MeOH): 265, 320, 345; IR (KBr) ν max cm -1 : 3400 (OH), 2900, 1650, 1600, 1450, 1360, 1180, 1130, 1100, 1050, 1020, 970, 940; FABMS m/z (rel. int.): 577 ([M-H] +, 20), 431 ([M-rhamnosyl] +, 14), 285 ([Mrhamnosyl-rhamnose] +, 35); 1 H NMR (CD 3 OD): δ 0.94 (3H, d, J = 5.6 Hz, H-6 ), 1.23 (3H, d, J = 6.0 Hz, H-6 ), 3.33 (1H, t, J = 9.5 Hz, H-4 ), 3.36 (1H, t, J = 9.5 Hz, H-4 ), 3.40 (1H, m, H-5 ), 3.61 (1H, dd, J = 9.5, 3.2 Hz, H-3 ), 3.63 (1H, m, H-5 ), 3.86 (1H, dd, J = 9.5, 3.2 Hz, H-3 ), 3.98 (1H, dd, J = 3.2, 1.6 Hz, H-2 ), 4.32 (1H, dd, J = 3.2, 1.6 Hz, H-2 ), 5.03 (1H, d, J = 1.6 Hz, H-1 ), 5.49 (1H, d, J = 1.6 Hz, H-1 ), 6.15 (1H, d, J = 2.0 Hz, H-6), 6.31 (1H,

6 146 Chin. Pharm. J. Vol. 56, No. 3, 2004 Chen et al. d, J = 2.0 Hz, H-8), 6.91 (2H, d, J = 8.8 Hz, H-3 and H-5 ), 7.72 (2H, d, J = 8.8 Hz, H-2 and H-6 ); 13 C NMR (CD 3 OD): see Ta ble 1. AC KNOWL EDG MENT This in ves ti ga tion was sup ported by a grant from the Na tional Sci ence Coun cil of Re pub lic of China (NSC B ). REFERENCES 1. Hu, T. W.; Lin, Y. T.; Ho, C. K. Nat u ral vari a - tion of chem i cal com po nents of the leaf oil of Cinnamomum osmophloeum Kaneh. Bull. Tai - wan For. Res. Inst. Eng. 1985, 78, Chang, S. T.; Cheng, S. S. Antitermitic ac tiv ity of leaf es sen tial oils and com po nents from Cinnamomum osmophleum. J. Agr. Food Chem. 2002, 50, Singh, H. B.; Srivastava, M.; Singh, A. B.; Srivastava, A. K. Cin na mon bark oils, a po tent fungitoxicant against fungi caus ing re spi ra tory tract mycoses. Al lergy 1995, 50, Azumi, S.; Tanimura, A.; Tanamoto, K. I. A novel in hib i tor of bac te rial endotoxin de rived from cin na mon bark. Biochem. Bioph. Res. Co. 1997, 234, Hili, P.; Ev ans, C. S.; Veness, R. G. Anti - microbial ac tion of es sen tial iol: the ef fect of dimethylsulphoxide on the ac tiv ity of cin na mon oil. Lett. Appl. Microbiol. 1997, 24, Lee, H. S.; Ahn, Y. J. Growth-inhibiting ef fects of Cinnamomum cassia bark-derived ma te ri als on hu man in tes ti nal bac te ria. J. Agr. Food Chem. 1998, 46, Chang, S. T.; Chen, P. F.; Chang, S. C. An ti bac - te rial ac tiv ity of leaf es sen tial oils and their con - stit u ents from Cinnamomum osmophloeum. J. Ethnopharmacol. 2001, 77, Wu, T. S.; Chen, Z. S. The Con stit u ents of Cinnamomum osmophloeum Kanehira. J. Tai - wan Pharmaceut. Assoc. 1977, 29, Hussain, R. A.; Kim, J.; Hu, T. W.; Pezzuto, J. M.; Soejarto, D. D.; Kinghorn, A. D. Iso la tion of a highly sweet con stit u ent from Cinna - momum osmophloeum leaves. Planta Med. 1986, Pauli, G. F. Higher or der and substituent chem i - cal shift ef fects in the pro ton NMR of glycosides. J. Nat. Prod. 2000, 63, Rao, K. V.; Damu, A. G.; Jayaprakasam, B.; Gunasekar, D. Flavonol glycosides from Cassia hir sute. J. Nat. Prod. 1999, 62,