Iridoid and Aromatic Glycosides from Scrophularia ningpoensisHemsl. and Their Inhibition of [Ca2+]i Increase Induced by KCl

Iridoid and Aromatic Glycosides from Scrophularia ningpoensis Hemsl. andTheir Inhibition of [Ca2þ]i Increase Induced by KCl

by Bin Chena),Yan Liub),Hong-Wei Liuc),Nai-Li Wanga)d), Bao-Feng Yangb), andXin-Sheng Yao*a)

a) Department of Natural Products Chemistry 81#, Shenyang Pharmaceutical University,103 Wenhua Avenue, Shenyang 110016, P. R. China

(phone: þ86-24-23993994; fax: þ86-24-23993994; e-mail: [email protected])b) Department of Pharmacology, Harbin Medical University, Harbin 150081, P. R. China

c) School of Chemical Biology and Pharmaceutical Sciences, Capital University of Medical Science,Beijing 100069, P. R. China

d) Key Laboratory for Research & Development of New Drugs from Traditional Chinese Medicine &Natural Products in Shenzhen, Shenzhen 518057, P. R. China

Bioassay-guided fractionation of EtOH extract of the roots of Scrophularia ningpoensis Hemsl.

resulted in the isolation of three new iridoid glycosides, i.e., 6’’-O-caffeoylharpagide (1), 6’’-O-feruloylharpagide (2), and 6’’-O-b-glucopyranosylharpagoside (3), and five new aromatic glycosides, i.e.,2-(3-hydroxy-4-methoxyphenyl)ethyl O-a-arabinopyranosyl-(1!6)-O-a-rhamnopyranosyl-(1!3)-O-b-glucopyranoside (4), phenyl O-b-xylopyranosyl-(1!6)-O-b-glucopyranoside (5), 3-methylphenylO-b-xylopyranosyl-(1!6)-O-b-glucopyranoside (6), 6-O-cinnamoyl b-fructofuranosyl-(2!1)-O-a-glu-copyranosyl-(6!1)-O-a-glucopyranoside (7), and 6-O-feruloyl b-fructofuranosyl-(2!1)-O-a-gluco-pyranosyl-(6!1)-O-a-glucopyranoside (8), together with four known compounds, i.e., 6’’-O-a-d-galactopyranosyl harpagoside (9), 6’’-O-(p-coumaroyl) harpagide (10), harpagoside (11), and angoro-side C (12). Activity of the isolated compounds on [Ca2þ ]i increase induced by KCl was evaluated on ratcardiac myocytes using confocal laser scanning microscopy. Iridoid glycosides 1, 10, and 11, and aromaticglycosides 5 and 6 significantly inhibited the increase of [Ca2þ ]i induced by KCl at 100 mm.

Introduction. – Calcium (Ca2þ ), as an important second messenger and mediator,plays a central role in the regulation of many biological functions, such as genetranscription, cell reproduction, and apoptosis. [Ca2þ ]i (intracellular Ca2þ concen-tration) critically regulates cardiac excitation–contraction (E–C) coupling in thenormal heart. Accumulation of [Ca2þ ]i (Ca2þ overload) is directly responsible forvarious myocardial damages [1–4].

The roots of Scrophularia ningpoensis Hemsl. (Scrophulariaceae) have been usedas a famous traditional herbal medicine named CXuanshenD in China for the treatmentof hypertension, inflammation, fever, dry cough, laryngitis, pharyngitis, and pulmonarytuberculosis [5–9]. A number of Scrophularia species have been chemicallyinvestigated, and various secondary metabolites, mainly iridoid and aromatic glyco-sides, have been reported [10–13]. These classes of substances are considered to beresponsible to a large extent for the therapeutic properties of plants of the genusScrophularia [14].

In our study of bioactive compounds from traditional Chinese herbal medicine, theBuOH-soluble fraction of the 60% EtOH extract of the roots of Scrophularia

CHEMISTRY & BIODIVERSITY – Vol. 5 (2008) 1723

H 2008 Verlag Helvetica Chimica Acta AG, ZKrich

ningpoensis was found to inhibit the [Ca2þ ]i increase induced by KCl in rat cardiacmyocytes. Bioassay-guided isolation afforded twelve iridoid and aromatic glycosides.We describe here the isolation and structure elucidation of three new iridoid and fournew aromatic glycosides. The activity of the isolated compounds has been investigatedusing confocal laser-scanning microscopy. This is the first report on the inhibitoryproperties of secondary metabolites from Scrophularia ningpoensis against KClinduced [Ca2þ ]i increase in cardiac myocytes.

Results and Discussion. – 1. Structure Elucidation. The 60% EtOH extract of theroots of S. ningpoensis was suspended in H2O, and partitioned with AcOEt and BuOHsuccessively. The BuOH-soluble fraction exhibited significant inhibition of [Ca2þ ]iincrease induced by KCl in rat cardiac myocytes (p<0.05, compared to control). It wassubjected to bioassay-guided fractionation with repeated column chromatography onsilica gel and ODS, and reversed-phase HPLC to afford compounds 1–12.

Compound 1 was obtained as pale brown amorphous powder. Its molecularformula, C24H30O13, was determined on the basis of HR-ESI-MS ([MþNa]þ at m/z549.1592). The UV spectrum of 1 showed absorption at 203, 227, and 312 nm. The IRspectrum of 1 showed the characteristic absorption bands for OH groups (3394 cm�1

(br.)), for ana,b-unsaturated ketone (n(C¼O) 1697, n(C¼C) 1607, n(C�O) 1033 cm�1),and for an aromatic moiety (1605, 1520 cm�1). In the 1H-NMR spectrum of 1 (Table 1),the H-atom resonances at d 6.28 (d, J ¼ 15.9, 1 H) and 7.56 (d, J ¼ 15.9, 1 H) indicatedthe presence of a (E)-C¼C bond conjugated with an aromatic ring; a typicalAMX spinsystem for a 1,3,4-trisubstituted aromatic ring was deduced from the H-atom signals at d

7.03 (d, J ¼ 2.0, 1 H), 6.93 (dd, J¼8.2, 2.0, 1 H), and 6.76 (d, J¼8.2, 1 H); an anomericH-atom signal at d 4.59 (d, J¼7.9, 1 H) together with H-atom signals at d 3.25 (t, J ¼8.9, 1 H), 3.39 (t, J¼7.4, 1 H), 3.40 (t, J¼7.4, 1 H), 3.54–3.57 (m, 1 H), 4.48 (dd, J ¼12.0, 2.1, 1 H), and 4.35 (dd, J¼12.0, 5.8, 1 H) indicated the presence of a sugar moiety.In addition, a group of characteristic signals for an iridoid skeleton were observed at d

6.32 (d, J ¼ 6.4, H�C(3)), 5.65 (d, J ¼ 1.2, H�C(1)), 4.93 (dd, J¼6.4, 1.4, H�C(4)),3.67 (t, J ¼ 3.9, H�C(6)), 2.54 (s, H�C(9)), 1.85 (dd, J¼13.7, 4.6, Ha�C(7)), 1.75 (dd,J¼13.8, 3.0, Hb�C(7)), and 1.18 (s, H�C(10)). The 13C-NMR spectrum of 1 (Table 1)in combination with the HSQC spectrum displayed resonances for a (E)-C¼C bond atd 147.2 and 114.8, for an aromatic ring at d 149.6, 146.8, 127.7, 123.1, 116.5, 115.2, and fora sugar moiety at d 99.2, 77.4, 75.7, 74.4, 71.7, and 64.5, for an acyl group at d 169.1, for apair of olefinic C-atoms at d 142.5 and 108.4. In addition, signals of three methine C-atoms at d 93.1, 78.3, and 59.5, of a methylene C-atom at d 46.9, and of two oxygenatedquaternary C-atoms at d 72.8 and 78.4 were observed. The 1H- and 13C-NMR datasuggested that 1 should be an iridoid glycoside with a structure similar to that of theknown iridoid glucoside 8-O-feruloyl harpagide (feruloyl¼3-(4-hydroxy-3-methoxy-phenyl)prop-2-enoyl) previously isolated from this plant [15]. The partial structures ofthe iridoid skeleton, caffeoyl (¼ 3-(3,4-dihydroxyphenyl)prop-2-enoyl) group, andsugar moiety were assigned by detailed analysis of 1H,1H-COSY, HSQC, and HMBCspectra. In the HMBC spectrum, the correlations of H�C(1’’)/C(1) and H�C(6’’)/C(9’)were observed, confirming the positions of the sugar moiety and the caffeoyl group in1 (Fig. 1). Acid hydrolysis, followed by optical rotation measurement and GC analysisof the aldononitrile peracetate derivatives using authentic sample as references,

CHEMISTRY & BIODIVERSITY – Vol. 5 (2008)1724

confirmed the presence of d-glucose. The configuration of the anomeric H-atom wasdeduced to be b from the coupling constant of the anomeric H-atom at d 4.59 (J¼7.9 Hz). On the basis of the above evidence, the structure of 1 was established as 6’’-O-caffeoyl harpagide.


Fig. 1. Significant HMBC (H!C) correlations of 1


Table 1. NMR Data for Iridoid Glucosides 1–3 (at 400 and 100 MHz in CD3OD); d in ppm, J in Hz.

Positiona) 1 2 3

d(H) d(C) d(H) d(C) d(H) d(C)

1 5.65 (d, J ¼ 1.2) 93.1 5.65 (d, J ¼ 1.2) 93.1 6.19 (d, J ¼ 1.2) 94.33 6.32 (d, J ¼ 6.4) 142.5 6.30 ( d, J ¼ 6.4) 142.5 6.39 (d, J ¼ 6.4) 143.84 4.93 (dd, J¼6.4, 1.4) 108.4 4.93 (dd, J¼6.4, 1.5) 108.4 4.93 (dd, J ¼ 6.4, 1.6) 107.05 72.8 72.7 73.66 3.67 (t, J¼3.9) 78.3 3.67 (t, J¼3.9) 78.3 3.74 (t, J¼3.4) 77.77 1.85 (dd, J¼13.7, 4.6), 46.9 1.84 (dd, J¼13.7, 4.6), 46.9 2.24 (d, J ¼ 15.1), 46.3

1.75 (dd, J¼13.8, 3.0) 1.73 (dd, J¼13.7, 3.2) 1.99 (dd, J¼15.1 4.4)8 78.4 78.4 88.59 2.54 (s) 59.5 2.54 (s) 59.5 2.92 (s) 55.410 1.18 (s) 25.0 1.17 (s) 25.0 1.53 (s) 22.91’ 169.1 169.1 168.32’ 6.28 (d, J¼15.9) 114.8 6.38 (d, J¼15.9) 115.2 6.50 (d, J¼16.0) 120.33’ 7.56 (d, J¼15.9) 147.2 7.62 (d, J¼15.9) 147.1 7.70 (d, J¼16.0) 146.04’ 127.7 127.6 136.05’ 7.03 (d, J¼2.0) 115.2 7.16 (d, J¼1.9) 117.7 7.57–7.61 (m) 129.26’ 146.8 149.3 7.36–7.40 (m) 130.07’ 149.6 150.6 7.39–7.41 (m) 131.38’ 6.76 (d, J¼8.2) 116.5 6.79 (d, J¼8.2) 116.5 7.36–7.40 (m) 130.09’ 6.93 (dd, J¼8.2, 2.0) 123.1 7.05 (dd, J¼8.2, 1.9) 124.2 7.57–7.61 (m) 129.210’ 3.87 (s) 56.41’’ 4.59 (d, J¼7.9) 99.2 4.59 (d, J¼7.9) 99.2 4.63 (d, J¼7.9) 99.32’’ 3.25 (t, J¼8.9) 74.4 3.24 (t, J¼9.0) 74.4 3.23 (t, J¼9.1) 74.43’’ 3.39 (t, J¼7.4) 77.4 3.41 (t, J¼6.2) 77.4 3.43–3.46 (m) 77.74’’ 3.40 (t, J¼7.4) 71.7 3.40 (t, J¼6.2) 71.7 3.38 (t, J¼7.4) 71.75’’ 3.54–3.57 (m) 75.7 3.54–3.58 (m) 75.6 3.52–3.56 (m) 77.76’’ 4.48 (dd, J¼12.0, 2.1), 64.5 4.48 (dd, J¼12.0, 2.2), 64.5 4.20 (dd, J¼12.0, 1.8), 69.6

4.35 (dd, J¼12.0, 5.8) 4.36 (dd, J¼12.0, 5.8) 3.89 (dd, J¼12.0, 6.5)1’’’ 4.59 (d, J¼7.9) 104.62’’’ 3.23 (t, J¼9.1) 75.43’’’ 3.40–3.42 (m) 77.74’’’ 3.38 (t, J¼7.4) 71.75’’’ 3.53 (d, J¼9.1) 77.76’’’ 3.90 (dd, J¼12.0, 2.3), 62.8

3.68 (dd, J¼12.0, 5.8)

a) C-Atom numbering as indicated in the Formulae.

Compound 2 was isolated as brown amorphous powder with the molecular formulaC25H32O13 established by HR-ESI-MS. The 1H- and 13C-NMR data (Table 1) of 2 werequite similar to those of 1 except for the presence of a MeO group. The structure of 2was determined by detailed analysis of 1H,1H-COSY, HSQC, and HMBC spectra. Theposition of the MeO group (d 3.87 (s)) was indicated by its HMBC correlation withC(2’) and NOE correlation with H�C(2’). Thus, compound 2 was determined to be 6’’-O-feruloyl harpagide.

Compound 3 was obtained as pale brown amorphous powder and showed UVabsorption at 204, 216, 222, and 279 nm. The HR-ESI-MS spectrum of 3 revealed themolecular formula C30H40O16 ([MþNa]þ at m/z 679.2192). The IR spectrum showedthe characteristic absorption bands corresponding to OH, acyl, and COO groups, andalkenes at 3421, 1701, 1632, 1601, and 1516 cm�1, respectively. Preliminary inspection ofthe 1H- and 13C-NMR spectra of 3 led to the identification of the following structuralfragments [16]: an iridoid skeleton, a cinnamoyl group, a (E)-C¼C bond, an acyl grouptogether with two C6 sugar moieties (1H-NMR: signals of b-anomeric H-atoms at d 4.63(d, J ¼ 7.9, H�C(1’’)), 4.59 (d, J ¼ 7.9, H�C(1’’’)); 13C-NMR: signals of two anomericC-atoms at 99.3 (C(1’’)) and 104.6 (C(1’’’))). Acid hydrolysis, followed by GC analysisof the aldononitrile peracetate derivatives, indicated the presence of glucose. b-Configurations of the anomeric H-atoms were inferred from the values of the couplingconstants of H�C(1’) and H�C(1’’) for two glucopyranosyl units (7.9 and 7.9 Hz). Thepositions of the sugar moiety at C(1) and the interglycosidic linkage were deduced fromHMBC experiments, in which H�C(1’’) and H�C(1’’’) were correlated with C(1)(94.3) and C(6’’) (69.6). Finally, the structure of 3 was elucidated as 6’’-O-b-glucopyranosyl harpagoside.

Compound 4 was isolated as white amorphous powder. The molecular formula of 4was determined to be C26H40O16 ([MþNa]þ at m/z 631.2177 in HR-ESI-MS).Compound 4 showed UVabsorption at 219 and 281 nm. The IR spectrum of 4 showedabsorption bands for OH groups (3337 cm�1 (br.)) and an aromatic moiety (1635,1516 cm�1). The 1H-NMR data of 4 showed the presence of a 2-(3-hydroxy-4-methoxyphenyl)ethoxy moiety in the structure, as indicated by H-atom signals at d 6.73(d, J¼2.0, H�C(2)), 6.80 (d, J¼8.2, H�C(5)), 6.66 (dd, J¼8.2, 2.0, H�C(6)), 2.80 (t,J¼7.5, CH2(7)), 3.98–3.99 (m, H�C(8)), and 3.80 (s, MeO), and three anomeric H-atom signals appearing at d 4.28 (d, J¼7.9, H�C(1’)), 4.30 (d, J¼6.8, H�C(1’’)), and5.15 (d, J¼1.5, H�C(1’’’)). The 13C-NMR spectra of 4 (Table 2) displayed resonancesfor an aromatic ring at d 133.0 (C(1)), 117.0 (C(2)), 147.3 (C(3)), 147.5 (C(4)), 112.9(C(5)) and 121.2 (C(6)), for two CH2 C-atoms at d 36.5 (C(7)) and 72.0 (C(8)), for aMeO C-atom d 56.5 (C(9)), for three anomeric C-atoms at d 105.1 (C(1’)), 104.2(C(1’’)), and 102.6 (C(1’’’)), and for 14 oxygenated C-atoms belonging to the sugarmoiety. In the NOESY spectrum, the correlations between MeO and H�C(5)confirmed the position of the aromatic MeO group. Acid hydrolysis, followed by GCanalysis of the aldononitrile peracetate derivatives, confirmed the presence of glucose,arabinose, and rhamnose in a ratio of 1 :1 : 1. The configurations for anomeric H-atomsof glucose and arabinose were determined to be b and a, respectively, from the largecoupling constants (7.9 and 6.8 Hz, resp.). The a-configuration of the anomeric H-atomof the rhamnose was secured by comparison of the chemical-shift values of C(3) and


C(5) with those of the corresponding C-atoms of methyl a- and b-rhamnopyranoside[17–19]. In the HMBC spectrum, the anomeric H-atoms at d 4.28 (glucose), 4.30(arabinose), and 5.15 (rhamnose) showed long-range correlations with C-atom signalsat d 72.0 (C(8) of the aglycone), 69.4 (C(6) of the glucosyl), and 84.0 (C(3) of theglucosyl), respectively. The H- and C-atom signals of sugar moiety were fully assignedby the analysis of the 1H,1H-COSY, HSQC, and HMBC spectral data. Consequently,the structure of 4 was determined as 2-(3-hydroxy-4-methoxyphenyl)ethyl O-a-arabinopyranosyl-(1!6)-O-a-rhamnopyranosyl-(1!3)-O-b-glucopyranoside.

Compound 5 was isolated as white amorphous powder. The molecular formula ofcompound 5 was deduced as C17H24O10 by HR-ESI-MS. The UV spectrum showedabsorption bands at 209, 261, 267, and 273 nm. Its IR spectrum showed absorptionbands at 3421, 1597, and 1493 cm�1, suggesting the presence of OH groups and anaromatic moiety. The 1H-NMR spectrum displayed signals for an aromatic ring at d

7.09–7.12 (m, H�C(2), H�C(6)), 7.26–7.30 (m, H�C(3), H�C(5)), and 6.99 (t, J¼7.4, H�C(4)), and for two anomeric H-atoms at d 4.89 (d, J¼7.6, H�C(1’)) and 4.31 (d,J¼7.4, H�C(1’’)). The 13C-NMR spectrum of 5 (Table 2) displayed resonances for anaromatic ring at d 158.9 (C(1)), 117.7 (C(2), C(6)), 130.4 (C(3), C(5)), 123.3 (C(4)),and for two anomeric C-atoms at d 102.2 (C(1’)) and 105.3 (C(1’’)). Acid hydrolysis of 5,followed by GC analysis of the aldononitrile peracetate derivatives, indicated thepresence of glucose and xylose in a ratio of 1 :1. The positions of the sugar chain andinterglycosidic linkage were established by the HMBC experiment, in which theanomeric H-atom signals at d 4.89 (glucose) and 4.31 (xylose) were correlated with d

158.9 (C(1) of the aglycone) and 69.7 (C(6) of the glucose), respectively. The confi-gurations for two anomeric H-atoms were determined to be b from the large couplingconstants of anomeric H-atoms (7.6 and 7.4 Hz). From the above evidence, the struc-ture of 5 was concluded as phenyl O-b-xylopyranosyl-(1!6)-O-b-glucopyranoside.

Compound 6 was isolated as white amorphous powder. The molecular formula ofcompound 6 was deduced as C18H26O10 by HR-ESI-MS. The UV spectrum showedabsorption at 205, 269, 276, and 287 nm. The 1H-NMR spectrum displayed signals foran aromatic ring at d 6.90–6.91 (m, H�C(2), H�C(6)), 6.81 (t, J¼7.7, H�C(4)) and7.12–7.15 (m, H�C(5)), for a Me group at d 2.30 (s, Me), and for two anomeric H-atoms at d 4.85 (d, J¼7.6, H�C(1’)) and 4.30 (d, J¼7.4, H�(1’’)). The correlations ofMe/C(2), C(3), and C(4) in the HMBC spectrum revealed a 1,3-disubstituted benzenering. The 13C-NMR spectrum of 6 was very similar to that of 5, and the signals of thesugar moiety were identical to those of 5. Acid hydrolysis also confirmed the presenceof glucose and xylose by GC analysis of the aldononitrile peracetate derivatives. Basedon above analysis, compound 6 was identified to possess the same sugar chain as that of5. The attachment of sugar moiety at C(1) was established by an HMBC correlation ofH�C(1’) (d 4.85)/C(1) (d 159.0). Thus the structure of 6 was determined to be 3-methylphenyl O-b-xylopyranosyl-(1!6)-O-b-glucopyranoside.

Compound 7 was isolated as white amorphous powder. The molecular formula wasdetermined to be C27H38O17 based on the HR-ESI-MS. The UV spectrum showedabsorptions at 205, 216, 222, and 277 nm. The IR spectrum indicated the presence ofOH groups (3379 cm�1 (br.)), an a,b-unsaturated ketone (n(C¼O) 1701, n(C¼C)1635, n(C�O) 1033 cm�1), and an aromatic moiety (1505, 1454 cm�1). The 1H-NMRspectrum showed signals due to two (E)-olefinic H-atoms at d 7.70 (d, J¼16.0,



Table2.NMRDataforPhenylpropanoidGlycosides4–8(at40

0an

d100MHzin

CD

3OD);

din

ppm,J

inHz.

Position

a )4

56

78

d(H

)d(C

)d(H

)d(C

)d(H

)d(C

)d(H

)d(C

)d(H

)d(C

)

113

3.0

158.9

159.0

135.6

127.7

26.73

(d,J¼

2.0)

117.0

7.09

–7.12

(m)

117.7

6.90

–6.91

(m)

118.4

7.60

–7.63

(m)

129.3

7.19

(d,J¼

1.8)

112.0

314

7.3

7.26

–7.30

(m)

130.4

140.6

7.30

–7.42

(m)

130.0

149.4

414

7.5

6.99

(t,J¼

7.4)

123.3

6.81

(d,J¼

7.7)

124.2

7.30

–7.42

(m)

131.5

150.6

56.80

(d,J¼

8.2)

112.9

7.26

–7.30

(m)

130.4

7.12

–7.15

(m)

130.3

7.30

–7.42

(m)

130.0

6.83

(d,J¼

8.2)

116.6

66.66

(dd,J¼

8.2,

2.0)

121.2

7.09

–7.12

(m)

117.7

6.90

–6.91

(m)

114.7

7.60

–7.63

(m)

129.3

7.07

(dd,J¼

8.2,

1.8)

124.3

72.80

(t,J¼

7.5)

36.5

7.70

(d,J¼

16.0)

146.6

7.65

(d,J¼

15.9)

147.4

83.98

–3.99

(m)

72.0

6.56

(d,J¼

16.0)

118.6

6.37

(d,J¼

15.9)

115.2

93.80

(s)

56.5

168.4

169.4

Me

2.30

(s)

21.6

MeO

3.90

(s)

56.7

1’4.28

(d,J¼

7.9)

105.1

4.89

(d,J¼

7.6)

102.2

4.85

(d,J¼

7.5)

102.2

4.96

(d,J¼

2.3)

100.9

4.92

(d,J¼

2.4)

100.9

2’3.26

–3.28

(m)

75.6

3.45

–3.47

(m)

74.8

3.46

–3.49

(m)

74.8

3.78

–3.80

(m)

70.9

3.80

–3.82

(m)

70.9

3’3.98

–3.99

(m)

84.0

3.45

–3.47

(m)

77.8

3.46

–3.49

(m)

78.1

3.83

–3.85

(m)

74.3

3.85

–3.86

(m)

74.3

4’3.40

–3.42

(m)

70.1

3.38

–3.42

(m)

71.4

3.37

–3.41

(m)

71.4

3.93

–3.94

(m)

71.5

3.95

(d,J¼

2.9)

71.5

5’3.42

–3.44

(m)

76.8

3.63

–3.67

(m)

77.3

3.61

–3.65

(m)

77.2

4.17

(t,J¼

5.5)

70.2

4.16

(t,J¼

5.5)

70.2

6’4.08

(dd,J¼

11.3,1

.5),

3.75

–3.76

(m)

69.4

4.10

(dd,J¼

11.6,1

.9),

3.80

–3.83

(m)

69.7

4.10

(dd,J¼

11.6,1

.9),

3.78

–3.81

(m)

70.0

4.35

(t,J¼

5.2)

65.1

4.33

–4.36

(m)

65.1


Table2(con

t.)

Position

a )4

56

78

d(H

)d(C

)d(H

)d(C

)d(H

)d(C

)d(H

)d(C

)d(H

)d(C

)

1’’

4.30

(d,J¼

6.8)

104.2

4.31

(d,J¼

7.4)

105.3

4.30

(d,J¼

7.4)

105.4

5.44

(d,J¼

3.8)

94.2

5.38

(d,J¼

3.7)

94.2

2’’

3.55

–3.59

(m)

72.4

3.20

(t,J¼

7.3)

74.9

3.21

(t,J¼

7.4)

74.9

3.38

(dd,J¼

9.8,

3.8)

73.3

3.39

(dd,J¼

9.8,

3.8)

73.3

3’’

3.48

–3.49

(m)

74.1

3.27

(t,J¼

8.7)

77.6

3.26

(t,J¼

8.7)

77.8

3.69

(d,J¼

9.2)

74.7

3.69

(d,J¼

9.7)

74.7

4’’

3.47

–3.48

(m)

69.5

3.45

–3.47

(m)

71.1

3.45

–3.47

(m)

71.2

3.29

–3.30

(m)

71.2

3.30

–3.31

(m)

71.2

5’’

3.85

–3.87

(m),

3.82

–3.83

(m)

66.8

3.80

–3.83

(m),

3.11

(t,J¼

10.3)

66.8

3.80

–3.82

(m),

3.11

(t,J¼

10.3)

66.8

3.78

–3.80

(m)

70.1

3.86

–3.87

(m)

70.1

6’’

3.93

–3.94

(m),

3.63

–3.65

(m)

69.2

3.91

–3.94

(m),

3.69

–3.71

(m)

69.2

1’’’

5.15

(d,J¼

1.5)

102.6

3.78

–3.80

(m),

3.63

–3.65

(m)

62.3

3.80

–3.82

(m),

3.61

–3.62

(m)

63.9

2’’’

3.93

–3.94

(m)

72.3

105.0

105.4

3’’’

3.69

–3.70

(m)

72.2

3.78

–3.80

(m)

83.4

3.69

–3.73

(m)

83.3

4’’’

3.36

–3.39

(m)

73.9

4.03

(t,J¼

8.0)

75.4

4.05

(t,J¼

8.4)

75.4

5’’’

3.47

–3.48

(m)

69.9

4.14

(t,J¼

8.2)

79.8

4.12

(t,J¼

8.5)

79.8

6’’’

1.23

(d,J¼

6.2)

17.9

3.72

–3.74

(m)

63.1

3.69

–3.73

(m)

63.4

a )C-A

tom

numbe

ring

asindicatedin

theFormulae.

H�C(7)) and 6.56 (d, J¼16.0, H�C(8)), five aromatic H-atoms at d 7.60–7.63 (m,H�C(2), H�C(6)) and 7.30–7.42 (m, H�C(3), H�C(4), H�C(5)), together with twoanomeric H-atom signals at d 4.96 (d, J¼2.3, H�C(1’)), 5.44 (d, J¼3.8, H�C(1’’)) anda group of H-atom signals derived from sugar units. Besides eight sp2-C-atom signalsfrom a cinnamoyl group, 18 C-atom signals corresponding to three hexose units wereobserved. Three anomeric C-atom signals appeared at d 94.2, 100.9, and 105.0, of whichthe signal at d 105.0 was confirmed as belonging to a quaternary C-atom from DEPTand HSQC spectral analysis, indicating the presence of two aldohexoses and oneketohexose in the structure. Glucose and fructose were detected by TLC after acidhydrolysis of 7. All H- and C-atom signals (Table 2) were assigned by the analysis of1H,1H-COSY, HSQC, and HMBC spectra. Configuration of both glucose residues wasdetermined to be a from the coupling constant of the anomeric H-atoms (2.3 and3.8 Hz). The b-configuration of the anomeric H-atom of fructose was determined fromNOE correlation of H�C(1’’’)/H�C(5’’’) and NMR comparison [20] [21]. The positionof the sugar moiety on aglycon and interglycosidic linkages were determined byHMBC-spectral analysis. In the HMBC spectrum, correlations of the d 4.35 (H�C(6’))with 168.4 (C(9)), d 4.96 (H�C(1’)) with 69.2 (C(6’’)), and d 5.44 (H�C(1’’)) with 105.0(C(2’’’)) were observed. Thus, the structure of 7 was elucidated as 6-O-cinnamoyl b-fructofuranosyl-(2!1)-O-a-glucopyranosyl-(6!1)-O-a-glucopyranoside.

Compound 8, white amorphous powder, had the molecular formula C28H40O19 asdetermined by HR-ESI-MS. A feruloyl group was deduced from the H-atom signals atd 7.19 (d, J¼1.8, H�C(2)), 6.83 (d, J¼8.2, H�C(5)), 7.07 (dd, J¼8.2, 1.8, H�C(6)),7.65 (d, J ¼ 15.9, H�C(7)), 6.37 (d, J¼15.9, H�C(8)), and 3.90 (s, MeO), and C-atomsignals at d 127.7 (C(1)), 112.0 (C(2)), 149.4 (C(3)), 150.6 (C(4)), 116.6 (C(5)), 124.3(C(6)), 147.4 (C(7)), 115.2 (C(8)), 169.4 (C(9)), and 56.7 (MeO). In the 13C-NMRspectrum of 8, the signals of the sugar moiety were identical to those of 7. Acidhydrolysis also confirmed the presence of glucose and fructose (TLC). Based on aboveanalysis, compound 8 was identified to possess the same sugar chain as 7. The structureof 8 was assigned by detailed analysis of 1H,1H-COSY, HSQC, and HMBC spectra, anddetermined as 6-O-feruloyl b-fructofuranosyl-(2!1)-O-a-glucopyranosyl-(6!1)-O-a-glucopyranoside.

In addition, four known compounds were isolated from the active fraction andidentified as 6’’-O-a-d-galactopyranosyl harpagoside (9) [22], 6’’-O-(p-coumaroyl)harpagide (10) [23], harpagoside (11) [16], and angoroside C (12) [24] by spectroscopicanalysis and comparison of data with those reported. Compounds 9 and 10 are reportedfrom this plant for the first time.

2.Biological Studies. The pharmacological activities of all compounds, isolated fromS. ningpoensis, on [Ca2þ ]i increase induced by KCl in rat cardiac myocytes wereevaluated by confocal laser scanning microscopy. In normal Tyrode solution or Ca2þ-free solution, all the compounds were tested to determine whether they were able tomodify [Ca2þ ]i at resting levels and found not to induce any detectable alteration in thebasal level [Ca2þ ]i (p>0.05 compared to control group, data not shown). Then, weexamined whether these compounds could affect KCl-mediated Ca2þ mobilization.

Among the iridoid glucosides, compounds 1, 10, and 11 showed significantinhibitory effects on the KCl-induced elevation of [Ca2þ ]i at the concentration of100 mm, causing a decrease in Fmax/F0 by nearly 30, 10, and 20%, respectively (n¼6, p<


0.01, compared to the KCl group). As for aromatic glycosides, compounds 5 and 6 alsosubstantially decreased KCl-induced elevations of [Ca2þ ]i in rat cardiac myocytes(Fig. 2).

Several iridoid and aromatic glycosides have been identified from this plant before.Our study indicates that iridoid glycosides in S. ningpoensis might contribute toprophylactic and therapeutic effects on myocardial damage through regulating [Ca2þ ]iin cardiac myocytes.

The authors are grateful to the Shanghai Institute of Materia Medica of Chinese Academy of Sciencefor recording the HR-ESI-MS. Thanks are also due to Xinluan Wang and Ling Li for ESI-MS, and toJinghui Huang and Sanlin Jin for NMR experiments.

Experimental Part

General. Column chromatography (CC): silica gel H (200–300 mesh; Qingdao Haiyang ChemicalGroup Corporation, P. R. China) and C18 reversed-phase (RP) silica gel (60–80 mesh; Merck),macroporous adsorptive resins D101 (250–300 mm; Tianjin Pesticide Factory, P. R. China). Anal. TLC:pre-coated silica gel plates (Merck; silica gel GF254, 1 mm). Optical rotations: Jasco P1030 polarimeter.UV Spectra: Shimadzu UV2401PC spectrophotometer; lmax in nm (log e). IR Spectra: ShimadzuFTIR8400 spectrometer; in cm�1. 1H-, 13C-, and 2D-NMR Spectra: Bruker AVANCE-400 NMRspectrometer; chemical shifts d in ppm rel. to residual solvent peaks (CD3OD: d(H) 3.30, d(C) 49.3), orrelative to Me4Si as internal standard, J in Hz. ESI-MS: Bruker Esquire-2000 mass spectrometer; in m/z(rel. %). HR-ESI-MS: Finnigan MAT95 mass spectrometer; in m/z (rel. %). Prep. HPLC (10 ml/min):


Fig. 2. Effects of iridoid glycosides and aromatic glycosides on [Ca2þ ]i increase induced by KCl in wistarrat cardiomyocytes. Iridoid glycosides 1–3 and 9–11; aromatic glycosides 4–8 and 12 ; concentration ofall compounds is 100 mm ; verapamil (Ver) was used as a positive control (10 mm). Mean�SME. n¼6.

*: p<0.05, **: p<0.01 vs. control (KCl).

Shimadzu Pak with UV detector; a Shim-pack PREP-ODS column (˘250�20 mm, 10 mm). GC/MS:Shimadzu GCMS-QP2010 plus gas chromatograph mass spectrometer with AOC-20i auto injection andAOC-20s auto sample. Fluorescence intensity (FI) of [Ca2þ ]i : laser-scanning confocal microscope(Olympus Fluo-view 300, Japan).

Animals.Our study was in accordance with the Principles of Laboratory Animal Care and Protocolsas well as the Animal Care Committee of the Harbin Medical University. Wistar rats of either sexweighting 200–250 g were provided by the Experimental Animal Center of Harbin Medical University.

Plant Material. The dried roots of Scrophularia ningpoensis Hemsl. (Scrophulariaceae) wereobtained from Guangzhou Qingping Medical Material Market, China, in March 2005, and identified bythe Shenzhen Institute for Drug Control, Shenzhen, China. Voucher specimens have been deposited withthe Research Center of the Traditional Chinese Medicine and Natural Products, Shenzhen, China.

Extraction and Isolation. Dried roots of S. ningpoensis (18 kg) were cut into small pieces andextracted with 60% EtOH (3�90 l) using a reflux apparatus for 2 h each time. The 60% EtOH extractwas concentrated in vacuo to afford a dark-brown residue that was suspended in H2O (20 l) and extractedwith AcOEt (3�20 l) and BuOH (3�20 l) successively.

The bioactive BuOH-soluble fraction was subjected to CC (macroporous adsorptive resins D101;EtOH/H2O 0 :100, 30 :70, 50 :50, 95 :5). The 50% EtOH eluate (50.2 g) was fractionated by CC (silicagel; CHCl3/MeOH 95 :5!70 :30): Fr. 1–7. Fr. 3 (3.9 g) was separated by CC (ODS;MeOH/H2O 40 :60),and purified by RP-HPLC (MeOH/H2O 35 :65): 2 (tR 36.4 min; 28.2 mg) and 10 (tR 34.1 min; 15.6 mg).Fr. 5 (6.1 g) was purified by RP-HPLC (MeOH/H2O 50 :50): 3 (tR 27.6 min; 237.7 mg) and 9 (tR 32.6 min;110.3 mg). Fr. 6 (1.7 g) was separated by CC (ODS ; MeOH/H2O 50 :50): 11 (384.6 mg). Fr. 4 (2.9 g) waspurified by RP-HPLC (MeOH/H2O 40 :60): 1 (tR 15.2 min; 170.3 mg) and 12 (tR 22.7 min; 435.4 mg). The30% EtOH eluate (51.4 g) was separated by CC (silica gel; CHCl3/MeOH 90 :0!60 :60): Fr. 1–7. Fr. 4(13.2 g) was subjected to CC (ODS ; MeOH/H2O 20 :80!60 :40): Fr. A–H. Fr. D (257.6 mg) waspurified by RP-HPLC (MeOH/H2O 30 :70): 6 (tR 12.8 min; 32.3 mg). Fr. 5 (10.7 g) was subjected to CC(ODS; MeOH/H2O 35 :65), and then purified by RP-HPLC (MeOH/H2O 25 :75): 5 (tR 26.9 min;42.6 mg) and 8 (tR 21.6 min; 16.4 mg). Fr. 6 (5.6 g) was fractionated by CC (ODS ; MeOH/H2O 20 :80!60 :40): Fr. A–J. Fr. H (152.3 mg) was purified by RP-HPLC (MeOH/H2O 25 :75): 4 (tR 20.5 min;60.2 mg) and 7 (tR 23.2 min; 20.2 mg).

6’’-O-Caffeoyl Harpagide (¼ (1S,4aS,5R,7S,7aR)-1,4a,5,6,7,7a-Hexahydro-4a,5,7-trihydroxy-7-meth-ylcyclopenta[c]pyran-1-yl 6-O-[(E)-3-(3,4-Dihydroxyphenyl)prop-2-enoyl]-b-d-glucopyranoside ; 1).Pale brown amorphous powder. [a]26D ¼ �37.6 (c ¼ 1.0, MeOH). UV (MeOH): 203 (2.56), 227 (2.48),312 (2.60). IR (KBr): 3394 (OH), 1697 (C¼O), 1607 (C¼C), 1605, 1520 (phenyl nucleus), 1033 (C�O).1H- and 13C-NMR: see Table 1. ESI-MS: 525 ([M – H]� ), 549 ([MþNa]þ ). HR-ESI-MS: 549.1592([MþNa]þ , C24H30NaOþ13 ; calc. 549.1584).

6’’-O-Feruloyl Harpagide (¼ (1S,4aS,5R,7S,7aR)-1,4a,5,6,7,7a-Hexahydro-4a,5,7-trihydroxy-7-methyl-cyclopenta[c]pyran-1-yl 6-O-[(E)-3-(4-Hydroxy-3-methoxyphenyl)prop-2-enoyl]-b-d-glucopyranoside ; 2).Brown amorphous powder. [a]26D ¼ �35.2 (c ¼ 1.0, MeOH). UV (MeOH): 204 (2.58), 235 (2.48), 325(2.61). IR (KBr): 3417 (OH), 1601, 1516 (phenyl nucleus). 1H- and 13C-NMR: see Table 1. ESI-MS: 539([M – H]� ), 563 ([MþNa]þ ). HR-ESI-MS: 563.1752 ([MþNa]þ , C25H32NaOþ13 ; calc. 563.1741).

6’’-O-b-Glucopyranosyl Harpagoside (¼ (1S,4aS,5R,7S,7aR)-1-[(6-O-b-d-Glucopyranosyl-b-d-glu-copyranosyl)oxy]-1,4a,5,6,7,7a-hexahydro-4a,5-dihydroxy-7-methylcyclopenta[c]pyran-7-yl (E)-3-Phe-nylprop-2-enoic Acid ; 3). Pale brown amorphous powder. [a]26D ¼ þ11.6 (c ¼ 1.0, MeOH). UV(MeOH): 204 (2.37), 216 (2.31), 222 (2.27), 279 (2.35). IR (KBr): 3421 (OH), 1701 (C¼O), 1632,1601, 1516 (phenyl nucleus). 1H- and 13C-NMR: see Table 1. ESI-MS: 655 ([M�H]� ), 679 ([MþNa]þ ).HR-ESI-MS: 679.2192 ([MþNa]þ , C30H40NaOþ16 : calc. 679.2214).

2-(3-Hydroxy-4-methoxyphenyl)ethyl O-a-d-Arabinopyranosyl-(1!6)-O-[6-deoxy-a-l-mannopy-ranosyl-(1!3)]-O-b-d-glucopyranoside (4). White amorphous powder, [a]26D ¼ �81.6 (c¼1.0, MeOH).UV (MeOH): 219 (3.86), 281 (3.50). IR (KBr): 3337 (OH), 1635, 1516 (phenyl nucleus). 1H- and13C-NMR: see Table 2. ESI-MS: 607 ([M�H]� ), 631 ([MþNa]þ ). HR-ESI-MS: 631.2177 ([MþNa]þ ,C26H40NaOþ16 ; calc. 631.2214).

Phenyl 6-O-b-d-Xylopyranosyl-b-d-glucopyranoside (5). White amorphous powder. [a]26D ¼ �57.6(c¼0.3, MeOH); UV (MeOH): 209 (2.15), 261 (1.33), 267 (1.42), 273 (1.36). IR (KBr): 3421 (OH),


1597, 1493 (phenyl nucleus). 1H- and 13C-NMR: see Table 2. ESI-MS: 387 ([M�H]� ), 411 ([MþNa]þ ).HR-ESI-MS: 411.1256 ([MþNa]þ , C17H24NaOþ10 ; calc. 411.1267).

3-Methylphenyl 6-O-b-d-Xylopyranosyl-b-d-O-b-glucopyranoside (6): White amorphous powder.[a]26D ¼ �56.7 (c¼1.0, MeOH). UV (MeOH): 205 (2.40), 269 (1.73), 276 (1.75), 287 (1.65). IR (KBr):3417 (OH), 1597, 1516 (phenyl nucleus). 1H- and 13C-NMR: see Table 2. ESI-MS: 401 ([M�H]� ), 425([MþNa]þ ). HR-ESI-MS: 425.1406 ([MþNa]þ , C18H26NaOþ10 ; calc. 425.1424).

b-d-Fructofuranosyl O-6-O-[(E)-3-Phenylprop-2-enoyl]-a-d-glucopyranosyl-(1!6)-a-d-glucopyra-noside (7). White amorphous powder. [a]26D ¼ �76.7 (c¼1.0, MeOH). UV (MeOH): 205 (2.74), 216(2.75), 222 (2.68), 277 (2.85). IR (KBr): 3379 (OH), 1701 (C¼O), 1635 (C¼C), 1505, 1454 (phenylnucleus), 1033 (C�O). 1H- and 13C-NMR: see Table 2. ESI-MS: 633 ([M�H]� ), 657 ([MþNa]þ ). HR-ESI-MS: 657.2008 ([MþNa]þ , C27H38NaOþ17 ; calc. 657.2007).

b-d-Fructofuranosyl O-6-O-[(E)-3-(4-Hydroxy-3-methoxyphenyl)prop-2-enoyl]-a-d-glucopyrano-syl-(1!6)-a-d-glucopyranoside (8). White amorphous powder. [a]26D ¼ �74.5 (c¼1.0, MeOH). UV(MeOH): 204 (2.52), 235 (2.39), 325 (2.57). IR (KBr): 3364 (OH), 1519, 1458 (phenyl nucleus). 1H- and13C-NMR: see Table 2. ESI-MS: 679 ([M�H]� ), 703 ([MþNa]þ ). HR-ESI-MS: 703.2073 ([MþNa]þ ,C28H40NaOþ19 ; calc. 703.2061).

Acid Hydrolysis. Each sample (5 mg) was heated in an ampule with 5 ml of 2m HCl at 1008 for 2 h.The aglycone was extracted with AcOEt three times, and the aq. residue was evaporated under reducedpressure. Pyridine (1 ml) and 2 mg of NH2OH·HCl were added to the residue, and the mixture washeated at 1008 for 1 h. After cooling, Ac2O (1.5 ml) was added, and the mixtures were heated at 1008 for1 h. The mixtures were evaporated under reduced pressure, and the resulting aldononitrile peracetateswere analyzed by GC/MS using authentic samples as reference samples [25]. GC/MS Analysisconditions: column DB-50, 25 mm�30 m; column oven temp. 1508 (2 min)!3008 (10 min); rate 158/min; injection temp. 2508 ; carrier gas He (1.0 ml/min); tR: Glc 7.824 min, Gal 8.012 min, Ara 5.989 min,Rha 5.859 min, Xyl 6.107 min). TLC Conditions: BuOH/acetone/H2O 10 :10 :2.5, Rf : Glc 0.32, Fru 0.38;spray reagent on TLC (phenylamine/o-phthalic acid/BuOH), 1058, 10 min.

Cell Isolation. SingleWistar rat ventricular myocytes were isolated as described in [26]. Briefly, adultWistar rats of either sex were sacrificed by a blow on the head. Then, the heart was quickly removed andcannulated on a Langendorff apparatus and retrogradely perfused through the aorta with standardTyrodeDs soln. (126 mm NaCl, 5.4 mm KCl, 10 mm HEPES, 0.33 mm NaH2PO4 ·2 H2O, 1.0 mm MgCl2 ·6 H2O, 1.8 mm CaCl2, 10 mm glucose, pH 7.4) for 5 min, and Ca2þ free TyrodeDs soln. until it stoppedbeating. The heart was then enzymatically digested with Ca2þ free TyrodeDs soln. containing collagenasetype II and BSA. The ventricular tissue was minced after it had been softened and placed in KB medium(70 mm glutamic acid, 15 mm taurine, 30 mm KCl, 10 mm KH2PO4, 10 mmHEPES, 0.5 mmMgCl2 ·6 H2O,10 mm glucose, 0.5 mm EGTA, pH 7.4). Single cells were obtained by gentle pipetting and stored at 48 for1–2 h, then gassed with 95%O2 and 5%CO2, and warmed to (37�0.5)8. Only rod-shapedmyocytes withclear cross-striations were studied.

Ca2þ Fluorescence Measurements. Fluorescence measurements in cardiomyocytes were described in[27]. Isolated cells were attached to the coverslips of the chamber with ConA and incubated with 10 mm

Fluo-3/AM, 0.03% Pluronic F-127 for 45 min at 378, followed by 10 min in TyrodeDs soln. containing1.8 mm Ca2þ . The fluorescent change of the Fluo-3/AM-loaded cell was detected by a confocal laser-scanning microscope (Fluoview-FV300, Olympus, Japan; excitation: 488 nm from an Ar ion laser;emission: 530 nm with 20� objective). Fluorescence images were collected every 10 s within 5 min, and30 successive images were obtained. KCl (final concentration 60 mm), which was used as a stimuli toevoke [Ca2þ ]i increment, was added between the 2nd and 3rd image. The data obtained from the 1st and2nd scan was considered the basal data of cardiomyocytes (FI0). Increases of [Ca2þ ]i are expressed as theratio of fluorescence intensity of Fluo-3 over baseline (FI/FI0). The effect of testing compounds on [Ca2þ ]iwas investigated by incubating them with loaded cardiomyocytes in TyrodeDs soln. containing 1.8 mm

Ca2þ TyrodeDs soln. for 5 min at r.t. before imaging. In the confocal microscope, [Ca2þ ]i increasedgradually after application of 60 mm KCl in the presence of 1.8 mm Ca2þ . The ratio FI/FI0 of the KClcontrol group was normalized as 1 (peak value) (n ¼ 6, p<0.01 compared with resting value).Acquisition rate was 1 frame (512 ·512) per 15 s, and [Ca2þ ]iwas monitored for at least 600 s. Increases of[Ca2þ ]i were expressed as the ratio of fluorescence intensity of Fluo-3 over baseline (FI/FI0).


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Received September 11, 2007


Documents

Iridoid and Aromatic Glycosides from Scrophularia ningpoensisHemsl. and Their Inhibition of [Ca2+]i Increase Induced by KCl