3720 Vol. 35 (1987)

Chem. Pharm. Bull. 35( 9 )3720-3725(1987)

Studies on the Constituents of Scutellaria Species. IX.1) On the Flavonoid Constituents of the Root of

Scutellaria indica L.2)

YUKINORI MIYAICHI,a YOSHITAKA IMOTO,a TSUYOSHI Tomimom*,a

and CHUN-CHING LIN b

School of Pharmacy, Hokuriku University,a 3 Ho, Kanagawa-machi, Kanazawa, 920-11, Japan and School of Pharmacy, Kaohsiung Medical College,b

Kaohsiung, Taiwan, Republic of China

(Received February 23. 19871

Four new flavanones (I-IV) and a new flavone (V) were isolated from the root of Scutellaria

indica L., together with 2(S)-5,2'-dihydroxy-7,8,6'-trimethoxyflavanone, (•} )-5,2'-dihydroxy-

6,7,6'-trimethoxyflavanone, 5,7-dihydroxy-8,2'-dimethoxy flavone, rivularin, 5,2',6'-trihydroxy-

7,8-dimethoxyflavone, scutevurin, 5,7,4'-trihydroxy-8-methoxyflavone, wogonin, alpinetin, cal-

damomin and wogonin 7-O-glucuronide. Compounds I-V (Fig. 1) were identified based on

spectral data and simple chemical modifications.

Keywords Scutellaria indica; Labiatae; flavonoid; flavone; flavanone; chalcone; structure

elucidation

Scutellaria indica L. is a perennial herb of the family Labiatae, which is widely distributed

in Japan, Taiwan, China, Korea and Indo-China. The dried whole herb of this plant is a crude

drug which is known as "han xin cao" (ŠØ•M‘•) in China and has been used as an antidotic,

analgesic and hemostatic for the treatment of hemoptysis, hematemesis and other diseases.3)

So far, only a flavone glycoside, scutellarin, has been isolated as a constituent of this plant4)

As a part of our studies on the flavonoid constituents of Scutellaria species, we have now

examined this plant.

As described in the experimental part, four new flavanones (I-IV) and a new flavone

(V) were isolated together with eleven known flavonoids (VI-XVI) from the ethanol extract

of the root of this plant which was collected in Taiwan. This paper deals with their structural

identification.

Compound I was obtained as colorless needles, mp 197 •Ž (dec.), C16H14O6, Mg-HCl test

( +). It gave the absorption bands of hydroxyl and conjugated carbonyl groups and benzene

rings in the infrared (IR) spectrum. The ultraviolet (UV) spectrum of I was characteristic of

the 5,7-dihydroxy flavanone series.5) The proton nuclear magnetic resonance (1H-NMR)

spectrum of I showed the signals of one methoxyl (3.67 ppm), two hydroxyls (9.6 and

10.8 ppm), one chelated hydroxyl (11.93 ppm) and an ABX type grouping due to the C-2

(5.76 ppm) and C-3 protons (2.76 and 3.23 ppm). In the aromatic region of the spectrum, the

signals of the remaining five protons occurred as a singlet (6.01 ppm, 1H) due to the A-ring

proton, and a double doublet (7.23 ppm, 1H, J= 7.9 and 7.5 Hz), a broad triplet (6.89 ppm,

1H, J=7.5 Hz) and two doublets (7.48 ppm, 1H, J=7.5 Hz; 6.92 ppm, 1H, J=7.9 Hz) due to

the B-ring protons.

On methylation by Kuhn's method,6) I gave a trimethyl ether, mp 174 •Ž (dec.),

C19H20O6, which was identical with 5,7,8,2'-tetramethoxyflavanone, prepared from 2(S)-5,7-

dihydroxy-8,2'-dimethoxyflavanone.7)

No. 9 3721

Compound I was, therefore, considered to be a monomethyl ether of 5,7,8,2'-tetrahy-

droxyflavanone. In the carbon-13 nuclear magnetic resonance (13C-NMR) spectrum of I,

the methoxyl carbon signal appeared downfield at 60.4 ppm, which indicated the methoxyl

to be on the C-8 carbon, being di-ortho-substituted by two oxygen functions.8)

It is known that flavanones having 2(S)-configuration exhibit a positive Cotton effect due

to n-n* transition ( •` 330 nm) and a negative Cotton effect due to n-n* transition (270

290 nm) in the circular dichroism (CD) spectra.9) The CD curve of I exhibited positive and

negative maxima at 309 and 288 nm, respectively, which established the 2(S)-configuration.

Based on the above findings, compound I was determined to be 2(S)-5,7,2'-trihydroxy-8-

methoxyflavanone.

Compound II was obtained as colorless needles, mp 202 °C, C15H1205, Mg-HC1 test ( + ).

The IR spectrum gave absorption bands corresponding to hydroxyl and conjugated carbonyl

groups and aromatic rings. The UV spectrum and diagnostic shifts suggested II to be a 5,7-

dihydroxyflavanone derivative.5) The 1H-NMR spectrum showed the presence of one chelated

hydroxyl (12.15 ppm), and C-2 (5.72 ppm) and C-3 (2.11, 3.23 ppm) protons. In the aromatic

region of the spectrum, a pair of doublets (J=2.0 Hz) (5.94 ppm, 1H, 5.91 ppm, 1H) for the A-

ring protons and two double doublets (7.21 ppm, 1H, J= 7.6 and 7.8 Hz; 6.87 ppm, 1H, J=7.6

and 7.8 Hz) and two doublets (6.89 ppm, 1H, J= 7.8 Hz; 7.44 ppm, 1H, J=7.8 Hz) due to the

B-ring protons were seen. These chemical shifts and splitting patterns of the B-ring protons

suggest that II possesses the same B-ring (2'-OH) as I. This was also supported by the 13C-

NMR spectrum, in which the carbon signals due to the B-ring of II were observed to be

almost superimposable on those of I. The 2(S)-configuration was confirmed in the same way

as in the case of I. From these results, the structure of II was determined to be 2(S)-5,7,2'-

trihydroxyflavanone.

Compound III was obtained as colorless needles, mp 192 °C, C17H1607, Mg-HC1 test

(+), and gave the absorption bands of hydroxyl and conjugated carbonyl groups and benzenerings in the IR spectrum. The UV spectrum and diagnostic shifts suggested the presence of a hydroxyl group at the C-5 position and the absence of a free hydroxyl at the C-7 position.5) The H-NMR spectrum of III showed the signals of two methoxyls (3.66 and 3.87 ppm), two hydroxyls (8.87 and 9.11 ppm), one chelated hydroxyl (12.03 ppm) and an ABX type grouping due to the C-2 (5.70 ppm) and C-3 protons (2.80 and 3.18 ppm). The mass spectrum of III exhibited a fragment ion peak originating from the B-ring at m/z 136 (Ci8H802 +). These findings indicated III to be a flavanone possessing one hydroxyl (on C-5) and two methoxyls in the A-ring and two hydroxyls in the B-ring.. In the aromatic region of the 1H-NMR spectrum, the remaining four protons were observed as a singlet (6.25 ppm, 1H) a double doublet (6.62 ppm, 1H, J= 2.0 and 9.3 Hz) and two doublets (6.75 ppm, 1H, J=9.3 Hz; 6.92 ppm, 1H, J=2.0 Hz). The former singlet could be assigned to the C-6 proton by long-range selective proton decoupling (LSPD)1°) in the 13C-NMR spectrum as follows. In the H non-decoupling 13C-NMR spectrum of III, the signal of the carbon attached to an isolated aromatic hydrogen was observed at 93.0 ppm in the form of a double doublet (J=163.2 and 7.4 Hz), which changed to a doublet when the chelated hydroxyl proton at the C-5 position was selectively irradiated, indicating that the isolated aromatic proton was present at the

position ortho (C-6) to the chelated hydroxyl (C-5). These data indicated that the substitution pattern of the A-ring was 5-hydroxy-7,8-dimethoxy. This was further confirmed by the 13C-NMR spectrum of III, in which the signal pattern of the A-ring was almost identical with that of 5,2'-dihydroxy-7,8,6'-trimethoxyflavanone (VI).7) The latter three signals were assigned to the C-3', C-4' and C-6' protons, respectively, from their chemical shifts and coupling

patterns. This was further confirmed by the 13C-1H shift correlation spectrum (COSY) and the 13C-1H long-range COSY.11)

The 2(S)-configuration of III was confirmed in the same way as in the case of I.

3722 Vol. 35 (1987)

Compound III was, therefore, determined to be 2(S)-5,2',5'-trihydroxy-7,8-dime-

thoxyflavanone.

Compound IV was obtained as pale yellow needles, mp 143 °C (dec.), C24H26O13, Mg-

HC1 test positive. It gave the absorption bands of hydroxyl and conjugated carbonyl groups

and benzene rings in the IR spectrum. The UV spectrum and diagnostic shifts suggested the

presence of a hydroxyl at the C-5 position and the absence of a free hydroxyl at the C-7

position.5)

On methanolysis, IV yielded 5,2 '-dihydroxy-7,8,6 '-trimethoxyflavanone (VI),7) 5,2'-

dihydroxy-6,7,6'-trimethoxyflavanone (VII),7) methyl glucuronopyranoside methylester and

the methyl glycoside of glucurono-6,3-lactone.

The absence of a substituent at the C-6 position in IV was confirmed by the LSPD

method.10) The aglycone of IV is, therefore, VI, and VII was considered to be a secondary

product formed from VI by ring isomerization (similar to the interconvertibility of hemi-

phloin and isohemiphloin by acid).12)

In the and 13C-NMR spectra of IV, the anomeric proton signal at 5.00 ppm (d,

J= 6.3 Hz) and a set of carbon signals between 71.4 ppm and 170.3 ppm including an anomeric

carbon signal at 101.1 ppm (d, 164.7 Hz) indicated the presence of a f3-glucuronopyranosyl

unit.

The 2(S)-configuration of IV was confirmed in the same way as in the case of I. Thus,

the structure of IV was established as 2(S)-5,2'-dihydroxy-7,8,6'-trimethoxyflavanone 2'-

O-f3-glucuronopyranoside.

Compound V was obtained as yellow needles, mp 247 °C (dec.), C231122012, Mg-HC1 test

positive, and gave the absorption bands of hydroxyl and conjugated carbonyl groups and

benzene rings in the IR spectrum. The UV spectrum and diagnostic shifts suggested the

presence of a hydroxyl at the C-5 position and the absence of a free hydroxyl at the C-7

position.5)

On methanolysis V yielded 5,7-dihydroxy-8,2'-dimethoxyflavone (VIII),7) methyl glucuro-

nopyranoside methylester and the methylglycoside of glucurono-6,3-lactone. In the "C-

NMR spectrum of V, the signals due to the sugar moiety indicated the presence of a fl-

glucuronopyranoside unit in V.

Hence V was determined to be 5,7-dihydroxy-8,2'-dimethoxyflavone 7-O-ƒÀ-

glucuronopyranoside. This was confirmed by its "C-NMR spectrum, in which the signal

patterns of the A-ring and the B-ring were almost identical with those of wogonin

glucuronopyranoside (XVI)13) and 2'-O-methylskullcapflavone I,7,14) respectively.

Compounds VI—XVi are known flavonoids and were identified as 2(S)-5,2'-dihydroxy-

7,8,6 '-trimethoxyflavanone,7) (•} )-5,2 '-dihydroxy-6,7,6 '-trimethoxyflavanone,7) 5,7-dihy-

droxy-8,2 '-dimethoxyflavone,7) rivularin,' 5) 5,2 ',6 '-trihydroxy-7,8-dimethoxyflavone,16) scu-

Fig. 1

No. 9 3723

tevurin,16) 5,7,4'-trihydroxy-8-methoxyflavone,17) wogonin,7 alpinetin,1) caldamomin1) and

wogonin 7-0-glucuronide,7) respectively, by direct comparison with authentic samples.

Experimental

General Procedures All melting points were determined on a Yanagimoto micro melting point apparatus and

are recorded uncorrected. UV spectra were determined with addition of diagnostic reagents by standard procedures')

on a Hitachi recording spectrophotometer, type 323. IR spectra in KBr disk were run on a JASCO IR-A-2

spectrometer. NMR spectra were taken in dimethyl sulfoxide-d6 (DMSO-d6) on a JEOL JNM-FX-100 spectrometer

(1H-NMR at 100 MHz and 13C-NMR at 25 MHz), and chemical shifts are given in 6 (ppm) with tetramethylsilane

(TMS) as an internal standard (s, singlet; d, doublet; m, multiplet; br, broad). Electron impact-mass spectra (EI-MS)

were taken on a JEOL JMS-DX-300 mass spectrometer. CD spectra were run on a JASCO J-20A automatic

recording spectropolarimeter. Optical rotations were measured with a JASCO DIP-4 digital polarimeter. Gas-liquid

chromatography (GLC) was run on a Shimadzu GC-6AM unit with a flame ionization detector using a glass column

(2 m x 4 mm i.d.) packed with 5% SE-30 on Chromosorb W (60-80 mesh); column temperature, programmed from

150 °C (20 min hold) to 240 °C at 5 •Ž/min. Thin layer chromatography (TLC) was carried out on Kieselgel 60 F 254

(Merck) with the following solvent systems: CHC13-Me0H-H20-AcOH (100 : 4 : 0.2 : 0.1) (TLC-1), n-hexane-

acetone-AcOH (60 : 40 : 0.1) (TLC-2), CHC13-Me0H-H2O-HCOOH (25 : 8 : 1 : 1) (TLC-3), AcOEt-methyl ethyl

ketone-H20-HCOOH (18 : 9 : 1 : 1) (TLC-4). Spots were detected by spraying of dil. H2SO4 followed by heating.

Extraction and Separation The dried root (330 g) of Scutellaria indica L., collected in Taiwan in 1985, was

extracted with boiling EtOH. The EtOH extract was concentrated to dryness to give a residue (110.7 g), which was

suspended in H2O and successively extracted with Et20 and n-BuOH. The Et20 layer was concentrated and the

residue (6.1 g) was chromatographed on silica gel (500 g) using n-hexane-acetone (10: 1 -+ 1: 1) as an eluent to give

five fractions, fr. 1-5, in the order of elution. Fraction 1 gave VI. Fraction 2 was rechromatographed on silica gel

(solvent: benzene) to give VII, VIII and XIII. Fraction 3 was subjected to rechromatography on silica gel [solvent:

benzene-CHC13 (1 : 1)] to give I, II and IX. Fraction 4, containing a mixture of two flavonoids, was passed through a

silica gel column [solvent: benzene-CHC13 (1: 2)] to give III and XI. Fraction 5 was subjected to repeated

chromatography on silica gel [solvent: benzene-AcOEt (10: 1)] to give X, XII, XIV and XV. The n-BuOH-soluble

portion was concentrated and the residue (18.3 g) was chromatographed on silica gel (500 g) with a gradient of

CHC13-Me0H-H2O-HCOOH (100: 10 : 1 : 0.1 -+ 100 : 30 : 3 : 0.3) as an eluent to give IV, V and XVI. Yields: I

(50 mg), II (30 mg), III (100 mg), IV (20 mg), V (20 mg), VI (100 mg), VII (15 mg), VIII (30 mg), IX (6 mg), X (4 mg),

XI (6 mg), XII (5 mg), XIII (40 mg), XIV (10 mg), XV (5 mg), XVI (30 mg).

2(S)-5,7,2'-Trihydroxy-8-methoxyflavanone (I) Colorless needles (MeOH/H20), mp 197 °C (dec.). Anal.

Calcd for C161-11406: C, 63.57; H, 4.67. Found: C, 63.53; H, 4.69. Mg-HC1(+). Rf : 0.24 (TLC-1), 0.28 (TLC-2). UV

Aler nm (loge): 240 sh (3.82), 291 (4.21), 342 (3.63); An Na nm (log E): 241 sh (4.12), 329 (4.36); 41ear - AIC1, nm

(loge): 240 sh (3.91), 277 sh (3.56), 316 (4.34), 400 (3.60); MeOHA1CI3HC1 nm (log e): 240 sh (3.91), 313 (4.31), 398

(3.58); emear'Ac nm (loge): 253 sh (3.74), 283 (3.59), 330 (4.35); Miler - Na0Ac - H3B03 nm (loge): 292 (4.10), 333

(4.01). IR 0,2,r, cm-1: 3488 (OH), 1641 (conjugated CO), 1614 (arom. C=C). 41-NMR: 3.67 (3H, s, OCH3), 2.76

(1H, dd, J=17.0, 3.2 Hz, cis 3-H), 3.23 (1H, dd, J=17.0, 12.3 Hz, trans 3-H), 5.76 (1H, dd, J=12.3, 3.2 Hz, 2-H), 6.01

(1H, s, 6-H), 6.89 (1H, br t, J=7 .5 Hz, 5'-H), 6.92 (1H, br d, J=7.9 Hz, 3'-H), 7.23 (1H, dd, J=7.9, 7.5 Hz, 4'-H),

7.48 (1H, d, J= 7.5 Hz, 6'-H), ca. 9.6 (1H, br s, 2'-OH), ca. 10.8 (1H, br s, 7-OH), 11.93 (1H, s, 5-OH). "C-NMR:

74.3 (C-2), 41.1 (C-3), 196.4 (C-4), 158.6 (C-5), 95.9 (C-6), 159.9 (C-7), 128.5 (C-8), 154.6 (C-9), 101.8 (C-10), 124.9

(C-1'), 154.3 (C-2'), 115.6 (C-3'), 129.4 (C-4'), 119.1 (C-5'), 126.8 (C-6'), 60.4 (C-8-OCH3). MS m/z (%): 302 (M +,

70), 269 (C15H905, 100), 167 (C7H3O5, 73). CD (c=0.005, Me0H) [O]14 (nm): + 16717 (309) (positive maximum),

-59554(288) (negative maximum).Methylation of I by Kuhn's Method: CH316) (0.2 ml) and Ag20 (50 mg) were added to a solution of I (8 mg) in

N,N-dimethylformamide (DMF) (0.3 ml), and the reaction mixture was left for 20 h with occasional shaking. Then

CHC13 was added, and after removal of the resulting precipitate by filtration, the filtrate was evaporated to dryness.

The residue was chromatographed on silica gel (10 g) using benzene as an eluent to give crude Ia, which was

recrystallized from Me0H to give Ia, colorless needles, mp 174 •Ž (dec.). This product was identical (UV, IR, 1H- and

i3GNMR, mixed fusion) with 5,7,8,2'-tetramethoxyflavanone.7)

2(S)-5,7,2'-Trihydroxyflavanone (II) Colorless needles (MeOH/H20), mp 202 •Ž. Anal. Calcd for C15111205:

C, 66.17; H, 4.44. Found: C, 66.32; H, 4.46. Mg-HC1( + ). Rf : 0.19 (TLC-1), 0.29 (TLC-2). UV A nm (log e): 239 sh

(3.83), 290 (4.23), 330 sh (3.56);rviAeaoft-Na0Me nm (loge): 241 sh (4.11), 325 (4.40); 42'1'3 nm (log e): 239 sh (3.81),

275 sh (3.54), 313 (4.35), 380 (3.53); rtlea0FI - AlC13 - HCI nm (log e): 239 sh (3.83); 275 sh (3.62), 311 (4.32), 379 (3.53);

MeOH-NaOAcλmax

nm (logs): 253 (3.69), 278 sh (3.59), 326 (4.32); 2.,s,leaoH-Na0Ac-1131303 .nm (logƒÃ): 291 (4.16), 323 sh (3.90).

IR vr1Eal,r, cm': (OH), 1639 (conjugated CO), 1597 (arom. C=C). 1H-NMR: 2.71 (1H, dd, J= 17.1, 3.4 Hz, cis 3- H), 3.23 (1H, dd, J= 17.1, 12.7 Hz, trans 3-H), 5.72 (1H, dd, J= 12.7, 3.4 Hz, 2-H), 5.91 (1H, d, J=2.0 Hz, 6-H), 5.94

(1H, d, J=2.0 Hz, 8-H), 6.87 (1H, dd, J=7.8, 7.6 Hz, 5'-H), 6.89 (1H, brd, J=7.8 Hz, 3'-H), 7.21 (1H, dd, J=7.8,

3724 Vol. 35 (1987)

7.6 Hz, 4'-H), 7.44 (1H, d, J= 7.8 Hz, 6'-H), 12.15 (1H, s, 5-OH), 13C-NMR: 74.0 (C-2), 41.1 (C-3), 196.5 (C-4), 163.7

(C-5), 95.9 (C-6), 166.8 (C-7), 95.1 (C-8), 163.4 (C-9), 101.8 (C-10), 124.9 (C-1'), 154.4 (C-2'), 115.6 (C-3'), 129.5 (C-

4'), 119.2 (C-5'), 127.1 (C-6'). MS nil z (%): 272 (M +, 60), 254 (C15H1004, 98), 153 (C6H105, 100). CD (c= 0.005,

MeOH) [O]14 (nm): +9301 (309) (positive maximum), -62006 (283) (negative maximum).

2(S)-5,2',5'-Trihydroxy-7,8-dimethoxyflavanone (III) Colorless needles (Me0H/H20), mp 192 •Ž. Anal.

Calcd for C,,H,607: C, 61.44; H, 4.85. Found: C, 61.49; H, 4.87. Mg-HC1(+). Rf: 0.15 (TLC-1), 0.21 (TLC-2). UV

λMeOhmax nm (1o9ε) : 240 sh (3.89), 292 (4.13); A ivier - NaOMe nm (logs): 243 (4.11), 288 (4.00), 315 sh (3.76), 390 (3.75);MeOH-AICi3λmax

nm (log E): 314 (4.22), 404 (3.56); A n?H_AIC13 - HC1 nm (log E): 310 (4.21), 396 (3.49); A max - NaWkc nm

(log E): 292 (4.10), 345 (3.48); A MeOH-NaOAc - H3B03 nm (log E): 292 (4.11), 345 (3.50). IR v:,lahx. cm -1: 3400 (OH), 1639 (conjugated CO), 1580 (arom. C =C). 111-NMR: 3.66, 3.87 (each 3H, each s, OCH3 x 2), 2.80 (1H, dd, J = 19.0, 3.9 Hz, cis 3-H), 3.18 (1H, dd, J = 19.0, 12.9 Hz, trans 3-H), 5.70 (1H, dd, J = 12.9, 3.9 Hz, 2-H), 6.25 (1H, s, 6-H), 6.62

(1H, dd, J=9.3, 2.0 Hz, 4'-H), 6.75 (1H, d, J= 9.3 Hz, 3'-H), 6.92 (1H, d, J= 2.0 Hz, 6'-H), 8.87, 9.11 (each 1H, each s, 5' and 2'-OH), 12.03 (1H, s, 5-OH). 13C-NMR: 74.5 (C-2), 41.5 (C-3), 197.1 (C-4), 159.0 (C-5), 93.0 (C-6,

J(c-6) _ (6-H) =163.2 Hz, J (c_6) _ (5 _0H)= 7.4 Hz), 161.1 (C-7), 129.3 (C-8), 153.9 (C-9), 102.4 (C-10), 125.5 (C-1'), 146.5 (C-2'), 116.4 (C-3'), 115.9 (C-4'), 150.1 (C-5'), 113.2 (C-6'), 56.3 (C-7-OCH3), 60.5 (C-8-OCH3). MS m I z (%): 332 (M 58), 299 (C16H1106, 100), 136 (C8H802+, 25) CD (c = 0.005, Me0H) [0114 -nm): + 9501 (308) (positive maximum),-54292(285)(negative　 maximum).

2(S)-5,2'-Dihydroxy-7,8,6'-trimethoxyflavanone 2'-04-Glucuronopyranoside (IV)--Pale yellow needles

(MeOH/H20) mp 143 °C (dec.). [4,4 - 71.8° (c= 0.03, MeOH). Anal. Calcd for C24H26013: C, 55.18; H, 5.03. Found:

C, 55.05; H, 5.06. Mg-HC1 (+). Rf: 0.41 (TLC-3), 0.32 (TLC-4). UV At,r nm(log e): 241 sh (3.97), 289 (4.19), 347

(3.55); A- Na0Me nm (loge): 250 sh (4.14), 287 (4.18), 370 (3.84); Anr_mo, nm (loge): 280 sh (3.72), 314 (4.30),

404 (3.58); A nr_mc,,_HC1nm (log e): 280 sh (3.74), 312 (4.29), 400 (3.60); Arver- a°Ac nm (loge): 289 (4.17), 349

(3.53); Arnear_Na0Ac -H,B03 nm (log e): 289 (4.17), 346 (3.54). IR v 21 cm': 3452 (OH), 1740 (COOH), 1637

(conjugated CO), 1603 (arom. C =C). 1H-NMR: 3.55, 3.80, 3.85 (each 3H, each s, OCH3 x 3), 2.44 (1H, dd, J =13.7,

2.7 Hz, cis 3-H), 3.8-4.2 (m, trans 3-H), 5.00 (1H, d, J =6.3 Hz, anomeric H of glucuronic acid unit), 6.07 (1H, dd,

J =13.9, 2.7 Hz, 2-H), 6.22 (1H, s, 6-H), 6.82 (2H, d, J=8.3 Hz, 3',5'-H), 7.38 (1H, t, J=8.3 Hz, 4'-H), 12.16 (1H, s, 5-

OH). 13C-NMR: 71.5 (C-2), 39.4 (C-3), 198.2 (C-4), 159.3 (C-5), 92.6 (C-6, J(C-6)- (6-H) =162.5 Hz, J(C-6)- (5-0H) =

6.6 Hz), 161.1 (C-7), 129.2 (C-8), 154.8 (C-9), 102.5 (C-10), 114.8 (C-1'), 156.8 (C-2'), 108.6 (C-3'), 131.0 (C-4'), 106.4

(C-5'), 159.0 (C-6'), 101.1 (C-1", J = 164.7 Hz), 73.2 (C-2"), 75.7 (C-3"), 71.4 (C-4"), 75.9 (C-5"), 170.3 (C-6"), 56.2

(C-6'-OCH3), 56.4 (C-7-OCH3), 60.6 (C-8-OCH3). MS m/z (%): 346 (C18111807, 67), 328 (C18H1606, 50), 313

(C17H1306, 100). CD (c = 0.005, MeOH) [O]14 (nm): + 6812 (309) (positive maximum), - 34058 (285) (negative

Maximum).

Methanolysis of IV: A solution of IV (10 mg) in 10% HC1-MeOH (2 ml) was heated under reflux on a water bath

for 3 h. The reaction mixture was neutralized with Ag2CO3. The precipitates were filtered off and the filtrate was

concentrated to give the residue. The residue was crystallized from MeOH/H20 to give a mixture of two types of

crystals, which was chromatographed on silica gel (10 mg) using benzene as an eluent to give pale yellowneedles

(MeOH), mp 202 °C (dec.) and colorless needles (MeOH), mp 221 •Ž (dec.). They were identified as compounds VI

and VII, respectively, by direct comparisons (TLC, UV, IR, 41- and 13C-NMR, mixed fusion), with authentic

specimens. The mother liquor of crystallization was shown to contain methyl glucuronopyranoside methyl ester [tR

13 min 24 s (both a and [3)] and the methyl glycoside of glucurono-6,3-lactone [tR 6 min 05 s (a, trace), 6 min 48 s (/3)] by

GLC (as the trimethylsilylether derivatives).

5,7-Dihydroxy-8,2'-dimethoxyflavone 7-O-jJ-Glucuronopyranoside (V) Yellow needles (MeOH) mp 247 •Ž

(dec.). [a]b4: - 45.9° (c= 0.04, MeOH). Anal. Calcd for C23H22012: C, 58.23; H, 4.68. Found: C, 58.29; H, 4.66. Mg-HC1

(+). Rf: 0.35 (TLC-3), 0.26 (TLC-4). UV A,1",:,?" nm (loge): 275 (4.41), 333 (4.04); MaOH - Na0Me nm (loge): 276 (4.39),

283 sh (4.37), 320 sh (4.33), 380 (3.74); 2,Nntear-mci, nm (loge): 255 (3.94) 286 (4.37), 295 sh (4.34), 345 (4.07), 404

(3.84); 2 Nnifear - AlC13 -HCI nm (loge): 284 (4.36), 295 sh (4.31), 342 (4.07), 405 (3.78);trvi,1-101-1- Na0Ac nm (log e): 275 (4.40),

333 (4.03); A MeOH - Na0Ac - H3B03 nm (log e): 275 (4.40), 333 (4.03). IR v mBX cm-1: 3500 (OH), 1714 (COOH), 1653

(conjugated CO), 1607, 1565 (arom. C =C). 1H-NMR: 3.86, 3.91 (each 3H, each s, OCH3 x 2), 5.29 (1H, br s,

anomeric H of glucuronic acid unit), 6.90 (1H, s, 3-H), 6.70 (1H, s, 6-H), 7.15 (1H, br t, J= 7 .6 Hz, 5'-H), 7.22 (1H,

brd, J=7.9 Hz, 3'-H), 7.57 (1H, br t, J=7.9 Hz, 4'-H), 7.86 (1H, dd, J=7.6, 1.2 Hz, 6'-H), 12.54 (1H, s, 5-OH). "C-

NMR: 161.9 (C-2), 109.8 (C-3), 182.5 (C-4), 156.2 (C-5), 98.7 (C-6, J(c_6)_ (6_1i) =163.3 Hz, J(c_6)_ (5_0H) = 7.4 Hz), 156.2

(C-7), 129.5 (C-8), 149.5 (C-9), 105.4 (C-10), 119.5 (C-1'), 158.1 (C-2'), 112.8 (C-3'), 133.5 (C-4'), 121.1 (C-5'), 129.1

(C-6'), 99.9 (C-1", J =163.3 Hz), 73.1 (C-2"), 75.5 (C-3"), 71.4 (C-4"), 75.9 (C-5"), 170.2 (C-6"), 56.0 (C-2'-OCH3),

61.5 (C-8-OCH3). MS m/z (%): 314 (C17H1406, 100), 299 (C16H1106, 70).

Methanolysis of V: V (10 mg) was methanolyzed and worked up in the same way as described for IV to give

compound VIII and a sugar fraction, which was shown to contain methyl gluctronopyranoside methyl ester and the

methyl glycoside of glucurono-6,3-lactone by GLC.

Identification of VI—XVI VI (mp 202 °C (dec.)), VII (mp 221 •Ž (dec.)), VIII (mp 231 °C), IX (mp 259 °C), X

(nip 286 °C (dec.)), XI (mp 278 °C (dec.)), XII (mp 302 •Ž), XIII (mp 203 °C), XIV (mp 222 °C), XV (mp 198 °C), XVI

(mp 270 °C (dec.)) were identified as 2(S)-5,2'-dihydroxy-7,8,6'-trimethoxyflavanone, ( + )-5,2'-dihydroxy-6,7,6'-

No. 9 3725

trimethoxyflavanone, 5,7-dihydroxy-8,2'-dimethoxyflavone, rivularin, 5,2',6'-trihydroxy-7,8-dimethoxyflavone, scutevurin, 5,7,4'-trihydroxy-8-methoxyflavone, wogonin, alpinetin, caldamomin and wogonin 7-O-glucuronide, respectively, by direct comparisons with authentic specimens (UV, IR, and 13C-NMR, mixed fusion).

Acknowledgement We thank Prof. T. Ohmoto, Dr. T. Nikaido and Dr. K. Koike (Toho University) for the measurement of 400 MHz NMR spectra. We are also grateful to Mrs. R. Igarashi and Miss H. Shimomura of this university for elementalanalysis and El-mass measurement.

References and Notes

1) Part VIII: T. Tomimori, Y. Miyaichi, Y. Imoto and H. Kizu, Shoyakugaku Zasshi, 40, 432 (1986). 2) Presented at the 106th Annual Meeting of the Pharmaceutical Society of Japan, Chiba, April 1986.3) Chiang Su New Medical College (ed.), "Dictionary of Chinese Crude Drugs (中葯大 辞典)," Shanghai

Scientific Technological Publishers, Shanghai, 1977, p. 2303. 4) K. shibata, S. Iwata and M. Nakamura, Acta Phytochim., 1, 105 (1923). 5) T. J. Mabry, K. R. Markham and M. B. Thomas, "The Systematic Identification of Flavonoids," Springer-

Verlag, New York, 1970, Chapter V. 6) R. Kuhn, Angew. Chem., 67, 32 (1955). 7) T. Tomimori, Y. Miyaichi, Y. Imoto, H. Kizu and T. Namba, Chem. Pharm. Bull., 33, 4457 (1985). 8) K. S. Dhami and J. B. Stothers, Can. J. Chem., 44, 2855 (1966); K. Panichpol and P. G. Waterman,

Phytochemistry, 17, 1363 (1978). 9) W. Gaffield, Tetrahedron, 26, 4093 (1970).

10) Y. Shirataki, I. Yokoe, M. Endo and M. Komatsu, Chem. Pharm. Bull., 33, 444 (1985); H. Komura, K. Mizukawa, H. Minakata, H. Huang, G. Qin and R. Xu, ibid., 31, 4206 (1983); H. Komura, K. Mizukawa and H. Minakata, Bull. Chem. Soc. Jpn., 55, 3053 (1982).

11) The spectra were measured in DMSO-d6 on a JEOL GX-400 spectrometer. 12) W. E. Hills and D. H. S. Horn, Aust. J. Chem., 18, 531 (1965). 13) T. Tomimori, Y. Miyaichi and H. Kizu, Yakugaku Zasshi, 102, 388 (1982). 14) T. Tomimori, Y. Miyaichi, Y. Imoto and H. Kizu, Shoyakugaku Zasshi, 38, 249 (1984). 15) C. J. Chou, J. Taiwan Pharm. Assoc., 30, 36 (1978). 16) T. Tomimori, Y. Miyaichi, Y. Imoto, H. Kizu and C. Suzuki, Yakugaku Zasshi, 104, 529 (1984). 17) T. Tomimori, Y. Miyaichi, Y. Imoto, H. Kizu and T. Namba, Chem. Pharm. Bull., 34, 406 (1985).