Sterol glucosides from Prunella vulgaris

Phytochemistry, Vol. 29, No. 7, pp. 2351-2355, Printed in Great Britain.

1990. 0

STEROL GLUCOSIDES HISASHI Medicinal

Plant

Garden,

School

KOJIMA,

NORIKO

FROM PRUNELLA

SATO,? AKIKO

HATANot

Sciences, Kitasato University, Sagamihara, Kanagawa Sciences, Kitasato University, Minato-Ku, Tokyo 108, Japan (Received 11 September

Key Word Index-Prunella

VULGARIS*

and HARUO OGURAt

of Pharmaceutical

Pharmaceutical

vulgaris; Labiatae;

phytosterols;

0031-9422/90 $3.00 + 0.00 1990 Pergamon Press plc

228, Japan;

Wchool

of

1989)

steryl glucosides.

‘H and 13C NMR spectra of sitosterol, stigmasterol, stigmast-7-en-3p-ol and spinasterol have been unambiguously assigned by use of HOMCOR and HETCOR techniqeus. The extensive application of these results leads to the direct identification of a mixture of the four B-D-glucopyranosides of the sterols, which were obtained from Abstract-The

Prunella vulgaris.

INTRODUCTION It

has been reported previously that the stems and leaves of Prunella vulgaris L. var. lilacina Nakai contain nine triterpenes and two sterols [l, 21. Before the above studies, Shimano et al. [3] had assumed the presence of the glucoside of ursolic acid, provisionally named prunellin, in the spikes of the same plant while Sendra [4] had found saponins having oleanolic acid as an aglycone in the herb P. vulgaris. Our further investigation of the more polar constituents of the chloroform and n-butanol extracts has yielded a mixture of steryl glucosides, against the expectation of triterpene glucoside. The steryl glucosides have been frequently isolated from the plant kingdom and their chromatographic separation is an unresolved problem. However, their direct identification may be achieved by ‘H and 13CNMR spectroscopy. We completely assigned the ‘H and 13CNMR spectra of the sterols deduced as their aglycones, most of our assignments were compared with the published spectral data (‘HNMR) [S, 61 and 13CNMR [7-93 and it was found necessary that a part of the latter data is interchanged or revised. Consequently, it has been possible to directly identify the components of the intractable steryl glycoside mixture. RESULTS

AND DISCUSSION

Exhaustive column chromatography [l] of the fractions more polar than those containing the triterpenes obtained from the chloroform and n-butanol extracts of the leaves and stems of P. vulgalis afforded plates, mp 267-270”, TLC R, 0.12 (violet) [l]. The mass spectrum showed fragments at m/z 576 [M] + and 574 [M] +,

*Part 5 in the series, ‘Constituents of the Labiatae Plants’. For part 4 see ref. [2]. *Unpublished data; compounds la and lb have been identified as the minor sterols of P. vulgaris by comparison with authentic samples (co-GLC and co-TLC).

and 414 and 412 (both, [M-glucose+H,O]‘). The ‘HNMR spectrum taken in pyridine-d, indicated the characteristic methyl singlet signals at 60.57, 0.59, 0.66 and 0.67, and anomeric protons as two doublets at 4.93 and 4.95. These data suggested that the crystals were a mixture of at least four steryl glucosides. As the further purification of this mixture was very difficult, we proposed to directly identify them by high-resolution NMR spectroscopy. The published ‘H and 13C NMR spectral data of phytosterols contain a few inconsistent or doubtful assignments. The complete assignments of sitosterol (la)*, stigmasterol (lb)*, stigmast-7-en-3/I-01 (4a) and spinasterol (4b) [l] were examined, and the results are summerized in Tables 1 and 2. Though the proton signals of C-19 (singlet), C-27 (doublet) and C-29 (triplet) in compound 4b overlapped each other around 60.8, their ‘H and 13CNMR assignments were mutually determined by their coupling pattern of J-resolved 2D NMR spectra. The ‘HNMR chemical shift assignments of the methyl protons were almost in accord with those of refs [S, 81, except for that of Me-19 in lb of ref. [S]. Furthermore, those for H-22 and H-23 in lb of ref. [6] should be contrariwise assigned. The 13C NMR chemical shifts given for 4a are the first assignment, and the assignments for la and lb were almost the same as those of ref. [7], whereas those for C18, and C-29 in lb of ref. [S] should be interchanged, and the signals of C-21, C-26 and C-27 in 4b of ref. [9] should be assigned to C-27, C-21 and C-26, respectively. The chemical shift differences between A5-sterol and A’-sterol were more than 0.7 ppm for C-3, C-4, C-9, C- 10, C- 13 C14, C-15 and C-19, as well as for C-5 through C-8. On the basis of the above data and the glycosylation shifts [lo], the ‘H and 13CNMR signals of the mixture (2a, 2b, 5a and 5b in pyridine-d,) and its acetate (3a, 3b, 6a and 6b in chloroform-d) were assigned, and are compiled in Tables 1 and 2. The anomeric protons (H-l’) influenced by the A5- or A’-double bond were observed as doublets at 64.95 and 4.93 in the former and at 4.59 [11] and 4.61 in the latter. These results, and the 13C absorption at

2351

*Overlapped signal. The figures in parentheses are coupling -Unassigned for overlapping with the signal of H,O. tJ=6.5 Hz. $J=7.5 Hz. $Signal with shoulder.

OAc

6;

6:

5

constants

4.13 dd (12, 2.5)

t

4.12 dd

(12, 5)

in Hz except for W,,, values in m.

4.42 dd

4.59 dd*

4.03 m*

t

(12, 5) 2.01, 2.03, 2.05,2.09

(12, 2.5)

t*

(9)

4.26 dd

(9.5) 3.69 ddd (9.5, 5, 2.5)

4.31

0.91 d* 0.86 d* 0.87 t*

-

1.07 d* -

0.67 s

4

(8)

4.95 d

0.94 s

(23)

2.38 m 2.63 m

3.97 m*

2b

(9.5) 5.08

0.90d* 0.87 d* 0.89 t’

0.99 d*

0.66 s

2a

0.90 d 0.87 d 0.89 t

0.99 d

0.57 s

5a

t

(12, 2)

4.61 dd

(9)

4.34

(8)

4.93 d

0.72 s

-

4.01 m*

(2a, Zb, 5a and 5b) in pyridine-d,

(8) 4.28

(8)

(1599) 0.85 d 0.80 d 0.81 t

(15,9) 5.02 m

1.03 d 5.16 m

0.55 s

6b

and steryl glycosides

(9.5, 8) 5.21 t§

4.61 d

0.77 s

5.15 m*

(8)

0.84 d 0.82 d 0.85 t

0.93 d

0.53 s

3.56 m (22)

4.59 d

0.85 d* 0.80 d* 0.81 t’

5.01 m*

1.03 d* 5.15 m*

0.70 s

6a

(3a, 3b, 6a and 6b) in chloroform-d

3

0.84 d* 0.82 d* 0.85 t*

0.99 s

(9)

5.37 m

3.49 m*

3b

acetates

4.08 t

0.83 d 0.81 d 0.84 t

0.93 d*

0.67 s

3a

of steryl glycosyl

4.96 dd§

0.83 d 0.81 d 0.84 t

(15,9) 0.85 d 0.80 d 0.80 t

s s d dd

(15, 9) 0.84 d 0.79 d 0.80 t

0.55 0.80 1.02 5.15

5.15 m*

(22)

3.59 m

4b

(15, 9) 5.02 dd

(9.5) 0.53 s 0.79 s 0.92 d

5.16 m

(22)

3.60 m

4a

la, lb, 4a and 4b, a mixture

(15, 9) 5.01 dd

0.69 s 1.01 s 1.02d 5.15 dd

(9)

(9)

0.68 s 1.01 s 0.92 d

5.35 m

5.35 m

3.52 m

(23)

3.52 m

lb

shifts of compounds

(23)

la

1. ‘H Chemical

2

Me-26t Me-27t Me-29$ 1’

23

Me-18 Me-19 Me-2lt 22

I

6

4.4 4,

3

H

Table

0.91 d 0.86 d 0.87 t

-

1.07 d -

0.59 s

5b

37.1 31.4 71.0 38.0

4b

39.7 42.3 56.7 24.3 28.2 56.0 11.8*

3a 37.2 29.4 80.1 38.9 140.3 122.2 31.8 31.9 50.1 36.7 21.0 39.6 42.2 56.8 24.4 28.9 55.9 12.0*

3b

117.2 139.7

6a

*Overlapped signal.

19.0

19.4

56.8

25.4 12.3

40.5 21.2 138.3 129.2 51.2 31.9 21.1

24.4 28.9 55.9 12.0

22.9 11.9

36.6 18.9 33.8 26.1 45.8 29.1 19.8

27.9 56.0 11.8

39.5 43.4 55.0

139.6

19.0

13.0

23.0

49.4 34.2 21.5

40.2 29.6 117.4

25.4 12.3

40.8 21.4 138.7 129.4 51.2 31.9 21.1

28.5 55.8 12.0

39.4 43.3 55.1

139.5

23.1* 12.0*

36.1 18.8 33.9 26.0 45.8* 29.1* 19.8; 19.0

19.4

25.4* 12.3;

40.5 21.2 138.3 129.3 51.2* 31.9* 21.12 23.0 12.0

36.6 18.9 33.9 26.2 45.8 29.1 19.8

28.0 56.1 11.8

39.5 43.4 55.0

20.8, 20.7, 20.6, 20.6

39.7 42.2

31.9

4a

MeCO,

37.2 31.6 71.8 42.3 140.7 121.7 31.9 31.9 50.1 36.5 21.1

lb

99.72 71.5 72.9. 68.5 71.7 62.1 170.7, 170.4, 169.4, 169.3

23.0 12.0

36.1 18.8 33.9 26.0 45.8 29.1 19.8

24.3 28.2 56.0 11.9

39.8 42.3

la

1 2 3 4 5 6 I 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 21 28 29 1’ 2 3 4 5 6 MeCO,

C

99.7 71.5 72.9 68.6 71.6 62.1

19.0

13.0

23.1

49.4 34.3 21.5

37.1 29.2 79.6 34.5 40.2 29.7

25.4 12.3

40.8 21.4 138.1 129.4 51.2 31.9 21.1

28.5 55.9 12.0

39.4 43.3 55.1

117.3 139.6

6b

23.4* 12.2*

36.5 19.1 34.3 26.4 46.1* 29.5* 19.5’

40.0 42.6 57.0 24.6 28.7 56.3* 12.0

2a

102.6 75.4f 78.7 71.7 78.5* 62.9

20.1*

19.3

37.6 30.3 78.3 39.4 141.0 122.0 32.2 32.1 50.4 37.0 21.4;

25.8* 12.6*

40.9 21.7* 138.9 129.5 51.5* 32.2* 21.4*

39.9 42.4 57.1 24.7 29.4 562 12.3*

2b

23.4 12.2

36.9 19.2 34.2 26.6 46.1 29.5 19.5

28.4 56.3 12.1

39.8 43.7 55.3

Sa

102.3 75.4 78.7 71.8 78.5 62.9

20.1

13.1

23.5

37.4 30.1 77.3 34.8 40.2 30.1 117.9 139.7 49.7 34.6 21.8

25.8 12.6

41.2 21.7 138.7 129.7 51.5 32.2 21.4

29.0 56.1 12.3

39.7 43.5 55.4

Sb

Table 2. 13C Chemical shifts of compounds la, lb, 4a and 4b, a mixture of steryl glycosyl acetates (3a, 3b, 6a and 6b) in chloroform-d and steryl glycosides (Za, 2b, 5a and 5b) in pyridine-d,

2354

Short Reports

R2 RIO ~

R2

Side chain ( R2 )

a

StO~ I

Rl =

H

4

Rl =

H

2

RI =

glcH4

5

Rl =

glcH4

3

Rl =

glcAc4

6

Rl =

glcAc4

glcH4 = 8 -D- glucopyranosyl

3 102.6 a n d 102.3 for the a n o m e r i c c a r b o n s of the former a n d 99.7 in the latter, indicated t h a t four glycoside b o n d s of the mixture were the same, a n d thus indicated u n a m biguously a /~-D-configuration a n d the p y r a n o s e form. F u r t h e r m o r e , each p r o t o n of C-18, C-19 a n d H-6' was also affected by the A 5- or AT-double b o n d a n d in the case of the C-18 protons, the four signals were revealed by the additional effect of the A 2z double bond. The n u m b e r of 13C_resonances of the same position, observed as four signals by the different effects of the three double b o n d s was seven c a r b o n s in the former a n d ten in the latter, whereas the overlapped signals were reversely more in the former. The relative height of the four signals for C-13 in the latter (3a; 19, 3b; 13, 6a; 41, 6b; 27) was found to correlate with the relative a m o u n t revealed by G C - M S (the percentage c o m p o s i t i o n of the four aglycones described below were la; 20, lb; 12, 4a; 40, 4b; 28). T h e 13C N M R spectral d a t a of 3a were almost identical with those of ref. [12], b u t its assignments for H-3', a n d H-4' should be interchanged. F u r t h e r m o r e , the d a t a of 2a were different from those of refs [13, 14] in the assignments for C-4 a n d C-12, a n d C - Y a n d C-5', which were the reverse. A l t h o u g h the observed d a t a of 3b, a n d the partial, a n d a few ambiguous, assignments of 5a have been described in refs 1-15] a n d [16], respectively, the complete assignments are n o w determined for b o t h compounds. The assignments for C-I (626.1) a n d C-27 (21.9) s h o w n in the data on 5b of ref. [17] are uncertain. F u r t h e r m o r e , the signals from C-21, C-26 a n d C-27 in 6b of ref. [9] were misassigned as for 4b. In order to c o r r o b o r a t e the above results, acid hydrolysis of the mixture gave D-glucose a n d a sterol mixture, whose c o m p o s i t i o n was analysed by G C - M S (see Experimental). F r o m the a b o v e data, the presence of the four steryl monoglucosides, sitosteryl, stigmasteryl, stigmast-7-enyl a n d spinasteryl fl-D-glucopyranoside was directly established by high resolution N M R spectroscopy. Furthermore, the a m o u n t of the glucosylated AT-sterols was ca twice t h a t of the glucosylated AS-sterols. If a mixture of these glucosides is isolated as one spot on a thin layer c h r o m a t o g r a m , its acetate is marginally preferable for the identification a n d semi-quantitative estimation.

EXPERIMENTAL Mp: uncorr. Plant extraction, isolation and TLC: unless otherwise indicated, see ref. [1]. Isolation of a mixture of steryl glucosides. After the five triterpene fractions eluted from silica gel CC of CHCI 3 and nBuOH extracts, the more polar fraction using CHCI3-Me2CO (1:1) as eluant was concd and the residue recrystalized from

b

MeOH-n-BuOH (1 : 1) to give 114.5 mg of plates, mp 267-270 °. ElMS m/z (rel. int.): 576 [M] + (24), 574 [M] + (13), 414 (17), 412 (7), 397 (100), 396 (71), 383 (10), 273 (8), 271 (5), 255 (30), 229 (11). HRMS re~z: 576.437 [M] ~, C35H6oO 6 (calc. for 576.439) and 574.423 [M] +, C35Hs80 6 (calc. for 574.423). 1H NMR: see Table 1 and 13CNMR: see Table 2. Acetylation of the mixture. The mixture (35 mg) was acetylated overnight with Ac20-pyridine at room temp. Crystalization of product from MeOH gave needles (30 mg). 1H and J3C NMR: see Table 1 and Table 2, respectively. Source ofsterots. The authentic sitosterol (la) and stigmasterol (lb) were purchased from Nakarai Chemical Ltd and Sigma, respectively; stigmast-7-en-3/~-ol (4a) and spinasterol (4b) were gifts from Dr K. Takeda (Shionogi Research Laboratories). Acid hydrolysis of the mixture. A soln of the mixture (40 rag) in 15% H2SO4 MeOH (1:1) (20ml) was heated for 4hr. The reaction mixture was added to H20 and extracted with Et20. The Et20 soln, after washing with H20 and drying over Na/SO, was evapd to yield a mixture of aglycones (25 mg), powder (recrystallized from MeOH. D-Glucose, from the aq. phase, was identified by co-TLC in the solvent system Me2CO-nBuOH-HaO (5:4: 1). GC-MS of the sterol mixture. Mass spectra (ELMS, 70 eV) were taken with instruments equipped with a direct inlet system (>m/z 50. Fused silica capillary column; SPB-I, 30 m x 0.25 mm I.D.; column temp 260°; He flow rate, 80 ml min 1). Compound Ib {Rt 20.1 min and rel. amount 12%) re~z:412 [M] + (35), 397 (5), 379 (5), 369 (10), 351 (16), 300 (18), 271 (23), 255 (42), 231 (5), 229 (12), 213 (16), 83 (80), 55 (100). Compound 4b (22.4 rain and 28%): 412 [M] + (35), 397 (12), 379 (3), 368 (19), 351 (9), 300 (18), 271 (100), 255 (41), 246 (25), 231 (13), 229 (21), 213 (14), 81 (76), 69 (40). Compound la (22.8 min and 20%1:414 [M] + (100), 399 (42), 396 (69), 381 (44), 329 (55), 303 (64), 273 (26), 255 (33), 246 (25), 231 (32), 229 (13), 213 (50), 69 (56), 55 (89). Compound (25.5 min and 40%): 414 [M] + (100), 399 (32), 381 (7), 273 (25), 255 (83), 246 (15), 231 (33), 55 (50). Two compounds la and 4b could not separated by the following conditions; column: 3% OV-I, 1 m × 3 mm I.D.; He flow rate: 20 ml rain- 7. The above data were identical with those of corresponding authentic samples. The presence of the A2/-double bond was distinguished by two unique peaks at m/z 351 and 300 [18], and others at 412, 397, 279 and 271, whereas different fragmentations between A5 and A vsterol could not be specifically observed, except for the rel. int. (the former was about a half of the latter) at m/z 229; C17H25. Conditions for N M R measurements. The IH (400 MHz) and 13C (1130MHz) NMR spectral data were recorded as 6 values at room temp in CDCI 3 or CsDsN (TMS as int. standard), which were assigned by DEPT pulse sequence or, if necessary, by comparative studies of 1H-IH and IH-~3C 2D COSY, and Jresolved 2D NMR experiments. In ~3C NMR spectral measurements of the acetylated mixture (ca 3 mg per 0.6 ml) the signals of

4a

Short Reports

2355

major compounds 6s and 6b were obtained by number of transient; 8192, and those of compounds 3a, 3b, 6a and 6b by 100 224.

8. Akihisa, T., Thakur, S., Rosenstein, F. U. and Matsumoto, T. (1986) Lipids 21, 39. 9. Henry, M. and Chantalat-Dublanche, I. (1985) Planta Med.

Acknowledgements-The authors thank Dr K. Takeda for generously providing authentic samples. Thanks are also due to the members of analytical centre of Kitasato University for GC-MS.

10. Seo, S., Tomita, Y., Tori, K. and Yoshimura, Y. (1978) J. Am. Chem. Sot. 100, 3331. 11. Dzizenko, A. K., Isakov, V. V., Uvarova, N. I., Oshitok, G. I. and Elyakov, G. B. (1973) Carbohydr. Res. 27, 249. 12. Isobe, T., Noda, Y., Ohasaki, A., Sakanaka, S., Kim, M. and Taniguchi, M. (1989) Yakugaku Zasshi 109, 175. 13. Sakakibara, J., Kaiya, T., Fukuda, H. and Ohki, T. (1983)

322.

REFERENCES

1. Kojima, H. and Ogura H. (1986) Phytochemistry 25, 729. 2. Kojima, H. and Ogura H. (1989) Phytochemistry 28, 1703.

3. Shimano, T., Mizuno, M., Okamoto, H. and Adachi, K. (1957) Jap. J. Pharm. Chem. 29, 109. 4. Sendra, J. (1963) Diss. Pharm. 15, 333. 5. Rubinstein, I., Goad, L. J., Clague, A. D. H. and Mulhern, L. J. (1976) Phytochemistry 15, 195. 6. Heupel, R. C., Sauvaire, Y., Le, P. H., Parish, E. J. and Nes, W. D. (1986) Lipids 21, 69. 7. Wright, J. L. C., McInnes, A. G., Shimizu, S., Smith, D. G., Walter, J. A., Idler, D. and Khalil, W. (1978) Can. J. Chem. 56, 1898.

Phyrochemisfry.

Phytochemistry 22, 2553. 14. Iribarren, A. M. and Pomilio, A. B. (1983) J. Nat. Prod. 46, 752. 15. Asakawa, Y., Toyota, M. and Harrison, L. J. (1985) Phytochemistry 24, 1505.

16. Arisawa, M., Kinghorn, A. D., Cordell, G. A., Phoebe, C. H. and Fansworth, N. R. (1985)Planta Med. 544. 17. Rauwald, H.-W., Sauter, M. and Schilcher, H. (1985) Phytochemistry 24, 2746. 18. Gershengorn, M. C., Smith, A. R. H., Goulston, G., Goad, L. J., Goodin, T. W. and Haines, T. H. (1968) Biochemistry 7, 1698.

Vol. 29, No. 7, pp. 2355 2356, 1990 Britain.

0

Printed in Great

A NAPHTHOQUINONE

CARBOXYLATE FROM FUNGAL DIOSPYROS MONTANA

M. PARDHASARADHI*

and B.

NAGESWARA

003 I -9422/90 $3.00 + 0.00 1990 Pergamon Press plc

INFESTED

RAO

Indian Institute of Chemical Technology, Hyderabad 500007, India (Received in revised form 21 November 1989) Key Word Index-Diospyros

montana; Ebenaceae; fungal-infested; yerrinquinone;

6-carbomethoxy-2

(or 3), 5-

dimethoxy-8-hydroxy-1,4-napthoquinone.

Abstract-Yerrinquinone assigned by ‘H NMR.

has been isolated from fungal-infested

INTRODUffION

Diospyrin, 6,2’-bis-7-methyljuglone [l], is known to occur in the bark and wood of Diospyros montana (Yerragatha, Telugu language). Compounds corresponding to the successive stages of reduction of diospyrin, i.e. /Idihydrodiospyrin and tetrahydrodiospyrin have been reported from the fresh bark of this tree [2, 31. When the

RRL-H communication No. 2266.

stems of Diospyros

montana.

Its structure

was

bark is chopped off from the stems, the exposed stems develop fungal infestation on standing. We report herein, the isolation and structure determination of a highly oxygenated naphthoquinone carboxylate, yerrinquinone (l), from such fungal-infested wood. RESULTS AND DISCUSSION

Yerrinquinone (I), ([M] + 292) gave a crimson colour with aqueous NaOH, exhibited reversible oxidation-reduction with Na,S,O,, and yielded methylether 2 ([Ml’ 306) on methylation. That it is an ester of a peri-hydroxy-