A spirostane hexaglycoside from Agave cantala fruits

A spirostane hexaglycoside from Agave cantala fruits

Phyfochemrstry, Vol 30. No. 12. pp. 41874189, Printed in Great Bntain. A SPIROSTANE 1991 C HEXAGLYCOSIDE FROM AGAVE CANTALA G. C. UNIYAL, P. K. A...

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Phyfochemrstry, Vol 30. No. 12. pp. 41874189, Printed in Great Bntain.

A SPIROSTANE

1991 C

HEXAGLYCOSIDE

FROM AGAVE CANTALA

G. C. UNIYAL, P. K. AGRAWAL, 0. Central Institute of Medicinal

and Aromatic

Key Word Index-&ace

FRUITS*

P. SATI? and R. S. THAKUR:

Plants, Lucknow 226016, India; tDeqartment Garhwal, Srinagar 246 174. India (Receioed

@X-9422/91 $3.00+0.00 1991 Pergamon Press plc

5 February

of Chemistry,

H.N.B.,

University

of

1991)

cantala; Agavaceae, fruits; steroidal glycosides; agaveside D.

Abstract-A new steroidal glycoside, agaveside D, isolated from the fruits of Agaue cantala was characterized as 3fl-{a-L-rhamnopyranosyl-( l-2). b-D-glycopyranosyl-(l +3)-~-D-glucopyranosy~[~-D-xylopyransoyl-(l-+4)-a-L-rhamnopyranosyl-(l-+2)]-~-D-glucopyranosyl)-25R-5a-spirostane on the basis of chemical degradation and spectrometry.

INTRODUCIION

In continuation

of our chemical investigation studies on [l, 23, we now report the isolation and structure determination of a new spirostane hexaglycoside, agaveside D separated by reverse. phase HPLC. Agaoe

cantala

RESULTS AND DISCUSSION

Agaveside D, C,,H,,,03,, mp 256-260”, exhibited broad bands at 3397 and 1074 cm-’ in its IR spectrum. The ‘H NMR spectrum displayed two tertiary methyl groups at 60.65 and 0.76, and four secondary methyl groups at 0.75, 1.03, 1.06 and 1.07. IR absorption bands at 899 and 922 cm- ’ led to the prediction of the existence of spirostane skeleton which was, moreover, evident by the presence of a quaternary carbon resonance (C-22) at 6109.78 in its “%NMR spectrum [33. Its broad band ’ 3C NMR spectrum, in conjunction with the analysis of the DEPT spectrum, showed the presence of six methyl, 15 x methylene, 38 x methine and three quaternary car-

bon atoms. The presence of six anomeric ’ % resonances at 6102.91, 104.67, 104.81, 104.82, 104.97 and 105.45, along with six anomeric ‘H resonances at 64.78, 4.80, 5.05, 5.08, 5.17 and 5.44 led to the identification of a hexaglycoside moiety. On acid hydrolysis of compound 1, tigogenin was identified as the sapogenin residue Cl,43 whereas glucose, rhamnose and xylose in the proportion 3:2: I were determined (HPLC) as the sugar components. The positive ionization fast atom bombardment mass spectrum (+ve FAB-MS) confirmed its molecular formula by displaying a quasi-molecular ion [M + Na] + at m/z 1349. The presence of approximately equal abundant fragments at m/z 1217, 1203 and 1187 corresponding lo the loss of pentosyl (xylosyl), 6-deoxyhexosyl (rhamnosyl) and hexosyl (glucosyl) moiety, respectively, from the quasi molecular ion, led to the recognition of the terminal sugar residues. This was complemented by the presence of an ion at m/z 909 [M + Na)-440]+ due lo the loss of all the three terminal sugar moieties. Fragment ions at m/z 1071 and 879 corresponding lo the loss of disaccharide (Xyl

lCIMAP Communication No. 1096. SAuthor lo whom correspondence should be addressed. 4187

4188 Table

C I

2 3 4 5 6 7 8 9 IO II I2 I3 I4 15 16 I7 18 I9 20 21 22 23 24 25 26 27

Short I 13C NMR spectral

data ofcompound d in ppm)

Sapogenin moiety 1 37.15 29.82 79.34 34.74 44.64 28.85 32.30 35.19 54.42 35.19 21.17 40.10 41.89 56.37 32.02 80.52 62.95 16.45 12.19 41.93 14.87 109.13 31.76 29.14 30.50 67.07 17.19

1 (in pyridine-d,,

Sugar moiety GIG-I G-1 G-2 G-3 G-4 G-5 G-6 Glc-II G-l G-2 G-3 G-4 G-5 G-6 Rham I Rh-I Rh-2 Rh-3 Rh-4 Rh-5 Rh-6 Glc-III G-l G-2 G-3 G-4 G-5 G-6 Rham-II Rh-I Rh-2 Rh-3 Rh-4 Rh-5

Rh-6 Xyl- I xy- I xy-2 xy-3 xy-4 xy-5

102.91 81.69 86.22 69.46 78.93 61.14 104.67 80.42 88.72 69.46 78.93 61.14 104.81 71.13 73.04 73.04 67.34 17.78 104.67 72.47 77.92 72.00 75.82 63.43

Reports

moieties are involved in glycosidic linkage formation and the inter-glycosidic linkage is I + 3 [I, 21. The appearance of resonances at 680.42 x 2, and 81.69 suggested that C-2 of both glucose [7] and C-4 of the rhamnose [8] are also involved in glycosidic linkage formation. The rhamnose was substituted at C-2 of the inner glucose [I,71 as it resonated at 681.69 and xylose was substituted at C-4 of rhamnose as it appeared at fi80.42 [S]. The second rhamnose was substituted at C-2 of the outer glucose as it appeared at 680.42 [l,7]. The “C NMR shielding data of the sugar moiety of this compound closely resembled with that of agaveside C for which enzymatic and methanolysis studies were performed [2]. The methylated sugars were identified by PC, using R, values, colour reaction and authentic samples. The 2.3,4,6-tetra-Omethyl-D-glucose, 4,6-di-O-methyl-D-glucose. 2,3.4-tri0-methyl-L-rhamnose, 2,3-di-0-methyl-L-rhamnose and 2,3,4-tri-O-methyl-D-xylose gave R, values 1.0.0.46, 1.01. 0.91 and 0.94, respectively. 2,3,4,6-Tetra-O-methyl-D-glucase and 2,3,-di-O-methyl-t.-rhamnose were further confirmed by comparison with authentic samples. The idcntity of 4,6-di-O-methyl-D-glucose was further confirmed as it gave an intense pink colour with Wallenfel’s reagent. Hence agavesides D is 3fl-{z-L-rhamnopyranosyl-( l-+2), /?-D-&ICOpyranOSyl-(

105.45 73.38 75.82 71.13 67.34

I -2)1-p-

EXPERI?+lENTAL The isolation procedure and experimental same as reported elsewhere [ 1.21. HPLC C and D was carried out employing

conditions were the

of a mixt. of agavaeside

the following conditions.

C, B (30 cm x 3.9 mm i.d.), solvent:

Column: PBondapak

-H1O, 104.97 72.00 69.46 80.42 67.34 15.49

l-+3)-/I-D-glucopyranosyl-[P-D-

xylopyranosyl-( I +4)-z-L-rhamnopyranosyl-( D-glucopyranosyl}-25R-5z-spirostane (1).

agaveside

the isolation of agaveside D after repeated chromatography.

Agawwde v:Ecm-‘: 922,899

D (I).

C,,H,,,O,.

3397 (brOH)

powder.

2925.

and 866 (899,922).

mp

256-260

.

IR

1375. 1243. 1074 (br, C O-C.

ZSR-spirostane.

‘H NMR

(pyridine-

d,):0.65 (d, Me), 0.75 (d, J = 6.5 HI Me), 0.76 (d,. Me). 1.03 (d. J =6.5 Hz, MC), I.06 (d, J =6.5 Hz, CH), 1.07 (d, J =6.5 HL Me), 4.76, 4.80, 5.05, 5.08. 5.44 (anomerlc-H).

“C NMR:

see Tahle I.

Hydrolysis ofcompound 1. Compound I (IO mg) was treated wth 2 M HCI-dloxane (I : I, 5 ml) for 5 hr. under reflux and worked-up

as usual. The aglyconc was identified as tlgogcnin,

whereas the aq. hydrolysate

revealed the presence of glucose, 4: I : 5). The

rhamnose and xylose on PC (n-BuOH-HOAc-H,O, molar ratio (3:2: I) was determined x 3.9 mm.

+ Rha) and trisaccharide moieties (2 Glc+ Rha) and a trisaccharide moiety (2 Glc+ Rha) suggested the substitution of these to the internal glucosyl unit. Other fragment ions at m/z 747 and 601 were attributed to the loss of Xyl + 2 Glc + I and 2 Rha units, respectively, from the quasi molecular ion, hence a partial structure of the sugar residue could be deduced. The interglycosidic linkages and the position of attachment of the sugar chain to the aglycone were established by comparison of “C NMR data with those reported for the saccharide chain of related glycosides [2. 33. The chemical shifts of the anomeric carbon signals confirmed that ii11the monosaccharide units were substituted on Cl. Thus the chemical shifts of the terminal glucose rhamnose and xylose could be attributed. The absorption at 686.22 and 88.72 suggested that C-3 of two glucose

MeOH

2 12 nm. R,: agaveslde D = 4.39 min. C = 3.72 min. Flow rate 0.6 ml min ‘. This resulted in

(4: I), UV J.,,,

id.,

carbohydrate

by HPLC

analysis

(column: 30 cm

column)

MeCN-H,O

(73: 27) as solvent. Permethylation Compound

of compound

1 and hydrolysis

of the product.

1 (20 mg) in HMPA (5 ml) was treated with NaH

(300 mg) and Mel (5 ml) at room temp. for 3 hr. The reaction mixt. was worked-up prep.

as usual and the residue was purified by

TLC (petrol-EtOAc, 7: 1). Hydrolysis

of 1 was performed

5 ml), neutralized

by refluxing

of the permethylate

I M

5:I

:4)

2,3.4-tri-O-methyl-L-rhamnose.

xylose, 2,3-tri-O-methyl-L-rhamnose

HCI-MeOH

(I : I.

(PC sol2.3,4,6-terra-O-melhyl-o-

and the coned hydrolysate

vent, n-BuOH-EtOH-H,O: glucose,

with

showed

2,3.4-lri-O-methyl-D-

and 4,6-di-O-methyl-o-glu-

case. Partial

hydrolysis

of compound1 on

TLC. Compound

I was

gel TLC and left in a HCI atmosphere at room (30”) for I hr. HCI vapour was eliminated under hot

applied on silica temp.

ventilation

and

then

authentic

samples of the sugars were

Short Reports applied to the chromatoplate. The chromatoplate was developed with solvent system CHCI,-MeOH-Me&O-H,0 (3:3:3: 1) and spots were detected by spraying 10% aq. H,SO, followed by heating, glucose, rhamnose and xylose were identified. Acknowledgements-We

are grateful lo Finningan MAT, U.K.

for recording FAB mass spectra. The facilities provided by 500 MHz FT-NMR National Facility supported by the Depart-

ment of Science and Technology at TIFR, Bombay, are gratefully acknowledged.

REFERENCES 1. Uniyal, G. C., Agrawal, P. K., Thakur, R. S. and Sati, 0. P. (1990) Phytochemistry 29, 937.

4189

2. Uniyal, G. C., Agrawal, P. K., Sati, 0. P. and Thakur, R. S.

(1991) Phyrochemistry 30 (in press). 3. Agrawal, P. K., Jain, D. C., Gupta, R. K. and Thakur, R. S. (1985) Phytochemistry 24, 2479. 4. Patel, A. V., Blunden, B., Crabb, T. A., Sauvaire, Y. and

Baceou, Y. C. (1987) Fitoterapia S&67. 5. Agrawal, P. K. and Bansal, M. C. (1989) in Carbon-13 NMR of Flaoonoids (Agrawal, P. K., ed.), p. 283. Elsevier, Amsterdam 6. Agrawal, P. K., Srivastava, S. K. and Gaffield, G. (1990) in Alkaloids: Chemical and Biological Perspectives Vol. 7 (Pelletier, S. W., ed.). Wiley, New York (in press). 7. Nakano, K., Matsuda, E., Tsurumi, K., Yamasaki, T., Murakami, K. Takaishi, Y. and Tomimatsy T. (1988) Phytochemistry Zl, 3235. 8. Dutton, G. G. S., Merrifield, E. H., Laffite, C., Pratviel Sosa, F. and Wylde, R. (1982) Org. Magn. Reson. 20, 154.

Phyrochemisfry.Vol. 30, No. 12. PP. 4189 4190, 1991 Printed in Great Britain.

0031-9422191 $3.00+0.00 Pergamon Press plc

TWO PHENYLPROPANOIDS FROM TODAROA AUREA SUBSP. SUAVEOLENS ANTONIO G. GONZALEZ, JANE BERMUO BARRERA, LUIS ARANCIBIA L., JESUS G. DfAZ and PEDRO PEREZ DE PAZ* Centro de Productos Naturales Organicos Antonio Go&In, Consejo Superior de Investigaciones Cientilicas Carretera La Espe.ranza 2, La Laguna, 38206 Tenerife, Canary Islands, Spain; *Department0 de Both&a, Facultad de Farmacia, Universidad de La Laguna, Canary Islands, Spain (Received 22 April 1991)

Key Word Index-Todaroa

aurea; Umbelliferae; phenylpropanoids;

pmethoxytodadiol;

suavediol; falcarinol.

Abstract-Two new phenylpropanoids, p-methoxytodadiol and suavediol were isolated from the aerial part and roots of Todaroa aurea subsp. suaoeolens, together with the acetylenic compound, falcarinol.

INTRODUCITON

The genus Todaroa is endemic to the Canary Islands [l] and is presently considered to contain two subspecies: T. aurea subsp. aurea and T. aurea subsp. suaoeolens [2]. We have re-investigated the constituents of T. aurea subsp. suavelolens.

&I,-CHOH-CH,OH

&H,--cHOH-CH,~H

RESULTS AND DISCUSSION

The extract of the aerial part of the plant afforded /Iamyrin, scopoletin, luteolin, the phenylpropanoids myristicin, todadiol, elemicin, apiole [3], p-methoxytodadiol (1) and the acetylenic falcarinol [4], while falcarinol, stigmasterol, sitosterol, scopoletin and two phenylpropanoids, apiole and suavediol(2). were obtained from the root extract. Compound 1, p-methoxytodadiol, was isolated as an oil and had IR signals for a hydroxy group (3580 cm- ‘)

and an aromatic ring (1580, 1490 cm-‘). Its mass spectrum showed the molecular ion [M]’ at m/z 242, C12H1s05, and a base peak at m/z 181 (loss of C,H,O,). From the ‘HNMR spectrum (Table 1) and the mass spectral fragmentation, 1 was seen lo be an aromatic compound with a -CH,XHOHXH,OH side chain; a deformed doublet at 62.62 for an Ar-CH,-group. a deformed double doublet at 63.41 and a multiplet at