Phytochemktry, VoL 31, No. 7, pp. 243S2438. 1992 Rimed in Great Britain.

0

A y-PYRONYL-TRITERPENOID SEIJI TSURUMl,*t Wraduate

SAPONIN

TOSHIHIRO

TAKAGI~

FROM

PISUM

0031.9422p2 f5.00+0.tm 1992 PergsmonPressLtd

SATZVUM

and TOHRU HASHIMOTOQ

School of Science and Technology, Kobe University, Rokkodai, Nada-ku, Kobe 657, Japan; and $De.partment of Biology, Faculty of Science, Kobe University, Rokkodai, Nada-ku, Kobe 657, Japan (Received 4 September 1991) Key Word Index-Pisum satiuwn; Leguminosae; derivative; chromosaponin 1.

pea; phytochrome

inhibitor; triterpenoid saponin; soyasaponin

I

Abstract-A new triterpenoid saponin was isolated from Pisum satiuum and characterized as 3-0-[U-Lrhamnopyranosyl-(l+2)-fi-D-galactopyranosyl( 1-+2)+~-glucuronopyranosyl(1 +)I-22-0-[3’-hydroxy-2’-methyl5’,6’-dihydro-4’-pyrone(6’~)]-3B,22B,24-trihydroxyolean-l2-ene. The name chromosaponin I is proposed. Chromosaponin I yielded soyasaponin 1, known as phytochrome inhibitor, during extraction, but the latter was not found in the free form in this plant.

INTRODUCflON

UV-B light (280-320 nm) induces anthocyanin and flavonoid syntheses in plants by itself, or synergistically with red light [l-4]. The involvement of a UV-B photoreceptor has been suggested, based on action spectra [ 1, 4, 53. The current approach to discover the putative photoreceptor is to search for a compound which has absorption at this wavelength. This paper describes a new triterpenoid saponin which is a conjugate of a y-pyrone and soyasaponin I, and has an absorption maximum at 295 nm. We also report that soyasaponin I, known as a phytochrome inhibitor, is released from this saponin during extraction. RESULTS AND DlSCUS!3lON

The methanolic extract from etiolated pea seedlings was purified by Sephadex LH-20 column chromatography and reversed-phase HPLC to give a UV-B absorbing substance, compound 1, i”,g” nm (log E); 295 (3.89), mp 210-212” (dec.), C,,H,,O,, (found C. 60.40, H, 7.88%: requires C, 60.67, H, 7.87%). Compound 1 did not give a molecular ion in the FDMS spectrum, but the analysis of its hydrolysate indicated that 1 was a conjugate of a triterpenoid saponin and a 7-pyrone, confirmed by the ‘H and “C NMR spectra. Mild alkaline hydrolysis of 1 gave products 2 and 3. Compound 2, C,H,03 (found 126.0369; talc. 126.0317), was identified with 3hydroxy-2-methylA-pyrone (Aldrich) by comparison of IR, UV and NMR spectra as well as mp. Acid hydrolysis of 3 gave an adycone (4) and D-ghtcuronolactone, Dgalactose and L-rhamnose. The aglycone 4 was identified with soyasapogenol B by HR mass, IR and NMR spectra as well as mp and [zlo [68]. The FD mass spectrum of 3 gave a molecular ion at m/z 965 [M+Na]’ and fragment ions at m/z 819 [M+Na -Rha]+,657[M+Na-Rha-Gal]+and481 [M+Na

*Author to whom correspondence

should be addressed.

CHflH

3 R = GlcA(2--l)-Gal(2-1).Rha 4 R-H GlcA = f~UglUCufOnOpyranOSYl

Gal = lbwgalactopyranosyl Rha = tr-L-rhamnopyranosyl

- Rha -Gal - GlcA] +, indicating the presence of rhamnose-galactose-glucuronic acid-soyasapogenol B. Comparison of the 13CNMR data of 3 with those of 4 suggested that the sugar moiety in 3 was linked to the C-3 hydroxyl group of 4. The C-3 signal (680.1) of 4 was shifted downfield by 6 11.0 due to the glycosidation shift [9] (Table 1). Measurement of NOE difference spectra of

2435

S. TSURUMI et al.

2436

Table

I. 13C NMR chemical

C

1

I 2 3 4 5 6

I 8 9 10 II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 diHPyr-2’ 3’ 4 5’ 6 7’ GlcA- 1 2 3 4 5 6 Gal-l 2 3 4 5 6 Rha-I 2 3 4 5 6

38.5 t 26.6 t 91.1 d 43.8 s 55.9 d 18.5 I 33.0 t 40.0 s 41.1 d 36.3 s 24.0 t 122.6 d 144.1 s 41.9 s 26.2. f 27.7’ r 37.1 5 44.4 d 46.0 f 30.5 5 36.4 r 81.8 d 23.0 q 63.6 r 15.8 q 16.8 q 26.2 q 21.1 q 33.6 q 21.7 q 152.8 s 134.9 s 185.9 s 42.4 t 97.4 d 15.7 q 105.4 d 78.4t d 76.6t d 73.8: d 77.7: d 172.4 s 101.7 d 77.8 d 76.6 d 71.1 d 76.4 d 61.5 t 102.5 d 72.4 d 72.8 d 74.3 d 69.4 d 18.9 q

shifts of compounds as int. standard) 3 38.6 t 26.6 t 91.1 d 43.8 s 56.0 d 18.5 f 33.2 t 39.9 s 47.8 d 36.4 s 24.1 t 122.3 d 144.8 s 42.3 s 26.4* t 28.6* t 38.0 s 45.2 d 46.7 I 30.9 s 42.3 t 75.5 d 23.0 q 63.6 [ 15.8 q 16.9 q 25.7 q 21.1 q 33.3 q 28.6 q

14 (62.9 MHz, pyridine-d,,

4

TMS

2

38.9 t 28.4 t 80.1 d 43.2 s 56.3 d 19.1 t 33.5 t

40.0s 48.1 d 37.0 s 24.1 t 122.4 d 144.8 s 42.3 s 26.4’ [ 28.7’ t 38.0 s 45.3 d 46.8 r 30.9 s 42.3 t 75.5 d 23.6 q 64.6 [ 16.3 q 17.1 q 25.7 q 21.1 q 33.3 q 28.7 q Pyr-2 3 4 5 6 7

149.4 144.9 173.6 114.4 154.0 14.1

s s s d d q

105.4 d 78.4t d 76.6t d 73.9: d 77.7: d 172.5 s 101.7 d 77.8 d 76.6 d 71.1 d 76.4 d 61.5 t 102.4 d 72.4 d 72.7 d 74.3 d 69.4 d 18.9 q

diHFyr = 3’-hydroxy-2’-methyl-5’,6’-dihydroxy-4’-pyrone. Pyr = 3-hydroxy-2-methyl4pyrone. VSAssignments in each vertical column may bc reversed.

Triterpenoid saponins from Pisum Table 2. Distribution of chromosaponin

Organs Top part above 2nd node Plumular leaves &2 mm segment (hook) 2- 12 mm segment 12-22 mm segment 22-32 mm segment 2nd Internode Upper 15 mm segment Middle 15 mm segment Lower 15 mm segment 1st Internode Upper 15 mm segment Lower 15 mm segment Cotyledons Root 3&35 mm segment 5-10 mm segment (r5 mm segment

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satiuum

I (1) in seven-day-old pea seedlings

Fr. wt of plant (mg segment _ ‘)

Content (jlgg-’ fr. wt)

0.32 0.22 0.78 0.83 0.90

950 3438 195 339 337

2.15 2.20 2.10

238 311 351

2.00 2.05 342.0

506 451 792

0.11 0.11 0.10

891 1039 2473

Size of seedling: co 35 mm in the top part, 50 mm in the 2nd internode, 35 mm in the 1st internode and 80 mm in the root. The figures before segment in the top part and root represent the distance from the respective tip.

3 supported the linkage of sugars. Irradiation

at 63.37 (H3), 65.81 (Gal H-1)and 66.30(Rha H-1)resulted in signals at 64.98 (d, J = 8 Hz, GlcA H-l), 64.61 (m, GlcA H-2) and 64.58 (t, J = 8 Hz, Gal H-2), respectively. The configurations of the anomeric protons of the three sugars were assigned on the basis of their 3.1,2 values (Rha, 2.2 Hz; Gal, 7.6 Hz, GlcA, 8.0 Hz) measured by J resolved 2D analysis and the ‘J,-- of Rha C-l (169.8 Hz) [lo]. Compound 3 was deduced to be 3-O-[cr-L-rhamnopyranosyl( l-2)-/?-D-gaiactopyranosyl( l--+2)-B-D-g~ucuronopyranosyl(1 -+)I-soyasapogenol B, i.e. soyasaponin I [6, 11, 123. This was confirmed by comparison of the spectral data of 3 with those of a soyasaponin I sample supplied by Professor Yokota. Comparison of the 13C and ‘H NMR data of 2 with those of 1 revealed that the double bond between C-5 and C-6 in 2 was replaced by a single bond in 1. The signals of 2,6 114.4 (d, C-S) and 6 154.0 (d, C-6), were shifted upfield by 672.0 and 656.6, respectively. The ‘H NMR spectrum of 1 revealed an ABX structure, 62.84 (lH, dd, J= 16.6 and 3.5 Hz, H-5’). 63.13 (lH, dd, J= 16.6 and 3.5 Hz, H5’) and 6 5.49 (lH, t, J= 3.5 Hz, H-6’) instead of the double bond between 66.43 (lH, d, J = 5.5 Hz, H-5) and 67.72 (lH, d, J= 5.5 Hz, H-6) in 2. The ‘H-l% longrange COSY spectra ( J,-- = 7 and 11 Hz) of 1 exhibited cross peaks between C-2’ and H-6’, C-3’ and H-5’, C-3’ and H,-7’, C-4’ and H-6’, and C-6’ and H-22, respectively, suggesting that the 5’,6’-dihydroq-pyrone moiety is linked to C-22 hydroxy group of 3. The linkage was confirmed by the NOE difference spectrum of 1 and a comparison of the 13CNMR data of 1 with those of 3. Irradiation at d 3.57 (s, H-22) resulted in a signal at 6 5.49 (t, H-6’). The C-22 signal (675.5) of 3 was shifted downfield by 66.3, and the C-21 and C-17 signals were shifted upfield by 65.9 and 60.9, respectively, which has been observed in glycosidation [9]. The above spectral data

identified 1 as 3-0-[a-L-rhamnopyranosyl( 1+2)-/?-Dgalactopyranosyl( 1-r2)-fi-D-g~ucuronopyranosyl( 1+)I22-0-[3’-hydroxy-2’-methyl-5’,6’-dihydro-4’-pyrone (6’-+)]-3/?,22&24-trihydroxyolean-12-ene. Thus, compound 1 is new and the name chromosaponin I is proposed for it. In seven-day-old etiolated pea seedlings, 1 was found in all parts of seedlings, but at higher concentration in the hook and root tip than in other non-growing tissues (Table 2). On the other hand, 3, which was reported as a phytochrome inhibitor in pea shoots [6], was almost undetectable. Extraction of frozen tissues or prolonged extraction of unfrozen tissues produced 3, accompanied by reduction of 1.Compound 3 may be an artefact derived from 1 during extraction. EXPERIMENTAL Mps: uncorr. ‘H and 13CNMR spectra were measured at 250 MHz and 62.9 MHZ respectively, with TMS as int. standard. FDMS and EIMS were obtained with a direct inlet system. HPLC was performed using a TSK gel ODS 120T column (300 x 7.8 mm) at co 60 kgcm-‘, detected at 210 nm or 290 nm. Assignment of ‘H and “C NMR spectra was made by the data from DEPT. and ‘H-‘H, IH-“C and long-range 1H-‘3C COSY. Plant muterid. Pea seeds (Pisum satiuum L. cv Alaska purchased from Watanabe Saishujo, Kogota, Japan) were sown and seedlings were grown in wet vermiculite in the dark at 25” for 7 days). Extraction and isolation. Seedlings (140 g fr. wt) were homogenized with 750 ml of cold 80% aq. MeOH in a homogenizer and the homogenate was centrifuged at 8000 8 for 10 min. The supernatant was concd under red. pres. and chromatographed with a Sephadex LH-20 column eluted with 80% MeOH-O.l% HOAc. The eluate containing 1 was repeatedly subjected to

2438

S.

TSURUMI et al.

reversed-phase HPLC eluted with 80% MeOH~.OS% HOAc. The eluate was further purified by reversed-phase HPLC eluted with 72% MeOH-0.05% HOAc to give compound I (36.6 mg). Compound I. White powder, M, 1068, mp 210-212’ (dec.). [zJ~’ --X2.6- (90% MeOH; c 0.5). (Found C, 60.40; H, 7.88%. C 54H “40 21 requires; C. 60.67; H, 7.87%.) IR I!::; cm-‘: 3468 (OH). 2944, 1736 (C=O), 1630, 1432, 1382. 1158. 1050. UV A$5)” nm (loge): 295 (3.89). FDMS 20 mA mjz (rel. int.): 965 [M +Na-diHPyrJ_ (100). 943 [M+H-diHPyr](43). 819 [M +Na-diHPyr-Rhaj’ (82). 797 [M+H-diHPyr-Rha]’ (50). 657 [M + Na -dlHPyr - Rha -Gal] * (I I), 481 [M + Na -diHPyr-Rha-Gal-GlcA]’ (IO). 459 [M+H-diHPyr -Rha-Gal-GlcA]‘(51). ‘HNMR(pyrldine-d,):60,69(3H,s. H-25), 0.89 (3H. s, H-26). 0.93 (3H, s, H-29). 1.01 (6H, s, H-28 and H-30). 1.25 (3H. s. H-27). 1.43 (3H. s. H-23). 1.78 (3H. d, J -6.0 H7, Rha H-6). 2.30 (3H, s, diHPyr H-7’), 2.84 (IH. dd, J H-5’). 3.13 (IH, dd, 5=16.6. 3.5Hz. -16.6, 3.5117.. diHPyr diHPyr H-5’), 3.25 (IH, d. J = 11.4 Hz. H-24), 3.36 (IH. dd. J = 10.8, 3.0 Hz. H-3). 3.57 (IH. hr. H-22), 3.95 (IH, t, 5-6.1 Hz, Gal H-5), 4.11 (IH, dd, J=9.6, 3.1 Hz. Gal H-3), 4.82 (IH, d, J =3.3 Hz, Rha H-2). 4.95-5.08 (ZH, m. GlcA H-l and Rha H-5), 5.11 (IH, hr. H-12). 5.49 (IH, I, J=3.5 Hz, diHPyr H-6’). 5.81 (IH.d,J=7.4H~.GalH-1).6.29(lH..s.RhaH-l).”CNMRsee Table I ./l/ka/inr hydrolys~.s ofcompound 1. A soln of l(76.4 mg) in 10 ml of 80% McOH contaming 26 mM NaOH was kept at 4” overnight. The hydrolysis was stopped by acidification with HOAc, and hydrolysatcs were subjected to reversed-phase HPLC eluted with 76% MeOH-0.1 % HOAc. The cluate (12 to 20 ml) was further purified through the same column eluted with 30% McOHa.I% HOAc to give compound 2 (4.4 mg). The eluate (42.-50 mll from the former HPLC gave compound 3 (57.4 mp). Compound 2. Brown powder. mp 15X- 160” (needles crystallized from 60% MeOH, mp 16t&161”), HRMS 70eV m/z: 126.0309 CM] * (talc. for C,HBO,, 126.0317). EIMS 70 eV m/z (rel. int.): I26 LM]’ (100). 97 (22). 71 (54). 55 (35). 43 (45). UVi~~~“‘“” nm (logc): 276 (3.95) at pH 5. 320 (3.90) at pH 8. IR viz cm ‘: 3264 (OH), 1658 (C=O), 1622. 1562. 1462. 1256, 1222. 1IYX. 916, 842, 506. ‘H NMR (CDCI,); 62.37 (3H. s, H-7). 6.43 (IH. d, J=5.5Hz. H-5. 7.72 (lH, d, J=5.5Hz, H-6). “CNMR (CDCI,); 6 14.3 (4. C-7). 112.9 (d, C-5). 143.1 (s, C-3). 148.7 (s. C-2). 154.3 (d, C-6). 172.9 (s, C-4). Compound 3. White powder, mp 216 217’ (plates crystallized from 70% MeOH, mp 220-221 ), Lz]b’ -6.0’ (MeOH: c 1.0). (Found C. 58.95: H, 8.26”/0. Calc. for C,,H,,O,,.ZH,O: C, 58.90: H, X.38%.) IR Y::: cm- ‘: 3452 (OH). 2Y44. 1728 (C=O), 1460. 1430. 1384. 1254, 1226. 1044. FDMS 20 mA m:‘z (rel. int.): 965[M+Na]‘(100),943[M+H]‘(22),819[M+Na-Rha]’ (41),797 [M+H-Rha]+ (33).657[M+Na-Rha-Gal](13). 481 [M+Na-Rha-Gal-GlcA]’ (13), 459 [M+H-Rha -Gal -GlcA](14). ‘H NMR (pyridine-d,); 6; 0.70 (3H, s, H25), 0.96 (3H, s. H-26). 1.00 (3H, s. H-29). 1.23 (3H. s, H-28), 1.30 (6H, s. H-27 and H-30). 1.44 (3H. s, H-23). 1.7Y (3H. d, J = 6.1 Hz, Rha H-6).2.19(1tf.d,J=9.1 HIH-~).~.~O(IH,~,J=I~.~H~, H-18). 3.25 (IH, d. J-11.3Hz. H-24). 3.37 (IH, dd, J=ll.O, 3.7 HI H-3). 3 75 (IH. t, H-22), 3.95 (IH, t. J=5.9 HL Gal H-5), 4.12(1H,dd.J=9.5,3.1 Hz,GalH-3),4.83(lH,d,J=3.2Hz,Rha H-2). 4.96. 5.09 (ZH, m, GlcA H-l and Rha H-5), 5.30 (IH, hr, H12). 5.81 (IH, d, J-7.4Hz. Gal H-l). 6.30 (IH, s, Rha H-I). ‘+Z NMR. see Table I Acid hydrolysis offcompound 3. A soln of 3 (30.7 mg) in 5 ml of 2 M HCI-MeOH was heated at 100” for 40 min. and then partitioned with CHCl,-H,O. The CHCI, layer was washed with H,O and coned to dryness. The residue was subjected to reversed-phase HPLC elutcd with 79% MeOH lo give compound 4 (9.3 mg).

Compound 4. White powder, mp 258-260”. [XI;” +81.2’ (CHCI,; c 05). HRMS 20 eV m/z: 458.3752 [MJ’. (Calcd for C,OH,,,O,, 458.3757.) EIMS 20 eV m,/z (rel. int.): 458 [M]’ (2), 440(1),234(100),224(9),219(24).1Rv~~:m~’:3400(0H).2948, 1464. 1382, 1038, 752. ‘H NMR (pyridine-d,); ci 0.97 (3H, s. H25). 1.01 (3H, s. H-29). 1.03 (3H, s. H-26). 1.26 (3H. s, H-27). 1.26 (3H, s, H-28), 1.33 (3H, s, H-30). 1.59 (3H, s. H-23). 2.44 (I H. d. J H-24and H-22),4.55(lH, = I2 Hz, H-18),3.65-3.78(3H,m.H-3, dd,5=10.9,2.3Hz,H-24),5.36(1H,r,J=3.6Hz.H-12),5.46(lH, dd, J-8.1, 2.3 Hz, OH-24). 5.77 (IH, d. J-4.4 Hz. OH-22). 6.78 see Table 1. (IH, d, J=4.8 Hz_ OH-3). “CNMR An&is ofsugars. A soln of 3 (3.0 mg) in 1 ml of 2 M HCI-50% dioxane was heated at 100’ for I hr and coned to dryness. The products were suspended in1.5 ml of CHCI, and extracted with H,O (2 ml x 5). The aq. fraction was analysed on a TSK gel Amido 80 column (250 x 4.6 mm) eluted with 80% aq. M&N. DGlucuronolactonc, u-galactose and L-rhamnose were detected at a molar ratio of 0.6: 1.0: 1.0. Quanriforiur anulysis of 1 and 3. Seven-day-old etiolated pea seedlings were divided as shown in Table 2. Each part of them was collected and homogenized with 80% aq. MeOH. The homogenate was filtered, coned and loaded to a reversed-phase column (250 x 4.6 mm, TSK gel ODS 120T) eluted with 80% MeOH~.OS% HOAc at cu 100 kgcm-‘. Compound I was quantified at 290 nm. and 3 at 210 nm.

Acknowledgements--We thank Professor A. Sera. Department of Chemistry, Kobe University, Kobe, Japan for his valuable criticism; Professor T. Yokota. Teikyo University. Utsunomiya. Japan for a gift of soyasaponin I; Dr K. Saiki, Kobc Women’s College of Pharmacy, Kobe, Japan for measuring MS: Mrs Nishinaka, Kobe University. Kobe, Japan for elementary analysis and Dr Y. Takeuchi. the Aburahi Laboratories, Shionogi Seiyaku Ltd, Aburahi Japan for technical advlce. This work was partly supported by a grant-in-ald 10 T. H., no. 63480015, from the Ministry of Education, Japan. and a grant to T. H., no. 861152. from the Yamada Science Foundation. Osaka, Japan.

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