Limonoid glucosides in calamondin seeds

003l- 9422/92S5.00+ 0.00 PergamonPressplc

Vol.31,No. 3, pp. 10441046,1992 Phytochemistry, Primedin Great Bntam.

LIMONOID

GLUCOSIDES

IN CALAMONDIN

SEEDS

MASAKI MIYAKE,* YOSHIHIKOOZAKI,* SHIGERUAYANO,*RAYMONDD. BENNETT, ZAREB HERMAN and SHIN HASEGAWA United States Department of Agriculture, Agricultural Research Service, Fruit and Vegetable Chemistry Laboratory, 263 South Chester Avenue, Pasadena, CA 91106, U.S.& *Wakayama Agricultural Biological Research Institute, Momoyama-cho, Wakayama 649-61, Japan (Received in revised form 24 July 1991)

Key Word Index-Citrus pyranoside;

limonoid

reticulata glucosides.

var. austera

x Fortunellu

sp.; Rutaceae;

calamondin;

calamm

17-/?-D-ghCO-

Abstract-Three new limonoid glucosides were isolated from seeds of calamondin (Citrus reticulata var. austera x Fortunella sp.). They were the 17-/?-o-glucopyranosides of calamin, methyl deacetylnomilinic acid and 6-keto-7/ldeacetylnomilol. In addition, the 17-p-D-glucopyranosides of nomilinic acid and deacetylnomilinic acid were also isolated.

INTRODUCTION Limonoids are a group of chemically related triterpenoids present in Rutaceae and Meliaceae. They are one of the bitter principles in citrus juices. Recently, limonoids have been shown to be present as glucoside derivatives in Citrus [l, 23. The glucosidation of lirnonoids occurs in fruit tissues and seeds during late stages of fruit growth and maturation, and is considered to be a natural debittering process in Citrus [3]. Calamondin contains, in addition to the citrus limonoids, a calarnin group of limonoids which differ from citrus limonoids. In the calamin group, either the carboxyl group at C-3 is methylated or the B-ring is oxygenated at C-6 or both [4]. In this study, we have analysed seeds of calamondin for limonoid glucosides. RESULTSAND

R’

R=

2

H

Me

5

AC

H

1H

H

DISCUSSION

Five limonoid glucosides were isolated from calamondin seeds, three are novel glucosides. Compounds 1 and 5 were identified by their NMR spectra [2] as the 17+-Dglucopyranosides of deacetylnomilinic acid and nomilinic acid, respectively. Compound 2 showed ‘H and 13C NMR spectra almost identical to those of deacetylnomilinic acid glucoside (l), which we had previously isolated from grapefruit seeds [2]. The only significant difference from 1 was the presence of a three-proton singlet at 63.62 in the ‘HNMR spectrum, assignable to a methyl ester group, and a corresponding methyl signal in the 13CNMR spectrum at 6 50.9. As methyl deacetylnomilinate is known to be a constituent of calamondin seeds [4], the new compound is clearly its glucoside, methyl deacetylnomilinate 17-#&Dglucopyranoside (2). The ‘H and r3CNMR spectra of the next isolated compound (3) were quite similar to those of 2, except that one of the methylene carbons had been replaced by an oxygenated methine. This suggested that it is a glucoside of calamin [4], which is the 6-keto-7/l-hydroxy analogue of 2. In fact, the resonances of carbons 1-12 of the new 1044

0

4

Short Reports

compound were almost identical to those of calamin, while those of carbons 13-23 showed the shifts expected for a 17-glucoside Cl]. The point of attachment was confirmed to be the 17-position by the observation of a strong cross peak between H-17 and glucose H-l in the ‘H-lH NOESY spectrum. Furthermore, all of the expected connectivities were observed in ‘H-lH COSY, ‘H-‘H NOESY, ‘HI-‘% COSY and long range ‘H-13C COSY spectra. In particular, cross peaks between H-7 and H-15 in the ‘H-lH NOESY spectrum and between C-6 and both H-5 and H-7 in the long range ‘H-13C COSY spectrum showed the presence of the 6-keto-7hydroxy moiety. As the glucose resonances in the *‘CNMR spectrum were identical to those of the previous limonoid glucosides Cl, 21, we assign the structure calamin 17-b-D-glucopyranoside (3) to this compound. The ‘H and 13C NMR spectra of 4 showed the presence of all of the functional groups of 3 except the methyl ester. An upfield shift of the H-l resonance and a downfield shift of the C-4 resonance were consistent with the formation of an A-ring lactone, which would lead to a structure of 6-keto-7/?-deacetylnomilol for the aglycone. This compound was previously isolated from calamondin seedlings [S], and in its 13C NMR spectrum the resonances of carbons 1-12 closely matched those of the glucoside. All of the NMR spectral data, including ‘H--lH COSY, ‘H-‘H NOESY and lH-‘jC COSY spectra, of the latter were completely consistent with the structure 6-keto-7B-deacetylnomilol 17$-Dglucopyranoside (4) for this compound. The biosynthetic pathways of limonoids in calamondin have been well established [6,7]. In this study, we found that calamondin seeds contained limonoid glucosides of both the citrus and calamin groups. It is of interest to note that these glucosides are derivatives of limonoid aglycones that are involved in the early stages of the biosynthetic pathways. This trend has been observed in other citrus seeds [S], suggesting that two reactions, the glucosidation of aglycones and the biosynthesis of aglycones, are competing against each other. This explains why seeds accumulate both aglycones and glucosides in large quantities [S, 91. In fruit tissue only the glucosides accumulate in relatively large quantities [3].

Gene&.

Calamondin

fruit

(Citrus

reticuluta

x Fortundu sp.) was obtained from the University

var. austera of California,

Riverside. Pectin= (Aspergilhs niger) and Amberlite XAD-2 were obtained from Sigma, A prep. HPLC column, C-18 reversephase (Partisil

ODS-3,2.2

Each fr. was refractionated on the same prep. column, eluting linearly with a gradient of MeGH-Hz0 (3: 17-11:9) for 150 min. Each fr. was analysed by TLC and HPLC by the procedures described previously [1, 2, 81. TLC plates were developed with EtOAc-MeCOEt -HCO,H (88%)-H,O (5: 3: 3: 1). The plate was then sprayed with Ehrlich’s reagent and the colour developed in a HCl gas chamber. The isolates were analysed also by HPLC using a C-18 analytical column. The column was eluted by using a linear gradient starting with 10% and ending at 60 min with 26% MeCN in 3 mM H3P04. Compounds were detected by UV absorption at 210 nm. 13CNMR assignments were made on the basis of DEPT, ‘H-13C COSY and long rang e ‘H-l 3C COSY spectra, and comparison with spectra of related compounds for which assignments had previously been made, Methyl deUCetyinOmiiinUte 17-j%D-ghCOp~UnOSide (2).

IHNMR (270 MHz, DMSO& 900): 60.79 (3H, s, Me-8), 0.98 (3H, s, Me-lo), 1.07 (3H, s, Me+), 1.16 (3H, s, Me&), 1.42 (3H, s,Me-13),2.79(1H,s,H-15),3.62(3H,s,Meester),4.12(1H,m,H1),4.14(1H,d,J=7 Hz,GlcH-1), 5_18(lH,s, H-17),6.52(1H,s, H-22), 7.39 (IH, s, H-23), 7.48 (lH, s, H-21); ‘“CNMR (67.8 MHz, DMSO-I,, 90”): 615.3 (Me-lo), 16,3 (Me-8), 16.8

(C-11), 24.6 (Me-13), 26.9 (C-12), 27.7 (Me+), 32.8 (Me-44,37.9 (C-2), 39.5 (C-6), 39.6 (C-9), 43.7 (C-13), 44.8 (C-lo), 47.0 (C-5), 50.9 (Me ester), 5 1.3(C-8), 58.3 (C- 15), 61.6 (Glc C-6), 70.4 (C- 14), 70.7 (Glc C-4), 73.2 (C-l), 73.3 (C-4), 74.2 (Glc C-Z), 76.2 (Glc C-5), 77.1 (Glc C-3), 77.6 (C-17), 104.4 (Glc C-l), 112.7 (C-22), 125.8(C20), 140.6 (C-23), 141.5 (C-21) 169.2 (C-16), 173.2 (C-3), 213.2 (C7). Calamin 178-D-glucopyrunoside (3). ‘H NMR (270 MHz, DMSO-d,, W): SO.50 (3H, s, Me-8), 0.90 (3H, s, Me-lo), 1.11 (3H, s, Me-4ol), 1.36 (3H, s, Me-13), 1.37 (3H, s, Me-4#?), 2.94 (lH, s, H-5), 3.59 (3H, s, Me ester), 3.86 (lH, s, H-15), 4.14 (lH, d, J =7 Hz, Glc H-l), 4.27 (lH, s, H-7), 4.58 (lH, m, H-l), 5.26(1H, s, H-17) 6.50 (lH, s, H-22), 7.36 (IH, s, H-23), 7.44 (lH, s, H-21); t3CNMR (67.8 MHz, DMSO-d,, 900): 613.1 (Me-s), 16.0

(Me-lo), 18.0 (C-11), 23.3 (Me-13), 26.8 (C-12), 27.7 (Me+, 32.8 (Me-rla), 37.3 (C-2), 41.0 (C-9), 43.6 (C-13), 49.8 (C-8), 50.7 (Me ester), 52.2 (C-lo), 58.2 (C-15), 61.7 (Glc C-6), 63.6 (C-5), 70.8 (Glc C-4), 71.6 (C-4), 72.4 (C-l), 74.3 (Glc C-2), 75.5 (C-14), 76.1 (Glc C-5), 77.1 (Glc C-3), 77.1 (C-17), 81.1 (C-7), 104.2 (Glc C-l), 112.6 (C-22), 126.2 (C-20), 140.4(C-23), 141.3(C-21), 170.2 (C-16), 172.4 (C-3), 210.7 (C-6). 6-Keto-7j?-deacetylnomilol

EXPERIMENTAL

x 25 cm, particle

size 10 pm) and an

analytical HPLC column, C-18 reverse-phase (4.6 x 250 mm, particle size 5 pm) were used. Silica gel HLF plates were obtained from Analtech (Newark, DE). Isolation ofglucosides. Calamondin se&s (200 g) were ground in 3 1 H,O with a Polytron homogenizer, and the pH was then adjusted to 4.0. Pectinase was added and the homogenate was stirred for 20 hr. The mixture was then centrifuged at 13 Ooo g for 15 min, and the supematant filtered. The clear filtrate was transferred onto the top of an XAD-2 column (3.0 x 75 cm), and the column was washed thoroughly with HzO. The iimonoid glucosides were eluted with MeOH. After evapn of the MeOH, the residue was dissolved in H,O and fractionated on a C-18 reverse phase prep. HPLC column. The column was eluted at 3 ml min-l with a linear gradient system of MeCN-Hz0 (3 : 17-l 1: 19) for 150 min. The glucosides were collected in 5 frs.

1045

178~D-glucopytQnosids

(4).

‘HNMR (270 MHz, DMSO-de, 90”): SO.54 (3H, s, Me-8), 0.85 (3H, s, Me-lo), 1.26 (3H, s, Me-&), 1.35 (3H, s, Me-13), 1.65 (3H, s, Me+)), 3.49 (lH, s, H-5), 3.63 (lH, m, H-l), 3.85 (lH, s, H-15X 4.14 (IH, d, J =7 Hz, Glc H-l), 4.36 (lH, s, H-7’),5.30 (lH, s, H17), 6.51 (lH, s, H-22), 7.37 (lH, s, H-23), 7.45 (lH, s, H-21); 13CNMR (67.8 MHz, DMSO-d,, 90”): 6 13.2 (Me-8), 14.4 (Melo), 15.5 (C-11), 23.1 (Me-13), 24.7 (Me+I), 26.5 (Me-12), 32.2 (Me-h), 37.2 (C-2), 38.6 (C-S), 44.0 (C-13), 49.6 (C-lo), 50.7 (C-8), 58.3 (C-15), 59.6 (C-5), 61.7 (Glc C-6), 66.7 (C-l), 70.8 (Glc C-4), 74.3 (Glc C-2), 75.3 (C-14), 76.1 (Glc C-S), 76.9 (Glc C-3), 77.2 (C-17), 80.8 (C-4), 81.3 (C-7), 104.2 (Glc C-l), 112.5 (C-22), 126.2 (C-20), 140.5 (C-23), 141.2 (C-21), 170.1 (C-16), 170.4 (C-3), 209.5 (C-6).

REFERENCES 1. Hasegawa, S., Bennett, R. D., Herman, Z, Fong, C. H. and

Ou, P. (1989) Pkytochemistry B, 1717. 2. Bennett, R. D., Hasegawa, S. and Herman, Z (1989) Phytochemistry 28, 2777.

1046

Short Reports

3. Hasegawa, S., Ou, P., Fong, C. H., Herman, Z., Coggins, W. C., Jr. and Atkin, D. R. (1990) J. Agric. Food Own. 39,262. 4. Bennett, R. D. and Haaegawa, S. (1981) Tetrahedron37, 17. 5. Hasegawa, S., Bennett, R. D. and Herman, Z. (1986)Phytochemistry25, 1984. 6. Herman, Z., Bennett, R. D., Ou, P., Fon8 C. H. and Hasegawa, S. (1987) Phytochemistry26, 2247.

7. Hasegawa, S., Herman, Z., Ou, P. and Fong, C. H. (1988) Phytochemistry27, 1349. 8. Ozaki, Y., Miyakc, M., Maeda, H., Ifuku, Y., Bennett, R. D., Herman, Z., Fong, C. H. and Hasegawa,S. (1991) Agric. Biol. Chem. 55, 137. 9. Hasegawa, S., Bennett, R. D. and Verdon, C. P. (1980) J. Agric. Food Chem. 28,922.

Phytoclzemistry,Vol. 31, No. 3, pp. 1046-1048,1992 Printed in Great Bntain.

0031-9422/92SS.OC+O.OO 0 1992Pergamon Press plc

A BIDESMOSIDIC OLEANOLIC ACID SAPONIN PANAX PSEUDO-GINSENG*

FROM

YOGENDRA N. SHUKLA, RAGHUNATH S. THAKUR and PETER PACHALY~ Central Institute of Medicinal and Aromatic Plants, Lucknow 226016, India; tPharmaceutica1 Germany

Institute, University of Bonn,

(Received 3 June 1991)

Key Word Index-Panax

pseudo-ginseng subsp. himalaicus var. angustifolius;

Araliaceae; rhizomes; triterpenoid

saponin pseudo-ginsenoside-RI,.

Abstract- A novel triterpenoid saponin, pseudo-ginsenoside-RI,, isolated from the rhizomes of Panax pseudo-ginseng subsp. himaluicus var. angustifolius has been characterized as 3-O-/3-D-glucopyranosyl( 1+2)-B-D-glucuronopyranosyl

(l-+6)-/3-D-glucopyranosyl

280-/I-D-xylopyranosyl-olean-12-en-2%oic

acid ester by physicochemical methods.

INTRODUCTION

In continuation of our work on the rhizomes and leaves of three varieties of Indian pseudo-ginseng [l], we have recently established that rhizome saponins possess promising adaptogenic and immunostimulant activities which are comparable with Korean ginseng [2]. Further work on the active fraction of Panax pseudoginseng subsp. kimalaicus var. angustifolius (Burk.) Li has led to the isolation and characterization of a further new saponin, pseudo-ginsenoside-RI,. RESULTSAND

DBCUSSION

Repeated column chromatography followed by preparative TLC of the saponin mixture obtained from the rhizomes afforded 1, whose structure was deduced as 3O-p-D-glucopyranosyl( l-+2)-j?-D-glucuronopyranosyl(l-+6)-/?-D-glucopyranosyl 28-0-~-D-XylOpyranOSylolean-12-en-2%oic acid ester. The IR spectrum of 1 indicated the presence of hyroxy, ester, gem-dimethyl and double bond functions. Acid hydrolysis yielded oleanolic acid (2), D-ghCOSe, D-XylOSe and D-glucuronic acid (identified by comparison with authentic samples). Its ‘H NMR spectrum exhibited four anomeric signals at *Part 13 in the series ‘Studies on Indian ginseng’. For Part 12 see ref. [l] CIMAP Publication No. 49/91.

R 1 Xyl 3 H 65.72 (d, 5=7 Hz), 5.22 (d, J=6 Hz), 4.90 (d, J=7 Hz) and 5.10 (d, J = 6.5 Hz) which were consistent with the j?-

configuration for D-XylOSe, D-glucuronic acid, D-glucose and D-ghCOSe. The “C NMR spectrum of 1 showed four anomeric signals at 6 103.6, 103.9, 103.4 and 94.1 for four sugar residues. The signal at 694.1 indicated that one of the sugars was linked to C-28 of the aglycone as an ester. This was further confirmed by the alkaline hydrolysis of 1, which afforded xylose and prosaponin (3). Twenty-three carbon signals were seen for the sugar moieties indicating the presence of three hexoses and one pentose, the