- Email: [email protected]
Limonoid glucosides inCitrus aurantium
Short Reports
3803
Acknowledgement--We thank The Agricultural and Food Re-
EXPERIMENTAL
Trichoderma harzianum (401 broth) was grown as described previously I-1]. Chromatography of the broth ext. on silica gel (Merck 9385) in EtOAc-petrol (3:7) gave harzianolide (35 mg) as an oil. [~t]2° + 1.3° (EtOH; c 1). MS found 222.125 C13HlsO 3, requires 222.125; 204.115 [ M - H 2 0 ] ÷, 189.091 [ M - M e , H20] +, 178.099 [ M - C 2 H 4 0 ] +. IRv~x ,ujol 3445, 1747, 1671, 1620 cm-1. For 1H and 13C NMR data see Table 1.
search Council for financial support in this work. REFERENCES
1. Dickinson, J. M., Hanson, J. R., Hitchcock, P. B. and Claydon, N. (1989) d. Chem. Soc., Perkin Trans 1 1885. 2. Evidente, A., Randazzo, G. and Ballio, A. (1986) J. Nat. Prod. 49, 593.
00314422/91 $3.00+0.00 Pergamon Press plc
Phytochemistry, Vol. 30, No. 11, pp. 3803 3805, 1991
Printed in Great Britain.
L I M O N O I D GLUCOSIDES IN CITRUS A U R A N T I U M RAYMOND D. BENNETT, MASAKI MIYAKE,* YOSHIHIKO OZAKI* and SHIN HASEGAWA USDA, ARS, Fruit and Vegetable Chemistry Laboratory, 263 South Chester Avenue, Pasadena, CA 91106, U.S.A.; *Wakayama Agricultural Biological Research Institute, Momoyama, Wakayama, Japan (Received 4 March 1991)
Key Word Index--Citrus aurantium; Rutaceae; limonoids; glucosides; 17-fl-D-glucopyranosides.
Abstraet--17-fl-D-Glucopyranosides of limonin, obacunone, deacetylnomilin, nomilin, deacetylnomilinic acid, nomilinic acid, isolimonic acid, ichangin and 19-hydroxydeacetylnomilinic acid were isolated from sour orange seeds. The aglycone of the latter compound is a possible intermediate in the biosynthesis of limonin.
INTRODUCTION
Since our initial reports of the finding of 17-fl-D-glucopyranosides of the major citrus limonoids in grapefruit seeds [1, 2], we have extended our investigations to other citrus species. Of particular interest is Citrus aurantium (sour orange); this species contains high concentrations of two unique limonoids, isolimonic acid (2) and ichangin (3), which are found only in trace amounts in most other species [3]. Here we report the isolation and characterization of glucosides of these two compounds and also of a third, previously unknown, limonoid from sour orange seeds. RESULTS
AND DISCUSSION
Nine limonoid glucosides were isolated from sour orange seeds. Six of them were shown to be limonin glucoside (4), obacunone glucoside (5), deacetylnomilin glucoside (6), nomilin glucoside (7), deacetylnomilinic acid glucoside (8) and nomilinic acid glucoside (9) by tH N M R spectroscopy, while the other three were new compounds. The ~H N M R spectrum of the first of these showed four C-methyl signals, indicating that C-19 is oxygenated, as in limonin (1), rather than methyl as in most other limonoids. As expected, a pair of AB doublets was observed in the ~H N M R spectrum between 6 3.5 and 4, assignable to the two H-19 protons. However, the coupling constant was 11 Hz rather than the 13Hz
normally observed for most limonin derivatives which contain a 3 to 19 lactone linkage. This suggested that the compound is a glucoside of 2. In 2 C-19 is linked to C-4 as a cyclic ether, and the coupling constant between the H19 protons is 11 Hz. The remainder of the 1 H N M R spectrum was completely consistent with an isolimonic acid glucoside structure, as were 1H-1H COSY and NOESY spectra. From the latter spectrum the point of attachment of the sugar was confirmed to be the 17position, by the observation of a strong cross peak between H-17 and glucose H-1. In the 13CNMR spectrum the chemical shifts of C-1 to C-12 were very similar to those of 2 methyl ester, while those for C-13 to C-17 and the furan ring showed the shifts characteristic of limonoid fl-D-glucosides [1, 2]. The sugar 13CNMR resonances were also identical to those observed previously for limonoid glucosides. Thus, we assign the structure isolimonic acid 17-fl-D-glucopyranoside (10) to this compound. The next new compound also showed four C-methyl signals in its 1H N M R spectrum, as well as the downfield AB pair of doublets characteristic of limonoids oxygenated at C-19. In this case, however, the coupling constant between the two H-19 resonances was 12.5 Hz, which is consistent with the presence of a 3,19-1actone system, rather than a cyclic ether. The position of the C-4 signal (672.5) in the 13CNMR spectrum showed the absence of an A'-ring (1,4-cyclic ether, C-4 6 78, 79). This
3804
Short Reports
23 o
2o ~ t . ~ /
! :tt
! ;
, a
-°
t
:
O
~:
2
0 0
(/~'0~~ O/GIc ~CO2H
3
4
o/O,c
O
~
oR
02H 0 ~ ~ 0 0
C02H
R
OR /'~ i A /GIc co2. j. 8 9
.k°
6
H
7
Ac
OH ~ i ~ /GIc o3.
k°" 10
R H Ae
o
o.
.o\ .2c ( - - W - - o /
H O ~ 11
CO2H
~
CO2H
12
suggested that the aglycone was 3. All of the signals in the 1H and x3C N M R spectra were consistent with an ichangin glucoside, as were the couplings observed in the 1 H J H COSY spectrum and the NOEs in the 1H-1H NOESY spectrum. A strong cross peak in the latter spectrum between H-17 and glucose H-1 confirmed the 17-position as the site of glucosidation. The exact correspondence of the sugar 13CNMR resonances to those of the previous limonoid glucosides then enabled us to assign the structure ichangin 17-fl-D-glucopyranoside (11) to this compound. The third new compound also showed four C-methyl resonances in its 1H N M R spectrum and a downfield pair of AB doublets, thus classifying it as a 19-oxygenated
limonoid like the previous two. However, this compound was unstable under the NMR conditions, being gradually converted to a product whose tH and t3CNMR spectra were identical to those of 11. This reaction could most plausibly be explained as a closing of the 3,19-1actone ring. Confirmation was provided that this was indeed the case by opening the lactone ring of 11 by treatment with base. The product was identical (NMR, HPLC) with the isolated compound. Thus, we can assign the structure 19hydroxydeacetylnomilinic acid 17-/~-D-glucopyranoside (12) to this compound. All of the NMR data are consistent with this assignment. In our previous papers on limonoid glucosides [1, 2] we were not able to assign the C-methyl signals in the 1H and 13CNMR spectra. For the present compounds we have made these assignments by a combination of 2D N M R techniques. The signals in the 1H NMR spectra were assigned first and then correlated with the ~3C NMR spectra by ~H- t 3C COSY spectra. The position of the 13methyl resonance is readily apparent in a ~H-tH NOESY spectrum, by cross peaks between it and the H-17 and glucose H-1 signals. The two 4-methyl signals are identifiable by their mutual coupling in a IH-~H COSY spectrum. They are differentiated by a cross peak between one of the H-19 signals and the 4/~-methyl signal in a I H - t H NOESY spectrum, while the 4~t-methyl shows a cross peak to H-5. With three of the four methyl signals assigned, the remaining one must then be attributed to the 8-methyl group, which is substantiated by a cross peak between it and the other H-19 signal in a tH-~H NOESY spectrum. The easy conversion of 12 to 11 raised the possibility that the latter could be an artifact produced during the isolation procedures, some of which involve acidic conditions. However, when the isolation was repeated under neutral conditions, making lactone ring closure impossible, 11 was still obtained, although in slightly lower yield. Therefore, both 11 and 12 are natural constituents of sour orange. The aglycone of 12 has never been isolated from any citrus species, although it has been postulated as an intermediate in the biosynthesis of limonin I-3]. Our findings of 12 in the present work supports this hypothesis. Another group has isolated two polar limonoids from lemon peel and reported that they were glucosides of ichangin and nomilinic acid, but with the glucose attached to C-4 rather than C-17 [4]. However, a close examination of their 13CNMR data shows the same anomalous shifts for the C-13 to C-17 and furan signals as in our 17-glucosides. Their evidence for a linkage to C-4 was the downfield shifts of the C-4 signals in the glucosides compared to the aglycones, but this assumes that their identifications of the latter are correct. In fact, our nomilin 17-glucoside (7) [11 when run in methanol-d, as their compounds were, had IH and t3CNMR spectra identical to those reported for their nomilinic acid 4glucoside. The attachment of the glucose in this compound is clearly at C-17, as shown by cross peaks between H-17 and glucose H-1 in the t H - t H N O E S Y spectrum and between H-17 and glucose C-1 in the long range 13C-tH COSY spectrum. The downfield position of the C-4 signal is then explainable by its inclusion in the Aring lactone of nomilin. By the same methods their ichangin 4-glucoside was shown to be identical to our limonin 17-glucoside (4) and dearly different from the ichangin 17-glucoside reported here.
Short Reports EXPERIMENTAL
General. NMR spectral assignments were made on the basis of 1H-1H COSY and NOESY, DEPT and l sC-1H COSY spectra. Isolation of glucosides. Sour orange seeds (200 g) obtained from the University of California at Riverside, were ground in 3 1 of H20 and the pH adjusted to 4. Pectinase (ex Aspergillus niger) was added and the homogenate stirred for 20 hr. The mixt. was then centrifuged at 13000g for 15 rain and the supernatant filtered. The filtrate was transferred to the top of an XAD-2 column (3 x 75 cm). The column was washed thoroughly with H20 and the limonoid glucosides eluted with MeCN. After the solvent was evapd, the residue was dissolved in H20 and fractionated on a C-18 reverse-phase prep. HPLC column. The column was eluted at 3 ml min- 1 with a linear gradient starting with 15% and ending with 45% MeCN in H20 at 150 min. Frs containing each limonoid glucoside were refractionated on the same prep. column, eluting linearly with a gradient starting with 15% and ending with 55% Me2CO in H20 at 150 min. HPLC analysis on a C-18 reverse-phase analytical column showed Rzs for 19-hydroxydeacetylnomilinic acid glucoside, isolimonic acid glucoside and ichangin glucoside of 11.0, 15.0 and 16.9 min, respectively. The column was eluted linearly starting with 15% and ending with 30% MeCN in 3 mM HaPO4 at 45 min. The flow rate was 1 ml min-1 with detection at 210 nm. Isolimonic acid 17-fl-D-glucopyranoside (10). 1H NMR (270 MHz, DMSO-d6, 90°); 60.84 (3H, s, Me-8), 0.95 (3H, s, Me4fl), 1.17 (3H, s, Me-4~t), 1.40 (3H, s, Me-13), 2.76 (1H, s, H-15), 3.62 (1H, d, J = 11 Hz, H-19), 3.71 (1H, d, J = 11 Hz, H-19), 4.13 (1H, d, J = 7 Hz, Glc H-l), 4.13 (1H, m, H-I), 5.22 (IH, s, H-17), 6.53 (IH, s, H-22), 7.41 (1H, s, H-23), 7.49 (1H, s, H-21). 13C NMR (67.8 MHz, DMSO-d6, 90°): 616.4 (Me-8), 19.2 (C-11), 23.3 (Me4fl), 24.8 (Me-13), 26.6 (C-12), 28.0 (Me-4~t), 35.8 (C-9), 38.6 (C-2), 39.3 (C-6), 44.2 (C-13), 48.5 (C-5), 50.5 (C-10), 53.0 (C-8), 57.4 (C15), 61.7 (GIc C-6), 68.1 (C-19), 70.0 (C-14), 70.7 (Glc C-4), 73.7 (C1), 74.2 (Glc C-2), 76.2 (Glc C-5), 77.1 (Glc C-3), 77.6 (C-17), 83.3 (C-4), 104.4 (Glc C-1), 112.6 (C-22), 125.6 (C-20), 140.7 (C-23), 141.4 (C-21), 169.2 (C-16), 173.1 (c-a), 2!1.7 (c-7). Ichangin 17-fl-D-glucopyranoside (11). 1HNMR (270 MHz, DMSO-d6, 90°): 60.83 (3H, s, Me-8), 1.01 (3H, s, Me-4fl), 1.20 (3H, s, Me-4~t), 1.40 (3H, s, Me-13), 2.80 (1H, s, H-15), 3.92 (1H, d,
3805
J = 12.5 Hz, H-19), 4.05 (1H, m, H-I), 4.13 (1H, d, J=7.5 Hz, Glc H-I), 4.68 (1H, d, J = 12.5 Hz, H-19), 5.28 (1H, s, H-17), 6.52 (IH, s, H-22), 7.42 (1H, s, H-23), 7.49 (1H, s, H-21). 13CNMR (67.8 MHz, DMSO-d~, 90°): 615.2 (Me-8), 17.7 (C-11), 24.2 (Me13), 25.6 (Me-4#), 27.1 (C-12), 33.7 (Me-4~t), 3.80 (C-2), 39.0 (C-6), 41.0 (C-9), 43.4 (C-13), 45.4 (C-5), 45.8 (C-10), 51.3 (C-8), 58.6 (C15), 61.7 (Glc C-6), 68.1 (C-19), 69.9 (C-14), 70.7 (GIc C-4), 72.5 (C4), 74.2 (GIc C-2), 76.2 (Glc C-5), 77.1 (GIc C-3), 77.4 (C- 17), 104.3 (Glc C-I), 112.6 (C-22), 125.6 (C-20), 140.7 (C-23), 141.6 (C-21), 168.9 (C-16), 170.9 (C-3), 211.8 (C-7).
19-Hydroxydeacetylnomilinic acid 17-fl-D-glucopyranoside (12). 1HNMR (270 MHz, DMSO-d6, 90°): 60.78 (3H, s, Me-8), 1.09 (3H, s, Me-4fl), 1.17 (3H, s, Me-4~t), 1.44 (3H, s, Me-13), 2.78 (1H, s, H-15), 3.47 (1H, d, J = l l . 5 Hz, H-19), 3.69 (IH, d, J = 11.5 Hz, H-19), 4.12 (1H, d, J = 7 Hz, Glc H-I), 4.68 (IH, m, H1), 5.21 (1H, s, H-17), 6.51 (1H, s, H-22), 7.40 (1H, s, H-23), 7.46 (IH, s, H-21). laC NMR (67.8 MHz, DMSO-d6, 90°): 615.7 (Me8), 18.2 (C-11), 24.8 (Me-13), 26.4 (Me-4p), 26.9 (C-12), 32.7 (Me4~t), 37.8 (C-2), 39.0 (C-6), 39.4 (C-9), 43.4 (C-13), 48.2 (C-5), 49.5 (C-10), 51.3 (C-8), 58.5 (C-15), 59.8 (C-19), 61.7 (Glc C-6), 70.5 (C14), 70.7 (Glc C-4), 71.0 (C-I), 73.8 (C-4), 74.2 (GIc C-2), 76.1 (GIc C-5), 77.1 (GIc C-3), 77.7 (C-17), 104.5 (GIc C-I), 112.6 (C-22), 125.8 (C-20), 140.6 (C-23), 141.5 (C-21), 169.0 (C-16), 173.8 (C-3), 212.2 (C-7). Conversion of compound 11 to 12. To a soln of 5 mg of 11 in 1 ml MeOH was added 20 #l of 5 M KOH soln. After 16 hr at 25 ° the MeOH was removed in vacuo. The residue was taken up in 1 ml H20, acidified to pH 6 with 1 M HCI, and passed through a Cls Sep-Pak cartridge. The product was eluted with MeOH and shown to be identical to 12 by 1HNMR and HPLC. REFERENCES 1. Hasegawa, S., Bennett, R. D., Herman, Z., Fong, C. H. and Ou, P. (1989) Phytochemistry 28, 1717. 2. Bennett, R. D., Hasegawa, S. and Herman, Z. (1989) Phytochemistry 28, 2777. 3. Bennett, R. D. and Hasegawa, S. (1980) Phytochemistry 19, 2417. 4. Matsubara, Y., Sawabe, A. and Iizuka, Y. (1990) Aoric. Biol. Chem. 54, 1143.
3803
Acknowledgement--We thank The Agricultural and Food Re-
EXPERIMENTAL
Trichoderma harzianum (401 broth) was grown as described previously I-1]. Chromatography of the broth ext. on silica gel (Merck 9385) in EtOAc-petrol (3:7) gave harzianolide (35 mg) as an oil. [~t]2° + 1.3° (EtOH; c 1). MS found 222.125 C13HlsO 3, requires 222.125; 204.115 [ M - H 2 0 ] ÷, 189.091 [ M - M e , H20] +, 178.099 [ M - C 2 H 4 0 ] +. IRv~x ,ujol 3445, 1747, 1671, 1620 cm-1. For 1H and 13C NMR data see Table 1.
search Council for financial support in this work. REFERENCES
1. Dickinson, J. M., Hanson, J. R., Hitchcock, P. B. and Claydon, N. (1989) d. Chem. Soc., Perkin Trans 1 1885. 2. Evidente, A., Randazzo, G. and Ballio, A. (1986) J. Nat. Prod. 49, 593.
00314422/91 $3.00+0.00 Pergamon Press plc
Phytochemistry, Vol. 30, No. 11, pp. 3803 3805, 1991
Printed in Great Britain.
L I M O N O I D GLUCOSIDES IN CITRUS A U R A N T I U M RAYMOND D. BENNETT, MASAKI MIYAKE,* YOSHIHIKO OZAKI* and SHIN HASEGAWA USDA, ARS, Fruit and Vegetable Chemistry Laboratory, 263 South Chester Avenue, Pasadena, CA 91106, U.S.A.; *Wakayama Agricultural Biological Research Institute, Momoyama, Wakayama, Japan (Received 4 March 1991)
Key Word Index--Citrus aurantium; Rutaceae; limonoids; glucosides; 17-fl-D-glucopyranosides.
Abstraet--17-fl-D-Glucopyranosides of limonin, obacunone, deacetylnomilin, nomilin, deacetylnomilinic acid, nomilinic acid, isolimonic acid, ichangin and 19-hydroxydeacetylnomilinic acid were isolated from sour orange seeds. The aglycone of the latter compound is a possible intermediate in the biosynthesis of limonin.
INTRODUCTION
Since our initial reports of the finding of 17-fl-D-glucopyranosides of the major citrus limonoids in grapefruit seeds [1, 2], we have extended our investigations to other citrus species. Of particular interest is Citrus aurantium (sour orange); this species contains high concentrations of two unique limonoids, isolimonic acid (2) and ichangin (3), which are found only in trace amounts in most other species [3]. Here we report the isolation and characterization of glucosides of these two compounds and also of a third, previously unknown, limonoid from sour orange seeds. RESULTS
AND DISCUSSION
Nine limonoid glucosides were isolated from sour orange seeds. Six of them were shown to be limonin glucoside (4), obacunone glucoside (5), deacetylnomilin glucoside (6), nomilin glucoside (7), deacetylnomilinic acid glucoside (8) and nomilinic acid glucoside (9) by tH N M R spectroscopy, while the other three were new compounds. The ~H N M R spectrum of the first of these showed four C-methyl signals, indicating that C-19 is oxygenated, as in limonin (1), rather than methyl as in most other limonoids. As expected, a pair of AB doublets was observed in the ~H N M R spectrum between 6 3.5 and 4, assignable to the two H-19 protons. However, the coupling constant was 11 Hz rather than the 13Hz
normally observed for most limonin derivatives which contain a 3 to 19 lactone linkage. This suggested that the compound is a glucoside of 2. In 2 C-19 is linked to C-4 as a cyclic ether, and the coupling constant between the H19 protons is 11 Hz. The remainder of the 1 H N M R spectrum was completely consistent with an isolimonic acid glucoside structure, as were 1H-1H COSY and NOESY spectra. From the latter spectrum the point of attachment of the sugar was confirmed to be the 17position, by the observation of a strong cross peak between H-17 and glucose H-1. In the 13CNMR spectrum the chemical shifts of C-1 to C-12 were very similar to those of 2 methyl ester, while those for C-13 to C-17 and the furan ring showed the shifts characteristic of limonoid fl-D-glucosides [1, 2]. The sugar 13CNMR resonances were also identical to those observed previously for limonoid glucosides. Thus, we assign the structure isolimonic acid 17-fl-D-glucopyranoside (10) to this compound. The next new compound also showed four C-methyl signals in its 1H N M R spectrum, as well as the downfield AB pair of doublets characteristic of limonoids oxygenated at C-19. In this case, however, the coupling constant between the two H-19 resonances was 12.5 Hz, which is consistent with the presence of a 3,19-1actone system, rather than a cyclic ether. The position of the C-4 signal (672.5) in the 13CNMR spectrum showed the absence of an A'-ring (1,4-cyclic ether, C-4 6 78, 79). This
3804
Short Reports
23 o
2o ~ t . ~ /
! :tt
! ;
, a
-°
t
:
O
~:
2
0 0
(/~'0~~ O/GIc ~CO2H
3
4
o/O,c
O
~
oR
02H 0 ~ ~ 0 0
C02H
R
OR /'~ i A /GIc co2. j. 8 9
.k°
6
H
7
Ac
OH ~ i ~ /GIc o3.
k°" 10
R H Ae
o
o.
.o\ .2c ( - - W - - o /
H O ~ 11
CO2H
~
CO2H
12
suggested that the aglycone was 3. All of the signals in the 1H and x3C N M R spectra were consistent with an ichangin glucoside, as were the couplings observed in the 1 H J H COSY spectrum and the NOEs in the 1H-1H NOESY spectrum. A strong cross peak in the latter spectrum between H-17 and glucose H-1 confirmed the 17-position as the site of glucosidation. The exact correspondence of the sugar 13CNMR resonances to those of the previous limonoid glucosides then enabled us to assign the structure ichangin 17-fl-D-glucopyranoside (11) to this compound. The third new compound also showed four C-methyl resonances in its 1H N M R spectrum and a downfield pair of AB doublets, thus classifying it as a 19-oxygenated
limonoid like the previous two. However, this compound was unstable under the NMR conditions, being gradually converted to a product whose tH and t3CNMR spectra were identical to those of 11. This reaction could most plausibly be explained as a closing of the 3,19-1actone ring. Confirmation was provided that this was indeed the case by opening the lactone ring of 11 by treatment with base. The product was identical (NMR, HPLC) with the isolated compound. Thus, we can assign the structure 19hydroxydeacetylnomilinic acid 17-/~-D-glucopyranoside (12) to this compound. All of the NMR data are consistent with this assignment. In our previous papers on limonoid glucosides [1, 2] we were not able to assign the C-methyl signals in the 1H and 13CNMR spectra. For the present compounds we have made these assignments by a combination of 2D N M R techniques. The signals in the 1H NMR spectra were assigned first and then correlated with the ~3C NMR spectra by ~H- t 3C COSY spectra. The position of the 13methyl resonance is readily apparent in a ~H-tH NOESY spectrum, by cross peaks between it and the H-17 and glucose H-1 signals. The two 4-methyl signals are identifiable by their mutual coupling in a IH-~H COSY spectrum. They are differentiated by a cross peak between one of the H-19 signals and the 4/~-methyl signal in a I H - t H NOESY spectrum, while the 4~t-methyl shows a cross peak to H-5. With three of the four methyl signals assigned, the remaining one must then be attributed to the 8-methyl group, which is substantiated by a cross peak between it and the other H-19 signal in a tH-~H NOESY spectrum. The easy conversion of 12 to 11 raised the possibility that the latter could be an artifact produced during the isolation procedures, some of which involve acidic conditions. However, when the isolation was repeated under neutral conditions, making lactone ring closure impossible, 11 was still obtained, although in slightly lower yield. Therefore, both 11 and 12 are natural constituents of sour orange. The aglycone of 12 has never been isolated from any citrus species, although it has been postulated as an intermediate in the biosynthesis of limonin I-3]. Our findings of 12 in the present work supports this hypothesis. Another group has isolated two polar limonoids from lemon peel and reported that they were glucosides of ichangin and nomilinic acid, but with the glucose attached to C-4 rather than C-17 [4]. However, a close examination of their 13CNMR data shows the same anomalous shifts for the C-13 to C-17 and furan signals as in our 17-glucosides. Their evidence for a linkage to C-4 was the downfield shifts of the C-4 signals in the glucosides compared to the aglycones, but this assumes that their identifications of the latter are correct. In fact, our nomilin 17-glucoside (7) [11 when run in methanol-d, as their compounds were, had IH and t3CNMR spectra identical to those reported for their nomilinic acid 4glucoside. The attachment of the glucose in this compound is clearly at C-17, as shown by cross peaks between H-17 and glucose H-1 in the t H - t H N O E S Y spectrum and between H-17 and glucose C-1 in the long range 13C-tH COSY spectrum. The downfield position of the C-4 signal is then explainable by its inclusion in the Aring lactone of nomilin. By the same methods their ichangin 4-glucoside was shown to be identical to our limonin 17-glucoside (4) and dearly different from the ichangin 17-glucoside reported here.
Short Reports EXPERIMENTAL
General. NMR spectral assignments were made on the basis of 1H-1H COSY and NOESY, DEPT and l sC-1H COSY spectra. Isolation of glucosides. Sour orange seeds (200 g) obtained from the University of California at Riverside, were ground in 3 1 of H20 and the pH adjusted to 4. Pectinase (ex Aspergillus niger) was added and the homogenate stirred for 20 hr. The mixt. was then centrifuged at 13000g for 15 rain and the supernatant filtered. The filtrate was transferred to the top of an XAD-2 column (3 x 75 cm). The column was washed thoroughly with H20 and the limonoid glucosides eluted with MeCN. After the solvent was evapd, the residue was dissolved in H20 and fractionated on a C-18 reverse-phase prep. HPLC column. The column was eluted at 3 ml min- 1 with a linear gradient starting with 15% and ending with 45% MeCN in H20 at 150 min. Frs containing each limonoid glucoside were refractionated on the same prep. column, eluting linearly with a gradient starting with 15% and ending with 55% Me2CO in H20 at 150 min. HPLC analysis on a C-18 reverse-phase analytical column showed Rzs for 19-hydroxydeacetylnomilinic acid glucoside, isolimonic acid glucoside and ichangin glucoside of 11.0, 15.0 and 16.9 min, respectively. The column was eluted linearly starting with 15% and ending with 30% MeCN in 3 mM HaPO4 at 45 min. The flow rate was 1 ml min-1 with detection at 210 nm. Isolimonic acid 17-fl-D-glucopyranoside (10). 1H NMR (270 MHz, DMSO-d6, 90°); 60.84 (3H, s, Me-8), 0.95 (3H, s, Me4fl), 1.17 (3H, s, Me-4~t), 1.40 (3H, s, Me-13), 2.76 (1H, s, H-15), 3.62 (1H, d, J = 11 Hz, H-19), 3.71 (1H, d, J = 11 Hz, H-19), 4.13 (1H, d, J = 7 Hz, Glc H-l), 4.13 (1H, m, H-I), 5.22 (IH, s, H-17), 6.53 (IH, s, H-22), 7.41 (1H, s, H-23), 7.49 (1H, s, H-21). 13C NMR (67.8 MHz, DMSO-d6, 90°): 616.4 (Me-8), 19.2 (C-11), 23.3 (Me4fl), 24.8 (Me-13), 26.6 (C-12), 28.0 (Me-4~t), 35.8 (C-9), 38.6 (C-2), 39.3 (C-6), 44.2 (C-13), 48.5 (C-5), 50.5 (C-10), 53.0 (C-8), 57.4 (C15), 61.7 (GIc C-6), 68.1 (C-19), 70.0 (C-14), 70.7 (Glc C-4), 73.7 (C1), 74.2 (Glc C-2), 76.2 (Glc C-5), 77.1 (Glc C-3), 77.6 (C-17), 83.3 (C-4), 104.4 (Glc C-1), 112.6 (C-22), 125.6 (C-20), 140.7 (C-23), 141.4 (C-21), 169.2 (C-16), 173.1 (c-a), 2!1.7 (c-7). Ichangin 17-fl-D-glucopyranoside (11). 1HNMR (270 MHz, DMSO-d6, 90°): 60.83 (3H, s, Me-8), 1.01 (3H, s, Me-4fl), 1.20 (3H, s, Me-4~t), 1.40 (3H, s, Me-13), 2.80 (1H, s, H-15), 3.92 (1H, d,
3805
J = 12.5 Hz, H-19), 4.05 (1H, m, H-I), 4.13 (1H, d, J=7.5 Hz, Glc H-I), 4.68 (1H, d, J = 12.5 Hz, H-19), 5.28 (1H, s, H-17), 6.52 (IH, s, H-22), 7.42 (1H, s, H-23), 7.49 (1H, s, H-21). 13CNMR (67.8 MHz, DMSO-d~, 90°): 615.2 (Me-8), 17.7 (C-11), 24.2 (Me13), 25.6 (Me-4#), 27.1 (C-12), 33.7 (Me-4~t), 3.80 (C-2), 39.0 (C-6), 41.0 (C-9), 43.4 (C-13), 45.4 (C-5), 45.8 (C-10), 51.3 (C-8), 58.6 (C15), 61.7 (Glc C-6), 68.1 (C-19), 69.9 (C-14), 70.7 (GIc C-4), 72.5 (C4), 74.2 (GIc C-2), 76.2 (Glc C-5), 77.1 (GIc C-3), 77.4 (C- 17), 104.3 (Glc C-I), 112.6 (C-22), 125.6 (C-20), 140.7 (C-23), 141.6 (C-21), 168.9 (C-16), 170.9 (C-3), 211.8 (C-7).
19-Hydroxydeacetylnomilinic acid 17-fl-D-glucopyranoside (12). 1HNMR (270 MHz, DMSO-d6, 90°): 60.78 (3H, s, Me-8), 1.09 (3H, s, Me-4fl), 1.17 (3H, s, Me-4~t), 1.44 (3H, s, Me-13), 2.78 (1H, s, H-15), 3.47 (1H, d, J = l l . 5 Hz, H-19), 3.69 (IH, d, J = 11.5 Hz, H-19), 4.12 (1H, d, J = 7 Hz, Glc H-I), 4.68 (IH, m, H1), 5.21 (1H, s, H-17), 6.51 (1H, s, H-22), 7.40 (1H, s, H-23), 7.46 (IH, s, H-21). laC NMR (67.8 MHz, DMSO-d6, 90°): 615.7 (Me8), 18.2 (C-11), 24.8 (Me-13), 26.4 (Me-4p), 26.9 (C-12), 32.7 (Me4~t), 37.8 (C-2), 39.0 (C-6), 39.4 (C-9), 43.4 (C-13), 48.2 (C-5), 49.5 (C-10), 51.3 (C-8), 58.5 (C-15), 59.8 (C-19), 61.7 (Glc C-6), 70.5 (C14), 70.7 (Glc C-4), 71.0 (C-I), 73.8 (C-4), 74.2 (GIc C-2), 76.1 (GIc C-5), 77.1 (GIc C-3), 77.7 (C-17), 104.5 (GIc C-I), 112.6 (C-22), 125.8 (C-20), 140.6 (C-23), 141.5 (C-21), 169.0 (C-16), 173.8 (C-3), 212.2 (C-7). Conversion of compound 11 to 12. To a soln of 5 mg of 11 in 1 ml MeOH was added 20 #l of 5 M KOH soln. After 16 hr at 25 ° the MeOH was removed in vacuo. The residue was taken up in 1 ml H20, acidified to pH 6 with 1 M HCI, and passed through a Cls Sep-Pak cartridge. The product was eluted with MeOH and shown to be identical to 12 by 1HNMR and HPLC. REFERENCES 1. Hasegawa, S., Bennett, R. D., Herman, Z., Fong, C. H. and Ou, P. (1989) Phytochemistry 28, 1717. 2. Bennett, R. D., Hasegawa, S. and Herman, Z. (1989) Phytochemistry 28, 2777. 3. Bennett, R. D. and Hasegawa, S. (1980) Phytochemistry 19, 2417. 4. Matsubara, Y., Sawabe, A. and Iizuka, Y. (1990) Aoric. Biol. Chem. 54, 1143.