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Ecdysteroids from Ajuga iva
Phytochemistry, Vol. 31, No. Printedin Great Britain.
11,pp. 3785-3788,1992
0031-9422/92 $5.00+0.00 0 1992PergamonPressLtd
ECDYSTEROIDS
FROM AJUGA IL4
MARC WESSNER,BRIGI~E CHAMPION,*JEAN-PIERREGIRAULT,* NAGUIB KAOUADJI, BASSIMASAIDI and RENE LAFONT~ Ecole Normale Su$rieure, Wpartement de Biologie,Laboratoire de Biochimie et Physiologie du Wveloppement (CNRS URA 686X 46 rue d’Ulm, F-75230 Paris Cedex 05, France; *Universitb Re& Descartes, Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologique-s(CNRS URA 400), 45 rue des Saints-P&es, F-75006 Paris, France (Received in
revisedform 7 April 1992)
Key Word Index-Ajuga iva; Labiatae; eaiysteroids; 22-oxocyasterone; 24,25-dehydroprecyasterone.
Abstract--Ajuga iva contains large amounts of three major ecdysteroids (makisterone A, 20-hydroxyecdysone and cyasterone) together with several minor compounds including 24,28&hydromakisterone A and two new phytoecdysteroids (22-oxocyasterone and 24,25_dehydroprecyasterone) which have been identified by spectrometric procedures.
INTRODUCTION Ajuga
iua
is a medicinal plant (‘chendgour&‘) used by inhabitants from North Africa [l]. It shows several interesting (e.g. antifebrile, anthelmintic, vulnmary) properties and also displays efficient hypoglyciemic effects, finding a use by diabetic people, but to our knowledge the active principles of this plant have not yet been investigated. Many plants contain phytoecdysteroids [2-41, and the genus Ajuga comprises many representatives which contain large amounts of these steroids (Table 1) [6-211. In addition, it has been shown that ecdysteroids have significant effects on induced hyperglycremia (induced with glucagon or alloxan but not with anti-insulin antiserum) in experimental mammals [22], and more recently ecdysteroids were shown to potentiate the effects of insulin [23]. All these facts prompted us to investigate this plant for ecdysteroid content. Ajuga iuu has been reported to contain large amounts of 20-hydroxyecdysone, cyasterone and makisterone A [13-15-J. In the present study we confirm the presence of large amounts of these ecdysteroids together with two new compounds which are fully described.
proportion of makisterone A in the present case. Besides these major compounds, several smaller peaks were observed, which were purified by two successive HPLC steps using solvent systems 2 and 3 (see Experimental and Fig. 1). The retention times of individual peaks are reported in Table 2. Each peak was analysed by CI/D mass and ‘H NMR (Table 3) spectrometry and these data were compared with those of available reference ecdysteroids. Three minor compounds were identified as 24,28dehydro-makisterone A and two new ecdysteroids (1 and 2).
RESULTSANDDISCUSSION Major and minor ecdysteroids of Ajuga iva
The analysis of a semi-purified sample (prior to silica LC) by NP- and RP-HPLC showed the presence of three major ecdysteroids in the plant extract, which were easily identified as cyasterone, makisterone A and 20_hydroxyecdysone by comparison of their retention time (HPLC) and mass spectral data with reference compounds. Their respective concentrations relative to plant dry weight were ca 1.3% for cyasterone and 1.6960for makisterone A and 20-hydroxyecdysone. These values are in the same range as those previously reported [15], with a higher TAuthor to whom correspondence should be addressed.
0
A”5-l’RBCYASTBRONB (2)
M. WESSNERet al.
3786 Table 1. Ecdysteroids in the genus Ajuga Sp!CkS
Ecdysteroid
Ref.
Ajwga chamtzpitys
Ajugalactone Cyasterone 20-Hydroxyecdysone Makisterone A
WI PI C63 I33
A. chmeacistus
Ajugasterone C Cyasterone 20-Hydroxyecdysone
r71 t71
A.
Cyasterone
C81
Ajugalactone Ajugasterone B Ajugasterone C Cyasterone 20-Hydroxyecdysone
PI WI WI WI WI
A. incisa Maximowicz
Ajugasterone B Cyasterone 2GHydroxyecdysone
WI WI WI
A. hi-a
20-Hydroxyecdysone Cyasterone Makisterone A
L-13-15-J c14, 151 rJ4, 151
chia
A. decu&em
A. japonica
Thunberg
Miquel
A. nipptib
A.
Makino
mmota
A. reptans
A. twkestanka
Briq.
(Rgl.)
PI
Ajugasterone C
Cl13
Ajugasterone A ( = polypodine B) Ajugasterone D Cyasterone 20-Hydroxyecdysone
Cl21
Ajugasterone C Cyasterone 2OHydroxyecdysone
c71 c71 C71
Ajugalactone Cyasterone 20-Hydroxyecdysone Polypodine B 29-Norcyasterone 29-Norsengosterone 2-Acetyl-norcyasterone 3-Acetyl-norcyasterone
CVI WI Cl73 Cl71 Cl81 Cl81 Cl91 Cl91
Cyasterone 20-Hydroxyecdysone Turbesterone Cyasterone 22-acetate
WI WI CW I211
Compound 1 was identified as 22-oxocyasterone, M, 518 (536 [M+H+NH,]+, 519 [M+H]+ and 501 [M + H - H,O] ‘) C29H1420B,corresponding to the loss of 2 amu with respect to cyasterone (iW, 520). Its IR spectrum exhibited carbonyl absorptions at v (KBr) 1652, 1702 and 1750 cm-l (y-la&one). From ‘H 1D and 2D NMR experiments the main features of the A, B and C rings of common ecdysteroids [24] were observed; with respect to the side-chain of cyasterone, the H-22 signal has disappeared. The signal at 6 4.39 (d, 4, J = 9,6 Hz) (in D,O), 4.22 (m) (in CD,OD) presented a coupling pattern (ID) and correlations in the 2D ‘H CUSY spectrum equivalent to the ones observed for cyasterone, but the correlations H-23-H-22 were missing. Moreover, the H23 signal was ,shifted downfield to ca 6 2.9, a value in agreement with the ccproton of a carbonyl group such as 22-oxo-ecdysteroids (in the case of 22-0x0-20hydroxyecdysone, the signal is at 6 2.8 in D,O). Compound 2 was identified as 24,25-dehydroprecyasterone, M, 518 (536 [M + H +NH,] ‘, 5 19 [M + HI] + and 501 [M + H - H,O]+). The IR spectrum showed carbony1 absorptions at v (KBr) 1652 and 1695 cm-’ which excludes the presence of a y-lactone ring as in cyasterone. ‘H 1D and 2D COSY NMR experiments show no major modification of the A, B and C rings. A methyl signal at 5 1.83 (d, J= 1 Hz, CD,OD) was found but no ethylenic proton signal was observed, in agreement with a fragment X=C(Me)-. For the four other signals, three singlets and one doublet at S 1.32 (d, 3 = 6 Hz) coupled to the signal at 6 4.93 (4, J = 6 Hz) were found, consistent with a MeCH < 0- group. The signal at S 4.34 (a, J = 11.5, 4.5 Hz) could be assigned to the H-22 signal, and was correlated with H-23 signals (2D COSY), but the latter were not correlated with a H-24 signal as observed in the case of cyasterone. As a consequence, the H-24 is missing, and all these elements are in agreement with a A24*25dehydro structure. In ajugalactone, there was a yczo 1695 cm - ’ signal for the a&dehydro S-lactone carbony1 [9). Thus structure 2 is in agreement with all the data. Moreover, NOEs were observed between H-28, Me26 and Me-29 and also between these two methyls, An alternative structure with the methyl group at position 24 would also fit with these data can be excluded from biological considerations (lack of any such parent sterol) and from the absence of NOE between the ethylenic methyl signal (6 1.83) and H-23 signals. This new compound contains the same lactone ring as ajugalactone [9] and it shows close structural similarities with precyasterone [ 25).
Table 2. Retention times of the major ecdysteroids from A+iua
Fraction number
Solvent 1
Solvent 2
Solvent 3
Compound
8-l 8-2-l = 9-1 8-3-2 8-4-l = 9-2 S-4-2 = 9-3 8-5 =9-4
13.6 16.8 21.1 32.0 38.4 45.6
8.2 8.4 9+6 12.6 15.1 16.9
35.4 8.9 9.2 8.1 9.8 5.7
22-Oxocyasterone* (1) Cyasterone A24*2*-Makisterone A Makbterone A A24~2S-Precyasterone* (2) XI-Hydroxyecdysone
Ecdysteroids from Ajuga iva
3781
1 = 22-oxocyasterone
8-4
8-21
cyasterone
8-22
(too low amount)
8-41
makisterone A
8-42
2 = 24(25)_dehydroprecyasterone
HPLc2
20-hydroxyecdysone
a-5
Fig. 1. The last steps of ecdysteroid isolation from LC fraction number 8. See Experimental for details on HPLC systems 1 and 2. Bold names refer to new ecdysteroids.
Table 3. ‘HNMR
spectral data of newly isolated ecdysteroids 22-Oxo-cyasterone
A24.25-Precyasterone
Cyasterone H
(CD,OD)
la le 2a 3e 4a 4a 5 I 9a lla lle 16a 168 17 22 23a 23b 24a 25 Me-18 Me-19 Me-21 Me-26 28 Me-29
(CD,OD)
3.83 m ( W,,, =22) 3.95 m (W1,2=8)
3.82 m (IV,,, = 22) 3.94 m (W,,,=8)
2.36 5.82 d (25) 3.14 m (W,,,=22)
5.8 d (2.5) 3.16 m (FVI,a=22)
3.53 d (11)
264 -
t (9)
2.9 dd (18, 6) 2.98 a’d (18, 5.6) 2.5 m 0.9 s 0.97 s 1.2 s 1.31 d (7) 4.20 m 1.42 d (6)
*Under HDO or CD,OH
2.5 m 0.87 s 0.96 s 1.39 s 1.19 d (7) 4.22 dq (9, 6) 1.36 d (6) signals.
(D20)
1.4 1.85 3.99 m (W1,2==22) 4.07 m (IV,,, = 8) 1.75 1.75 2.36 5.96 d (2.5) 3.12 m (W,,,=22) 1.75 1.85
2.54 2.95 2.95 2.35 26m 0.87 s 1.00 s 1.49 s 1.20 d (7) 4.39 dq (9, 6) 1.42 d (6)
(CD,OD)
P20)
-
1.4 1.85 3.99 m ( W1,2 = 22) 4.07 m ( W,,, = 8) 1.75 1.75 2.36 5.99 d (25) 3.12 m (W,,,=22) 1.7 1.8 1.95 1.75 26 4.34 dd (11.5.4.5) 2.4 2.55 -
0.89 s 0.96 s 1.33 s 1.84 d (1) 4.92 1.29 d (6)
0.87 s 1.00 s 1.33 s 1.83 s, b 4.93 q (6) 1.32 d (6)
3.83 m (IV,,, = 22) 3.95 m ( W1,2 = 8)
5.8 d (2.5) 3.15 m (W,,,=22)
4.13 dd (13, 3)
M. WESSNER et al.
3788 EXPERIMENTAL
Plants. Plants were collected in the area of Wilaya de Tiaret (Djebel Tissemssilet, Algeria) in September 1989 and were airdried. The dried whole plant including roots was used. Chemicals. Reference ecdysteroids were from previous work [24, 261. Most solvents were from Carlo Erba (Milano, Italy), with the exception of MeCN and CH,Cl, (HPLC grade) which were from Prolabo (Paris, France). Extraction and prepurijcation. They were performed as previously described [24,27]. Briefly, plants (15 g dry wt) were tinely ground and extracted overnight with 300 ml EtOH in a flask under constant agitation with a magnetic stirrer, then the extract was filtered over a glass fibre filter and evapd to dryness. The residue was partitioned between hexane-MeOH-EtOH-H,O (3: 1: 1: 1) and the aq. phase evapd to dryness. The residue was dissolved in 20 ml MeOH, then 50 ml Me,CO added and the resulting ppt. discarded by centrifugation (10 min at 5000 g). The supematant was adsorbed on to Celite (10 g) during evapn with a rotatory evaporator, then applied as a slurry into CHCl, (30 ml) on to a silica column (30 g) preconditioned with CHCl,. The column was then eluted with a step-gradient of MeOH in CHCl,: CHCl, (100 ml), then CHCl,-MeOH, 19:l (lOOml), 9: 1 (100 ml), 4: 1 (300 ml) and 1: 1 (100 ml). Fourteen frs of 50 ml were collected, then assayed by TLC on silica plates (Merck Si6OF254) (CHCl,-MeOH, 4: 1). Major ecdysteroid-containing frs (frs 8 and 9) were further analysed by HPLC (see below and Fig. 1). HPLC. HPLC was performed with DuPont (Normal-Phase HPLC) or a Waters (Reverse-Phase HPLC) equipment. Normalphase HPLC used Zorbax@-Sil columns (25 cm long, 4.6 or 9.4mm i.d.) eluted with one of the following solvents: CH,Cl,-isopropanol-H,0, 125 : 25 : 2 (solvent system 1) or 125:40:3 (solvent system 2). Some controls used Reverse-phase HPLC on a Waters Novapak@ Cl8 cartridge (10 cm long, 8 mm i.d.) eluted with MeCN-1% trifluoroacetic acid (23: 77) (solvent system 3). The flow-rate was set at 1 ml min- ’ with analytical columns and 4 ml min- ’ with the semi-prep. NP column. Spectrometric procedures. Mass spectrometry used chemicalionization @I/D) with NH, as the reagent gas as previously described [28]. ‘HNMR was used for new ecdysteroids only. Samples were lyophilized in 99.5% D,O, then dissolved in CD,OD and analysed as previously described [25,26,29]. FTIR spectrometry (in KBr) was performed with a Nicolet 5DX apparatus. The vco frequencies were compared with data from the literature [30, 313.
Acknowledgement-The authors thank Dr Nicole Morin for mass spectroscopic analyses. REFERENCES
Floc’h, E. (1983) Contribution li une itude ethnobotanique de laf!ore tunisienne, pp. 203-204. Publications Scientiques
Jx
Tunisiennes, Imprimerie Oflicielle de la R&publique Tunisienne. Bergamasco, R. and Horn, D. H. S. (1983) in Invertebrate Endocrinology Vol. 1 Endocrinology of Insects (Downer, R. G. H. and H. Laufer, H., eds), pp. 627-654. Liss, New York. Lafont, R. and Horn, D. H. S. (1989) in Ecdysone (Koolman, J. A., ed.), pp. 39-64. Georg Thieme, Stuttgart.
4. Camps, F. (1991) in Ecological Chemistry and Biochemistry of Plant Terpenoids (Harborne, J. B. and Tomas-Barberan,
F. A., eds), pp. 331-376. Clarendon Press, Oxford. 5. Lafont, R., Bouthier, A. and Wilson, I. D. (1991) in Insect Chemical Ecology (Hrdy, I., ed.), pp. 197-214. Academia
Praha. 6. Camps, F., Coll, J. and Dargallo, 0. (1985) An. Quim. BlC, 74. 7. Kubo, I., Klocke, 3. A., Ganjian, I., Ichikawa, N. and Matsumoto, T. (1983) J. Chromatogr. 257, 157. 8. lkan, R. and Ravid, U. (1971) Phytochemistry IO, 1659. 9. Koreeda, M., Nakanishi, K. and Goto, M. (1970) J. Am.
Chem. Sot. 9X 7512. 10. lmai, S., Fujioka, S, Murata, E., Otsuka, K. and Nakanishi, K. (1969) J. Chem. Sot., Chem. Commun. 82.
11. [mai, S., Murata, E., Fujioka, S., Koreeda, M. and Nakanishi, K. (1969) J. Chem. Sot., Chem. Commun. 546. 12. Imai, S., Toyosato, T., Sakai, M., Sato, Y., Fujioka, S., Murata, E. and Goto, M. (1969~)Chem. Pharm. Bull. 17,340. 13. Ikan, R. and Ravid, U. (1970) Planta Med. 20, 33. 14. Khafagy, S. M., Sabri, N. N., El-Sebakhy, N. and Blessington, B. (1979) Planta Med. 35, 184. 15. Nazmi Sabri, N., Asaad, A. and Khafagy, S. M. (1981) Planta Med. 42, 293. 16. Chou, W.-S. and Lu, M.-S. (1980) in Progress in Ecdysone Research (Hoffmann, J. A., cd.), pp. 281-297. Elsevier/ North-Holland, Amsterdam. 17. Camps, F., Coll, J. and Cortel, A. (1981) Rev. Lutinoam. Quim. l&81. 18. Camps, F., Coll, J. and Cartel, A. (1982) Chem. Letters 1313. 19. Camps, F., Coll, J., Cortel, A., Molins, E. and Miravitelles, C. (1985) J. Chem. Res. Synop. (l), 14. 20. Usmanov, B. Z., Gorovits, M. B. and Abubakhirov, N. K. (1975) Khim. Prir. Sodin. 466. 21. Usmanov, B. Z., Rashkes, Ya. V. and Abubakhirov, N. K. (1978) Khim. Prir. Soedin. 215. 22. Yoshida, T., Otaka, T., Uchiyama, M. and Ogawa, S. (1971) B&hem.
Pharmacol. 20, 3263.
23. Kosovskii, M.I., Syrov, V. N., Mirakhmedov, M. M., Katkova, S. P. and Khushbatkova, 2. A. (1989) Probl, Endokrinol. X(77.
24. Girault, J.-P., Biithori, M., Varga, E., Szendrei, K. and Lafont, R. (1990) J. Nat. Prod. 53, 279. 25. Hikino, H., Nomoto, K., Ino, R. and Takemoto, T. (1970) Chem Pharm. Bull. 18, 1078. 26. Girault, J.-P., Lafont, R., Varga, E., Hajdu, Zs., Herke, I. and Szendrei, K. (1988) Phytochemistry 27, 737. 27. Rudel, D., @thori, M., Gharbi, J., Girault, J.-P., Racz, I., Melis, K., Szendrei, K. and Lafont, R. (1992) Planta Med. (in press). 28. Lafont, R., Beydon, P., Mauchamp, B., Somm&Martin, G., Andrianjafintrimo, M. and Krien, P. (1981) in Regulation of Insect Development and Behaviour (Sehnal, F., Zabza, A. and Cymborowski, B., eds), pp. 125-144. Technical University of Wroclaw Press. 29. Girault, J.-P. and Lafont, R. (1988) J. Insect Physiol. 34,701. 30. Dolphin, D. and Wick, A. (1977) Tabulation of Inpared Spectral Data, p. 356. J. Wiley, New York. 31. Horn, D. H. S. and Bergamasco, R. (1985) in Comprehensive Insect Physiology, Biochemistry and Pharmacology Vol. 7 (Kerkut, G. A. and Gilbert, L. I., eds), p. 217. Pergamon Press, Oxford.
11,pp. 3785-3788,1992
0031-9422/92 $5.00+0.00 0 1992PergamonPressLtd
ECDYSTEROIDS
FROM AJUGA IL4
MARC WESSNER,BRIGI~E CHAMPION,*JEAN-PIERREGIRAULT,* NAGUIB KAOUADJI, BASSIMASAIDI and RENE LAFONT~ Ecole Normale Su$rieure, Wpartement de Biologie,Laboratoire de Biochimie et Physiologie du Wveloppement (CNRS URA 686X 46 rue d’Ulm, F-75230 Paris Cedex 05, France; *Universitb Re& Descartes, Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologique-s(CNRS URA 400), 45 rue des Saints-P&es, F-75006 Paris, France (Received in
revisedform 7 April 1992)
Key Word Index-Ajuga iva; Labiatae; eaiysteroids; 22-oxocyasterone; 24,25-dehydroprecyasterone.
Abstract--Ajuga iva contains large amounts of three major ecdysteroids (makisterone A, 20-hydroxyecdysone and cyasterone) together with several minor compounds including 24,28&hydromakisterone A and two new phytoecdysteroids (22-oxocyasterone and 24,25_dehydroprecyasterone) which have been identified by spectrometric procedures.
INTRODUCTION Ajuga
iua
is a medicinal plant (‘chendgour&‘) used by inhabitants from North Africa [l]. It shows several interesting (e.g. antifebrile, anthelmintic, vulnmary) properties and also displays efficient hypoglyciemic effects, finding a use by diabetic people, but to our knowledge the active principles of this plant have not yet been investigated. Many plants contain phytoecdysteroids [2-41, and the genus Ajuga comprises many representatives which contain large amounts of these steroids (Table 1) [6-211. In addition, it has been shown that ecdysteroids have significant effects on induced hyperglycremia (induced with glucagon or alloxan but not with anti-insulin antiserum) in experimental mammals [22], and more recently ecdysteroids were shown to potentiate the effects of insulin [23]. All these facts prompted us to investigate this plant for ecdysteroid content. Ajuga iuu has been reported to contain large amounts of 20-hydroxyecdysone, cyasterone and makisterone A [13-15-J. In the present study we confirm the presence of large amounts of these ecdysteroids together with two new compounds which are fully described.
proportion of makisterone A in the present case. Besides these major compounds, several smaller peaks were observed, which were purified by two successive HPLC steps using solvent systems 2 and 3 (see Experimental and Fig. 1). The retention times of individual peaks are reported in Table 2. Each peak was analysed by CI/D mass and ‘H NMR (Table 3) spectrometry and these data were compared with those of available reference ecdysteroids. Three minor compounds were identified as 24,28dehydro-makisterone A and two new ecdysteroids (1 and 2).
RESULTSANDDISCUSSION Major and minor ecdysteroids of Ajuga iva
The analysis of a semi-purified sample (prior to silica LC) by NP- and RP-HPLC showed the presence of three major ecdysteroids in the plant extract, which were easily identified as cyasterone, makisterone A and 20_hydroxyecdysone by comparison of their retention time (HPLC) and mass spectral data with reference compounds. Their respective concentrations relative to plant dry weight were ca 1.3% for cyasterone and 1.6960for makisterone A and 20-hydroxyecdysone. These values are in the same range as those previously reported [15], with a higher TAuthor to whom correspondence should be addressed.
0
A”5-l’RBCYASTBRONB (2)
M. WESSNERet al.
3786 Table 1. Ecdysteroids in the genus Ajuga Sp!CkS
Ecdysteroid
Ref.
Ajwga chamtzpitys
Ajugalactone Cyasterone 20-Hydroxyecdysone Makisterone A
WI PI C63 I33
A. chmeacistus
Ajugasterone C Cyasterone 20-Hydroxyecdysone
r71 t71
A.
Cyasterone
C81
Ajugalactone Ajugasterone B Ajugasterone C Cyasterone 20-Hydroxyecdysone
PI WI WI WI WI
A. incisa Maximowicz
Ajugasterone B Cyasterone 2GHydroxyecdysone
WI WI WI
A. hi-a
20-Hydroxyecdysone Cyasterone Makisterone A
L-13-15-J c14, 151 rJ4, 151
chia
A. decu&em
A. japonica
Thunberg
Miquel
A. nipptib
A.
Makino
mmota
A. reptans
A. twkestanka
Briq.
(Rgl.)
PI
Ajugasterone C
Cl13
Ajugasterone A ( = polypodine B) Ajugasterone D Cyasterone 20-Hydroxyecdysone
Cl21
Ajugasterone C Cyasterone 2OHydroxyecdysone
c71 c71 C71
Ajugalactone Cyasterone 20-Hydroxyecdysone Polypodine B 29-Norcyasterone 29-Norsengosterone 2-Acetyl-norcyasterone 3-Acetyl-norcyasterone
CVI WI Cl73 Cl71 Cl81 Cl81 Cl91 Cl91
Cyasterone 20-Hydroxyecdysone Turbesterone Cyasterone 22-acetate
WI WI CW I211
Compound 1 was identified as 22-oxocyasterone, M, 518 (536 [M+H+NH,]+, 519 [M+H]+ and 501 [M + H - H,O] ‘) C29H1420B,corresponding to the loss of 2 amu with respect to cyasterone (iW, 520). Its IR spectrum exhibited carbonyl absorptions at v (KBr) 1652, 1702 and 1750 cm-l (y-la&one). From ‘H 1D and 2D NMR experiments the main features of the A, B and C rings of common ecdysteroids [24] were observed; with respect to the side-chain of cyasterone, the H-22 signal has disappeared. The signal at 6 4.39 (d, 4, J = 9,6 Hz) (in D,O), 4.22 (m) (in CD,OD) presented a coupling pattern (ID) and correlations in the 2D ‘H CUSY spectrum equivalent to the ones observed for cyasterone, but the correlations H-23-H-22 were missing. Moreover, the H23 signal was ,shifted downfield to ca 6 2.9, a value in agreement with the ccproton of a carbonyl group such as 22-oxo-ecdysteroids (in the case of 22-0x0-20hydroxyecdysone, the signal is at 6 2.8 in D,O). Compound 2 was identified as 24,25-dehydroprecyasterone, M, 518 (536 [M + H +NH,] ‘, 5 19 [M + HI] + and 501 [M + H - H,O]+). The IR spectrum showed carbony1 absorptions at v (KBr) 1652 and 1695 cm-’ which excludes the presence of a y-lactone ring as in cyasterone. ‘H 1D and 2D COSY NMR experiments show no major modification of the A, B and C rings. A methyl signal at 5 1.83 (d, J= 1 Hz, CD,OD) was found but no ethylenic proton signal was observed, in agreement with a fragment X=C(Me)-. For the four other signals, three singlets and one doublet at S 1.32 (d, 3 = 6 Hz) coupled to the signal at 6 4.93 (4, J = 6 Hz) were found, consistent with a MeCH < 0- group. The signal at S 4.34 (a, J = 11.5, 4.5 Hz) could be assigned to the H-22 signal, and was correlated with H-23 signals (2D COSY), but the latter were not correlated with a H-24 signal as observed in the case of cyasterone. As a consequence, the H-24 is missing, and all these elements are in agreement with a A24*25dehydro structure. In ajugalactone, there was a yczo 1695 cm - ’ signal for the a&dehydro S-lactone carbony1 [9). Thus structure 2 is in agreement with all the data. Moreover, NOEs were observed between H-28, Me26 and Me-29 and also between these two methyls, An alternative structure with the methyl group at position 24 would also fit with these data can be excluded from biological considerations (lack of any such parent sterol) and from the absence of NOE between the ethylenic methyl signal (6 1.83) and H-23 signals. This new compound contains the same lactone ring as ajugalactone [9] and it shows close structural similarities with precyasterone [ 25).
Table 2. Retention times of the major ecdysteroids from A+iua
Fraction number
Solvent 1
Solvent 2
Solvent 3
Compound
8-l 8-2-l = 9-1 8-3-2 8-4-l = 9-2 S-4-2 = 9-3 8-5 =9-4
13.6 16.8 21.1 32.0 38.4 45.6
8.2 8.4 9+6 12.6 15.1 16.9
35.4 8.9 9.2 8.1 9.8 5.7
22-Oxocyasterone* (1) Cyasterone A24*2*-Makisterone A Makbterone A A24~2S-Precyasterone* (2) XI-Hydroxyecdysone
Ecdysteroids from Ajuga iva
3781
1 = 22-oxocyasterone
8-4
8-21
cyasterone
8-22
(too low amount)
8-41
makisterone A
8-42
2 = 24(25)_dehydroprecyasterone
HPLc2
20-hydroxyecdysone
a-5
Fig. 1. The last steps of ecdysteroid isolation from LC fraction number 8. See Experimental for details on HPLC systems 1 and 2. Bold names refer to new ecdysteroids.
Table 3. ‘HNMR
spectral data of newly isolated ecdysteroids 22-Oxo-cyasterone
A24.25-Precyasterone
Cyasterone H
(CD,OD)
la le 2a 3e 4a 4a 5 I 9a lla lle 16a 168 17 22 23a 23b 24a 25 Me-18 Me-19 Me-21 Me-26 28 Me-29
(CD,OD)
3.83 m ( W,,, =22) 3.95 m (W1,2=8)
3.82 m (IV,,, = 22) 3.94 m (W,,,=8)
2.36 5.82 d (25) 3.14 m (W,,,=22)
5.8 d (2.5) 3.16 m (FVI,a=22)
3.53 d (11)
264 -
t (9)
2.9 dd (18, 6) 2.98 a’d (18, 5.6) 2.5 m 0.9 s 0.97 s 1.2 s 1.31 d (7) 4.20 m 1.42 d (6)
*Under HDO or CD,OH
2.5 m 0.87 s 0.96 s 1.39 s 1.19 d (7) 4.22 dq (9, 6) 1.36 d (6) signals.
(D20)
1.4 1.85 3.99 m (W1,2==22) 4.07 m (IV,,, = 8) 1.75 1.75 2.36 5.96 d (2.5) 3.12 m (W,,,=22) 1.75 1.85
2.54 2.95 2.95 2.35 26m 0.87 s 1.00 s 1.49 s 1.20 d (7) 4.39 dq (9, 6) 1.42 d (6)
(CD,OD)
P20)
-
1.4 1.85 3.99 m ( W1,2 = 22) 4.07 m ( W,,, = 8) 1.75 1.75 2.36 5.99 d (25) 3.12 m (W,,,=22) 1.7 1.8 1.95 1.75 26 4.34 dd (11.5.4.5) 2.4 2.55 -
0.89 s 0.96 s 1.33 s 1.84 d (1) 4.92 1.29 d (6)
0.87 s 1.00 s 1.33 s 1.83 s, b 4.93 q (6) 1.32 d (6)
3.83 m (IV,,, = 22) 3.95 m ( W1,2 = 8)
5.8 d (2.5) 3.15 m (W,,,=22)
4.13 dd (13, 3)
M. WESSNER et al.
3788 EXPERIMENTAL
Plants. Plants were collected in the area of Wilaya de Tiaret (Djebel Tissemssilet, Algeria) in September 1989 and were airdried. The dried whole plant including roots was used. Chemicals. Reference ecdysteroids were from previous work [24, 261. Most solvents were from Carlo Erba (Milano, Italy), with the exception of MeCN and CH,Cl, (HPLC grade) which were from Prolabo (Paris, France). Extraction and prepurijcation. They were performed as previously described [24,27]. Briefly, plants (15 g dry wt) were tinely ground and extracted overnight with 300 ml EtOH in a flask under constant agitation with a magnetic stirrer, then the extract was filtered over a glass fibre filter and evapd to dryness. The residue was partitioned between hexane-MeOH-EtOH-H,O (3: 1: 1: 1) and the aq. phase evapd to dryness. The residue was dissolved in 20 ml MeOH, then 50 ml Me,CO added and the resulting ppt. discarded by centrifugation (10 min at 5000 g). The supematant was adsorbed on to Celite (10 g) during evapn with a rotatory evaporator, then applied as a slurry into CHCl, (30 ml) on to a silica column (30 g) preconditioned with CHCl,. The column was then eluted with a step-gradient of MeOH in CHCl,: CHCl, (100 ml), then CHCl,-MeOH, 19:l (lOOml), 9: 1 (100 ml), 4: 1 (300 ml) and 1: 1 (100 ml). Fourteen frs of 50 ml were collected, then assayed by TLC on silica plates (Merck Si6OF254) (CHCl,-MeOH, 4: 1). Major ecdysteroid-containing frs (frs 8 and 9) were further analysed by HPLC (see below and Fig. 1). HPLC. HPLC was performed with DuPont (Normal-Phase HPLC) or a Waters (Reverse-Phase HPLC) equipment. Normalphase HPLC used Zorbax@-Sil columns (25 cm long, 4.6 or 9.4mm i.d.) eluted with one of the following solvents: CH,Cl,-isopropanol-H,0, 125 : 25 : 2 (solvent system 1) or 125:40:3 (solvent system 2). Some controls used Reverse-phase HPLC on a Waters Novapak@ Cl8 cartridge (10 cm long, 8 mm i.d.) eluted with MeCN-1% trifluoroacetic acid (23: 77) (solvent system 3). The flow-rate was set at 1 ml min- ’ with analytical columns and 4 ml min- ’ with the semi-prep. NP column. Spectrometric procedures. Mass spectrometry used chemicalionization @I/D) with NH, as the reagent gas as previously described [28]. ‘HNMR was used for new ecdysteroids only. Samples were lyophilized in 99.5% D,O, then dissolved in CD,OD and analysed as previously described [25,26,29]. FTIR spectrometry (in KBr) was performed with a Nicolet 5DX apparatus. The vco frequencies were compared with data from the literature [30, 313.
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