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Insect allelochemicals from Ajuga plants
Phytochemisby,Vol. 32, No. 6, pp. 1361.1370, 1993 Printedin Great Britain.
0031 9422/93 $24.00 + 0.00 Q 1993 PergamonPress Ltd
REVIEW ARTICLE NUMBER 75 INSECT ALLELOCHEMICALS FRANCISCO CAMPS
FROM AJUGA PLANTS and
JOSEP COLL
Department of Biological Organic Chemistry, C.I.D., C.S.I.C. Jordi Girona, 18-26. 08034-Barcelona, Spain (Receioed 4 August 1992)
Key Word Index-Ajuga; hormone.
Labiatae; clerodane diterpene; phytoecdysteroid;
antifeedant;
moulting
Abstract-Clerodane diterpenoids and phytoecdysteroids with potential insect antifeedant and moulting hormone activities, respectively, have been isolated from Ajuga plants. Some clerodanes were active against larvae of Egyptian cotton leafworm, Spodoptera littoralis, when present in the diet at 3 ppm doses. Structure-antifeedant activity relations were established. Likewise, first stage larvae of the greenhouse whitefly, Trialeurodes uaporariorum, exhibited complete mortality when fed on A. reptans. This effect was mainly originated by 29-norsengosterone and ajugalactone, two phytoecdysteroids occurring in this plant. For biotechnological production of phytoecdysteroids its total content in different parts of normally grown or in vitro micropropagated A. reptans plants was studied. Great quantitative and qualitative differences were observed. For comparison of these qualitative differences, a dealkylation ratio (Dr = C,,/C,, phytoecdysteroid content) and a C-5 hydroxylation ratio (5Hr = 5-OH/5-H phytoecdysteroid content) were established. The 5Hr values appeared to be quite constant ranging from 0.2 to 0.4, whereas Dr values oscillated from 2.3 in whole plants to 12 in root cultures. Production of phytoecdysteroids was highest ( N 5000 ppm/dry wt) in cultures of roots in an hormone supplemented solid medium.
INTRODUCTION
More than one hundred species and fifty varieties and subspecies of Ajugu plants are unevenly distributed over the world [ 11. While in America there are recorded only three newly introduced species, and two endemic species in Australia, these Labiatae are especially abundant in China, Korea and Japan and also widespread in Europe. They have been used in folk medicine in various cultures and several interesting medicinal properties, such as antifebrile, anthelmintic, hypoglycaemic and vulnerary effects have been attributed to them. Traditionally, in China some Ajuga plants have been used to treat inflammation [2]. Many representatives of the genus Ajuga contain phytoecdysteroids [3], polyhydroxysteroids with a 5fl-H-7ene-6-one system, exhibiting well established physiological activities in insects and also in mammals which might explain some of the successful applications of these plants in folk medicine. The observation that Ajuga remota leaves, collected in Kenya, were not attacked by African armyworms led to the isolation of three moderately strong antifeedants named ajugarins I-III with diterpene clerodane structures [4], closely related to clerodin, the first compound known of this series isolated from Clerodendron infirtunatum (Verbenaceae) [S]. The absolute configuration of this compound was incorrectly assigned at first
[6]. After some years, this assignment was reversed on the basis of new X-ray diffraction studies [7, 81. Since then, the nomenclature of nco-clerodanes has been proposed for those diterpenes of this series with absolute configurations coincident with the revised structure of clerodin [8]. At that time, our group initiated research on new biorational insecticides from natural origin. One particular line of interest was the search of insect allelochemicals in plants. Based on the above precedents, we selected some Mediterranean Ajuga species for isolation of both phytoecdysteroids and insect antifeedants. In this communication we review our work in this area and compare it with the results reported by other authors. CLEBODANX DITERPENOIDS FROM AJUGA PLANT% INSECT’ ANTIFEEDANT ACTIVITY
Ajugarins I-III were the first insect antifeedants, isolated by I. Kubo and coworkers [4], from Ajuga remota. At a later stage, the same authors isolated from the same plant two other related structures, ajugarins IV and V, and also clerodin [9-111. The structures of these compounds were elucidated by spectral, X-ray diffraction and circular dichroism studies. The main structural characteristics are the but-13-en-15,16-olide ring and oxygen substituents at particular sites of the decalin system. As we will comment later, these patterns are important to elicit antifeedant activity.
1361
F. CAMPS and J. COLL
1362
(Substituentsin pmmbeses follow the order R’; R*; R3)
i)R’ Ajugachin A (MePr) B (AcMeBu) Ajugapitin (MeBu)
Ajugacmhin A oig; AC) B (Tis; H) AJu&ll I (AC: AC) II (AC; H)
i)R Ajugacumbin C (Tig) E (HOMeBu)
Ajugacumbm D
OR3
OR’
i)Ac
bR’
Ajugacumbin F (Tig; H) Ajupti III (AC; Ac)
Ajugmwin Al (H) AZ (AC) Gl (McBu)
Ajugmwin B 1 (AC; AC; H) 82 (AC; AC: AC) B3 (AC; H: H) B4WAcH) B5 (H; AC; AC) HI (AC; AC: IIg)
6AC
OAc Ajugammin Dl
Ajugmwin Fl (H; H) FL (H: AC) F3 (AC; H) F4 (AC; AC)
The antifeedant activities of ajugarins I-III were investigated by the host-plant leaf disk method [12] using Zea mays (maize) for the monophagous Spodoptera exempta and Ricinus communis for the polyphagous S.
Ajugrmptansin
Ajugamarin Cl (AC; AC; H) El (H; Ac; McBu) E2 (AC; H; MeBu) E3 (AC; AC; MeBu)
6Ac Ajugareptansone A
littoralis. Activity levels of 100 ppm against S. exempta and 300 ppm against S. littoralis were found. The other two ajugarins exhibited no antifeedant activities, and only moderate insecticidal and growth inhibitory activit-
Insect allelochemicals from Ajuga plants
AjUgdllIV
1363
AjqarbV
IvainI(oH)
2-G%
I (OAC)
ies were reported for ajugarin IV against different insects. The first plant we studied was Ajuga reptans, collected in the Montseny mountains near Barcelona. From this plant we isolated a new neo-clerodane, ajugareptansin (500ppm from whole dry plant). The structure of this compound was established by chemical and spectral means [ 131 and its absolute configuration was confirmed by X-ray diffraction analysis of the corresponding pbromobenzoate [ 141. This absolute configuration, unambiguously established by the Bijvoet method, was the same as that of clerodendrin A p-bromobenzoate chlorohydrin [8]. Relevant structural characteristics of this compound were the ester and the hexahydrofurofuran substituents at C-l and C-9, respectively, that caused
some distortion due to mutual steric interaction. Also from A. reptans we isolated two minor components (with the yields from dry plant indicated) ajugarep tansone A (8 ppm) and ajugareptansone B (6 ppm) with neo-clerodane structures [ 1S] closely related to ajugarins. These structures were established by study of the corresponding spectral data and fully confirmed by X-ray diffraction analysis [ 16,17J The close structural relationship between both structures suggested the possibility of ajugareptansone B being an artifact formed during the isolation procedure. In fact, a partial transformation (55%) was observed when ajugareptansone A was left standing at room temperature for 48 hr in an ethyl acetate--n-hexane mixture in the presence of alumina
1364
F. CAMPS and J. COLL
(Brockmann grade I). This result, however, was not conclusive enough to exclude the occurrence of ajugareptansone B in the plant. The oxygen substitution at C-l and the occurrence of compounds with hexahydrofurofuran and butenolide substituents at C-9 are the most relevant features of the neo-clerodane structures isolated from A. reptans. This latter coincidence was also observed in A. remota. We also studied Ajuga iva collected in Beer-Sheva (Israel) and isolated four new neo-clerodanes named ivains I-IV (300,10,40 and 60 ppm yield from dry plant, respectively). The structural elucidation of these compounds was carried out by spectral methods and comparison with spectral characteristics of other clerodane structures previously determined in our laboratory [ 181. In the present case, mass spectral data suggested the presence of hexahydrofurofuran moiety in ivains I and IV, (m/z 113), whereas mass spectral fragmentation (m/z 1.57) and ‘H NMR spectroscopy revealed the occurrence of a 1Sethoxyhexahydrofurofuran moiety in ivain III. Almost at the same time, Spanish colleagues isolated from Ajuga chamaepitys two compounds with structures closely related to ivains, which were named 14,15-dihydroajugapitin (2-epi-ivain IV) and ajugapitin (14,15dehydro-2-epi-ivain-IV), respectively, by these authors [ 191. At a later stage, we identified in this plant three new minor neo-clerodanes lSethoxy-14_hydroajugapitin, 14hydro-1 S-hydroxyajugapitin and chamaepitin [3-( 3’acetoxy)-14-hydro-15-hydroxyajugapitin] [20, 211. Recently, from A. chamaepitys var. psewdochia collected in Bulgaria the isolation of two neo-clerodane diterpenoids, ajugachins A and B, has been reported. These structures
Table
1. Clerodane
differ from that of ajugapitin only in the side chain of ester substituent at C-3 [22]. In our ongoing research, we found unexpectedly that Ajuga pseudoiva contains both types of C-2 epimers, as deduced from the structures of the compounds isolated, 2-acetylivain I and the known 14,15_dihydroajugapitin
c231. With all the clerodane structures isolated in our laboratory and some model synthetic compounds we undertook a thorough study on antifeedant activity-structure relationships [24]. These activities were evaluated by a choice test using newly ecdysed fifth instar larvae of Egyptian cotton leafworm, Spodoptera littoralis, and leaf disks of lettuce (Lactuca satira). Consumed areas of treated (CTD) and control disks (CCD) were simultaneously measured at regular intervals to evaluate the corresponding feeding ratios (FR = CTD/CCD). For comparative purposes, we recommend the use of FR,,, the ratio when 50% area of control disk is consumed. Most of the natural products assayed exhibited good to excellent antifeedant activities (FR,, ~0.5) at 30 ppm with the remarkable exceptions of ajugareptansin and ajugareptansone A with activities at 300 ppm or higher. The most active compounds were found in the ajugapitin series in which some products exhibited activity at 0.3 ppm dose, whereas FR,, ratios observed in the ivain series were 10 100-fold lower. These results agreed with suggestions of previous authors [25,26] that the presence in the clerodane structure of one spiroepoxide substituent at C-4 and two acetate groups at C-6 and C-19, together with the hexahydrofurofuran moiety at C-9 was important to elicit antifeedant activity. However, when free
diterpenes
in the genus Ajuga
Species
Clerodanes
References
A. ciliata var. villosior
Ajugamarms B4, B5, El, E2, E3, Fl, F2, F3; deacetylajugarin IV; ajugarin IV Ajugapitin; 14,1%dihydroajugapitin 15-ethoxy-14-hydroajugapitin; 14-hydro-15hydroxyajugapitin Chamaepitin Ajugachms A, B; ajugapitin; 14,15_dihydroajugapitin AJugamarms A2, Gl. Hl, F4, B2 Ajugacumbins A, B, C, D Ajugacumbins E, F; ajugamarin Al Ajugavensins A, B, C Ivams I-IV Ajugamarin Al AJugamarin Bl Ajugamarins Cl, B2, B3, Dl; ajugarin-I 2-Acetylivain I; 14,15dihydroajugapitin Ajugarins I-III Clerodin Ajugarin IV Ajugarin V Ajugareptansin Ajugareptansones A, B
30
A. chamaepitys
A chamaepitys var. pseudochia A. decumbens Thunberg
A. genevensls A. iva A. nipponensis Makino A. pseudoiva A. remota
A. reptans
19 20 21 22 31 33 32 34 18 27, 29 28, 29 29 23 4 11 9 10 13 15
Insect allelochemicals from Ajuga plants rotation of this moiety and conformational distortion of the trans decalin can occur by the presence of bulky substituents at C-l, such as in ajugareptansin, that activity is drastically reduced. On the other hand, ajugareptansone A with the same substitution at C-9 as ajugarins exhibited poor antifeedant activity. One important result found in our research was the enhancement of activity originated by a-OH substitution at C-2, that so far to our knowledge has not been con8rmed by other authors. Japanese and Chinese researchers have studied the neoclerodane diterpenoids from three Ajuga species of that geographic region: A. nipponensis, A. ciliata var. villosior and A. decumbens. Ajugamarin Al, isolated some years ago from A. nipponensis [2fl, is the parent compound of different series of structures (ajugamarins A-H), recently characterized in those plants [28-321. These structures are closely related to ajugarins except for the 12Soxygenated substitution and, in some of these series, the presence of /?-oxygenated substitution at C-l. In fact, ajugarin I was also isolated from A. nipponensis [29] and ajugarin IV and deacetylajugarin IV from A. ciliata [30]. Likewise, another series of neo-clerodane diterpenoids ajugacumbins A-F was characterized in A. decumbens [32, 331. The structures of ajugacumbins are also very similar to those of ajugarins except usually for the presence of t&late and related moieties as substituents at C-19 instead of the acetate group. Although the insect antifeedant activity of these series of compounds is presumed from their structural similitudes with ajugarins, only the activities of ajugacumbins A-C are mentioned in the literature [33]. In the hostplant leaf disk method using Boehmeria nivea with larvae of Pureda vesta Fabricius, the lowest effective concentration for antifeedant activity was 50 ppm for ajugacumbin A and 200 ppm for the other two compounds. Very recently, ajugavensins A-C have been isolated from the European species A. genevensis [34]. Like ajugareptansin, the first two bear bulky substituents at Cl, such as methylbutanoate in ajugavensin A or tiglate in ajugavensin B, whereas ajugavensin C has one hydroxyl at C-l and the acetate moiety at C-19 is replaced by one tiglate group. Although different configurational assignments for these substituents at C-l in the three compounds were first made, X-ray diffraction studies showed this configuration to be the same as in ajugareptansin c351. Extensive comparative insect antifeedant bioassays under rigorously standardized conditions should be carried out with all the above neo-clerodane diterpenoids against different insects to establish more general structure-activity relationships. PHYTOECDYSTEUOIDS
FROM
AJVGA
GROWTH REGULATOR
PLANTS:
INSECT
ACTIVITY
The screening of plants by simple insect moulting bioassays such as the Chilo dipping and fly pupation tests [36, 371, revealed the presence of ecdysteroids. In the genus Ajuga, a variety of such compounds have been isolated or identified [38-551 (Table 2). Among them,
1365
cyasterone and 20-hydroxyecdysone are the most abundant but ajugalactone is the most characteristic with unique structural features, such as a C-12 keto group and an unsaturated d-lactone ring. This compound exhibits dual bioactivity: moulting hormone activity when injected into diapausing pupae of Manduca sexta and antiponasterone A activity in the Chilo dipping test. Other phytoecdysteroids have so far only been found in this genus. Four new C-28 phytoecdysteroids, two major components, 29-norsengosterone and 29-norcyasterone [SO], and two minor ones, 2-acetyl- and 3-acetyl-29norcyasterone [5 l] were isolated in our laboratory from A. reptans. Also isolated from this plant were the known ajugalactone, cyasterone, 20-hydroxyecdysone and polypodine B [SO]. The structures of the major new phytoecdysteroids were assigned by spectral means in comparison with the corresponding data of cyasterone and confirmed by X-ray diffraction analysis of crystals of 29norcyasterone-2,3 : 20,22-diacetonide [56]. The structures of the minor components were established respectively by acetylation of 29-norcyasterone to give the corresponding 2-acetyl derivative and X-ray diffraction analysis of 3acetyl-29-norcyasterone [5 11. Large amounts of makisterone A, 20-hydroxyecdysone and cyasterone have been reported to be present in Ajuga iva and a minor component tentatively identified as 23hydroxycyasterone on mass spectral data basis only [46]. Recently, the presence of these major compounds has been confirmed, and also two new phytoecdysteroids, 22oxocyasterone and 24,25_dehydroprecyasterone, and one already described, 24,28dehydromakisterone A, have been isolated, and fully identified, as minor components from this plant [47]. Structural assignments for these compounds were carried out by different spectral procedures taking cyasterone for comparison as related structure. From in vivo and in vitro tests with phytoecdysteroids, different structural requirements have been deduced for insect moulting activity [57]. However, the results of biological studies reveal that there may be a wide variation in the susceptibility of different insects to these compounds. Insects possess diverse abilities to absorb, detoxify and efficiently eliminate phytoecdysteroids, although in some cases it has been shown that the incorporation of these compounds in artificial diets affect insect growth and reproduction. Thus, Kubo et al. [58] have reported that extracts of A. remota, containing cyasterone and 20_hydroxyecdysone, added to the diet of Bombyx mori and Pectinophora gossypiella inhibited ecdysis, resulting in the larval retention of the exuvial head capsules and exuviae, and leading to the final death of the insects. Likewise, the incidental observation that first stage larvae of the greenhouse whitefly, Trialeurodes vaporariorum exhibited complete mortality when fed on A. reptans plant growing under greenhouse conditions led to the development of a rapid and reliable bioassay studying the effects of phytoecdysteroids on larval development of this economically important pest [59]. Leaf fragments of Nicotiana glauca were infested in vitro with whitefly eggs, disinfected and cultivated on MS medium and the ecdys-
F.
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CAMPS
and J.
COLL
Table 2. Ecdysteroids in the genus Ajuga Species
Bcdysteroid
References
A. chamaepitys
Ajugaiactone; cyasterone; 2Ghydroxyecdysone; makisterone A Ajugasterone C; cyasterone; 20-hydroxyecdysone Cyasterone 20-Hydroxyecdysone Ajugalactone Ajugasterone B Ajugasterone C Cyasterone; 2~hydroxy~dysone Ajugasterone B Cyasterone; Mhydroxyecdysone 2~Hydroxy~dysone Cyasterone; makisterone A 23-Hydroxycyasterone 24(28)-Dehydromakisterone A; 22-oxocyasterone; 24.25-dehydroprecyastel rone Ajugasterone C Polypodine B (= ajugasterone A); cyasterone; 20-hydroxyecdysone Ajugasterone D AJugasterone C; cyasterone: 20-hydroxyecdysone Ajugalactone; cyasterone; 20-hydroxyecdysone; polypodine B 29-Norcyasterone; ZPNorsengosterone Z-Acetylnorcyasterone; 3-a~ty~norcy~terone Sengosterone 20-Hydroxyecdysone; cyasterone; 29norcyasterone; isocyasterone Ajugalactone; cyasterone; 20-hydroxy~dysone; turkesterone 22-Acetylcyasterone
38
A. chameacistus
A. chia A. decumbens Thunberg
A. incisa Maximowicz A. iva
A. jap~~~~~
Miquel
A. nippaaensis Makino
A. remora A. reptans
A. reptans var. atropurpurea A. turkestanica (Rgl.) Briq.
tecoids investigated applied by incorporation in the culture medium. The addition of 29norsengosterone and ajugalactone produced the highest mortalities on the first stage larvae, whereas 29-norcyasterone and polypodine B showed no appreciable differences when compared with untreated controls; 20-hydroxyecdysone was only effective when applied in the last stages of development.
~OTECHNOL~GICAL
PRODUCTION
ALL~LOCHEMICA~ FROM AJIG
To better understand
plant-insect
OF
INSECT PLANTS
interactions in A. production of insect allelochemicals of this plant by in vitro cultures, we studied the development of appropriate analytical methodology for rapid isolation and detection of both clerodanes and phytoecdysteroids in samples from diverse sources. So far, we have failed to detect clerodanes in samples of callus cultures of A. repruns which exhibited low levels of antifeedant activity as measured by the choice test commented above. However, we established efficient conditions for HPLC determination of eight phytoecdysteroids isolated from A. reptuns and Po~~p~j~ v~~~#e, using Spherisorb ODS-2 columns, ultraviolet detection, reptans, as well as for biotechnological
39
40 41 36 42 43 44 42 44 45-47 46,47 46 47 43 44 48 39 49 50 51
52 53 54 55
isopropanol-water as the mobile phase and tem~rature control [60]. Furthermore, HPLC-TSP-MS coupling permitted not only the quantification but also the identification of minor phytoecdysteroids, especially if previously derivatized as acetonides [61]. Both positive and negative ion acquisition modes were used, exhibiting the negative ion detection higher sensitivity and less fragmentation. We applied this HPLC analytical procedure to detect the most productive organs in the plant before establishment of appropriate in vitro cultures for ecdysteroid production. The occurrence of ecdysteroids in callus cultures was first described for seedling callus tissues from several Aehyruntes species [62] and Triunt~e~u portulucastrum [63]. The isolation of ecdysteroids from culture filtrates of gametophytes of Pteriditrm aquilinum [64] as well as callus and suspension cultures of this fern has been reported 1651. In this context, we have recently obtained promising results with in vitro cultures from Polypodium vulgare [66]. Related work in Ajuga plants has only been reported in A. turkestunica [67] and A. reptuns var. a~ropurpureu [53]. In the first case, in vitro production by callus and cell cultures of 20-hydroxyecdysone and turkesterone was
Insect allelochemicals from Ajuga plants
observed. While the first compound was produced in quantities two- to six-fold higher than those found in roots and aerial parts, the corresponding amount of turkesterone was slightly lower than that isolated from roots. Hairy root cultures of A. reptans var. atropurpurea transformed with Agrobacterium rhizogenes produced four phytoecdysteroids 20-hydroxyecdysone, 29-norcyasterone, cyasterone and isocyasterone, which were also present in a methanol extract of fresh roots [53]. When we investigated the total content and relative composition of phytoecdysteroids in different parts of normally grown or in oitro micropropagated A. reptans plants [68], that content was extremely low
PHYTO32:6-B
1367
( N 60 ppm/dry wt) in leaves of these micropropagated plants, whereas that of the corresponding roots was the highest detected in our experiments ( N 4000 ppm/dry wt). The relative composition was very variable in the different materials analysed, the major components being ajugalactone (45-57X) in leaves of wild plants and 29-norcyasterone (51%) in roots of micropropagated plants, respectively. Apparently, callus cultures obtained from leaves or roots lose their capacity to produce ecdysteroids, since we could not find these compounds in those cultures at the lowest detection level of our analytical method (5 ppm/dry wt). Fortunately, we were more successful in our study of in vitro cultures of root and shoot tissues of
1368
F. CAMPS
A. reptans [69]. While ecdysteroids were not produced in shoot tissues, after six weeks, root cultures in an hormone supplemented solid medium produced more than 5000 ppmjdry wt of phytoecdysteroids. This amount was higher than those produced in the same period in a basal medium (-3000 ppm) and in liquid medium (-2000 ppm). In these cases 29-norcyasterone was also the main component of the mixture. These cultures seem very appropriate for biosynthetic studies. From all the data obtained of our in vivo and in vitro experiments, we conclude that ecdysteroid production is related to organised macrostructures in the plant and occurs in the roots. The contents in the different materials studied seem to indicate a relationship between growth and ecdysteroid production. As far as the biosynthesis of phytoecdysteroids in A. reptans is concerned, two major biosynthetic pathways appear to be operating, side chain dealkylation and 5-hydroxylation, leading to two series of related compounds, namely cyasterone/29-norC,&,,/CZ, cyasterone/20-hydroxyecdysone sengosterand one/29-norsengosterone/5,20-dihydroxyecdysone (polypodine B), both series being related by the presence or absence of one hydroxyl at C-5. On the other hand, the unique structural features of ajugalactone make this C,, phytoecdysteroid unamenable to such comparisons and suggest that it may be formed as a dead end in the biosynthetic pathway. Five C2s and CZ9 compounds, cyasterone (CY), 29norcyasterone (29NC), sengosterone (SG), 29-norsengosterone (29NS) and ajugalactone (AJL) represent a mean content higher than 95% in all the samples studied. For comparison of the qualitative differences among the diverse plant materials studied, we established a dealkylation ratio [Dr = (29NS + 29NC)/(SG + CY + AJL)]. Likewise, we considered an index of hydroxylation at C-5 [SHr = (SG + 29NS + 520E)/(CY + 29NC + 20E)]. The values of the 5Hr index appeared to be quite constant with values usually ranging from 0.2 to 0.4 except in greenhouse plants and some isolated roots which exhibited a high dispersion of results due to very high values observed in some samples. In contrast, the Dr values varied with culture types. While tissues belonging to a whole plant had values near 2.3 or even lower for wild plants, root cultures can reach values above 12. When analyses were individually considered, in most cases the leaves had Dr values lower than the corresponding root materials, suggesting that dealkylation can take place preferentially in the roots.
CONCLUSIONS
In summary, we have reviewed the isolation and characterization of different secondary metabolites of Ajuga plants with insect allelochemical activities. Although it has been indicated that chemical defence in plants is multifaceted [70], we have separately investigated the activities of clerodane diterpenoids affecting the ‘insect behavior as antifeedants and phytoecdysteroids interfering with insect development. Ongoing research on
and
J. COLL
biosynthetic studies and biotechnological production of these metabolites has also been commented on. We hope that, in the future, further research of this so far hardly investigated plant genus will be carried out, not only looking for new insect allelochemicals which might clarify unexplored insect-plant interactions, but also to confirm if any of the above classes of compound are responsible of their interesting activities recorded in folk medicine. In fact, it has been shown that phytoecdysteroids exhibited different activities in vertebrates [3]. Among them, it is worth mentioning: the stimulation of protein, an anabolic activity in murine liver [71]. the suppression of the hyperglycaemia induced by either glucagon or alloxan in diabetic mice [72], the inhibition of the development of hypercholesterolaemia and hyperlipidaemia produced by Triton WR-1339 in rats and antiatherosclerotic action in rabbits [73], the increase of bile acids and bilirubin levels and decrease of cholesterol content in the bile secretion of normal rats or rats with induced toxic hepatitis [74], the normalization of the respiratory chain and the terminal pathway of electron transport in hepatocytes of rats with induced hepatitis [75] and the potentiation of the effects of insulin [76]. Acknowledgements-This research was supported by funds from Spanish CAICYT (Grants 3708/79 and 1664/82) and CICYT (BIO 88-0230) and predoctoral fellowships from the Spanish Ministry of Education and Science. Special acknowledgements should be expressed to the Ph. D. students A. Cortel, 0. Dargallo, M. P. Marco and J. Tom&s for their enthusiasm, dedication and important contributions to this research, Drs F. J. Sinchez-Baeza and A. Llebaria and Mrs. J. Estremera (Dep. Biological Organic Chemistry, CID, CSIC) for preparation of drawings and typing the manuscript, respectively. REFERENCES
1. Darvas, B. (1991) Niivenyvedelem Budapest 27, 481. 2. Shimomura, H., Sashida, Y. and Ogawa, K. (1992) Chem. Pharm. Bull. 37, 988. 3. Camps, F. (1991) in Ecological Chemistry and Biochemistry of Plant Terpenoids (Harborne, J. B. and Tom&s-Barberin, F. A., eds), pp. 331-376. Clarendon Press, Oxford. 4. Kubo, I., Lee, Y.-W., Balogh-Nair, V., Nakanishi, K. and Chapya, A. (1976) J. Chem. Sot., Chem. Commun. 949. 5. Barton, D. H. R., Cheung, H. T., Cross, A. D., Jackmann, L. M. and Martin-Smith, M. (1961) J. Chem. Sot. 5061. Paul, I. C., Sim, G. A., Hamor T. A. and Robertson, J. M. (1962) J. Chem. Sot. 4133. Kato, N., Munakata, K. and Katayama, C. (1973) J. Chem. Sot., Perkin Trans II 69. Rogers, D., Unal, G. C., Williams, D. J., Ley, S. V., Sim, G. A., Joshi, B. S. and Ravindranath, K. R. (1979) J. Chem. Sot., Chem. Commun. 97. 9. Kubo, I.. Klocke, J. A., Miura, I. and Fukuyama, Y. (1982) J. Chem. Sot., Chem. Commun. 618.
Insect allelochemicals from Ajuga plants 10. Kubo, I., Fukuyama, Y. and Chapya, A. (1983) Chem. Letters 223. 11. Kubo, I., Kido, M. and Fukuyama, Y. (1980) J. Chem. Sot., Chem. Commun. 897. 12. Kubo, I. and Nakanishi, K. (1977) in Host Plant Resistance to Pests (Hedin, P.A., ed.), pp. 165-178. ACS Symposium Series 62. American Chemical Society, Washington DC. 13. Camps, F., Coil, J., Cortel, A. and Messeguer, A. (1979) Tetrahedron Letters 1709. 14. Solans, X., Miravitlles, C., Declercq J. P. and Germain, G. (1979) Acta Cryst. B 35, 2732. 15. Camps, F., Coll, J. and Cortel, A. (1981) Chem. Letters 1093. 16. Miravitlles, C., Solans, X., Germain, G. and Declercq, J. P. (1982) Acta Cryst. B 38, 188. 17. Solans, X., Miravitlles, C., Declercq, J. P. and Germain, G. (1983) Acta Cryst. C 39, 307. 18. Camps, F., Coil, J. and Cortel, A. (1982) Chem. Letters 1053. 19. Hernandez, H., Pascual, C., Sanz, J. and Rodriguez, B. (1982) Phytochemistry 21, 2909. 20. Camps, F., Coll, J. and Dargallo, 0. (1984) Phytochemistry 23, 2577. 21. Camps, F., Coll, J., Dargallo, O., Rius, J. and Miravitlles, C. (1987) Phytochemistry 26, 1475. 22. Boneva, I. M., Mikhova, B. P., Malakov, P. Y.,
Papanov, G. Y., Duddeck, H. and Spassov, S. L. (1990) Phytochemistry 29, 2931. 23. Camps, F., Coll, J. and Dargallo, 0. (1984) Phytochemistry 23, 387. 24. Bell&s, X., Camps, F., Coll, J. and Piulachs, M. D. (1985) J. Chem. Ecol. 11, 1439. 25. Kojima, Y. and Kato, N. (1981) Tetrahedron 37,2527. 26. Geuskens, R. B. M., Luteijn, J. M. and Schoonhoven, L. M. (1983) Experientia 39, 403. 27. Shimomura, H., Sashida, Y., Ogawa, K. and Iitaka, Y. (1981) Tetrahedron Letters 22, 1367. 28. Shimomura, Y., Sashida, Y., Ogawa, K. and Iitaka, Y.
(1983) Chem. Pharm. Bull. 31, 2192. 29. Shimomura, H., Sashida, Y. and Ogawa, K. (1989) Chem. Pharm. Bull. 37, 354. 30. Shimomura, H., Sashida, Y. and Ogawa, K. (1989) Chem. Parm. Bull. 37, 988. 31. Shimomura, H., Sashida, Y. and Ogawa, K. (1989) Chem. Parm. Bull. 37, 996. 32. Zhi-da, M., Mizuno, M., Shi-qiang, W., Iinuma, M. and Tanaka, T. (1990) Chem. Pharm. Bull. 38, 3167. 33. Zhi-da, M., Shi-qiang, W. Qi-tai, Z., Bing, W., Mizuro, M., Tanaka, T. and Inuma, M. (1989) Chem. Pharm. Bull. 37, 2505. 34. Malakov, P. Y., Papanov, G. Y., de la Torre, M. C. and Rodriguez, B. (1991) Phytochemisbry 30, 4083. 35. Malakov, P. Y., Papanov, G. Y., Perales, A., de la Terre, M. C. and Rodriguez, B. (1992) Phytochemistry 31, 3151. 36. Koreeda, M., Nakanishi, K. and Goto, M. (1970) J. Am. Chem. Sot. 92,7512. 37. Thompson, J. A. (1974) in Invertebrate Endocrinology and Hormonal Heterophylly (Burdette, W. J., ed.),
pp. 121-129. Springer, Berlin.
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38. Camps, F., Coll, J. and Dargallo, 0. (1985) An. Quim. UC, 74. 39. Kubo, I., Klocke, J. A., Ganjian, I., Ichikawa, N. and Matsumoto, T. (1983) J. Chromatogr. 257, 157. 40. Ikan, R. and Ravid, U. (1971) Phytochemistry 10, 1659. 41. Abubakirov, N. K. (1982) Proc. Indian Natl. Sci. Acad., 48& Suppl. 1, 122.
42. Imai, S., Fujioka, S., Murata, E., Otsuka, K. and Nakanishi, K. (1969) Chem. Commun. 82. 43. Imai, S., Murata, E., Fujioka, S., Koreeda, M. and Nakanishi, K. (1969) Chem. Commun. 546. 44. Imai, S., Toyosato, T., Sakai, M., Sato, Y., Fujioka, S., Murata, E. and Goto, M. (1969) Chem. Pharm. Bull. 17, 340. 45. Ikan, R. and Ravid, U. (1971) Planta Med. u), 33. 46. Nazmi Sabri, N., Asaad, A and Khafagy, S. M. (1981) Planta Med. 42, 293. 47. Wessner, M., Champion, B., Girault, J. P., Kaouadji, N., Saidi, B. and Lafont, R. (1992) Phytochemistry 31 3785. 48. Chou, W. S. and Lu, M. S. (1980) in Progress in Ecdysone Research (Hoffmann, J. A., ed.),
pp. 281-297. Elsevier/North-Holland, Amsterdam. 49. Camps, F., Coll, J. and Cortel, A. (1981) Rev. Latinoam. Quim. 12, 8 1. 50. Camps, F., Coll, J. and Cortel, A. (1982) Chem. Letters 1313. 51. Camps, F., Coll, J., Cortel, A., Molins, E. and Miravitlles, C. (1985) J. Chem. Res. (S) 14. 52. Calcagno, M. P., Camps, F., Coil, J., Melt, E., Messeguer, J. and Tom&, J. (1992) Xth Ecdysone Workshop (Liverpool) Abstracts p. 83. 53. Matsumoto, T. and Tanaka, N. (1991) Agric. Biol. Gem. 55, 1019. 54. Usmanov, B. Z., Gorovits, M. B. and Abubakhirov, N. K. (1975) Khim. Prir. Soedin. 466. 55. Usmanov, B. Z., Rashkes, Ya..V. and Abubakhirov, N. K. (1978) Khim. Prir. Soedin. 215. 56. Miravitlles, C., Solans, X., Germain, G. and Declercq, J. P. (1982) Cryst. Struct. Commun. 11, 1683. 57. Bergamasco, R. and Horn, D. H. S. (1980) in Progress in Ecdysone Research (Hoffmann, J. A., ed.),
pp. 299-324. Elsevier North-Holland, Amsterdam. 58. Kubo, I., Klocke, J. and Asano, S. (1981) Agric. Biol. Chem. 45, 1925. 59. Melt, E., Messeguer, J., Gabarra, R., Tom&, J., Coll, J. and Camps, F. (1992) Entomol. Exp. Appl. 62,163. 60. Camps, F., Coll, J., Marco, M. P. and Tomis, J. (1990) J. Chromatogr. 514, 199. 61. Camps, F., Coil, J., Marco, M. P. and Sanchez, F. J. (1992) J. Chromatogr. (submitted). 62. Hikino, H., Jin, H. and Takemoto, T. (1971) Chem. Pharm. Bull. 19, 439. 63. Ravishankar, G. A. and Metha, A. R. (1911) J. Nat. Prod. 42, 152. 64. MC Morris, T. C. and Voeller, V. (1971) Phytochemistry 10, 3253.
65. Vanek, T., Macek, T., Vaisar, T. and Breznovito, A. (1990) Biotechnol. Letters 12, 727. 66. Camps, F., Claveria, E., Coll, J., Marco, M. P., Mel&
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E. and Messeguer, J. (1990) Phytochemistry 29,38 19. 67. Lev, S. V., Zakirova, R. P., Saatov, Z., Gorovits, B. and Abubakirov, N. K. (1990) Khim. Prir. Soedin. 5 1. 68. Tomas, J., Camps, F., Claveria, E., Coil, J., Mel& E. and Messeguer, J. (1992) Phytochemistry 31, 1585. 69. Tomas, J., Camps, F., Coil, J., Mel& E. and Messeguer, J. (1992) Phytochemistry 31 (in press). 70. Kubo, I. and Klocke, J. (1986) in Natural Resistance of Plants to Pests: Roles of Allelochemicals (Green, M. B. and Hedin, P. A., eds), pp. 206-219 ACS Symposium Series 296, American Chemical Society, Washington DC. 71. Syrov, V. N. (1984) Biol. Nauk. 9, 37.
72. Uchiyama, M. and Yoshida, T. (1974) in Invertebrate Endocrinology and Hormonal Heterophylly (Burdette, W. J., ed.), pp. 375400. Springer, Berlin. 73. Syrov, V. N., Khushbaktova,Z. A., Abzalova, M. Kh. and Sultanov, M. B. (1983) Dokl. Akad. Nauk. UzSSR 9, 44. 74. Syrov, V. N. (1986) Farmakol. Toksikol. 49, 100. 75. Tashmukhamedova, M. A., Almatov, K. T., Khushbaktova, Z. A., Syrov, V. N. and Sultanov, M. B. (1986) Vopr. Med. Khim. 32, 81. 76. Kosovskii, M. I., Syrov, V. N., Mirakhmedov, M. M., Katkova, S. P. and Khushbatkova, Z. A. (1989) Probl. Endokrinol. 35, 77.
0031 9422/93 $24.00 + 0.00 Q 1993 PergamonPress Ltd
REVIEW ARTICLE NUMBER 75 INSECT ALLELOCHEMICALS FRANCISCO CAMPS
FROM AJUGA PLANTS and
JOSEP COLL
Department of Biological Organic Chemistry, C.I.D., C.S.I.C. Jordi Girona, 18-26. 08034-Barcelona, Spain (Receioed 4 August 1992)
Key Word Index-Ajuga; hormone.
Labiatae; clerodane diterpene; phytoecdysteroid;
antifeedant;
moulting
Abstract-Clerodane diterpenoids and phytoecdysteroids with potential insect antifeedant and moulting hormone activities, respectively, have been isolated from Ajuga plants. Some clerodanes were active against larvae of Egyptian cotton leafworm, Spodoptera littoralis, when present in the diet at 3 ppm doses. Structure-antifeedant activity relations were established. Likewise, first stage larvae of the greenhouse whitefly, Trialeurodes uaporariorum, exhibited complete mortality when fed on A. reptans. This effect was mainly originated by 29-norsengosterone and ajugalactone, two phytoecdysteroids occurring in this plant. For biotechnological production of phytoecdysteroids its total content in different parts of normally grown or in vitro micropropagated A. reptans plants was studied. Great quantitative and qualitative differences were observed. For comparison of these qualitative differences, a dealkylation ratio (Dr = C,,/C,, phytoecdysteroid content) and a C-5 hydroxylation ratio (5Hr = 5-OH/5-H phytoecdysteroid content) were established. The 5Hr values appeared to be quite constant ranging from 0.2 to 0.4, whereas Dr values oscillated from 2.3 in whole plants to 12 in root cultures. Production of phytoecdysteroids was highest ( N 5000 ppm/dry wt) in cultures of roots in an hormone supplemented solid medium.
INTRODUCTION
More than one hundred species and fifty varieties and subspecies of Ajugu plants are unevenly distributed over the world [ 11. While in America there are recorded only three newly introduced species, and two endemic species in Australia, these Labiatae are especially abundant in China, Korea and Japan and also widespread in Europe. They have been used in folk medicine in various cultures and several interesting medicinal properties, such as antifebrile, anthelmintic, hypoglycaemic and vulnerary effects have been attributed to them. Traditionally, in China some Ajuga plants have been used to treat inflammation [2]. Many representatives of the genus Ajuga contain phytoecdysteroids [3], polyhydroxysteroids with a 5fl-H-7ene-6-one system, exhibiting well established physiological activities in insects and also in mammals which might explain some of the successful applications of these plants in folk medicine. The observation that Ajuga remota leaves, collected in Kenya, were not attacked by African armyworms led to the isolation of three moderately strong antifeedants named ajugarins I-III with diterpene clerodane structures [4], closely related to clerodin, the first compound known of this series isolated from Clerodendron infirtunatum (Verbenaceae) [S]. The absolute configuration of this compound was incorrectly assigned at first
[6]. After some years, this assignment was reversed on the basis of new X-ray diffraction studies [7, 81. Since then, the nomenclature of nco-clerodanes has been proposed for those diterpenes of this series with absolute configurations coincident with the revised structure of clerodin [8]. At that time, our group initiated research on new biorational insecticides from natural origin. One particular line of interest was the search of insect allelochemicals in plants. Based on the above precedents, we selected some Mediterranean Ajuga species for isolation of both phytoecdysteroids and insect antifeedants. In this communication we review our work in this area and compare it with the results reported by other authors. CLEBODANX DITERPENOIDS FROM AJUGA PLANT% INSECT’ ANTIFEEDANT ACTIVITY
Ajugarins I-III were the first insect antifeedants, isolated by I. Kubo and coworkers [4], from Ajuga remota. At a later stage, the same authors isolated from the same plant two other related structures, ajugarins IV and V, and also clerodin [9-111. The structures of these compounds were elucidated by spectral, X-ray diffraction and circular dichroism studies. The main structural characteristics are the but-13-en-15,16-olide ring and oxygen substituents at particular sites of the decalin system. As we will comment later, these patterns are important to elicit antifeedant activity.
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F. CAMPS and J. COLL
1362
(Substituentsin pmmbeses follow the order R’; R*; R3)
i)R’ Ajugachin A (MePr) B (AcMeBu) Ajugapitin (MeBu)
Ajugacmhin A oig; AC) B (Tis; H) AJu&ll I (AC: AC) II (AC; H)
i)R Ajugacumbin C (Tig) E (HOMeBu)
Ajugacumbm D
OR3
OR’
i)Ac
bR’
Ajugacumbin F (Tig; H) Ajupti III (AC; Ac)
Ajugmwin Al (H) AZ (AC) Gl (McBu)
Ajugmwin B 1 (AC; AC; H) 82 (AC; AC: AC) B3 (AC; H: H) B4WAcH) B5 (H; AC; AC) HI (AC; AC: IIg)
6AC
OAc Ajugammin Dl
Ajugmwin Fl (H; H) FL (H: AC) F3 (AC; H) F4 (AC; AC)
The antifeedant activities of ajugarins I-III were investigated by the host-plant leaf disk method [12] using Zea mays (maize) for the monophagous Spodoptera exempta and Ricinus communis for the polyphagous S.
Ajugrmptansin
Ajugamarin Cl (AC; AC; H) El (H; Ac; McBu) E2 (AC; H; MeBu) E3 (AC; AC; MeBu)
6Ac Ajugareptansone A
littoralis. Activity levels of 100 ppm against S. exempta and 300 ppm against S. littoralis were found. The other two ajugarins exhibited no antifeedant activities, and only moderate insecticidal and growth inhibitory activit-
Insect allelochemicals from Ajuga plants
AjUgdllIV
1363
AjqarbV
IvainI(oH)
2-G%
I (OAC)
ies were reported for ajugarin IV against different insects. The first plant we studied was Ajuga reptans, collected in the Montseny mountains near Barcelona. From this plant we isolated a new neo-clerodane, ajugareptansin (500ppm from whole dry plant). The structure of this compound was established by chemical and spectral means [ 131 and its absolute configuration was confirmed by X-ray diffraction analysis of the corresponding pbromobenzoate [ 141. This absolute configuration, unambiguously established by the Bijvoet method, was the same as that of clerodendrin A p-bromobenzoate chlorohydrin [8]. Relevant structural characteristics of this compound were the ester and the hexahydrofurofuran substituents at C-l and C-9, respectively, that caused
some distortion due to mutual steric interaction. Also from A. reptans we isolated two minor components (with the yields from dry plant indicated) ajugarep tansone A (8 ppm) and ajugareptansone B (6 ppm) with neo-clerodane structures [ 1S] closely related to ajugarins. These structures were established by study of the corresponding spectral data and fully confirmed by X-ray diffraction analysis [ 16,17J The close structural relationship between both structures suggested the possibility of ajugareptansone B being an artifact formed during the isolation procedure. In fact, a partial transformation (55%) was observed when ajugareptansone A was left standing at room temperature for 48 hr in an ethyl acetate--n-hexane mixture in the presence of alumina
1364
F. CAMPS and J. COLL
(Brockmann grade I). This result, however, was not conclusive enough to exclude the occurrence of ajugareptansone B in the plant. The oxygen substitution at C-l and the occurrence of compounds with hexahydrofurofuran and butenolide substituents at C-9 are the most relevant features of the neo-clerodane structures isolated from A. reptans. This latter coincidence was also observed in A. remota. We also studied Ajuga iva collected in Beer-Sheva (Israel) and isolated four new neo-clerodanes named ivains I-IV (300,10,40 and 60 ppm yield from dry plant, respectively). The structural elucidation of these compounds was carried out by spectral methods and comparison with spectral characteristics of other clerodane structures previously determined in our laboratory [ 181. In the present case, mass spectral data suggested the presence of hexahydrofurofuran moiety in ivains I and IV, (m/z 113), whereas mass spectral fragmentation (m/z 1.57) and ‘H NMR spectroscopy revealed the occurrence of a 1Sethoxyhexahydrofurofuran moiety in ivain III. Almost at the same time, Spanish colleagues isolated from Ajuga chamaepitys two compounds with structures closely related to ivains, which were named 14,15-dihydroajugapitin (2-epi-ivain IV) and ajugapitin (14,15dehydro-2-epi-ivain-IV), respectively, by these authors [ 191. At a later stage, we identified in this plant three new minor neo-clerodanes lSethoxy-14_hydroajugapitin, 14hydro-1 S-hydroxyajugapitin and chamaepitin [3-( 3’acetoxy)-14-hydro-15-hydroxyajugapitin] [20, 211. Recently, from A. chamaepitys var. psewdochia collected in Bulgaria the isolation of two neo-clerodane diterpenoids, ajugachins A and B, has been reported. These structures
Table
1. Clerodane
differ from that of ajugapitin only in the side chain of ester substituent at C-3 [22]. In our ongoing research, we found unexpectedly that Ajuga pseudoiva contains both types of C-2 epimers, as deduced from the structures of the compounds isolated, 2-acetylivain I and the known 14,15_dihydroajugapitin
c231. With all the clerodane structures isolated in our laboratory and some model synthetic compounds we undertook a thorough study on antifeedant activity-structure relationships [24]. These activities were evaluated by a choice test using newly ecdysed fifth instar larvae of Egyptian cotton leafworm, Spodoptera littoralis, and leaf disks of lettuce (Lactuca satira). Consumed areas of treated (CTD) and control disks (CCD) were simultaneously measured at regular intervals to evaluate the corresponding feeding ratios (FR = CTD/CCD). For comparative purposes, we recommend the use of FR,,, the ratio when 50% area of control disk is consumed. Most of the natural products assayed exhibited good to excellent antifeedant activities (FR,, ~0.5) at 30 ppm with the remarkable exceptions of ajugareptansin and ajugareptansone A with activities at 300 ppm or higher. The most active compounds were found in the ajugapitin series in which some products exhibited activity at 0.3 ppm dose, whereas FR,, ratios observed in the ivain series were 10 100-fold lower. These results agreed with suggestions of previous authors [25,26] that the presence in the clerodane structure of one spiroepoxide substituent at C-4 and two acetate groups at C-6 and C-19, together with the hexahydrofurofuran moiety at C-9 was important to elicit antifeedant activity. However, when free
diterpenes
in the genus Ajuga
Species
Clerodanes
References
A. ciliata var. villosior
Ajugamarms B4, B5, El, E2, E3, Fl, F2, F3; deacetylajugarin IV; ajugarin IV Ajugapitin; 14,1%dihydroajugapitin 15-ethoxy-14-hydroajugapitin; 14-hydro-15hydroxyajugapitin Chamaepitin Ajugachms A, B; ajugapitin; 14,15_dihydroajugapitin AJugamarms A2, Gl. Hl, F4, B2 Ajugacumbins A, B, C, D Ajugacumbins E, F; ajugamarin Al Ajugavensins A, B, C Ivams I-IV Ajugamarin Al AJugamarin Bl Ajugamarins Cl, B2, B3, Dl; ajugarin-I 2-Acetylivain I; 14,15dihydroajugapitin Ajugarins I-III Clerodin Ajugarin IV Ajugarin V Ajugareptansin Ajugareptansones A, B
30
A. chamaepitys
A chamaepitys var. pseudochia A. decumbens Thunberg
A. genevensls A. iva A. nipponensis Makino A. pseudoiva A. remota
A. reptans
19 20 21 22 31 33 32 34 18 27, 29 28, 29 29 23 4 11 9 10 13 15
Insect allelochemicals from Ajuga plants rotation of this moiety and conformational distortion of the trans decalin can occur by the presence of bulky substituents at C-l, such as in ajugareptansin, that activity is drastically reduced. On the other hand, ajugareptansone A with the same substitution at C-9 as ajugarins exhibited poor antifeedant activity. One important result found in our research was the enhancement of activity originated by a-OH substitution at C-2, that so far to our knowledge has not been con8rmed by other authors. Japanese and Chinese researchers have studied the neoclerodane diterpenoids from three Ajuga species of that geographic region: A. nipponensis, A. ciliata var. villosior and A. decumbens. Ajugamarin Al, isolated some years ago from A. nipponensis [2fl, is the parent compound of different series of structures (ajugamarins A-H), recently characterized in those plants [28-321. These structures are closely related to ajugarins except for the 12Soxygenated substitution and, in some of these series, the presence of /?-oxygenated substitution at C-l. In fact, ajugarin I was also isolated from A. nipponensis [29] and ajugarin IV and deacetylajugarin IV from A. ciliata [30]. Likewise, another series of neo-clerodane diterpenoids ajugacumbins A-F was characterized in A. decumbens [32, 331. The structures of ajugacumbins are also very similar to those of ajugarins except usually for the presence of t&late and related moieties as substituents at C-19 instead of the acetate group. Although the insect antifeedant activity of these series of compounds is presumed from their structural similitudes with ajugarins, only the activities of ajugacumbins A-C are mentioned in the literature [33]. In the hostplant leaf disk method using Boehmeria nivea with larvae of Pureda vesta Fabricius, the lowest effective concentration for antifeedant activity was 50 ppm for ajugacumbin A and 200 ppm for the other two compounds. Very recently, ajugavensins A-C have been isolated from the European species A. genevensis [34]. Like ajugareptansin, the first two bear bulky substituents at Cl, such as methylbutanoate in ajugavensin A or tiglate in ajugavensin B, whereas ajugavensin C has one hydroxyl at C-l and the acetate moiety at C-19 is replaced by one tiglate group. Although different configurational assignments for these substituents at C-l in the three compounds were first made, X-ray diffraction studies showed this configuration to be the same as in ajugareptansin c351. Extensive comparative insect antifeedant bioassays under rigorously standardized conditions should be carried out with all the above neo-clerodane diterpenoids against different insects to establish more general structure-activity relationships. PHYTOECDYSTEUOIDS
FROM
AJVGA
GROWTH REGULATOR
PLANTS:
INSECT
ACTIVITY
The screening of plants by simple insect moulting bioassays such as the Chilo dipping and fly pupation tests [36, 371, revealed the presence of ecdysteroids. In the genus Ajuga, a variety of such compounds have been isolated or identified [38-551 (Table 2). Among them,
1365
cyasterone and 20-hydroxyecdysone are the most abundant but ajugalactone is the most characteristic with unique structural features, such as a C-12 keto group and an unsaturated d-lactone ring. This compound exhibits dual bioactivity: moulting hormone activity when injected into diapausing pupae of Manduca sexta and antiponasterone A activity in the Chilo dipping test. Other phytoecdysteroids have so far only been found in this genus. Four new C-28 phytoecdysteroids, two major components, 29-norsengosterone and 29-norcyasterone [SO], and two minor ones, 2-acetyl- and 3-acetyl-29norcyasterone [5 l] were isolated in our laboratory from A. reptans. Also isolated from this plant were the known ajugalactone, cyasterone, 20-hydroxyecdysone and polypodine B [SO]. The structures of the major new phytoecdysteroids were assigned by spectral means in comparison with the corresponding data of cyasterone and confirmed by X-ray diffraction analysis of crystals of 29norcyasterone-2,3 : 20,22-diacetonide [56]. The structures of the minor components were established respectively by acetylation of 29-norcyasterone to give the corresponding 2-acetyl derivative and X-ray diffraction analysis of 3acetyl-29-norcyasterone [5 11. Large amounts of makisterone A, 20-hydroxyecdysone and cyasterone have been reported to be present in Ajuga iva and a minor component tentatively identified as 23hydroxycyasterone on mass spectral data basis only [46]. Recently, the presence of these major compounds has been confirmed, and also two new phytoecdysteroids, 22oxocyasterone and 24,25_dehydroprecyasterone, and one already described, 24,28dehydromakisterone A, have been isolated, and fully identified, as minor components from this plant [47]. Structural assignments for these compounds were carried out by different spectral procedures taking cyasterone for comparison as related structure. From in vivo and in vitro tests with phytoecdysteroids, different structural requirements have been deduced for insect moulting activity [57]. However, the results of biological studies reveal that there may be a wide variation in the susceptibility of different insects to these compounds. Insects possess diverse abilities to absorb, detoxify and efficiently eliminate phytoecdysteroids, although in some cases it has been shown that the incorporation of these compounds in artificial diets affect insect growth and reproduction. Thus, Kubo et al. [58] have reported that extracts of A. remota, containing cyasterone and 20_hydroxyecdysone, added to the diet of Bombyx mori and Pectinophora gossypiella inhibited ecdysis, resulting in the larval retention of the exuvial head capsules and exuviae, and leading to the final death of the insects. Likewise, the incidental observation that first stage larvae of the greenhouse whitefly, Trialeurodes vaporariorum exhibited complete mortality when fed on A. reptans plant growing under greenhouse conditions led to the development of a rapid and reliable bioassay studying the effects of phytoecdysteroids on larval development of this economically important pest [59]. Leaf fragments of Nicotiana glauca were infested in vitro with whitefly eggs, disinfected and cultivated on MS medium and the ecdys-
F.
1366
CAMPS
and J.
COLL
Table 2. Ecdysteroids in the genus Ajuga Species
Bcdysteroid
References
A. chamaepitys
Ajugaiactone; cyasterone; 2Ghydroxyecdysone; makisterone A Ajugasterone C; cyasterone; 20-hydroxyecdysone Cyasterone 20-Hydroxyecdysone Ajugalactone Ajugasterone B Ajugasterone C Cyasterone; 2~hydroxy~dysone Ajugasterone B Cyasterone; Mhydroxyecdysone 2~Hydroxy~dysone Cyasterone; makisterone A 23-Hydroxycyasterone 24(28)-Dehydromakisterone A; 22-oxocyasterone; 24.25-dehydroprecyastel rone Ajugasterone C Polypodine B (= ajugasterone A); cyasterone; 20-hydroxyecdysone Ajugasterone D AJugasterone C; cyasterone: 20-hydroxyecdysone Ajugalactone; cyasterone; 20-hydroxyecdysone; polypodine B 29-Norcyasterone; ZPNorsengosterone Z-Acetylnorcyasterone; 3-a~ty~norcy~terone Sengosterone 20-Hydroxyecdysone; cyasterone; 29norcyasterone; isocyasterone Ajugalactone; cyasterone; 20-hydroxy~dysone; turkesterone 22-Acetylcyasterone
38
A. chameacistus
A. chia A. decumbens Thunberg
A. incisa Maximowicz A. iva
A. jap~~~~~
Miquel
A. nippaaensis Makino
A. remora A. reptans
A. reptans var. atropurpurea A. turkestanica (Rgl.) Briq.
tecoids investigated applied by incorporation in the culture medium. The addition of 29norsengosterone and ajugalactone produced the highest mortalities on the first stage larvae, whereas 29-norcyasterone and polypodine B showed no appreciable differences when compared with untreated controls; 20-hydroxyecdysone was only effective when applied in the last stages of development.
~OTECHNOL~GICAL
PRODUCTION
ALL~LOCHEMICA~ FROM AJIG
To better understand
plant-insect
OF
INSECT PLANTS
interactions in A. production of insect allelochemicals of this plant by in vitro cultures, we studied the development of appropriate analytical methodology for rapid isolation and detection of both clerodanes and phytoecdysteroids in samples from diverse sources. So far, we have failed to detect clerodanes in samples of callus cultures of A. repruns which exhibited low levels of antifeedant activity as measured by the choice test commented above. However, we established efficient conditions for HPLC determination of eight phytoecdysteroids isolated from A. reptuns and Po~~p~j~ v~~~#e, using Spherisorb ODS-2 columns, ultraviolet detection, reptans, as well as for biotechnological
39
40 41 36 42 43 44 42 44 45-47 46,47 46 47 43 44 48 39 49 50 51
52 53 54 55
isopropanol-water as the mobile phase and tem~rature control [60]. Furthermore, HPLC-TSP-MS coupling permitted not only the quantification but also the identification of minor phytoecdysteroids, especially if previously derivatized as acetonides [61]. Both positive and negative ion acquisition modes were used, exhibiting the negative ion detection higher sensitivity and less fragmentation. We applied this HPLC analytical procedure to detect the most productive organs in the plant before establishment of appropriate in vitro cultures for ecdysteroid production. The occurrence of ecdysteroids in callus cultures was first described for seedling callus tissues from several Aehyruntes species [62] and Triunt~e~u portulucastrum [63]. The isolation of ecdysteroids from culture filtrates of gametophytes of Pteriditrm aquilinum [64] as well as callus and suspension cultures of this fern has been reported 1651. In this context, we have recently obtained promising results with in vitro cultures from Polypodium vulgare [66]. Related work in Ajuga plants has only been reported in A. turkestunica [67] and A. reptuns var. a~ropurpureu [53]. In the first case, in vitro production by callus and cell cultures of 20-hydroxyecdysone and turkesterone was
Insect allelochemicals from Ajuga plants
observed. While the first compound was produced in quantities two- to six-fold higher than those found in roots and aerial parts, the corresponding amount of turkesterone was slightly lower than that isolated from roots. Hairy root cultures of A. reptans var. atropurpurea transformed with Agrobacterium rhizogenes produced four phytoecdysteroids 20-hydroxyecdysone, 29-norcyasterone, cyasterone and isocyasterone, which were also present in a methanol extract of fresh roots [53]. When we investigated the total content and relative composition of phytoecdysteroids in different parts of normally grown or in oitro micropropagated A. reptans plants [68], that content was extremely low
PHYTO32:6-B
1367
( N 60 ppm/dry wt) in leaves of these micropropagated plants, whereas that of the corresponding roots was the highest detected in our experiments ( N 4000 ppm/dry wt). The relative composition was very variable in the different materials analysed, the major components being ajugalactone (45-57X) in leaves of wild plants and 29-norcyasterone (51%) in roots of micropropagated plants, respectively. Apparently, callus cultures obtained from leaves or roots lose their capacity to produce ecdysteroids, since we could not find these compounds in those cultures at the lowest detection level of our analytical method (5 ppm/dry wt). Fortunately, we were more successful in our study of in vitro cultures of root and shoot tissues of
1368
F. CAMPS
A. reptans [69]. While ecdysteroids were not produced in shoot tissues, after six weeks, root cultures in an hormone supplemented solid medium produced more than 5000 ppmjdry wt of phytoecdysteroids. This amount was higher than those produced in the same period in a basal medium (-3000 ppm) and in liquid medium (-2000 ppm). In these cases 29-norcyasterone was also the main component of the mixture. These cultures seem very appropriate for biosynthetic studies. From all the data obtained of our in vivo and in vitro experiments, we conclude that ecdysteroid production is related to organised macrostructures in the plant and occurs in the roots. The contents in the different materials studied seem to indicate a relationship between growth and ecdysteroid production. As far as the biosynthesis of phytoecdysteroids in A. reptans is concerned, two major biosynthetic pathways appear to be operating, side chain dealkylation and 5-hydroxylation, leading to two series of related compounds, namely cyasterone/29-norC,&,,/CZ, cyasterone/20-hydroxyecdysone sengosterand one/29-norsengosterone/5,20-dihydroxyecdysone (polypodine B), both series being related by the presence or absence of one hydroxyl at C-5. On the other hand, the unique structural features of ajugalactone make this C,, phytoecdysteroid unamenable to such comparisons and suggest that it may be formed as a dead end in the biosynthetic pathway. Five C2s and CZ9 compounds, cyasterone (CY), 29norcyasterone (29NC), sengosterone (SG), 29-norsengosterone (29NS) and ajugalactone (AJL) represent a mean content higher than 95% in all the samples studied. For comparison of the qualitative differences among the diverse plant materials studied, we established a dealkylation ratio [Dr = (29NS + 29NC)/(SG + CY + AJL)]. Likewise, we considered an index of hydroxylation at C-5 [SHr = (SG + 29NS + 520E)/(CY + 29NC + 20E)]. The values of the 5Hr index appeared to be quite constant with values usually ranging from 0.2 to 0.4 except in greenhouse plants and some isolated roots which exhibited a high dispersion of results due to very high values observed in some samples. In contrast, the Dr values varied with culture types. While tissues belonging to a whole plant had values near 2.3 or even lower for wild plants, root cultures can reach values above 12. When analyses were individually considered, in most cases the leaves had Dr values lower than the corresponding root materials, suggesting that dealkylation can take place preferentially in the roots.
CONCLUSIONS
In summary, we have reviewed the isolation and characterization of different secondary metabolites of Ajuga plants with insect allelochemical activities. Although it has been indicated that chemical defence in plants is multifaceted [70], we have separately investigated the activities of clerodane diterpenoids affecting the ‘insect behavior as antifeedants and phytoecdysteroids interfering with insect development. Ongoing research on
and
J. COLL
biosynthetic studies and biotechnological production of these metabolites has also been commented on. We hope that, in the future, further research of this so far hardly investigated plant genus will be carried out, not only looking for new insect allelochemicals which might clarify unexplored insect-plant interactions, but also to confirm if any of the above classes of compound are responsible of their interesting activities recorded in folk medicine. In fact, it has been shown that phytoecdysteroids exhibited different activities in vertebrates [3]. Among them, it is worth mentioning: the stimulation of protein, an anabolic activity in murine liver [71]. the suppression of the hyperglycaemia induced by either glucagon or alloxan in diabetic mice [72], the inhibition of the development of hypercholesterolaemia and hyperlipidaemia produced by Triton WR-1339 in rats and antiatherosclerotic action in rabbits [73], the increase of bile acids and bilirubin levels and decrease of cholesterol content in the bile secretion of normal rats or rats with induced toxic hepatitis [74], the normalization of the respiratory chain and the terminal pathway of electron transport in hepatocytes of rats with induced hepatitis [75] and the potentiation of the effects of insulin [76]. Acknowledgements-This research was supported by funds from Spanish CAICYT (Grants 3708/79 and 1664/82) and CICYT (BIO 88-0230) and predoctoral fellowships from the Spanish Ministry of Education and Science. Special acknowledgements should be expressed to the Ph. D. students A. Cortel, 0. Dargallo, M. P. Marco and J. Tom&s for their enthusiasm, dedication and important contributions to this research, Drs F. J. Sinchez-Baeza and A. Llebaria and Mrs. J. Estremera (Dep. Biological Organic Chemistry, CID, CSIC) for preparation of drawings and typing the manuscript, respectively. REFERENCES
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