Phytoecdysteroid and Clerodane Content in Three Wild Ajuga Species

7 days ago - (1,2) Three Ajuga species (Ajuga chamaepitys, Ajuga orientalis, and ..... However, Axyris amaranthoides seeds collected from the UK and ...
0 downloads 0 Views 3MB Size
Download PDF
This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.

Article Cite This: ACS Omega 2019, 4, 2369−2376

http://pubs.acs.org/journal/acsodf

Phytoecdysteroid and Clerodane Content in Three Wild Ajuga Species in Israel Leena Taha-Salaime,†,‡ Rachel Davidovich-Rikanati,§ Asaf Sadeh,∥ Jackline Abu-Nassar,‡ Sally Marzouk-Kheredin,‡ Yahyaa Yahyaa,§ Mwafaq Ibdah,§ Murad Ghanim,⊥ Efraim Lewinsohn,§ Moshe Inbar,† and Radi Aly*,‡ †

Department of Evolutionary and Environmental Biology, The Faculty of Natural Science, University of Haifa, Haifa 31905, Israel Department of Plant Pathology and Weeds Research, Newe Ya’ar Research Center and §Department of Plant Sciences Research, Newe Ya’ar Research Center, Agricultural Research Organization, Ramat Yishay 30095, Israel ∥ Department of Natural Resources, Agricultural Research Organization, The Volcani Center, Ramat Yishay 30095, Israel ⊥ Department of Entomology, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel

Downloaded via 185.46.86.172 on February 5, 2019 at 18:11:24 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

ABSTRACT: Many species of the genus Ajuga (family Lamiaceae) contain phytoecdysteroids and clerodane diterpenes. Phytoecdysteroids are triterpene-derived analogues of steroid hormones that control molting and metamorphosis in arthropods, whereas clerodanes deter phytophagous insects. We identified and quantified phytoecdysteroid and clerodane contents in three Ajuga plant species in Israel. Leaves and roots of Ajuga iva, Ajuga chamaepitys (Ajuga chia), and Ajuga orientalis were collected from three different populations. Using liquid chromatography−time of flight−mass spectrometry analysis, we identified three phytoecdysteroids: 20hydroxyecdysone (ecdysterone), makisterone A, and cyasterone and two clerodanes: dihydroajugapitin and columbin. Their contents varied significantly among plant species, organs, and populations. The highest concentrations of 20hydroxyecdysone, makisterone A, and cyasterone were recorded in leaves and roots of A. iva. Cyasterone content tended to be higher in leaves of A. chamaepitys. Clerodane concentrations were generally negligible or nonexistent. Dihydroajugapitin concentrations were highest in A. iva leaves but were lower or undetectable in the roots and in the other two species. Columbin concentration was similar in all species and organs. Phytoecdysteroid contents also varied among populations within species. Because phytoecdysteroids have disruptive effects on phytophagous insect growth, the potential role of extracts of A. iva in pestmanagement programs is of interest.

■

INTRODUCTION Ajuga is a genus in the family Lamiaceae that includes over 300 species of annual and perennial herbs with worldwide distribution.1,2 Three Ajuga species (Ajuga chamaepitys, Ajuga orientalis, and Ajuga iva) (Figure 1) grow naturally in Israel. A. orientalis is a perennial plant with dentate leaf margins, found in northern and central Israel. A. chamaepitys is a low semishrub or perennial herb, with entire leaf margins, restricted to the Mediterranean region. Occasional specimens are also found in the crevices of smooth-faced rocks in other parts of the country. A. iva is a perennial plant with entire leaf margins that is widely distributed along the Mediterranean coast, the central mountain ridge, and the arid Negev. Many Ajuga plants contain bioactive compounds, including phytoecdysteroids (20-hydroxyecdysone, cyasterone, makisterone A), anthocyanins, carotenoids, diterpenes (the neoclerodanes ajuganane and ajujalactone), flavonoids, iridoids, diterpenoids, triterpenoids, ursolic acid, phenylethanoid glycosides, sphingolipids, tocopherols, fatty acids (triglycerides), and anolides.3−9 © 2019 American Chemical Society

Insects are major ubiquitous pests of many grains and crops, constraining and constantly threatening yield quality and quantity. Insect ecdysones are steroid hormones that control molting and effect several changes during metamorphoses in arthropods. They have different roles depending on insect developmental stage, that is, first in repression and later in activation of stem cell and primordial germ cell differentiation. In adults, ecdysone stimulates the proliferation of germline stem cells and inhibits insect development.10 The same molecules (phytoecdysteroids) have been discovered in several Ajuga plant species. Phytoecdysteroids are natural plant steroids, mainly with 19−29 carbon molecules. Most of them possess a cholest-7-en-6-one carbon skeleton (C27), synthesized from phytosterols in the cytosol through the mevalonic acid pathway.11,12 They display a wide Received: October 31, 2018 Accepted: January 2, 2019 Published: January 31, 2019 2369

DOI: 10.1021/acsomega.8b03029 ACS Omega 2019, 4, 2369−2376

ACS Omega

Article

when fed on Ajuga reptans plants.30 Effective ecdysis inhibition has been observed in Pectinophora gossypiella with ponasterone A.31 Phytoecdysteroids such as ecdysterone, polypodine B, and ponasterone A, applied at 25−250 ppm in the diet, induced ecdysial failure associated with the appearance of larvae having two head capsules and developmental anomalies during metamorphosis in larvae of Acrolepiopsis assectella.32 In our previous study,14 A. iva extract fractionated on a silica gel column yielded two fractions that showed high activity against the sweet potato whitefly, Bemisia tabaci and the persea mite, Oligonychus perseae. A dose of 5 mg AI L−1 of A. iva phytoecdysteroids significantly reduced fecundity, fertility, and survival of these pests. Clerodanes belong to the group of diterpenoids sometimes termed diterpenoids of the cascarillin group, ent-clerodanes, neoclerodanes, or clerodanes. This is a large group of 20carbon terpene compounds derived from geranylgeranyl diphosphate that are biosynthesized through the deoxyxylulose phosphate pathway in the cytoplasm, mostly in the leaves and stems of species of the Lamiaceae and Asteraceae families.3,33−35 Diterpenoids with a neoclerodane skeleton have antiinsect activity,35 especially as antifeedants.35 Clerodanes have antimicrobial, antifungal, antiviral, antitumor, cytotoxic, antibiotic, amoebicidal, cancer chemopreventive, and hypoglycemic effects. Neoclerodane diterpenes isolated from A. remota are known as antifeedants for insects,34 and isolation of ajugarins (I−III) from A. remota confirmed their strong antifeedant activity.36 Apart from insect antifeedant properties, several clerodane diterpenes display other effects against insects. Insecticidal activity has been reported for ajugarins I and IV.37 Besides insect antifeedant and antifungal activities, many clerodane diterpenes have pharmacological activities that are beneficial to humans, including action as opioid receptor probes, as well as nerve growth factor-potentiating, antiulcer, cytotoxic, anti-inflammatory, antiparasitic, and antibacterial activities.37 Koul38 showed that the most active compounds among diterpenes were those with a clerodane skeleton, such as dihydroclerodin, and clerodin hemiacetal from Caryopteris divaricata, which exhibited 100% antifeedant activity at 50 ppm concentration.38 In comparison to the activity of phytoecdysteroids and clerodanes on insects, chemical insecticides act as toxicants39 and inhibit development.40−42 Currently, mainly insecticides are used for insect control, with high risk to food security, human health, and the environment.10,11 The negative aspects associated with their application have put great pressure on farmers and regulators to reduce or ban their use. Therefore, botanical pesticides are better alternatives as these are less expensive, eco-friendly, cost-effective, and biodegradable.43 In this work, we analyzed the content of three important phytoecdysteroids (20-hydroxyecdysone, makisterone A, and cyasterone) and two clerodanes (dihydroajugapitin and columbin) in three Ajuga plant species (A. iva, A. chamaepitys, and A. orientalis) growing naturally in Israel. To the best of our knowledge, this is the first comprehensive report dealing with quantification of phytoecdysteroids and clerodanes in Israeli Ajuga plants.

Figure 1. Ajuga species in Israel. (A) A. iva, (B) A. chamaepitys, and (C) A. orientalis.

array of benefits in agriculture and in folk medicine and are readily available in large amounts. 11,13 In fact, their concentrations in plants are generally higher than those found in arthropods.14−16 They can be found in the leaves, roots, fruit, flowers, bark, rhizomes, and seeds, with their contents varying among organs, developmental stages, seasons, and habitats.17,18 The phytoecdysteroids in Ajuga species exhibit physiological activity in insects;10 they affect a wide range of insects at very low concentrations and are not harmful to human or animal cells.19 Insects that ingest phytoecdysteroids and have not adapted to these defense molecules are subject to serious adverse effects, including weight loss, molting disruption, and/ or mortality.11,20 These phytoecdysteroids mimic the ecdysteroids that control insect development at all stages of the life cycle; their addition can therefore disrupt normal ecdysteroid levels or actions, severely impairing the insect’s development.21 Several studies have investigated the effects of phytoecdysteroids on phytophagous insects. Application of phytoecdysteroids to a number of pest species resulted in strong disruption of growth and developmental stages, including inhibition of growth and induced death before or after molting.8,12,14,19,22−24 Phytoecdysteroids inhibited feeding in Pieris brassicae and Mamestra brassicae larvae when given at 200 mg kg−1 fresh weight (FW) in sucrose solution25 and inhibited drinking in Dysdercus koenigii, Dysdercus fulvoniger, and Spilostethus pandurus adults at 100 mg kg−1.26 Jones and Firn27 reported that ecdysone and 20-hydroxyecdysone deter feeding in P. brassicae when incorporated above 5 mg kg−1 diet. Chilo partellus, Phyllobius pyri, and Pteropus argentatus were deterred from feeding by 20-hydroxyecdysone at concentrations above 50−70 mg kg−1.28 Zhang and Kubo29 reported that an extract of Ajuga remota containing 20-hydroxyecdysone and cyasterone inhibits ecdysiast in the moth Bombyx mori, resulting in larval retention of the exuvial head capsules and ultimately, death.29 Nymphs of the greenhouse whitefly died

■

RESULTS Three phytoecdysteroids (20-hydroxyecdysone, makisterone A, and cyasterone) and two clerodanes (columbin and dihydroajugapitin) were identified in different Israeli Ajuga variants and species (A. iva, A. chamaepitys, and A. orientalis), 2370

DOI: 10.1021/acsomega.8b03029 ACS Omega 2019, 4, 2369−2376

ACS Omega

Article

Figure 2. Chromatograms of (+) extracted ion chromatogram LC−MS analysis of methanolic extracts from Israeli Ajuga species (leaves and roots), and standards of phytoecdysteroids (C27H44O7, C28H46O7, and C29H44O8) and clerodanes (C20H22O6 and C29H44O10). Compounds were identified by comparison of exact mass (m/z) and retention time (Rt) to commercial standards. (A,B) Crude extracts from A. iva (leaves and roots, respectively). (C,D) Crude extracts from A. chamaepitys (leaves and roots, respectively). (E) Commercial standards of phytoecdysteroids and clerodanes. Peak 1, 20-hydroxyecdysone C27H44O7 (Rt = 3.961 min, m/z = 481.3160); peak 2, makisterone A C28H46O7 (Rt = 4.353 min, m/z = 495.3316); peak 3, cyasterone C29H44O8 (Rt = 4.4844 min, m/z = 521.3109); peak 4, columbin C20H22O6 (Rt = 6.157 min, m/z = 359.1489); and peak 5, dihydroajugapitin C29H44O10 (Rt = 8.942 min, m/z = 553.3007). (F,H,J,L, and N) Mass spectra of commercial standards (20hydroxyecdysone, makisterone A, cyasterone, columbin, and dihydroajugapitin, respectively). (G, I, K, M, and O) Mass spectra of methanolic extracts from A. iva leaves (20-hydroxyecdysone, makisterone A, cyasterone, columbin, and dihydroajugapitin, respectively).

Identification and Quantification of Phytoecdysteroids in Leaves and Roots of Ajuga Plants. Both leaves and roots of all Ajuga species examined contained 20hydroxyecdysone, makisterone A, and cyasterone (Figure 2). We found considerable variation in 20-hydroxyecdysone and makisterone A content among the species and between leaves and roots within species (Figure 3). In general, A. iva contained higher concentrations of 20-hydroxyecdysone, makisterone A, and cyasterone (up to 10 mg g−1 FW) than the other species. We found significant differences in 20-hydroxyecdysone content among species, in both leaves (Figure 3A; F2,8 = 43.349; p > 0.01) and roots (Figure 3B; F2,8 = 18.249; p > 0.01). The highest concentrations were found in A. iva, in both tissues, and its content was negligible in A. orientalis. There was

in leaves and roots that were collected in April from three populations in different geographical areas. Methanolic extracts of the plant tissues were analyzed by liquid chromatography (LC)−mass spectrometry (MS). The most abundant compounds identified were the phytoecdysteroids, mainly 20hydroxyecdysone, makisterone A, and cyasterone. Besides these major compounds, we observed different phytoecdysteroid derivatives and smaller peaks of clerodanes. Here, we focus on the quantification of the three main phytoecdysteroids (20-hydroxyecdysone, makisterone A, and cyasterone) that have been shown to possess physiological activity in insects and control molting and metamorphosis in arthropods and two clerodanes (columbin and dihydroajugapitin) shown to possess antifeedant activity in insects. 2371

DOI: 10.1021/acsomega.8b03029 ACS Omega 2019, 4, 2369−2376

ACS Omega

Article

Figure 3. Phytoecdysteroid compounds from different populations of three Ajuga plant species in Israel. Leaves and roots of A. iva (hatched bars, n = 15), A. chamaepitys (full bars, n = 15), and A. orientalis (empty bars, n = 14) were collected from different locations in Israel. Detected phytoecdysteroid compounds were: 20-hydroxyecdysone from leaf (A) and root (B) extracts; makisterone A from leaf (C) and root (D) extracts; cyasterone from leaf (E) and root (F) extracts. Error bars in all graphs represent standard error of the mean (mean ± SE, μg g−1 FW) and significance is indicated in each experiment. Different letters indicate statistically significant (p ≤ 0.05) differences between species. Collection locations: BO, Bet Oren in Mount Carmel; Shfar, Shfaram in the lower Galilee; TAN, Tel Arad in the Negev; GJN, Goral Junction in the Negev; YHN, Heran Forest in the Negev; YLN, Lahav Forest in the Negev; AHG, Aloni Habashan in the Golan; WAG, Wadi Abo Saeed in the Golan; and WJG, Waset Junction in the Golan.

Clerodane Variability among Ajuga Plant Species and between Organs (Leaves and Roots). In the current study, we identified two clerodanes: columbin and dihydroajugapitin, together with several minor compounds, including put_ivains I−IV, put_2-acetylivain, put_bracteonin A, put_14,15-dehydroajugareptansin, put_clerodendrin, put_ajugarin II, put_15-ethoxy-14-hydroajugapitin, put_carioptin, put_carioptinol, put_dihydrocarioptinol, and put_cascarillin. All Ajuga species (leaves and roots) contained columbin in trace amounts, close to the threshold of detection (5 μg g−1 FW) (Figure 4A,B). Columbin concentrations in the leaves and roots were similar in all species (Figure 4). Variation among populations within species was low in roots and nonexistent in leaves (20 and 0% of total variance components, respectively). One sample of A. iva leaves was excluded from the data set as an extreme outlier (17 times higher than the mean of all samples). The leaf dihydroajugapitin content was significantly higher in A. iva (Figure 4C; F2,8 = 57.866; p < 0.01), demonstrating the highest concentration (mean 9.7 μg g−1 FW), whereas its content was negligible in the other two species. Amongpopulation variation was nonexistent (0% of total variance components). However, in roots, the compound was not detected in A. chamaepitys or in two (out of three) populations of A. orientalis. In A. iva roots, dihydroajugapitin concentration was negligible (Figure 4D). One sample of A. iva roots was

substantial variation in 20-hydroxyecdysone concentration among populations within species (33% of total variance components) in both plant tissues. Makisterone A content varied significantly among Ajuga species, in both leaves (Figure 3C; F2,8 = 13.203; p > 0.01) and roots (Figure 3D; F2,8 = 119.374; p < 0.01). Again, the highest concentrations were found in A. iva, in both tissues (leaves and roots), and the content was negligible in A. orientalis leaves (less than 1 mg g−1 FW). There was substantial variation in makisterone A concentration among populations in the leaves but not in the roots (52 and 0% of total variance components, respectively). Cyasterone content was not statistically significant among Ajuga species in leaves (Figure 3E; F2,8 = 3.242; p > 0.01) because of large population variation (73% of total variance components). However, A. chamaepitys leaves seemed to have the highest concentration of this compound (Figure 3E). In contrast, cyasterone content was significantly higher in the roots of A. iva (Figure 3F; F2,8 = 46.470; p < 0.01). The variation in root cyasterone concentrations among populations within species was very low (5.5% of total variance components). Several smaller peaks were observed, which were identified as the compounds: put_abutasterone, put_ponasterone A, put_polypodine B, put_muristerone A, and put_sidisterone by comparison of their retention times and mass spectral data with reference compounds. 2372

DOI: 10.1021/acsomega.8b03029 ACS Omega 2019, 4, 2369−2376

ACS Omega

Article

Figure 4. Clerodane compounds from different populations of three Ajuga plant species in Israel. Leaves and roots of A. iva (hatched bars, n = 15), A. chamaepitys (full bars, n = 15), and A. orientalis (empty bars, n = 14) were collected from different locations in Israel. Detected clerodane compounds were: columbin from leaf (A) and root (B) extracts and dihydroajugapitin from leaf (C) and root (D) extracts. Error bars in all graphs represent the standard error of the mean (mean ± SE, ng g−1 FW), and significance is indicated in each experiment. Different letters indicate statistically significant (p ≤ 0.05) differences between species. Collection locations: BO, Bet Oren in Mount Carmel; Shfar, Shfaram in the lower Galilee; TAN, Tel Arad in the Negev; GJN, Goral Junction in the Negev; YHN, Heran Forest in the Negev; YLN, Lahav Forest in the Negev; AHG, Aloni Habashan in the Golan; WAG, Wadi Abo Saeed in the Golan; and WJG, Waste Waset Junction in the Golan.

with much lower concentrations associated with the aerial portions (leaves and stems). Conversely, Atriplex oblongifolia seeds collected from Germany contained highest ecdysterone concentrations in the aerial portions, with an almost uniform concentration along the stem.49 In this study, A. iva leaves were found to contain high concentrations of the three phytoecdysteroids: 0.54 ± 0.05% of plant FW for 20-hydroxyecdysone (2.42 ± 0.2% of DW), 1.14 ± 0.16% of FW for makisterone A (5.12 ± 0.72% of DW), and 0.81 ± 0.13% of FW for cyasterone (3.63 ± 0.57% of DW). However, other studies in A. iva have shown respectively lower concentrations: A. iva collected from Egypt contained makisterone A at 0.005% of plant FW and cyasterone at 0.00007% of FW.50 Another study showed concentrations relative to plant DW of 1.6% for 20-hydroxyecdysone and makisterone A and 1.3% for cyasterone.51 In general, we found considerable variation among species in phytoecdysteroid content. A. iva had the greatest concentrations of phytoecdysteroids in both leaves and roots and of dihydroajugapitin in leaves; A. orientalis had the lowest content of phytoecdysteroids (Figure 3). The occurrence and content of phytoecdysteroids vary not only in the different parts, or between plants at the same stage of development, but also within season and plant geographical distribution.52 In the current study, the content of 20-hydroxyecdysone and cyasterone from Israeli A. iva plants was twice and three times the amount in Algerian A. iva plants, respectively. All three species of Ajuga showed very low contents of clerodane compounds compared to their phytoecdysteroid content. In contrast, recent research has reported higher contents of clerodanes other than dihydroajugapitin or columbin in A. chamaepitys and other Ajuga species, such as Ajuga pseudoiva, A. remota, Ajuga bracteosa, and A.

excluded from the data set as an extreme outlier (32 times higher than the mean concentration in all samples).

■

DISCUSSION

The three phytoecdysteroids (20-hydroxyecdysone, makisterone A, and cyasterone) and two clerodanes (dihydroajugapitin and columbin) identified here in the leaves and roots of three Israeli wild Ajuga species have been previously identified in different Ajuga species.14,16,44 Our results are in agreement with studies that investigated Ajuga plants in different habitats and showed very high levels of ecdysteroids and also showed a special and unique quantity of ecdysteroids. A. iva leaves contained about 2.49 ± 0.34% [mean ± standard error (SE)] total phytoecdysteroids out of the plant FW, equivalent to 11.17 ± 1.49% of dry weight (DW); A. chamaepitys contained about 2.2 ± 0.45% of FW, equivalent to 11 ± 2.23% DW, and A. orientalis contained about 0.01% of FW, equivalent to 0.09 ± 0.02% DW. Phytoecdysteroid-containing species typically contain 0.01−1.2% of DW.11,45−47 Recent studies have shown that ecdysteroids are present at 5−6% of plant DW in plant species,15 and in A. iva leaves collected from Algeria, phytoecdysteroids were 0.001−3% of the plant DW.43,46 Other species in the tribe Beteae had a different accumulation of phytoecdysteroids: Beta patellaris seeds collected from Denmark contained 1.2 mg g−1 DW, Beta webbiana seeds collected from the Netherlands contained 0.442 mg g−1 DW, and Beta procumbens seeds collected from the Netherlands did not contain any ecdysteroids.48 High concentrations of ecdysterone were found in leaves and stems of Rhagodia candolleana seeds collected from Spain (9 mg g−1 DW). However, Axyris amaranthoides seeds collected from the UK and Belgium contained very high ecdysteroid concentrations in their roots (1.3 mg g−1 DW), 2373

DOI: 10.1021/acsomega.8b03029 ACS Omega 2019, 4, 2369−2376

ACS Omega

■

reptans.34,35,53 A. remota collected from Kenya showed a low concentration of dihydroajugapitin and clerodane ajugarin I at 0.60% of plant DW in aerial parts (leaves and stems).54 In a previous study, columbin was isolated from the chloroform extract of Tinospora cordifolia plants collected from India.55 We found the highest contents of columbin in the A. iva population from Lahav Forest in the Negev, whereas dihydroajugapitin was found in the leaves of all three populations of A. iva but not in A. chamaepitys or A. orientalis. However, in another study on A. orientalis collected from Turkey, a new neoclerodane, ajugorientin, was isolated, and two other known clerodanesajugapitin and 14,15-dihydro15-hydroxyajugapitinwere found in Ajuga australis collected from Australia.56 We did not observe any variation in phytoecdysteroid concentration among populations, and variations in clerodane concentration were negligible. In the three species of Ajuga, phytoecdysteroid concentrations were similar between leaves and roots; however, higher accumulation of cyasterone was observed in A. iva roots versus leaves. Concentrations were generally high in the leaves, particularly for cyasterone (73% of variance) but not in the roots (up to 33% of total variance for 20-hydroxyecdysone). High concentrations of 20-hydroxyecdysone and makisterone A were quantified in A. iva leaves. Ecdysteroid levels have been recently shown to fluctuate during plant development and are most dominant in the aerial parts during periods of intensive growth.57,58 Leaves of Silene viridiflora represent the largest part of the plant’s biomass (30− 70% of DW), and they also contain the largest proportion of 20-hydroxyecdysone (40−70% of leaf DW).59 Although the identity of the ecdysone-synthesizing tissue is unknown,60 this might be explained by differences in leaf structure or different types of trichomes that contain secondary metabolites.60 Several lines of evidence indicate that phytoecdysteroids are produced mainly in the roots and then transported to the leaves, as in the case of some alkaloids.61 Similarly, a study of ecdysteroid distribution in A. reptans plant organs showed significant accumulation of 20-hydroxyecdysone in roots versus leaves at the end of the vegetative phase.62 Phytoecdysteroid biosynthesis occurs in developing tissues, and these compounds are then transported to other organs.62 Knowledge of the biosynthetic pathway(s) for ecdysteroids and their regulation is rather limited.11,22 This is due, in part, to a lack of convenient study systems, a situation which is beginning to change with the application of amenable in vitro systems, such as hairy-root or cell-suspension cultures. In vitro culture studies of root and shoot tissues of A. reptans have revealed that the roots produce phytoecdysteroids when isolated from the rest of the plant organs (root cultures), whereas phytoecdysteroids are not detected in shoot cultures in the absence of root tissue. These results strengthen the hypothesis that the ecdysteroids in A. reptans are biosynthesized in the root system.10,59 To the best of our knowledge, there is no evidence of the presence of clerodanes exclusively in the roots. Our results describe the bioactive content and concentration of phytoecdysteroids in Ajuga plant species and provide further evidence of their previously reported activities. We assume that phytoecdysteroids protect plants from phytophagous insects and can be applied in an integrated pest management program. However, further research is needed to characterize and isolate these and other active compounds from Ajuga species to help alleviate insect-related ecological, agricultural, and economic challenges.

Article

EXPERIMENTAL SECTION

Plant Material and Extraction. Leaves and roots of A. iva, A. chamaepitys, and A. orientalis were collected during the blooming season in April from several locations in Israel in 2017. Each species was sampled from three geographical areas. We sampled three populations from each area, five plants per population. A. orientalis was collected from Waset Junction in the Golan (33°9′43″N 35°43′8″E), Wadi Abo Saeed in the Golan (033°13′41″N 35°46′37″E), and Aloni Habashan in the Golan (33°4′19″N 35°49′45″E). A. chamaepitys was collected from Shfaram in the lower Galilee (32°47′43″N 35°8′45″E), Bet Oren in Mount Carmel (32°44′77″N 35°2′74″E), and Tel Arad in the Negev (31°21′2″N 35°7′6″E). A. iva was collected from Lahav Forest in the Negev (32°42′33″N 35°8′34″E), Heran Forest in the Negev (31°18′15″N 34°59′1″E), and Goral Junction in the Negev (31°18′28″N 34°48′1″E). The samples were washed, air-dried, and stored at −80 °C until extraction. Leaves and roots (0.1 g samples) were ground separately with liquid nitrogen to a uniform powder. The powder was placed in a 20 mL disposable scintillation vial containing 5 mL of 80% (w/v) MeOH and 0.125 ppm hyoscyamine as an internal standard, then sealed and homogenized with shaking (1200 rpm). After 1 h, the extract was centrifuged, filtered, and analyzed by LC−time of flight (TOF)−MS. LC−TOF−MS Analysis. Methanolic extracts of the plant material (1 μL) were injected into an Agilent 1290 Infinity series liquid chromatograph coupled with an Agilent 1290 Infinity diode array detector and Agilent 6224 Accurate Mass TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). The analytical column was a Zorbax Extend-C18 Rapid Resolution HT column (2.1 × 50.0 mm, 1.8 μm, Agilent Technologies, Waldbronn, Germany). The gradient-elution mobile phase consisted of H2O with 0.1% (v/v) formic acid (eluent A) and acetonitrile containing 0.1% (v/v) formic acid (eluent B). The column was equilibrated with 5% B for 2 min. Eluent B was then increased to 95% over 9 min and restored to 5% B at 14.5 min. The flow rate of the mobile phase was 0.3 mL min−1, and the column oven temperature was 40 °C. Eluting compounds were subjected to a dual-sprayer orthogonal electrospray ionization (ESI) source, with one sprayer for analytical flow and one for the reference compound (Agilent Technologies, USA). The ESI source was operated in positive mode with the following settings: gas temperature of 300 °C with a flow of 8 L min−1 and nebulizer set to 35 psig, VCap set to 3000 V, fragmentor to 110 V, and skimmer to 65 V. Scan mode of the mass detector was applied (100−1700 m/z) with a rate of 1 spectrum s−1. The [M + H] ions of the target compounds were detected using the “find compound by formula” function and analyzed by MassHunter qualitative and quantitative analysis software version B.07.00 (Agilent Technologies). Compounds were identified by comparison of exact masses and retention times to purchased standards: 20hydroxyecdysone (cat. no. 5289, Sigma-Aldrich, Israel), makisterone A (cat. no. AG-CN2-0073-M005, A.G. Scientific Inc., San Diego, CA, USA), cyasterone (cat. no. 00003960-001, ChromaDex Analytics Inc., Irvine, CA, USA), dihydroajugapitin (cat. no. QP-3369, 87480-84-0, Quality Phytochemicals LLC, East Brunswick, NJ, USA), and columbin (cat. no. QP787, 546-97-4, Quality Phytochemicals). Statistical Analysis. To estimate variation among populations in phytoecdysteroid (20-hydroxyecdysone, makis2374

DOI: 10.1021/acsomega.8b03029 ACS Omega 2019, 4, 2369−2376

ACS Omega

Article

(9) Yoshida, T.; Otaka, T.; Uchiyama, M.; Ogawa, S. Effect of ecdysterone on hyperglycemia in experimental animals. Biochem. Pharmacol. 1971, 20, 3263−3268. (10) Belles, X.; Piulachs, M. D. Ecdysone signalling and ovarian development in insects: from stem cells to ovarian follicle formation. Biochim. Biophys. Acta 2015, 1849, 181−186. (11) Dinan, L. Phytoecdysteroids: biological aspects. Phytochemistry 2001, 57, 325−339. (12) Adler, J. H.; Grebenok, R. J. Occurrence, biosynthesis, and putative role of ecdysteroids in plants. Crit. Rev. Biochem. Mol. Biol. 1999, 34, 253−264. (13) Lafont, R.; Dinan, L. Practical uses for ecdysteroids in mammals including humans: an update. J. Insect Sci. 2003, 3, 1−30. (14) Aly, R.; Ravid, U.; Abu-Nassar, J.; Botnick, I.; Lebedev, G.; Gal, S.; Ziadna, H.; Achdari, G.; Smirov, E.; Meir, A.; Ghanim, M. Biological activity of natural phytoecdysteroids from Ajuga iva against the sweetpotato whitefly Bemisia tabaci and the persea mite Oligonychus perseae. Pest Manage. Sci. 2011, 67, 1493−1498. (15) Dinan, L. A strategy for the identification of ecdysteroid receptor agonists and antagonists from plants. Eur. J. Entomol. 1995, 92, 271−283. (16) Ikan, R.; Ravid, U. The isolation and identification of ecdysterone from Ajuga iva. Planta Med. 1971, 20, 33−35. (17) Grebenok, R. J.; Adler, J. H. Ecdysteroid distribution during development of spinach. Phytochemistry 1993, 33, 341−347. (18) Dinan, L. The analysis of phytoecdysteroids in single (preflowering stage) specimens of fat hen, Chenopodium album. Phytochem. Anal. 1992, 3, 132−138. (19) Dinan, L.; Lafont, R. Effects and applications of arthropod steroid hormones (ecdysteroids) in mammals. J. Endocrinol. 2006, 191, 1−8. (20) Dinan, L.; Savchenko, T.; Whiting, P. On the distribution of phytoecdysteroids in plants. Cell. Mol. Life Sci. 2001, 58, 1121−1132. (21) Dinan, L. Ecdysteroid structure and hormonal activity. In Ecdysone: From Chemistry to Mode of Action; Koolman, J., Ed.; George Thieme Verlag: Stuttgart, Germany, 1989; Chapter 36, pp 345−354. (22) Dinan, L. The Karlson Lecture. Phytoecdysteroids: what use are they? Arch. Insect Biochem. Physiol. 2009, 72, 126−141. (23) Dinan, L.; Savchenko, T.; Whiting, P.; Sarker, S. D. Plant natural products as insect steroid receptor agonists and antagonists. Pestic. Sci. 1999, 55, 331−335. (24) Ray, D. P.; Dutta, D.; Srivastava, S.; Kumar, B.; Saha, S. Insect growth regulatory activity of Thevetia nerifolia Juss. against Spodoptera litura (Fab.). J. Appl. Bot. Food Qual. 2012, 85, 212−215. (25) Ma, W. C. Dynamics of feeding responses in Pieris brassicae L. as a function of chemosensory input: a behavioural, ultrastructural and electrophysiological study. Mdedelingen Landbouwhogeschool Wageningen 1972, 72, 1−162. (26) Kubo, I.; Klocke, J. A.; Asano, S. Effects of ingested phytoecdysteroids on the growth and development of two lepidopterous larvae. J. Insect Physiol. 1983, 29, 307−316. (27) Jones, C. G.; Firn, R. D. The role of phytoecdysteroids in bracken fern, Pteridium aquilinum (L.) Kuhn as a defense against phytophagous insect attack. J. Chem. Ecol. 1978, 4, 117−138. (28) Riddiford, L. M.; Cherbas, P.; Truman, J. W. Ecdysone receptors and their biological actions. Vitam. Horm. 2000, 60, 1−73. (29) Zhang, M.; Kubo, I. Metabolic fate of ecdysteroids in larval Bombyx mori and Heliothis virescens. Insect Biochem. Mol. Biol. 1993, 23, 831−843. (30) Darvas, B.; Defu, C.; Polgar, L. A.; Kormendy, C.; Vidal, E.; Pap, L.; Coll, J. Effects of some materials extracted from Ajuga reptans var.reptans on Aedes aegypti and Dysdercus cingulatus larvae. Pestic. Sci. 1997, 49, 392−395. (31) Nakanishi, K.; Koreeda, M.; Sasaki, S.; Chang, M. L.; Hsu, H. Y. Insect hormones. The structure of ponasterone A, insect-moulting hormone from the leaves of Podocarpus nakaii Hay. Chem. Commun. 1966, 915−917.

terone A, and cyasterone) and clerodane (dihydroajugapitin and columbin) contents in the Ajuga species, analyses included a mixed-model analysis of variance for each tissue separately, with populations of origin as random nested factor within species, using restricted maximum likelihood estimation. When significant (p < 0.05) species effects were detected, we followed with Tukey honestly significant difference pairwise comparisons among species. For these pairwise comparisons, the nonindependent population samples were averaged to avoid pseudo-replication (n = 3 conservative replicates per species). One of these conservative comparisons (20hydroxyecdysone between A. chamaepitys and A. orientalis roots) resulted in p = 0.077, despite a three orders of magnitude difference between these species’ means, and we therefore considered this difference significant. In all other pairwise comparisons, statistical significance was reported at p < 0.05. Data were analyzed using JMP software 13.1.0 (SAS Institute Inc., Cary, NC, USA).

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (R.A.). ORCID

Radi Aly: 0000-0001-6717-613X Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by the Israeli Ministry of Science and Technology (research grant no. 3-14496). We thank Prof. Uzi Ravid and Dr. Alona Sheachter for their fruitful advice and comments. We thank Jomaa Zabarqa and his team for his assistance to collect Ajuga plants from several regions in Southern Israel.

■

REFERENCES

(1) Atay, I.; Kirmizibekmez, H.; Kaiser, M.; Akaydin, G.; Yesilada, E.; Tasdemir, D. Evaluation ofin vitroantiprotozoal activity ofAjuga laxmanniiand its secondary metabolites. Pharm. Biol. 2016, 54, 1808− 1814. (2) Sivanesan, I.; Jeong, B. R. Silicon promotes adventitious shoot regeneration and enhances salinity tolerance of Ajuga multiflora Bunge by altering activity of antioxidant enzyme. Sci. World J. 2014, 2014, 521703. (3) Hussain, J.; Begum, N.; Hussain, H.; Khan, F. U.; Rehman, N. U.; Al-Harrasi, A.; Ali, L. Ajuganane: a new phenolic compound from Ajuga bracteosa. Nat. Prod. Commun. 2012, 7, 615−616. (4) Israili, Z. H.; Lyoussi, B. Ethnopharmacology of the plants of genus Ajuga. Pak. J. Pharm. Sci. 2009, 22, 425−462. (5) Kayani, W. K.; Rani, R.; Ihsan-ul-Haq; Mirza, B. Seasonal and geographical impact on the morphology and 20-hydroxyecdysone content in different tissue types of wild Ajuga bracteosa Wall. ex Benth. Steroids 2014, 87, 12−20. (6) Ni, B.; Dong, X.; Fu, J.; Yin, X.; Lin, L.; Xia, Z.; Zhao, Y.; Xue, D.; Yang, C.; Ni, J. Phytochemical and biological properties of Ajuga decumbens (Labiatae): a review. Trop. J. Pharm. Res. 2015, 14, 1525− 1536. (7) Riaz, N.; Nawaz, S. A.; Mukhtar, N.; Malik, A.; Afza, N.; Ali, S.; Ullah, S.; Muhammad, P.; Choudhary, M. I. Isolation and enzymeinhibition studies of the chemical constituents from Ajuga bracteosa. Chem. Biodiversity 2007, 4, 72−83. (8) Speranza, A. Into the world of steroids: a biochemical ″keep in touch″ in plants and animals. Plant Signaling Behav. 2010, 5, 940− 943. 2375

DOI: 10.1021/acsomega.8b03029 ACS Omega 2019, 4, 2369−2376

ACS Omega

Article

(32) Arnault, C.; Sláma, K. Dietary effects of phytoecdysones in the leek-moth, Acrolepiopsis assectella Zell. (Lepidoptera: Acrolepiidae). J. Chem. Ecol. 1986, 12, 1979−1986. (33) Arns, S.; Barriault, L. Cascading pericyclic reactions: building complex carbon frameworks for natural product synthesis. Chem. Commun. 2007, 2211−2221. (34) Camps, F.; Coll, J. Insect allelochemicals from Ajuga plants. Phytochemistry 1993, 32, 1361−1370. (35) Klein Gebbinck, E. A.; Jansen, B. J. M.; de Groot, A. Insect antifeedant activity of clerodane diterpenes and related model compounds. Phytochemistry 2002, 61, 737−770. (36) Kubo, I.; Lee, Y.-W.; Balogh-Nair, V.; Nakanishi, K.; Chapya, A. Structure of ajugarins. J. Chem. Soc., Chem. Commun. 1976, 949−950. (37) Li, R.; Morris-Natschke, S. L.; Lee, K.-H. Clerodane diterpenes: sources, structures, and biological activities. Nat. Prod. Rep. 2016, 33, 1166−1226. (38) Koul, O. Insect feeding deterrents in plants. Indian Rev. Life Sci. 1982, 2, 97−125. (39) Beckel, H.; Lorini, I.; Lazzari, S. M. N. Resistance of Oryzaephilus surinamensis (L.) (Coleoptera: Silvanidae) to insecticides pyrethroids and organophosphates used in stored wheat. In Summaries and Minutes of III Technical Seminar of Trigo/XVII Reuniao of the Commission I South-Center Brazilian of Inquiry of Wheat, Portuguese, 2002; p 44. (40) Chaubey, M. K. Insecticidal activity of Trachyspermum ammi (Umbelliferae), Anethum graveolens (Umbelliferae) and Nigella sativa (Ranunculaceae) against stored-product beetle Tribolium castaneum Herbst (Coleoptera: Tenebrionidae). Afr. J. Agric. Res. 2007, 2, 596−600. (41) Chaubey, M. K. Fumigant toxicity of essential oils and pure compounds against Sitophilus oryzae L. (Coleoptera: Curculionidae). Biol. Agric. Hortic. 2012, 28, 111−119. (42) Dhadialla, T. S.; Carlson, G. R.; Le, D. P. New insecticides with ecdysteroidal and juvenile hormone activity. Annu. Rev. Entomol. 1998, 43, 545−569. (43) Chaubey, M. K. Role of phytoecdysteroids in insect pest management: a review. J. Agron. 2017, 17, 1−10. (44) Yilmaz, A.; Crowley, R. S.; Sherwood, A. M.; Prisinzano, T. E. Semisynthesis and kappa-opioid receptor activity of derivatives of columbin, a furanolactone diterpene. J. Nat. Prod. 2017, 80, 2094− 2100. (45) Syrov, V. N.; Shakhmurova, G. A.; Khushbaktova, Z. A. Effects of phytoecdysteroids and bemithyl on functional, metabolic, and immunobiological parameters of working capacity in experimental animals. Eksp. Klin. Farmakol. 2008, 71, 40−43. (46) Thiem, B.; Kikowska, M.; Maliński, M. P.; Kruszka, D.; Napierała, M.; Florek, E. Ecdysteroids: production in plant in vitro cultures. Phytochem. Rev. 2016, 16, 603−622. (47) Cheng, D. M.; Yousef, G. G.; Lila, M. A. Variation in phytoecdysteroid accumulation in seeds and shoots of Spinacia oleracea L. accessions. HortScience 2010, 45, 1634−1638. (48) Dinan, L. Distribution and levels of phytoecdysteroids within individual plants of species of the Chenopodiaceae. Eur. J. Entomol. 1995, 92, 295−300. (49) Khafagy, S.; Sabri, N.; El-Sebakhy, N.; Blessington, B.; Asaad, A. A C−28 ecdysone−like substance from Ajuga iva. Planta Med. 1979, 35, 184−185. (50) Wessner, M.; Champion, B.; Girault, J.-P.; Kaouadji, N.; Saidi, B.; Lafont, R. Ecdysteroids from Ajuga iva. Phytochemistry 1992, 31, 3785−3788. (51) Castro, A.; Coll, J.; Arfan, M. neo-Clerodane diterpenoids from Ajuga bracteosa. J. Nat. Prod. 2011, 74, 1036−1041. (52) Galbraith, M. N.; Horn, D. H. S. An insect-moulting hormone from a plant. Chem. Commun. 1966, 24, 905−906. (53) Coll, J.; Tandrón, Y. Isolation and identification of neoclerodane diterpenes from Ajuga remota by high-performance liquid chromatography. Phytochem. Anal. 2005, 16, 67.

(54) Sivasubramanian, A.; Narasimh, K. K. G.; Rathnasamy, R.; Oliveira Campos, A. M. F. A new antifeedant clerodane diterpenoid from Tinospora cordifolia. Nat. Prod. Res. 2013, 27, 1431−1436. (55) de la Torre, M. C.; Rodríguez, B.; Bruno, M.; Piozzi, F.; Vassallo, N.; Bondí, M. L.; Servettaz, O. Neo-clerodane diterpenoids from Ajuga australis and A. orientalis. Phytochemistry 1997, 45, 121− 123. (56) Mamadalieva, N. Z. Phytoecdysteroids from Silene plants: Distribution, diversity and biological (antitumour, antibacterial and antioxidant) activities. Boletin Latinoamericano y del Caribe de Plantas Medicinales y Aromaticas 2012, 11, 474−497. (57) Meng, Y.; Whiting, P.; Zibareva, L.; Bertho, G.; Girault, J.-P.; Lafont, R.; Dinan, L. Identification and quantitative analysis of the phytoecdysteroids in Silene species (Caryophyllaceae) by highperformance liquid chromatography: novel ecdysteroids from S. pseudotites. J. Chromatogr. A 2001, 935, 309−319. (58) Zibareva, L. Distribution and levels of phytoecdysteroids in plants of the genus Silene during development. Arch. Insect Biochem. Physiol. 2000, 43, 1−8. (59) Rees, H. H. Edcdysteroid biosynthesis and inactivation in relation to function. Eur. J. Entomol. 1995, 92, 9−39. (60) Sakurai, S.; Warren, J. T.; Gilbert, L. I. Ecdysteroid synthesis and molting by the tobacco hornworm, Manduca sexta, in the absence of prothoracic glands. Arch. Insect Biochem. Physiol. 1991, 18, 13−36. (61) Bourgaud, F.; Gravot, A.; Milesi, S.; Gontier, E. Production of plant secondary metabolites: a historical perspective. Plant Sci. 2001, 161, 839−851. (62) Tomás, J.; Camps, F.; Claveria, E.; Coll, J.; Melé, E.; Messeguer, J. Composition and location of phytoecdysteroids in Ajuga reptans in vivo and in vitro cultures. Phytochemistry 1992, 31, 1585−1591.

2376

DOI: 10.1021/acsomega.8b03029 ACS Omega 2019, 4, 2369−2376