The Sesquiterpenes β-Caryophyllene and Caryophyllene Oxide Isolated from Senecio salignus Act as Phytogrowth and Photosynthesis Inhibitors

The n-hexane extract of S. salignus plants inhibited ATP synthesis and two sesquiterpenes, the β-caryophyllene (1) and caryophyllene oxide (2) were isolated from this nonpolar fraction. Compound 1 inhibited by 42% the root elongation of Physalis ixocarpa seedlings at 50 µg/mL and by 53% at 150 µg/mL, whereas at 150 µg/mL this compound only inhibited root elongation of Echinochloa crus-galli by 30%. On the other hand, compound 2 had no effect on either germination or root and stem growth of E. cruss galli and P. ixocarpa. However, 1 and 2 inhibited the dry biomass of P. ixocarpa plants grown for 18 days previous to treatment and it was found that 1 was the most active biomass inhibitor. The Chl a fluorescence transient in vivo experiment indicates that 1 (100 µg/mL) has a major effect at 72 h after treatment on leaves of P. ixocarpa plants by inhibiting photosystem II (PS II) transforming active reaction centers to “heat sinks” or the formation of silent reaction centers unable to reduce QA. β-Caryophyllene also induces chlorosis on treated leaves.


Introduction
The genus Senecio includes approximately 1,500 species widely distributed in Mexico and Central America [1]. Among the many species, the synonymous genus Barkleyanthus only has a single species: B. salicifolius or Senecio salignus (H.B.K.) Rob. and Brett. Quercetin was isolated from S. salignus (B. salicifolius) [2]. Furoeremophilane epoxides were also isolated from the aerial parts of S. salignus and 7-ketofuranoeremophilane from the roots in 1976 [3]. Later, the same authors found the pyrrolizidine alkaloid 7-angelylheliotridine [4]. S. salignus leaves are used against intermittent fever and rheumatism [5]. Our search for secondary metabolites in plants that affect photosynthesis, identified two sesquiterpenes: β-caryophyllene (1) and caryophyllene oxide (2) in S. salignus. It is well known that β-caryophyllene has anti-inflammatory, insecticidal and fungicidal [6] activities. In this work, compound 1 inhibited the germination and growth of Physalis ixocarpa, as well as the growth (root and stem elongation) of Echinocloa crus-galli. In vitro assays showed that sesquiterpene 1 also inhibited photosynthetic activities; however, compound 2 only inhibited Physalis ixocarpa plant growth, root and stem elongation. Light reactions of photosynthesis were performed by fluorescence induction curves of chlorophyll a of photosystem II (PS II), as shown by a JIP test.

Sesquiterpene Isolation and ATP Synthesis Determination
In the screening bioassay (ATP synthesis) it was found that the n-hexane extract (leaves and stems) of S. salignus, exhibited the major inhibition result (I 50 79.3 µg/mL) (Figure 1), indicating that it might contain secondary metabolites with inhibitory activity on photosynthesis. F30-F54 of the primary fraction obtained from a chromatographic column inhibited ATP synthesis with an I 50 value equal to that obtained for the extract. In order to know the compounds involved in this active fraction, a secondary chromatographic fractionation eluting with an ascendant gradient of n-hexane-EtOAc 80:20, and then with acetone 100%, gave a mixture of compounds, spectroscopically identified as β-caryophyllene (1, 3 mg) and caryophyllene oxide (2, 2 mg) [7] (Figure 2).   (1), and caryophyllene oxide (2).

Effects of 1 and 2 on Germination and Root and Stem
Growth of E. cruss-galli and P. ixocarpa As concentration of caryophyllene oxide increased root elongation of E. crus-galli was partially inhibited (23 and 30% at 100 and 150 µg/mL respectively); the significance values were 0.014 and 1.21 × 10 −4 (obtained from the statistical t test for two populations method). The root growth of P. ixocarpa plants was inhibited by 42 to 53%, when concentrations of 1 were varied from 50 to 150 µg/mL with p values of 0.0046 and 1.94 × 10 −4 (Table 1). These results are in agreement with the previously reported inhibition of seed germination, root and shoot growth of Brassica. campestris, Raphanus sativus, Lactuca sativa, Mikania micrantha and Achyranthes japonica [8]. On the other hand, compound 2 had no effect on either germination and root and stem growth of E. cruss-galli and P. ixocarpa.   Table 2). Compound 1 (100 µM) inhibited the dry biomass of P. ixocarpa plants by 37%, and 2 only by 22%, suggesting that the double bond between carbons 4-5 in the β-caryophyllene molecule is necessary for activity. L. perenne plants were less inhibited by both sesquiterpenes. Figure 3A shows bleached leaves of P. ixocarpa plants treated with 1, after one week and the elongation of the plant decreased ( Figure 3B), which are in agreement with the dry biomass and bleaching effect results reported for monoterpene derivatives of another Asteraceae species plant family [9] which inhibits phytoene desaturase, a key enzyme in carotenoid pigment biosynthesis. Since carotenoids protect chlorophyll from photooxidation, their lack would result in loss of chlorophyll. The bleaching of P. ixocarpa leaves caused by -caryophyllene suggests that it could act as a carotenoid biosynthesis inhibitor. L. perenne plants were less inhibited (15% and 4%) with 1 and 2 (100 µg/mL).   From the kinetics data of the fluorescence curve measured at 24, 48 and 72 h after treatment on leaves and compared with the control, various parameters were calculated and plotted as a radar graph, a circular graphic with a series of spokes or rays projecting from a central point, with each ray label representing a different variable ( Figure 5). Data values were normalized to a common specific range or percentile values. Figure 5 shows the parameters (Strasser et al. [11]) of P. ixocarpa plants affected with 1, Panel A shows the effects at 24 h of treatment. Performance index decreased 25% and 10% at 50 and 100 µg/mL, respectively; and the absorption per reaction center (ABS/RC), the quantum yield for energy dissipation PHI(D 0 ), the trapped energy flux per reaction center TR 0 /RC and the indicator of the water splitting enzyme function (dV/dt 0 ) all were increased 10% at 100 µg/mL. Furthermore, at 72 h of treatments of P. ixocarpa plants with 1 (100 µg/mL): The electron transport per cross section (ET 0 /CS) and the maximum quantum yield of primary photochemistry [PHI(P 0 )] decreased 50% ( Figure 5, Panel C). The probability that a trapped exciton moves an electron into the electron transport chain beyond Q A − (PSI 0 ), the density of the reaction centers (RC/CS 0 ), the trapping and the electron transport per cross section (TR 0 /CS and ET 0 /CS) decreased around 50%; while the following parameters increased up to 20%: dV/dt 0 , ABS/RC and TR 0 /RC. These observations suggested that some PSII RCs were transformed to "heat sinks" or "silent reaction centers"; these centers can neither reduce Q A and their excitation energy is dissipated as heat [11], this is the reason why PHI(D 0 ) values increase. The mechanism of action of 1 is similar to other natural products like robustaflavone [12]. In panel D the effects of 50 µM of DCMU on the parameters are shown to compare them.

Reagents
Reagents were purchased from Sigma-Aldrich and the salts from J. T. Baker.

Tested Material
Aerial parts of S. salignus were collected in the state of Guerrero, Mexico in 2004 (R. Santiago, collection No R1995) and a voucher specimen was deposited at Facultad de Ciencias UNAM. No 115634. The aerial parts of S. salignus were dried, powdered and extracted exhaustively by maceration at room temperature with n-hexane. The resultant crude extract (22 g) inhibited ATP synthesis (I 50 = 79.3 µg/mL) ( Figure 1) and was fractionated via column chromatography on silica gel (0.0063-0.200 mm), and eluted with a mixture of increasing polarity gradient of n-hexane-EtOAc. The fractions eluted with n-hexane-EtOAc 90:10 were subjected to a second fractionation and eluted with mixtures of n-hexane-EtOAc of ascendant polarity, and β-caryophyllene and caryophyllene oxide were isolated with hexane-EtOAc 80:20 ( Figure 2). The compounds structures were identified by spectroscopic methods with a Bruker DRX 400spectrometer for NMR, for mass spectra an Shimadzu QP5050A device for electron impact (70 eV) low-resolution (EI-MS) was used and for gas chromatography/mass (GC/MS) spectrometry an Shimadzu QP-5050A, equipped with a PTE™-5 was used, to give values consistent with data reported in the literature [7].

Chloroplast Isolation and Chlorophyll Determination
Intact chloroplasts were isolated from market spinach leaves (Spinacea oleracea) as reported [13,14]. Chloroplasts were suspended in a small volume of the following solution: 400 mM sucrose, 5 mM MgCl 2 , 10 mM KCl, and 30 mM N-Tris[hydroxymethyl)methyl]glycine (tricine) pH 8.0 with KOH addition. Chloroplasts were stored as concentrated suspension in the dark for 1 h at 4 °C. The chlorophyll (Chl) concentration was measured according to Strain et al. [15].

Determination of ATP Synthesis
Intact chloroplasts (20 μg of Chl /mL) were broken before each assay by osmotic rupture in a solution (3 mL) containing: 100 mM sorbitol, 10 mM KCl, 5 mM MgCl 2 , 0.5 mM KCN, 1 mM tricine-KOH at pH 8.0 in the presence of 50 μM methylviologen (MV) and 1 mM adenosine diphosphate (ADP) at pH 6.5. The pH was adjusted to 8.0 with 50 mM KOH and ATP synthesis was titrimetrically determined using a microelectrode (Orion Mod. 8103 Ross) connected to a potentiometer (Corning Medical, model 12, Acton, MA, USA) with an expanded scale and a Gilson recorder (Kipp & Zonen, Bohemia, NY, USA). Alkalization rates were measured in the linear part during illumination. The reaction was calibrated by back titration with saturated HCl. The reaction started when turning the light on in the presence of chloroplasts (20 µg of chlorophyll per mL) [14,16]. The I 50 value (concentration producing 50% inhibition) for each activity was determined from plots of the activity at different concentrations of compound.

Chlorophyll a Fluorescence of PS II
Chlorophyll fluorescence induction curves were measured at room temperature with a Hansatech Handy PEA (Plant Efficient Analyzer) as previously described [12]. The maximum fluorescence yield of PS II was generated using three light-emitting diodes (broad 650 nm). The pulse duration was 2 s, with intensity of 2830 nm and gain of 0.7. Chlorophyll fluorescence determination in vivo, in intact and dark adapted (30 min) leaves, was performed for control and sprayed plants with concentrations of 25, 50 and 100 µg/mL of 1 and 2, after 24, 48 an 72 h of treatments. Data were analyzed and processed with Handy PEA and Biolyzer programs to obtain different photosynthetic parameters associated to PSII, according to the O-J-I-P test equations [14,17]

Seed Germination Bioassays
Monocot seeds of Echinochloa crus galli L. P. Beauv. an annual grass and dicotyledon seeds of Physalis ixocarpa L. (green tomato) were purchased from Semillas Berentsen, S. A. de C. V. (Celaya, Guanajuato, Mexico). Germination tests were run in triplicate with 40 E. crus galli seeds and 40 P. ixocarpa seeds for each concentration of sample for five days (three days for germination and two days more for root and shoot growth). The number of seeds used for each experiment was selected for that show an appreciable change in O 2 uptake that could be detected by the oxygraph (Yellow Spring Instrument) Model 5300. The test seeds were held in the dark at 28 °C in 9.0-cm Petri dishes containing an 8.5-cm sheet of Whatman No. 1 filter paper and 10.0 mL of test or control solution. Dishes were wrapped with Parafilm foil and incubated at 28 °C in the dark. The number of germinated seeds was determined according to the criterion of 1 mm extrusion of the radicle. Germination rates were counted at 72 and 120 h later for root and shoot growth measurements. 1 and 2 were initially dissolved in dimethyl sulfoxide (DMSO). The maximum final concentration of DMSO was less than 0.1% and the same DMSO concentration was used in the control solution [18].

Plant Material for in Vivo Assays
The seeds of P. ixocarpa and L. perenne, were sown in 12 cm diameter pots and were watered daily in the greenhouse at 27 ± 2 °C. After 15 and 18 days of emergence for P. ixocarpa and L. perenne, respectively, the plants were selected for similar size and were sprayed manually with the 1 and 2 at concentrations of 25, 50 and 100 µg/mL (one stock of 10 mg/mL of each compound was prepared in DMSO). An aliquot of the stock solution was taken to obtain the desired concentration in an aqueous suspension containing 0.05% w/v of polyoxyethylenesorbitan monolaurate (Tween-20). The control samples were sprayed with distilled water containing the same amount of DMSO and Tween-20 [19].

Conclusions
Allelochemicals play a role in mediating interspecific interactions [20,21], and various plants enhance competitiveness and fitness [22,23]. β-caryophyllene (1), a well-known volatile sesquiterpene with allelopathic potential, has been reported to inhibit development of seedlings of various plant species [24,25]. In this work, for the first time from the n-hexane extract of aerial parts of S. salignus plants, were isolated -caryophyllene and caryophyllene oxide. Compound 1 inhibited 42% the root elongation of Physalis ixocarpa seedling at 50 µg/mL and 53% at 150 µg/mL and it inhibited the root elongation of Echinochloa crus-galli to a lesser extent (30% at 150 µg/mL). On the other hand, compound 2 was inactive in both seedlings. Both compounds inhibited the dry biomass of Physalis ixocarpa plants, being -caryophyllene the most active. The Chl a fluorescence transient in vivo indicated that 100 µg/mL of -caryophyllene presented a major effect on photosynthesis of P. ixocarpa plants 72 h after treatment, inhibiting PS II by transforming active reaction centers to "heat sinks" or the formation of silent reaction centers, and also induces chlorosis on treated leaves.