1 Area of Ecology, Faculty of Science, University of Extremadura, 06071 Badajoz, Spain; e-mail: email@example.com.
2 Department of Organic Chemical, Faculty of Science, University of Cadiz, 11510, Purto Real, Cádiz, Spain.
The leaves and photosynthetic stems of Cistus ladanifer secrete a sticky substance called labdanum. Under natural conditions, together with other functions, this exudate presents allelopathic activity. In the present study, labdanum was separated into various fractions, and the majority diterpene constituents were purified, identifying oxocativic acid, labdanolic acid, 6-acetoxy-7-oxo-8-labden-15-oic acid, and 7-oxo-8-labden-15-oic acid. Then two laboratory bioassays STS (Standard Target Species) and coleoptile bioassays were carried out to determine the allelopathic activity of these compounds. The results showed there to be diterpenes present in the C. ladanifer exudate with allelopathic capacity.
Ecological potential activity of the four majority diterpenes present in the exuded of Cistus ladanifer L.
The leaves and photosynthetic stems of Cistus ladanifer, a shrub typical of Mediterranean scrublands of southern Europe, secrete a sticky substance called labdanum (Braun-Blanquet, 1941). The secretion is greatest in summer and under situations of stress. Amongst other activities, it presents a clear allelopathic activity under natural conditions. But which are the compounds in the labdanum responsible for this activity?
To begin to reply to this question, the aim of the present work was to isolate, purify, and identify a series of terpene compounds that absorb UV radiation and that are present in proportionately great amounts in the labdanum. Two bioassays were then applied to the purified compounds to test firstly their potential activity, and secondly their phytotoxic capacity.
Extraction of the labdanum
Fresh C. ladanifer leaves were immersed in chloroform in the proportion 1:2 to extract the exudate. The chloroform was evaporated off, and the residue was re-suspended in methanol and stored frozen at -20°C for 12 hours to allow the waxes to settle out. These were then removed by centrifugation.
Purification of the compounds
Before the actual purification of the compounds, the labdanum was separated into various fractions by chromatography in a Sephadex LH-20 column (Vogt, 1991) using methanol as eluent. The fraction that had a majority presence of diterpenes was then assayed by HPLC using a Spherisorb 5μ (C-18 of 4.6x250mm) reverse phase analytical column with water/acetonitrile under gradient conditions (Chaves, 1993; Chaves, 1999). The four majority compounds that were detected were collected and analyzed by NMR to check their correct purification and facilitate their subsequent identification.
Wheat seeds (Triticum sp.) were put to germinate in distilled water in total darkness at 22°C. After 4 days, the coleoptiles were ready to be cut into 4 mm sections. These were placed into test tubes containing a pH 5.6 buffer of sucrose (20g/l), citric acid (1.02g/l), and potassium phosphate dibasic (2.9 g/l). The medium used to dissolve the compounds and prepare the different concentrations (10-3-10-6M) was DMSO, at a proportion of 5μl per ml of buffer. Five coleoptiles in 5ml of solution at different concentrations were put into each test tube, and these were placed in a rotor that maintained the culture under continual shaking in a chamber in total darkness at 22°C for 24 hours (Macías, 2000). Their lengths were then measured using the computer program, Fotomed (Castellano, 2002).
STS (Standard Target Species) bioassay
Tomato (Lycopersicum esculentum), onion (Allium cepa), wheat (Triticum sp.), garden cress (Lepidium sativum), and lettuce (Lactuca sativa) seeds were used for the STS bioassay. Different concentrations of the purified compounds were prepared for each seed. For the compounds with a greater amount available, the complete series of concentrations 10-3M to 10-6M were prepared. As with the coleoptile bioassay, the medium used to dissolve the compounds was DMSO at the proportion of 5μl per ml of MES buffer solution (10mM, pH 6). Twenty-five seeds of each species were put on paper in Petri dishes (4 replicates), and 5ml of solution was added. The dishes were maintained at 25°C in darkness for 3 (L. sativum), 5 (L. esculentum, L. sativa, and Triticum sp.), and 7 days (A. cepa). The number of germinated seeds were then counted, and the root and cotyledon lengths were measured (Macías 2000; Castellano 2002).
Identification of compounds
After the task of purifying the compounds, complicated due to the great complexity of the C. ladanifer exudate, they were identified on the basis of their nuclear magnetic resonance (NMR) spectra. The identified compounds belonging to the diterpene group were:
2. Labdanolic acid. Mr:324
3. 6-acetoxy-7-oxo-8-labden-15-oic acid. Mr:378. λ:255
4. 7-oxo-8-labden-15-oic acid. Mr:320. λ:249
As one observes in Fig. 1, the activity of the four compounds studied was high (-80% with respect to the control) at the 10-3M concentration, being reduced to a half (-40%) but still significant in the case of compounds 1 and 4 at the 10-4M concentration. For the rest of the compounds, and at lower concentrations, the inhibition in the elongation of the coleoptile sections was not significant.
Figure 1. Effect of the four diterpenes identified in the exudate of C. ladanifer (1.- oxocativic acid; 2.- labdanolic acid; 3.- 6-acetoxy-7-oxo-8-labden-15-oic acid; 4.- 7-oxo-8-labden-15-oic acid) on the growth of wheat (Triticum sp.) coleoptiles. The results are expressed with respect to the control.
The results show that the effects depended on the compound and on the species. Oxocativic acid (1) provoked significant inhibition only on the cress, affecting both germination: -47% (10-3M), -23% (10-4M), and -32% (10-6M), and root and cotyledon lengths -50% (10-6M), -51% (10-3M), respectively. The tomato cotyledon length was also inhibited (-46%) at the 10-3M concentration. Figure 2 shows that, as in the previous case, labdanolic acid (2) significantly inhibited only the germination of the cress seeds at the same concentrations: -79% (10-3M), -14% (10-4M), and -18% (10-6M). This compound's inhibition of root and cotyledon lengths, however, was general for all the seeds, but only at the 10-3M concentration (Figs. 3 and 4). With respect to 6-acetoxy-7-oxo-8-labden-15-oic acid (3), it must be noted that the series of concentrations tested was only from 10-4M to 10-6M due to the small amount of purified compound available. At these concentrations, significant negative effects were not observed in the germination of any species, but there was inhibition of the root lengths of cress and onion: -30% (10-6M) and -53% (10-4M), respectively, and in the cotyledon lengths of these same two species: -32% (10-6M) and -58% (10-4M), respectively.
Figure 2. Effect of labdanolic acid on the germination of the five species tested. Results expressed as a percentage with respect to the control.
Figure 3. Effect of labdanolic acid on the root length of the five species tested. Results expressed as a percentage with respect to the control.
Figure 4. Effect of labdanolic acid on the cotyledon length of the five species tested. Results expressed as a percentage with respect to the control.
In view of the results, one can conclude that the phytotoxic activity of the diterpenes was not uniform, the activity of each compound depended on the species and on the concentration used in the trial. Nevertheless, the coleoptile trials clearly showed a high, concentration-dependent, inhibitory effect. Also, the STS trials showed that the influence of these compounds, mainly of labdanolic acid, on root and cotyledon growth was greater than on germination, with the growth of the seedling clearly being inhibited after the seed had germinated. The following step will be to determine the concentration of these compounds in the medium where they naturally exert their action, the soil. It has to be taken into account that these compounds are synthesized and act together, so that their individual activity could well be altered in the soil. It also has to be considered that the soil and climate conditions of the natural medium may enhance or reduce that activity.
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