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Heliespirones C-E, new spiroterpenes from Helianthus annuus

F.A. Macías1, Ascensión Torres, J.L.G. Galindo, Rosa M. Varela and José M.G. Molinillo

1 Cadiz Allelopathy Group (GAC), Dept. of Organic Chemistry, Faculty of Sciences University of Cadiz. Apdo. 40, 11510 – Puerto Real, Cádiz, Spain. Email


The discovery of new allelochemicals from plants or microbes has attracted much attention in the last 20 years. Allelochemicals are involved as biocommunicators, and are also potential sources of new structural types of herbicides with new modes of action which may be less harmful than those presently used in agriculture. From the medium polar active fraction of leaf aqueous extract of Helianthus annuus L., cv. Peredovick® we have isolated three compounds with a heliespirone skeleton: heliespirones C, D, and E. The structure of heliespirone C, a potential allelopathic agent, was elucidated by homo- and hetero-nuclear 2D-NMR spectroscopy and crystallographic data. Heliespirones D and E are still under study.

Media summary

Heliespirones C-E have been isolated from medium polar active fractions of sunflower leaves.

Key Words

Allelopathy, bioactivity, heliespirone, spiroterpene, Helianthus annuus, sunflower, etiolated coleoptiles bioassays


Sunflower species are native to North America and many examples of their allelopathic activity in wild and agricultural ecosystems have been already reported: Helianthus rigidus exhibits autotoxicity and sunflower (Helianthus annuus) has a great allelopathic potential and inhibits seedling growth of weeds, including velvetleaf, thorn apple, morning glory, and wild mustard, among others (Leather 1987).

The indiscriminate use of herbicides has resulted in: a) increasing incidence of resistance in weeds to some herbicide classes such as triazines and dinitroanilines; b) shifts in weed population to species that are more closely related to the crop they infest; and c) environmental pollution and potential health hazards. Allelopathy, an emerging branch of applied sciences, may help in overcoming such problems through development of crop varieties having greater ability to smother weeds, use of natural phytotoxins from plants or microbes as herbicides and use of synthetic derivatives of natural products as herbicides (Macias 1996).

We have carried out systematic allelopathic studies on cultivar sunflowers to evaluate their potential as source of allelopathic agents and natural herbicide templates. Previously, we have described and characterized from sunflower simple phenolics, triterpenes and steroids (Macias 1997), sesquiterpene lactones, mainly germacranolides and guaianolides (Macias 1993; 1996b), flavonoids (Macias 1997b), heliannuols (Macias 1993b; 1994) and the new family of spiroterpenes, the heliespirones (Macias 1998; Figure 1). Here in we describe the isolation and structural characterization of three new heliespirones C-E.

Figure 1. Heliespirones previously isolated from Helianthus annuus


Plant material and Isolation

The methodology used is a biodirected isolation using a wheat coleoptile bioassay for discriminating fractions was a Coleoptile’s bioassay. This is a fast and sensitive bioassay to a wide range of bioactivities including plant growth regulators, herbicides, antimicrobials, mycotoxins, and assorted pharmaceuticals (Hancok 1964).

Fresh leaves (25.0 Kg) of sunflower cv. Atila were soaked with H2O (wt plant: V. solvent 1: 3) for 24 h at 25°C in the dark. The aqueous phase was extracted with 0.5 L of CH2Cl2 per 1.0 L of H2O, the combined extracts were dried over Na2SO4 and evaporated in vacuum to yield 11.5 g of crude extract. This material was fractionated by CC on silica gel using hexane, ethyl ether, EtOAc, methanol, and water yielding fractions A1 (0.3 g), A2 (4.7 g), A3 (3.1 g), A4 (1.9 g), A5 (1.0 g) and A6 (0.8 g), respectively.


Coleoptiles were obtained from 3-day-old wheat seedlings sown on 15 cm diameter Petri dishes fitted with Whatman #1 filter paper and grown at 24 °C in the dark. The etiolated seedlings were removed from the dishes and selected for size uniformity under a green safelight. The selected etiolated seedlings were placed in a Van der Wij guillotine, and the apical 2 mm were cut off and discarded. The next 4 mm portions were selected for bioassay and kept in an aqueous nutritive buffer for 1 h before being used to synchronize the growth.

Fractions were tested at 1000, 500, 250 and 125 ppm in a buffered nutritive aqueous solution (citric acid-sodium hydrogenphosphate buffer, pH 5.6; 2% sucrose). Mother solutions were prepared in DMSO and diluted to the proper concentration with the buffer to a 0.5% v/v DMSO final maximum concentration. Following dilutions were prepared maintaining the same buffer and DMSO concentrations. Bioassays were performed in 10 mL test tubes as follows: five coleoptiles were placed per tube containing 2 mL of test solution each; three replicates were prepared for each test solution and the experiments were run in duplicate. Test tubes were placed in a roller tube apparatus and rotated at 6 rpm for 24 h at 22 °C in the dark. Increments of coleoptile elongation were measured by digitalization of their photographic images and data were statistically analysed.

Results & Discussion

All fractions were assayed with the coleoptile bioassay being fractions A2 and A3 the more active (Figure 2). The bioactive fraction A3 was chromatographed using silica gel and eluting with CHCl3:acetone 9:1. After purification of the low polar fractions by HPLC with a Hibar Si 60 (Merck) column (CHCl3:methanol 19:1) yielded heliespirones C (22 mg), D (5 mg) and E (2 mg) were isolated.

Figure 2. Bioactivity of fractions A2-A6

Heliespirone C (figure 3) showed a HRMS with a molecular ion at m/z 268.167, and absorptions at 3448 and 3554 cm-1 in the IR spectrum, suggest the presence of one hydroxyl group. Absorption at 1677 cm-1 indicated the presence of a conjugated carbonyl group.

Figure 3. Structure of heliespirone C

The signals observed in the 1H-NMR (figure 4), 13C-HNMR, gCOSY, gHSQC, gHMBC were in agreement heliespirone-type structure. The 1H-NMR spectrum showed a deshielded signal (H-3; δ6.6s) corresponding to a proton attached to a double bond conjugated with a carbonyl group, and another signal (H-15; δ1.96; 3H) which was assigned to a methyl group attached to double bond.

Figure 4. 1H-NMR of heliespirone C.

This structure and the presence of dimmers with hydrogen bond between them, was confirmed with the X-Ray data (Figures 5 and 6).

Figure 5. X-Ray structure of heliespirone C

Figure 6. X-Ray structure with dimer of heliespirone C


Hancok CR, Barlow HW, Lacey HJ (1964). The east malling coleoptile straight growth test method. Journal of Experimental Botany 15, 166-176.

Leather, GR (1987). Weed control using allelopathic sunflowers and herbicide. Plant Soil, 98,17-23

Macias FA, Varela RM, Torres A, Molinillo JMG (1993). Potential allelopathic guaianolides from cultivar sunflower leaves, var. SH-222. Phytochemistry 34, 669-674.

Macias FA, Varela RM, Torres A., Molinillo JMG (1993b). Novel sesquiterpene from bioactive fractions of cultivar sunflower. Tetrahedron Letters 34, 1999-2002.

Macias FA, Molinillo JMG, Varela RM, Torres A (1994). Structural Elucidation and Chemistry of a Novel Family of Bioactive Sesquiterpenes: Heliannuols. Journal of Organic Chemistry 59, 8261-8266.

Macias FA, Varela RM, Torres A, Molinillo JMG (1996). Field Crops as Source of Natural Herbicide Models: Sunflowers. In ‘Allelopathy in Pests Management for Sustainable Agriculture. Vol 2’ (Eds SS Narwal, P Tauro) pp. 77-88, (Scientific Publishers: Jodhpur, India).

Macias FA, Torres A, Molinillo JMG, Varela RM, Castellano D (1996b). Potential allelopathic sesquiterpene lactones from sunflower leaves. Phytochemistry 43, 1205-1215.

Macias FA, Molinillo JMG, Varela RM, Torres A, Galindo JCG (1997). Bioactive Compounds from the Genus Helianthus.In ‘Recent Advances in Allelopatjhy. A Science for the Future, Vol I’. (Eds FA Macias, JMG Molinillo, JCG Galindo, HG Cutler) pp 121-148, (CAB Publishers: UK).

Macias FA. Molinillo JMG, Torres A, Varela RM, Castellano D (1997b). Bioactive flavonoids from Helianthus annuus cultivars. Phytochemistry 45, 683-687.

Macias FA, Varela RM, Torres A, Molinillo JMG (1998). Heliespirone A. The first member of a novel family of bioactive sesquiterpenes. Tetrahedron Letters 39, 427-430.

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