isolated from the Stella variety of Helianthus annuus. This compound was ..... M.; Foy, C. L.) Southwest Missouri University, Springfield, MO, USA (1999). - 68 -
Solvent Extraction Research and Development, Japan, Vol. 18, 55 – 68 (2011)
Identification of Major Compounds Extracted by Supercritical Fluids from Helianthus Annuus L Leaves Lourdes CASAS,1* Casimiro MANTELL,1 Miguel RODRÍGUEZ,1 Ascensión TORRES,2 Francisco A. MACÍAS2 and Enrique J. MARTÍNEZ DE LA OSSA1 1
Department of Chemical Engineering and Food Technology, Faculty of Science, University of Cadiz, Spain 2
Department of Organic Chemistry, Faculty of Science, University of Cadiz, Spain (Received May 21, 2010; Accepted July 2, 2010)
In the present study, sunflower leaves were extracted using an analytical-scale SFE under a wide range of extracting conditions with the objective of determining not only the best extraction conditions but also the variables that control the process in terms of the extraction yields and bioactivities of the extracts obtained. The results showed that 15 % of water as a modifier of carbon dioxide is the most efficient solvent to obtained the best extraction yields. However, the best activity profiles were obtained for the samples extracted with supercritical carbon dioxide and 5% water at 500 bar. The extract obtained in a pilot plant with CO2 and 5 % H2O at 400 bar and 50 ºC was fractionated and 5 major compounds were identified: tambulin, heliannone A, kaempferol-3-glucoside, niveusin B, and pinoresinol.
1. Introduction Significant progress in the field of natural products chemistry can involve advances in technology, new molecules of substantial interest or changes in ethical principles for the collection of organisms [1]. During the last two decades, there have been numerous changes in natural products chemistry and these include plant selection and collection, isolation techniques, structure elucidation, biological evaluation, semi-synthesis and biosynthesis. There are two important fields in which technical advances have an influence on natural products chemistry in general. These aspects concern analytical techniques and structure-elucidation techniques. The development of new analytical techniques allows the isolation of new compounds in very small amounts
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from more and more complex matrices. The natural products chemist can use a wide range of methods for separation: droplet counter current chromatography, rotation locular counter-current chromatography, centrifugal partition chromatography and, of course, high performance liquid chromatography (HPLC). These new techniques have changed our laboratories and the routines of our daily work. Many different factors now exist to fine-tune separations: the solvent, gradients and the stationary phase – or a combination of these factors. On the other hand, the isolation of small amounts of minor compounds would be useless if we did not have appropriate structure-elucidation techniques available. As a result, the development of these techniques has been impressive, especially in the field of nuclear magnetic resonance (NMR). The range of NMR techniques that are available is almost bewildering. Since active compounds in natural products are usually present in low concentrations, a great deal of research has been carried out to develop more effective and selective extraction methods for the recovery of these compounds from the raw materials. The development of alternative extraction techniques with better selectivity and efficiency is therefore highly desirable. Consequently, supercritical fluid extraction (SFE) as an environmentally friendly and efficient extraction technique for solid materials was introduced and has been extensively studied for the separation of active compounds from herbs and other plants [2]. The high solvation power of supercritical fluids (SF) was first reported over a century ago. In recent years, SFE has received a great deal of attention as the full potential of this technology in analytical applications has begun to emerge. Today, SFE has become an acceptable extraction technique for use in many areas. SFE of active natural products from herbal or, more generally, from plant materials has become one of the most important application areas. The increasing public interest in herbal medicines and natural products has led to the publication of numerous SFE-related research papers in herbal or natural product studies in recent years [2]. Several studies have been published concerning the isolation and identification of bioactive compounds from sunflowers using maceration with organic solvents and/or water as a technique to obtain extracts [3-8]. The influence of various extraction conditions on the supercritical extraction process on this raw material has also been studied [9-12]. The resulting extracts were found to have high bioactivity and it was observed that the addition of water to the CO2 improved the extraction yields [10]. However, the chemical identification of the compounds extracted by this technology has not been carried out to date. The work described here concerned the identification of the major compounds responsible for the bioactivity of the extracts obtained by SFE with CO2 and water as cosolvent. The extraction conditions are also discussed briefly because in previous studies the conditions had not been optimized for the process with CO2 and water.
2. Experimental
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2.1 Raw material and extraction at high pressure The raw materials used in the extraction process were leaves of Helianthus Annuus L (variety Aitana) and these were provided by the Center for Agricultural Research Station (CIFA), Junta de Andalucía, Jerez, Spain. Previous studies concerned the supercritical extraction process of sunflower leaves using various cosolvents, with water giving the highest yields and best bioactivity profiles [10]. However, in this previous study only 5% (v/v) water as cosolvent was tested and it was therefore appropriate to evaluate the extraction yields and bioactivity profiles of extracts obtained using different percentages of water. The extractions were carried out with 10 and 15% (v/v) water as a cosolvent. After obtaining the best percentage of cosolvent, the effect of pressure and temperature on the extraction system was studied. These experiments were carried out in an ISCO extractor (Nebraska, USA, model SFX 220). A schematic representation of the equipment and further details can be found in a previous publication [10]. The experiments were carried out in duplicate in order to confirm the results. After the best extraction conditions (temperature, pressure, and percentage of cosolvent) had been obtained the extractions were carried out in a pilot plant from Thar Technology (Pittsburgh, PA, USA, model SF2000) to obtain larger quantities and to characterize the major compounds in the extracts. A schematic representation of the equipment and further details can be found in a previous publication [11]. Bioassays constitute an important tool to evaluate the inhibitory or stimulatory activity in terms of growth for the substances extracted. In this case, coleoptile bioassays were carried out on crude extracts and fractionated components. A description of the bioassay technique used can be found in a previous publication [4]. The aim of this study was not to determine specific values, but to attempt to obtain activity profiles on the basis that an extract will be more bioactive when its activity levels persist as the sample is diluted. The data are expressed as percentage differences from the control, which means that a value of zero represents an identical value to the control. On the other hand, a positive value represents stimulation of the parameter in question and a negative value represents inhibition of the wheat coleoptiles under the given experimental conditions. 2.2 Cluster analysis Statistical treatments were performed using the program Statgraphics Plus 5.1 (Statistical Graphics Corp.). Association analysis of the data based on the bioactivity profile was performed for each of the different sets of extraction conditions. To further clarify the relationships between the clusters and those individuals forming the clusters, a dendrogram was generated by hierarchical cluster analysis. The squared Euclidean distance between normalised data and the nearest neighbor method were to measure the similarity between samples. This method is selected because it is the best method to resolves these kinds of problems, also this method is used by the most usual commercial statistical packages making this study more easy [13].
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2.3 Separation and identification of compounds Dried ground plant material (190 g) was extracted with CO2 and H2O at 400 bar, 50 ºC and 5% H2O for 120 min. The extract was collected in acetone and the solvent was evaporated under reduced pressure. A total of 6.76 g of extract was subjected to open column chromatography on silica gel 60 (0.063–0.2 mm) and eluted using hexane/acetone mixtures of increasing polarity. Fractions were pooled according to their behaviour (Rf values) on thin layer chromatography (TLC). Six pooled fractions were subsequently bioassayed. Two of these fractions showed improved bioactivity over the control. The active fractions were subjected to repeated flash and column chromatography in order to isolate and identify the active compounds. The fractionation process involved column and thin layer chromatographic techniques. Silica gel column chromatography was conducted using silica gel 60 (0.06–0.2 mm). TLC was carried out on 0.25 mm pre-coated (SIL-25 UV254) glass-backed plates. The plates were first viewed under UV light and then developed using sulfuric acid/water/acetic acid (1:4:20) and then heat.
.
For HPLC, LiChrosorb silica 60 was used in the normal-phase mode with differential refractometer (RI) and UV detectors in conjunction with a Hitachi L-6020A HPLC system. All solvents were of spectroscopic grade or were distilled from glass prior to use. Compounds were identified using spectroscopic data such as 1H and
13
C NMR spectra and by direct comparison with published data and
structures. NMR spectra were recorded in CDCl3 using a Varian 400 MHz Unity spectrometer. 3. Results and Discussion 3.1 Selection of extraction conditions The extraction yields expressed as mg of extract/100 g of dry leaves obtained with pure CO2 and CO2 with different percentages of water as cosolvent [5, 10 and 15% (v/v)] for dried and congealed samples are represented in Fig. 1. The data obtained with pure CO2 and CO2 + 5% (v/v) water as cosolvent were reported in a previous publication [9, 10] but are included in Fig. 1 for the sake of comparison.
Figure 1. Influence of the percentage of water for different extraction conditions (pre-treatment/percentage of water used as cosolvent). (
) data previously published in reference 10
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The effects of temperature, pressure and pre-treatment of the samples when the extraction was carried out with CO2 + 10% and 15% (v/v) of water as cosolvent are similar to those obtained with CO2 + 5% (v/v) of water [9, 10]. It can be seen from Fig. 1 that, at a constant temperature and pressure, an increase in the percentage of water as cosolvent from 5% to 15% leads to an increase in the extraction yield. Fig. 2 shows the activity profiles, with respect to the control for different extraction conditions. A value in this kind of graphics of -100 means that there is a 100% inhibition in the growth of the coleoptiles, i.e. there is no growth, and a value of 0, means zero inhibition, i.e. the coleoptiles grow in the same way that of the control. In this figure the data shown that all the extracts present an inhibitory activity. The sample extracted with CO2+5% water as cosolvent is more bioactive because its activity levels persist as the sample is diluted. An increase in the percentage of water as cosolvent leads to a reduction in the selectivity of the process to obtain bioactive substances. The congealed samples extracted with 10 and 15 % water as coslvent proved detrimental to the extraction process in terms of the bioactivity with respect to dried sample extracted with the same cosolvent. 35/D/5%
50/D/5%
35/C/5%
50/C/5%
35/D/10%
50/D/10%
35/C/10%
50/C/10%
35/D/15%
50/D/15%
35/C/15%
50/C/15%
0
-10
-20
Activity (% control)
-30
-40
-50
-60
-70 1000 ppm 500 ppm 250 ppm 125 ppm 75 ppm
-80
-90
-100 Extraction conditions
Figure 2. Bioactivities of extracts obtained at 500 bar [temperature/pre-treatment (D: dried and C: congealed)/percentage of water used as cosolvent].
The results of the cluster analysis are shown in Fig. 3 and the order in which the experiments for the general bioactivity assay are grouped can be seen. The dendrogram can be broken at different levels to yield different clusterings of the data, but it is recommended to make a cut in the largest line, that is at the distance between 2 and 3 in squared Euclidean distance [13]. According to this analysis, two groups are formed. The first group includes: -
the extraction carried out with CO2 + 5% (v/v) water as cosolvent
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-
dried samples extracted with 10 and 15% (v/v) of water as cosolvent
-
congealed samples extracted with 15% (v/v) of water at 50 ºC.
This group corresponds to a small diminution in the activity on increasing the dilution. The second group is formed by samples arising from other extraction conditions and these show a marked decrease in activity as the dilution is increased. Dendrogram Nearest Neighbor Method,Squared Euclidean
4
Distance
3 2 1
35/C/15%
50/C/10%
35/C/10%
35/D/10%
50/C/15%
50/D/10%
35/D/15%
35/C/5%
50/D/15%
50/D/5%
50/C/5%
35/D/5%
0
Figure 3. Cluster analysis for the bioactivities of extracts obtained with different percentages of water as a modifier [temperature/pre-treatment (D: dried and C: congealed)/percentage of water used as cosolvent].
The results of the general bioactivity assay for the extracts obtained with CO2 and 5% (v/v) of water are good and the variables considered in the analysis do not significantly affect the results. It was therefore decided to continue the study into the influence of temperature and pressure with the chosen solvent system. However, it must be borne in mind that the yields are higher on increasing the percentage of water in the carbon dioxide. Such an increase would lead to extracts with higher levels of water and, as a result, the extract would require concentration and this, in turn, would make the process more expensive.
Figure 4. Extraction yields for different values of pressure and temperature with CO2 + 5% water as cosolvent. (
) data previously published in reference 10
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The effects of pressure and temperature on the SFE efficiency for the bioactive compounds were assessed by carrying out extractions at pressures of 100, 200, 300, 400 and 500 bar and at temperatures of 35, 40, 45 and 50 ºC. According to the experimental data (Fig. 4(A) for dried samples and 4(B) for congealed samples), the best extraction yields were obtained on using dried samples. It is very important to study the behaviour of systems at 300 bar because the extraction yield is independent of temperature. The activity levels of the extracts obtained at 300 bar and at temperatures of 35 and 50 ºC are shown in Fig. 5. When comparing the results of Fig 5. with those obtained, for extracts obtained with 5% water as cosolvent (Fig. 2) it can be seen that there is a clear difference between the activity levels of the extracts obtained at the different pressures selected. The extracts obtained at 500 bar exhibited -100 % activity at 1000 ppm and values near to -30 % for 125 ppm. Theses extracts showed the best bioactivity profiles in that the activity level did not decrease markedly upon dilution. The activity profiles of the extracts obtained at 300 bar show a marked decrease in the activity level on dilution. At 1000 ppm the samples only exhibited -70 % activity with decreased to -10% approximately, at 125 ppm. The statistical results of the cluster analysis applied to the activity data for 300 and 500 bar are shown in Fig. 6. Analysis of Figs. 5 and 6 led us to infer that it is advisable to perform the extraction at 500 bar because the activity levels of these extracts show greater persistence on increasing dilution. 35ºC/D/300
50ºC/D/300
35ºC/C/300
50ºC/C/300
35ºC/D/500
50ºC/D/500
35ºC/C/500
50ºC/C/500
0
Activity (% control)
-20 -40 -60 -80 -100
1000 ppm 500 ppm 250 ppm
-120
125 ppm Extraction conditions
Figure 5. Bioactivities of extracts obtained at 300 and 500 bar [temperature/pre-treatment (D: dried and C: congealed)/ pressure].
Having selected the best conditions in terms of pressure, temperature and percentage of cosolvent, the extraction was carried out on the pilot plant in order to obtain sufficient quantities for chemical characterization of the major components in the extract.
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Dendrogram Nearest Neighbor Method,Squared Euclidean
8
Distance
6 4 2
35/C/300
50/C/300
50/D/300
35/D/300
35/C/500
50/D/500
50/C/500
35/D/500
0
Figure 6. Cluster analysis for the bioactivities of extracts obtained at 300 and 500 bar [temperature/pre-treatment (D: dried and C: congealed)].
3.2 Identification of major compounds The extract obtained on the pilot plant with CO2 and 5% H2O at 400 bar (the pilot plant could not operate at pressures above 400 bar) and 50 ºC was chromatographed on a column of silica gel using hexane-acetone mixtures of increasing polarity and finally methanol, and 6 fractions were obtained according to their behaviour (Rf values) on TLC. It was necessary to perform a general bioassay in order to select the fractions that provide the extracts with the best bioactivity levels. The activity profiles are shown in Fig. 7. It can be seen that fractions I and II inhibit the growth of coleoptiles by about 60% at the highest concentration tested (1000 ppm), but these bioactivity profiles decrease rapidly with increasing dilution. I
II
III
IV
V
VI
Actitivy (% control)
0 -20 1000ppm
-40
500ppm
-60
250ppm
-80
125ppm
-100 -120 Fractions
Figure 7. Bioactivities of the fractions of crude extracts
The major compound found in fraction I was myrcenol, as corroborated by the spectroscopic data. This compound was first isolated from the fungus Ascoidea hylecoeti [14] but has not been isolated previously from sunflower. However, myrcene – a derivative of myrcenol – is a common compound in helianthus and has been isolated from sunflower seeds and flowers [15].
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Fraction II yielded two major compounds. The spectroscopic data for one of these compounds are the same as those described in the literature for 9,12-octadecadienoic acid or linoleic acid [16]. The study of the fatty acid composition of sunflower varieties shows that this acid is present in notable proportions, which is consistent with this product being obtained as a major component in this fraction. The other major compound in fraction II was isolated as a crystalline solid. The 1H-NMR and 13CNMR spectra both contain signals that are characteristic of a kaurane-type diterpene, with a double bond between positions C-16 and C-17. The spectroscopic data are consistent with those described in the literature [17] for (–)-kaur-16-en-19-oic acid, which was previously isolated from the same species. The monoterpenes have two features that enable the evaluation of their activity – firstly, they are volatile and can therefore be released into the environment by simple volatilization and, secondly, they have low solubility and so the thresholds of activity of these compounds are mostly below the solubility limit [18]. These characteristics were corroborated by the low levels of bioactivity and the profiles obtained (Fig. 7) for this fraction in the general bioactivity test. Fraction III showed inhibitory activity on coleoptiles of greater than 80% at the highest concentration tested (1000 ppm), but this fraction didn’t maintained good levels of activity on increasing dilution. Analysis of the 1H-NMR spectrum and TLC behaviour is consistent with this fraction being a complex mixture of different products. The 1H-NMR and
13
C-NMR spectra exhibited typical signals of two guaianolide sesquiterpene
lactones, with the spectroscopic data for one compound matching those reported [19] for annuolida H isolated from the Stella variety of Helianthus annuus. This compound was tested by Macias et al. [19] using the coleoptile bioassay and the inhibitory activity was 75% from 10–3 M. The other sesquiterpene lactone showed chemical shifts very similar to those of this compound, but with the signal due to the hydroxyl group at C-2 absent from the spectrum. All spectroscopic data matched the 8β-angeloyloxycumambranolide, which was first isolated by Gershenzon and Mabry (1984) [20] from Helianthus Maximilian. Sesquiterpene lactones bearing germacranolides, guaianolides, eudesmanolides and melampolides are the most abundant compounds from this genus of Helianthus – along with the diterpenes. Apart from the α-methylene-γ-lactone system, the most common feature is the presence of an oxygenated group (mainly angelate esters) at C-8. Sesquiterpene lactones exhibit a wide range of activities: e.g. allelopathic [3-5], deterrent [21], cytotoxic [22], bacteriacidal [23] and fungicidal [24]. Some of these activities can be correlated to their ecological roles in the plant, but others are mainly characterized by their toxic properties and their ability to bind to important biomolecules [25]. Structure-activity studies have shown that the presence of an α-methylene-γ-lactone system and/or an α-methylene group in a cyclopentenone ring may be necessary for the activity. The presence of additional functional groups and their relative configuration can also have a significant influence on the activity.
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Fraction III exhibited another majority flavonoid-type compound. The 1H-NMR data were similar to spectroscopic data in the literature [26] and showed that the major compound was 3,5-dihydroxy-4',7,8trimethoxyflavone, which in the literature is also called tambulin. This substance is very common in sunflower and has been isolated from several varieties of Helianthus annuus including VYP [27] and Peredovik [28]. Finally, another major sesquiterpene component was also identified in this fraction and this had a heliannane skeleton, as confirmed by the spectroscopic data [29, 30], which are consistent with a ring with the basic structure of a cyclic aromatic ether with seven members and an ether function at C-10. This corresponds to heliannuol D, which was isolated from the SH-222 [27] variety of Helianthus annuus. Sesquiterpenes are usually stored in glandular trichomes of leaves along with monoterpenes. There are reports of the isolation of a family of heliannuols from sunflowers with plant growth regulator activity. In this case, heliannuol D exerted a potent inhibitory effect on onion root and shoot growth [3, 6]. Fractions IV, V and VI showed good activity profiles, with VI showing the best results (Fig. 7). Fraction IV presented a maximum inhibition of almost 70% at 1000 ppm and this decreases to 40% at 500 ppm. This fraction mainly contains tambulin and another flavonoid – the 1H-NMR spectrum of which shows that this has a chalcone-type skeleton. Comparison of the spectroscopic data with those published by Macías et al. [28] shows this compound to be 2',4-dihydroxy-3',4'-dimethoxychalcone (heliannone A), which was first isolated from Helianthus annuus var. VYP. Structure-activity studies with several flavones show that the cytotoxic effects seem to be related to the number and position of free hydroxy groups [31]. Flavonoids reported from sunflower have shown moderate germination and growth activity on Lycopersicon esculentum and Hordeum vulgare seeds [28]. The observed effects on the growth inhibition of coleoptiles were also significant on fraction V, with almost 70% at 1000 ppm, but in this case showed a more uniform activity profile than fraction IV. This fraction also yielded heliannone A and a mixture of sesquiterpene lactones – two compounds that have a germacranolide skeleton with a tetrahydrofuran ring, niveusine B and 1-methoxy-4,5-dihydroniveusina A, which were previously isolated from glandular trichomes of Helianthus annuus L [32, 33] and leaves of the varieties Stella and SH-222 [19, 27]. Furthermore, the lactone mentioned above with a guaianolide skeleton was also identified (8β-angeloyloxycumambranolide) along with leptocarpin, a sesquiterpene lactone with a 1,10-dihydroheliangolide skeleton, which was previously isolated from the leaves of Helianthus annuus of varieties Peredovick and VYP [7, 27]. Attention is increasingly focused on the possible role of sesquiterpene lactones as plant growth regulators. The ability of these compounds to block the synthesis of the complex IAA-receptor has been proposed in the case of the activity of niveusin B [34]. Fraction VI proved to be the most successful in coleoptiles bioassays and the best inhibition profile was maintained even on dilution. At 1000 ppm the activity is 100% and at 500 ppm it was 70%. Even at
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concentrations of 250 and 125 ppm the fraction showed nearly 50% inhibition. The proton nuclear magnetic resonance spectrum of fraction VI led to the identification of one major product, a glycosylated flavone. This compound has a glycosylated flavonol-type skeleton as the 1H-NMR spectrum showed a typical signal of the anomeric carbon and the other characteristic signals match those described in the literature for D-glucose [35]. The spectroscopic data obtained from both the characteristic signals of the flavone and the sugar are consistent with those reported for kaempferol-3-glucoside, which is present in the leaves of many species of the genus Helianthus (H. divaricatus, H. giganteus, H. hisutus, H. resinosus, H. tuberosus [35], H. decapetalus) but not previously isolated from Helianthus annuus L. Fraction VI was chromatographed on a column of silica gel using hexane-acetone mixtures of increasing polarity and finally methanol. The compounds identified were tambulin, heliannona A, niveusin B and a compound that belonged to the lignan family. These latter compounds are characteristically formed by two units of phenylpropane (C3-C6) and constitute a complex family of functionalized skeletons. The 1H-NMR spectrum for this compound showed that it was a symmetrical molecule, as seen from the integrals, with three aromatic hydrogens – a pattern consistent with a 1,3,4-trisubstituted ring. The structure identified for this compound is described in the literature for pinoresinol, which was previously isolated from the leaves of Helianthus annuus of varieties VYP and Stella [8]. Reported cases of allelopathy appear to involve complex mixtures of chemicals, which interact in an additive manner [37] or synergistically [6,7,38]. Scant knowledge on the constituents of the chemical complex, the concentration and the modes of action of the components prevail. These facts, together with techniques that are far removed from natural systems (e.g., in this case, the use of SFE with a wide range of extraction conditions – pre-treatment of the sample, pressure, temperature and considering the addition of different percentages of water as modifier), need to be considered. The main objective in this field must be to ascertain not only the best extraction conditions but also the variables that control the process in terms of the extraction yields and bioactivities of the extracts and the fractions obtained. These issues will contribute to overcoming any reservations about the significance of allelopathy research. In summary, the fractions I, II, III, IV, V and VI were chromatographed on silica gel columns (Flash chromatography) using hexane/acetone mixtures of increasing polarity, and yielded 13 compounds: myrcenol, linoleic acid, (–)-kaur-16-en-19-oic acid, tambulim, heliannone A, heliannuol D, annuolide H, leptocarpin, kaempferol-3-glucoside, 8β-angeloyloxycumambranolide, niveusin B, 1-methoxy-4,5dihydroniveusin A and pinoresinol. A summary of the major compounds identified in each fraction is
shown in Table 1. Table 1. Major compounds identificated in each fraction.
Fraction I II
Compounds
myrcenol linoleic acid, (-)-kaur-16-en-19-oic acid
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III IV V VI
8β-angeloyloxycumambranolide, tambulin, heliannuol D, annuolide H, tambulin, heliannone A heliannone A, niveusine B, 1-metoxy-4,5-dihydroniveusina A, leptocarpin, 8β-angeloyloxycumambranolide kaempferol-3-glucoside, tambulin, heliannone A, niveusine B, pinoresinol
The structures of these compounds are shown in Fig. 8. O HO lin o leic acid
Figure 8. Compounds from sunflower.
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4. Conclusions The crude extract obtained at 400 bar and 50 ºC using CO2 and 5% water as cosolvent was fractionated to give six fractions. The major components of these fractions were characterized. The best activity profile on coleoptiles bioassay was obtained for fraction VI and this contained 5 major components: tambulin, heliannone A, kaempferol-3-glucoside, niveusin B and pinoresinol. Synergistic or additive interactions are usually not comparable to the activity of a single active compound, unless such a compound already participates in the combination. The specific structural factors that operate and determine the activity of a particular combination of compounds still remains unclear. The same holds true for the combined effect; the character of such an effect cannot be predicted on the basis of individual compounds acting alone. In some cases, a noninhibitory concentration of a specific compound inhibits growth when this compound acts additively or synergistically with other allelochemicals that are present and such joint action is the most common situation. In order to explain allelopathy it is essential to recognize the stress interactions involving allelochemicals in plants.
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