Phytochemical responses to herbicide exposure and effects on herbivorous insects
2. Identification and quantitative analysis of selected phenolic compounds
The aim of this chapter is to identify the most important phenolic compounds in F. convolvulus leaves and to develop a reliable quantitative method for detection of these compounds in plant tissues.
2.1 Materials and methods
2.1.1 Plant material
F. convolvulus was grown in a greenhouse. Leaf material was lyophilised and stored dry at - 20° C until extractions were made. The compounds extracted, identified and quantified in this chapter are numbered from 1 to 6.
2.1.2 General techniques
HPLC was run initially on a Pharmacia chromatograph equipped with a Pharmacia LKB VWM 2141 dual wavelength UV detector. Reversed phase C18 columns (5m m particle size, 4.6 ´ 250 mm) were used, and the final column choice was a Phenomenex Prodigy ODS3 column, since it could tolerate the low pH values which were necessary for the analyses. Solvents were: 0.5% aqueous trifluoracetic acid (A) and acetonitrile (B). The following gradient was used: 0-5.0 min 5% B isocratic, 5.0 - 25.0 min: linear gradient 5 - 20% B, 25.0 - 40.0 min: linear gradient 20 - 35% B; 40.0 - 55.0 min: linear gradient: 35 - 60% B; 55-60 min: linear gradient 60-80% B; 60-85 min: 80% B isocratic; 65-75 min: 80-5% B; 75-85 min: 5% B isocratic. The flow rate was 0.8 ml min-1. In 1999 and 2000, HPLC was run on a Shimadzu LC-10AT liquid chromatograph equipped with a Shimadzu SPD M10 AVP Diode array detector. The Prodigy C18 column was used and solvents were: 0.5% aqueous trifluoracetic acid (A) and acetonitrile (B). The following gradient was used: 0-5.0 min 5% B isocratic, 5.0 - 25.0 min: linear gradient 5 - 30 % B, 25.0 - 40.0 min: linear gradient 30 - 50% B; 40.0 - 50.0 min: linear gradient: 50 - 80% B. The flow rate was 0.8 ml min-1. High voltage electrophoresis was run on Whatman No. 3 chromatography paper in a buffer, Pyridin-HOAc-H2O (25:1:500) at pH 6.5; 50 min at 5 kV and 90 mA.
2.1.3 Hydrolysis
Acid hydrolysis was performed in 1 M HCl in 50% methanol for 2 hours (reflux). Each hydrolysate was evaporated to dryness. N-trimethylsilyl (TMSi) imidazole was added to hydrolysates of compounds 2, 3 and 5, and the resulting TMSi ethers were analysed by GC-MS. TMSi ethers of meso and racemic dl tartaric, as well as glucose, galactose and mannose were used as reference compounds. The hydrolysate of compound 6 (2 mg) was redissolved in H2O (1 ml) and extracted with ethylacetate (3 ´ 2 ml) in a separation funnel. The aqueous and ethylacetate phases were concentrated to small volumes and analysed by paper chromatography together with authentic compound 6 (unhydrolysed), kaempferol, quercetin, glucuronic, galacturonic acid and glucuronolactone formed by acid treatment of glucuronic acid.
Enzymatic hydrolysis of compound 6 (2 mg) was performed in 500 m l H2O and 2.5 mg glucuronidase (Sigma) was added. The mixture was left at room temperature for 3 hours. The hydrolysate was then evaporated to dryness, redissolved in H2O (1 ml) and extracted with ethylacetate (3 ´ 2 ml) in a separation funnel. The aqueous and ethylacetate phases were concentrated to small volumes and analysed by paper chromatography together with authentic compound 6 (unhydrolysed), kaempferol, quercetin, glucuronic, and galacturonic acid.
2.1.4 Extraction and isolation
Lyophilised leaves (100 g batch-1) were transferred to boiling 70% aqueous ethanol (4 l) and homogenised for 5 minutes with an Ultra-Turrax homogeniser. After filtration, the residue was extracted ´ 2 with 70% aqueous ethanol (2.5 l) at room temperature. The filtrates were combined, evaporated to a small volume (ca. 400 ml) and extracted in a separation funnel with equal amounts of chloroform (´ 3) and later with ethylacetate (´ 3). The aqueous phase was further concentrated, centrifuged for 10 minutes at 15000 rpm before it was transferred to a column (10 ´ 23 cm) containing Polygosil C18 60-4063 (Macherey Nagel). The column was rinsed first with H2O and later with increasing concentrations of aqueous ethanol. Fractions (200 - 500 ml) were collected according the UV-absorption of the effluent. All compounds were finally purified on MCI GEL CHP20P (2.6 ´ 100 cm, Mitsubishi Chemical Co.). Fractions containing compounds 4 and 5, which eluted early from the C18 column were separated on the MCI GEL column using 5% acetic acid in 15% aqueous ethanol. Fractions containing compound 1, which eluted later from the C18 column were treated similarly. Fractions containing compounds 2, 3 and 6 were purified on the MCI GEL column rinsed with 10 - 25% aqueous ethanol. The purity of the compounds and fractions was controlled by HPLC.
2.1.5 Quantitative analysis
Lyophilised plant material (25 - 100 mg) was homogenised in 70% aqueous ethanol (5
ml) in a centrifuge tube at room temperature. After centrifugation, the supernatant was
removed and the pellet was extracted twice with 70% aqueous ethanol using the same
procedure. Kaempferol-3-O-J-D-[J-D-glucopyranosyl(1®2)glucopyranoside]-7-O-J-D-
glucopyranoside isolated from cabbage leaves (Nielsen et al., 1993) was added as an
internal standard during the first homogenisation step. 0.50 m
mole internal standard was added to samples of leaves of 60 to 100 mg, while 0.25 mmole was added to smaller samples. The combined extracts were
evaporated to a small volume and transferred to a volumetric flask (5.0 ml). 3.0 ml from
the volumetric flask was purified by solid phase extraction using a 500 mg C18 column
(International Sorbent Technology). The effluent from the C18 column (ca. 3 ml) was
collected and combined with the effluent from the same column using 40% aqueous ethanol to
a total volume of 10.0 ml (volumetric flask). The effluent from the C18 column (100 ml sample-1) was analysed by HPLC. Response factors (at
330 nm) were 1.00 for the flavonoid (compound 6) and 0.72 for the hydroxycinnamoyl esters.
Response factors for the hydroxycinnamoyl esters were determined by comparison of
published 
-values for caffeic acid derivatives (18500
at 330 nm) (Clifford 1999) with those for flavonol glycosides (14.000 at ca. 350 nm » 13.000 at 330 nm).
2.2 Results
2.2.1 Identification of the phenolic compounds
Six phenolic compounds were isolated and identified from F. convolvulus leaves (Fig. 1). Compound 3 was isolated from leaves treated with the herbicide, chlorsulfuron, while other compounds were isolated from untreated leaves. Compounds 1, 2, 4 and 5 were esters of caffeic acid while compound 3 was an ester of p-coumaric acid, and compound 6 was a flavonoid. The identification of these compounds involving FAB mass spectrometry and 1D and 2D NMR and various chemical techniques is unequivocal (Nielsen, Olsen & Kjær, unpublished).
Figure 2.1
Phenolic compounds isolated from leaves of F. convolvulus
The compound 1 was found to be a caffeoylester of quinic acid, but retention times on HPLC as well as NMR spectra demonstrated that the compound was different from chlorogenic acid, which is the most common caffeoyl quinic acid ester. Comparison of 1D and 2D NMR of 1 with those published previously for this compound and its isomers (Corse et al., 1966; Flores-Parra et al., 1989; Scholz-Böttcher et al., 1991) demonstrated that esterification had occurred in the 3-position of quinic acid in 1, while it occurs in the 5-posion in chlorogenic acid (5-CQA). Compound 1 is therefore neochlorogenic acid (3-CQA) which is also a rather common plant constituent. The naming of the caffeoylquinic acids follow the new conventions from which is opposite to the one used in earlier investigations. As an example neochlorogenic acid is 3-CQA according to the new conventions, while is was named 5-CQA in older literature, for example in .
Compound 2 was found to be 1-caffeoyl-b -D-glucose. The glucose moiety was identified after acid hydrolysis of the parent compound, while the attachment between caffeic acid and glucose was determined by NMR. Similar analyses demonstrated that compound 3 was 1-p-coumaroyl-b -D-glucose.
Mobility studies using high voltage electrophoresis demonstrated that compounds 4 and 5 contained two free dicarboxylate groups suggesting that they were caffeoyl esters of hydroxydicarboxylic acids (Fig 2.1). Compound 4 was found to be 2-caffeoyltartronic acid, while compound 5 was found to be 2-caffeoyl-meso-tartaric acid. The identification of the acyl moiety as meso-tartaric acid as opposed to one of the optically active forms of tartaric acid was confirmed by GC-MS after acid hydrolysis.
UV- and NMR-data demonstrated that compound 6 is a flavonoid, and identified the aglycon as quercetin. Mobility studies using high voltage electrophoresis and several chemical techniques demonstrated the presence of a glucuronic acid moiety. The attachment between quercetin and glucuronidc acid was confirmed by NMR. Compound 6 could therefore be identified as quercetin-3-O-b -D-glucuronide. None of the compounds had previously been found in leaves of F. convolvulus, but they had been reported from other plant species (see later). The compounds constituted more than 90% of the phenolic found in a typical sample from F. convolvulus leaves (Fig. 2.2).
Figure 2.2
HPLC chromatogram showing peaks at 330 nm originating from six phenolic compounds
isolated from F. convolvulus leaves. Numbers are according to Fig. 1; is: internal
standard.
Quantitative analyses have been based on HPLC throughout the studies, but considerable developments have occurred in the actual methods used. When the project started, the Pharmacia HPLC equipped with a dual wavelength detector was available. This system was more or less satisfactory for identification of the major compounds although severe overlap between the identified compounds and some minor compounds occurred from time to time. With the increasing interest in detection of sometimes trace amounts of compound 3, this system proved to be insufficient, since it was not possible to distinguish between trace amounts of compound 3 and other minor phenolic compounds. It was therefore a big improvement that a Shimadzu HPLC equipped with a diode-array detector became available in 1999. The advantage of the diode-array detector is that it is possible to obtain full UV spectra of all the peaks. Since UV spectra of compound 3 are clearly different from UV spectra of caffeoyl derivatives (compounds 1, 2, 4, 5 and several minor compounds), the identification of 3 could be made with much higher certainty (Fig 2.3).
Figure 2.3
UV spectra of compounds 2 (top) and 3 (bottom) demonstrating the typical difference
between caffeoyl esters (UVmax higher than 320 nm) and p-coumaroyl esters (UVmax below 320
nm).
None of the compounds identified from F. convolvulus are new natural products, but they have not previously been found in Polygonaceae. Neochlorogenic acid (compound 1) is less common than its isomer, chlorogenic acid, but has nevertheless been identified from a variety of plants including cabbage and coffee beans (Clifford, 1999) and a variety of ripe fruits (Möller and Herrmann, 1983). The glucose esters, compounds 2 and 3, are also widely distributed in the plant kingdom (Mølgaard and Ravn, 1988), and they are often precursors for biosynthesis of other hydroxycinnamic acid esters (Strack et al., 1987). 2-caffeoyl-tartronic acid (compound 4) seems occur more rarely in plants, since it has only been reported from three plant species, Vigna radiata (Fabaceae) (Strack et al., 1985), Chondrilla juncea (Asteraceae) (Terencio et al., 1993) and Nepeta cataria (Lamiaceae) (Snook et al., 1993) . 2-caffeoyl-meso-tartaric acid (compound 5)has previously been reported from Equisetum arvense (Hohlfeld et al., 1996), while the isomeric compound 2-caffeoyl-L-tartaric acid (caftaric acid) has been reported from grapes and wine as well as from grapevine leaves, where it occurs together with quercetin-3-O-b -D-glucuronid acid (compound 6) (Goetz et al., 1999; Singleton et al., 1978). Caffeoyl esters of D-tartaric acid have been reported from Cichorium species (Compositae) (Wöldecke and Herrmann, 1974). The mixture of hydroxycinnamoyl derivatives found in F. convolvulus has not been found previously in other plant species. A characteristic mixture of different natural compounds may be more important than any single compound for the ability of phytophagous insects to recognise their host plants (Feeny et al., 1988). The phenolic compounds isolated from F. convolvulus may therefore be important for the ability of the leaf beetle, G. polygoni, to recognise F. convolvulus as one of its major host plants.
Some of the compounds from F. convolvulus as well as their isomers have previously been reported to be involved in biological interactions. Neochlorogenic acid (compound 1) inhibited growth of Spodoptera litura larvae in the same way as its more common isomer, chlorogenic acid (Stevenson et al., 1993). Chlorogenic acid has been reported as a deterrent or growth reducing compound for several insects (Hoover et al., 1998; Stamp and Yang, 1996), but at the same time it is part of a behavioural active mixture which stimulate oviposition by black swallowtail butterflies (Papilio polygenes) on carrot leaves. Other caffeoyl quinic acid derivatives are important oviposition stimulants or synergists for other swallowtail butterflies (Carter et al., 1999; Haribal et al., 1998). Typically, there is a tight linkage between chemical structure and biological activity in these interactions, and it is characteristic that the caffeoylquinic acids are more active in combination with other host plant compounds for example flavonoids (Carter et al., 1999; Feeny et al., 1988). Flavonoids are often involved in biological interactions and deterrent as well as stimulatory effects on insect behaviour and growth have been reported depending on the type of insect and the particular chemical structure (Harborne and Grayer, 1993). However, no effect on insects has yet been attributed to quercetin glucuronid (compound 6), but this compound may be involved in resistance to grey mould in grape berries together with caffeoyl-L-tartaric acid (caftaric acid) (Goetz et al., 1999). Caffeoyl-tartronic acid (compound 4) has previously been identified as a precursor for catechol in a grasshopper, Romalea guttata (Snook et al., 1993). The grasshoppers obtain the precursor from its host plant, catnip, and metabolise it into a component of their own defensive secretion.