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FAIR-CT96-1436
Improvement of efficiency and reduction of application rates of preferable naturally grown biocides by complexing with gamma cyclodextrin |
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Proposal No: | FAIR-CT96-1436 |
| Date Prepared: | May 2001, February 1998 | |
| Source: |
Final Report Executive Summary
First Annual Progress Report |
Introduction
This project that started in 1996 aimed to use the following naturally occurring pesticides in a new and sophisticated manner in conjunction with the novel compound gamma-cyclodextrin. The compounds concerned were:
Initially the project focused on complexes formed with the more delicate Scilliroside. However, the work has gradually shifted to Pyrethrum complexes in the latter years (1999 and 2000). In general these activities kept on track with the initial proposed research activities and hence the final report can report four years of scientific studies covering cultivation, scale up trials and patenting (in preparation) for gamma-cyclodextrin complexed Scilliroside and Pyrethrins.
Abbreviations
g-CD or gamma-CD = gamma Cyclodextrin
CD = Cyclodextrin
NOAEL = no-observed-adversed-effect level
DOC = dissolved organic carbon
PBO = Piperonylbutoxide
IUPAC nomenclature for compounds under investigation:
Scilliroside (C32H44O12): 3 beta-(beta-D-glucopyranosyloxy)- 17 beta-(2-oxo-2H-pyran-5-yl)- 14 beta-androst- 4-ene- 6 beta,8,14-triol 6-acetate
Cinerin I (C20H2803): (Z)-(S)-3-(but-2-enyl)-2-methyl-4-oxocyclopent-2-enyl (IR)-trans-2,2- dimethyl-3- (2-methylprop-1-enyl)cyclopropanecarboxylate
Cinerin II (C21H2805): (Z)-(S)-3-(but-2-enyl)-2-methyl-4-oxocyclopent-2-enyl (E)-(IR)-trans-3-(2- methoxycarbonylprop-1-enyl)-2,2-dimethylcyclopropanecarboxylate
Jasmolin I (C21H3003): (Z)-(S)-2-methyl-4-oxo-3-(pent-2-enyl)cyclopent-2-enyl (IR)-trans-2,2- dimethyl- 3-(2-methylprop-1-enyl)cyclopropanecarboxylate
Jasmolin II (C22H3005): (Z)-(S)-2-methyl-4-oxo-3-(pent-2-enyl)cyclopent-2-enyl (E)-(IR)-trans-3- (2- methoxycarbonylprop- I -enyl)-2,2-dimethylcyclopropanecarboxylate
Pyrethrin I (C21H2803): (Z)-(S)-2-methyl-4-oxo-3-(penta-2,4-dienyl)cyclopent-2-enyl (+)-trans- chrysanthemate
Pyrethrin II (C22H2805): (Z)-(S)-2-methyl-4-oxo-3-(penta-2,4-dienyl)cyclopent-2-enyl pyrethrate 8
Activities
This project kicked-off in January 1997. During this year various activities were carried out, including cultivation, extraction and purification, toxicity studies for gamma-CD and scheduling of toxicity studies for the active compounds. Taking into account the strict principles and laws for toxicity studies investigations on cyclodextrins were performed by AC TNO, Netherlands, and monitored by CR Wacker, Germany. The progress of carcinogenicity studies and chronic feeding studies was in line with the anticipated schedule. Other tasks and related studies were carried out using pure active ingredients of. both scilliroside and pyrethrins. Cyclodextrin complex formation with and extraction methods using Cyclodextrines and the isolation of active ingredients directly from drug material by the use of gamma Cyclodextrins were all investigated. Scilliroside was purified for the first time up to a concentration of 99% active substance by gama-Cyclodextrin extraction and several recrystallization steps.
The product formed the basis for the physical-chemical studies as well as the toxicity investigations and the analysis of standard preparations. Toxicological studies were scheduled and first preliminary application studies were carried out, although these gave contradictory results. Characterisation of pyrethrins was possible after the isolation of six naturally occurring esters by HPLC. Preparatively isolated Pyrethrum I and II were complexed with gamma-Cyclodextrins. This material was used for the physical-chemical studies. A method for the purification of pyrethrins with gamma-Cyclodextrins was evaluated.
The isolation of active principles from the plant Pyrethrum cinerariifolium by gamma-cylodextrin was not carried out in 1997 due to the bad weather that impeded cultivation in Italy. These tests were re-scheduled for 1998. Studies on scilliroside extraction from plant material of Urginea maritima (L.) Baker were carried out. Plant material for these tests had been available since mid 1997. First results directly indicated that currently used methods needed re-evaluation and that information in the available literature was inappropriate.
During 1998 a one year toxicity study of gamma-cyclodextrin was completed. The chronic oral toxicity of gamma cyclodextrin was examined in rats by administration in the diet (7 days/week). It was concluded that gamma cyclodextrin is tolerated without any specific adverse effect at all dose levels tested. Therefore, the no-observed-adverse-effect level (NOAEL) of gamma-cyclodextrin in the present study was placed at the highest dose level. The carcinogenicity studies with gamma-cyclodextrin in rats were also carried out during 1998.
A method of isolation of scilliroside from the raw material (extract, 20 %) had been set up by 1998 and optimised The method starts with the formation of a complex of scilliroside with gamma-cyclodextrin, followed by a selective extraction with ether/butanol, and, finally by recrystallisation. An alternative extraction procedure for obtaining scilliroside from powdered Urginea maritima by complex formation with gamma-cyclodextrin was also developed. Maceration of the dried plant material with aqueous gamma-cyclodextrin led to formation of scillilroside: gamma-cyclodextrin complexes. By using organic solvents, scilliroside was extracted from this complex, leading to 100-fold enrichment, the final extract containing 15 % scilliroside.
All samples obtained as above were analysed by HPLC. This included investigation of other samples of squills from Algeria which were also analysed to determine their content of scilliroside, using UV and HPLC methods.
It was found that the aglycon of scilliroside, scillirosidine, could have a higher acceptance by rats when used in bait formulations as scilliroside is rather bitter. Therefore scillirosidine was prepared by enzymatic hydrolysis of the sugar moiety by beta-glucuronidase and was analysed by physico-chemical methods. The stability of scillirosidine was examined in various solvents and at different pH values . To characterise the pure compound, 1H-NMR and 13C-NMR spectra were recorded and interpreted. Theoretical methods for the prediction of association ability of guest molecules were developed, since the experimental determination is often difficult, mainly due to the low solubility of the guest molecules.
Scilliroside: gamma cyclodextrin pilot production has been carried out. Solid complexes of (host:guest) mole ratios of 1:2 and 1:3 were manufactured using a kneading method. The complexes were analysed by HPLC, NMR and TGA/DSC spectra, to show that the complexes obtained were real inclusion complexes and not only adsorbates.
Genotoxicity studies of scilliroside were carried out in accordance with the OECD guideline 471 and EEC guideline 92/69, part B.14. Scilliroside was tested at concentrations ranging from 62 micro gr to 5000 micro gr per plate according to the 'direct plate incorporation method'. In the test systems various strains of Salmonella typhimurium were used. The results indicate that scilliroside is 'non-mutagenic' in the Salmonella typhimurium reverse mutation test at amounts up to 5000 micro gr per plate.
New bait formulations have been developed, based on gamma-cyclodextrin:scilliroside complexes and various ingredients. The aim was to compare different scilliroside compositions with and without gamma cyclodextrin. All tests again showed high rat acceptance, but usually low acceptance for house and field mice. Baits, packed into envelopes have been prepared, which appear to be more appetising than other types of baits. These tests are still in progress.
To supply pyrethrins for analysis (by means of physico-chemical methods), for biological tests and formulation experiments, different extraction methods have been worked out. The extractions were performed with dichlormethane and n-hexane from the pulverised drug (flowers of pyrethrum). During precipitation in hexane, an ethanol-soluble precipitate was formed, which contained fatty and waxy materials; these were separated by centrifugation. From the purified extract the single compounds were isolated by preparative HPLC.
As an alternative method pyrethrins were extracted using supercritical C02. A method was also established for the enrichment of commercial 25 % (Fluka) and a 50 % (Hentschke) pyrethrum extracts. The extraction were accomplished with hexan:acetonitrile (1: 1) and heptone:acetonitrile (1: 1) solvent mixtures. With the, hexan:acetonitrile (1: 1) solvent mixture, starting from the 25 % extract, an increase up to 72 % could be achieved after a 2-step extraction, with the heptone:ocetonitrile (1:1) solvent mixture the increase was even higher (up to 82 %).
In order characterise the pure compounds, 1H-NMR and 13C-NMR spectra were recorded for jasmolin I and II and for cinnerin I + II.
Using theoretical models, the stability constants of the pyrethrins were predicted. Experimentally determined constants are not yet available, since their measurement is difficult (very low solubility of the pyrethrins), but this work is in progress. For all pyrethrins quantum-mechanical ab inition calculations were performed at HF/3-21 G level, starting from fully minimised (MM2) geometries. To get an insight to the geometries of the host-guest complexes, dynamical Monte Carlo simulation, taking into account salvation by a continuum approximation, were done. In the typical low-energy geometry the complex adopts a conformation where the guest is inserted into the cyclodextrin cavity in a way that maximises its van der Waals contacts to the host.
The stability of the pyrethrum extract, the pyrethrum-beta-cyclodextrin complex and the pyrethrum-gamma cyclodextrin complex against UV radiation were analysed by exposing respective probes to UV light, at 254 nm and 366 nm.
During 1999 the 24 month carcinogenicity study with gamma cyclodextrin in rats was finished During the study, the test substance (gamma-CD) was administered via the diet, and clinical signs, growth, food intake, hematological parameters were monitored. No treatment-related differences in general condition, behaviour, or incidence of palpable masses to the control group could be observed. The overall mortality was not affected by the test substance. Haematology after 12, 18 and 24 months did not reveal any changes in red and white blood or thrombocyte cell counts. It was concluded that the test substance does not show any tumourigenic properties. Ready Biodegradability Modified OECD Screening test showed that Scilliroside can not be classified as readily biodegradable although there is clear evidence that biodegradation has started within 28 days of incubation. This test was done by measuring the DOC (dissolved organic carbon) at frequent intervals over a 28 day period.
As preliminary studies for the extraction of scilliroside from the plant, the partition coefficients between different organic solvents and water were determined. Isolation of scilliroside form the plant showed that including a complexing step with gamma-CD leads to surprisingly high contents of the active substance: the plant powder was extracted with a mixture of organic solvents with water and the polysaccharides were precipitated with ether. Complexes of this extract with gamma-CD results in further purification.
To obtain a better understanding of the host-guest interaction, linear and non-linear free energy relationships for formation of complexes alpha, beta and gamma cyclodextrin with guest molecules were developed, with the aim of free energy prediction and interpretation of the association process. Highly predictive models were obtained although the structural variability of the compounds used for their deduction was large. The significant correlation of steric descriptors suggests that van der Waals interactions are important to complex formation in the case of all three CDs. In the case of alpha-CD dipole-dipole interactions might also stabilise the complexes. For the beta-CD:guest complexes the hydrophobic effect seems to have an important contribution to their stabilisation, the hydrogen bond donor capacity of the guest playing only a minor role in the complex formation process. This is in agreement with the fact that in beta-CD most of the hydrogen bonds are intramolecular, forming a band around the molecule (therefore also the lowest solubility of O-CD, compared to alpha- and gamma-CD). In contrast, the gamma-CD:guest association appears to be crucially influenced by the hydrogen bond acceptor and donor capacity of the guest, while the hydrophobic effect seems to be of lower importance.
Various ready to use baits using talcum as carrier and the scilliroside-gamma-CD complex as the active ingredient have been formulated. Results showed that all formulations appeared more appetising as compared to the untreated. The comparison has been done with a commercial check (Rattofene from the company Leica) packaged in 10 g little envelopes. Baits prepared by hot melt coating containing the scilliroside-gamma-CD complex were tested. Five rats were offered their usual food and baits. In all cases the acceptance of the baits was good and all animals died within 36 hours after uptake.
Pyrethrum cinnerariifolium plants, as well as a crude extract were used in further purification of the active ingredient. The crude extract has been obtained by extraction with supercritical C02. From the purified extract the single compounds have been isolated by preparative HPLC, with hexane-ethylacetate as mobile phase. Around 70 pyrethrin containing samples from different experiments were analysed and the 1H-NMR and 13C-NMR spectra obtained have been calculated for the various pyrethrins, and compared to the previously recorded experimental spectra . Comparison of the measured with the calculated spectra shows that the calculated low energy structures of the pyrethrins (conformational search by means of force fields, followed by ab initio quantum chemical method) in vacua are good estimates of the solution conformations.
Investigations on the stability of pyrethrum in solution upon UV irradiation, showed that formation of complexes did not have a significant protective effect. In contrast, the stability of pyrethrum in solid form upon irradiation was influenced by complex formation. While a rapid degradation of the pyrethrins could be observed after irradiation with an UV lamp, the complexes (beta- and gamma-CD) were significantly more stable under the same conditions. Irradiated with a sun lamp they were stable for one day, after three days 50-70 % of the pyrethrins remained not degraded. The sun lamp mimics the most relevant conditions concerning outdoor use of these pesticides.
Attempts were made to determine the stability constants of the pyrethrins with gamma-CD, employing the solubility method. Various set-ups were tried, but the reproducibility of the solubility isotherms obtained was not good, leading to highly uncertain values for the stability constant.
The biological activity of pyrethrum was tested in vivo on the grain weevil (Sitophilus granarius), using various concentrations of pyrethrum and synergist (PBO). A commercial product, "GRANEX®" , was also tested under the some conditions for comparison. GRANEX and the gamma-CD-pyrethrum complex show a similar toxicity on the grain weevil. After 2 days all animals displayed signs of paralysis and on the seventh day all were dead. Further experiments established that PBO alone has no pesticide properties.
In 1999 cultivation of pyrethrum did not present any particular problems. Plants survived the winter period well and gave enough flowers. The first harvest was done in July, and the second in September. Compared to the area Mercato Saraceno (470 kg/ha) Cesena's fields gave a slightly better harvest (550 kg/ha). By the end of 1999 and in early 2000 it became obvious that toxicological studies would have to be re-scheduled and some of them cancelled.
Studies originally intended to be carried out on the active scilliroside, namely subchronic toxicology, reproductive toxicology and teratogenicity studies, were cancelled. Instead studies on pyrethrum toxicology were initiated, to obtain the, so far missing, acute toxicology data as well as exotoxicology, that was unknown.
Changes in the project tasks as well as the extended time frame were reported to the EU and accepted. Some of the above studies were still in progress during the preparations of the final report.
It should be noted that one essential bait trial, with the active Scilliroside has been carried out by the Austrian Federal Office and Research Centre for Agriculture during the year 2000. This is intented to be prolonged in 2001. Other trials are under Federal Office guidance and so far have not been prohibited or limited. They are running as field studies and providing additional information concerning effects of Scilliroside. These toxicity studies should be finalised during 2001.
In accordance with the principles and legislation relating to toxicity studies investigations on cyclodextrins were performed and monitored in areas of carcinogenicity and chronic feeding studies. The other tasks and studies carried out included:
Scilliroside was purified up to a content of 99 percent active ingredient by the use of gamma-CD and followed by recrystalisation. This provided the necessary material for further pysical-chemical property studies, for studies of acute toxicity and for use as a analytical standard. The method purifying scilliroside using with gamma-CD was evaluated and introduced as a standard procedure.
Scheduled toxicological studies using scilliroside are under discussion. Preliminary application studies with baits containing scilliroside gave contradictory results. Further tests are required.
Characterisation of pyrethrins became possible after isolation of six naturally occurring esters, using HPLC. Pyrethrum I and II were used to form gamma-CD complexes for further physical-chemical studies. The method of purification for pyrethrins with gamma-CD is being evaluated further. Extraction tests using plant material were made impossible by unexpected weather conditions in Italy. New cultivations have been planted.
Studies on gamma-CD extraction of scilliroside from the plant Urginea maritima are also continuing. Plant material has been available since July 1997, however it was necessary to evaluate the extraction method before making use of the plant material.
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