Monocrotaline extraction

Pyrrolizidine alkaloids have long been known to be antitumor active and, more recently, have become of interest as anti-cancer agents. They occur naturally in several plant species, but are often difficult to extract and isolate from the plant material without degradation or the use of toxic solvents. The extraction of a model pyrrolizidine alkaloid, monocrotaline, from the seeds of Crotalaria spectabilis was investigated in this work. 

The crushed seeds of Crotalaria spectabilis were first contacted with supercritical carbon dioxide and, as expected, the oils comprising the bulk of the seed material were preferentially extracted. The addition of ethanol and water as co-solvents in the fluid phase led to the appearance of monocrotaline in the extract. Monocrotaline contents as high as 24% of the total extract could be obtained with carbon dioxide-ethanol mixtures. 

In order to increase the extract purity further, two additional processes were developed. The temperature-solubility cross-over point was utilized to obtain extracts containing as much as 50% monocrotaline. A second novel process incorporating ion exchange resins was also studied and yielded extracts containing 94 to 100% monocrotaline. Because this technique depends only on the basic character common to the pyrrolizidine alkaloids, it is expected to be equally effective in the extraction and isolation of the other members of this class and, in fact, could be extended to the other classes of basic alkaloids. 

The seeds of Crotalaria spectabilis were obtained in Clarke County, Georgia in November, 1984. These seeds were analyzed and found to contain 1.9 wt.% monocrotaline and 2.5 wt.% hexane-extractable lipid material. The seeds (5mm indiameter) were milled to 1 mm to break the hard outer coat and to expose the inner seed material containing the monocrotaline. The seed fragments were sieved to remove the 850-1- micron fraction (predominantly outer coat fragments) and the 850- micron fraction was packed in the equilibrium cell. 

Before investigating the extraction of monocrotaline from Crotalaria spectabilis, the solubility of pure monocrotaline was measured. This was done to determine the magnitude of the solubility, to evaluate the effect of co-solvents, and to confirm the integrity of the extracted monocrotaline. 

Due to the relative success of the pure component solubility studies, the same series of experiments were carried out using the complex seed material. Three systems were investigated to evaluate the ability of supercritical fluids to extract monocrotaline from the seeds of Crotalaria spectabilis. Pure carbon dioxide was studied with the expectation that the oils would be preferentially extracted. Ethanol was added as a co-solvent to increase the solubility of monocrotaline. Also, due to its success in the extraction of caffeine and nicotine, water was used as a co-solvent. 

The solubility of the seed material in carbon dioxide was defined as the intial concentration prior to observation of these depletion effects. The solubility was found to range from 0.016 wt.% to 0.6 wt.% (Figure 4). Analysis of the extracts revealed that the monocrotaline content was very low (<0.05 wt.%), and that the extracts were predominantly lipid material as expected. 

Carbon Dioxide - Ethanol - Crotalaria Spectabilis System. The solubility of the seed material increased by as much as 20 fold in the presence of ethanol (Figure 5). The presence of monocrotaline in the extracts became detectable at 2-4 mol% ethanol and increased markedly as ethanol concentration increased. The solubility of monocrotaline when extracted from the seed material was from 50% to 93% less than the comparable solubility (same ethanol concentration, temperature, and pressure) of monocrotaline. This is in contrast to the observations of Dobbs (10) and Kurnik et al. (11) who found that the solubility of a component is generally enhanced in the presence of other components. As the extraction proceeded and the seed bed became depleted of soluble components, the overall exit fluid phase concentration again decreased. However, the... 

Figure 4 Overall solubility of Crotalaria spectabilis extract in carbon dioxide. No monocrotaline was detected in die extract...

The objective of any separation process is to obtain high purity product. Monocrotaline selectivities as high as 40 wt.% were obtained in the above studies. Clearly, significant post-processing would be required to obtain pure monocrotaline from the extract. Two alternative supercritical fluid-based processes were therefore considered in order to obtain pure monocrotaline. 

The converse of this process, namely extraction at 10 mol% ethanol, 308.15K, 14.77 MPa followed by an increase in the temperature, should result in only the lipid material being deposited. By operating the second bath at 363.15 K no monocrotaline was deposited as expected. The fraction of monocrotaline in the supercritical phase could thus be increased to 49 wt% in this fashion. 

Ion Exchange Adsorption Process. Supercritical fluid extracts of Crotalaria spectabilis contain monocrotaline, a basic alkaloid, and non-polar lipid material. In the separation of caffeine from coffee, Zosel (3) recommended separation of the caffeine from the fluid phase by either adsorption onto activated carbon or absorption into liquid water. Activated carbon adsorption would be undesirable in the present case because the lipids would also be adsorbed and because desorption from activated carbon is quite difficult. Liquid water would absorb... 

Figure 10 Lipid material and monocrotaline solubility in 92 mole% carbon dioxide 4- 8 mole% ethanol. The overall extract without the monocrotaline is assumed to consist of lipid material.

Monocrotaline (63) from Crotalaria spectabilis (Legumy is responsible for the activity of Crotalaria extracts against adenocarcinoma-755 in mice. Similar alkaloids have been screened for anti-tumour activity by other researchers in general, however, pyrrolizidine alkaloids have liver toxicity and it is questionable whether they can be used in chemotherapy. Furthermore, cissampareine (64) from Cissampelos pareira (Menisperm.) and coronaridine (65) from Ervata-mia dichotoma (Apocyn.) have been investigated by the Kupchan group. 

Monocrotaline, a pyrrolizidine alkaloid of chemotherapeutic interest, has been extracted from the seeds of Crotalaria spectabilis using supercritical carbon dioxide and carbon dioxide-ethanol mixtures (29). Other alkaloids that have been extracted using SFE include nicotine and caffeine. Environmental applications of supercritical fluids include regeneration of activated carbon, extraction of organic contaminants like polynuclear aromatic hydrocarbons and polychlorinated biphenyls from water and soils, and the newly emerging field of supercritical water oxidation. 

Extraction of monocrotaline from Crotalaria spectabilis Two kilograms of Florida grown Crotalaria spectabilis seed ground to 16-mesh was extracted continuously for 72 hours with 95% ethanol. The extract was acidified to Congo paper with citric acid, the solvent was removed in vacuo, and the residue was taken... 

Monocrotic acid, CiHnOt. A mixture of 20 g. of monocrotaline and 40 g. of barium hydroxide octahydrate in 250 cc. of water was refluxed for one hour. After cooling, the solution was saturated with carbon dioxide and the barium carbonate was filtered. The filtrate was made just acid to congo red with hydrochloric acid and was then submitted to continuous extraction with ether for twelve hours. The ether extract was dried, the ether was removed, and the residue was distilled in vacuo at 145-146° (18 mm.) yield, 4.2 g. (48%). 

Monocrotalic acid, CJIaOi. A solution of 10.8 g. of monocrotaline in a mixture of 10 cc. of glacial acetic acid and 40 cc. of ethanol was hydrogenated at 2-3 atm. with 0.1 g. of platinum oxide catalyst. Two moles of hydrogen were absorbed in 5 hours. The solution was filtered from the catalyst and evaporated in vacuo. The remaining sirup was taken up in 34 cc. of 1 iV hydrochloric acid and the aqueous solution thus obtained was extracted continuously with ether for 24 hours. The ether solution was dried, the ether was removed, and the crystalline residue of monocrotalic acid was purified by recrystallization from acetone-petroleum ether (b. p. 30-60°) white plates, m.p. 181-182°. 

Monocrotaline was extracted from the seeds of Crotalaria spectabilis in 3.2% yield and from those of Crotalaria retusa in 1.89% yield. 

The n.m.r. spectral assignments have been made for a number of pyrrolizidine alkaloids, including retronecine (51), platynecine, heliotrine, supinine, lasiocarpine, crispatine, monocrotaline (54), madurensine, and retror-sine. Some of the assignments reported for retrorsine have been revised. A H n.m.r. spectroscopic method for determination of the amount of pyrrolizidine alkaloids present in the extracts of small samples of a number of Senecio species... 

Certified pure reference standards are essential for accurate quantitation, particularly in LC-MS and LC-MS/MS. Standards of monocrotaline and retrorsine have long been commercially available at relatively low cost. The poor availability of most other PAs has handicapped identification of sample extracts and made quantification less accurate than desired. Additional PA standards are now becoming... 

Antibodies raised to a retrorsine hemisuccinate-BSA conjugate have been used to determine total alkaloids including PANOs in reduced Senecio extracts [54]. Antibodies to a conjugate between the retronecine part of PAs and bovine serum albumin (BSA) detected retronecine and monocrotaline [55]. Antibodies that are specific to monocrotaline but which did not cross-react with retrorsine, retrorsine-N-oxide, riddelliine or retronecine have been reported [56]. Antibodies prepared to date react only to PAs that have related 12-membered macrocyclic... 

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