AU2009280165B2

AU2009280165B2 - Fractionation and modification of natural essential oils containing beta-triketones

Info

Publication number: AU2009280165B2
Application number: AU2009280165A
Authority: AU
Inventors: Robert Arthur Franich
Original assignee: CHEMIPRENEUR Ltd
Current assignee: CHEMIPRENEUR Ltd
Priority date: 2008-08-08
Filing date: 2009-08-07
Publication date: 2014-09-25
Anticipated expiration: 2029-08-07
Also published as: NZ590535A; WO2010016777A1; AU2009280165A1

Abstract

The invention describes a process in which β-triketones are extracted from essential oils. The essential oil is fractionated into its component parts such that any one of the component parts may be further used. The component parts may be modified with chitosan, or its tertiary amino or quaternary amino derivatives to form salts. These salts may find a number of uses. One such use may, for example, be in the delivery of antimicrobial activity.

Description

5 Patents Form No. 5 PATENTS ACT 1953 .0 COMPLETE SPECIFICATION FRACTIONATION AND MODIFICATION OF NATURAL ESSENTIAL OILS CONTAINING @ .5 TRIKETONES 1, Robert Arthur Franich, a New Zealand citizen, of 11 Awatea Terrace ROTORUA, New Zealand, hereby declare this invention, for which I pray that a patent may be granted to me, and the method by which it is to be performed, to be particularly described in and by the 20 following statement: 1 FIELD OF THE INVENTION The invention relates to a process of fractionating an essential oil containing P-triketone compounds .5 into its component parts, such that each part may be used or modified for one or more specific applications. In particular, the invention provides a method of separating P-triketones from non enolic compounds in essential oils by formation of a hydrolysable intermediate solid salt, or chelate, of the p-triketones, in an anhydrous state, and admixed with all the non-enolic compounds present in the essential oil. Using apparatus for solid-liquid separation, a facile and efficient separation 0 process is enabled using a solvent in which the solid intermediate salt, or chelate, is insoluble and in which the non-enolic compounds are miscible. The extracted salt, or chelate, of the 3-triketones may be then hydrolysed and the p-triketones recovered by distillation. The non-enolic compounds may be fractionated using column chromatography. 15 BACKGROUND TO THE INVENTION Natural volatile oils isolated from plant materials, often referred to as essential oils, may have value as medicinal preparations in their own right, or as components of such preparations. Essential oils may also have value as fragrance ingredients or as flavours in foods and beverages. An example of such an oil with well-known medicinal value is Manuka Oil, derived from the leaves and twigs of New [0 Zealand Manuka, Leptospermum scoparium. The antibacterial properties of Manuka Oil having a high content (>30% by weight) of p-triketone compounds (leptospermone, isoleptospermone, flavesone, grandiflorone in Manuka Oil) are well-documented and the value of the oil is understood and appreciated by users. Other essential oils, such as Manuka Oils having low 3-triketones content or oil derived from New 45 Zealand Kanuka (Kunzea ericoides) are not recognised as having exceptional value for their antimicrobial properties, but can be used as fragrance components for soaps and personal care products. Many of the species of the Leptospermum genus have 3-triketones in their essential oils to varying degrees. Nonetheless, essential oils with low to modest 3-triketones content could have value for supply of p-triketone compounds by separation of these from other, non-enolic, 50 compounds in the oil. The essential oils non-enolic fraction, after 3-triketones separation, could be 2 used for other applications, such as for fragrance materials, and many other applications for which essential oils are recognized as being useful. p-Triketone compounds, such as those listed above, are able to undergo tautomerism, to exist both as the ketone and enol chemical structures. The enol functional group in organic chemistry is 15 ionisable to give an enolate anion in the presence of an alkali, such as sodium hydrogen carbonate or sodium hydroxide. In science laboratory studies on Manuka Oil, 3-triketones preparations have been achieved by their extraction from the whole essential oil by using sodium hydrogen carbonate or sodium hydroxide aqueous solution to form the water-soluble p-triketones enolate sodium salts, then, by using a liquid-liquid partition process, separate the aqueous solution of the p-triketones iO salts from the oily non-enolic compounds. Acidifying the aqueous phase re-generates the original triketones, which can be distilled to recover, or which can be extracted into a suitable solvent, such as methylene dichloride. Drying this extract and removal of the solvent affords the 3-triketone fraction. Such a separation method is suitable for laboratory work, but can be problematic for industrial-scale manufacture of p-triketones from essential oils owing to the formation of emulsions i5 through use of the strong alkali solution, which can result in reduction of the p-triketone compounds yield owing to their being emulsified with the oily, non-enolic fraction layer in the liquid-liquid separation process. An industrial-scalable process is required to effectively fractionate 3-triketones from essential oils containing these in various proportions, (such as from commercial Manuka Oils from various sources 'O and referred to in the industry as high-p-triketone and low-p-triketone oils) in order to provide a p triketones product and non-enolic products, such as those compounds without a p-triketone functional group, and to use or modify the fractions for practical applications. OBJECT OF THE INVENTION 75 It is therefore an object of the invention to provide a process for the fractionation and/ or modification of natural essential oils containing p-triketones and non-enolic compounds on a useful scale, or to at least provide the public with a useful choice. 80 SUMMARY OF THE INVENTION 3 The invention provides a process of separating an essential oil which contains 3-triketones into a fraction containing p-triketones of high functional group purity, and a fraction containing all other non-enolic compounds. In particular, the invention provides a process for fractionation of essential oils which contain P 15 triketone compounds together with other natural compounds which are predominantly non-enolic, in which the p-triketone compounds are converted by chemical reaction with a bivalent alkali earth metal oxide into an anhydrous mixture of solid salts, or chelate of the p-triketone compounds enol tautomer, together with non-enolic compounds present which do not undergo the chemical reaction; and Q0 the anhydrous solid salts, or chelate, of the p-triketone compounds are separated from the non enolic compounds by using a solid-liquid Soxhlet separation process and a suitable solvent in which the solid p-triketone salts, or chelate, are insoluble and with which the non-enolic compounds in the essential oil are miscible; and the extracted anhydrous solid salts, or chelate, of the p-triketone compounds are hydrolysed with )5 aqueous acid solution and the liberated p-triketone compounds are subsequently recovered by steam distillation to afford a p-triketone compounds product with high functional group purity; and the non-enolic essential oil constituents are further separated into hydrocarbons and polar compounds. Preferably, the 3-triketones are selected from but not limited to the group comprising flavesone, )0 isoleptospermone, leptospermone and grandiflorone. Preferably the starting essential oil is one having a useful proportion of P-triketone compounds, such as Manuka Oil. The non-enolic compounds are preferably selected from the group consisting of hydrocarbons and polar compounds. 05 The hydrocarbons are preferably selected from the group consisting of monoterpenes and sesquiterpenes. The polar compounds are preferably selected from the group including those with ester, ether, alcohol, aldehyde, ketone and phenol functional groups. 4 The -triketones are preferably chemically reacted, as their enol tautomer, with a bivalent metal .0 oxide, preferably selected from the alkali earth metal oxides, and more preferably from the MgO or CaO oxides, most preferably using CaO. An example of an essential oil is Manuka Oil, derived from the plant Leptospermum scoparium. However, the starting essential oil could be from other species such as, but not limited to, other Leptospermum species or Kanuka oils from for example, Kunzea ericoides. .5 The product of the reaction between CaO and the 3-triketones of an essential oil is a neutral, solid calcium bis-(p-triketone) salt, or chelate, and water. The invention also provides a process in which a second equivalent of CaO is used in a reaction between CaO and p-triketones in an essential oil to form Ca(OH) 2 so that an anhydrous reaction product mixture between CaO and the essential oil P triketones is obtained. .0 The invention also provides a process in which a Soxhlet extraction method is applied to the mixture comprising the solid Calcium salt, or chelate, product (obtained from the chemical reaction of triketones in an essential oil with CaO), and the non-enolic compounds which are dispersed with, adhere to or are absorbed by the solid. The solvent used in the Soxhlet process is preferably a non-polar, and non-chemically reactive .5 solvent, and most preferably the solvent is a hydrocarbon solvent, preferably pure hexane or a mixture of hexane isomers such as those manufactured commercially for use as an industrial solvent. Other non- chemically-reactive solvents known for carrying out Soxhlet extraction processes may be used where advantage for using these is apparent. For an efficient separation of non-enolic compounds from the solid Calcium P-triketones salt, or 30 chelate, and Ca(OH) 2 , one or more Soxhlet extraction process steps may be carried out, using fresh solvent for second or subsequent extraction process steps to achieve a maximum extraction of non enolic compounds from the solid Calcium P-triketones salt, or chelate, and Ca(OH) 2 in the product mixture. The invention also provides a process of hydrolysing the extracted Calcium P-triketones salts, or 35 chelate, product, mixed with Ca(OH) 2 , with aqueous acid, such as aqueous sulphuric acid, in stoichiometric amount to chemically react with the Calcium P-triketones salts, or chelate, and to react with Ca(OH) 2 , and to form Calcium sulphate and release the P-triketones. 5 The invention also provides a process in which the P-triketones produced from the aqueous sulphuric acid hydrolysis reaction of their Calcium salts, or chelates, are recovered using steam [0 distillation, using an appropriate method for their separation, such as use of a long water column though which the p-triketones oily product falls. The invention also provides a process in which the mixture of non-enolic compounds extracted from the solid Calcium p-triketones salts, or chelate, are separated according to the polarity of the compounds using activated alumina column chromatography. [5 In the case of Manuka Oil, the non-enolic compounds separated using alumina column chromatography are a mixture of hydrocarbons (mainly comprised of monoterpene and sesquiterpene hydrocarbons) which may be eluted from the alumina column using a non-polar solvent such as a hydrocarbon solvent, and preferably using hexane or a mixture of hexane isomers as the eluting solvent and a fraction containing all the polar compounds (comprised of a complex io mixture of alcohols, aldehydes, ketones, esters, ethers and phenols) which may be eluted with a polar solvent, and preferably using ethanol as the eluting solvent. From the fractions obtained from the alumina column chromatography, the hydrocarbon fraction may be further fractionated to give useful sub-fractions or pure compounds using methods known in chemistry, such as fractional distillation and argentation chromatography using silver nitrate 15 absorbed onto silicagel. Similarly, the complex mixture of polar compounds obtained from the ethanol eluant may be modified by hydrolysis (using either acid or alkaline conditions) to remove esters (such as 3-methylbutyl 3-methylbutanoate) to give a mixture comprising mainly sesquiterpene alcohols, aldehydes, ketones, ethers and phenols. This hydrolysis product may be yet further modified by using oxidation chemistry (e.g., chromic acid oxidation using so-called Jones' reagent) to 60 give a mixture of sesquiterpene derivatives comprising aldehydes, ketones, phenols and tertiary alcohols only. The invention thus provides a process which enables the large-scale preparation of 3-triketones fractions of high functional group purity, containing compounds such as flavesone, leptospermone, isoleptospermone and grandiflorone, (and other compounds which may have a p-triketone structure 65 as part of the molecule), from essential oils containing these, such as Manuka Oil. The essential oil fractionation process may be applied to other essential oils which contain 3 triketones, examples of those oils being obtained from species of Leptospermum or Kunzea or other species, in addition to the genus Leptospermum and Kunzea, and whose essential oils contain 3 triketones. 6 '0 The invention also provides essential oil non-enolic mixtures which have no 3-triketones, and which may be further fractionated into useful mixtures or pure compounds using separation methods such as fractional distillation and/or chromatography. In the case of commercial Manuka Oil, the non-enolic fraction from which the 3-triketones has been separated consists mainly of sesquiterpene hydrocarbons and a complex polar mixture of esters, '5 ethers, and sesquiterpene derivatives, such as alcohols, aldehydes, ketones and phenols. The invention provides an industrial-scale separation of 3-triketones from essential oils containing these, and it also provides an essential oil non-enolic fraction from the process which are deficient in p-triketones and which may be further fractionated or modified into useful mixtures or pure compounds. In the case of Manuka Oil, these are sesquiterpene hydrocarbons and polar 10 compounds, including sesquiterpene derivatives such as those in Manuka Oil. The separated fractions derived from essential oils containing 3-triketones, such as Manuka Oil, may be used as such or modified to give products with utility value. The invention also provides preparations of P-triketones, when separated from essential oils containing these according to the methods in this specification. The invention also provides mixtures 15 of hydrocarbons and polar compounds isolated and/or modified according to the processes of this invention. BRIEF DESCRIPTION OF THE DRAWINGS The invention will now be described, by way of example only and with reference to the drawings, in 90 which: Figure 1 shows leptospermone keto-enol tautomer equilibria. Figure 2 is a chemical reaction of two leptospermone tautomer with CaO to form the salt, or chelate, product and water. The enolate tautomer structure allows the negative charge to be shared between the coordinated oxygen atoms by way of electron shifts, thereby imparting a bidentate 95 ligand property to the 3-triketone enolate anion, and a chelate-like molecular structure of the product. Figure 3a is a total ion GCMS chromatogram of a commercial Manuka Oil sample 7 Figure 3b is a partial chromatogram (32-45 min retention time) of a commercial Manuka Oil. Peaks 1,2 and 3 are flavesone, isoleptospermone and leptospermone respectively. Peak 4 is calamenene, a )0 sesquiterpene hydrocarbon. Figure 4a is a total ion GCMS chromatogram of the supernatant liquid at the conclusion of the chemical reaction of CaO and manuka essential oil, which shows an absence of peaks of the three p triketones shown in Figure 3a,b. Figure 4b is a partial chromatogram (32-45 min retention time) of the supernatant liquid at the )5 conclusion of the chelate formation chemical reaction, highlighting the absence of peaks of the three p-triketones (cf Figure 3b). Peak 4 is calamenene. Figure 5a is a total ion GCMS chromatogram of 3-triketones mixture preparation, 99.7% pure with respect to p-triketone functional group, from hydrolysis of their calcium salt or chelate derivative and steam distillation recovery of the product. .0 Figure 5b is a partial chromatogram (32-45 min retention time) of the 3-triketones mixture obtained from hydrolysis of the calcium salts or chelate of the p-triketones and steam distillation purification of the product. Peaks 1,2 and 3 are of flavesone, isoleptospermone and leptospermone respectively. Figure 6a is a total ion GCMS chromatogram of the hydrocarbon fraction of Manuka Oil non-enolic .5 extract obtained from the alumina column chromatography using hexane or a mixture of hexane isomers as the eluant . Figure 6b is a partial chromatogram (32-45 min retention time) of the hydrocarbon fraction of Manuka Oil residual extract obtained from the alumina column chromatography. Peak 4 is calamenene. 20 Figure 7 is a total ion GCMS chromatogram of the fraction comprising the polar compounds of Manuka Oil non-enolic extract obtained from the alumina column chromatography by using ethanol as the eluant. Figure 8 is a total ion chromatogram of the product mixture obtained after the fraction comprising polar compounds obtained from the alumina column chromatography by using ethanol as the eluant 25 has been subjected to alkaline hydrolysis in order to remove the small quantities C4, C5 and C6 esters, such as 3-methylbutyl 3-methylbutanoate. 8 Figure 9 is a total ion chromatogram of the product mixture obtained after the product obtained from alkaline hydrolysis of the polar fraction from the alumina chromatography has been subjected to Jones' oxidation in order to convert primary alcohols to aldehydes and secondary alcohols to !0 ketones. The Gas Chromatography Mass Spectrometry data shown in Figures 3-9 display the retention time for the compounds eluting from the gas chromatography column, and the ion abundance for each of the compounds. Using a commercial Manuka Oil as the example, no individual compound mass spectrometry data are included, but for each GCMS file, the mass spectrum for each separated 5 compound may be obtained, and, with published Retention Index data for compounds, be used for individual GCMS peak identification. DETAILED DESCRIPTION OF THE INVENTION The invention provides a process in which extraction methods using a Soxhlet apparatus and hexane [0 solvent are applied to the anhydrous solid Calcium salts, or chelate, product obtained from reaction of CaO and p-triketones in an essential oil to extract all the un-reacted, non-enolic compounds, such as hydrocarbons and polar compounds, which are dispersed with or adhere to or are adsorbed by the p-triketones Calcium salts, or chelate, solid and Ca(OH) 2 . The solvent used for the Soxhlet method is preferably pure hexane or a mixture of hexane isomers. [5 For an efficient separation of non-enolic compounds from the solid Calcium 3-triketones salts, or chelate, one or more Soxhlet extraction process steps are preferably carried out, using fresh hexane for the second or subsequent extraction step, to achieve a maximum extraction of non-enolic compounds from the solid Calcium p-triketones salts, or chelate, product and Ca(OH) 2 . The invention also provides a process of hydrolysing the extracted Calcium 3-triketones salts, or 50 chelate, product with aqueous acid, such as aqueous sulphuric acid, in stoichiometric amount to chemically react with the Calcium p-triketones salts, or chelate, and to react with Ca(OH) 2 to form Calcium sulphate and release the p-triketones. The invention also provides a process in which the 3-triketones produced from the aqueous sulphuric acid hydrolysis of the Calcium p-triketones salts, or chelates, are recovered using steam 55 distillation. 9 The invention also provides a process in which the mixture of non-enolic compounds extracted from the solid Calcium p-triketones salts, or chelate, produced using the Soxhlet method are separated using activated alumina column chromatography. In the case of Manuka Oil, the non-enolic compounds separated using alumina column chromatography are a mixture of hydrocarbons i0 (mainly comprised of sesquiterpene hydrocarbons), eluted with hexane or a mixture of hexane isomers and a polar compounds fraction (comprised of a complex mixture of alcohols, aldehydes, ketones, esters, phenols), eluted with ethanol. While two fractions (hydrocarbon and mixture of polar compounds) are described in this invention, those skilled in chromatography may use the well known process of gradient solvent elution to separate the mixture of polar compounds into multiple i5 sub-fractions. In the first aspect, the p-triketone compounds, such as leptospermone, (Figure 1), which are found as components in essential oils of Manuka, Kanuka, and many essential oils derived from the genus Leptospermum, are chemically reacted with a bivalent metal oxide to form neutral salts, or chelate, which, as a solid p-triketone derivative, can be separated from the non-enolic compounds of the '0 essential oil using the Soxhlet extraction method. In the case of Manuka Oil, the non-enolic compounds are mainly sesquiterpene hydrocarbons and polar sesquiterpene compounds with lesser amounts of alkyl esters, among other compounds. Such a bi-valent metal oxide can be selected from any oxide of the periodic table of the elements, but is preferably a metal oxide derived from Group 2 (Be, Mg, Ca, Sr, Ba). More preferably, the metal oxide selected is from Mg, Ca or Ba, and most '5 preferably, the metal oxides are MgO and CaO, with CaO the most preferred metal oxide. Calcium oxide in the form of burnt lime, or quicklime, is a cheap industrial product, readily available, and of a consistent quality according to the source of limestone or CaCO 3 used for its manufacture. Preferably the CaO is ground to pass a 40 mesh sieve in order to facilitate the diffusion and reactivity of the CaO particles in the p-triketones-containing essential oil. The chemical reaction of 80 leptospermone with CaO is shown in Figure 2. Similar chemistry occurs for isoleptospermone, flavesone, and grandiflorone and is likely to occur with all compounds containing the p-triketone molecular structure which have an enolic tautomer, with CaO. Hereafter, the most-preferred option of using CaO is described. Use of CaO is the most preferred oxide for facile and quantitative chemical reaction with p-triketones, such as those in Manuka Oil, to form salts, or chelate, as shown, which 85 are insoluble in non-polar solvents, such as hexane or hexane isomers, and also insoluble in the essential oil residual mixture of compounds, which in the case of Manuka Oil, comprise sesquiterpene hydrocarbon and a mixture of polar compounds such as alcohols, esters, aldehydes, ketones and phenols. 10 The by-product of the chemical reaction, water, can be further chemically reacted with a second )0 mole of CaO to form Ca(OH) 2 , so that the whole reaction mixture of essential oil and CaO (as burnt lime or quicklime) is anhydrous, an outcome achieved according to the measured weight of oil taken, the proportion of p-triketones in the oil (determined by analysis, such as gas chromatography or gas chromatography-mass spectrometry), the quality of the CaO, burnt lime or quicklime used and the yield of water produced. By preferably using CaO for the chemical reaction (Figure 2), a )5 quantitative yield of p-triketones salts, or chelate, is readily obtained, compared with the slower and lower yield of chelate formation reaction when using MgO, explained by the much slower reaction of MgO with 3-triketones and with the water produced in the reaction, Figure 2. This is an important aspect of the invention which facilitates the separation of the solid p-triketones Calcium salts, or chelate, from the liquid, non-enolic, mixture of essential oil molecules. Depending on the 3 )0 triketones content of the essential oil used, a non-polar solvent such as pure hexane or a mixture of hexane isomers may be used in order to keep the reaction mixture of CaO and essential oil fluid. For a high-p-triketones content commercial oil Manuka Oil (e.g., 36% 3-triketones content), hexane up to 50% of the weight of oil used may be added to maintain a well-stirred mixture which might otherwise become near-solid. Heat is evolved during the chemical reaction, so cooling the reaction )5 vessel is required to keep the temperature below the boiling point of the non-polar solvent, such as hexane, used. In a second aspect of the invention, the method for separation of the solid p-triketones Calcium salts, or chelate, from the non-enolic compounds of the essential oil mixture (in the case of Manuka Oil) uses a Soxhlet extraction process and a non-polar solvent such as pure hexane or a mixture of .0 hexane isomers. Prior to adding the solid 3-triketones Calcium salts, or chelate, into an extraction thimble or bag, any liquid on the surface of the mixture can be decanted and kept. In order to maximise the extraction yield of the non-enolic compounds from the Soxhlet process, more than one extraction step may be required. In a first solvent extraction step, the solid is extracted using Soxhlet solvent cycling for a number of hours (typically 5) required to produce a consistent vapour phase 15 composition, which comprises the extraction solvent (e.g., pure hexane or mixture of hexane isomers), and, in the case of Manuka Oil, residual monoterpene and sesquiterpene hydrocarbons, co-distilling with the solvent, and which comprise less than 5% of the total volatile solvent mixture cycling in the Soxhlet process. After the first Soxhlet extraction step, the solid 3-triketones Calcium salts, or chelate, may contain a constant residual non-enolic fraction content of ca. 4-5%. The 20 boiling flask is then emptied and a fresh charge of pure solvent is added, and the Soxhlet extraction process continued for a further period of time, typically, 5 hours. At the end of this period, the solid p-triketones Calcium salts, or chelate, contains less than 1% non-enolic fraction. The solvent 11 extracts and any decanted liquid, described above, are combined and concentrated to constant weight. This fraction contains the mixture of non-enolic compounds which did not chemically react .5 with CaO, and in the case of Manuka Oil, comprise mainly sesquiterpene hydrocarbons and polar sesquiterpenoid products, and small amounts of alkyl esters, aldehydes, monoterpene hydrocarbons and monoterpene alcohols and ethers (e.g., cineole). Alternatively, a modified Soxhlet may be used wherein the boiling solvent is first passed through a packed column (e.g., containing rings or saddles), such that the monoterpenes and sesquiterpenes 10 vapourised with the solvent are condensed and returned to the boiling flask, with the result that pure solvent vapour is delivered to the top of the column and condenser for delivery to the Soxhlet extraction thimbles or bags. This modification of the simple Soxhlet device enables a continuous solvent cycling extraction process with the same outcome as described above but without requiring a second, or subsequent, solvent charge. 15 In a third aspect of the invention, the extracted p-triketones Calcium salts, or chelate, product together with Ca(OH) 2 is isolated from the Soxhlet extraction thimble or bag as a dry, friable powder. This product is placed in a large container and water is added to make a uniform, smooth slurry, followed by aqueous sulphuric acid (50% v/v) added in a stoichiometric amount according to the weight of CaO used, to quantitatively form CaSO 4 , and to hydrolyse the p-triketones Calcium salts, or [0 chelate, and to allow the p-triketones to separate as a pale-yellow to orange oil. Heat is evolved during the acidification step. The pH of the mixture is adjusted if necessary to 3-5, preferably 4, and the mixture is then steam-distilled to recover the pure p-triketones product as a near-colourless to light-gold coloured oil, with relative density of 1.082 at 20'C in the case of the Manuka Oil triketones, flavesone, isoleptospermone and leptospermone. The 3-triketones product is obtained in 45 90-95% yield of theoretical, and is better than 99% (typically 99.7%) pure according to p-triketones content by GC or GCMS analysis. The ratio of each of the 3-triketones in the derived product is identical with that measured in the original essential oil. The by-product from the hydrolysis reaction, CaSO 4 , is dried to a solid suitable for disposal as the naturally-occurring mineral, gypsum. In a fourth aspect of the invention, the combined non-enolic compounds from the Soxhlet extraction 50 step of the Calcium salts, or chelates, of the p-triketones may be separated using fractional distillation and/or preparative column chromatography. In the case of the non-enolic compounds extracted from the Soxhlet separation of the Calcium salts, or chelate, of the p-triketones in Manuka Oil, viz., monoterpene and sesquiterpene hydrocarbons and polar compounds, these may be added to an alumina chromatography column in a ratio of 1 part of extract concentrate to 10 parts, more 55 preferably 3-8 parts and most preferably 5 parts of alumina (activated at an elevated temperature, 12 e.g., 400*C for a period of time, e.g., 5 hours, in order to oxidise any organic material adsorbed to the alumina and to minimise or remove adsorbed water). Column chromatography provides a better separation of these compounds than vacuum fractional distillation, for example, since groups of the sesquiterpene hydrocarbons and some of the polar compounds in the extract have overlapping i0 boiling point ranges. Alumina is a better-performing adsorbent than is silica gel for the chromatographic separation of the compounds according to functional groups. The column is eluted with pure hexane or hexane isomer mixture (typically three column volumes), and then the retained solvent in the column is removed by applying vacuum to the exit of the column. The combined solvent eluants from the alumina column are concentrated at atmospheric pressure and steam i5 temperature and using a fractionation column, such as a Vigreux column, to give a clear, mobile liquid comprised of monoterpene and sesquiterpene hydrocarbons, as shown by GCMS analysis. The sesquiterpene hydrocarbons are obtained in an approximate 98% yield of theoretical calculated on the calamenene content of the original Manuka essential oil. The hexane or hexane isomers mixture solvent is recovered in the distillate for re-use. '0 The alumina column is then eluted with ethanol (95%, typically three volumes), followed by application of vacuum, as described above, to remove the ethanol retained in the column. The ethanol eluants from the alumina column are combined and concentrated at atmospheric pressure and steam temperature and using a fractionation column, such as a Vigreux column, to give a light red-brown viscous oil comprised of a complex mixture of the polar fraction of sesquiterpenoids and '5 some minor alkyl esters. The ethanol solvent is recovered in the distillate for re-use. Finally, the alumina column is eluted with pure water (typically three column volumes), prior to the alumina column being emptied and the adsorbent dried and activated at an elevated temperature (e.g., 400*C) for re-use. To recover any useful compounds from the water eluants, these may be freeze-dried. Alternatively, to the water eluant is added NaCl to saturation, and then the mixture is 80 extracted with methylene dichloride. Drying and concentration of this extract gives, in the case of Manuka Oil, a small amount (<0.3% of the total oil) of a brown, viscous, resinous product which has a naphthalene-like odour. In a fifth aspect of the invention, the mixture of polar compounds obtained by elution of the alumina column with ethanol may be hydrolysed under alkaline conditions in order to remove the small 85 amount of methylbutyl esters which impart a 'butyric' olfactory note to the product. In addition, the product from the hydrolysis modification step may be subjected to oxidation chemistry in order to convert primary alcohols in the mixture to aldehydes and secondary alcohols to ketones, thereby 13 providing a product comprised of polar sesquiterpene derivatives stabilised towards further oxidation when in use. 0 This invention therefore provides a process whereby essential oils containing various 3-triketones, such as those known in Manuka essential oil, can be processed into separate fractions through using Calcium salts, or chelate, formation of the p-triketones in the oil to facilitate their separation from the non-enolic mixture of compounds. In the case of Manuka essential oil used to demonstrate the separation process, the non-enolic compounds are sesquiterpene hydrocarbons, esters and polar )5 sesquiterpenoids. Hydrolysis of the Soxhlet-extracted 3-triketones Calcium salts, or chelate, product by acidification with aqueous sulphuric acid, and then steam-distillation enables recovery of a p triketones product. Simple activated alumina column chromatographic separation of the mixture of non-enolic compounds, such as those from Manuka Oil, in the Soxhlet process extract gave a fraction of hydrocarbons (mainly sesquiterpenes) and a fraction of polar compounds. The sequence )0 of steps described has enabled preparation of essential oil components or fractions, with Manuka essential oil as an example, where the fractions can be used directly or further modified into products with utility value. Specific examples describing the invention. )5 1. Chemical reaction of p-triketones flavesone, isoleptospermone, leptospermone (and, if present, grandiflorone) in Manuka essential oil with Calcium oxide to form solid Calcium salts, or chelate. The chemical reaction between 3-triketones and Calcium oxide, CaO, is shown in Figure 2. One mole 10 of CaO combines with two moles of p-triketones, with a further mole of CaO used for chemical reaction with the water by-product, thereby providing an anhydrous reaction product mixture. The p-triketones mixture in a typical New Zealand East Cape Manuka essential oil, having a 3-triketones content of 36% m/m of oil, and a p-triketones distribution of 20.6% flavesone, 18.6% isoleptospermone, 58.9% leptospermone, 1.9% grandiflorone, has an average molecular weight of 15 264, for the purpose of calculating the stoichiometry of the chemical reaction. While the chemical reaction between CaO and p-triketones may proceed according to the stoichiometry, the reaction mixture is heterogeneous, and the yield is dependent on the accessibility of the p-triketones to the CaO particles surface and diffusion into the particles and this is dependent on CaO particle size and 14 particle porosity. Thus, for complete chemical reaction of P-triketones and CaO to form solid salts, .0 or chelate, a quantity of CaO over that calculated according to the reaction stoichiometry may be required. A known weight (e.g., 1 kg) of Manuka essential oil having a known content of 3-triketones (e.g., 36% m/m, as determined by GC or GCMS) is placed in a container fitted with an efficient paddle stirrer and a source of cooling, if required. The oil is stirred, and Calcium oxide, CaO (0.212 kg, 5 ground to pass a 40 mesh sieve), is then added in portions. After approximately 30 minutes, the mixture becomes warm. Cooling is required if the temperature of the mixture exceeds 50*C. Stirring is continued until the mixture cools, typically a further 2 hours. Stirring is stopped, and a small sample of the supernatant liquid is analysed for assessment of completion of the salts, or chelate, formation, as shown by absence of the p-triketones GC or GCMS peaks. An example GCMS analysis !0 of the supernatant liquid at the conclusion of chemical reaction to form the salts, or chelate, is shown in Figure 4a,b, which shows an absence of peaks due to the three 3-triketones which were initially present in the starting essential oil. In the event of incomplete chemical reaction and significant P-triketones GC peaks are evident, further CaO is added until complete chemical reaction is achieved and maximum yield of Calcium salts, or chelate, of p-triketones is obtained. The total 15 weight of CaO used is recorded. 2. Separation of the p-triketones Calcium salts, or chelate, from the non-enolic essential oil compounds by Soxhlet extraction. The reaction product from 1, above, is allowed to settle, and any liquid above the solid 3-triketones 40 calcium chelate is decanted, and kept for combining with the extract obtained, as described here. The solid product is transferred to a Soxhlet extraction thimble or extraction bag, and loaded into the extraction cylinder of the Soxhlet apparatus. The boiling flask is charged with a non-polar solvent, typically pure hexane (often referred to as 'hexanes' when derived from petroleum fractionation), and the apparatus assembled for Soxhlet extraction. After a period of time, e.g., 1-12 45 hours, and typically 5 hours of extraction process with efficient cycling of the solvent, the boiling flask is cooled, and the extract is removed. A sample of solid from the Soxhlet thimble or bag may be removed and tested for any residual non-enolic compounds present. In the case of incomplete extraction of non-enolic compounds from the solid product, a second charge of pure hexane is added, and extraction process continued for a further period of time, typically 5 hours. At the 50 conclusion of the hexane extraction sequence, any liquid recovered and kept at the beginning of this 15 process is added to the extract, and the hexane solvent is removed by distillation using an efficient fractionation column, such as a Vigreux column. To ensure best quality of the recovered extract, the boiling flask is heated at atmospheric pressure and at steam temperature so that the extract is not heated to a temperature above 100*C. Use of a fractionation column enables a maximum yield of 15 extract recovered, and best quality of hexane solvent recovered for re-use. The solvent-free extract is obtained, in the case of Manuka essential oil, as a pale yellow-brown liquid, typically 62-64% of the weight of the original Manuka oil. The GCMS analysis of the Soxhlet extracted product is shown in Figure 4a,b, confirming the absence of the 3-triketones in the extracted non-enolic oil compounds mixture. iO 3. Hydrolysis of 3-triketones Calcium salts, or chelate with aqueous sulphuric acid and recovery of the p-triketones fraction by steam distillation. The dry, solid product obtained from 2, above, is removed from the extraction thimble or bag and transferred to a container equipped with an efficient paddle stirrer. Water equal to the weight of i5 the extract is added, the solid broken up and stirred to form a uniform slurry. Aqueous sulphuric acid (50% v/v) is added while monitoring the temperature of the reaction, to ensure the temperature does not reach boiling point (e.g., 100*C), as this could cause some degree of steam distillation of p-triketones as these are liberated from the Calcium salts, or chelate, and thereby possibly lower the yield of p-triketones expected. Cooling is used as required to preferably keep the 'O temperature less than 100*C. The weight of sulphuric acid added is that required to completely react with the weight recorded of the CaO used in step 1, above, to quantitatively release all the p triketones from the Calcium salts, or chelate, and to form insoluble, solid calcium sulphate as the hydrolysis reaction by-product. The pH of the reaction mixture is checked to ensure that the pH is less than 7, and preferably close to 4. The 3-triketones appear as a yellow to orange oil partly 75 floating on the aqueous mixture, and partly dispersed throughout the aqueous mixture. The total product from the hydrolysis step, above, is transferred to a boiling flask set up for steam distillation, using an efficient condenser and a long column receiver. Since the p-triketones product from Manuka essential oil has a relative density of 1.082 (20*C), the steam-distilled 3-triketones sink in the water column, enabling the product to be obtained by drawing off from the bottom of the 80 receiver. The supernatant water phase in the receiver may be in the form of a hydrosol, in which a small quantity of p-triketones products may be dispersed. To recover 3-triketones from the hydrosol, the water phase is allowed to percolate through a small bed of solid-phase adsorbent in a 16 column, such as a polystyrene bead resin, to effect a solid-phase extraction process. Pure water is recovered from the eluant of this column, and may be re-used for steam distillation. The bead resin, 15 when saturated, is extracted with ethanol (95%) to recover the adsorbed p-triketones. In this process, the yield of p-triketones from the steam distillation process is maximised. The 3-triketones product is obtained as a pale yellow to light gold oil, and can be dried using phase separation filtration or by use of a drying agent. The 3-triketones product is obtained in 90-95% of the theoretical yield. An example of the GCMS analysis of the 3-triketones product obtained is shown in )0 Figure 5a,b. Peak area percent calculation shows the 3-triketones product to be 99.7% pure with respect to, in the case of Manuka Oil, the flavesone, isoleptospermone and leptospermone content, with a trace of calamenene and other minor sesquiterpene hydrocarbons detectable. The residue in the steam distillation boiling flask may contain a very small quantity of non-steam distilled p-triketones, such as grandiflorone (if present in the original Manuka essential oil). This may )5 be recovered using a standard laboratory solvent extraction procedure, if required, or may be discarded. The solid residue, Calcium sulphate, in the boiling flask is readily filtered, and can be simply dried to gypsum mineral for disposal. 4. Alumina column chromatography of the non-enolic mixture extract obtained from the )0 Soxhlet extraction, step 2, above. A column (e.g., 60mm diameter, 35 cm length) is dry-packed with activated alumina (e.g., 60 mesh, 750g, previously heated at an elevated temperature, e.g., 400*C for a period to time, e.g., 5 hours and cooled in absence of moisture). The extract obtained from the Soxhlet extraction, Example 2, above, is uniformly added to the top of the column, in a ratio of 5:1 of adsorbent : extract, and 05 allowed to adsorb, at atmospheric pressure and ambient temperature (e.g., 20'C), into the alumina. Pure hexane, or hexanes isomer mixture, is used to elute the hydrocarbon fraction, typically three column volumes being used to exhaustively remove all the hydrocarbons. The residual hexane solvent adsorbed in the column is drawn out by applying vacuum to the column exit, and the hexane eluants combined and the solvent removed by distillation at atmospheric pressure and steam 10 temperature and using a fractionation column, such as a Vigreux column, to ensure maximum yield of the hydrocarbon fraction and to recover best quality hexane for re-use. The monoterpene and sesquiterpene hydrocarbon fraction yield is better than 90% of the theoretical yield, estimated from the calamenene peak in the GCMS analysis of the original Manuka essential oil. An example of the GCMS analysis of the hydrocarbon fraction obtained is shown in Figure 6a,b. 17 .5 The alumina column is then eluted using ethanol (95%), typically three column volumes being used to exhaustively remove all the polar adsorbed compounds from the column. The residual ethanol adsorbed in the column is drawn out by applying a vacuum to the column exit. The ethanol eluants are combined, and the solvent removed by distillation at atmospheric pressure and steam temperature and using a fractionation column, such as Vigreux column, to ensure maximum yield of .0 the mixture of polar compounds, and to recover the best quality ethanol for re-use. The polar compounds fraction is obtained in a yield of 9% of the original Manuka Oil used. An example of the GCMS analysis of the polar compound mixture fraction is shown in Figure 7. Finally, the alumina column is eluted with pure water, typically three column volumes being used to exhaustively remove the residual ethanol and any highly-polar compounds adsorbed on the alumina. .5 To remove water residual in the column, vacuum is applied at the column exit. In the case of chromatography of the non-enolic compounds from Manuka essential oil, the water eluants can be extracted to give a resinous product having a naphthalene-like odour. The water-wet alumina adsorbent can be re-activated for re-use by heating at an elevated temperature, e.g., 400*C for a period of time, e.g., typically 5 hours. io 5. Hydrolysis of the polar compound mixture obtained from the alumina chromatography The polar compound mixture (25g), obtained from the ethanol eluant from the alumina chromatography, is dissolved in ethanol (95%, 100mL) and to the solution is added a solution of sodium hydroxide (1g) in water (5mL), sufficient to hydrolyse the esters in the mixture. The solution 15 is heated and stirred under reflux for a period to time sufficient to effect the hydrolysis reaction, typically 3h. At the end of this period, the mixture is cooled and added to water (400mL), and the pH brought to 6-7 by the addition of an acid, e.g., hydrochloric acid. This mixture is then extracted into pure hexane (3 x 30mL), and the combined hexane extracts are washed with water (3 x 100mL) to remove low molecular weight acids and alcohols from the extract. The hexane layer is then dried 40 (e.g., over exsiccated magnesium sulphate), filtered and concentrated in vacuo to give an oily product (24.2g, 96%), the GC and GCMS analysis of which showed the absence of the C4, C5 and C6 esters (Figure 8) originally present in the Manuka Oil used. 6. Chromic acid oxidation (Jones' oxidation) of the hydrolysis product from 5., above. 18 5 The hydrolysis product (20g) is dissolved in pure acetone (100mL), and the solution is cooled to 0-5*C. To the solution is added Jones' reagent, in portions, keeping the reaction mixture temperature below 5*C. Typically for this oxidation reaction, 2.5 - 3 mL of reagent is added, until the formation of the green chromium Ill salts precipitate is completed and a distinct red tinge of colour remains in the acetone layer. At the conclusion of the reaction, water is added io (200mL) and solid sodium metabisulphite (3g) added in order to reduce any residual chromium VI to chromium Ill. The oxidation reaction product is extracted into hexane (3 x 30mL), and the combined hexane extract is washed with water (3 x 100mL) to remove all inorganic salts. The hexane layer is then dried (e.g., over exsiccated magnesium sulphate), filtered and concentrated in vacuo to give an oily product (16,8g, 84%) containing aldehydes, ketones, tertiary alcohols and 15 phenols, the GCMS analysis of which is shown in Figure 9. It will be appreciated by those in the art that whilst the invention has been described with reference to the above specific embodiments, various modification and improvements may be made without departing from the scope of the invention as set out in this specification: iO Industrial Application The invention provides a process of separation of 3-triketones from non-enolic compounds of essential oils in which these compounds occur. Hydrocarbons and polar compounds fractions are also produced according to the process and these compounds may be separately fractionated and modified as required. The separated P-triketones may be further modified to 3)5 produce other useful compounds. 19

Claims

1. A process for fractionation of essential oils which contain 3-triketone compounds 'O together with other natural compounds which are predominantly non-enolic, in which the p-triketone compounds are converted by chemical reaction with a bivalent alkali earth metal oxide into an anhydrous mixture of solid salts, or chelate of the p-triketone compounds enol tautomer, together with non-enolic compounds present which do not undergo the chemical reaction; and '5 the anhydrous solid salts, or chelate, of the p-triketone compounds are separated from the non-enolic compounds by using a solid-liquid Soxhlet separation process and a suitable solvent in which the solid p-triketone salts, or chelate, are insoluble and with which the non-enolic compounds in the essential oil are miscible; and the extracted anhydrous solid salts, or chelate, of the p-triketone compounds are i0 hydrolysed with aqueous acid solution and the liberated p-triketone compounds are subsequently recovered by steam distillation to afford a p-triketone compounds product with high functional group purity; and the non-enolic essential oil constituents are further separated into hydrocarbons and polar compounds. 15

2. A process according to claim 1 in which the 3-triketones are selected from, but not limited to, the group comprising flavesone, isoleptospermone, leptospermone and grandiflorone.

3. A process according to claim 1 or claim 2 in which the essential oil initial ingredient is 90 typical of one containing 3-triketones together with non-enolic natural compounds, an example of which is Manuka Oil.

4. A process according to any preceding claim in which the 3-triketones in an essential oil are chemically reacted with a bivalent alkali earth metal oxide to form an insoluble salt, or chelate, and water. 95 5. A process according to claim 4 in which the bivalent alkali earth metal oxides are selected from the alkali earth metal oxides. 20

6. A process according to claim 5 in which the alkali earth metal oxides are MgO or CaO oxides.

7. A process according to claim 6 in which the oxide is CaO. )0 A process according to any preceding claim in which a second equivalent of CaO is used to chemically react with the water by-product from the chemical reaction between CaO and p-triketones of an essential oil to form Ca(OH) 2 and thereby an anhydrous reaction product between CaO and the essential oil p-triketones is obtained. )5 A process according to claim 8 in which a Soxhlet solvent extraction method is applied to the solid p-triketone Ca salts, or chelate, product in an essential oil and using a non-polar solvent to substantially extract all the un-reacted, non-enolic compounds which are dispersed with, adhered to or are absorbed by the solid mixture comprising Calcium salts, or chelate, of P-triketones, and Ca(OH) 2 . .0 10. A process according to claims 9, in which the non-enolic compounds are hydrocarbons and more polar compounds.

11. A process according to claim 9 in which the solvent used is a non-polar solvent.

12. A process according to claim 11 in which the solvent is a hydrocarbon solvent.

13. A process according to claim 12 in which the solvent is pure hexane or a mixture of .5 hexane isomers.

14. A process according to any one of claims 9-13 in which one or more Soxhlet extraction process steps are carried out, using fresh solvent for second or subsequent extraction process steps.

15. A process according to any preceding claim in which the extracted, anhydrous P 20 triketones salts, or chelate, product, mixed with Ca(OH) 2 , is hydrolysed with aqueous sulphuric acid, in stoichiometric amount to chemically react with the Ca P-triketones salts, or chelate, and to react with Ca(OH) 2 , and to form the Calcium salt, such as Calcium sulphate, and release the P-triketones compounds in their un-ionised form. 21

16. A process according to claim 15 in which the P-triketones produced from the .5 aqueous acid hydrolysis of the Calcium salts, or chelate, are recovered using steam distillation, using an appropriate method for their separation and recovery.

17. A process according to claim 16 in which the separation step comprises the use of a long water column through which the p-triketones oily product falls.

18. A process according to claim 14 in which the mixture of non-enolic compounds io extracted from the solid Ca p-triketones salts, or chelate, using the Soxhlet method, are separated according to the polarity of the compounds using activated alumina column chromatography.

19. A process according to claim 18, in which the initial starting essential oil is Manuka Oil, and wherein the non-enolic compounds separated using alumina column 5 chromatography are a mixture of hydrocarbons (mainly comprised of sesquiterpene hydrocarbons with lesser amounts of monoterpene hydrocarbons) which are eluted from the alumina column using a non-polar solvent, and a fraction containing all the polar compounds (comprised of a complex mixture of alcohols, aldehydes, ketones, esters, ethers and phenols), which are eluted with a polar solvent. [0 20. A process according to claim 19 in which the eluting solvent is a hydrocarbon solvent.

21. A process according to claim 20 in which the eluting solvent is hexane or a mixture of hexane isomers.

22. A process according to claim 19 in which the polar eluting solvent is ethanol. 45 23. A process according to claim 21 in which, from the fractions obtained from the alumina column chromatography, the pure hydrocarbon fraction is further fractionated to provide useful sub-fractions or pure compounds.

24. The process according to claim 23 in which the fractionation used is fractional distillation and/or argentation chromatography using silver nitrate absorbed onto 50 silicagel.

25. A process according to claim 22 in which the complex mixture of polar compounds obtained from the ethanol eluant from activated alumina chromatography is modified by hydrolysis to remove esters to give a mixture, in the example of Manuka 22 essential oil, comprising mainly sesquiterpene alcohols, aldehydes, ketones and 5 phenols.

26. A process according to claim 25 in which the hydrolysis step is carried out under either acid or alkaline conditions.

27. A process according to claim 25 or claim 26 in which the hydrolysis product is further modified using oxidation chemistry to give a mixture of sesquiterpenoid derivatives O0 comprising aldehydes, ketones, phenols and tertiary alcohols only.

28. A process according to claim 27 in which chromic acid (Jones') oxidation is used. 23