Field of the invention:

The invention relates to a method of separating and/or fractionating of waxes from plants.

Background to the Invention

Natural waxes are extensively used in the cosmetic industry for numerous applications including creams and lipsticks. However, as the world's supplies of natural waxes, such as Carnauba and Candelilla begin to run short of demand, it would be highly desirable to examine natural and sustainable alternatives. By-products and waste-products of the agricultural and forest industries represent a potential supply of waxes for many industries. For instance, wheat straw waxes have been demonstrated to exhibit ideal properties for the cosmetic industry. In addition, such waxes could potentially be used for fruit/cheese coating and a number of other applications in the food industry where petrochemically-derived waxes are forbidden.

In the scope of the present invention, the term "wax" includes complex mixtures of families such as, but not exclusively, sterols, alkanes, fatty acids, fatty alcohols, ketones, aldehydes and wax esters.

A number of high-value fractions can also be isolated from wheat straw waxes. Odd-numbered alkanes are well known for their semio-chemical properties. A semiochemical is a generic term used for a chemical substance or mixture that carries a message (pheromones, allomones, kairomones, attractants and repellants). Semiochemicals can be used as components of insect pest management strategies, n-tricosane (C23H48) and n-pentacosane (C25H52) for instance, have been demonstrated to lower parasitism rates of the pea aphid, Acyrthosiphon pisum, on broad bean plants. (Y. Nakashima, Birkett, M.A., Pye B.J., Pickett J.A., Powell W., The role of semiochemicals in the avoidance of the seven-spot ladybird, Coccinella septempunctata, by the aphid parasitoid, Aphidius ervi, Journal of Chemical Ecology, 30 (2004) 1103-1116)

Primary long chain aliphatic alcohols typically having carbon chain lengths ranging from C ₂₀ to C ₃₅ and are generically refered to as polycosanols. Typical aliphatic alcohols of the mixture are tetracosanol (C24), hexacosanol (C26), octacosanol (C28), triacontanol (C30), and dotriacontanol (C32). Common distribution and concentration ranges of the various components of polycosanol are disclosed in US Patent 5,856,316 and European Patent Application EPl 121 928 Al. Among these aliphatic alcohols, octacosanol (C28) is of particular interest since it has been reported to exhibit a number of beneficial effects such as an increase in physical stamina and as a remedy of damaged nerve cells. Likewise, antilipaemic effects have been also further attributed to hexacosanol (C26). Also, polycosanols are known to have attractive and useful biological activity. They have been shown to effectively reduce cholesterol levels in blood stream by blocking the synthesis of cholesterol in the liver. Polycosanols have also been shown to promote the degradation of LDL (low density lipoprotein) cholesterol, (see PCT patent WO 2004/041257; Menendez et al., Biol. Res., 27 (1999) 199; Brit. J. Nutrition, 77 (1997) 923; Gouni-Berthold I. et al., Am. Heart J., 143 (2002) 356-365; Torres O. et al., treatment of hypercholesterolemia in NIDDM with polycosanol, 18 (1995) 393-396). In addition, polycosanol can also be used for the treatment of hypercholesterolemia, and atherosclerotic complications such as platelet hyperaggregabiulity, ischemia and thrombosis, and to prevent drug induced gastric ulcer, and to improve male sexual activity as disclosed in US patent 5,856,316.

It has been known for a number of years that plant sterols, especially β-sitosterol, can lower the absorption of cholesterol from the intestines and therefore reduce blood cholesterol. (Pollak OJ., successful prevention of experimental hypercholesterolemia and cholesterol atherosclerosis in the rabbit, Circulation, 7 (1953) 696-701; Farquhar J. W., Smith R.E., Dempsey M.E., the effect of β-sitosterol in serum lipids of young men with arteriosclerotic heart disease, 14 (1956) 77-82; US patent 5,958,913). It has also been demonstrated that modified sterols could effectively lower blood cholesterol (Heinemann T., Leiss O., von Bergamann K., effect of low-dose sitostanol on serum cholesterol in patients with hypercholesterole mia, 61 (1986) 219-223); Ikeda I., Sugano M., comparison of absorption and metabolism of beta-sitosterol and beta-sitostanol in rats, 30 (1978) 227-237). Straub

disclosed in patent US 5,244,887 the use of hardened sterols (hereafter referred to as stanols) to reduce cholesterol absorption from food. Miettenen reported the use of β-sitostanol fatty acid ester for lowering high cholesterol level in serum, as disclosed in patent US 5,502,045 and US 5,958, 913.

A number of patents also disclosed a composition containing both phytosterols and polycosanols and derivatives thereof for reducing blood cholesterol. US patent 5,952,393 discloses a purportedly synergistic anti-cholesterol effect of a composition comprising phytosterols and polycosanols. European Patent Application EP 1 108 364 A2 discloses the admixture of long chain alcohols in sterol compounds including sterols, stanols, stanol esters and sterol esters. US patent 6,197,832 discloses the method of administering a composition of phytosterols and polycosanols. European Patent Application EP 1 121 928 Al discloses compositions containing phytosterol and polycosanol esters for reducing blood cholesterol and triglycerides.

Statements of Invention

The invention relates to a method of isolating wax from a plant. More particularly, the invention relates to a method of isolating long chain alkane, long chain aliphatic alcohol, and sterol fractions and selected one or more alkanes, alcohols, sterols contained therein from plants.

The present invention relates to a method of isolating wax from a biomass source, for example animals, insects, plants (such as flowers, cereals, vegetables). Such a biomass source (referred to herein as "source") is subjected to appropriate physical and/or chemical modification/extraction/separation techniques for isolation and purification of wax. The source is preferably a plant source.

Where the source of wax is a plant, the plant may be selected from the group of families consisting of chenopodiaceae, compositae, cruciferae, cucurbitaceae, gramineae, liliaceae, linaceae, umbebelliferae, urticaceae and others. The source may be a by-product of the

process of growing sugar beets, linseed, sunflower, rapeseed, flax, barley, rice, wheat, millet, sorghum, corn, maize, teff, oats, rye, spelt, energy crops and others. Alternatively, the source may be a by-product of the process of harvesting cereals. Preferably the source is straw. The term straw mentioned herein is referred to as any part left over following the harvesting of crops, i.e. leaves, stems and the like. Preferably the straw is wheat straw.

The present invention relates to a method of extracting polycosanols from plants. Such sources are subjected to appropriate physical and/or chemical modifϊcation/extraction/separation techniques for isolation and purification of polycosanols.

The present invention relates to a method of extracting long chain alkanes, for example Cl 5- C40 alkanes, from a source. Such sources are subjected to appropriate physical and/or chemical modification /extraction/separation techniques for isolation and purification of alkanes.

The present invention relates to a method of extracting sterols from plants. Such sources are subjected to appropriate physical and/or chemical modification/extraction/separation techniques for isolation and purification of the sterols.

More generally, the present invention relates to a method of fractionating wax into desirable fractions and further into individual compounds. Fractions may include polycosanols, octacosanol, hexacosanol, sterols, and alkanes, for example stanols, stanol esters, sterol esters, polycosanol esters, octacosanol esters, hexacosanol esters.

As used herein the term "wax" is intended to include, but is not limited to, wax fractions such as those described herein.

The invention relates to a mixture of higher primary aliphatic alcohols (referred to as polycosanols), containing alcohols ranging from 20 to 40 carbon atoms, especially those ranging from 22 to 38, for example 24 to 34. The invention relates to a mixture of alkanes

ranging from 15 to 45 carbon atoms, for example 21 to 45 carbon atoms. The invention relates a mixture of sterols, especially beta-sitosterol, campesterol, and stigmasterol. The invention also relates to any mixtures of polycosanols, alkanes, and sterols and derivatives thereof resulting from (bio)chemical modifications of the extracted wax and/or (bio)chemically modified wax. The alkane, polycosanol, sterol and modified version compositions of the present invention are unique in that they are derived from novel source materials.

The sources of waxes may initially be subjected to solid-liquid extraction. Examples of suitable solvents are hexane, toluene, ethanol, acetone, and others including solvent mixtures. The purity of the obtained wax is at least about 10%, more preferably at least 50%, most preferably at least 90%. Following the initial extraction, a second-stage purification procedure may be performed to purify the wax. The purification can be carried out using several different procedures, including solid-liquid extraction, subcritical/supercritical fluid extraction, normal phase chromatography, reverse phase chromatography, gel permeation chromatography, multiple crystallisation, molecular distillation, complexation and combinations of such.

In a preferred aspect of the invention there is provided a method of fractionating wax wherein the fractionation is performed using a subcritical/supercritical fluid. Thus the method of the invention comprises the step of subjecting a source to a fluid at subcritical/supercritical pressure and temperature extraction conditions. The expression "supercritical fluid" is intended to define a substance above its critical temperature and critical pressure. The expression "subcritical fluid" is intended to define a substance below its critical temperature and critical pressure. The critical point represents the highest temperature and pressure at which the substance can exist as a vapour and liquid in equilibrium. The critical point for carbon dioxide is at a pressure of 73.8 bar and 31.1 ⁰ C. Thus the expression "supercritical fluid" with respect to carbon dioxide as the solvent is intended to mean carbon dioxide above its critical point. The expression "subcritical extraction" with respect to carbon dioxide as the solvent is intended to mean carbon dioxide below the critical point for carbon dioxide.

In the method of the invention, any fluid can be used as extractant. However, the use of CO ₂ is preferred.

In the case of CO ₂ as the extractant, the extraction temperature may be in the range between - O ⁰ C and 200°C (e.g 56.6 ⁰ C to 200 ⁰ C). Preferably the extraction temperature is in the range of 0 to 200 ⁰ C, for example 1O ⁰ C to 200 ⁰ C, such as 4O ⁰ C to 200 ⁰ C. The corresponding pressure range may extend from 5.17 to 1000 bar, for example 60 to 300 bar, such as 75 to 300 bar e.g. 80 to 300 bar.

Thus the method of the invention provides for the extraction of wax from a source using subcritical or supercritical, preferably supercritical, carbon dioxide at different temperatures and pressures. In a preferred method of the invention the flow rate of the subcritical/supercritical fluid is constant (for example 5kg/hour) during the extraction process.

In a preferred aspect of the invention, the subcritical/supercritical extraction is carried out stage-wise by changing the extraction pressure and/or temperature with time, for example by progressively increasing the extraction pressure and/or temperature as the extraction process progresses with time. By increasing the extraction temperature and/or pressure, to increase the solvent (e.g. carbon dioxide) density, the solvent strength of the extractant fluid is increased.

Preferably the method of the invention comprises first and second extraction steps, to obtain first and second fractions respectively. Preferably the first and second extraction steps are performed using carbon dioxide at subcritical or supercritical temperatures and/or pressures. Preferably the second extraction step is performed after the first extraction step and wherein the second extraction step is performed at a higher temperature and/or pressure than the first extraction step. Subsequent extraction steps, for example, third, fourth, fifth etc. (referred to herein as "subsequent extraction steps) extraction steps, may be performed after the second extraction step wherein each subsequent extraction step is performed at a higher pressure and/or temperature than the previous extraction step i.e. the extraction pressure and/or

temperature is/are progressively increased with time as subsequent extraction steps are performed.

Preferably the second extraction step is performed using carbon dioxide at a higher pressure than in the first extraction step.

Preferably still the second extraction step is performed using carbon dioxide at a higher temperature and pressure than in the first extraction step.

The first, second, an optionally subsequent, extraction step(s) may be a subcritical extraction or a supercritical extraction. The expression "subcritical extraction" with respect to carbon dioxide as the solvent is intended to mean extraction which takes place below the critical point for carbon dioxide (as defined hereinbefore), for example the subcritical extraction may be performed with carbon dioxide at a temperature of between less than 30 ⁰ C, such as between O ⁰ C and 3O ⁰ C, for example at between 1O ⁰ C and 30 ⁰ C, such as at about 10°C-20°C or 2O ⁰ C- 3O ⁰ C and/or at a pressure of less than 73 bar, for example between 0 and 70 bar, including between 10 and 60 bar such as at about 30-40 bar, 40-50 bar, 50 to 60 bar. The expression "supercritical extraction" with respect to carbon dioxide is intended to mean extraction which takes place above the critical point for carbon dioxide (as defined hereinbefore), for example the supercritical extraction may be performed with carbon dioxide at a temperature of between 32 ⁰ C and 300 ⁰ C, for example at between 35 ⁰ C and 200 ⁰ C, such as at between about 4O ⁰ C and 8O ⁰ C, for example at about 4O ⁰ C to 6O ⁰ C including 40-50 ⁰ C, 50-60 ⁰ C, 60-70 ⁰ C and 70-80 ⁰ C, and/or at a pressure of greater than 74 bar, for example between 75 and 100 bar, including 100 to 500 bar such as 100-300 bar for example about 100-200 or200-300 bar.

Preferably the first extraction step is a subcritical extraction. Preferably the second, and optionally subsequent, extraction step(s) is a supercritical extraction. Where the first extraction is a subcritical extraction, the first extraction step may be performed with carbon dioxide at a temperature of between 10 and 3O ⁰ C, for example at about 1O ⁰ C and/or at a pressure of up to 70 bar, for example at a pressure of between 10 and 60 bar, such as 60 bar.

Where the second, and optionally subsequent, extraction step is a supercritical extraction, the second extraction step may be performed with carbon dioxide at a temperature of 35 to 7O ⁰ C, for example between 40 and 7O ⁰ C including about 45 ⁰ C, 5O ⁰ C, 55 ⁰ C, 6O ⁰ C or 65 ⁰ C and/or at a pressure of between 75 and 300 bar, for example 100 to 300 bar, including about 100, 200 or 300 bar.

In the method of the invention, the first and second, and optionally subsequent, extraction steps may be supercritical extractions. The first and/or second extraction step may be performed at a temperature of 35 to 7O ⁰ C, for example between 40 and 7O ⁰ C including about 45 ⁰ C, 5O ⁰ C, 55 ⁰ C, 6O ⁰ C or 65 ⁰ C and/or at a pressure of between 75 and 300 bar, for example 100 to 300 bar, including about 100, 200 or 300 bar.

Preferably the first or second extraction step is performed at about 4O ⁰ C. Preferably the first extraction is performed at about 100 bar or 300 bar.

A subsequent extraction step may be performed at a temperature of up to 40 to 8O ⁰ C, for example at least 60, 65 ⁰ C, 7O ⁰ C or 75 ⁰ C, and/or at a pressure of between 100 and 300 bar, for example about 100, 200 or 300 bar

In a preferred embodiment of the invention the first or second, preferably first, extraction step is performed at a temperature of about 4O ⁰ C and a pressure of about 100 bar.

In a preferred embodiment of the invention the first or second, preferably first, extraction step is performed at a temperature of about 4O ⁰ C and a pressure of about 300 bar.

In a preferred embodiment of the invention the first or second, preferably second, extraction step is performed at a temperature of about 6O ⁰ C and a pressure of about 100 bar.

Ina preferred embodiment of the invention a subsequent extraction step is performed at a temperature of about 8O ⁰ C and a pressure of about 300 bar.

The extraction steps can be carried out using a plurality of separators in series by stage-wise change of the pressure and/or temperature with time. Thus the invention further provides apparatus for extracting wax in accordance with the method of the invention wherein the apparatus comprises a first and second separator wherein the first and second extraction processes are performed in the first and second separators respectively. Preferably the separators are connected in series to a vessel containing the source.

One or more further fractionation(s) of the fractions obtained can be carried out thereby separating the fractions into individual compounds or families of compounds. Further fractionation(s) can be carried out using several different procedures, including solid-liquid extraction, subcritical/supercritical fluid extraction, normal phase chromatography, reverse phase chromatography, gel permeation chromatography, multiple crystallisation, molecular distillation, complexation and combinations of such.

The method of the invention may be performed in the presence of a co-solvent. The co- solvent serves to increase the polarity of the subcritical/supercritical fluid, thus increasing its ability to extract more polar compounds. Preferably the co-solvent is an alcohol such as ethanol, acetone or a fluorinated solvent. The ethanol may be present in the extraction process at a maximum concentration of 20 v/v ethanol.

Extraction/fractionation of waxes from wheat straw can also underpin the development of biorefineries which, on the model of a petrochemical refinery, will use all parts of the crops to generate fuels and a range of higher-value chemicals and materials. As well as its economic and environmental benefits (supercritical extraction is an environmentally-friendly alternative to liquid solvent extraction), the removal of wheat straw waxes can add value to the residue for applications such as paper-making and co-firing, Economical losses associated with pitch (wax) deposit in Kraft paper mills often amount to 1% sales (Allen L.H., TAPPI press, 2000). Co-extraction of water during supercritical CO ₂ extraction drastically increases the calorific value of the residue (water content typically drops from ~ 10 to 3%). Thus the invention provides a residue, for example a wheat straw residue, obtainable following the extraction of

wax from straw in accordance with the method of the invention. Such a residue may have applications as described herein.

The invention further provides a wax fraction obtainable by the extraction method of the invention. A wax fraction obtainable by the method of the invention will typically contain polycosanols, alkanes, and/or sterols as described herein. Thus the invention provides a product comprising a wax obtainable by the method of the invention. Thus a wax fraction obtainable by the invention may be useful as a pharmaceutical, nutraceutical, agrochemical, cosmetic (such as lipsticks, hair gel, hair wax, or other cosmetic products of the like), polish or coating.

In a further aspect of the invention there is provided a product comprising wax obtainable by the method of the invention.

The product according to the invention may be a medicament. The medicament may be useful as a cholesterol lowering agent. Thus the medicament may be useful in the treatment or prevention of a cardiovascular, or related, disorder including hypertension, heart failure, coronary heart disease, hyperlipidemia, dyslipidemia, hypercholesterolemia, atherosclerosis, arteriosclerosis, vascular disease or diabetes.

The product according to the invention may be a cosmetic.

The product according to the invention may be a nutraceutical, for example a food or beverage ingredient, constituent or additive.

The product according to the invention may be a polish.

The product according to the invention may be a coating. The coating may be a food coating for example a coating for fruit or cheese.

In a preferred embodiment, the source of waxes is subjected to a preliminary mechanical treatment in order to increase the efficiency of the extraction and/or obtain a wax-enriched material, i.e. pressing, grinding, filtering, component fractionation and the like.

In one embodiment, the polycosanols of the invention are combined with one or more phytochemicals such as vitamins, sterols, saponins, phytoestrogens, flavonoids, polyphenols, catechins and the like, as disclosed in patent US 6,261,565Bl. Preferably, one or more of the phytochemicals consists of one or more modified or/and unmodified sterols of the invention. The synergistic combination of polycosanols and sterols is expected to provide an enhanced anti-cholesterol effect, whether in foods, beverages, or health supplement/nutraceutical applications. Preferably, one or more of the phytochemicals consists of one or more modified sterols of the invention.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

Examples

Examples of the method of the invention are given below for the purpose of illustration only and with reference to the drawings in which:

Figure 1 shows crude and wax yields achieved by Soxhlet extraction of wheat straw (Consort variety) using different solvents

Figure 2 shows crude and wax yields achieved by Soxhlet extraction of 5 varieties of wheat straw using hexane

Figure 3 shows the 3 botanical components of wheat straw Figure 4 shows crude and wax yields achieved by Soxhlet extraction of the 3 components of wheat straw (leaves, intemodes and nodes)

Figure 5 shows a comparison of crude and wax yields achieved by Soxhlet extraction with hexane and supercritical extractions with neat carbon dioxide of wheat straw.

Figure 6 shows a comparison of crude and wax yield achieved by Soxhlet extraction with hexane and supercritical extractions with carbon dioxide with or without a modifier of wheat straw.

Figure 7 is a comparison of differential scanning calorimetry curves of commercially available waxes and wheat straw wax

Figure 8 is a comparison of differential scanning calorimetry curves of crude and purified hexane extracts and supercritical fluid extract

Figure 9 and 10 corresponds to chromatograms of fractions of wheat straw wax obtained by column chromatography of a Soxhlet extract using hexane.

Figure 11 shows HT-GC/MS chromatograms of fractionated waxes obtained using carbon dioxide. The identity of major compounds is shown on the chromatogram.

Example 1. Isolation of wheat straw waxes by organic solvents

Ground Consort variety wheat straw (30 g) was extracted with 900 mL of the desired solvent for 5 h. The recovered extracts were filtered and concentrated to dryness by rotary evaporator.

They were further maintained under vacuum overnight in a dessicator and weighed (crude yield). Crude extracts were then eluted on silica gel with a (85:15 v.v) mixture of hexane/ether

to yield to a wax fraction (wax yield). Results are summarised in Figure 1. The yield of crude extract based on the weight of dried extract has been found to lie between 1.2 and 2.8% of dry weight of wheat straw (Consort variety). However, it can be seen that the wax yield remains constant for each solvent. It can therefore be assumed that the straw wax loading of the Consort variety is around 0.8% of the dry matter. This experiment was repeated and a error of less than 6% was observed.

Example 2. Variation in crude yield and wax yield of wheat straw wax within varieties of wheat 5 varieties of wheat were grown under controlled field conditions namely Marris Widgeon, IMP, Xil9, Wallace, and Squarehead Master. Wheat straw of the desired variety (30 g) was extracted with 900 mL of hexane for 5 h. The recovered extracts were filtered and concentrated to dryness by rotary evaporator. They were further maintained under vacuum overnight in a dessicator and weighed (crude yield). Crude extracts were then eluted on silica gel with a (85:15 v:v) mixture of hexane/ether to yield to a wax fraction (wax yield). Results are summarised in Figure 2. This experiment was repeated and a error of less than 6% was observed.

Example 3. Variation in crude yield and wax yield of wheat straw wax within botanical components

Wheat straw (Consort variety) was physically fractionated into its major botanical components: nodes, internodes and leaves. The internodes are separated by nodes, which are the starting point of the leaves as depicted in Figure 3. The 3 fractions were further chopped and extracted with 900 mL of hexane for 5 h. The recovered extracts were filtered and concentrated to dryness by rotary evaporator. They were further maintained under vacuum overnight in a dessicator and weighed (crude yield). Crude extracts were then eluted on silica gel with a (85:15 v:v) mixture of hexane/ether to yield to a wax fraction (wax yield). Results are summarised in Figure 4. This experiment was repeated and a error of less than 6% was observed.

Example 4. Isolation of wheat straw waxes by supercritical carbon dioxide and comparison with Soxhlet extraction.

Ground wheat straw was extracted by supercritical carbon dioxide at different pressures and temperatures (40°C/10 MPa (lOObar), 60°C/20 MPa (200 bar), 80°C/30 MPa (300 bar) respectively). SFE (what does SFE stand for?) flow rate was maintained at 5ml/min. Extracts were left under vacuum overnight in a dessicator and weighed. Results are summarised in Figure 5. For comparison purposes, the results achieved by Soxhlet extraction with hexane (most selective liquid solvent) are also provided. At minimum pressure and temperature (40°C and 10 MPa (100 bar)), 100% selectivity was achieved but only half of the wax loading was recovered. By progressively increasing pressure and temperature and therefore the solvent strength, an increase of the wax recovery could be achieved. Conditions of 60 ⁰ C and 20 MPa (200 bar) gave a pure wax extract corresponding to 88% of the wax loading.

Example 5. Isolation of wheat straw waxes by supercritical carbon dioxide. Effect of a modifier

Ground wheat straw was extracted by supercritical carbon dioxide at 40 ⁰ C/ 10 MPa (100 bar) and 80°C/30 MPa (300 bar) in the presence of ethanol (20%, v:v). SFE flow rate was maintained at 5ml/min. Extracts were left under vacuum overnight in a desiccator and weighed. Results are summarised in figure 6. For comparison purposes, the results achieved by Soxhlet extraction with hexane (most selective liquid solvent) and supercritical CO ₂ without a modifier as in example 4 are also provided.

Example 6. Identification of the individual components present in wheat straw

Wheat straw wax as in example 1 was subjected to trimethylsilylation. 200 μl bis- (trimethylsilyl)-trifluoro-acetamide, 1% TCMS and 100 μL toluene were added to the extract (~ 3 mg). The reaction was completed by warming the stoppered vial on a heating block at 75°C for 30 min. The solution was analysed by EI-GC/MS ((DB5-HT, 6m), temperature- programmed from 40 ⁰ C to 16O ⁰ C (6min) at 10°C/min and from 160 ⁰ C to 395°C (lOmin) at

15°C/min) and identified by computer comparison with the NIST library, by interpretation of the mass spectrum and when possible by comparison with standard compounds. Wheat straw wax was found to be mainly composed of fatty acids, fatty alcohols, alkanes, and sterols. Table 1 gathers the identified individual components present within wheat straw wax.

Example 7. Analysis of wax samples by DSC

Wheat straw Consort variety (30 g) was extracted with 900 mL of hexane for 5 h. The recovered extract was filtered and concentrated to dryness by rotary evaporator. It was further eluted on silica gel with a (85:15 v.v) mixture of hexane/ether to yield a waxy product. 2 commercially available waxes were also investigated: Carnauba wax and Lanolin. The desired sample under investigation was weighed in an aluminium pan and further placed in the test cell along with the empty reference pan. The test cell was heated from 20 ⁰ C to 105 ⁰ C at 10°C/min, and cooled down to -10 ⁰ C at 10°C/min. After holding the cell at -10 ⁰ C for 1 min, it was heated from -10 to 105 ⁰ C at 10 °C/min and held for 1 min at 105 ⁰ C. DSC curve of the last heating cycle was recorded. As shown in figure 7, an endothermic transition occurs when the 3 waxes were heated to 105 ⁰ C, corresponding to the melting of the waxes. The melting point of wheat straw wax Soxhlet extracted with hexane was found to lie between the ones of the 2 commercial waxes.

Example 8. Determination of the melting points of a wheat straw wax extract before and after purification and comparison with a supercritical carbon dioxide extract.

Thermal transition temperatures of a wheat straw wax extracted with hexane were determined, before and after purification by column chromatography as in example 1. In addition, the thermal transition temperature of a supercritical carbon dioxide extract of wheat straw (40 ⁰ C and 10 MPa) was measured (see Figure 8). The desired sample was weighed in an aluminium pan and placed in the test cell along with the empty reference pan. The test cell was heated from 20 ⁰ C to 105 ⁰ C at 10°C/min, and cooled down to -10 ⁰ C at 10°C/min. After holding the cell at -10 ⁰ C for 1 min, it was heated from -10 to 105 ⁰ C at 10 °C/min and held for 1 min at

105 ⁰ C. DSC curve of the last heating cycle was recorded. When extracted by solvent, a slight shift in the melting point of the wheat straw wax fraction was measured after purification (47.3 ⁰ C for the crude extract against 47.6°C for the purified wax extract).

Example 9. Isolation of alkanes and polycosanols from wheat straw wax

A crude hexane extract of wheat straw (lOOmg) obtained by Soxhlet extraction as in example

1 was chromatographed on a 20 cm x 1 cm silica gel K60 column. The solvent system was as follows:

3 times the volume of hexane (45 mL) 6 times the volume of hexane/diethyl ether (99 : 1 , v.v) (90 mL)

5 times the volume of hexane/diethyl ether (95:5, v:v) (75 mL)

5 times the volume of hexane/diethyl ether (92:8, v:v) (75 mL)

8 times the volume of hexane/diethyl ether (85:15, v:v) (120 mL)

5 times the volume of diethyl ether (75 mL)

32 fractions of 15 mL were collected and concentrated to dryness by rotary evaporator. A number of these fractions were silylated and further analysed by EI GC-MS,. (DB5-HT, 6m), temperature-programmed from 4O ⁰ C to 16O ⁰ C (6min) at 10°C/min and from 160 ⁰ C to 395°C

(lOmin) at 15°C/min. The 2 ^nd fraction (7.3 mg) was found to be mainly alkanes ranging from 25 to 33 carbon atoms, nonacosane and hentriacontane being the most abundant compounds (see GC-MS trace in

Figure 9).

The 23 ^rd fraction (5.4 mg) was found to be a pure fraction of polycosanol containing alcohols ranging from 24 to 32 carbon atoms, octacosanol being the most abundant compound (see GC- MS trace in Figure 10).

Example 10

A pilot scale trial was carried out in collaboration with Botanix Ltd. In this study 5 extractions were performed using supercritical carbon dioxide (at fixed CO2 flow rate = 5Kg/h):

Conditions wwaaxx } yield (% of charge)

4O ⁰ C / 100 bar 0.45

40 ⁰ C / 300 bar 0.55 7O ⁰ C / 100 bar 0.004

7O ⁰ C / 300 bar 0.40

55 ⁰ C / 200 bar 0.35

We carried out a statistical analysis (factorial design) to determine the best conditions of extractions (4O ⁰ C / 300 bar). These optimal conditions were further used during the following plant scale trial.

We also carried out a fractionation of wheat straw waxes by stage- wise increase of extraction pressures and temperatures (using the same raw material (charge)).

10 ⁰ C / 60 bar (liquid CO2) = fraction mostly composed of low molecular weight fatty acids and sterols 4O ⁰ C / 100 bar (near critical point) = fraction mostly commonly made of low molecular weight fatty acids and alkanes.

40 ⁰ C / 300 bar = fraction mostly commonly made of high molecular weight fatty acids and fatty alcohols (polycosanols) (see Figure 11)

We have recently carried out a commercial trial using 74.19Kg of wheat straw (coarse-milled form) yielding 77Og of wax.

Pressure (bar): 300

Temperature ( ⁰ C): 40

Charge weight (kg): lβ.O(average)

Number of charges: 4.5

CO2 Flow (kg h-l): 350 Total Input (kg) : 74.19

Total wax yield (% of charge): 1.04%

The yield doubled through scale-up (from 0.55% to 1.04%) due to a higher extraction efficiency of the equipment. The final product (formulation composition = fatty acids, fatty alcohols, alkanes, sterols, sterol esters, and wax esters) is tested in cosmetic products such as lipsticks.