Methods and uses relating to obtaining natural products from plants

Abstract

The present invention provides a method of obtaining petroselinin from Smyrnium olusatrum. The method comprises the steps of: (a) removing one or more terpene compounds from a portion of the plant Smyrnium olusatrum or an oil component extracted therefrom; and (b) obtaining petroselinin from the material remaining after step (a) or an extract obtained therefrom. Step (a) is carried out by direct distillation or extraction with, for example, liquid and / or supercritical carbon dioxide. Step (b) may comprise crystallizing petroselinin from the material remaining after step (a) or an extract obtained therefrom. The method may further comprise hydrolysing petroselinin to petroselinic acid.

Description

[0001] Methods and uses relating to obtaining natural products from plants

[0002] The present invention relates to the isolation of natural compounds from plants.

[0003] In particular the invention relates to a process able to separate and isolate individual terpenes, a solid unsaturated triglyceride and protein components from Smyrnium olusatrum.

[0004] Smyrnium olusatrum L. is an umbellifer of the Apiaceae family and is commonly called Alexanders or Horse Parsley. It is a biannual plant with black seeds formed in mid-summer.

[0005] It is widely found around the coastline in the UK and was brought over by the Romans for its culinary and medicinal properties with all parts of the plant being edible, and widely eaten. It is also widespread along the coastline of the Mediterranean.

[0006] A number of publications have described the isolation of essential oils from Smyrnium olusatrum L., mainly using hydrodistillation, steam distillation or supercritical extraction, together with the characterisation of the complex mixtures of compounds in these oils. The compounds fall into different classes with the furanosesquiterpenes being the most prominent followed by monoterpenes. Among the studies reported for these complex mixtures, significant components that are commonly reported are the furanosesquiterpenes, isofuranodiene, which rearranges on heating to curzerene, 1 p-acetoxyfurano-eudesm-4(15)-ene and glechomafuran.

[0007] The oils and some of the terpenoid components separated by chromatography (sometimes from other plant oils) show a number of biological properties. The many publications for the biological activity of the essential oil from Alexanders describe its effect on bacteria, weeds, fungi, mosquitos and African trypanosomiasis.

[0008] The furanosesquiterpenes present in Alexanders seed have been characterised in a number of publications with isofuranodiene and curzerene often being the most prominent. Isofuranodiene can be converted into curzerene by heating via a Cope rearrangement, and isolation of it has been achieved by stabilization as metal complexes. Isofuranodiene and curzerene are biologically active components occurring in several species of the genus Commiphora, from which myrrh is obtained, and of the genus Curcuma. Both are used in traditional Chinese medicine and Ayurveda, and as flavourings, food additives and ingredients in perfume. Together with other furanosesquiterpenes, they are responsible for the characteristic myrrh and turmeric like odours. Isofuranodiene is reported to possess significant anticancer and anti-angiogenesis activity, together with analgesic effects. It has also shown neuritogenic, and hepatoprotective effects and has been reported to treat ischemia. It is also active against stored product insects. Curzerene is an anti-oxidant, reported to have anticancer, antifungal and antimicrobial properties and has been shown to control leishmaniasis. 1 p-Acetoxy-furanoeudesm-4(15)-ene shows strong anti-cancer effects and is a powerful radical scavenger. Glechomafuran is an anti-oxidant.

[0009] The seeds of Smyrnium olusatrum have a high concentration of petroselinin, or TPS, a triglyceride of petroselinic acid, or PSA, a monounsaturated fatty acid. Petroselinin can be converted into PSA by typical hydrolytic procedures.

[0010] Other oils rich in petroselinin, for example, from coriander seeds, have been used in food applications, as described for example in US 6365175. PSA has been used in cosmetic and nutraceutical applications where anti-inflammatory and anti-ageing properties are described, for example in EP1013178. Similar oils have been isolated by appropriate methods from Smyrnium olusatrum.

[0011] Petroselinic acid has a melting point of 30 - 33°C whereas its more commonly found isomer, oleic acid, has a melting point of just 13 -14°C. This difference is reflected in the triglyceride, TPS, having a melting point of 33 °C while the oleic equivalent melts at 5°C. Such high melting point oils, typically palm and coconut oils, are widely preferred in food applications giving the desired texture. TPS, comprising the monounsaturated PSA, can provide a health benefit over palm and coconut oils, which contain significant amounts of saturated fats.

[0012] Although there are a number of advantageous applications of petroselinic acid or the triglyceride oil obtained from Smyrnium olusatrum, its use has previously been limited due to the complex steps needed to recover it from natural sources. Similarly, the fact that terpenes have only previously been obtained from Smyrnium olusatrum in complex mixtures or in small quantities, has limited their utility. Separation methods of the prior art are complex and laborious limiting any commercial usefulness.

[0013] The present inventors have developed a novel method by which both individual terpenes and petroselinic acid-based triglycerides can be obtained from Smyrnium olusatrum in significant quantities.

[0014] According to a first aspect of the present invention, there is provided a method of obtaining petroselinin from Smyrnium olusatrum, the method comprising the steps of:

[0015] (a) removing one or more terpene compounds from a portion of Smyrnium olusatrum or an oil component extracted therefrom; and

[0016] (b) obtaining petroselinin from the material remaining after step (a) or an extract obtained therefrom; wherein step (a) is carried out by direct distillation or extraction with, for example, liquid and / or supercritical carbon dioxide.

[0017] Optionally, the method further comprises hydrolysing petroselinin to petroselinic acid.

[0018] The present invention also provides a method of obtaining petroselinin from Smyrnium olusatrum, the method comprising the steps of:

[0019] (a) removing one or more terpene compounds from a portion of Smyrnium olusatrum or an oil component extracted therefrom; and

[0020] (b) crystallizing petroselinin from the material remaining after step (a) or an extract obtained therefrom; wherein step (a) is carried out by direct distillation or extraction with, for example, liquid and / or supercritical carbon dioxide.

[0021] Optionally, the method further comprises hydrolysing petroselinin to petroselinic acid.

[0022] Step (a) involves removing one or more terpene compounds from a portion of the Smyrnium olusatrum plant or an oil component extracted therefrom; and involves direct distillation or extraction with, for example, liquid and / or supercritical carbon dioxide.

[0023] Step (b) involves obtaining petroselinin from the material remaining after step (a) or an extract obtained therefrom.

[0024] Step (b) may involve crystallization of petroselinin from the material remaining after step (a) or an extract obtained therefrom.

[0025] In some embodiments step (a) involves extraction of one or more terpene compounds with liquid and / or supercritical carbon dioxide. In such embodiments step (b) suitably involves obtaining petroselinin from an extract obtained from the remaining plant material. In such embodiments step (b) may suitably involve crystallization of petroselinin from an extract obtained from the remaining plant material. The extract used in step (b) is suitably obtained by extraction with liquid and / or supercritical carbon dioxide.

[0026] In some embodiments step (a) involves removing one or more terpene compounds from an oil component extracted from a portion of the Smyrnium olusatrum plant. In such embodiments step (a) suitably involves direct distillation of the extracted oil component and step (b) suitably involves obtaining petroselinin from the remainder of the oil component following the removal of one or more terpene compounds by direct distillation in step (a). In such embodiments step (b) may suitably involve crystallization of petroselinin from the remainder of the oil component following the removal of one or more terpene compounds by direct distillation in step (a). In such embodiments there is suitably a step prior to step (a) of obtaining an oil component from the portion of the plant.

[0027] The present invention is carried out on a portion of the plant Smyrnium olusatrum, which is also known as Alexanders or Horse Parsley.

[0028] The portion of the plant may comprise the whole plant.

[0029] The portion of the plant preferably comprises the seeds of the plant.

[0030] The seeds are grown inside a shell or husk.

[0031] In some preferred embodiments the whole seed including the husk may be used in step (a).

[0032] In some embodiments the seeds may be dehulled to remove or partially remove the outer husk prior to step (a). In such embodiments the seeds may be separated from the husk.

[0033] In some embodiments mixed de-husked seeds and husks are used in step (a).

[0034] In some embodiments the husk and de-husked seeds are separated. One or both components may be used in the method of the invention.

[0035] In some embodiments the seeds (preferably including the husks) may be milled, crushed or pulverized prior to use in step (a).

[0036] In some preferred embodiments whole seeds are milled to an average particle size range of 0.01 to 5 mm, preferably 0.05 to 2.5 mm, preferably 0.1 to 1 mm. Particle size may suitably be determined by sieving techniques.

[0037] In some embodiments step (a) involves extraction of one or more terpene compounds with liquid and / or supercritical carbon dioxide.

[0038] In such embodiments the portion of the plant preferably comprises milled seeds. The carbon dioxide used suitably has a pressure of at least 70 bar and a temperature of at least 10°C.

[0039] Preferably the carbon dioxide used in step (a) has a pressure of 70 to 100 bar and a temperature of 15 to 40°C.

[0040] Preferably the carbon dioxide used in step (a) has a pressure of 75 to 85 bar and a temperature of 15 to 25°C.

[0041] In step (a) contacting the portion of the plant with liquid and / or supercritical carbon dioxide provides an extract which contains one or more terpene compounds. If the temperature and pressure of the carbon dioxide used in step (a) are increased slowly it is possible to enhance the purity of individual terpene fractions.

[0042] Surprisingly, the fractions collected during supercritical extraction were light in colour, with little odour, in contrast to the oil extracted by traditional methods which are dark in colour and possess the typical myrrh / turmeric odour.

[0043] In such embodiments step (b) suitably involves isolation of petroselinin from an extract obtained from the remaining plant material. The extract used in step (b) is suitably obtained by extraction with liquid and / or supercritical carbon dioxide.

[0044] Thus the present invention may involve the steps of:

[0045] (a) removing one or more terpene compounds from a portion of the plant Smyrnium olusatrum by extraction with liquid and / or supercritical carbon dioxide;

[0046] (x) further contacting the portion of the plant with liquid and / or supercritical carbon dioxide to obtain an oil comprising petroselinin; and optionally

[0047] (b) crystallization of the oil obtained in step (x) to provide petroselinin in solid form.

[0048] Step (b) also involves contacting the portion of the plant with liquid and / or supercritical carbon dioxide.

[0049] The present inventors have surprisingly found that by slowly increasing the temperature and pressure of the liquid and / or supercritical carbon dioxide used different compounds can be extracted from the portion of the plant.

[0050] Preferably the temperature and pressure of the liquid and / or supercritical carbon dioxide used in step (x) are greater than those used in step (a). The carbon dioxide used in step (x) suitably has a pressure of at least 200 bar and a temperature of at least 40°C.

[0051] Preferably the carbon dioxide used in step (x) has a pressure of 150 to 450 bar and a temperature of 20 to 60°C.

[0052] Preferably the carbon dioxide used in step (x) has a pressure of 150 to 450 bar and a temperature of 30 to 55°C.

[0053] A particular advantage of embodiments of the invention which involve the use of supercritical carbon dioxide is that the product obtained is light in colour and has low odour. This can be highly advantageous in a number of applications and may reduce the need for further processing steps of decolouration and deodorisation.

[0054] In some preferred embodiments of the present invention step (a) involves direct distillation of an oil extracted from Smyrnium olusatrum.

[0055] Thus in some embodiments the present invention involves the steps of:

[0056] (y) obtaining an oil component from a portion of the plant Smyrnium olusatrum;

[0057] (a) directly distilling the oil component obtained in step (y) to remove one or more terpene compounds; and optionally

[0058] (b) crystallization of the oil obtained in step (a) to provide petroselinin in solid form.

[0059] Step (y) may be carried out on any portion of the plant. Preferably step (y) is carried out on seeds of the plant. Step (y) may involve standard techniques known in the art for the extraction of oils from seeds.

[0060] In some embodiment step (y) may involve pressing the seeds of the plant.

[0061] In some embodiments in step (y) the seeds may be ground and extracted with a solvent. Suitable solvents include petroleum and hexane. The solvent is suitably evaporated to give the oil component.

[0062] Step (a) suitably involves direct distillation of the oil component obtained in step (y).

[0063] By direct distillation, we mean to refer to simple distillation of the oil component. We do not mean to include hydrodistillation or steam distillation.

[0064] The direct distillation suitably involves heating the oil component, preferably under vacuum. Suitably the direct distillation of step (a) involves heating the material using appropriate short path equipment and a pressure of d mbar to a temperature of at least 60°C, preferably at least 80°C, more preferably at least 100°C, at reduced pressure. This produces a distillate containing the major terpenes, including a fraction enriched in isofuranodiene from which it crystallised. By carrying out the distillation using standard equipment at a temperature 140- 180°C and a pressure of <1 mbar the first fraction to distill is largely p-phellandrene and the second fraction is >85% curzerene, the thermal rearrangement product of isofuranodiene. The residue is triglyceride together with two major sesquiterpenes, glechomafuran and 1 p- acetoxyfurano-3-eudesmene.

[0065] By slowly increasing the distillation temperature and / or slowly reducing the pressure it is possible to extract one or more terpene compounds in excellent purity and yield.

[0066] In embodiments, step (b) of the method of the present invention involves crystallization of petroselinin from the material remaining after step (a) or an extract obtained therefrom.

[0067] Step (b) suitably involves inducing crystallization of petroselinin directly from an oil which is rich in petroselinin. Such an oil is suitably obtained following steps (a) and (x) or steps (y) and (a).

[0068] By petroselinin we mean to refer to a triglyceride of petroselinic acid.

[0069] Step (b) may involve any standard crystallization technique. Such techniques will be known to the person skilled in the art and include, for example, cooling and / or seeding the oil.

[0070] In some embodiments the oil may crystallize on standing.

[0071] In some embodiments a recrystallization step may be carried out to further purify the petroselinin.

[0072] In some embodiments the oil component rich in petroselinin is suitably hydrolysed by conventional hydrolytic methods to give petroselinic acid

[0073] The petroselinin may be crystallised and subsequently hydrolysed to petroselinic acid. However, embodiments in which an oil comprising petroselinin is hydrolysed and then petroselinic acid is crystallised therefrom are also within the scope of the invention as are embodiments in which petroselinin is hydrolysed to petroselinic acid without crystallisation. The present invention provides simple methods by which a crystalline solid comprising petroselinin and / or petroselinic acid can be obtained in high yield and purity.

[0074] Advantageously, the invention may also provide components rich in terpene compounds, also in high yield and purity.

[0075] These materials have numerous applications in foods and cosmetics and as naturally sourced materials are desirable to consumers. The antimicrobial properties of these materials means that they are very useful in food and cosmetic applications. The separation of the terpene components according to the invention provides petroselinin and / or petroselinic acid having desirable organoleptic properties.

[0076] The invention also makes use of a plant generally considered to be a weed. This is a hardy plant which can grow under a variety of conditions and can be a useful further crop for farmers particularly as the effects of climate change drive diversification.

[0077] The residual plant material which remains after extraction of the terpene and petroselinin is high in protein is useful as animal feed. Advantageously, because this material comprises residual terpene and petroselinin components, it may have antimicrobial properties. This offers further benefits as the use as animal feed may lead to a reduction in the need to use antibiotics in animals fed with this material.

[0078] Further aspects of specific embodiments of the invention will now be described.

[0079] Initially the inventors were able to obtain enough seed to investigate the extraction and refining of the oil by wild harvesting and the agronomy of growing this crop on agricultural land to provide larger quantities of seed was successful. The seeds were shown to contain up to 20% oil and the oil contained about 65% TPS. Food industry approved refining methods for the oil provided a green-coloured oil and this was shown to be a replacement, partly or totally, for palm oil in foods. The extracted or pressed seed solid residues contained around 16 % protein.

[0080] There are a number of options for extracting oil from the seed. The seeds may be cold pressed to provide an oil. Alternatively, the seeds may be ground and directly extracted with a solvent such as petroleum or hexane, and the solvent evaporated to give an oil.

[0081] Surprisingly, simple controlled removal of the outer husk from the seed and extraction of the two portions gave an enhancement of around a factor of 10 as determined by proton NMR for triglyceride relative to terpenes between the core and husk fractions, the husk containing predominantly the terpenes. The triglyceride and terpene rich materials could each then separately be treated as above, enhancing the efficiency of the separation.

[0082] The initial separation of terpenoid and triglyceride fractions may involve a supercritical process. The approach was to first extract the finely ground seed at lower temperatures and pressures to remove the lighter, temperature sensitive volatile oils, and then to extract the remaining material at a higher pressure and temperature to remove the remaining oils, waxes, etc. This approach provided a range of fractions, the early fractions containing only terpenes, the later fractions containing triglycerides. In this case, all the major four terpenes were in the early fractions, so the sequence of supercritical extraction, followed by distillation of the early fractions can be used in place of cold pressing of the seed followed by distillation to provide the four isolated terpenes.

[0083] Surprisingly, simple distillation of the pressed oil under vacuum (not hydrodistillation or steam distillation) produced two significant fractions, the first containing just two monoterpenes, the second, curzerene, of around 90% purity. If the initial distillation of the pressed oil, or oil extracted from the outer husk of the seeds was carried out at high vacuum using short path distillation equipment, the second fraction was isofuranodiene, rather than curzerene. This oil crystallised on standing at -4°C.

[0084] Also, surprisingly, in addition, glechomafuran crystallised from the oil and could be isolated by filtration. The non-distilling material could then be recrystallized at low temperature to produce high purity TPS. The residue from this process was enriched in 1 p-acetoxyfuranoeudesm- 4(15)-ene which could be separated pure by low temperature crystallization of the remaining triglyceride from acetone or by hydrolysis of the triglyceride with aqueous base to provide the sodium salts of the free fatty acids, plus glycerol and the acetoxy-compound in organic solvent.

[0085] The sequence of extraction and crystallization may be reversed.

[0086] The invention provides a method that produces significant quantities of separated monoterpenes, of isofuranodiene, glechomafuran, 1 p-acetoxyfuranoeudesm-4(15)-ene, curzerene, and in addition, of tripetroselinin, optionally in a crystalline form, and of protein rich solids. The linked process is initiated by pressing, solvent or super-critical extraction of the whole seed, but more particularly of the seed separated into outer shell and inner core. The ratio of terpenes to triglycerides in the bulk material for pressing or solvent extraction could be controlled by removal of the outer seed husk using a ball mill, and collection of this and the remaining seed core; this changed the ratio by a factor of at least 10. Surprisingly, a combination of simple distillation under vacuum (not hydrodistillation or steam distillation), or selective supercritical extraction of terpene and triglyceride fractions, followed by direct crystallization from the distillate or the residual oils then provides the individual separated components.

[0087] The present invention provides methods of obtaining terpenoid compounds of high purity from the oil extracted from the seed of Smyrnium olusatrum, together with a crystalline triglyceride and residual protein cake.

[0088] The initial separation of terpenes and triglycerides may be achieved by separation of seed husk and seed core.

[0089] The separation may be achieved without the use of solvents.

[0090] The separation may be achieved without the use of organic solvents.

[0091] The single isolated terpenes may include p-phellandrene, glechomafuran, 1 p- acetoxyfuranoeudesm-4(15)-diene and isofuranodiene (or curzerene).

[0092] Monoterpenoid compounds isolated may include beta-phellandrene and alpha-pinene.

[0093] A mixture of curzerene and germacrene D at >95 % may be provided.

[0094] Curzerene at >85 % may be provided.

[0095] The terpenoid compound curzerene at >95 % may be provided.

[0096] The terpenoid compound isofuranodiene at >85 % may be provided.

[0097] The terpenoid compound isofuranodiene at >95 % may be provided.

[0098] The terpenoid compound glechomafuran at >85 % may be provided.

[0099] The terpenoid compound glechomafuran at > 95 % may be provided.

[0100] The terpenoid compound 1 p-acetoxyfuranoeudesm-4(15)-diene at >95 % may be provided.

[0101] The method may provide a purified oil rich in petroselinin, the triglyceride of petroselinic acid.

[0102] The purified oil suitably provides a solid product, comprising mainly petroselinin. Suitably the triglyceride compound is petroselinin at >85 %.

[0103] Suitably the triglyceride compound is petroselinin at >95 %.

[0104] The solid product can be used to produce baked goods with texture similar to such goods produced with solid fats such as palm oil and coconut oil.

[0105] The solid product can be converted into petroselinic acid as a solid product. Both petroselinin and petroselinic acid can be used in skincare agents and in food supplements.

[0106] The method may produce significant yields of individual components, isofuranodiene, curzerene, 1 p-acetoxyfuranoeudesm-4(15)-diene and glechomafuran shown to be active in human and animal health applications.

[0107] The petroselinin and / or petroselinic acid provided by the present invention may be used in the manufacture of margarine or chocolate.

[0108] Features, integers, characteristics, compounds, molecules, 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. All of the features disclosed in this specification (including any accompanying claims, abstract and figures), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0109] The present invention will now be further described with reference to the following figures which show:

[0110] Figure 1 shows the HPLC / ELSD traces for TPS, isofuranodiene, glechomafuran, 1 p- acetoxyfurano-eudesm-4(15)-ene.

[0111] ELSD conditions - Tube Temp: 45°C, Gas Flow: 2 L / min, Gain: 8 HPLC - THF / water gradient method, 25cm C18 column.

[0112] Sample concentrations and injection volumes were as follows: Fig 1 A: TPS, 5f l

[0113] Fig 1 B: isofuranodiene, 22g / L; 20 |_il

[0114] Fig 1C: glechomafuran, 3g / L; 10 |_il

[0115] Fig 1 D: 1 p-acetoxyfurano-5eudesm-4(15)-ene 10g / L; 5 |_il

[0116] Figure 2 shows MIC results for antimicrobial testing of various compounds against E. coli and S. aureus.

[0117] The invention will also be further described with reference to the following non-limiting examples.

[0118] Example 1

[0119] Wild harvesting of seed was carried out on field verges using a rapeseed harvester. This seed was drilled in rows at a rate of 35 kg / Ha in a 3 Ha plot and harvested 2 years later.

[0120] Example 2

[0121] The initial separation of terpenoid and triglyceride fractions in the seed was enhanced by rolling in a mill to separate the outer husk and the internal seed. Thus, 100 g of seed was coarse ground and sieved to give larger solid (seed core, 66.2 g) and fines (shell, 31 .6 g). Both were subjected to a single extraction with petrol, filtered and the solvent evaporated. The coarse material gave an amber oil (4.22 g). The fines gave a light brown waxy / solid / oil mix (4.16 g). The ratio of triglyceride to terpene mixture could be measured approximately by NMR; in the coarse fraction it was ca. 2:1 .2, while in the fines it was ca. 1 :6. By adjusting the extent of removal of the husk, these ratios could be changed. When the amount of seed coat removed as fines was reduced to 25% of the total seed weight, the ratio of triglyceride to terpene mixture in the fines changed to ca. 1 :8.

[0122] Example 3

[0123] The raw seed, harvested from the field rows, was flaked using flaking rolls prior to being pressed on a mini press to form a press cake, this press cake was then further passed through the mini press a second time to extract further oil. The pressing produced 26.5 Kg (3.6 %) and 18 Kg (2.8%) of press oil from the 1st and 2nd pressing respectively. A further pressing of the press cake from the second run collected some oil (5 kg). A total oil yield of 6.2% weight of the seed was obtained, with 79.6% of the available oil recovered from the intake seed. The oil was characterised using standard bulk oil analytical methods. (Acid Value (mg KOH / g) 21.06; Peroxide Value (meq / Kg) 3.96). The oil contained both the mixture of terpenes and fatty acid derivatives.

[0124] Example 4

[0125] 1100 g of milled cake in 2500 ml of petroleum ether, bp 40-60°C, was mixed with a mechanical stirrer and refluxed for 5 h. The mixture was filtered under vacuum suction. Following solvent removal, using a rotary evaporator, 120.6 g (10.96%) of dark yellow oil was recovered. Reclaimed petroleum ether (1800 ml) was added to the residue and the mixture was heated for 3 h. After cooling the solvent was decanted, filtered and removed gaining 38.0 g (3.45%) of dark yellow oil, resulting in a total 158.6 g (14.4%).

[0126] Example 5

[0127] The seed was milled to a particle size of 0.2-0.5 mm and 999.3 g was packed into a 2000 ml extraction vessel. This is a bulk density of 0.499. It was first extracted with liquid CO2 at 80 bar and 20°C and once the fraction weight had fallen to 1 g / 15 minutes the pressure was increased to 200 bar and 40°C (mild supercritical). Fractions were again collected every 15 minutes until the fraction weight had fallen to 1 g / 15 minutes. The final fractions were collected at 400 bar and 50°C. The fractions collected at 200 bar and 40°C (ca. 5 %) were principally terpenes by GC-MS and NMR; those collected at 400 bar and 50°C (6 %) were principally triglycerides by NMR. The early fractions contained all the major terpenes isolated using the linked protocol (beta-phellandrene, isofuranodiene, glechomafuran and 1 p- acetoxyfuranoeudesm-4(15)-ene). All the fractions were isolated as yellow to light brown oils.

[0128] Example 6

[0129] The oil from examples 2 to 4 was distilled under vacuum using standard laboratory equipment with the flask temperature set at 140°C, head temperature at 48°C to give fraction A, a clear colourless liquid and then increased to 180°C (head temperature at 105-110°C) to give fraction B, a clear, light yellow, free flowing oil. Fraction A was about 7% and Fraction B about 10% of the oil. Fraction A was a mixture of a-pinene and p-phellandrene in ca. 1 :3 ratio. This is evident on the basis of GCMS and NMR spectra. Fraction B was principally curzerene, with germacrene D as a minor component by GCMS and1H and13C NMR.

[0130] The remaining non-distilled oil was largely triglyceride, together with less volatile terpenes, principally glechomafuran and 1 p-acetoxyfuranoeudesm-4(15)-ene.

[0131] Example 7 A portion of the pressed oil (900 g) from example 3 was subjected to short path distillation using a thin wipe-film distillation apparatus (Pope Scientific wiped still, 2” diameter) under high vacuum. An initial fraction (28 g) was obtained with the distillation temperature at 80°C and vacuum at 0.5 mbar, flow rate of 1 L / hr for the input, analysis of the fraction indicated it predominantly consisted of p-phellandrene. The temperature of the still was increased and the residue re-processed, a second fraction was obtained (20 g) collected at 100°C and 0.6 mbar, flow rate 1 L / hr, analysis indicated a mixture of p-phellandrene and isofuranodiene. The temperature of the still was further increased and the residue re-processed, a third fraction (46 g) was collected at 120°C and 0.8 mbar, flow rate 1 L / hr, analysis indicated isofuranodiene as a main component. Analysis of the final residue by NMR indicated a mixture of TPS containing the less volatile furanosesquiterpenes; glechomafuran and 1 p- acetoxyfuranoeudesm-4(15)-ene.

[0132] Further processing of the third fraction from the short path distillation, holding the distillate at - 20°C resulted in the isolation of isofuranodiene as a crystalline white solid (15 g). The HPLC of this is shown in Figure 1. This rearranged rather rapidly in deuterated chloroform, or in the presence of acid, and so NMR spectra were run in DMSO. The rearrangement products were two isomeric eudesmafurans.

[0133] Example 8

[0134] When the oil, after removal of the volatile terpenes of example 3, was allowed to stand at room temperature, a white solid settled at the bottom of the flask. This was removed by filtration and characterized as glechomafuran, which is surprisingly insoluble in the oil. HPLC of the isolated glechomafuran is shown in Figure 1.

[0135] Example 9

[0136] The fines, as in Example 4, from removal of the husk from the seeds (30 g) in example 2 were extracted with petroleum for 8 h at ambient temperature and 4 h at 40°C. This gave a yellow / brown oil from which a solid separated on standing. The solid (0.24 g) was glechomafuran; the remaining oil (1 .9 g) was mixed terpenes.

[0137] Example 10

[0138] The oil obtained in example 7, was dissolved in acetone and held in a freezer at -20°C for 18 hours. Crystals were formed, filtered, washed with chilled (-20°C) acetone and dried by evaporation to give a waxy tan solid. The tan solid product could be further purified by dissolving in hexane and passed through a silica gel pad and the hexane removed under vacuum to provide an off white solid, m.pt. 35°C. The carbon NMR of the crystalline triglyceride showed only two peaks in the alkene region, corresponding to the two unsaturated carbons of petroselinic acid as the ester of glycerol; the signals for linoleate and oleate that were present in the crude oil had been removed. The proton NMR was also that of tripetroselinin.

[0139] The strong smell of the pressed oil was not present in the triglyceride. This was used to prepare cakes that had a similar texture and taste to those made with palm oil.

[0140] Example 11

[0141] The oil remaining after low temperature recrystallization of example 9 contained around 35 % of 1 p-acetoxyfuranoeudesm-4(15)-ene as well as triglycerides, largely not tri-petroselinin. It was dissolved in acetone, kept in a -80°C freezer overnight and then rapidly filtered to remove solid triglyceride. The process was repeated, when evaporation of the remaining acetone solution gave 1 p-acetoxyfuranoeudesm-4(15)-ene.

[0142] In an alternative separation of the sesquiterpenes the triglyceride was hydrolysed using 5 % aqueous sodium hydroxide and extracted with an organic solvent. This gave an aqueous fraction containing the sodium salts of fatty acids, predominantly petroselinic acid, and an organic fraction containing 1 p-acetoxyfuranoeudesm-4(15)-ene and glechomafuran.

[0143] The two sesqiterpenes were separated by column chromatography and characterized by gems, Ic and proton or carbon NMR. The HPLC of the 1 p-acetoxyfuranoeudesm-4(15)-ene is shown in Figure 1 .

[0144] Example 12

[0145] The seed used in example 1 , was analysed for moisture and protein levels, which were found to be 8.8 and 18.4% respectively. After solvent extraction according to Example 4 these values were 15.2 and 17.7% respectively. The analyses were carried out at the Institute of Aquaculture, University Of Stirling, Stirling, Scotland.

[0146] Example 13

[0147] Hydrolyses of petroselinin and isolation of petroselinic acid

[0148] Alexanders seed oil, after pressing (30.8 g) was weighed into a 250ml flask, 2N sodium hydroxide (100 ml) was added followed by MeOH (50 ml). The reaction mixture was refluxed for 3h, cooled down and extracted with hexane. The aqueous phase was acidified to pH 1 with a 10% sulfuric acid solution.

[0149] The mixture was extracted with hexane; the organic phase was then washed with water and dried over magnesium sulfate, filtered and concentrated under reduced pressure to give an oil (26.5 g, 86.03%).

[0150] Analysis, 1 H-NMR and 13C-NMR, showed that the oil is mainly Petroselinic Acid.

[0151] In this example, petroselinic acid was obtained from crude oil containing petroselinin, with no distillation steps. However, the crude oil could be distilled to remove terpenes prior to hydrolysis of petroselinin to petroselinic acid. Equally, terpenes could be removed from the crude oil by extraction with liquid and / or supercritical carbon dioxide before hydrolysis of petroselinin to petroselinic acid.

[0152] Example 14

[0153] Chocolate preparation

[0154] In a first example of chocolate preparation, tripetroselinin (50 g) is melted in a bowl at 50°C until a clear oil then cacao powder (25 g) and powdered sugar (20 g) added and the ingredients mixed with a spatula until a homogeneous smooth mixture is obtained. The mixture is poured into a silicone mould and allowed to cool gradually to room temperature then cooled in the freezer to solidify. The mould is inverted to free the solid bar which is stored in the refrigerator.

[0155] In a second example (producing a darker chocolate) TPS (25 g) and cocoa butter (25 g) are melted in a bowl at 50°C until a clear oil then cacao powder (25 g) and powdered sugar (20 g) added and the ingredients mixed with a spatula until a homogeneous smooth mixture is obtained. The mixture is poured into a silicone mould and allowed to cool gradually to room temperature to solidify. The mould is inverted to free the solid bar which is stored in the refrigerator.

[0156] The chocolate samples had good taste and good melt properties in the mouth.

[0157] Example 15

[0158] Margarine preparation

[0159] Samples of margarine, based on a standard supermarket recipe containing palm fat and vegetable oil, were prepared with Tripetroselinin (TPS) replacing palm fat at 25-100% of palm fat. Viscosity tests were carried out using a Brookfield Engineering Labs instrument to measure hardness, work done, adhesive force and adhesiveness. The composition of the various margarines is shown below:

[0160] (Components wt% shown)

[0161] E471 is mono- and diglycerides of fatty acids and vegetable oil is rapeseed oil.

[0162] The physical testing of the margarine samples showed acceptable properties up to 75% TPS.

[0163] Example 16

[0164] Palm oil free sponge cake preparation

[0165] The oil obtained by the method of the present invention can be used to produce baked goods with a texture and taste similar to such goods produced with solid fats such as palm oil and coconut oil.

[0166] Collaboration with an UK cake company, who produce and supply iced sponge cakes to the ‘free from’ segment of the food industry, resulted in developing a recipe using the refined oil as a total replacement of palm oil. The refined oil-based cake had the desired texture and taste properties as their standard, palm oil-based cake. Samples of the cake were fed to volunteers at an UK University under controlled and ethically approved conditions and the feedback on taste was very positive. The recipe used is considered proprietary by the cake company. These results demonstrate that the oil obtained by the method of the present invention can be used as a total replacement for palm oil in baked goods without compromising on taste or texture.

[0167] Observations on the refined Alexanders seed oil and palm oil containing cakes over time showed that the Alexanders seed oil cake did not develop any mould while the palm oil cake did so after a few weeks.

[0168] Example 17

[0169] Antimicrobial activity The antimicrobial activity of the various fractions isolated were investigated using two species, E. coli and S. aureus, with activity noted for some fractions.

[0170] Bacterial overnight cultures were created by inoculating 5 ml of MHB broth with a single colony from a petri dish. These were incubated at 37°C with shaking for 16-20 hours. Serial dilutions of the compounds were created in MHB from 0.03-2 mg / ml, including a DMSO control at the same concentrations. 50 pl of the serial dilution was added to a 96-well U-bottom plate, including positive and negative controls containing no compound and no bacteria respectively. 2 ml of MHB was inoculated with 100 pl of bacterial overnight culture, and grown at 37°C with shaking until OD 600 nm ~ 0.07. This was performed in biological triplicate for both E. coli and S. aureus. After reaching the desired optical density, cultures were diluted 1 :10 in MHB, and then 50 pl of diluted bacterial culture added to the 96-well plates as appropriate. The plates were incubated at 37°C with shaking for 16 hours before reading OD600 nm using a plate reader. The percentage growth at each concentration of compound was calculated in comparison to growth in absence of compound to find inhibitory values.

[0171] The compounds tested were as follows:

[0172] MIC results are shown in Figure 2. The results show more activity of the compounds against S. aureus than E. coli, particularly for Acetoxy Eudesmene and PSA with PSA showing interesting biphasic activity. These results demonstrate the antimicrobial effects of petroselinic acid and other terpene compounds extracted from Smyrnium olusatrum.

[0173] The forgoing embodiments are not intended to limit the scope of the protection afforded by the claims, but rather to describe examples of how the invention may be put into practice.

Claims

Claims1 . A method of obtaining petroselinin from Smyrnium olusatrum, the method comprising the steps of:(a) removing one or more terpene compounds from a portion of the plant Smyrnium olusatrum or an oil component extracted therefrom; and(b) obtaining petroselinin from the material remaining after step (a) or an extract obtained therefrom; wherein step (a) is carried out by direct distillation or extraction with, for example, liquid and / or supercritical carbon dioxide.

2. The method according to claim 1 wherein step (b) comprises crystallizing petroselinin from the material remaining after step (a) or an extract obtained therefrom.

3. The method according to claim 1 or 2 wherein the method further comprises hydrolysing petroselinin to petroselinic acid.

4. The method according to any preceding claim wherein the portion of the plant Smyrnium olusatrum comprises the seed.

5. The method according to claim 4 wherein the seed is dehulled.

6. The method according to claim 5 wherein the seed husk and core are separated.

7. The method according to any of claims 4 to 6 wherein the seed is milled, crushed or pulverised.

8. The method according to any preceding claim which involves the steps of:(a) removing one or more terpene compounds from a portion of the plant Smyrnium olusatrum by extraction with liquid and / or supercritical carbon dioxide;(x) further contacting the portion of the plant with liquid and / or supercritical carbon dioxide to obtain an oil comprising petroselinin; and(b) crystallization of the oil obtained in step (x) to provide petroselinin in solid form.

9. The method according to claim 8 wherein step (x) also involves contacting the portion of the plant Smyrnium olusatrum with liquid and / or supercritical carbon dioxide.

10. The method according to any of claims 1 to 7 which involves the steps of:(y) obtaining an oil component from a portion of the plant Smyrnium olusatrum(a) directly distilling the oil component obtained in step (y) to remove one or more terpene compounds; and(b) crystallization of the oil obtained in step (a) to provide petroselinin in solid form.

11. The method according to claim 10 wherein step (y) involves cold pressing the seeds of the plant or extraction with a solvent.

12. The method according to any preceding claim wherein step (b) suitably involves inducing crystallization of petroselinin directly from an oil which is rich in petroselinin.

13. The method according to any of claims 1 to 11 wherein step (b) involves crystallization of petroselinic acid following hydrolyzation of the oil component rich in petroselinin.

14. The method according to any preceding claim which produces a sample of petroselinin and / or petroselinic acid which is light in colour.

15. The method according to any preceding claim which produces a sample of petroselinin and / or petroselinic acid which has a low odour.

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