WO2016193755A1

WO2016193755A1 - Process for recovering terpenes from plant material

Info

Publication number: WO2016193755A1
Application number: PCT/GB2016/051651
Authority: WO
Inventors: Jason ORNSTEIN; Yuan DAI
Original assignee: Synshark LLC
Current assignee: Synshark LLC
Priority date: 2015-06-04
Filing date: 2016-06-03
Publication date: 2016-12-08
Anticipated expiration: 2017-12-04

Abstract

The invention provides a process for isolating one or more terpene from tobacco- derived biomass, the process comprising introducing the biomass and an organic solvent into a reactor, decomposing the biomass with the organic solvent at a first temperature, and recovering a liquid product and a solid product from the reactor, wherein the liquid product contains one or more terpene. The invention also relates to associated processes and products.

Description

Process for recovering terpenes from plant material Field

The invention relates to processes for the recovery or isolation of terpenes, such as squalene, from plant material and in particular from tobacco-derived plant material. The processes may also include the isolation of alkaloids. The invention also relates to associated processes and products.

Background

Biological material derived from living, or recently living, organisms is referred to as biomass and this material may contain a wide range of potentially useful components.

Fractionation is a very important process used industrially in order to isolate valuable components or constituents from biomass, such as that derived from tobacco.

Terpenes such as squalene, and alkaloids such as nicotine, are naturally produced in tobacco, but the ability to efficiently recover or isolate these high value products from the biomass determines their marketability. Further, as new processes are developed to genetically increase the production of terpenes and alkaloids, novel methods are needed to improve fractionation yields.

Squalene is widely found in marine animal oils as a trace component. It is suggested that the demand for squalene is the cause for livering' three to five million deep-water sharks a year. This number is expected to increase significantly as the preventative effect in many human diseases of squalene is further explored. Because of this, new sources for squalene such as plants are being developed. Tobacco is extensively farmed and could be a candidate for squalene production, but fractionation yields must be high to deliver cost competitive tobacco squalene. Alkaloids are a group of naturally occurring chemical compounds that contain mostly basic nitrogen atoms. Alkaloids are produced by a large variety of organisms including bacteria, fungi, plants, and animals and they have previously been purified from such sources by acid-base extraction. Alkaloids have a wide range of pharmacological activities including anti-malarial (e.g. quinine), anti-asthma (e.g. ephedrine), anti- cancer (e.g. homoharringtonine), cholinomimetic (e.g. galantamine), vasodilatory (e.g. vincamine), anti-arrhythmic (e.g. quinidine), analgesic (e.g. morphine), antibacterial (e.g. chelerythrine), and anti-hyperglycemic (e.g. piperine). Other alkaloids are well- known stimulants (e.g. caffeine and nicotine).

Summary

According to a first aspect of the invention, there is provided a process for isolating one or more terpenes from tobacco-derived biomass, the process comprising: a)

introducing the biomass and an organic solvent into a reactor, b) decomposing the biomass with the organic solvent at a first temperature, and c) recovering a liquid product and a solid product from the reactor, wherein the liquid product contains one or more terpenes.

In some embodiments, the process comprises fractionation of the liquid product to isolate one or more terpenes. In some embodiments, the process comprises mechanical decomposition of the biomass by centrifugation, shaking or stirring of the biomass and the organic solvent in the reactor.

In some embodiments, the pressure in the reactor is elevated compared to the ambient pressure outside the reactor.

In some embodiments, the first temperature is between o°C and 350°C.

In some embodiments, the biomass has a residence time within the reactor of less than 360 minutes.

In some embodiments, the solvent is selected from the group consisting of: alcohols, supercritical fluids, hydrocarbons including volatile purified hydrocarbons, alkali metal salts including sodium hydroxide and potassium hydroxide, animal-derived fats and oils, vegetable-derived fats and oils, liquid carbon dioxide, water, acetone, and chloroform, and combinations thereof. In some embodiments, the solvent comprises water, hexane, supercritical carbon dioxide, supercritical methanol, or a combination of two or more thereof.

In some embodiments, the process further comprises extracting oil from the solid product. In some embodiments, the process further comprises: d) introducing the solid product into a reactor with an organic solvent; e) further decomposing the solid product with the organic solvent at a second temperature; and f) recovering a second liquid product and a second solid product from the reactor, wherein the second liquid product contains additional terpene.

In some embodiments, the process comprises fractionation of the second liquid product to isolate one or more terpenes.

In some embodiments, the process comprises further treating the second solid product to isolate further terpene.

In some embodiments, the liquid product contains greater than 0.05 wt % solids derived from the biomass.

In some embodiments, the first temperature is between o°C and 150°C and the second temperature is between about o°C and 350°C. In some embodiments, the biomass is prepared by physically reducing the tobacco plant material. In some embodiments, the biomass is prepared by grinding the tobacco plant material. For example, the grinding may be mechanical grinding or cryogenic grinding. In some embodiments, the physical reduction of the plant material is carried out in the presence of one or more solvents.

In some embodiments, the biomass is prepared by drying the tobacco plant material.

In some embodiments, water or supercritical water is added as a facilitator for fractionation.

In some embodiments, the terpene is a hemiterpene, monoterpene, sesquiterpene, diterpene, sesterterpene, triterpene, tetraterpene or polyterpene. In some

embodiments, the terpene is a triterpene, for example, the terpene may be the linear triterpene squalene. In some embodiments, the process is a process for isolating one or more terpenes and one or more alkaloids from tobacco-derived biomass, the liquid product recovered from the reactor containing one or more terpenes and one or more alkaloids. In some embodiments, the process comprises fractionation of the liquid product to isolate one or more terpenes and/or one or more alkaloids.

In some embodiments, the biomass is decomposed in an acidic or polar environment to optimize alkaloid recovery.

In some embodiments, the biomass has a residence time within the reactor of up to 360 minutes to optimize terpene recovery.

In some embodiments, the biomass has a residence time within the reactor of between 3 minutes and 720 minutes to optimize alkaloid recovery.

In some embodiments, the alkaloid is selected from the group consisting of: a pyrrolidine, tropane, pyrrolizidine, piperidine, quinolizidine, indolizidine, pyridine, isoquinoline, oxazole, isoxazole, thiazole, quinazoline, acridine, quinoline, indole, imidazole, purine, β-phenylethylamine, colchicine, muscarine, benzylamine, putrescine, spermidine, sperine, peptide, diterpenes, and a steroid. In some embodiments, the alkaloid is a pyridine, selected from the group consisting of:

trigonelline, ricine, arecoline, nicotine, nornicotine, anabasine, anatabine, actinidine, gentianine and pediculinine. For example, the alkaloid may be nicotine.

According to a second aspect of the invention, there is provided a process of manufacturing a product, the process comprising a process of the first aspect of the invention, and further comprising isolating one or more terpenes from the liquid product and incorporating at least one of the isolated terpenes into the product.

In some embodiments, the product is a cosmetic or pharmaceutical product.

According to a third aspect of the invention, there is provided a product manufactured according to a process of the second aspect of the invention. According to a fourth aspect of the invention, there is provided a use of squalene obtained by a process according to the first aspect of the invention in a cosmetic or pharmaceutical product. According to a fifth aspect of the invention, there is provided a use of nicotine obtained by a process as according to the first aspect of the invention in pharmaceuticals, supplements or smoking experiences.

Brief Description of the Drawings

The various features, advantages and other uses of the present apparatus will become more apparent by referring to the following detailed description and drawing, in which: Figure l is a flow diagram summarizing the process for isolating one or more terpenes from tobacco biomass according to an embodiment of the present invention.

Figure 2 is a GC-MS analysis run on GCMS-QP2010SE (Shimadzu) of a fractioned liquid produced in accordance with an embodiment of the process of the present invention set out in Example 1.

Figure 3 is a graph showing squalene yield from samples produced in accordance with an embodiment of the process of the present invention set out in Example 2, and a GC- MS analysis of a fractioned liquid.

Figure 4 is a graph showing squalene yield from samples produced in accordance with an embodiment of the process of the present invention set out in Example 3, and a GC- MS analysis of a fractioned liquid.

Figure 5 is a graph showing squalene yield from samples produced in accordance with an embodiment of the process of the present invention set out in Example 3, and a GC- MS analysis of a fractioned liquid.

Figure 6 is a graph showing squalene yield from samples produced in accordance with an embodiment of the process of the present invention set out in Example 4, and a GC- MS analysis of a fractioned liquid.

Figure 7 is a graph showing squalene yield from samples produced in accordance with an embodiment of the process of the present invention set out in Example 5, and a GC- MS analysis of a fractioned liquid.

Figure 8 is a graph showing squalene yield from samples produced in accordance with an embodiment of the process of the present invention set out in Example 6, and a GC- MS analysis of a fractioned liquid.

Detailed Description Fractionation is a separation process in which a mixture of components is divided during a phase transition into a number of fractions which are collected based on differences in a specific property of the individual components. Fractionation allows the isolation of multiple components in a mixture in a single run. A typical protocol to isolate a pure chemical agent from natural origin is bioassay-guided fractionation, which involves the step-by-step separation of extracted components based on differences in their physicochemical properties, and assessing the biological activity, followed by next round of separation and assaying. The starting material from which desired components including terpenes and alkaloids are to be isolated include biomass derived from plants. Suitable plants include tobacco. In some embodiments, plants are selected which are a good source of the desired components to be isolated. Plants may, for example, be bred to have high levels of the desired components and/or maybe genetically modified to have high levels of those components.

Biomass is organic matter derived from plants and it includes lignocellulosic biomass, for example, harvested plant matter such as leaf and stalk. The biomass may be virgin biomass derived (directly) from plants, or it may be waste biomass, which is a low value by-product of biomass processing. In preferred embodiments, the biomass is tobacco- derived. In some embodiments, it comprises fresh, dried and/or cured leaves of a tobacco plant. For clarity, it is confirmed that seeds and oils extracted from plants, for example from their seeds, are not suitable for use as the biomass starting material in the processes of the present invention.

In some embodiments of the present invention, a process for recovering terpenes, and optionally alkaloids, from a tobacco-derived biomass is provided, the process including feeding the biomass and an organic solvent into a reactor, decomposing the biomass with organic solvent, or with an organic solvent and water or supercritical water, at a temperature between o°C and 350°C and recovering a liquid product and solid product from the reactor, wherein the liquid product contains at least one terpene. In some embodiments, the liquid product further includes one or more alkaloid. In some embodiments, the liquid product includes squalene and nicotine. The term "reactor" is used to refer to a container or vessel within which the process occurs. In some embodiments, the reactor is an apparatus that allows the temperature or the pressure within it to be controlled and/ or adjusted. Alternatively or in addition, the reactor is an apparatus that allows the contents to be agitated, for example by shaking, stirring, blending or milling. Thus, according to another aspect of the invention, there is provided a process for isolating one or more terpenes from tobacco-derived biomass, the process comprising: a) introducing the biomass and an organic solvent into a vessel, b) physically reducing the biomass with the organic solvent at a first temperature, and c) recovering a liquid product and a solid product from the reactor, wherein the liquid product contains one or more terpenes.

In some embodiments, the solvent may be added to the biomass after it has been physically reduced. Optionally, an additional step of physical reduction of the biomass and solvent may be carried out, or the mixture may be agitated, such as by shaking or stirring.

In some embodiments of the present invention, a process is provided for isolating one or more terpenes and/or one or more alkaloids from a biomass starting material. In some embodiments, the terpene to be isolated is squalene. In some embodiments, the alkaloid to be isolated is nicotine.

For decomposing of tobacco plant with organic solvent, the objective is to develop a process which maximizes terpene, or terpene and alkaloid yield in liquid phase. In order to decompose the maximum amounts of terpene, specifically squalene, from the tobacco leaves biomass in a liquid phase.

Processes according to the present invention may include one or more steps of treating the biomass to enhance removal of the terpenes from the biomass, referred to as "decomposition" or "decomposing" the plant material herein. In some embodiments, the biomass is treated to physically reduce the biomass, thereby disrupting the physical structure of the biomass. The treatment may involve, for example, separating parts of the biomass and/or disrupting individual plant cells to access the interior of such cells. Such treatment may involve cutting, chopping, milling, grinding, crushing, blending, and the like. Additionally or alternatively, the biomass may be physically reduced or decomposed by adjusting the pressure and/ or temperature. In some embodiments, the processes of the invention may also include a step of drying the biomass. The biomass may be dried by any suitable method, including air drying, oven drying at elevated temperatures or freeze-drying. The drying step may be carried out prior to contacting the biomass with a solvent and before any decomposition step to further physically reduce the biomass.

Solvents suitable for use in the processes of the invention include those in which terpenes such as squalene are soluble. In some embodiments, the solvent is selective for terpenes, so that upon contact with the biomass, terpenes are solubilized whilst other components of the biomass are not solubilized or are solubilized to a lesser degree. Suitable solvents include organic solvents. Solvents used may be selected from: alcohols including methanol, ethanol, propanol (such as 2-propanol);

supercritical fluids including supercritical carbon dioxide and supercritical methanol; hydrocarbons including volatile purified hydrocarbons such as petroleum ether and hexane; alkali metal salts including sodium hydroxide, potassium hydroxide; animal- derived fats and oils; vegetable-derived fats and oils; liquid carbon dioxide; water; acetone; and chloroform. In some embodiments, combinations of solvents may be used. For example, the solvent used may comprise a combination of supercritical carbon dioxide and an alcohol (such as ethanol or methanol) or a hydrocarbon solvent.

In some embodiments, the treatment to physically reduce or decompose the biomass may be conducted in the presence of one or more solvents. For example, the biomass may be milled, chopped or blended in the presence of a solvent such as water, hexane or supercritical carbon dioxide. This results in a slurry of biomass from which terpenes may be separated, preferably from the liquid phase.

The process may, in some embodiments, include further steps to break down and/or remove unwanted constituents from the biomass or from an extract isolated from the biomass, for example the liquid phase. Such steps may include, for example:

adsorption of components using an adsorbent material; absorption of components using an absorbent material; saponification; and cracking.

Figure 1 is a flow diagram of a process for isolating one or more terpenes from a tobacco-derived biomass 1. As illustrated in step 2, the biomass is subjected to physical reduction treatment. This step may comprise one or more of freeze-drying, grinding and drying. The physically reduced biomass resulting from step 2 is next contacted with a solvent in step 3. Suitable solvents include water, hydrocarbons such as hexane and supercritical fluids such as supercritical carbon dioxide, or combinations thereof. The solvents can be used to extract oil from the biomass after physical reduction in step 2. Extraction can be performed using a reactor vessel, as a non-limiting example.

In order to increase the efficacy and/ or efficiency of the decomposition of the biomass and of the solvent extraction, the combination of the physically reduced biomass and solvent may be subjected to further processing, as shown in step 4. This processing may include adjusting the pressure within the reactor, adjusting the temperature within the reactor or agitation, such as stirring, blending or otherwise creating turbulence within the slurry comprising the physically reduced biomass and solvent. The liquid phase and solid phase of the slurry are separated after the extraction in step 5, with the liquid phase being terpene oil 6 which can be used as a cosmetic or pharmaceutical precursor. The solid phase is a wet cake which can be used as bio char or which can optionally undergo further processing to extract more terpenes or other components.

Present in tobacco biomass with terpenes are alkaloids. It improves the economic feasibility of tobacco terpene production if alkaloids, specifically nicotine, are also recovered in the fraction process. The nicotine oil market is growing in response to the shift towards electronic and other alternative smoking experiences.

Examples

Example 1

In order to determine how to isolate the maximum amounts of terpene, specifically squalene, from the liquid phase of a decomposed biomass derived from tobacco leaves, a set of experiments were performed at varying residence time and temperature in a reactor as described below in Table 1.

Table 1

I Process parameters I Run Temperature (°C) Residence time (minutes)

Exp l 0 150

Exp 2 30 120

Exp 3 6o 90

Exp 4 90 60

Exp 5 120 30

For all five experiments, puree derived from tobacco leaves (Nicotiana Tabacum L. 1068) was used. To obtain a uniform and more flowable material, additional liquid nitrogen was added to the tobacco leaves. The prepared puree measured as 14 wt % solids. The tobacco leaves puree (500 mg), hexane (3 mL) and cedrene (27 μΐ at 1000 ppm) were placed in a shaker reactor at 200 rpm for two hours. The cedrene was added at a final concentration (9 ppm) to serve as an internal control. Extracts (2 mL) were withdrawn and loaded onto a silica column (0.5 g) in a glass pipette, collecting the flow through. Additional hexane (4 mL) was added to the column, to collect the flow through.

The compositional analysis on the flow through (2 mL) was performed and the results are shown in Figure 2. One microliter of sample was injected into the GC-MS using an AOC-201 auto-sampler in 10:1 split mode (injector 28o°C) onto a ZB-5MS1 fused silica capillary column (30 m x 0.25 mm x 0.25 μπι thickness). The initial oven temperature was 40°C, which was ramped to 120°C at 20°C/min, then ramped to 200°C at 6°C/min, then ramped to 26o°C at 20°C/min, and finally ramped to 3io°C for 3 min at 5°C/min. Helium was used as the carrier gas. The ion source was set to 230°C and the interface was 28o°C.

Squalene quantification was performed using selected ions. Peak identification of the compound was performed using direct comparison of the sample mass chromatogram with those of commercially available standard compounds. The quantitative calculations of squalene concentration were based on the peak area ratios relative to those of the standard.

Example 2 In a further set of experiments, tobacco leaves (8.35 g) were dried in an oven at 65.6°C (i50°F) for different time periods. The drying times are listed in Table 2.

Table 2

The biomass had reached a stable weight after six hours in the oven. It was therefore concluded that fresh tobacco leaves could be dried in 6 hours in an oven at 65.6°C, and that longer drying periods provided no additional benefit. The drying time will vary under different experiment conditions, such as drying temperature, amount of leaves and capacity of the oven.

2.5 month-old Gi leaf was dried in a 65.6°C (i50°F) oven for 6 hours. Samples of approximately 0.5 g of the dried leaf were ground in the presence of liquid nitrogen to produce a puree, and were then analyzed using the standard protocol (as set out in Example 1). As a comparison, 0.5 g samples of fresh leaf were also processed to form a puree and were then analyzed using the same standard protocol.

The squalene yields are shown in the graph of Figure 3. The black bars (Samples 1 to 3) represent the squalene yield from the fresh leaf samples, and the grey bars (Samples 4 to 6) represent the squalene yield from the samples of dried tobacco leaves. The amounts of squalene are provided as g of squalene per g of Fresh Weight (FW) leaf and the dry weight was converted to fresh weight for dried samples according to the dry weight vs fresh weight ratio to facilitate the comparison of squalene amounts between the treatment and control.

From the results we can see that the squalene yields from the fresh samples and dried samples were comparable. This result suggests that squalene was not severely degraded or lost as a result of the fast drying process used to prepare the dried samples, and it confirms that this fast drying process could be adopted when extracting squalene from tobacco-derived biomass. Extracting squalene from dried biomass is attractive as squalene is not expected to be completely stable in plant material and is expected to be gradually degraded over time by enzymes. Removing water from the plant material by drying can reduce or halt enzyme activity.

Example 3

In a further set of experiments, tobacco leaves (10 g) were blended in a household blender with hexane at different volumes for ι minute. The volume of hexane used is listed in Table 3.

Table 3

The blended tobacco leaves were centrifuged at 4000 rpm and the supernatant was loaded onto a silica column and the extracted squalene was analyzed as described in Example 1. As a comparison, 0.5 g samples of fresh leaf were prepared in liquid nitrogen as described in Example 1 and were also analyzed using the standard protocol.

The squalene yields are shown in the graph of Figure 4. The black bars (Samples 1 to 3) represent the squalene yield from the fresh leaf samples, and the grey bars (Samples 4 to 6) represent the squalene yield from the leaf which had been blended for 1 minute with 60 ml hexane (6: 1 ratio).

Because the squalene yields achieved using these two extraction methods are comparable (see the graphs of Figures 3 and 4), the efficiency of these methods are the same. In other words, the wet blending or milling method could be adopted when extracting squalene from tobacco-derived biomass.

The effects of reducing the amount of hexane used in wet milling extraction were also assessed and the results are shown in Figure 5. The black bars (Samples 1 to 3) show the squalene yield from fresh leaf samples measured using the standard protocol, the grey bars (Samples 4 to 6) show the squalene yield from samples blended with hexane in a ratio of 3:1 (30 ml hexane for 10 g of tobacco leaf), and the white bars (Samples 7 and 8) show the squalene yield from samples blended with hexane in a ratio of 2:1 (20 ml hexane for 10 g of tobacco leaf). Because the results were similar, the squalene extraction efficiency was not affected by reducing the volume of hexane used. In other words, the hexane amount used for wet milling could be reduced to 2 ml per gram tobacco leaf without adversely affecting the squalene yield.

Example 4

In yet another set of experiments, tobacco leaves (10.5 g) were blended with 21 mL water in a household blender for 1 minute. Samples of approximately 3 to 4 g of blended tobacco leaves were withdrawn at different times after blending (o, 0.5, 1, 2, 3, 4, 6, 24 hours).

After withdrawal, the water blended tobacco leaves were frozen to -8o°C for storage. 0.5 g samples of the frozen tobacco material were weighed and dried with a moisture meter at 105°C for approximately 30 minutes. The dried material was mixed with liquid nitrogen and the puree was processed and the squalene yields calculated as described in Example 1. The results are shown in the graph of Figure 6. Whilst it was expected to see a linear change in the squalene yields the longer the samples were kept at room temperature following blending, this was not reflected in the results. The results suggest that the squalene is not significantly degraded over time when aqueous blended samples are kept for up to 24 hours at room temperature following blending. The results show less than a 30% reduction in squalene yield when comparing the sample withdrawn 24 hours after blending to the sample withdrawn immediately (24 hour and o hour results in graph).

The aqueous blends of tobacco leaf provide one physical format to process the leaf for downstream extraction process. The aqueous blends enable efficient size expansion of the biomass and facilitate extraction process that requires biomass particle to exceed certain diameters.

Example 5

About 10 g of 3 month TP5 tobacco leaves were first dried in a 65.6°C (i50°F) oven overnight. Then, the dried materials were ground in a blender with 30 ml of hexane (3:1 ratio). The samples were analyzed using the standard protocol set out in Example 1.

The squalene yields are shown in the graph of Figure 7. The black bars (Samples 1 to 3) represent the squalene yield from the fresh leaf samples prepared using the standard method as set out in Example 1, and the grey bars (Samples 4 to 6) represent the squalene yield from the samples of wet milled dried tobacco sample. Because the yields are similar, the efficiency of wet milling method for dried tobacco leaf is similar with the standard squalene extraction method.

Example 6

50 g of tobacco leaves were incubated with supercritical (SC) C0₂ (pressure: 7446.3 kPa (1080 psi), temperature: 50°C). The leaf samples were extracted 3 times with SCF C0₂ (Extracts 1, 2 and 3), and the incubation time was 30 minutes, 30 minutes and 1 hour, respectively. After extraction, plant debris was removed from the extraction vessel and the extraction vessel was washed with 20 ml of hexane. The extraction samples and wash samples were all tested using standard method.

The squalene yields are shown in the graph of Figure 8. The yields were disappointing. Although squalene could be detected following the supercritical fluid extraction, the yields were only about 20% of those seen using the standard protocol. However, this is indicative of a need to refine the supercritical fluid extraction process to enhance the squalene yield. It is possible, for example, that the experiment was not performed under proper supercritical conditions. The person skilled in the field of supercritical fluid extraction would have no difficulty in identifying how to adapt the basic process used in this example in order to increase the squalene yield, using the teaching herein.

Indeed, it is known that supercritical carbon dioxide can be effective in extracting terpenes such as squalene. For example, in Kraujalis, P. et al, The Journal of

Supercritical Fluids, Vol 80, August 2013, pp 78-85 "Supercritical carbon dioxide extraction of squalene and tocopherols from amaranth and assessment of extracts antioxidant activity", a study is described in which tocopherols and squalene are extracted from a lipophilic fraction of amaranth seeds using supercritical fluid extraction with carbon dioxide (SCE-C0₂) under different pressure conditions, optionally adding 2 and 5% of co-solvent ethanol, which provides excellent yields. The experiments demonstrate that it is possible to achieve commercially significant yields of terpenes including squalene from tobacco-derived biomass using the processes of the present invention. The terpene yield may be enhanced by exposure of biomass starting material to one or more solvents, in combination with one or more steps to physically reduce the biomass starting material.

The entirety of this disclosure shows by way of illustration various embodiments in which the claimed invention(s) may be practiced. The advantages and features of the disclosure are of a representative sample of embodiments only, and are not exhaustive and/ or exclusive. They are presented only to assist in understanding and teach the claimed features. It is to be understood that advantages, embodiments, examples, functions, features, structures, and/or other aspects of the disclosure are not to be considered limitations on the disclosure as defined by the claims or limitations on equivalents to the claims, and that other embodiments may be utilized and

modifications may be made without departing from the scope and/ or spirit of the disclosure. Various embodiments may suitably comprise, consist of, or consist essentially of, various combinations of the disclosed elements, components, features, parts, steps, means, etc. In addition, the disclosure includes other inventions not presently claimed, but which may be claimed in future.

Claims

1. A process for isolating one or more terpenes from tobacco-derived biomass, comprising:

a) introducing the biomass and an organic solvent into a reactor,

b) decomposing the biomass with the organic solvent at a first temperature, and

c) recovering a liquid product and a solid product from the reactor, wherein the liquid product contains one or more terpenes.

2. A process of claim 1, comprising fractionation of the liquid product to isolate one or more terpenes.

3. A process of claim 1 or claim 2, comprising mechanical decomposition of the biomass by centrifugation, shaking or stirring of the biomass and the organic solvent in the reactor.

4. A process of any one of the preceding claims, wherein the pressure in the reactor is elevated compared to the ambient pressure outside the reactor.

5. A process of any one of the preceding claims, wherein the first temperature is between o°C and 350°C.

6. A process of claim 1, wherein the biomass has a residence time within the reactor of less than 360 minutes.

7. A process of any one of the preceding claims, wherein the solvent is selected from the group consisting of: alcohols, supercritical fluids, hydrocarbons including volatile purified hydrocarbons, alkali metal salts including sodium hydroxide and potassium hydroxide, animal-derived fats and oils, vegetable-derived fats and oils, liquid carbon dioxide, water, acetone, and chloroform, and combinations thereof.

8. A process of any one of the preceding claims, wherein the solvent comprises water, hexane, supercritical carbon dioxide, supercritical methanol, or a combination of two or more thereof.

9. A process of any one of the preceding claims, further comprising extracting from the solid product.

A process of any one of the preceding claims, further comprising:

d) introducing the solid product into a reactor with an organic solvent;

e) further decomposing the solid product with the organic solvent at a second temperature; and

f) recovering a second liquid product and a second solid product from the reactor, wherein the second liquid product contains additional terpene.

11. A process of claim 10, comprising fractionation of the second liquid product to isolate one or more terpenes.

12. A process of claim 10, comprising further treating the second solid product to isolate further terpene.

13. A process of any one of the preceding claims, wherein the liquid product contains greater than 0.05 wt % solids derived from the biomass.

14. The process of claim 10, wherein the first temperature is between o°C and 150°C and the second temperature is between about o°C and 350°C.

15. A process of any one of the preceding claims, wherein the biomass is prepared by physically reducing the tobacco plant material.

16. A process of claim 15, wherein the biomass is prepared by grinding the tobacco plant material.

17. A process of claim 16, wherein the grinding is mechanical grinding or cryogenic grinding.

18. A process of any one of claims 15 to 17, wherein the physical reduction of the plant material is carried out in the presence of one or more solvents.

19. A process of any one of the preceding claims, wherein the biomass is prepared by drying the tobacco plant material.

20. A process of claim 2, wherein water or supercritical water is added as a facilitator for fractionation.

21. A process of any one of the preceding claims, wherein the terpene is a hemiterpene, monoterpene, sesquiterpene, diterpene, sesterterpene, triterpene, tetraterpene or polyterpene.

22. A process of claim 21, wherein the terpene is a triterpene.

23. A process of claim 21, wherein the terpene is the linear triterpene squalene.

24. A process of any one of the preceding claims, for isolating one or more terpenes and one or more alkaloids from tobacco-derived biomass, the liquid product recovered from the reactor containing one or more terpenes and one or more alkaloids.

25. A process of claim 24, comprising fractionation of the liquid product to isolate one or more terpenes and/or one or more alkaloids.

26. A process of claim 24, wherein the biomass is decomposed in an acidic or polar environment to optimize alkaloid recovery.

27. A process of claim 24, wherein the biomass has a residence time within the reactor of up to 360 minutes to optimize terpene recovery.

28. A process of claim 24, wherein the biomass has a residence time within the reactor of between 3 minutes and 720 minutes to optimize alkaloid recovery.

29. A process of claim 24, wherein the alkaloid is selected from the group consisting of: a pyrrolidine, tropane, pyrrolizidine, piperidine, quinolizidine, indolizidine, pyridine, isoquinoline, oxazole, isoxazole, thiazole, quinazoline, acridine, quinoline, indole, imidazole, purine, β-phenylethylamine, colchicine, muscarine, benzylamine, putrescine, spermidine, sperine, peptide, diterpenes, and a steroid.

30. A process of claim 29, wherein the alkaloid is a pyridine, selected from the group consisting of: trigonelline, ricine, arecoline, nicotine, nornicotine, anabasine, anatabine, actinidine, gentianine and pediculinine.

31. A process of claim 30, wherein the alkaloid is nicotine.

32. A process of manufacturing a product, comprising a process of claim 1 and further comprising isolating one or more terpenes from the liquid product and incorporating at least one of the isolated terpenes into the product.

33. A process of claim 32, wherein the product is a cosmetic or pharmaceutical product.

34. A product manufactured according to a process of claim 32.

35. Use of squalene obtained by a process as claimed in any one of claims 1 to 31 in a cosmetic or pharmaceutical product.

36. Use of nicotine obtained by a process as claimed in any one of claims 1 to 31 in pharmaceuticals, supplements or smoking experiences.