Maca extract and preparation thereof

Abstract

Described herein is an oil-based composition comprising a maca extract, in particular an extract comprising macamides and macaenes, and uses thereof. Also described herein is a process of producing an oil-based composition comprising a maca extract, and uses of the oil-based composition comprising a maca extract.

Description

MACA EXTRACT AND PREPARATION THEREOFFIELD OF THE INVENTION

[0001] The present invention relates to an oil-based composition comprising a maca extract, in particular an extract comprising macamides and macaenes, and uses thereof. The present invention also relates to a process of producing an oil-based composition comprising a maca extract.BACKGROUND TO THE INVENTION

[0002] The maca (Lepidium meyenii) plant is native to South America and also grown in China. Maca is also known as Peruvian ginseng, and is grown for consumption of the root, which is generally dried and powdered. As a root vegetable native to the Andes of Peru, it has been used as a traditional medicine by the indigenous people of the region for centuries for its health properties associated with improving physical strength, resilience, endurance, and enhancement of reproductive functions. Recent clinical studies have shown maca to be beneficial in managing hormonal imbalances, improving reproductive function, improving mental health, improving neurological function, reducing inflammation, and reducing fatigue (Zhou et al. 2017).

[0003] Maca roots occur as three main phenotypes, differentiated by the colouration of their hypocotyl and stem: red, black, and yellow. These colours are produced from the same seed crop but in different ratios and each exhibit unique properties. Yellow maca is the most abundant of the three, constituting approximately 65% of the harvest. Black maca is the rarest phenotype of maca, constituting approximately 10% of the harvest and is considered the most sacred by the indigenous Andean people. It has been shown to be effective in supporting male fertility and sexual function, and in improving cognitive function and memory. It has also been shown to reduce blood glucose levels and combat oxidative stress. Red maca is slightly more common than black maca, constituting the remaining 25% of the harvest. It is known to improve prostate function in men, possesses anti-inflammatory properties to support wound healing and immunity, and has been shown to effectively improve mood and support female fertility. Furthermore, both black and red maca have been shown to be effective at improving bone composition and strength.

[0004] Macamides and macaenes are bioactive secondary metabolites unique to maca and have thus been the focus of much of the literature concerned with understanding the mechanisms behind the health benefits of the root. Macamides are benzylamide or methoxybenzylamide derivatives formed from their combination with a free-fatty acid of varying length and degree of saturation, while macaenes are derived from the oxidation of free-fatty acids.

[0005] Emerging evidence suggests maca has an additional benefit of supporting the endocannabinoid system (ECS). The ECS is a critical neuromodulatory system, important for regulating a broad spectrum of functions, including pain, appetite, digestion, sleep, inflammation, and mood (Zou & Kumar, 2018). The endocannabinoid system comprises the cannabinoid receptors CB1, CB2 and possibly others; the fatty acid derivatives which act as their endogenous ligands; and the proteins responsible for the synthesis, reuptake and degradation of these endogenous ligands (Scotter et al. BrJ Pharmacol. 2010 Jun; 160(3): 480-498.) CB1 receptors are predominantly found in the central nervous system, whereas CB2 receptors are located on immune cells. Macamides are structurally similar to an endocannabinoid, anandamide (AEA) (Hajdu et al. 2014; Zhu et al. 2020). AEA is critically important for modulating nerve conduction and the immune system. Activation of cannabinoid receptors by AEA leads coupling to Gi proteins, resulting in reduced intracellular cyclic AMP levels, and subsequent physiological responses (Pertwee, 1997).

[0006] One problem with current methods of extraction of macamide and macaenes from maca root is relatively poor yield. Another problem is providing maca extracts in a desirable, natural, and concentrated form. Maca extracts are often provided in the form of dried powders, which can be difficult to ingest in sufficient quantity when taken orally, as it is typically blended with other ingredients. The powders furthermore tend to have low macamide and macaene levels and are therefore of more limited efficacy in the body.

[0007] Methods of extracting macamides and macaenes from dry maca root or dried maca powders using ethanol-based solvents are common in the art. As macamides are lipophilic in nature, their solubility in a polar-based solvent such as ethanol is limited, resulting in a low concentration solution. Furthermore, such ethanolic extracts are poorly soluble in oil.

[0008] Methods of isolating macamides and macaenes at high concentration are described in the prior art using chromatography-based separation. This is however best suited for small-scale extractions and for quantitative analysis of macamides andmacaenes (Xia et al. 2019) as it is not practical to process larger amounts of maca in this fashion. Furthermore, reversed-phase chromatography introduces the risk of poor elution of macamides from the column due to the presence of fatty chains in their structure.

[0009] Another method of providing a high-concentration of a macamide is through direct chemical synthesis (Singh et al. 2020). This is however an expensive and impractical way of providing a single macamide, let alone multiple macamides, and may not be acceptable to consumers given the synthetic origin. Furthermore, the synthesis process, while perhaps appropriate for delivering a very high concentration of a single macamide as a drug via parenteral administration, may not be compatible with a consumer product that is intended to be taken orally, topically or sublingually.

[0010] There is therefore an ongoing need for improved natural maca extracts, and a safe, food-compatible, consumer-compatible and cost-effective process of producing them. It is an object of the present invention to go some way to meeting these needs and / or overcome some or all of the disadvantages of the prior art, and / or to at least provide the public with a useful choice.

[0011] Other objects of the invention may become apparent from the following description which is given by way of example only.

[0012] Any discussion of documents, acts, materials, devices, articles, or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date.SUMMARY OF THE INVENTION

[0013] In a first aspect, the present invention provides an oil-based composition comprising a maca extract, wherein the maca extract comprises at least about 50 mg / g maca compounds.

[0014] In a second aspect, the invention provides an oil-based composition comprising a maca extract, wherein the maca extract comprises: a. macamide / V-Benzyloctadec-9Z-enamide; b. macamide / V-Benzyloctadeca-9Z,12Z-dienamide;c. macamide / V-Benzyloctadeca-9Z,12Z,15Z-trienamide; and d. macamide / V-(3-Methoxybenzyl)octadeca-9Z,12Z-dienamide.

[0015] In a third aspect, the invention provides a process of producing an oil-based composition comprising a maca extract, the process comprising: providing a maca root; suspending the maca root in an oil; contacting the oil comprising the maca root with a solvent comprising an alkane at a temperature and pressure at which the solvent is liquid, thereby producing the composition.

[0016] In a fourth aspect, the invention provides an oil-based composition comprising the maca extract when produced by the process of the third aspect.

[0017] In a fifth aspect, the invention provides use of the composition of the first or second or fourth aspects for the treatment of diseases or disorders selected from the group consisting of acne, addiction, anxiety, arthritis, autism spectrum disorder, disorders stemming from autoimmunity, side effects resulting from cancer treatment, chronic pain, chronic fatigue syndrome, depression, digestive disorders, epileptic seizures, irritable bowel syndrome, Crohn's disease, ulcerative colitis, high blood pressure, hormonal imbalances, inflammation, insomnia, disorders of the libido, menopause, mental health disorders, muscular pain, neurological conditions, Parkinson's disease, post-traumatic stress disorder, seizures, disorders of the skin, injury to the skin, eczema, psoriasis, sexual function disorders, and stress.

[0018] The following embodiments and preferences may relate alone or in any combination of any two or more of any of the above aspects.

[0019] In various embodiments, the maca extract comprises at least about 50 mg / g total macamides.

[0020] In various embodiments, the maca extract comprises at least about 3 mg / g total macaenes.

[0021] In various embodiments, the maca extract comprises at least about 50 mg / g total macamides and at least about 3 mg / g total macaenes.

[0022] In various embodiments, the maca extract comprises: a. at least about 9 mg / g macamide / V-Benzyloctadec-9Z-enamide; and / or b. at least about 23 mg / g macamide / V-Benzyloctadeca-9Z,12Z-dienamide; and / or c. at least about 3 mg / g macamide / V-Benzyloctadeca-9Z,12Z,15Z- trienamide; and / or d. at least about 4 mg / g macamide / V-(3-Methoxybenzyl)octadeca-9Z,12Z- dienamide.

[0023] In various embodiments, the maca extract comprises at least one maca compound selected from the group consisting of a. macamide / V-Benzylpalmitamide, b. macamide / V-Benzyloctadecanamide, c. macamide / V-(3-Methoxybenzyl)octadeca-9Z,12Z,15Z-trienamide, d. macamide / V-(3-Methoxybenzyl)octadec-9Z-enamide, e. macamide / V-(3-Methoxybenzyl)hexadecanamide, f. macamide / V-(3-Methoxybenzyl)octadecanamide, g. macamide / V-Phenethyloctadeca-9Z,12Z-dienamide, h. macamide / V-Phenethyloctadeca-9Z,12Z,15Z-trienamide, i. macamide / V-Benzylicosa-5Z,8Z,llZ,14Z-tetraenamide, j. macamide / V-Benzylicosa-5Z,8Z,llZ,14Z,17Z-pentaenamide, k. macaene 9-oxo-octadeca-10,12-dienoic acid, and l. macamide / V-Benzyl-9-oxo-octadeca-10,12-dienamide.

[0024] In various embodiments, the maca extract comprises at least about 4 mg / g macamide / V-Benzylpalmitamide.

[0025] In various embodiments, the maca extract comprises at least about 0.2 mg / g macamide / V-Benzyloctadecanamide.

[0026] In various embodiments, the maca extract comprises at least about 0.5 mg / g macamide / V-(3-Methoxybenzyl)octadeca-9Z,12Z,15Z-trienamide.

[0027] In various embodiments, the maca extract comprises at least about 0.4 mg / g macamide / V-(3-Methoxybenzyl)octadec-9Z-enamide.

[0028] In various embodiments, the maca extract comprises at least about 0.2 mg / g macamide / V-(3-Methoxybenzyl)hexadecanamide.

[0029] In various embodiments, the maca extract comprises at least about 0.02 mg / g of / V-(3-Methoxybenzyl)octadecanamide, about 0.04 mg / g / V-Phenethyloctadeca- 9Z,12Z-dienamide, about 0.01 mg / g / V-Phenethyloctadeca-9Z,12Z,15Z-trienamide, about 0.01 mg / g / V-Benzylicosa-5Z,8Z,llZ,14Z-tetraenamide, and / or about 0.04 mg / g N- Benzylicosa-5Z,8Z,llZ,14Z,17Z-pentaenamide.

[0030] In various embodiments, the maca extract comprises least about 3 mg / g macaene 9-oxo-octadeca-10,12-dienoic acid.

[0031] In various embodiments, the maca extract comprises at least about 4 mg / g macamide / V-Benzyl-9-oxo-octadeca-10,12-dienamide.

[0032] In various embodiments, the composition comprises at least one terpene and / or terpenoid.

[0033] In various embodiments, the at least one terpene is selected from the group consisting of alpha bisabolol, alpha phellandrene, alpha pinene, beta caryophyllene, beta pinene, cadinene, camphene, citral, citronellol, delta 3 carene, eucalyptol, eugenol, gamma terpinene, geraniol, humulene, limonene, linalool, myrcene, nerol, nerolidol, ocimene, para-cymene, terpineol, terpinolene, and valencene.

[0034] In various embodiments, the composition comprises the at least one terpene and / or terpenoid in a total concentration of from about 0.1 to about 5% w / w%.

[0035] In various embodiments, the oil is selected from the group consisting of arnica oil, avocado oil, black seed oil, canola oil, coconut oil, flaxseed oil, frankincense oil, grapeseed oil, Kakadu plum seed oil, macadamia oil, medium chain triglyceride (MCT) oil, native sandalwood oil, olive oil, palm oil, rice bran oil, sesame oil, soya oil, sunfloweroil, vegetable oil, walnut oil, hemp seed oil, mineral oil, peanut oil, tung oil, bees wax, Danish oil, black cumin seed oil, coconut MCT oil, castor oil, and any combination of any two or more thereof.

[0036] In various embodiments, the oil is MCT oil.

[0037] In various embodiments, the composition comprises residual alkane.

[0038] In various embodiments, the alkane is selected from the group consisting of propane, butane, isobutane, pentane and isomers thereof, hexane and isomers thereof, and heptane and isomers thereof.

[0039] In various embodiments, the alkane is propane or butane.

[0040] In various embodiments, the alkane is propane.

[0041] In various embodiments, the process further comprises drying the maca root before contacting the maca root with the oil.

[0042] In various embodiments, the process further comprises comminuting the maca root to produce maca root particles before contacting the maca root with the oil.

[0043] In various embodiments, contacting the oil comprising the maca root with the solvent is performed at a pressure of from about 10 to about 50 bar.

[0044] In various embodiments, the process results in a yield of from about 0.2 to about 1.5% of macamides and macaenes.

[0045] In various embodiments, the process results in a yield of from about 0.5 to about 0.7 % of macamides and macaenes.

[0046] In various embodiments, the process provides a maca extract comprising a greater ratio of macamides to macaenes than the ratio found in the maca root.

[0047] In various embodiments, the composition is administered orally, sublingually, or topically.

[0048] The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in theart to which the invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

[0049] It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5, and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are hereby expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

[0050] In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

[0051] To those skilled in the art to which the invention relates, many changes in construction and widely differing embodiments and applications of the invention will suggest themselves without departing from the scope of the invention as defined in the appended claims. The disclosures and the descriptions herein are purely illustrative and are not intended to be in any sense limiting.

[0052] Although the present invention is broadly as defined above, those persons skilled in the art will appreciate that the invention is not limited thereto and that the invention also includes embodiments of which the following description gives examples.BRIEF DESCRIPTION OF THE FIGURES

[0053] The present invention will be described with reference to the accompanying figures, in which:

[0054] Figure 1A shows a representative chromatogram at 210 nm showing macamides 5, 6 and 7. Brown (second from top), blue (third from top) and pink (bottom)are the hexane extracts after 2, 4 and 24 hrs. Black (top) is the MC7 standard compound. The peaks labelled as are fatty acids.

[0055] Figure IB shows a representative chromatogram at 280 nm showing macaene peak and macamide 2. The peaks labelled as have the same MW as main peaks.

[0056] Figure 1C shows representative peak areas for the maca compounds of interest after extraction for 2, 4, and 24 hours. Peaks at 210 nm for the first 3 macamides, 280 nm for macaene and MC2.

[0057] Figure 2 shows a representative comparison between the yields in mg / g of three difference extraction processes (hexane, CO2 and propane).

[0058] Figure 3A shows representative analysis results for the two extractions as shown in Example 6.

[0059] Figure 3B shows combined amounts of tested macamides and macaenes for the clear and cloudy fractions from the two extractions as shown in Example 6.

[0060] Figure 4 shows representative binding data of macamides and maca extracts on (A) CB1 and (B) CB2 receptors. Compound of interest of their corresponding vehicle control (Veh) were incubated with 1 nM [3H] CP55,940, in the presence of either (A) 0.08 mg / mL ppls-3HA-hCBl HEK membrane preparation or (B) 0.03 mg / mL 3HA-hCB2 HEK membrane preparation for one hour. Data represent mean ± SD of technical triplicates. Veh = EtOH in binding buffer for AEA, or DMSO for macamides and maca extracts; AEA = anandamide; macamides 86 = / V-benzyl-(-9Z, 12Z)-octadecadienamide; macamide 15 = / V-3-methoxybenzyl-(9Z, 12Z)-octadecadienamide; macamide 58 = N- benzylpalmitamide.

[0061] Figure 5 shows representative concentration-response curves of cAMP formation in HEK293 cells stably expressing (A) 3HA-hCBl or (B) HA-hCB2 receptors. Following transfection with 5 pg pcDNA3L-His-CAMYEL plasmid, HEK293 cells were stimulated with 5 pM forskolin (FSK / F) and compounds of interest. Curves produced using area-under-curve analysis of approximately 25 minutes of cAMP CAMYEL BRET biosensor data and normalized to basal (0%) and FSK (100%). Data represent mean ± SD of technical duplicates. AEA = anandamide; macamides 86 = / V-benzyl-(-9Z, 12Z)- octadecadienamide; macamide 15 = / V-3-methoxybenzyl-(9Z, 12Z)- octadecadienamide; macamide 58 = / V-benzylpalmitamide

[0062] Figure 6 shows representative concentration-response curves of cAMP formation for (A) macamide 86, (B) macamide 15, (C) maca hexane extract and (D) maca propane extract (D). HEK293 cells wildtype (WT), or stably expressing 3HA-hCBl or HA-hCB2 receptors stimulated with 5 pM forskolin (FSK) and compounds of interest. Curves produced using area-under-curve analysis of approximately 25 minutes of kinetic CAMYEL BRET biosensor data and normalized to basal (0%) and FSK (100%). Data represent mean ± SD of technical duplicates. Macamides 86 = / V-benzyl-(-9Z, 12Z)- octadecadienamide; macamide 15 = / V-3-methoxybenzyl-(9Z, 12Z)-octadecadienamide.

[0063] Figure 7 shows representative concentration-response curves of cAMP formation in HEK293 cells stably expressing (A) 3HA-hCBl or (B) HA-hCB2 receptors. Varying AEA concentrations were added to HEK293 cells stimulated with 5 pM forskolin (FSK) and 31.6 pM of macamides or maca extracts. Curves were produced using area- under-curve analysis of approximately 25 minutes of kinetic CAMYEL BRET biosensor data and normalized to basal (0%) and FSK (100%). Data represent mean ± SD of technical duplicates. Vehicle (V) = FSK only for AEA, and FSK + compound for macamides and maca extracts; AEA = anandamide; macamides 86 = / V-benzyl-(-9Z, 12Z)- octadecadienamide; macamide 15 = / V-3-methoxybenzyl-(9Z, 12Z)- octadecadienamide; macamide 58 = / V-benzylpalmitamide.

[0064] Figure 8 shows the effect of macamides on CXCL10 levels in IFN-y- stimulated HaCaT cells. Cells were co-treated with 100 ng / mL IFN-y and non-toxic concentrations of macamide 15, macamide 58, and macamide 86 for 24 hours. Results are normalised to vehicle controls (veh Ctrl) and expressed as the mean percentage ± SEM from three biological replicates. Data was analysed by one-way ANOVA coupled with a post-hoc test for linear trend. * significant linear trend in mean values with increasing concentration (P<0.05).

[0065] Figure 9 shows the effect of maca products and extracts on MCP-1 levels in IFN-y-stimulated HaCaT cells. Cells were co-treated with 100 ng / mL IFN-y and non-toxic concentrations of macamide 15, macamide 58 and macamide 86 for 24 hours. Results are normalised to vehicle controls (veh Ctrl) and expressed as the mean percentage ± SEM from three biological replicates. Data was analysed by one-way ANOVA coupled with a post-hoc test for linear trend. * significant linear trend in mean values with increasing concentration (P<0.05).

[0066] Figure 10 shows the effect of maca products and extracts on TNF-a levels in LPS-stimulated THP-1 cells. Cells were co-treated with 20 ng / mL PMA, 1 pg / mL LPS, andnon-toxic concentrations of macamide 15, macamide 58 and macamide 86 for 24 hours. Results are normalised to vehicle controls (veh Ctrl) and expressed as the mean percentage ± SEM from three biological replicates. Data was analysed by one-way ANOVA coupled with a post-hoc test for linear trend. * significant linear trend in mean values with increasing concentration (P<0.05).

[0067] Figure 11 shows the effect of maca products and extracts on MCP-2 levels in stimulated HaCaT cells. Cells were co-treated with 20 ng / mL LTA, 10 ng / mL TNF-a, 10 ng / mL IFN-y, 10 ng / mL IL-4, 10 ng / mL IL-13, and non-toxic concentrations of (A) MCB oil, (B) MCT oil, (C) black maca powder, (D) red maca powder, (E) macamide 15, (F) macamide 58, (G) macamide 86, (H) maca propane extract, (I) maca hexane extract, (J) maca CO2 extract, and (K) controls and CBD for 24 hours. Results are normalised to vehicle controls (veh Ctrl) and expressed as the mean percentage ± SEM from three biological replicates. Data was analysed by one-way ANOVA coupled with a post-hoc test for linear trend. * significant linear trend in mean values with increasing concentration (P<0.05).

[0068] Figure 12 shows the effect of controls, MCB, MCT and vehicles on human COX-2 activity. Recombinant COX-2 enzyme was treated with (A) hydrocortisone (250 pg / mL), cannabidiol (CBD, 10 pM), (B) MCB oil, (C) MCT oil, (D) ethanol, (E) DMSO, or (F) 9: 1 DMSO:chloroform for a 10-minute co-incubation with the substrate TMPD and cosubstrates arachidonic acid and eicosapentanoic acid. Results are expressed as the mean, normalised percentage of COX-2 activity ± SD from conditions assayed in triplicate. Data was analysed using a one-way ANOVA followed by Dunnett's multiple comparisons test post-hoc. * significant increase in COX-2 activity compared to control (P<0.05).

[0069] Figure 13 shows the effect of macamides and maca extracts on human COX- 2 activity. Recombinant COX-2 enzyme was treated with (A) macamide 15, (B) macamide 58, (C) macamide 86, (D) maca propane extract, (E) maca hexane extract, or (F) maca CO2 extract for a 10-minute co-incubation with the substrate TMPD and cosubstrates arachidonic acid and eicosapentanoic acid. Results are expressed as the mean, normalised percentage of COX-2 activity ± SD from conditions assayed in triplicate. Data was analysed using a one-way ANOVA coupled with a post-hoc test for linear trend. * significant linear trend in mean values with increasing concentration (P<0.05).

[0070] Figure 14 shows the effect of maca products and extracts on p-endorphin release in HaCaT cells. Cells were treated with (A) HU-308 (1 and 10 pM), non-toxic concentrations of (B) MCB oil, (C) ethanol, (D) macamide 15, (E) macamide 58, (F)macamide 86, (G) DMSO, (H) maca propane extract, (I) maca hexane extract, and (J) maca CO2 extract, or (K) 9: 1 DMSO:chloroform for 45 minutes. Results are expressed as the mean amount of p-endorphin ± SD from two biological replicates. Dotted line represents the sensitivity limit of the assay of 9.38 pg / mL. Data was analysed using a one-way ANOVA followed by Dunnett's multiple comparisons test post-hoc. * significant increase in mean p-endorphin release compared to media control (P<0.05).

[0071] Figure 15 shows the effect of maca products and extracts on the activity of human recombinant FAAH. Recombinant FAAH enzyme was treated with (A) the known inhibitor JZL 195 (1 pM) and CBD (10 pM), (B) MCB oil, (C) ethanol, (D) macamide 15, (E) macamide 58, (F) macamide 86, (G) DMSO, (H) maca propane extract, (I) maca hexane extract, and (J) maca CO2 extract, or (K) 9: 1 DMSO:chloroform for a 30 minute co-incubation with the substrate 7-amino-4-methylcoumarin-arachidonamide. Results are expressed as the mean percentage FAAH inhibition ± SD from conditions assayed in duplicate. Data was analysed using a one-way ANOVA followed by Dunnett's multiple comparisons test post-hoc. * significant inhibition of FAAH activity compared to control (P<0.05).DETAILED DESCRIPTION OF THE INVENTIONDefinitions

[0072] The term "comprising" as used in this specification and claims means "consisting at least in part of". When interpreting each statement in this specification and claims that includes the term "comprising", features other than that or those prefaced by the term may also be present. Related terms such as "comprise", "comprised" and "comprises" are to be interpreted in the same manner.

[0073] The term "maca compounds" or "maca compound" as used herein refers to macaenes and / or macamides.

[0074] As used herein the term "and / or" means "and" or "or", or both.

[0075] As used herein "(s)" following a noun means the plural and / or singular forms of the noun.

[0076] The general chemical and biological terms used, for example, in the formulae herein have their usual meanings.Maca

[0077] The maca root material may be provided from red, black, and / or yellow maca varieties, and / or any combination of these varieties or subvariations including white or purple maca. In a particular embodiment, the maca is Peruvian maca grown in its native or customary altitude and / or native geographical range.

[0078] In certain embodiments, the maca is provided as a fresh root material. In other embodiments, the maca is provided as a dried root material. In a particular embodiment, the dry root material may be initially provided with a moisture level of less than 15%. The maca root material may then be stored, such as storage at relative humidity levels of less than 80% and / or temperatures less than 30 °C.Macamides and macaenes

[0079] The invention described herein relates to an oil-based composition comprising an extract comprising macamides and macaenes derived from natural maca. Known macamides and macaenes are listed in Table 1 below:Table 1. Macamide and macaene names and chemical structures

[0080] In various embodiments, the maca extract comprises an effective amount of maca compounds in the oil composition. In various embodiments, the maca extract comprises at least about 50 mg / g maca compounds, or at least 55 mg / g, 60 mg / g, 65 mg / g, 70 mg / g, 75 mg / g, 80 mg / g, 85 mg / g, 90 mg / g, 95 mg / g, 100 mg / g, 110 mg / g, 120 mg / g, 130 mg / g, 140 mg / g, 150 mg / g maca compounds. In various embodiments, the maca extract comprises from about 50 mg / g to about 150 mg / g maca compounds.

[0081] In various embodiments, the maca extract comprises at least about 50 mg / g total macamides, for example at least about 55mg / g, 60 mg / g, 65 mg / g, 70 mg / g, 80 mg / g / , 90 mg / g, 100 mg / g, 110 mg / g, 120 mg / g, 130 mg / g, 140 mg / g total macamides.

[0082] In various embodiments, the maca extract comprises at least about 3 mg / g total macaenes, for example at least about 5 mg / g, 6 mg / g, 7 mg / g, 8 mg / g, 9 mg / g, 10 mg / g, 12 mg / g, 14 mg / g, 15 mg / g total macaenes.

[0083] In various embodiments, the maca extract comprises at least about 5 w / w% maca compounds, or at least 5.5 w / w%, 6 w / w%, 7 w / w%, 7.5 w / w%, 8 w / w%, 8.5 w / w%, 9.0 w / w%, 9.50 w / w%, 10.0 w / w%, 11.0 w / w%, 12.0 w / w%, 13.0 w / w%, 14.0 w / w%, 15.0 w / w% maca compounds.

[0084] In various embodiments, the maca extract comprises at least about 5 w / w% total macamides, for example at least about 5.5 w / w%, 6 w / w%, 6.5 w / w%, 7 w / w%, 8 w / w%, 9 w / w%, 10 w / w%, 11 w / w%, 12 w / w%, 13 w / w%, 14 w / w% total macamides.

[0085] In various embodiments, the maca extract comprises at least about 0.3 w / w% total macaenes, for example at least about 0.5 w / w%, 0.7 w / w%, 1.0 w / w%, 1.2 w / w%, 1.4 w / w%, 1.5 w / w% total macaenes.

[0086] In various embodiments, the maca extract comprises:• macamide / V-Benzyloctadec-9Z-enamide; and / or• macamide / V-Benzyloctadeca-9Z,12Z-dienamide); and / or• macamide / V-Benzyloctadeca-9Z,12Z,15Z-trienamide5, MC5); and / or• macamide / V-(3-Methoxybenzyl)octadeca-9Z,12Z-dienamide); and / or• macaene 9-oxo-octadeca-10,12-dienoic acid; and / or• macamide / V-Benzyl-9-oxo-octadeca-10,12-dienamide; and / or• macamide / V-Benzylpalmitamide; and / or• macamide / V-Benzyloctadecanamide; and / or• macamide / V-Benzylicosa-5Z,8Z,llZ,14Z-tetraenamide,• macamide / V-Benzylicosa-5Z,8Z,llZ,14Z,17Z-pentaenamide,• macamide / V-(3-Methoxybenzyl)octadeca-9Z,12Z,15Z-trienamide; and / or• macamide / V-(3-Methoxybenzyl)octadeca-9Z-enamide; and / or• macamide / V-(3-Methoxybenzyl)hexadecanamide; and / or• / V-(3-Methoxybenzyl)octadecanamide; and / or• / V-Phenethyloctadeca-9Z,12Z-dienamide; and / or• / V-Phenethyloctadeca-9Z,12Z,15Z-trienamide.

[0087] In various embodiments, the maca extract comprises at least about 9 mg / g macamide / V-Benzyloctadec-9Z-enamide, for example at least about 10 mg / g 11 mg / g, 12 mg / g, 13 mg / g, 14 mg / g, 15 mg / g, 16 mg / g, 17 mg / g, 18 mg / g, 19 mg / g, 20mg / g, 21mg / g, 22 mg / g, 23 mg / g, 24 mg / g, 25 mg / g / V-Benzyloctadec-9Z-enamide.

[0088] In various embodiments, the maca extract comprises at least about 23 mg / g macamide / V-Benzyloctadeca-9Z,12Z-dienamide, for example at least about 31 mg / g, 32 mg / g, 33 mg / g, 34 mg / g, 35 mg / g, 40 mg / g, 45 mg / g, 50 mg / g, 55 mg / g2, 60 mg / g, 65 mg / g / V-Benzyloctadeca-9Z,12Z-dienamide.

[0089] In various embodiments, the maca extract comprises at least about 3 mg / g macamide / V-Benzyloctadeca-9Z,12Z,15Z-trienamide, for example at least about 4 mg / g, 5 mg / g, 6 mg / g, 7 mg / g, 8 mg / g, 9 mg / g, 10 mg / g, 15 mg / g, 20 mg / g, 21 mg / g N- Benzyloctadeca-9Z,12Z,15Z-trienamide.

[0090] In various embodiments, the maca extract comprises at least about 4 mg / g macamide / V-(3-Methoxybenzyl)octadeca-9Z,12Z-dienamide, for example at least about 5 mg / g, 6 mg / g 7 mg / g, 8 mg / g, 9 mg / g, 10 mg / g, 11 mg / g, 12 mg / g, 13 mg / g / V-(3- Methoxybenzyl)octadeca-9Z,12Z-dienamide.

[0091] In various embodiments, the maca extract comprises at least about 50 mg / g to about 140 mg / g total macamides.

[0092] In various embodiments, the maca extract comprises at least about 3 mg / g to about 15 mg / g total macaenes.

[0093] In various embodiments, the maca extract comprises at least about 50 mg / g to about 140 mg / g total macamides and at least about 3 mg / g to about 15 mg / g total macaenes.

[0094] In various embodiments, the maca extract comprises: a. at least about 9 mg / g to about 25 mg / g macamide / V-Benzyloctadec-9Z- enamide; and / or b. at least about 23 mg / g to about 65 mg / g macamide / V-Benzyloctadeca- 9Z,12Z-dienamide; and / or c. at least about 3 mg / g to about 21 mg / g macamide / V-Benzyloctadeca- 9Z,12Z,15Z-trienamide; and / or d. at least about 4 mg / g to about 13 mg / g macamide / V-(3- Methoxybenzyl)octadeca-9Z,12Z-dienamide.

[0095] In various embodiments, the maca extract comprises at least about 4 mg / g to about 65 mg / g macamide / V-Benzylpalmitamide.

[0096] In various embodiments, the maca extract comprises at least about 0.2 mg / g to about 5 mg / g macamide / V-Benzyloctadecanamide.

[0097] In various embodiments, the maca extract comprises at least about 0.5 mg / g to about 1.7 mg / g macamide / V-(3-Methoxybenzyl)octadeca-9Z,12Z,15Z- trienamide.

[0098] In various embodiments, the maca extract comprises at least about 0.3 mg / g to about 1.3 mg / g macamide / V-(3-Methoxybenzyl)octadec-9Z-enamide.

[0099] In various embodiments, the maca extract comprises at least about 0.2 mg / g to about 0.8 mg / g macamide / V-(3-Methoxybenzyl)hexadecanamide.

[0100] In various embodiments, the maca extract comprises at least about 0.02 mg / g to about 0.6g mg / g of / V-(3-Methoxybenzyl)octadecanamide, about 0.04 mg / g to about 0.11 mg / g / V-Phenethyloctadeca-9Z,12Z-dienamide, about 0.01 mg / g to about 0.02 mg / g / V-Phenethyloctadeca-9Z,12Z,15Z-trienamide, about 0.01 mg / g N- Benzylicosa-5Z,8Z,llZ,14Z-tetraenamide, and / or about 0.04 mg / g to about 0.27 mg / g / V-Benzylicosa-5Z,8Z,llZ,14Z,17Z-pentaenamide.

[0101] In various embodiments, the maca extract comprises least about 3 mg / g to about 15 mg / g macaene 9-oxo-octadeca-10,12-dienoic acid.

[0102] In various embodiments, the maca extract comprises at least about 4 mg / g to about 15 mg / g macamide / V-Benzyl-9-oxo-octadeca-10,12-dienamide.

[0103] In various embodiments, the maca extract comprises a ratio between unsaturated macamides and macaenes to saturated macamides of about 1: 1. In further embodiments, the ratio is about 2: 1. In yet further embodiments, the ratio is about 3: 1. In yet further embodiments, the ratio is about 4: 1. In yet further embodiments, the ratio is about 5: 1, In yet further embodiments, the ratio is about 6: 1. In yet further embodiments, the ratio is about 7: 1. In yet further embodiments, the ratio is about 8: 1. In yet further embodiments, the ratio is about 9: 1. In yet further embodiments, the ratio is about 10: 1.

[0104] The exact biological mode of action of macamides and macaenes themselves is yet to be determined as they have been shown to operate via several biological pathways, however, it is postulated that they exert their functions primarily within the endocannabinoid system (ECS). Studies have shown that macamides either bind and inhibit fatty acid amide hydrolase (FAAH), an enzyme which normally binds the endogenous cannabinoid, anandamide, or bind and transport anandamide itself. Anandamide is an important compound for managing responses to stress as it is knownto produce feelings of pleasure and pain relief. This compound typically has a short halflife owing to its frequent degradation by FAAH, which thus quenches its effects on the ECS. Inhibition of FAAH by macamides is thought to prevent the delay of this degradation and potentiate anandamide activity in vivo.

[0105] As previously noted, anandamide and macamides have structural similarities. It is therefore theorised that macamides are exogenous molecules that can increase circulating levels of or sensitivity to natural endocannabinoids like anandamide.

[0106] It is also theorised that macamides have adaptogenic properties. Adaptogens are natural substances that help the body adapt to stress by normalising and regulating the systems of the body. For example, in certain physiological circumstances, certain macamides (generally unsaturated) may work on FAAH inhibition, but they may also simultaneously work as antagonists or reverse agonists on CB1 and CB2 receptors. It may be expected that the higher concentrations of unsaturated macamides in the present invention would be more likely to bind and / or inhibit fatty acid amide hydrolase (FAAH) within the endocannabinoid system (ECS) as has been shown in the prior art, thus delaying the degradation of anandamide and thus potentiating its effect in the ECS. However, the composition of the invention shows surprisingly decreased anandamide activity on CB1 and CB2 receptors as compared with certain individual macamides alone (see Example 7). This result shows that the composition of the invention is likely to be adaptogenic, thus aiding in moderating the ECS and its downstream effects in the body. This adaptogenic behaviour of the various macamide and / or macaene compounds is thought to moderate the ECS, such as when the body is under physical or mental stress.

[0107] In the context of medicinal cannabis, synergistic activity of a variety of compounds derived from cannabis, such as cannabidiol and terpenes, is known as the "entourage effect". The entourage effect may occur through different mechanisms, e.g., by preventing the breakdown of the endogenous cannabinoids (ECBs) by enzymes that are responsible for their destruction, in which case the ECBs last longer in the body giving a prolonged and enhanced action, or by improving the binding of ECBs on various receptors, or by activating other receptors, e.g., opening up ion channels that cause a synergistic effect of the major molecule. Without wishing to be bound by theory, the inventor of the present invention considers the composition of the present invention may also display this entourage effect.

[0108] Macamides may also inhibit epoxide hydrolase (EH), an enzyme that converts epoxy fatty acids to less reactive diols. By inhibiting EH, macamides canmodulate the levels of epoxides and their corresponding diols, which may affect their biological activities. For example, macamides may increase the levels of epoxyeicosatrienoic acids (EETs), which are epoxides derived from arachidonic acid and have anti-inflammatory, analgesic, vasodilatory, and cardioprotective effects. Furthermore, macamides may also decrease the levels of dihydroxyeicosatrienoic acids (DHETs), which are diols derived from EETs and have pro-inflammatory, vasoconstrictive, and pro-fibrotic effects. Furthermore, Singh et al. 2020 showed that known macamides displayed excellent inhibitory potency towards EH, and demonstrated reduction of inflammatory pain in rodents. Therefore, macamides may be used as epoxide hydrolase inhibitors for the treatment or prevention of various diseases and conditions, such as hypertension, cardiovascular diseases, inflammation, fibrosis, pain, in particular inflammatory pain. The composition described herein is useful for the treatment of diseases or disorders responsive to inhibition of epoxide hydrolase.

[0109] The composition of the present invention is derived from a natural source, as opposed to compositions that are blends of synthetic or isolated macamides and macaenes. This is advantageous as there is resistance in the consumer market to synthetic compositions. Additionally, the invention provides for a suite of natural macamides and macaenes that are thought to be superior to high concentrations of bioactive macamides alone, through the entourage effect and / or adaptogenic behaviour. The maca compounds of the composition described herein are not synthetic.

[0110] The composition of the present invention contains a high concentration of bioactive maca compounds. This is advantageous as a lower dose of the composition is required to provide therapeutic effects, compared to compositions comprising lower concentrations of active maca compounds. For example, the recommended dose of a composition of the present invention is from 0.5 to 1 mL. For example, the composition may be administered in doses of 2 to 5 drops one to three times daily, with a drop containing approximately 0.05 mL. The use of a concentrated composition improves user compliance as it is easier to take a smaller dose.Oil

[0111] The composition of the present invention is an oil-based composition that comprises the maca extract. The oil acts as a carrier for the maca extract and comprises an oil or combination of oils. The oil carrier provides stability and fluidity to the composition, and is ideally not susceptible to oxidation.

[0112] Advantageously, the oil is provided in the composition by the process used. That is, the oil is not added after the extraction process, rather the product of the extraction process is the maca extract in the oil. This results in a composition ready for use either directly or for further formulation into a desired product. The process is therefore an efficient process of preparing a composition providing the user advantages in production costs and process efficiencies.

[0113] Any food-safe oil may be used. In various embodiments, the oil is selected from the group consisting of arnica oil, avocado oil, black seed oil, canola oil, coconut oil, flaxseed oil, frankincense oil, grapeseed oil, Kakadu plum seed oil, macadamia oil, medium chain triglyceride (MCT) oil, native sandalwood oil, olive oil, palm oil, rice bran oil, sesame oil, soya oil, sunflower oil, vegetable oil, walnut oil, hemp seed oil, mineral oil, peanut oil, tung oil, bees wax, Danish oil, black cumin seed oil, coconut MCT oil, castor oil, and any combination of any two or more thereof. In a preferred embodiment, the oil is MCT oil.

[0114] MCT oil is known to have a variety of beneficial effects such as for brain health, heart health, and anti-inflammatory properties.Terpenes

[0115] In various embodiments, the composition further comprises at least one terpene and / or terpenoid. These compounds are useful in modulating and / or complementing the natural aroma of the composition.

[0116] In various embodiments, the terpene is selected from the group consisting of alpha bisabolol, alpha phellandrene, alpha pinene, beta caryophyllene, beta pinene, cadinene, camphene, citral, citronellol, delta 3 carene, eucalyptol, eugenol, gamma terpinene, geraniol, humulene, limonene, linalool, myrcene, nerol, nerolidol, ocimene, para-cymene, terpineol, terpinolene, and valencene.

[0117] In various embodiments, the composition comprises the at least one terpene and / or terpenoid in a total concentration of 0.1-5% w / w%, for example from about 0.1 to about 4% w / w, or about 0.1 to about 3% w / w, or about 0.1 to about 2% w / w, or about 0.1 to about 1% w / w, or about 0.2 to about 5% w / w, or about 0.2 to about 4% w / w, or about 0.2 to about 3% w / w, or about 0.2 to about 2% w / w, or about 0.2 to about 1% w / w, or about 0.5 to about 5% w / w, or about 0.5 to about 4% w / w, or about 0.5 to about 3% w / w, or about 0.5 to about 2% w / w, or about 0.5 to about 1% w / w.Other components

[0118] In various embodiments, the composition comprises residual alkane. In various embodiments, the composition comprises at least about 0.01% w / w alkane. In various embodiments, the composition comprises from about 0.01% to about 0.5% alkane, for example from about 0.01% to about 0.4%, or about 0.01% to about 0.3%, or about 0.01% to about 0.2%, or about 0.01% to about 0.1%, or about 0.01% to about 0.05%, or about 0.01% to about 0.02%, or about 0.02% to about 0.5%, or 0.02% to about 0.4%, or about 0.02% to about 0.3%, or about 0.02% to about 0.2%, or about 0.02% to about 0.1%, or about 0.02% to about 0.05%.

[0119] In various embodiments, the alkane is selected from the group consisting of propane, butane, isobutane, pentane and isomers thereof, hexane and isomers thereof, and heptane and isomers thereof. In a preferred embodiment, the alkane is propane or butane. In a more preferred embodiment, the alkane is propane.

[0120] In various embodiments, the composition comprises additional components selected from flavours, colours, preservatives, stabilisers, amylase, lecithin, other active ingredients including but not limited to cacao, medicinal mushrooms, green tea extract, chamomile, valerian Root, curcumin, kava kava, and blue lotus.Process

[0121] In one aspect the invention provides a process of producing an oil-based composition comprising a maca extract, the process comprising: providing a maca root; suspending the maca root in an oil, contacting the oil comprising maca root with a solvent comprising an alkane at a temperature and pressure at which the solvent is liquid, thereby producing the composition.

[0122] The process comprises providing maca root, also known as maca bulb. In various embodiments, the maca root is fresh or dried. The fresh maca root may be comminuted, for example may be chopped or blended. The dried maca root may be comminuted, for example may be powdered or chopped. In various embodiments, the maca root is dried powdered maca root.

[0123] The process may include water washing of the maca root material. Optionally or additionally, the maca root material can be sanitised by washing with a sodium hypochlorite solution for 5-10 minutes.

[0124] The process may also comprise gelatinizing the maca root starting material under steam heat and pressure. This process may comprise boiling and pressurising the dried roots to break down the starch content and to neutralise enzymes. The leftover pulp may be dried and ground to create a gelatinised maca powder. The gelatinisation temperature may be carried out at temperatures of 90 °C to 150 °C and / or at pressures that allow water to achieve temperatures from 90 °C to 150 °C.

[0125] The process may further comprise a heat treatment step. In a particular embodiment, the heat treatment is carried out at a temperature between 70 °C and 120 °C and / or at a humidity of less than 10%.

[0126] The process may further comprise crushing, cutting, grinding, or otherwise comminuting the maca root material. In a particular embodiment, the maca root material granules may be able to pass through a 20-200 Mesh sieve, and in particular an 80 Mesh sieve. The dried powdered maca root may therefore have a particle size that can pass through about 20 mesh to about 200 mesh. In a particular embodiment, the particle size of the dried powdered maca root has a particle size that can pass through 80 mesh.

[0127] At any point in the process of preparing the maca root material, the maca material being processed may be stored at relative humidity levels of less than 80% and / or at temperatures less than 30 °C.

[0128] In various embodiments, the process further comprises comminuting the maca root to produce maca root particles before suspending the maca root in the carrier.

[0129] The process comprises contacting the carrier comprising maca root with a solvent comprising an alkane at a temperature and pressure at which the solvent is liquid. Contacting the oil comprising the maca root with a liquid solvent comprising an alkane results in extraction of the maca compounds in the maca root in to the oil r, i.e. an extraction step. The oil can be viewed as a co-solvent extracting the maca compounds in tandem with the solvent comprising an alkane.

[0130] If necessary, the process comprises removing residual alkane from the oil following the extraction step. Methods for removing residual alkane will be known to a skilled worker, such as evaporation under pressure.

[0131] In various embodiments, solvent comprising an alkane is a supercritical liquid. A supercritical liquid is a substance at a temperature and pressure where distinct liquid and gas phases do not exist. Advantageously, when the solvent comprising analkane is a supercritical liquid the process provides a concentrated maca extract that does not need to undergo any further processing steps, such as removal of residual alkane, before use.

[0132] In various embodiments, the alkane is selected from the group consisting of propane, butane, isobutane, pentane and isomers thereof, hexane and isomers thereof, and heptane and isomers thereof. In various embodiments, the alkane is selected from the group consisting of propane, butane, isobutane, pentane and isomers of pentane. In a preferred embodiment, the alkane is propane.

[0133] In various embodiments, the oil is selected from the group consisting of arnica oil, avocado oil, black seed oil, canola oil, coconut oil, flaxseed oil, frankincense oil, grapeseed oil, Kakadu plum seed oil, macadamia oil, MCT oil, native sandalwood oil, olive oil, palm oil, rice bran oil, sesame oil, soya oil, sunflower oil, vegetable oil, walnut oil, hemp seed oil, mineral oil, peanut oil, tung oil, bees wax, Danish oil, black cumin seed oil, coconut MCT oil, castor oil, and any combination of any two or more thereof. In a preferred embodiment, the oil is MCT oil.

[0134] In various embodiments, contacting the carrier comprising the maca root with the solvent is performed at a pressure of from about 10 to about 50 bar, for example from about 10 to about 40 bar, or about 20 to about 50 bar, or about 20 to about 40 bar, or about 30 to about 50 bar, or about 30 to about 40 bar.

[0135] In various embodiments, contacting the carrier comprising the maca root with the solvent is performed at a temperature of from about 20 to about 70°C, for example from about 20 to about 60°C, or about 20 to about 50°C, or about 30 to about 70°C, or about 30 to about 60°C, or about 30 to about 50°C.

[0136] In one embodiment, contacting the carrier comprising the maca root with the solvent is performed at a pressure of 40 bar and a temperature of 50°C. A skilled worker will appreciate that there is a relationship between pressure and temperature in regards to providing a liquid alkane or mixture of alkanes, and will be able to select the temperature and pressure required to provide a liquid.

[0137] In one embodiment, the extraction comprises a continuous recirculation of the liquid alkane throughout the extraction process. The liquid alkane may be pressurised to maintain its liquid phase and is mixed with the feed material in an extraction vessel. The resulting mixture is then depressurised in a separator, where the extract collects at the bottom and the solvent is recompressed and recirculated. In certain embodiments,the solvent:feed ratio in the process is about 6: 1. In various embodiments, the oil is present as a co-solvent during the extraction process.

[0138] In various embodiments, the process is carried out in an extraction apparatus. The extraction apparatus is capable of withstanding the pressure and temperature required for the extraction.

[0139] In various embodiments, the process comprises washing the extraction apparatus with an alcohol and retaining the alcohol portion. A skilled worker will appreciate the alcohol must be removed / evaporated before adding the alcohol portion to the composition of the invention. In a preferred embodiment, the alcohol is ethanol.

[0140] In various embodiments, the process results in a yield of at least about 0.2% of macamides and macaenes, for example, at least about 0.4%, about 0.5%, about 0.6%, about 0.8%, about 1.0%, about 1.1%, or about 1.2%.

[0141] In various embodiments, the process results in a yield of about 0.2 to about 1.5% of macamides and macaenes, for example from about 0.2 to about 1.4%, or about 0.2 to about 1.3%, or about 0.2 to about 1.2% or about 0.2 to about 1.1%, or about 0.2 to about 1.0%, or about 0.2 to about 0.9%, or about 0.2 to about 0.8%, or about 0.2 to about 0.7 %, or about 0.3 to about 1.5%, or about 0.3 to about 1.4%, or about 0.3 to about 1.3%, or about 0.3 to about 1.2%, or about 0.3 to about 1.1%, or about 0.3 to about 1.0%, about 0.3 to about 0.9%, or about 0.3 to about 0.8%, or about 0.3 to about 0.7 %, or about 0.4 to about 1.5%, or about 0.4 to about 1.4%, or about 0.4 to about 1.3%, or about 0.4 to about 1.2%, or about 0.4 to about 1.1%, or about 0.4 to about 1.0%, about 0.4 to about 0.9%, or about 0.4 to about 0.8%, or about 0.4 to about 0.7 %, or about 0.4 to about 1.5%, or about 0.4 to about 1.4%, or about 0.4 to about 1.3%, or about 0.4 to about 1.2%, or about 0.4 to about 1.1%, or about 0.5 to about 1.0%, about 0.5 to about 0.9%, or about 0.5 to about 0.8%, or about 0.5 to about 0.7 %, or about 0.6 to about 1.5%, or about 0.6 to about 1.4%, or about 0.6 to about 1.3%, or about 0.6 to about 1.2%, or about 0.6 to about 1.1%, or about 0.6 to about 1.0%, about 0.6 to about 0.9%, or about 0.6 to about 0.8%, or about 0.6 to about 0.7 %.

[0142] The process of the present invention results in a higher yield than prior art processes, which in turn provides a product with a high proportion of maca compounds.

[0143] The process may result in an extract comprising a greater ratio of macamides to macaenes than the ratio found in natural maca root, that is, the processmay preferentially extract macamides compared to macaenes. This is advantageous as macamides may have greater therapeutic effects than macaenes.

[0144] In one aspect, the invention provides an oil-based composition comprising a maca extract when produced by the process of the invention. In one aspect, the invention provides a propane-derived maca extract.Uses

[0145] The composition described herein is useful for the treatment of diseases or disorders mediated by the endocannabinoid system and / or diseases or disorders responsive to mediation of the endocannabinoid system. In various embodiments, the invention relates to a method of treating diseases or disorders mediated by the endocannabinoid system by administering an effective amount of the composition of the invention.

[0146] The composition described herein is useful for the treatment of diseases or disorders responsive to inhibition of epoxide hydrolase. In various embodiments, the invention relates to a method of treating diseases or disorders responsive to inhibition of epoxide hydrolase by administering an effective amount of the composition of the invention.

[0147] The maca compositions described herein are useful for the treatment of diseases or disorders selected from the group consisting of acne, addiction, anxiety, arthritis, autism spectrum disorder, disorders stemming from autoimmunity, side effects resulting from cancer treatment, chronic pain, chronic fatigue syndrome, depression, digestive disorders, epileptic seizures, irritable bowel syndrome, Crohn's disease, ulcerative colitis, high blood pressure, hormonal imbalances, inflammation, insomnia, disorders of the libido, menopause, mental health disorders, muscular pain, neurological conditions, Parkinson's disease, post-traumatic stress disorder, seizures, disorders of the skin, eczema, psoriasis, sexual function disorders, and stress.

[0148] The maca compositions described herein are useful for the treatment of topical indications such as scar treatment, wound healing, acne, and topical psoriasis.

[0149] Described herein is the use of the composition of the invention or prepared by the process of the invention in the manufacture of a medicament for treating diseases or disorders selected from the group consisting of acne, addiction, anxiety, arthritis, autism spectrum disorder, disorders stemming from autoimmunity, side effects resultingfrom cancer treatment, chronic pain, chronic fatigue syndrome, depression, digestive disorders, epileptic seizures, irritable bowel syndrome, Crohn's disease, ulcerative colitis, high blood pressure, hormonal imbalances, inflammation, insomnia, disorders of the libido, menopause, mental health disorders, muscular pain, neurological conditions, Parkinson's disease, post-traumatic stress disorder, seizures, disorders of the skin, injury to the skin, eczema, psoriasis, sexual function disorders, and stress.

[0150] Described herein is the use of the composition of the invention or prepared by the process of the invention in a non-therapeutic method of increasing cognitive function, increasing energy, increasing endurance, increasing libido, increasing mental clarity, improving mood, increasing muscle growth, increasing muscle strength, increasing physical performance, increasing sexual function, increasing stamina, improving thyroid function, improving hormonal balance, improving immune function, improving recovery times, increasing resilience to stress, improving mood, and improving neurological function.

[0151] Described herein is a composition of the invention or prepared by the process of the invention for use in treating diseases or disorders selected from the group consisting of acne, addiction, anxiety, arthritis, autism spectrum disorder, disorders stemming from autoimmunity, side effects resulting from cancer treatment, chronic pain, chronic fatigue syndrome, depression, digestive disorders, epileptic seizures, irritable bowel syndrome, Crohn's disease, ulcerative colitis, high blood pressure, hormonal imbalances, inflammation, insomnia, disorders of the libido, menopause, mental health disorders, muscular pain, neurological conditions, Parkinson's disease, post-traumatic stress disorder, seizures, disorders of the skin, injury to the skin, eczema, psoriasis, sexual function disorders, and stress.

[0152] Described herein is a method of treating diseases or disorders selected from the group consisting of acne, addiction, anxiety, arthritis, autism spectrum disorder, disorders stemming from autoimmunity, side effects resulting from cancer treatment, chronic pain, chronic fatigue syndrome, depression, digestive disorders, epileptic seizures, irritable bowel syndrome, Crohn's disease, ulcerative colitis, high blood pressure, hormonal imbalances, inflammation, insomnia, disorders of the libido, menopause, mental health disorders, muscular pain, neurological conditions, Parkinson's disease, post-traumatic stress disorder, seizures, disorders of the skin, injury to the skin, eczema, psoriasis, sexual function disorders, and stress. by administering to a patient in need thereof an effective amount of a composition of the invention or prepared by the process of the invention.

[0153] Described herein is a method of increasing cognitive function, increasing energy, increasing endurance, increasing libido, increasing mental clarity, improving mood, increasing muscle growth, increasing muscle strength, increasing physical performance, increasing sexual function, increasing stamina, improving thyroid function, improving hormonal balance, improving immune function, improving recovery times, increasing resilience to stress, improving mood, and improving neurological function in a subject in need thereof by administering to a patient in need thereof an effective amount of a composition of the invention or prepared by the process of the invention.

[0154] Advantageously, the composition of the invention is suitable for use in ketogenic diets and / or carbohydrate-restricted diets. Other maca compositions, especially powdered compositions or ethanol-based compositions are not suitable for use in ketogenic diets and / or carbohydrate-restricted diets due to their high carbohydrate content. As the composition of the present invention is oil-based, it is suitable for use in a ketogenic diet and / or carbohydrate restricted diets to provide the abovementioned benefits of the maca extract. In various embodiments, the composition is substantially free of carbohydrates.

[0155] Advantageously, the composition of the invention and / or produced by the process of the invention is in a form that can be used directly without further modification. However as will be known to a skilled person, standard formulation additives can of course be used if desired.

[0156] The compositions described herein are suitable for oral, sublingual, or topical administration. In various embodiments, the compositions of the invention are formulated for oral, sublingual, or topical administration.

[0157] In a particular embodiment, the maca composition is to be administered orally or sublingually at a dosage of 0.1 to 1.0 mL daily. In a preferred embodiment, the maca composition is to be administered at a dosage of 0.5 to 1.0 mL daily. The composition may be administered at least once a day, at least twice a day, or at least three times a day.

[0158] In a particular embodiment, described herein is a method of treating diseases or disorders selected from the group consisting of acne, addiction, anxiety, arthritis, autism spectrum disorder, disorders stemming from autoimmunity, side effects resulting from cancer treatment, chronic pain, chronic fatigue syndrome, depression, digestive disorders, epileptic seizures, irritable bowel syndrome, Crohn's disease,ulcerative colitis, high blood pressure, hormonal imbalances, inflammation, insomnia, disorders of the libido, menopause, mental health disorders, muscular pain, neurological conditions, Parkinson's disease, post-traumatic stress disorder, seizures, disorders of the skin, injury to the skin, eczema, psoriasis, sexual function disorders, and stress. by administering to a patient in need thereof an effective amount of a composition of the invention or prepared by the process of the invention, wherein the composition is administered at a dose of 0.1 to 1.0 mL daily.

[0159] In a particular embodiment, described herein is a method of increasing cognitive function, increasing energy, increasing endurance, increasing libido, increasing mental clarity, improving mood, increasing muscle growth, increasing muscle strength, increasing physical performance, increasing sexual function, increasing stamina, improving thyroid function, improving hormonal balance, improving immune function, improving recovery times, increasing resilience to stress, improving mood, and improving neurological function in a subject in need thereof by administering to a patient in need thereof an effective amount of a composition of the invention or prepared by the process of the invention, wherein the composition is administered at a dose of 0.1 to 1.0 mL daily.

[0160] In various embodiments, the composition of the invention provides an antiinflammatory effect mediated by C-X-C motif chemokine ligand 10 (CXCL-10), monocyte chemoattractant protein 1 (MCP-1), tumour necrosis factor- a (TNF-a), monocyte chemoattractant protein 2 (MCP-2), and / or fatty acid amide hydrolase (FAAH). The composition described herein is useful for the treatment of diseases or disorders responsive to inhibition of C-X-C motif chemokine ligand 10 (CXCL-10), monocyte chemoattractant protein 1 (MCP-1), tumour necrosis factor- a (TNF-a), monocyte chemoattractant protein 2 (MCP-2), and / or fatty acid amide hydrolase (FAAH). In various embodiments, the invention relates to a method of treating diseases or disorders responsive to inhibition of C-X-C motif chemokine ligand 10 (CXCL-10), monocyte chemoattractant protein 1 (MCP-1), tumour necrosis factor- a (TNF-a), monocyte chemoattractant protein 2 (MCP-2), and / or fatty acid amide hydrolase (FAAH) by administering an effective amount of the composition of the invention.

[0161] In various embodiments, the composition of the invention provides an antiinflammatory effect mediated by C-X-C motif chemokine ligand 10 (CXCL-10). Preferably, the composition of the invention may reduce CXCL-10 release. The composition described herein is useful for the treatment of diseases or disorders responsive to inhibition of C-X- C motif chemokine ligand 10 (CXCL-10).. In various embodiments, the invention relatesto a method of treating diseases or disorders responsive to inhibition of C-X-C motif chemokine ligand 10 (CXCL-10). by administering an effective amount of the composition of the invention.

[0162] In various embodiments, the composition of the invention provides an antiinflammatory effect mediated by monocyte chemoattractant protein 1 (MCP-1). Preferably, the composition of the invention may reduce MCP-1 chemokine levels. The composition described herein is useful for the treatment of diseases or disorders responsive to inhibition of monocyte chemoattractant protein 1 (MCP-1). In various embodiments, the invention relates to a method of treating diseases or disorders responsive to inhibition of monocyte chemoattractant protein 1 (MCP-1) by administering an effective amount of the composition of the invention.

[0163] In various embodiments, the composition of the invention provides an antiinflammatory effect mediated by tumour necrosis factor- a (TNF-a). Preferably, the composition of the invention may reduce TNF-a release. The composition described herein is useful for the treatment of diseases or disorders responsive to inhibition of tumour necrosis factor- a (TNF-a). In various embodiments, the invention relates to a method of treating diseases or disorders responsive to inhibition of tumour necrosis factor- a (TNF-a) by administering an effective amount of the composition of the invention.

[0164] In various embodiments, the composition of the invention provides an antiinflammatory effect mediated by monocyte chemoattractant protein 2 (MCP-2). Preferably, the composition of the invention may reduce MCP-2 release. The composition described herein is useful for the treatment of diseases or disorders responsive to inhibition of monocyte chemoattractant protein 2 (MCP-2). In various embodiments, the invention relates to a method of treating diseases or disorders responsive to inhibition of monocyte chemoattractant protein 2 (MCP-2) by administering an effective amount of the composition of the invention.

[0165] In various embodiments, the composition of the invention provides an analgesic and / or anti-inflammatory effect mediated by fatty acid amide hydrolase (FAAH). Preferably, the composition of the invention may inhibit FAAH activity. More preferably, the composition of the invention may more significantly inhibit FAAH activity relative to individual isolated macamides. The composition described herein is useful for the treatment of diseases or disorders responsive to inhibition of fatty acid amide hydrolase (FAAH). In various embodiments, the invention relates to a method of treatingdiseases or disorders responsive to inhibition of fatty acid amide hydrolase (FAAH) by administering an effective amount of the composition of the invention.

[0166] In various embodiments, the composition of the invention may moderate release of p-endorphins.

[0167] The following non-limiting examples are provided to illustrate the present invention and in no way limit the scope thereof.

[0168] It is not the intention to limit the scope of the invention to the abovementioned examples only. As would be appreciated by a skilled person in the art, many variations are possible without departing from the scope of the invention as set out in the appended claims.EXAMPLESEXAMPLE 1

[0169] Summary

[0170] A variety of commercial maca powders were extracted in n-hexanes for analysis by GCMS. A previously developed GCMS-based quantification method was used to quantify macamides in the extracts with the addition of an ethyl benzoate (EtOBz) internal standard. Concentrations of macamides in the gelatinised maca powders and the maca blends were found to be similar to previous with reduction of approximately 71.5% in total with the adjustment by the internal standard. The ethanolic maca extracts, however, were found to have less than 1 pg / g of all three macamides.

[0171] Experimental

[0172] The following commercial maca root powders were provided by Seleno Health: gelatinised yellow, gelatinised red, gelatinised black, red maca produced by ethanol extraction, black maca produced by ethanol extraction, a first powdered blend, and a second powdered blend. The ethanolic extracts were produced by extracting the coarse ground red or black maca in a solution of water and a 96% sugar cane ethanol solution. / V-benzyl-(9Z,12Z)-octadecadieneamide (Standard 1) and / V-(3- methoxybenzyl)-(9Z,12Z)-octadecadieneamide (Standard 2) were purchased from Ambeed (Arlington Hts, IL 60004, USA) for use as standards. / V-benzylhexadecanamide(Standard 3) was purchased from Sapphire Bioscience Pty. Ltd. (Redfern, NSW, Australia) for use as a standard. / V-hexanes and methanol (MeOH) were used as solvents and ethyl benzoate (EtOBz) was used an internal standard for GCMS.

[0173] Macamide extraction from maca powder

[0174] Maca powders were extracted with n-hexanes at a ratio of 3 g of powder to 15 mL of n-hexanes. The mixtures were stirred for 4 hours at room temperature. Each sample was separated into two, filtered using an Agilent PTFE (0.45 pm, 25 mm) syringe filter and the filtrate dried under reduced pressure. One half of each sample was analysed by GCMS and the other half intended to be analysed by HPLC. Each sample destined for GCMS analysis was re-suspended in 1000 pL of n-hexanes containing 10.5 pg / mL of EtOBz. Each sample destined for HPLC analysis was attempted to be resuspended in 1000 pL of MeOH.

[0175] Analysis by GCMS

[0176] Analysis was performed using a Shimadzu QP2010-Plus gas chromatograph (Shimadzu, Kyoto, Japan) fitted with an RXI-5SHMS column (30 m x 0.25 mm i.d. x 0.25 pm film thickness; Restek, Bellefonte, PA). Aliquots (5 pL) of sample were introduced using an AOC-20i autosampler with a 5: 1 split ratio at an injection temperature of 270°C as determined previously. Helium was the carrier gas set at a linear velocity of 43.4 cm / s (1.43 mL / min) at a constant flow. An electron-impact mass spectrometer operating at 70 eV in positive-ion mode was attached and set to scan from m / z 42.0 to 600.0 every 0.30 seconds.

[0177] Concentration of macamide standards 1-3 were determined using a calibration curve previously generated with the following method. Macamide standards 1 and 2 were dissolved in n-hexanes and macamide standard 3 was dissolved in chloroform to make the following concentrations: 5 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.1 mg / mL, 0.05 mg / mL, 0.01 mg / mL, 0.005 mg / mL, 0.001 mg / mL. These were then used with the preceding GCMS method to establish calibration curves and quantify corresponding macamides in the various samples of maca extracts.

[0178] Two samples of n-hexanes containing 10.5 pg / mL of EtOBz were injected with the above method and the mean area of the peak corresponding to the EtOBz calculated and used to normalise calculations for standards 1-3 in the maca extracts.

[0179] Results

[0180] Extractions of various commercial maca powders were performed in n- hexanes and their macamide content quantified using previously generated calibration curves for each standard. These results were then normalised to an EtOBz internal standard present in the n-hexanes, which saw a reduction in the final concentrations of each standard of approximately 71.5% each. As for the maca powders extracted using ethanol, these were all found to contain less than 1 pg / g of all three macamides combined. The results are shown in Table 2 below.Table 2. Summary of quantitative results of standards 1-3 for gelatinised maca sampled, maca blends and maca samples extracted using an ethanol solvent.EXAMPLE 2

[0181] Summary

[0182] A series of analyses were undertaken on a set of maca powders using a LCMS method to look for variation across a set of maca components. Singh et al. have recently reported a detailed LCMS analysis of a range of maca powders with analysis of 19 separate macamides. [Singh et al. J. Nat. Prod. 2020, 83, 12, 3689-3697]

[0183] Experimental

[0184] 17 samples were provided, all powders and commercially packaged.

[0185] A sample of each powder was weighed out (approx. 600 mg) and in dispersed in approx. 10 ml of methanol. Extraction was facilitated by placement in an ultrasonic bath (3 X 30 mins). The extracts were then centrifuged and the supernatant collected and volume measured.

[0186] Samples were then analysed directly on a Shimadzu LCMS system (+ve mode, ESI) using a Phenomenex Omega PS C18 column (150 mm X 2.1 mm). The instrument was setup to measure the levels of nineteen compounds using MRM values taken from Singh et al.

[0187] Quantification was performed by separate injection of a standard mixed solution of supplied standards ( / V-benzypalmitamide (1), / V-benzoyl linoleamide (4) and / V-(3-methoxybenzyl)octadeca-9Z,12Z-dienamide (11). The following macamides were used for compounds and standards:• / V-Benzylpalmitamide (1) [standard used for 1-3]• / V-Benzyloctadecanamide (2)• / V-Benzyloctadec-9Z-enamide (3)• / V-Benzyloctadeca-9Z,12Z-dienamide (4) [standard used for 4-7 and 18,19]• / V-Benzyloctadeca-9Z,12Z,15Z-trienamide (5)• / V-Benzylicosa-5Z,8Z,llZ,14Z-tetraenamide (6)• / V-Benzylicosa-5Z,8Z,llZ,14Z,17Z-pentaenamide (7)• / V-(3-Methoxybenzyl)hexadecanamide (8)• / V-(3-Methoxybenzyl)octadecanamide (9)• / V-(3-Methoxybenzyl)octadec-9Z-enamide (10)• / V-(3-Methoxybenzyl)octadeca-9Z,12Z-dienamide (11) [standard used for 8-17]• / V-(3-Methoxybenzyl)octadeca-9Z,12Z,15Z-trienamide (12)• / V-(3-Methoxybenzyl)icosa-5Z,8Z,llZ,14Z-tetraenamide (13)• / V-(3-Methoxybenzyl)icosa-5Z,8Z,llZ,14Z,17Z-pentaenamide (14)• / V-(3,4-Dimethoxybenzyl)hexadecanamide (15)• / V-(3,4-Dimethoxybenzyl)octadeca-9Z,12Z-dienamide (16)• / V-(3,4-Dimethoxybenzyl)octadeca-9Z,12Z,15Z-trienamide (17)• / V-Phenethyloctadeca-9Z,12Z-dienamide (18)• / V-Phenethyloctadeca-9Z,12Z,15Z-trienamide (19).

[0188] Results

[0189] See Table 3 below for values of 10 selected macamides expressed as ug / g powder. These are the same 10 compounds which were quantified in the Singh paper. As seen in that work the other 9 components were either not seen of below the realistic detection limit. The tested powders ranged in total macamide concentration from 34-882 ug / g.Table 3. Results as pg / g powder for selected macamide compounds 1-5, 8-9, 10-12.EXAMPLE 3

[0190] Summary

[0191] Supercritical CO2 extraction of 10 kg of dried yellow gelatinised maca powder was carried out at 300 bar and 45°C. The extraction yield obtained was 0.4% (i.e. 0.4 g of extract per 100 g of maca powder). The extract obtained was a dark green resin, solid at room temperature.

[0192] Experimental

[0193] Dry, yellow gelatinised maca root powder supplied by Seleno Health was used as received. The material was placed in two 10L extraction baskets with sintered filter discs at both ends, and the baskets loaded into pressure vessels. CO2 was passed through the vessels in series in upflow direction. The CO2 containing the dissolved extract (after passing through the bed) was depressurized to approximately 55 bar and 45°C into a separation vessel where the extract was accumulated. Gas phase CO2 was then condensed and recirculated. Extract accumulating in the separation vessel was manually recovered through a valve periodically during the run to determine the progress of the extraction. After extraction, the plant was depressurized and the residual marc was allowed to degas overnight before being unloaded. An ethanol wash was applied to the plant once the extraction was completed to determine the quantity of any additional extract retained in the plant, and kept separate from the extract. Extraction parameters for both extractions are listed in Table 4 below.Table 4. Extraction conditions

[0194] Results

[0195] The extraction yield (including the amount of extract recovered during the final ethanol wash) was 0.4%, i.e. 0.4 g of extract per 100 g of maca powder (see Table 5 below).Table 5. Extraction yields from supercritical CO2 extraction

[0196] The extract obtained was a dark green resin, solid at room temperature, with a characteristic, strong smell. Towards the end of the extraction, the extract became a bit more fluid, with a reddish / brown tinge.EXAMPLE 4

[0197] Summary

[0198] An extraction with mixed hexanes (petroleum spirits 40-60°C cut), was performed at room temperature for 24 hours, followed by filtration and re-extraction of the filter cake. The combined filtrates produced a 0.74% extraction yield once the solvent was removed. Macamides and macaenes of the different fractions were analysed by LCMS. The results indicate that extraction of total macamides was practically complete (91-100% extracted), while macaene and MC2 were less completely extracted (50-58% extracted).

[0199] Extraction

[0200] The extraction was carried out in a 20L glass round bottom flask equipped with internal baffles. 1 kg of dried maca root powder (as received) was placed in the flask and 5 L (3.35 kg) petroleum spirits (boiling point 40-60 °C) was added. The flask was then placed on a rotary evaporator with the vacuum and heat off and with stirring speed set to 30 rpm; this achieved good mixing with the solids being in suspension in the bowl. The temperature in the water bath was constant at 17-18 °C. Approx. 15 mL samples of the solvent phase were taken after 2 hrs, 4 hrs, and 24 hours, after which the extraction was stopped and filtered through a vacuum filter. The filter cake was then resuspended in additional solvent, stirred for 1 hour at room temperature (18 °C), and filtered. The filtrates were combined and evaporated under vacuum to dryness (partial vacuum at 30 °C followed by full vacuum at 60 °C for 30 minutes).

[0201] Analysis

[0202] The following samples were analysed:• The final dried extract• Samples of the marc and feed material• Filtered liquid samples (hexane) of the extract taken after 2, 4 and 24 hours of extraction.

[0203] The feed and marc samples were extracted using hexane (lOmL) by taking about 150 mg and extracting using 3 x 5-minute treatments in a sonic bath. The extracts were centrifuged, the supernatant extract evaporated under N2 to dryness before being taken up in 1 mL methanol.

[0204] The liquid samples (the three extraction time points) were analysed directly.

[0205] Analysis was carried out by LCMS (Shimadzu 8040 triple quad, electrospray interface, with Nexera UPLC and photodiode array detector) using a RP C18 column with detection using both UV and mass spectrometry. The MS data was used to identify the peaks based on known molecular masses.

[0206] Results

[0207] A total of 7.4 g of solvent-free extract was obtained from the combined filtrates, representing a 0.74% yield.

[0208] A sample of the C16 (palmitic acid) macamide was used as a quantitative standard. This was only useful for the macamides such as MC5, MC6 and MC7 that have similar structures. These macamides were quantified according to peaks areas observed at 210 nm (Figure 1A).

[0209] The major macamide containing a dienone group (MC2) and macaene (1) show peaks in the UV chromatogram at 280 nm (Figure IB). The amounts of these compounds could not be determined without a standard, but a comparative estimate was obtained using the peak at 280 quantified relative to the C16 macamide standard at 210 nm. Note smaller peaks are seen with the same MW as the two main compounds, these may be cis / trans isomers.

[0210] The compound analysis results (Table 6 below) and the mass balance results for total macamides (Table 7 below), which shows the amount of macamides in the extract and marc relative to the feed, was well over 100%. This can be caused by incomplete analytical extraction of macamides in the feed powder prior to analysis. This, coupled to the low level of remaining macamides in the marc, indicates that macamides were practically fully extracted by petroleum spirits in 24 hours. The mass balance of macaene + MC2, however, is lower, with only 91% of the total amount initially present in the feed being present in the extract and marc. In this case, the split between extract and marc is practically equal, indicating that macaene and MC2 are not fully extracted by petroleum spirits. The dienone moiety in these two compounds may be less stable and may degrade with light and heat exposure.Table 6. Maca compound analysis resultsTable 7. Mass balance for total macamides (A) and total macaene + MC2 (B)

[0211] Samples of the hexane extracts were provided for LCMS analysis, following the published method of Singh et al. J. Nat. Prod. 2020, 83, 12, 3689-3697. These were analysed directly and the peaks areas of the various compounds of interest were compared. The data in Figure 1C shows that there is not a large difference in extract concentration between any of the samples, suggesting that relatively short extraction times (e.g. 2-4 hrs) may be sufficient for good extraction. The small drop in concentration for the 24 hr sample may not be significant.EXAMPLE 5

[0212] Summary

[0213] An extraction with liquefied propane was performed using bench scale equipment. Total extraction yield was 1.0%. Macamides and macaenes of the different fractions were analysed by LCMS.

[0214] Extraction

[0215] The dry, yellow gelatinised maca root powder supplied by Seleno Health was used as received. The feed material was placed in a 1.6 L basket with sintered discs at both ends, and the basket was inserted into a 2L extraction vessel. The feed material (900g) filled the basket to approximately 95% full. Liquid propane (4.5kg) was then passed through the vessel in downflow direction until 9.5: 1 propane to feed mass had been circulated. At this point, the propane flow was stopped, and the extraction vessel was slowly depressurised. An ethanol wash of the plant was then carried out to recoverany residual extract remaining in the plant. Extraction parameters are listed in Table 8 below.Table 8. Extraction conditions

[0216] Analysis

[0217] The propane marc sample was extracted using methanol (10 ml) by taking about 630 mg and extracting using 3 x 5-minute treatments in a sonic bath. The extract was centrifuged and the supernatant analysed directly.

[0218] Samples of hexane and CO2 extracts obtained by in previous work were also analysed for comparison.

[0219] Analysis was carried out by LCMS as previously detailed, using a RP C18 column with detection using both UV and mass spectrometry. The MS data was used to identify the peaks based on known molecular masses.

[0220] Results

[0221] Propane extraction of 900 g of maca powder produced 6.1 g of extract (0.7% yield). An additional 2.5 g of extract was recovered during the ethanol wash, increasing the total extraction yield to 1.0% (see Table 9 below).Table 9. Extraction yields

[0222] The compound analysis results (Table 10 below, see also Figure 2) comparing the three different extracts (hexane, CO2 and propane).Table 10. Maca compound analysis results (mg / g extract for extract samples, mg / g powder for marc sample)EXAMPLE 6

[0223] Propane extraction was scaled up as follows:

[0224] Propane extraction

[0225] The feed material (dry, pre-ground maca root, batch nos. HSO661, MNB5K and MCH5K) was supplied by Seleno Health and used as received. Two extractions were carried out. In each extraction, approximately 100 kg of powder was placed in a 180 L extraction basket with sintered filter discs at both ends, filling up approximately 95% of the basket volume with a packing density of approximately 585 g / L. Food grade MCT oil C8 60% (supplied by Pure Nature, batch no. PNF2307) was added to the bottom of the vessel before inserting the basket. The amount of oil was calculated as 25 miykg of feed. The vessel was then pressurised to the extraction pressure and pressurised propane was circulated in upflow direction. The extract was accumulated in the separation vessel and manually recovered through a valve at the end of the extraction. The total ratio of propane to feed used was approximately 5.7: 1 (i.e. 5.7 kg of propane per kg of feed). At this point, the extraction was deemed complete, the plant was slowly depressurised, and the residual marc was allowed to degas overnight before being unloaded.

[0226] Additional MCT oil (25 mL / kg of feed) was used to wash the lines and control valve after the extraction and maximise extract recovery. This oil was kept separate from the bulk extract collected throughout the run.

[0227] Extraction parameters are listed in Table 11 below.Table 11. Extraction conditions

[0228] Analysis

[0229] A sample of each powder was weighed out (approximately 600 mg for maca and approximately 20 mg for extracts) and dispersed in approximately 10 mL of methanol. Extraction was facilitated by placement in an ultrasonic bath (3 x 30 mins). The extracts were then centrifuged and the supernatant collected and volume measured.

[0230] Samples were then analysed directly on a Shimadzu LCMS system (+ve mode, ESI) using a Phenomenex Omega PS C18 column (150 mm x 2.1 mm). The instrument was setup to measure the levels of nineteen macamide compounds using MRM values taken from Singh et al.

[0231] Quantification was performed by separate injection of a standard ( / V- benzylpalmitamide; compound 1). See list below for compounds (compounds 13-17 not present at measurable levels).

[0232] List of macamide compounds:• / V-Benzylpalmitamide (1) (MC7 in previous work)• / V-Benzyloctadecanamide (2)• / V-Benzyloctadec-9Z-enamide (3) (MC372 in previous work)• / V-Benzyloctadeca-9Z,12Z-dienamide (4) (MC6 in previous work) / V-Benzyloctadeca-9Z,12Z,15Z-trienamide (5) (MC5 in previous work)• / V-Benzylicosa-5Z,8Z,llZ,14Z-tetraenamide (6)• / V-Benzylicosa-5Z,8Z,llZ,14Z,17Z-pentaenamide (7)• / V-(3-Methoxybenzyl)hexadecanamide (8)• / V-(3-Methoxybenzyl)octadecanamide (9)• / V-(3-Methoxybenzyl)octadec-9Z-enamide (10)• / V-(3-Methoxybenzyl)octadeca-9Z,12Z-dienamide (11)• / V-(3-Methoxybenzyl)octadeca-9Z,12Z,15Z-trienamide (12)• / V-Phenethyloctadeca-9Z,12Z-dienamide (18)• / V-Phenethyloctadeca-9Z,12Z,15Z-trienamide (19)

[0233] Macaenes were also measured. These were determined from MS data for macamide MC2 ( / V-Benzyl-9-oxo-octadeca-10,12-dienamide, using the same standard as above). The macaene was then calculated from the MC2 concentration using the relative UV peak area at 280 nm.• 9-oxo-octadeca-10,12-dienoic acid (macaene)

[0234] Results

[0235] Propane extraction of 214.19 kg of maca powder produced 1.145 kg of extract (0.53% average yield); see Table 12. This is calculated based on "oil-free extract", which assumes all the MCT oil added to the extraction vessel is extracted. In reality, a small fraction of the added oil is likely to have remained in the marc, which would mean the actual yield was higher.

[0236] The yield obtained in the first extraction was higher, possibly due to the feed material used. Batches MNB5K and MCH5K (yellow gelatinised maca chip fragments), which made up approximately 38% of the feed used in extraction 2, was coarser in appearance and had a red colour, as opposed to batch HSO661, which was a pale yellow fine gelatinised maca powder, and which made up 100% of the feed in extraction 1 and approximately 62% of the feed in extraction 2.

[0237] The extract had a tendency to separate on standing and cooling, with a cloudy suspension being formed at the bottom. Samples of the extract were centrifuged prior to analysis and the two phases were analysed separately (see results in Figure 3A, and see percentages of each macamide or macaene relative to the total measured macamides and macaenes in Figure 3B). The cloudy fraction, once centrifuged, was a very small proportion of the total extract, and the composition of the bulk extract is expected to be very similar to that of the clear fraction. Interestingly, the composition of the cloudy fraction for the two extracts is fairly similar, indicating the cloudiness in the samples is due to selective dissolution of the macamides. If a clear product was desired then the cloudy layer could be removed by winterisation or centrifuging, with a small loss of total macamide content.

[0238] Results show that the feed used in extraction 2 was lower in macamides, with a total macamide concentration of 1.4 mg / g (vs 2.35 mg / g in extraction 1, see Table 13). This is in agreement with the higher extraction yield obtained in extraction 1 and likely caused by variability in the different batches of feed used in each extraction. In terms of macaenes, both feeds were relatively similar, but a higher proportion of these remain unextracted in the marc of the second extraction (0.87 mg / g vs 0.09 mg / g, see Table 13).

[0239] The wash fraction has a pale yellow colour and the analysis showed presence of macamides (1.87 mg / g in extraction 1 and 2.93 mg / g in extraction 2) and macaenes (0.30 mg / g in extraction 1 and 0.46 in extraction 2), see Table 13 below. This fraction could be combined with the bulk extract if required.

[0240] In terms of macamide yield, extraction 1 achieved excellent recovery, whereas macamide extraction was less complete in extraction 2. This could be in part due to the different quality feed being used, and also the larger particle size of the feed.Table 12. Extraction YieldsTable 13. Total macamides and macaenes (mg / g)EXAMPLE 7

[0241] Summary

[0242] This example aims to characterize the binding and functional profiles of macamides on CB1 and CB2 receptors. Three isolated macamides, abbreviated as macamides 86, 15 and 58, were used (macamides 5, 11, and 1, from Table 1, respectively). Two macamide-containing maca extracts (hexane and propane) were also used. The hypothesis is that macamides bind effectively and acts as agonists to CB1 but not CB2 receptors based on previous observations.

[0243] Materials and Methods

[0244] The compounds of interest included three macamides abbreviated as macamides 86 = / V-benzyl-(-9Z, 12Z)-octadecadienamide (CAS 18286-71-0), macamides 15 = / V-3-methoxybenzyl-(9Z, 12Z)-octadecadienamide (CAS 883715-22-8) and macamides 58 = / V-benzylpalmitamide (CAS 74058-71-2); and two maca extracts: maca hexane extracts containing 14 mg / g macamide 86, 20 mg / g macamide 15 and 25 mg / g macamide 58, and maca propane extracts containing 20 mg / g macamide 86 30 mg / g macamide 15 and 42 mg / g macamide 58. The concentrations for maca extracts were calculated using the molecular weight of the most concentrated macamides present. The endogenous cannabinoid, anandamide (AEA) was purchased from Sapphire Bioscience. AEA is used as a positive control due to known binding and agonist activity on CB1 and CB2 receptors.

[0245] Cell culture

[0246] Human embryonic kidney (HEK) 293 cells wildtype, and HEK293 3HA-hCBl and HEK-Flp HA-3TCS-hCB2 have all been previously described (Cawston et al., 2015). Cells were cultured in Dulbecco's Modified Eagle Medium supplemented with 10 % fetal bovine serum. 250 pg / mL Zeocin was added to HEK293 3HA-hCBl and 50 pg / mL Hygromycin to HEK293-Flp HA-3TCS-hCB2 to maintain selection pressure. Cells were then grown in 37°C with 5% CO2 until they are confluent.

[0247] Radioligand competition binding assay

[0248] A series of compound concentrations or matched compound vehicles with EtOH or DMSO, 1 nM [3H] CP55940, 0.08 mg / mL of ppls-3HA-hCBl pPEF4a HEK membrane preparation or 0.03 mg / mL 3HA-hCB2 HEK membrane preparation werediluted in binding buffer (50 mM HEPES pH 7.4, 1 mM MgCI2, 1 mM CaCI2, 2 mg / mL BSA). A mixture of 50 pL radioligand, 50 pL compound of interest or vehicle, and 100 pL membrane preparation were incubated for one hour at 30°C, and then transferred to 96 well Harvest Plate pre-soaked with 0.1% PEI and washed four times with 200 pL ice-cold wash buffer (50 mM HEPES pH 7.4, 500 mM NaCI, 1 mg / ml BSA). The harvest plates were left to dry at room temperature overnight prior to addition of 50 pL scintillation cocktail to each well. The amount of radioactivity in each well was then detected by scintillation counting for two minutes per well after 30 minutes delay using TriLux Liquid Scintillation Counter (Perkin Elmer).

[0249] CAMP CAMYEL assay

[0250] HEK293 wildtype, HEK293 cells stably expressing 3HA-hCBl or HA-hCB2 were seeded into 10 cm dishes. On the next day, fresh medium was applied, and the cells were transfected with 5 pg pcDNA3L-His-CAMYEL plasmid and 30 pL PEI. The following day, cells were trypsinised and plated into PDL prepared 96-well cell culture plates at a density of 60,000 cells / wells. Cells were serum-starved with phenol-free medium for at least 30 minutes before the assay. Cells were then incubated with coelenterazine-H at 37oC for 5 minutes, before adding forskolin (FSK) and the compound mixture. Luminescence was simultaneously detected at 475nm and 535 nm, in a BMG LUMIstar for 25 minutes and the ratio of emissions at 475 / 535 nm calculated in MARs.

[0251] Data analysis

[0252] All data analysis utilised GraphPad Prism 9.2.1. Binding affinity was determined utilising One site fit Ki (with [3H] CP55,940 Kd of 3.5nM and 1.16 nM for CB1 and CB2 respectively). Concentration response curves modelled by fitting a three- parameter, non-linear regression curve. Statistical evaluation performed on GraphPad Prism 9.2.1 for Windows. CAMYEL data was analysed with oneway ANOVA and Dunnett multiple comparisons test. Data are presented as mean ± SD.

[0253] Results

[0254] Unsaturated macamides, maca propane extracts and maca hexane extracts bind with selectivity towards CB2 receptors.

[0255] Radioligand competition binding assay measures the binding affinity (Ki) of compounds to CB1 and CB2 receptors. Ki is defined as the concentration of compound required to displace 50% of radioligand binding (measured as corrected counts perminute, CCPM; pKi is the negative log of this value). AEA is an endogenous cannabinoid and here displaced the radioligand with expected affinity for CB1 (pKi = 6.1 ± 0.1 log M) and CB2 receptors (pKi = 6.3 ± 0.1 log M) (see Table 14 and Figure 4). Saturated macamide 58 did not bind, while unsaturated macamides 86 and 15, maca propane extracts and maca hexane extracts bound CB1 and CB2 receptors, but with lower affinity than AEA (Table 14 and Figure 4). Table 14 shows macamides 86 and maca hexane extract has 4-fold selectivity towards CB2 receptors over CBi receptors; macamides 15 and maca propane extract has 3-fold selectivity towards CB2 receptors over CBi receptors.Table 14. Binding affinity of macamides and maca extracts.

[0256] Macamides and maca extracts are not agonists on CBI and CB2 receptors.

[0257] The CAMYEL assay measures the potency (IC50) and efficacy of compounds to modulate intracellular cAMP levels. IC50 is defined by the compound concentration required to cause 50% reduction in forskolin-stimulated cAMP levels. Figure 5 demonstrates that when the endogenous agonist, AEA, activates CBI and CB2 receptors, forskolin-stimulated cAMP levels are reduced in a dose-dependent manner. In contrast,high concentrations of macamides 86 and 15 cause increased cAMP levels, which is even more pronounced with maca hexane extract and maca propane extract. Macamide 58 has no effect on cAMP levels, which is consistent with binding data.

[0258] To investigate non-cannabinoid receptor mediated cAMP production, the cAMP assay was conducted with HEK293 wildtype cells lacking cannabinoid receptors. Figure 4C shows cAMP levels elevated both in the presence and absence of cannabinoid receptors. However, the degree of cAMP production differs in each cell line. Maca extracts produce noticeably higher cAMP levels in HEK293 cells stably expressing CB1 receptors than in wildtype HEK293 cells.

[0259] Unsaturated macamides, maca propane extract and maca hexane extract antagonise AEA effect

[0260] Since the compounds bound but not activate cannabinoid receptors, we investigated if they block AEA activity at the receptors instead (i.e., act as antagonists). An antagonist would produce a rightward shift of the AEA concentration-response curve, reducing the apparent ICso. AEA alone acts on CBi and CB2 receptors with pICso (log M) of 7.4 ± 0.1 and 6.5 ± 0.3, respectively (Table 15). Macamide 58 has no effect on AEA concentration-response curves (Figure 4). AEA potency on CBi receptors is significantly (p < 0.001) reduced by macamides 86 and 15, maca propane extracts and maca hexane extracts (Table 15, Figure 4). AEA activity at CB2 receptors is significantly (p < 0.05) reduced by macamide 86 (Table 15, Figure 5).Table 15. AEA potency on cAMP production in the presence and absence of macamides or maca extracts at CBi or CB2 receptors.

[0261] It was found from this data that macamides bind with moderate selectivity towards CB2 receptors, and may act as antagonists or inverse agonists.EXAMPLE 8

[0262] The macamides and macaenes extracted using supercritical propane and a food safe oil provide the composition of the invention. The oil is preferably but not limited to MCT oil. Terpenes are optionally added up to about 2% w / w concentration.

[0263] Total macamides and macaenes were determined by weighing a approx. 20 mg sample and dispersing in approximately lOmL of methanol. Extraction was facilitated by placement in a ultrasonic bath (3 x 30min). The extract was centrifuged and the supernatant collected and analysed directly on a Shimadzu LCMS system (+ve mode, ESI) using a Phenomenex Omega PS C18 column (150 mm x 2.1 mm).

[0264] Two different batches of extract provided the following results shown in Table 16 below:Table 16. Total maca compounds from two batch extractions.EXAMPLE 9

[0265] Prior to administration a group of volunteers are tested for a baseline measurement across a panel of cognitive and physical states. A suitably concentrated maca extract in an oil is administered to an experimental arm, and a control arm is administered the oil without a maca extract. Optionally a prior art maca extract in oil is administered in a second control arm. The administration may be topical, oral, or sublingual.

[0266] Measurements are then taken at a prescribed interval after administration. Results from the experimental arm are compared against the first control arm and / or the second control arm.

[0267] The experimental arm is expected to show an effect relating to increased cognitive function, improving mood, increased mental clarity, sustained state of serenity, tranquillity peace and / or equanimity, increased physical performance, increased libido, increased sexual function, increased energy, increased muscle strength, increased muscle growth, increased energy, increased stamina, increased endurance, increased analgesic effect and / or decreased inflammation.EXAMPLE 10

[0268] Summary

[0269] This example aims to characterise the impact of selected macamides on immune signalling in vitro, including testing for cytotoxicity and cell stimulation for C-X-C motif chemokine ligand 10 (CXCL-10), for monocyte chemoattractant protein 1 (MCP-1) and for tumour necrosis factor- a (TNF-a).

[0270] Cytotoxicity

[0271] HaCaT immortalised human keratinocytes were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco Inc., Brooklyn, NY, USA), supplemented with 10% heat-inactivated foetal bovine serum (FBS, Moregate Biotech, NZ), 50 U / mLpenici I li n-streptomyci n and 60 pg / mL kanamycin sulphate (Gibco Inc., Brooklyn, NY, USA).

[0272] To assess cytotoxicity, HaCaT cells were seeded in 96-well plates (7.5x104 cells / mL) and incubated for 24-hours in 5% CO2 at 37 °C. Cells were then treated with varying concentrations of maca extract or vehicle controls in triplicate for a further 24- hours. Following this period, the media was removed, each well was washed with phosphate-buffered saline (PBS) and the cells were fixed with ice-cold methanol for 15 minutes. A crystal violet stain was used to quantify cell viability based on the proportional relationship between viable, adherent cells and stained DNA content (Feoktistova et al., 2016). Briefly, cells were stained with crystal violet solution (0.01% (v / v), Sigma) for 10 minutes at room temperature, washed three times with distilled water, and the crystals were solubilised with 33% (v / v) glacial acetic acid (Merck). Absorbance was read at 590 nm, with the percentage of viable cells calculated relative to media-only control.

[0273] The cytotoxicity of macamides were assessed in HaCaT cells to obtain the maximum non-toxic concentrations for subsequent in vitro experiments. This was necessary to ensure that cell viability is sufficient to accurately measure biological processes, such as the production of cytokines and chemokines. The maximum non-toxic concentration was the concentration applied to cells for 24-hours that provided 80% cell viability or greater and had no significant statistical difference to media control treatment. Table 17 lists the maximum non-toxic concentrations for the macamides tested. A previous study noted that water, methanol, and chloroform extracts of black, red, grey and yellow maca were all well-tolerated (>86% viability) at 25-200 pg / mL in HaCaT cells (Kasprzak et al., 2024). The maximum non-toxic concentration for DMSO was 1% (v / v).Table 17. Cytotoxicity of macamides in HaCaT human keratinocytes.

[0274] CXCL10 and MCP-1 are chemokines that are upregulated in states of inflammation or tissue damage, such as multiple sclerosis and viral infection (Sorensen et al., 1999; Zink et a / ., 2001; Liu et al., 2011). As chemokines, these proteins influence the recruitment, migration and infiltration of immune cells, including activated T cells and monocytes, to the site of inflammation. To upregulate CXCL10 and MCP-1, HaCaT cells were treated with 100 ng / mL IFN-y. This stimulated HaCaTs to release an average of 23,021 pg / mL CXCL10 and 32,209 pg / mL MCP-1 (Table 18). TNF-a is a pro-inflammatory cytokine that is expressed during various inflammatory states in the body, including infection, tissue trauma, and autoimmune disease (Popa et al., 2007). To upregulate TNF-a, THP-1 cells were treated with 20 ng / mL PMA and 1 pg / mL LPS. This stimulated THP-ls to release an average of 6,953 pg / mL TNF-a (Table 18).Table 18. Positive and negative control values for ELISAs

[0275] Cell stimulation for C-X-C motif chemokine ligand 10 (CXCL-10) and monocyte chemoattractant protein 1 (MCP-1) enzyme-linked immunosorbent assays (ELISAs)

[0276] HaCaT cells were seeded in 24-well plates (7.5x104 cells / mL) and incubated for 24-hours in 5% CO2 at 37 °C. Cells were then co-treated with 100 ng / mL interferongamma (IFN-y, BioLegend, Inc., San Diego, CA, USA) and non-toxic concentrations of maca extract or vehicle controls. For cytokine and chemokine analyses, the treated cellculture medium was collected after 24 h of incubation with co-treatments of cytokine(s) and maca extracts or vehicle controls. Hydrocortisone (250 pg / mL, purity > 98%; Sigma, St. Louis, MO, USA) served as a positive control due to previously-documented suppression of cytokine and chemokine release in stimulated keratinocytes (Kok et al., 2023). The collected media was centrifuged at 1,800 rpm, with the supernatant aliquoted and stored at -80 °C.

[0277] BD-OptEIA™ CXCL-10 (Cat. No. 550926) and MCP-1 (Cat. No. 555179) enzyme-linked immunosorbent assay (ELISA) kits were purchased from BD Biosciences Pharmingen (San Diego, CA, USA). Chemokine levels within the conditioned medium from treated HaCaT cells were quantified in accordance with the manufacturer's instructions. CXCL-10 and MCP-1 amounts were normalised to IFN-y-stimulated or vehicle control conditions.

[0278] Cell stimulation for tumour necrosis factor- a (TNF-a) ELISA

[0279] Human monocytic leukaemia THP-1 cells were cultured in Roswell Park Memorial Institute 1640 medium (Gibco Inc., Brooklyn, NY, USA) with 10% heat- inactivated FBS (Moregate Biotech, NZ), 2 mM sodium pyruvate (Gibco Inc., Brooklyn, NY, USA), 50 U / mL penicillin-streptomycin, and 60 pg / mL kanamycin sulphate (Gibco Inc., Brooklyn, NY, USA).

[0280] In 24-well plates, THP-1 cells (1x106 cells / mL) were stimulated with 20 ng / mL phorbol myristate acetate (PMA, Abeam Ltd, Cambridge, UK), and 1 pg / mL lipopolysaccharide (LPS, Escherichia coli 055: B5 derived, purity >97%; Sigma, St. Louis, MO, USA) then co-treated with non-toxic concentrations of maca extract or vehicle control. The conditioned medium was collected by centrifugation (4,000 rpm) after 24 h incubation with the stimulant and maca extract co-treatment, then stored at -80°C.

[0281] Human TNF-a ELISA Max™ Deluxe kit (Cat. No. 430204) was purchased from BioLegend® (BioLegend, Inc., San Diego, CA, USA). Cytokine levels within the conditioned medium from treated THP-1 cells were quantified in accordance with the manufacturer's instructions. TNF-a release was normalised to stimulated or vehicle control conditions.

[0282] CXCL10 results

[0283] DMSO had no significant effect on CXCL10 levels in IFN-y-stimulated HaCaT cells.

[0284] Macamide 15 and macamide 86 both caused reductions in CXCL10 release when co-treated alongside IFN-y in HaCaT cells, reducing CXCL10 to 3 and 11% of control values at the highest concentrations tested, respectively (Figures 8A and 8C). Macamide 58 had no significant effect on IFN-y-stimulated CXCL10 release (Figure 8B).This is the first evidence of an anti-inflammatory effect on stimulated CXCL10 levels from macamide 15 and macamide 86 in vitro.

[0285] MCP-1 results

[0286] DMSO had no significant effect on MCP-1 levels in IFN-y-stimulated HaCaT cells. DMSO had similar effects on MCP-1 as was seen for CXCL10.

[0287] Macamide 15 and macamide 86 showed anti-inflammatory activity, significantly reducing MCP-1 levels with increasing concentration (Figures 9A and 9C, P<0.05). These findings provide an initial assessment of the effectiveness of macamides on reducing MCP-1 chemokine levels in IFN-y-stimulated HaCaT cells.

[0288] Cell stimulation for tumour necrosis factor- a (TNF-a) ELISA

[0289] Human monocytic leukaemia THP-1 cells were cultured in Roswell Park Memorial Institute 1640 medium (Gibco Inc., Brooklyn, NY, USA) with 10% heat- inactivated FBS (Moregate Biotech, NZ), 2 mM sodium pyruvate (Gibco Inc., Brooklyn, NY, USA), 50 U / mL penicillin-streptomycin, and 60 g / mL kanamycin sulphate (Gibco Inc., Brooklyn, NY, USA).

[0290] In 24-well plates, THP-1 cells (1x106 cells / mL) were stimulated with 20 ng / mL phorbol myristate acetate (PMA, Abeam Ltd, Cambridge, UK), and 1 pg / mL lipopolysaccharide (LPS, Escherichia coli 055: B5 derived, purity >97%; Sigma, St. Louis, MO, USA) then co-treated with non-toxic concentrations of maca extract or vehicle control. The conditioned medium was collected by centrifugation (4,000 rpm) after 24 h incubation with the stimulant and maca extract co-treatment, then stored at -80°C.

[0291] Human TNF-a ELISA Max™ Deluxe kit (Cat. No. 430204) was purchased from BioLegend® (BioLegend, Inc., San Diego, CA, USA). Cytokine levels within the conditioned medium from treated THP-1 cells were quantified in accordance with the manufacturer's instructions. TNF-a release was normalised to stimulated or vehicle control conditions.

[0292] TNF-a results

[0293] DMSO had no significant effect on TNF-a levels in LPS-stimulated THP-1 cells. Unlike CXCL10 and MCP-1, TNF-a levels were similar to stimulated control values across the tested concentration ranges for ethanol and DMSO. Higher concentrations of 9: 1 DMSO:chloroform appeared to increase TNF-a release from LPS-stimulated THP-1 cells.

[0294] Macamide 15 had significant anti-inflammatory effects on TNF-a release when co-treated alongside PMA and LPS in THP-1 cells (Figure 10A, P<0.05). Treatment of stimulated THP-1 cells with macamide 86 appeared to reduce TNF-a at higher concentrations, although this trend was not significant. These results provide the first in vitro evaluation of the anti-inflammatory effects of macamides on TNF-a levels.Ethanolic maca extract (200 and 400 mg / kg) and the macamide N-(3-Methoxybenzyl)- (9Z,12Z,15Z)-octadecatrienamide (5 and 25 mg / kg) have been previously reported to reduce TNF-a levels in the hippocampus of rats following corticosterone-induced neurotoxicity (Yu et al., 2020; Yu et al., 2021).EXAMPLE 11

[0295] Summary

[0296] This example aims to characterise the impact of selected maca products, extracts and macamides on immune signalling in vitro, including testing for monocyte chemoattractant protein 2 (MCP-2), Cyclooxygenase (COX)-2 activity, cell stimulation for P-endorphin and Fatty acid amide hydrolase (FAAH) activity / inhibition.

[0297] Cytotoxicity data for maca products and extracts

[0298] Following on from the cytotoxicity data for macamides in Example 9, the cytotoxicity of maca products, product components and extracts were assessed in HaCaT cells to obtain the maximum non-toxic concentrations for subsequent in vitro experiments. This was necessary to ensure that cell viability is sufficient to accurately measure biological processes, such as the production of cytokines and chemokines. The maximum non-toxic concentration was the concentration applied to cells for 24-hours that provided 80% cell viability or greater and had no significant statistical difference to media control treatment. Table 19 lists the maximum non-toxic concentrations for all products, components and extracts tested. A previous study noted that water, methanol,and chloroform extracts of black, red, grey and yellow maca were all well-tolerated (>86% viability) at 25-200 pg / mL in HaCaT cells (Kasprzak et al., 2024). The maximum non-toxic concentration for all vehicles (ethanol, DMSO, 9: 1 DMSO:chloroform) was 1% (v / v). It was necessary to assess the cytotoxicity of all vehicles to determine the toxicity of each maca product, product component and extract alone.Table 19. Cytotoxicity of macamides in HaCaT human keratinocytes.

[0299] Cell stimulation for monocyte chemoattractant protein 2 (MCP-2) ELISA

[0300] HaCaT cells were seeded in 24-well plates (7.5x104 cells / mL) and incubated for 24-hours in 5% CO2 at 37 °C. Cells were then co-treated with 10 ng / mL interferongamma (IFN-y, BioLegend, Inc., San Diego, CA, USA), 10 ng / mL TNF-a (Sino Biological, Inc. Beijing, China), 10 ng / mL interleukin-4 (IL-4, Sino Biological, Inc. Beijing, China),10 ng / mL interleukin-13 (IL-13, Sino Biological, Inc. Beijing, China), 20 ng / mL lipoteichoic acid (LTA, Staphylococcus aureus derived, purity >97%; Sigma, St. Louis, MO, USA) and non-toxic concentrations of maca extract or vehicle controls.

[0301] Human CCL-8 (MCP-2) ELISA Max™ Deluxe kit (Cat. No. 442204) was purchased from BioLegend® (BioLegend, Inc., San Diego, CA, USA). Chemokine levels within the conditioned medium of treated HaCaT cells were quantified in accordance with the manufacturer's instructions. MCP-2 release was normalised to stimulated or vehicle control conditions.

[0302] Cyclooxygenase (COX)-2 activity assay

[0303] The impact of maca products and extracts on the activity of COX-2 are determined following the methods of Petrovic & Murray (2010). COX peroxidase activity oxidises the substrate, N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), creating a blue product with a maximum absorbance at 590 nm (Petrovic & Murray, 2010). This reaction can be utilized for the high-throughput screening of potential COX inhibitors.

[0304] Recombinant COX-2 enzyme was purchased from In Vitro Technologies (Auckland, NZ). TMPD, arachidonic acid, eicosapentaenoic acid, and hematin were purchased from Sigma-Aldrich (Sigma, St. Louis, MO, USA). A range of concentrations of maca extracts or vehicle controls were screened in triplicate for COX-2 inhibition in accordance with the methods of Petrovic & Murray (2010). The percent COX-2 activity relative to an activity control was calculated.

[0305] Cell stimulation for (3-endorphin ELISA

[0306] Following previously published methods (Ibrahim et al., 2005; Gao et al., 2016), HaCaT cells were cultured in 24-well plates (7.5x 104 cells / mL) and incubated for 24-hours in 5% CO2 at 37 °C. Cells were then treated with non-toxic concentrations of maca extract or vehicle controls 30-60 minutes. A cannabinoid CB2 receptor selective agonist, HU-308, investigated as a positive control. At 45-minutes post-treatment, culture media was collected by pipetting and centrifuged for 5 minutes at 2,000 rpm. The supernatant was aliquoted and stored at -80 °C until required.

[0307] Human Beta Endorphin ELISA kit (Cat. No. EEL031) was purchased from Invitrogen™ (Thermo Fisher Scientific, Waltham, MA, USA). Beta-endorphin levels within the conditioned medium of treated HaCaT cells was quantified in accordance with the manufacturer's instructions.

[0308] Fatty acid amide hydrolase (FAAH) activity / inhibition assay

[0309] As outlined by Alasmari et al. (2019), maca extracts, product components, and vehicle controls are assessed for the ability to inhibit FAAH activity. The assay measures the hydrolysis of a substrate, 7-amino-4-methylcoumarin-arachidonamide, by the FAAH enzyme, to the fluorescent 7-amino-4-methylcoumarin (7-AMC) product (Wang et al., 2006). Subsequently, FAAH inhibitors are expected to decrease the fluorescence of the final solution by preventing hydrolysis.

[0310] Fatty Acid Amide Hydrolase Inhibitor Screening Assay kit (Cat. No. 10005196) was purchased from Cayman Chemical (Cayman Chemical, Ann Harbour, MI, USA). A range of concentrations of maca extracts or vehicle controls were screened in duplicate for FAAH inhibition in accordance with the manufacturer's instructions. The kit includes a known inhibitor of FAAH, JZL 195, as a positive control (Long et al., 2009). Drug treatments or solvent controls were preincubated with human recombinant FAAH enzyme for 5 minutes at 37°C. The reaction was initiated through addition of FAAH substrate and incubated for 30 minutes at 37°C. FAAH conversion of 7-amino-4- methylcoumarin-arachidonamide to 7-AMC was measured in a SpectraMax i3x multimode microplate reader (Molecular Devices, San Jose, CA, USA) using an excitation wavelength of 350 nm and an emission wavelength of 460 nm. The percent inhibition or percent FAAH activity relative to vehicle control will be calculated as a function of the inhibitor concentration. This will be used to determine the IC50 value (the concentration at which there is 50% inhibition).

[0311] Results and discussion

[0312] MCP-2 results

[0313] The chemokine MCP-2 (CCL8) recruits immune cells to sites of inflammation. Upregulation of MCP-2 has been linked to inflammatory conditions such as Crohn's Disease, allergy and viral infection (Gelzo et al., 2021; Irak et al., 2021). To upregulate MCP-2, HaCaT cells were treated with 20 ng / mL LTA, 10 ng / mL TNF-a, 10 ng / mL IFN-y, 10 ng / mL IL-4, and 10 ng / mL IL-13. This stimulated HaCaTs to release an average of 5,601 pg / mL MCP-2, compared to 409 pg / mL released in media control conditions.

[0314] Ethanol, DMSO and 9: 1 DMSO:chloroform vehicles had no significant effect on MCP-2 levels in stimulated HaCaT cells. MCP-2 levels were similar to stimulated control values across the tested concentration ranges for all three solvent controls.

[0315] MCB oil, black and red maca, macamides 15 and 86, hexane and CO2 maca extracts had significant, concentration-dependent effects on MCP-2 levels (Figure 11, P<0.05). At the highest concentrations tested, black and red maca powder, along with macamides 15 and 86 all exhibited MCP-2 levels lower than 5% of respective vehicle controls. MCT oil, macamide 58 and maca propane extract treatment did not significantly affect MCP-2 release in stimulated HaCaT cells (Figures 11B, 11F and 11H). CDB treatment at 10 and 1 pM (equivalent to 3 and 0.3 pg / mL) also did not significantly affect stimulated MCP-2 levels (Figure 11K). This is the first evidence of an anti-inflammatory effect on stimulated MCP-2 levels from maca products, extracts and macamides in vitro.

[0316] COX-2 activity results

[0317] COX catalyses the conversion of arachidonic acid to prostaglandin G2 and H2. Prostaglandin H2 is then converted to a range of eicosanoids. These eicosanoids mediate several inflammatory responses, including vasodilation, recruitment of immune cells, pain perception, and tissue repair (Sheppe & Edelmann, 2021). COX-2 inhibitors are important therapeutics for the relief of acute pain and inflammation due to the drugs' inhibition of prostaglandin synthesis, preventing the aforementioned inflammatory signalling cascade (Vane, 1971). Maca products, product components and extracts were assessed for the ability to directly affect COX-2 enzyme activity in vitro as a potential anti-inflammatory mechanism.

[0318] The phytocannabinoid cannabidiol (CBD) and corticosteroid hydrocortisone did not inhibit COX-2 activity relative to control (Figure 12A). These results differ to those previously published in the literature, where CBD (1 pM) reduced COX-2 activity by 22% relative to control in a similar, acellular assay using arachidonic acid as a substrate (Cosentino et al., 2023). Corticosteroids are known to repress COX-2 gene expression by destabilising the messenger RNA (Newton et al., 1998) but have less defined effects on direct enzyme activity. MCB and MCT oil treatments did not significantly inhibit COX-2 activity (Figure 12B and 12C). The four highest concentrations of MCT oil tested increased COX-2 activity compared to control (P<0.05). As polyunsaturated fatty acids serve as co-substrates in this assay (Petrovic & Murray, 2010), it is possible that the treatment of medium chain triglycerides provided an alternative substrate source, resulting in greater COX-2 activity. Ethanol as a vehicle had no significant effect on COX- 2 activity (Figure 12D). However, DMSO and 9: 1 DMSO:chloroform vehicles caused significant increases in COX-2 activity (Figure 12E and 12F). To account for the effect of these DMSO and 9: 1 DMSO:chloroform vehicle concentrations, COX-2 activity following treatment with macamides and maca extracts were subsequently normalised to eachcorresponding vehicle concentration. None of the macamides or maca extracts tested were able to inhibit COX-2 activity in vitro (Figure 13). Similarly to MCT oil treatment, the higher concentrations tested of macamide 15 seemed to increase COX-2 activity.

[0319] (3-endorphin results

[0320] P-endorphin is an endogenous opioid neuropeptide produced in both and central and peripheral nervous systems, p-endorphins have analgesic properties and are linked to the hypothalamic-pituitary-adrenal (HPA) axis, controlling a wide range of metabolic, immune and stress responses in the body (Pilozzi et al., 2020). Previous studies have shown that activation of the cannabinoid CB2 receptor in keratinocytes results in p-endorphin release, which inhibits nociception by primary afferent neurons (Ibrahim et al., 2005; Gao et al., 2016). Maca products, product components and extracts were assessed for the ability to induce P-endorphin release in vitro, as a potential mechanism for proposed analgesic and homeostatic applications.

[0321] In general, detected levels of P-endorphin were low across the conditions tested in this assay. The assay had a sensitivity limit of 9.38 pg / mL, as indicated by the dotted line on each figure (Figure 14), where many treatments elicited P-endorphin levels at or below the sensitivity threshold. The cannabinoid CB2 receptor-selective agonist HU- 308 did not significantly affect P-endorphin release at 1 or 10 pM (Figure 14A), despite CB2-selective agonists being used previously to cause P-endorphin release in keratinocytes (Ibrahim et al., 2005; Gao et al., 2016). MCB oil, macamides 15, 58 and 86, and their respective vehicles did not significantly affect P-endorphin release in HaCaT cells relative to media only controls (Figures 148-14G). Likewise, maca propane extract and 9: 1 DMSO:chloroform treatment did not alter p-endorphin levels with increasing concentrations, despite trending towards an increase. Maca hexane and CO2 extracts, at 32.75 and 37 pg / mL, respectively, significantly increased P-endorphin release relative to media controls (Figures 141 and 14J, P<0.05). These approximate 2-fold increases in p- endorphin are similar to previously reported results from CB2 receptor activation in rat paw skin and HaCaT keratinocytes (Ibrahim et al., 2005; Gao et al., 2016).

[0322] FAAH inhibition results

[0323] FAAH is a serine hydrolase that degrades the endocannabinoid arachidonoyl ethanolamide (AEA), producing arachidonic acid and ethanolamine (Cravatt et al., 1996). Inhibition of FAAH allows AEA to persist, providing enhanced analgesic and antiinflammatory effects (Ahn et al., 2009). JZL 195 is a potent inhibitor of FAAH (Long etal., 2009) and was used as a positive control in this assay, eliciting a mean inhibition of 98.4% (Figure 15A). CBD treatment, at a concentration equivalent to 10 pM, did not affect FAAH activity. This was expected as previous studies have reported IC50 values for CBD at >20 pM, making it a weak inhibitor of FAAH (Bisogno et al., 2001). Compared to the activity control, MCB oil, macamide 15, macamide 86, maca propane extract and maca hexane extract significantly inhibited FAAH activity at all concentrations tested (Figure 15, P<0.05). Ethanol, DMSO, and macamide 58 treatment significantly inhibited FAAH activity at four of the concentrations tested. 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Claims

CLAIMS1. An oil-based composition comprising a maca extract, wherein the maca extract comprises at least about 50 mg / g maca compounds.

2. An oil-based composition comprising a maca extract, wherein the maca extract comprises: a. macamide N-Benzyloctadec-9Z-enamide; b. macamide N-Benzyloctadeca-9Z,12Z-dienamide; c. macamide N-Benzyloctadeca-9Z,12Z,15Z-trienamide; and d. macamide N-(3-Methoxybenzyl)octadeca-9Z,12Z-dienamide.

3. The composition of claim 1 or claim 2, wherein the maca extract comprises at least about 50 mg / g total macamides.

4. The composition of any one of the preceding claims, wherein the maca extract comprises at least about 3 mg / g total macaenes.

5. The composition of any one of the preceding claims, wherein the maca extract comprises at least about 50 mg / g total macamides and at least about 3 mg / g total macaenes.

6. The composition of any one of the preceding claims, wherein the maca extract comprises: at least about 9 mg / g macamide N-Benzyloctadec-9Z-enamide; and / or at least about 23 mg / g macamide N-Benzyloctadeca-9Z,12Z-dienamide; and / or at least about 3 mg / g macamide N-Benzyloctadeca-9Z,12Z,15Z-trienamide; and / or at least about 4 mg / g macamide N-(3-Methoxybenzyl)octadeca-9Z,12Z- dienamide.

7. The composition of any one of the preceding claims, wherein the maca extract further comprises at least one maca compound selected from the group consisting of a. macamide N-Benzylpalmitamide,b. macamide N-Benzyloctadecanamide, c. macamide N-(3-Methoxybenzyl)octadeca-9Z,12Z,15Z-trienamide, d. macamide N-(3-Methoxybenzyl)octadec-9Z-enamide, e. macamide N-(3-Methoxybenzyl)hexadecanamide, f. macamide N-(3-Methoxybenzyl)octadecanamide, g. macamide N-Phenethyloctadeca-9Z,12Z-dienamide, h. macamide N-Phenethyloctadeca-9Z,12Z,15Z-trienamide, i. N-Benzylicosa-5Z,8Z,llZ,14Z-tetraenamide, j. N-Benzylicosa-5Z,8Z,llZ,14Z,17Z-pentaenamide, k. macaene 9-oxo-octadeca-10,12-dienoic acid, and l. macamide N-Benzyl-9-oxo-octadeca-10,12-dienamide.

8. The composition of any one of the preceding claims, wherein the maca extract further comprises at least about 4 mg / g macamide N-Benzylpalmitamide.

9. The composition of any one of the preceding claims, wherein the maca extract further comprises at least about 0.2 mg / g macamide N-Benzyloctadecanamide.

10. The composition of any one of the preceding claims, wherein the maca extract further comprises at least about 0.5 mg / g macamide N-(3-Methoxybenzyl)octadeca- 9Z,12Z,15Z-trienamide.

11. The composition of any one of the preceding claims, wherein the maca extract further comprises at least about 0.3 mg / g macamide N-(3-Methoxybenzyl)octadec- 9Z-enamide.

12. The composition of any one of the preceding claims, wherein the maca extract further comprises at least about 0.2 mg / g macamide N-(3- Methoxybenzyl) hexadeca namide.

13. The composition of any one of the preceding claims, wherein the maca extract further comprises at least about 0.01 mg / g of at least one macamide selected from the group consisting of N-(3-Methoxybenzyl)octadecanamide, N-Phenethyloctadeca- 9Z,12Z-dienamide, N-Phenethyloctadeca-9Z,12Z,15Z-trienamide, N-Benzylicosa- 5Z,8Z,llZ,14Z-tetraenamide, or N-Benzylicosa-5Z,8Z,llZ,14Z,17Z-pentaenamide.

14. The composition of any one of the preceding claims, wherein the maca extract further comprises at least about 3 mg / g macaene 9-oxo-octadeca-10,12-dienoic acid.

15. The composition of any one of the preceding claims, wherein the maca extract further comprises at least about 4 mg / g macamide N-Benzyl-9-oxo-octadeca-10,12- dienamide.

16. The composition of any one of the preceding claims, further comprising at least one terpene and / or terpenoid.

17. The composition of claim 16, wherein the at least one terpene is selected from the group consisting of alpha bisabolol, alpha phellandrene, alpha pinene, beta caryophyllene, beta pinene, cadinene, camphene, citral, citronellol, delta 3 carene, eucalyptol, eugenol, gamma terpinene, geraniol, humulene, limonene, linalool, myrcene, nerol, nerolidol, ocimene, para-cymene, terpineol, terpinolene, and valencene.

18. The composition of claim 16 or claim 17, wherein the maca extract comprises the at least one terpene and / or terpenoid in a total concentration of from about 0.1 to about 5% w / w%.

19. The composition of any one of the preceding claims, wherein the oil is selected from the group consisting of arnica oil, avocado oil, black seed oil, canola oil, coconut oil, flaxseed oil, frankincense oil, grapeseed oil, Kakadu plum seed oil, macadamia oil, medium chain triglyceride (MCT) oil, native sandalwood oil, olive oil, palm oil, rice bran oil, sesame oil, soya oil, sunflower oil, vegetable oil, walnut oil, hemp seed oil, mineral oil, peanut oil, tung oil, bees wax, Danish oil, black cumin seed oil, coconut MCT oil, castor oil, and any combination of any two or more thereof, preferably MCT oil.

20. The composition of any one of the preceding claims, further comprising residual alkane.

21. The composition of claim 20, wherein the alkane is selected from the group consisting of propane, butane, isobutane, pentane and isomers thereof, hexane and isomers thereof, and heptane and isomers thereof.

22. The composition of claim 20 or claim 21, wherein the alkane is propane or butane.

23. A process of producing an oil-based composition comprising a maca extract, the process comprising: providing a maca root; suspending the maca root in an oil; contacting the oil comprising the maca root with a solvent comprising an alkane at a temperature and pressure at which the solvent is liquid, thereby producing the composition.

24. The process of claim 23, wherein the process further comprises drying the maca root before contacting the maca root with the oil.

25. The process of claim 23 or claim 24, wherein the process further comprises comminuting the maca root to produce maca root particles before contacting the maca root with the oil.

26. The process of any one of claims 23 to 25, wherein the alkane is selected from the group consisting of propane, butane, isobutane, pentane and isomers thereof, hexane and isomers thereof, and heptane and isomers thereof, preferably propane.

27. The process of any one of claims 23 to 26, wherein contacting the oil comprising the maca root with the solvent is performed at a pressure of from about 10 to about 50 bar.

28. The process of any one of claims 23 to 27, wherein the oil is selected from the group consisting of arnica oil, avocado oil, black seed oil, canola oil, coconut oil, flaxseed oil, frankincense oil, grapeseed oil, Kakadu plum seed oil, macadamia oil, MCT oil, native sandalwood oil, olive oil, palm oil, rice bran oil, sesame oil, soya oil, sunflower oil, vegetable oil, walnut oil, hemp seed oil, mineral oil, peanut oil, tung oil, bees wax, Danish oil, black cumin seed oil, coconut MCT oil, castor oil, and any combination of any two or more thereof, preferably MCT oil.

29. The process of any one of claims 23 to 28, wherein the process results in a yield of from about 0.2 to about 1.5% of macamides and macaenes.

30. The process of any one of claims 23 to 29, wherein the process provides a composition comprising a greater ratio of macamides to macaenes than the ratio found in the maca root.

31. The process of any one of claims 23 to 30, wherein the process provides a composition comprising a greater ratio of unsaturated macamides to saturated macamides than the ratio found in the maca root.

32. An oil-based composition comprising the maca extract when produced by the process of any one of claims 23 to 31.

33. Use of the composition of any one of claims 1 to 23 or claim 32 for the treatment of diseases or disorders selected from the group consisting of acne, addiction, anxiety, arthritis, autism spectrum disorder, disorders stemming from autoimmunity, side effects resulting from cancer treatment, chronic pain, chronic fatigue syndrome, depression, digestive disorders, epileptic seizures, irritable bowel syndrome, Crohn's disease, ulcerative colitis, high blood pressure, hormonal imbalances, inflammation, insomnia, disorders of the libido, menopause, mental health disorders, muscular pain, neurological conditions, Parkinson's disease, post-traumatic stress disorder, seizures, disorders of the skin, injury to the skin, eczema, psoriasis, sexual function disorders, and stress.

34. The use of claim 33, wherein the composition is administered orally, sublingually or topically.

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