ENERGY DRINKS AND OTHER NUTRITIONAL AIDS DERIVED FROM AGAVE-BASED SPIRITS

Abstract

Compositions containing monoamine oxidase inhibitors prepared by removal of alcohol from agave-derived beverages are disclosed.

Claims

1. A rating method for agave derived beverages, comprising assigning each beverage a position on a scale of quality based on its content of MAO A inhibitors or MAO B inhibitors or both.

2. The rating method of claim 1, which is applied to a beverage in which ethanol has been removed.

3. The rating method of claim 2, wherein the method comprises a step of removing ethanol from a distilled alcoholic beverage derived from agave by a process that retains volatile compounds other than ethanol.

4. The rating method of claim 3, wherein the process is reverse osmosis or a spinning cone column.

5. The rating method of claim 1, which is applied to a beverage comprising ethanol.

6. The rating method of claim 5, wherein the method comprises a step of assaying for MAO inhibitors in the presence of ethanol, said assay for MAO inhibitors including alcohol dehydrogenase and NAD.

7. The rating method of claim 6, wherein said assay comprises the steps of: a) adding to the sample to be analyzed a substrate effective amount of kynuramine dihydrobromide, along with nicotinamide adenine dinucleotide and alcohol dehydrogenase; b) adding MAO A and/or MAO B; c) incubating at 37° C.; d) stopping the reaction with strong base; and e) assessing the results with an excitation wavelength of 310 nm and an emission wavelength of 405 nm.

8. The rating method of claim 1, wherein the quality of each beverage is assigned based solely on MAO A inhibitor content.

9. The rating method of claim 1, wherein the quality of each beverage is assigned based solely on MAO B inhibitor content.

10. The rating method of claim 1, wherein the quality of each beverage is assigned based on both MAO A and MAO B inhibitor contents.

11. A label displaying the quality of an agave derived beverage based on its content of MAO A inhibitors or MAO B inhibitors or both, wherein said quality has been assessed using the rating method of claim 1.

12. A composition prepared by removing ethanol from a distilled alcoholic beverage derived from agave, wherein MAO inhibitory activity of the composition is reduced compared to the distilled alcoholic beverage.

13. The composition of claim 12, wherein the ethanol is removed with the use of a vacuum.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 shows the results of assessment of MAO inhibition activity in a tequila at various ages as assessed by a standard commercial assay kit.

[0020] FIG. 2 shows the results of assessing the MAO inhibition activity of various brands and ages of tequila using kynuramine as a substrate.

[0021] FIG. 3 shows the results of an MAO inhibition assay with kynuramine as a substrate after separation of tequilas into various fractions.

[0022] FIG. 4 is a graph showing the MAO inhibition activity of various column fractions based on the kynuramine assay.

[0023] FIG. 5 shows the results of MAO inhibition assays in a sample of tequila that has been subjected to reverse osmosis and compares the activity in the concentrate with the activity in the filtrate.

MODES OF CARRYING OUT THE INVENTION

[0024] The non-alcoholic forms of alcoholic beverages made from the agave plant extracts are the subject of the present invention. These alcoholic beverages include pulque, tequila, mezcal, sotol, Bacanora or other ferments made from agave-plant-derived-sugars. In the present invention the alcohol has been removed under conditions where other volatile compounds are retained.

[0025] These compositions have monoamine oxidase (MAO) inhibitory properties and are thus useful for use as mood elevators, antidepressants or treatment for certain diseases such as Parkinson's disease. The invention also relates to a method of grading and preparation or mixing different extracts to select a desired physiological stimulant or antidepressant effect for use as an energy drink and/or mood elevator by assessing MAO activity in the presence of alcohol. In the grading/evaluation tool aspect of the invention, different agave-based beverages receive monoamine oxidase (MAO) inhibition ratings for each form of MAO, A+B, which are expressed as percent inhibition relative to a control with no inhibitor present. Examples of ratings for mescal, tequila, Bacanora, sotol and pulque are shown in Example 6. Example 7 shows similar results when the fractions obtained by reverse osmosis are analyzed.

[0026] As shown herein, the various agave-derived beverages can be analyzed directly for their contents of MAO A and MAO B inhibitors. This makes possible a rating system wherein the results for a particular beverage could be included on the label. The various beverages could be ranked on a scale based on their contents in comparison to other brands of the same beverage, for example. The ratings could be for combinations of inhibitors for both MAO A and MAO B or for each individually. Thus for a scale of 1-10, with 10 the best rating and 1 the worst, a beverage which shows 80% inhibition of MAO A compared to 40% inhibition shown by a rival brand in the same assays, could be given an 9 vs. 5 for the lower activity beverage. The correlation of inhibition with a particular scale can be designed to provide helpful information to the consumer. The scale used or rating selected need not, of course, be 1-10, but any other scale that might be arbitrarily selected, such as A-F.

[0027] The MAO assays used for measuring percent inhibition accept samples with high concentrations of ethanol. The assay method of the invention is necessary to accurately measure inhibition of MAO activity and must tolerate samples with greater than 40% ethanol, which is the main component of the samples of interest to be graded. The range of alcohol typically encountered with distilled agave spirits is 6% for pulque to about 60% for some tequilas and mescal, but more commonly encountered samples will have 40% alcohol (80 proof). Due to the volatility of some (but not all) of the MAO inhibitors derived from agave-based beverages, it is particularly important not to evaporate ethanol from the sample under reduced pressure or by distillation at atmospheric pressure.

[0028] The compositions of the invention may be used as a food supplement and/or included in foods or medicines, or used to treat depression, Parkinson's disease or general malaise. The composition may also be used as an adjunct or substitute for coffee or in decaffeinated products. The compositions may be mixed with two or more carriers, excipients and/or diluents that are pharmaceutically or nutritionally acceptable. Thus, in general, the compositions of the invention may be included in juices or other soft drinks such as colas or fruit flavored sodas, or can be consumed directly. The compositions may also be included in foodstuffs such as uncooked liquids, for example salad dressings, and consumed along with the solid components of the salad. The amounts of compositions to be consumed or administered to subjects is highly dependent on the nature of the condition to be treated as well as the concentrations of the MAO inhibitors determined to be in the composition itself. Levels of MAO inhibitors useful for various medical indications are known in the art and these guidelines can be followed. For use as food supplements, this is a matter of the judgment of the nutritionist or other practitioner.

[0029] Thus, the invention includes foodstuffs and drinks that contain the composition of the invention in amounts effective to have the desired physiological effect.

[0030] One successful method to remove the ethanol from the beverage starting material is reverse osmosis (RO). An RO membrane with a molecular weight cut-off of −100 Daltons separates ethanol from the inhibitors of MAO's formed by microorganisms or during distillation or inhibitors that may be naturally occurring in the starting material. Isolation of the MAO inhibitors is accomplished without the use of vacuum or distillation. Therefore the invention compositions may be prepared for example, by diluting with distilled water 0.75 liters of tequila (or other distilled agave beverage) to a volume of 7.5-75 liters, depending on the final desired ethanol concentration. For example, 0.75 liters of tequila at 80 proof (40% alcohol by volume) is diluted to 7.5 liters at which point the diluted material is 4% alcohol by volume. The diluted tequila is circulated in a reverse osmosis unit fitted with a membrane with molecular weight cut-off of ˜100 Daltons (Tangent Membranes, Inc., RO MINI) wherein the diluted material is concentrated back to a volume of 0.75 liters. Additional reduction of ethanol is achieved by diluting the material again to 7.5 liters or greater and continuing the circulation through the RO unit. For creation of the composition such as an energy drink according to the invention, dilution and subsequent circulation may be continued until the product is considered nonalcoholic. The active MAO inhibitors are then contained in the concentrated, non-alcoholic portion and may then be supplemented with vitamins, minerals, amino acids, protein or caffeine, where desired, and may be included in other food or drink preparations as noted above.

[0031] It is an important aspect in the creation of the compositions of the invention that some of the MAO inhibitors are volatile compounds and if the composition is created by evaporation of the ethanol through heated distillation or under reduced pressure, for example, loss of essential MAO inhibitors occurs.

[0032] The following examples are offered to illustrate but not to limit the invention.

Example 1

Assay of Various Tequilas Using Commercially Available Assays

[0033] A commercial MAO activity kit was employed to test samples of different commercially available tequilas or mezcal, S1, S1 DW, S2 and S3. The kit was purchased from Sigma-Aldrich (Cat. No. MAK136 Monoamine oxidase activity kit) which includes known MAO inhibitor compounds, clorgyline and pargyline, as controls and tyramine as the enzyme substrate. Horseradish peroxidase is also included in this kit to interact with hydrogen peroxide generated from the oxidation of tyramine in order to react with the included label, Amplex® red. The action of MAO A and/or B creates a cascade of events resulting in the fluorescence emission. S1 and S3 are tequilas with S1 an aged version of S3 and S1 DW a distilled water reconstituted sample of S1 after drying under a stream of argon. S2 is a sample of mezcal, which is made from versions of agave other than blue agave. DW is distilled water Fluorescence data are calculated for percent inhibition relative to a buffer control (no inhibitor). A known inhibitor of MAO A, clorgyline, and a known inhibitor of MAO B, pargyline, are included as controls. As shown in FIG. 1, depending upon the sample selective inhibition of MAO A or B may be achieved.

Example 2

Testing Various Tequilas Using a Kynuramine Only Assay

[0034] A different assay where kynuramine only is the substrate and assay label is used in this example. This assay uses kynuramine as the MAO substrate and it is a substrate for both MAO A and B. Thus is a more desirable method because effects on peroxidase or spectral interference are eliminated. Kynuramine oxidation results in an OD change at 360 nm and 320 nm. The product is also fluorescent and may be detected by an increase in emission intensity at 400 run with an excitation wavelength at 320 nm.

[0035] The assay was set up in clear polystyrene 96-well plates according to the following protocol: Phosphate buffer (205 μl/100 mM/pH 7.2) was added to the wells followed by 20 μl of each extract sample and control inhibitor clorgyline (for MAO A) and pargyline (for MAO B) at 10 μM. Recombinant human MAO A and MAO B (0.5 mg/ml protein) was purchased from Corning® and each assay was supplied with 3 μg of this protein. Some assays were run with a null protein as a control also supplied by Corning®. The inhibitor(s), buffer and proteins were allowed to pre-incubate in a PowerWave™ optical density reading plate reader set at 37° C. for 10 minutes. The kynuramine substrate was then added to each well and the plate was further incubated at 37° C. for 30 minutes. The plate was read at OD360 nm and the change in absorbance from time zero to 30 minutes was calculated and transformed to % inhibition. Samples S40, S41, S42 are blanco, reposado and añejo, respectively, and are tequilas from the same manufacturer at 0, 1 and 2 stages of aging. 51, S3 are tequilas as in Example 1 and S2 is mezcal. Percent inhibition is calculated from the optical density change at 360 nm from time 0 to 30 min.

[0036] The results are shown in FIG. 2.

(One tequila used as a sample (DJ1942) containing 40% ethanol was tested for interfering effects on the assay without the alcohol dehydrogenase and NAD and interference was shown.)

Example 3

Effect of Aging

[0037] The kynuramine assay was used to test three different ages of tequila, S40, S41, S42 and to compare aqueous and methylene chloride extractions of these samples. The aged tequilas show increased MAO inhibitory activity vs. the younger distillates, as shown in FIG. 3.

Example 4

Fractionation of MAO Inhibitor Activity

[0038] Tequila (1750 ml) was diluted to 5000 ml with deionized water and separated into three portions, and each portion was extracted with dichloromethane (4×100 ml). The extracts were combined, washed with deionized water (2×100 ml) and dried over anhydrous sodium sulfate. The sample was reduced in volume to 10 ml by evaporation of solvent using a rotary evaporator then dry silica was added and the solvent was removed at room temperature. The sample-adsorbed silica was dry loaded on a silica column with a 50 ml bed volume and the column was run with a step gradient using hexane:ethyl acetate (100% hexane, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, 100% ethyl acetate). During the column run 7 ml fractions were collected. The samples were assayed as follows:

[0039] The MAO assays for A and B are set up in black Thermo Scientific™ 96-well plates (#7205) with each assay containing 1 μl of sample dissolved in DMSO, 87 μl of 100 mM potassium phosphate buffer pH 7.4, 3 μl of a stock solution of 0.75 mM kynuramine. A set of controls with DMSO alone (0 inhibitor), 1 μl of 50 μmolar clorgyline in DMSO as a MAO A inhibition control and 1 μl of 50 μmolar pargyline in DMSO as a MAO B inhibition control were added on each plate. When optimizing the assays, a no-protein set was sometimes included on the plates.

[0040] Recombinant human MAO A and MAO B (Corning®, SUPERSOMES™) was added to the wells for each respective assay at 750 ng protein in 9 μl buffer.

[0041] The plate was incubated at 37° C. for 30 minutes and then 100 μl of 2N NaOH was added to each well. The plate was read in a fluorescence spectrophotometer at excitation wavelength 310 and emission wavelength 405.

[0042] To identify inhibitory activity in column fractions 25 μl of each fraction was placed in 1.5 ml microfuge tubes and evaporated to dryness in a Savant™ SpeedVac™. DMSO (5 μl) was added to each tube and 1 μl was assayed for MAO A and B inhibitory activity according to the method above. FIG. 4 shows MAO A and B inhibitory activity across the fractions collected.

[0043] Significant MAO inhibition is seen in fractions 6 through 13.

Example 5

Preparation of MAO Inhibitor-Containing Oils

[0044] Tequila (100 ml) was diluted to 200 ml with deionized water and extracted with dichloromethane (3×50 ml). The extracts were combined and passed over a column of anhydrous sodium sulfate. The solvent was evaporated using a rotary evaporator and 30 mg of oil was obtained. Samples of this oil inhibited MAO A by 75% and MAO B by 87%.

[0045] Tequila (100 ml) was diluted to 200 ml with deionized water and extracted with dichloromethane (3×50 ml). The extracts were combined and passed over a column of anhydrous sodium sulfate. The solvent was removed by evaporation in a Kuderna Danish apparatus warmed over a water bath at 70° C. resulting in 17 mg of an oil. Samples of the oil showed 55% MAO A inhibition and 88% MAO B inhibition.

Example 6

Assay of Various Tequila Samples, Per Se

[0046] As discussed above, samples with MAO inhibitory activity must not be evaporated under reduced pressure or the compounds will co-evaporate with ethanol. Therefore, the samples should be assayed without evaporation of ethanol to eliminate losses of MAO inhibiting compounds and in order to accurately grade the MAO inhibitory activity of the product analyzed. The assays were set up in black Thermo Scientific™ 96-well plates (#7205) as follows:

[0047] Each assay included 5 μl of sample, 0.975 m kynuramine dihydrobromide, 0.75 μmoles nicotinamide adenine dinucleotide (NAD) in a total volume of 93 μl with 100 mM phosphate buffered saline, pH 7.4.

[0048] Alcohol dehydrogenase was diluted to 0.25 units/ml and 3 μl was added to the wells. The plates were incubated at 25 to 37° C. for 15 minutes.

[0049] Human recombinant MAO A and B (Corning) supplied at 5 mg/ml was aliquoted in vials at 20 μl each and diluted for the assay with 246 μl of buffer, then 4 μl of each MAO was added to its respective wells.

[0050] The plates were incubated at 37° C. for 30 minutes and 2N NaOH (100 μl) was added and the plates were read at excitation wavelength 310 and emission wavelength 405.

[0051] Using this assay, various tequila samples were tested with the results shown in Table 1.

TABLE-US-00001 TABLE 1 Ratings of Various Agave-Derived Beverages % % Inhibition Inhibition Brand MAO A MAO B Suerte Blanco 5 6 Suerte Reposado 7 12 Suerte Anejo 14 19 Qui blanco 33 7 Don Julio 70.sup.th Anniversary 12 6 Grand Patron Piedra 32 49 Don Julio Blanco 9 8 Don Julio Repesado 18 22 Don Julio Anejo 10 26 Don Julio 1942 18 35 Patron Silver Roca 39 11 Patron Roca Anejo 21 40 Patron Grand Platinum 38 9 Esperanto 36 4 Avion Silver 31 7 1800 silver 11 11 Cuervo Silver −1 4 Cuervo Gold 4 40 “Cheap” Mezcal 43 2 “Expensive” Mezcal 40 8 Ketel One Vodka −2 −2 Jaegermeister 49 55 Tres Generacion 15 41 Clorgyline/Parglyine 97 82 (positive control) DMSO (negative control) 2 1 DMSO (negative control) 2 −1 Inhib (positive control) 98 96 Inhib (positive control) 98 95

Example 7

Effect of Reverse Osmosis (RO)

[0052] An RO Mini from Tangent Membranes, Inc. fitted with a ˜100 mwco filter was used to separate the ethanol from tequila while retaining MAO inhibiting compounds. Here, 1.5 liters añejo tequila (DJA) was diluted to 15 liters with distilled water and circulated in the RO Mini until the volume was reduced back to 1.5 liters. The MAO inhibitory activity of 10, 20, 30 and 40 μl of the concentrated sample and the filtrate was assessed. (FIG. 5.)

[0053] As shown in FIG. 5, the concentrate retains MAO A and B inhibitors while MAO inhibitors are not present in the filtrate which contains only ethanol and water from the diluted sample.

[0054] Table 2 shows the results for various beverages as well as diluted tequila starting material (labeled diluted RO Control), 1st RO Concentrate, 2nd RO Concentrate, 1st RO Filtrate and 2nd RO Filtrate. The filtrates are shown in the MAO inhibition assay to have less inhibitory activity, while most of the activity remains with the concentrate.

TABLE-US-00002 TABLE 2 MAO A % MAO B % Sample Inhibition Inhibition Kirkland Anejo (Costco brand) 23 32 Milagro Anejo 26 23 Don Julio Anejo 17 32 Don Julio 1942 19 35 Pulque 14 26 Sotol 71 58 Milagro Barrel Res Anejo 27 15 Diluted RO Control 17 35 1st RO Concentrate 23 52 2nd RO Concentrate 34 49 1st RO Filtrate 10 7 2nd RO Filtrate 3 8 Clorgyline/Parglyine 97 83 DMSO 1 1