Bioptersides, uses thereof, and related extraction methods

Abstract

Disclosed are compositions comprising about 0.5% to about 10% total bioptersides, or pharmaceutically acceptable salts thereof, and methods for preparing the same. In certain aspects, methods of improving, increasing, enhancing, or augmenting certain conditions are provided, comprising administering to a subject in need thereof an effective amount of a composition comprising an amount of total bioptersides.

Description

[0001] BIOPTERSIDES, USES THEREOF, AND RELATED EXTRACTION METHODS

[0002] RELATED APPLICATION

[0003] This application claims the benefit of priority to U.S. Provisional Patent Application No.: 63 / 656,768, filed June 6, 2024.

[0004] BACKGROUND

[0005] Biopterin (2-amino-6-(l,2-dihydroxypropyl)-lH-pteridin-4-one) and biopterin derivatives such as tetrahydrobiopterin [(6J?)-2-amino-6-[(l / ?,2S)-l,2-dihydroxypropyl]-5, 6,7,8- tetrahydropteridin-4( 1 H)-one], are important, naturally occurring pterin species involved in a number of important biochemical processes. In humans, for example, tetrahydrobiopterin is a cofactor of the Aromatic Amino Acid Hydroxylase (AAAH) enzymes, is used in the biosynthesis of multiple neurotransmitters (e.g., serotonin, melatonin, dopamine, norepinephrine, and epinephrine), and is a cofactor to Nitric Oxide Synthases for the production of nitric oxide.

[0006] Bioptersides, a class of molecules comprising biopterin, or a derivative thereof, attached to a glycosyl moiety, are promising bioactive compounds that can affect a variety of biophysical conditions, and may be obtained from natural sources e.g., naturally produced by cyanobacteria). However, even the natural sources that produce the highest quantities of bioptersides still produce only relatively low concentrations (e.g., less than 0.1 wt.%), which may or may not be useful in dietary supplementation or pharmaceutical contexts.

[0007] As such, there exists a need for compositions comprising higher concentrations of bioptersides and expedient methods for preparing compositions comprising bioptersides.

[0008] BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1 is a plot showing activation of human endothelial nitric oxide synthase (p-eNOS) compared to control (CTL) in human endothelial cells administered biopterside compositions of the present disclosure. CTL = Growth Supplement Only Control Treatment; SPI = CTL + 10.9 pg / mL Spirulina Treatment; BIO-ALO = CTL + 10.9 pg / mL Biopterside A Treatment; BIO- AHI = CTL + 86.8 pg / mL Biopterside A Treatment; BIO-BLO = CTL + 10.9 pg / mL Biopterside B Treatment; BIO-BHI = CTL + 86.8 pg / mL Biopterside B Treatment; BIO-ABLO = CTL + 10.9 pg / mL Biopterside A+B Treatment; BIO-ABHI = CTL + 86.8 pg / mL Biopterside A+B Treatment. All treatments were compared to CTL. * = significant up-regulation compared to CTL (p<0.05). *BIO-ABLO vs CTL = 1.41 + 0.35 (p=0.0259); *BIO-ABHI vs CTL = 1.40 + 0.23 (p=0.0075).

[0010] FIG. 2 is a plot showing activation of human endothelial nitric oxide synthase (p-eNOS) compared to spirulina (SPI) in human endothelial cells. CTL = Growth Supplement Only Control Treatment; SPI = CTL + 10.9 pg / mL Spirulina Treatment; BIO-ALO = CTL + 10.9 pg / mL Biopterside A Treatment; BIO-AHI = CTL + 86.8 pg / mL Biopterside A Treatment; BIO-BLO = CTL + 10.9 pg / mL Biopterside B Treatment; BIO-BHI = CTL + 86.8 pg / mL Biopterside B Treatment; BIO-ABLO = CTL + 10.9 pg / mL Biopterside A+B Treatment; BIO-ABHI = CTL + 86.8 pg / mL Biopterside A+B Treatment. All treatments were compared to SPI. t = significant up-regulation compared to SPI (p<0.05). jBIO-ABLO vs SPI = 1.41 ± 0.35 (p=0.0010); fBIO- ABHI vs SPI = 1.40 + 0.23 (p=0.0059).

[0011] FIG. 3 is a bar chart showing activation of human endothelial nitric oxide synthase [p- eNOS (Seri 177)] relative to total eNOS, displaying data obtained in the study detailed in Example 8. All treatments were compared to CONTROL (CTL). * = significant up-regulation compared to CTL (p<0.05). # = significant down-regulation compared to CTL (p<0.05).

[0012] FIG. 4 is a bar chart showing activation of human endothelial nitric oxide synthase [p- eNOS (Seri 177)] relative to L-Citrulline (L-CIT) p-eNOS response, displaying data obtained in the study detailed in Example 8. All treatments were compared to L-CITRULLINE (CIT). * = significant up-regulation compared to CIT (p<0.05). * = significant down-regulation compared to CIT (p<0.05).

[0013] FIG. 5 is a bar chart showing total NOx (Nitrate + Nitrite) Response in Media + Cells [detects NOx present in media (background + NOx released by HUVECs) + intracellular], displaying data obtained in the study detailed in Example 8. All treatments were compared to CONTROL (CTL). * = significant up-regulation compared to CTL (p<0.05). * = significant down-regulation compared to CTL (p<0.05).

[0014] FIG. 6 is a bar chart showing total NOx (Nitrate + Nitrite) Response in Media + Cells [detects NOx present in media (background + NOx released by HUVECs) + intracellular] relative to L-Citrulline (L-CIT) Total NOx response, displaying data obtained in the study detailed in Example 8. All treatments were compared to L-CITRULLINE (L-CIT). * = significant up-regulation compared to L-CIT (p<0.05). * = significant down-regulation compared to L-CIT (p<0.05).

[0015] FIG. 7 is a schematic overview of the randomized, positively controlled, acute crossover design study described in Example 9.

[0016] FIG. 8 shows effects of an exemplary biopterside composition on resting caloric expenditure and fuel oxidation, displaying data obtained in the study detailed in Example 9. Manufacturer’s software auto-selected a 5-minute interval that yielded the lowest variance (“Best 5 Minutes”) that was used for data analysis. C4 = Commercial Energy Drink Alone; C4+BHN = C4 in combination with 20 mg Biopterside. REE = Resting Energy Expenditure (kcal per day); FAT = Fat Oxidation Rate (kcal per day); CHO = Carbohydrate Oxidation Rate (kcal per day); Rf = Respiratory Frequency (breaths per minute). Rf is most affected by central driving factors.

[0017] FIG. 9 shows effects of an exemplary biopterside composition on graded exercise testing (GXT) response, displaying data obtained in the study detailed in Example 9. C4 = Commercial Energy Drink Alone; C4+BHN = C4 in combination with 20 mg Biopterside. OUES = Oxygen Uptake Efficiency Slope (mL of 02 per minute per L of inspired air per minute). OUES is directly correlated to cardiorespiratory fitness and inversely related to cardiovascular risk. V 02 / WR = Oxygen Utilization per Workload Slope [mL of 02 per minute per watt (W)]. V02 / WR is a surrogate measure of oxygen utilization efficiency (or, mitochondrial efficiency). Rf @ VT1 = Respiratory Frequency (breaths per minute) at Aerobic Energy Threshold (VT1). VT1 occurs when maintaining energy demands [intramuscular Adenosine Triphosphate (ATP) availability > ATP demand] shifts to relying almost exclusively on anaerobic metabolic pathways (i.e., fast glycolysis and lactate shuttling). Rf is most affected by central driving factors. EE @ FAT MAX = Energy Expenditure (kcal per minute) at the sub-maximal workload where the Maximal Fat Oxidation Rate (grams per minute) is observed. H+ - ATP = is the duration of time (minutes) spent within the stage of highest exercise intensity, calculated as being equal to Maximal Time to Exhaustion (TTE; minutes) minus the timepoint (minutes) at which Ventilatory Threshold (VT2) is observed. Maintaining energy demands during this stage of exercise (H+ - ATP) is impacted most by anaerobic metabolic pathways (i.e., fast glycolysis and lactate shuttling), buffering rate of free ion concentrations [e.g., hydrogen ions or protons (H+), calcium ions (Ca2+), etc.], and ATP resynthesis rate through the phosphocreatine (PCr) high-energy phosphate system. Power @ FAT MAX = sub-maximal Workload [Watts (W)] where the Maximal Fat Oxidation Rate (grams per minute) is observed.

[0018] SUMMARY

[0019] In certain aspects, the present application provides compositions, comprising about 0.5% to about 10% total bioptersides (BHn), wherein the total bioptersides (BHn) comprises a first biopterin (Bl) selected from biopterin xyloside (BX), biopterin glucoside (BGlc), and biopterin arabinoside (BA), or pharmaceutically acceptable salts thereof.

[0020] In other aspects, this application provides methods of improving, increasing, enhancing, or augmenting: weight loss, fat metabolism, physical energy, cognitive energy (e.g., mental energy or working memory), circulation, blood flow, lean mass, mitochondrial function or health, endothelial function (e.g., vascular homeostasis), neurocognitive function, cardiovascular function, muscle recovery, recovery from physiological stress, muscle performance, exercise capacity, insulin sensitivity, metabolism, blood glucose control, fat loss, weight management, oxidative stress, thermogenesis, reaction times, neuromuscular function, nitric oxide biosynthesis, L-DOPA biosynthesis, serotonin biosynthesis, neurohormone biosynthesis, sexual performance, male erectile dysfunction; tyrosine, phenylalanine, arginine, citrulline, and / or tryptophan metabolism; endothelial nitric oxide synthase phosphorylation, mood disorders, or the attenuation or reduction of reactive oxygen species (ROS) and oxidative tissue damage (e.g., effects of aging) or other forms of physiological stress), comprising administering to a subject in need thereof an effective amount of a composition, comprising an amount of total bioptersides (BHn), wherein the total bioptersides (BHn) comprises a first biopterin (Bl) selected from biopterin xyloside (BX), biopterin glucoside (BGlc) and biopterin arabinoside (BA), or pharmaceutically acceptable salts thereof.

[0021] In some aspects, this application provides a method of preparing a composition of the disclosure, comprising the steps of: contacting a precursor with an acid aqueous solution to form a mixture; agitating the mixture; filtering the mixture to obtain a filter cake and a filtrate solution; optionally adjusting the pH of the filtrate solution; and isolating the composition from the filtrate solution; wherein the precursor is selected from macroalgae, microalgae (e.g., cyanobacteria), blue-green algae, (Arthrospira) Spirulina (e.g., S. plantensis, Aphanizomenon flosaquae, Arthrospira maxima), Chlorella (e.g., C. vulgaris, C. pyrenoidosa, C. ellpsiodea, C. stigmatophora, C. zofingiensis), Haematococcus (e.g., H. pluvialis) Tetraselmis / Tetrasellimis (e.g., T. chuii, T. suecica), Isochryisis (e.g., I. galbana), Nanochloropsis (e.g., N. gaditana, N. oculata, N. salina , Euglena (e.g., E. gracilis), Schizachyrium, Crypthecodinium, Chlamydomonas (e.g., C. reinhardtii), Dunaliella (e.g., D. salina), Scenedesmus, Phaeodactylum, Ochromonas, Prymnesium, Coccomyxa, Skeletonema, Arthronema, Porphyra, Chlorococcum, Fucus, Microcystis, Glaucocystophyte, Lyngbya, Phaeodactylum, Haslea, Pavlova, Thalassiosira, Chaetoceros, Amphora, Annochloropsis, Pelagophyceae, Lyngbya, Anabaena, Porphyridium, Odentella, Undaria, Synechocystis, Synechococcus, Nostoc, Oscillatoria, Cylindrotheca, royal jelly, animal liver (e.g., beef liver), spinach, velvet bean (mucuna pruriens), fava bean (vicia faba), english broom (cytisus scoparius), banana, common purslane, Mexican rose, black caraway, black bean, soybean, watercress, water lotus, Buffalo gourd, pumpkin, acorn squash, sunflower, mung bean, black mustard, carrot, Acetobacter xyliunum, Achromobacter liquidum, Aerobacter aerogenes, Aeromonas hydrophilia, Bacillus spp. (e.g., B. alvei, B. cereus, B. coagulans, B. roseus, B. sphaericus), Brevibacterium acetylicum, Corynebacterium equi, Crithidia fasciculata, Erwinia carotovora, Eschericia coli, Flavobacterium aquatile, Gluconobacter spp (e.g., G. melanogenus, G. suboxydans), Lactobacillus casei, Micrococcus spp. (e.g., M.flavus, M. luteus, M. lysodeikiticus, M. ureae), Pseudomonas aeruginosa, P. aureofaciens, P. convexa, P. fluorescens, P. putrefaciens , Serratia spp. (e.g., S. indica, S. marcescens), Streptococcus faecalis, insects or insect cocoons (e.g., fruit fly, silkworm, honey bee), and a combination of any of them.

[0022] DETAILED DESCRIPTION

[0023] Biopterside Compositions

[0024] In certain aspects, the present application provides compositions, comprising about 0.5% to about 10% total bioptersides (BHn), wherein the total bioptersides (BHn) comprises a first biopterin (Bl) selected from biopterin xyloside (BX), biopterin glucoside (BGlc), and biopterin arabinoside (BA), or pharmaceutically acceptable salts thereof. In certain embodiments, the first biopterin (Bl) is biopterin xyloside. In some embodiments, the first biopterin (Bl) is biopterin arabinoside. In certain embodiments, the first biopterin (Bl) is biopterin glucoside (BGlc).

[0025] In certain embodiments, biopterin xyloside has a structure according to Formula la: la.

[0026] In some embodiments, biopterin arabinoside has a structure according to Formula lb:

[0027] In certain embodiments, biopterin glucoside has a structure according to Formula Ic:

[0028] Ic.

[0029] As will be understood by one of ordinary skill in the art, a plurality of structural isomers are possible for the molecules encompassed by Formulae la, lb, and Ic, and the presence or absence of these isomers is expressly anticipated, encompassed by, and incorporated in the various embodiments of the present invention set forth herein. The specific identities and collection of isomers of the total bioptersides may, in some embodiments, depend on the source of the total bioptersides.

[0030] In certain embodiments, the composition does not comprise additional biopterin glycosides (BG), or pharmaceutically acceptable salts thereof. In other embodiments, compositions of the disclosure further comprise one or more additional biopterin glycosides (BG), or pharmaceutically acceptable salts thereof. In certain embodiments, the one or more additional biopterin glycosides (BG) comprises at least one of biopterin xyloside (BX), biopterin glucoside (BGlc), and biopterin arabinoside (BA), or pharmaceutically acceptable salts thereof. In certain embodiments, the ratio of B 1 :BG is about 12: 1 to about 6: 1. In further embodiments, the ratio of B1:BG is about 10: 1 to about 8:1. In some embodiments, the ratio of B1:BG is about 10:1. In certain embodiments, the ratio of B1:BG is about 9.5:1. In some embodiments, the ratio of B 1 :BG is about 9: 1. In certain embodiments, the ratio of B 1 :BG is about 8.5: 1. In some embodiments, the ratio of B1:BG is about 8:1.

[0031] In some embodiments, compositions of the disclosure further comprise one or more pharmaceutically acceptable excipients.

[0032] In certain embodiments, the total bioptersides (BHn), biopterin xyloside (BX), biopterin glucoside (BGlc), biopterin arabinoside (BA), and, when present, the one or more additional biopterin glycosides (BG) were not derived from petroleum.

[0033] In some embodiments, compositions of the disclosure further comprise one or more additional components selected from the group consisting of folate, folic acid, 5- methyltetrahydrofolate, a compound derived from folate, folic acid, or 5 -methyl tetrahydrofolate, iron, one or more antioxidant compounds (e.g., Vitamin C, N-acetylcysteine (NAC), glutathione, and polyphenols), one or more dopaminergic compounds [e.g., tyrosine, phenylalanine, L- DOPA, Mucuna pruriens, Musa spp. (e.g., bananas and plantains), Persea americana (e.g., avocado), Citrus spp., eggs, dairy, animal organ tissue], caffeine, arginine, citrulline, ornithine, nitrates, vitamin B6, vitamin B12, zinc, and colostrum. In further embodiments, the one or more additional components are selected from folate, folic acid, 5 -methyl tetrahydrofolate, and a compound derived from folate, folic acid, or 5-methyltetrahydrofolate. In yet further embodiments, the one or more additional components comprise iron. In still further embodiments, the one or more additional components comprise one or more antioxidant compounds (e.g., Vitamin C, N-acetylcysteine (NAC), glutathione, and polyphenols). In certain embodiments, the one or more additional components comprise one or more dopaminergic compounds [e.g., tyrosine, phenylalanine, L-DOPA, Mucuna pruriens, Musa spp. (e.g., bananas and plantains), Persea americana (e.g., avocado), Citrus spp., eggs, dairy, animal organ tissue]. In further embodiments, the one or more additional components comprise caffeine. In yet further embodiments, the one or more additional components comprise arginine, citrulline, ornithine, or nitrates, or a combination thereof. In still further embodiments, the one or more additional components comprise Vitamin B6. In certain embodiments, the one or more additional components comprise Vitamin Bl 2. In further embodiments, the one or more additional components comprise zinc. In yet further embodiments, the one or more additional components comprise colostrum.

[0034] Uses of Biopterside Compositions

[0035] In other aspects, this application provides methods of improving, increasing, enhancing, or augmenting: weight loss, fat metabolism, physical energy, cognitive energy (e.g., mental energy or working memory), circulation, blood flow, lean mass, mitochondrial function or health, endothelial function (e.g., vascular homeostasis), neurocognitive function, cardiovascular function, muscle recovery, recovery from physiological stress, muscle performance, exercise capacity, insulin sensitivity, metabolism, blood glucose control, fat loss, weight management, oxidative stress, thermogenesis, reaction times, neuromuscular function, nitric oxide biosynthesis, L-DOPA biosynthesis, serotonin biosynthesis, neurohormone biosynthesis, sexual performance, male erectile dysfunction; tyrosine, phenylalanine, arginine, citrulline, and / or tryptophan metabolism; endothelial nitric oxide synthase phosphorylation, mood disorders, or the attenuation or reduction of reactive oxygen species (ROS) and oxidative tissue damage e.g., effects of aging or other forms of physiological stress), comprising administering to a subject in need thereof an effective amount of a composition, comprising an amount of total bioptersides (BHn), wherein the total bioptersides (BHn) comprises a first biopterin (Bl) selected from biopterin xyloside (BX), biopterin glucoside (BGlc), and biopterin arabinoside (BA), or pharmaceutically acceptable salts thereof.

[0036] In certain embodiments, the method improves, enhances, or augments neurocognitive function. In some embodiments, the method improves, enhances, or augments cardiovascular function. In certain embodiments, improving, enhancing, or augmenting cardiovascular function comprises increasing blood flow. In some embodiments, improving, enhancing, or augmenting cardiovascular function comprises inducing vasodilation. In certain embodiments, the method improves, enhances, or augments muscle recovery. In some embodiments, the method improves, enhances, or augments recovery from physiological stress. In certain embodiments, the method improves, enhances, or augments muscle performance. In some embodiments, the method improves, enhances, or augments exercise capacity. In certain embodiments, the method improves, enhances, or augments insulin sensitivity. In some embodiments, the method improves, enhances, or augments metabolism. In certain embodiments, the method improves, enhances, or augments blood glucose control. In some embodiments, the method improves, enhances, or augments fat loss. In certain embodiments, the method improves, enhances, or augments weight management. In some embodiments, the method reduces oxidative stress. In certain embodiments, the method improves, enhances, or augments thermogenesis. In some embodiments, the method improves, enhances, or augments reaction times. In certain embodiments, the method improves, enhances, or augments neuromuscular function. In some embodiments, the method improves, enhances, or augments nitric oxide biosynthesis. In certain embodiments, the method improves, enhances, or augments L-DOPA biosynthesis. In some embodiments, the method improves, enhances, or augments serotonin biosynthesis. In certain embodiments, the method improves, enhances, or augments neurohormone biosynthesis. In some embodiments, the method improves, enhances, or augments sexual performance. In certain embodiments, the method mitigates male erectile dysfunction. In some embodiments, the method improves, enhances, or augments metabolism of one or more amino acids selected from tyrosine, phenylalanine, arginine, citrulline, and tryptophan. In certain embodiments, the method improves, enhances, or augments endothelial nitric oxide synthase phosphorylation. In some embodiments, the method reduces the effects of aging. In certain embodiments, the method improves, enhances, or augments mood.

[0037] In some embodiments, the composition comprises about 0.5% to about 10% total bioptersides (BHn), wherein the total bioptersides (BHn) comprises a first biopterin (Bl) selected from biopterin xyloside (BX), biopterin glucoside (BGlc), and biopterin arabinoside (BA), or pharmaceutically acceptable salts thereof.

[0038] In certain embodiments, the composition further comprises one or more additional biopterin glycosides (BG), or pharmaceutically acceptable salts thereof.

[0039] In certain embodiments, the B1:BG ratio is about 12: 1 to about 6: 1.

[0040] In certain embodiments, the composition is a composition of the present disclosure.

[0041] Methods of Preparing Biopterside Compositions

[0042] In some aspects, provided herein are methods of preparing a composition of the disclosure, comprising the steps of: contacting a precursor with an acid aqueous solution to form a mixture; agitating the mixture; filtering the mixture to obtain a filter cake and a filtrate solution; optionally adjusting the pH of the filtrate solution; and isolating the composition from the filtrate solution; wherein the precursor is selected from macroalgae, microalgae (e.g., cyanobacteria), blue-green algae, (Arthrospira) Spirulina (e.g., S. plantensis, Aphanizomenon flosaquae, Arthrospira maxima), Chlorella (e.g., C. vulgaris, C. pyrenoidosa, C. ellpsiodea, C. stigmatophora, C. zofingiensis), Haematococcus (e.g., H. pluvialis) Tetraselmis / Tetrasellimis (e.g., T. chuii, T. suecica), Isochryisis (e.g., I. galbana), Nanochloropsis (e.g., N. gaditana, N. oculata, N. salina , Euglena (e.g., E. gracilis), Schizachyrium, Crypthecodinium, Chlamydomonas (e.g., C. reinhardtii), Dunaliella (e.g., D. salina), Scenedesmus, Phaeodactylum, Ochromonas, Prymnesium, Coccomyxa, Skeletonema, Arthronema, Porphyra, Chlorococcum, Fucus, Microcystis, Glaucocystophyte, Lyngbya, Phaeodactylum, Haslea, Pavlova, Thalassiosira, Chaetoceros, Amphora, Annochloropsis, Pelagophyceae, Lyngbya, Anabaena, Porphyridium, Odentella, Undaria, Synechocystis, Synechococcus, Nostoc, Oscillatoria, Cylindrotheca, royal jelly, animal liver (e.g., beef liver), spinach, velvet bean (mucuna pruriens), fava bean (vicia faba), english broom (cytisus scoparius), banana, common purslane, Mexican rose, black caraway, black bean, soybean, watercress, water lotus, Buffalo gourd, pumpkin, acorn squash, sunflower, mung bean, black mustard, carrot, Acetobacter xyliunum, Achromobacter liquidum, Aerobacter aerogenes, Aeromonas hydrophilia, Bacillus spp. (e.g., B. alvei, B. cereus, B. coagulans, B. roseus, B. sphaericus), Brevibacterium acetylicum, Corynebacterium equi, Crithidia fasciculata, Erwinia carotovora, Eschericia coli, Flavobacterium aquatile, Gluconobacter spp (e.g., G. melanogenus, G. suboxydans), Lactobacillus casei, Micrococcus spp. (e.g., M.flavus, M. luteus, M. lysodeikiticus, M. ureae), Pseudomonas aeruginosa, P. aureofaciens, P. convexa, P. fluorescens, P. putrefaciens , Serratia spp. (e.g., S. indica, S. marcescens), Streptococcus faecalis , insects or insect cocoons (e.g., fruit fly, silkworm, honey bee), and a combination of any of them.

[0043] In certain embodiments, the precursor is a cyanobacterium. In certain preferred embodiments, the cyanobacterium is (Arthrospira) Spirulina.

[0044] In some embodiments, the precursor is not derived from petroleum. In certain embodiments, the acid aqueous solution comprises a Bronsted acid (e.g., HC1, HBr) and deionized water. In further embodiments, the acid aqueous solution is about 0.1 N to about 2 N HC1.

[0045] In some embodiments, methods of the disclosure further comprise contacting a precursor with a solvent. In certain embodiments, methods of the disclosure further comprise contacting a precursor with supercritical CO2. In certain embodiments, the precursor is contacted with supercritical CO2, thereby forming an extract, and the extract is contacted with an aqueous acid solution to form a mixture.

[0046] In certain embodiments, the mixture has a pH of about 1.5 to about 6.9.

[0047] In some embodiments, agitating the mixture comprises mechanical stirring.

[0048] In certain embodiments, agitating the mixture comprises sonic agitation.

[0049] In some embodiments, the method further comprises heating the mixture while agitating the mixture. In certain embodiments, heating the mixture comprises microwave-assisted heating.

[0050] In certain embodiments, the mixture is agitated at about ambient temperature.

[0051] In some embodiments, the method comprises the step of adjusting the pH of the filtrate solution. In further embodiments, the pH of the filtrate solution is adjusted to about 4.0 to about 8.0. In yet further embodiments, the pH of the filtrate solution is adjusted to about 6. In still further embodiments, the pH of the filtrate solution is adjusted to about 7. In certain embodiments, the pH of the filtrate solution is adjusted to about 8.

[0052] In certain embodiments, isolating the composition from the filtrate solution comprises adding a second solvent to the filtrate solution. In further embodiments, the second solvent is a food grade solvent selected from ethyl acetate, methanol, ethanol, and 2-propanol.

[0053] In some embodiments, isolating the composition comprises recrystallization, reverse osmosis, lyophilization, spray drying, removing solvent from the filtrate solution, or any combination thereof.

[0054] In certain embodiments, isolating the composition comprises removing solvent from the filtrate solution.

[0055] In some embodiments, isolating the composition comprises heating the filtrate solution under reduced pressure.

[0056] In certain embodiments, filtering the mixture comprises tangential flow filtration, deadend filtration, nanofiltration, reverse osmosis, or a combination thereof. In some embodiments, filtering the mixture comprises ultrafiltration.

[0057] In certain embodiments, the ultrafiltration comprises passing the mixture through a 1 kDa to 100 kDa ultrafiltration membrane.

[0058] In some embodiments, isolating the composition further comprises contacting the mixture with a chromatography medium. In certain embodiments, the chromatography medium comprises size-exclusion chromatography (SEC) media. In some embodiments, the chromatography medium is a resin or membrane. In further embodiments, the chromatography medium is a resin (e.g., an ion exchange resin). In yet further embodiments, the chromatography medium is a membrane (e.g., an ion exchange membrane).

[0059] In certain embodiments, filtering the mixture comprises contacting the mixture with an ultrafiltration medium, thereby producing a filtrate solution, and isolating the composition from the filtrate solution comprises contacting the filtrate solution with a chromatography medium, preferably an ion exchange resin or ion exchange membrane.

[0060] Definitions

[0061] The term “bioptersides(s)” as used herein refers to a molecule that comprises a pterin moiety, for example, a radical of biopterin [2-amino-6-( 1 ,2-dihydroxypropyl)- 1 H-pteridin-4- one], or a derivative thereof, attached to an aglycone or a glycosyl moiety, e.g., via a single covalent bond. For example, certain compositions of the present disclosure comprise biopterin xyloside, which is a molecule comprising a biopterin radical attached to a xylosyl radical as shown in Formula la. See also the graphic below, which depicts: biopterin (la); 2’-0-(oc-D- glucopyranosyl) biopterin (lb); the corresponding p-D-ribofuranosyl analog (1c); and 2’-O-(2- acetamido-2-deoxy-|3-D-glucopyranosyl)biopterin (limipterin) (Id).

[0062]

[0063] Pharmaceutical Compositions

[0064] In certain embodiments, the present application is directed to a pharmaceutical composition. In certain embodiments, the pharmaceutical composition comprises the depot formulations as disclosed herein. In some embodiments, the depot formulation further comprises an additional therapeutic compound (i.e., agent) and a pharmaceutically acceptable carrier. At least one additional therapeutic agent can refer to an agent useful in the treatment of a mitochondrial disease, such as Leber's Hereditary Optic Neuropathy. The pharmaceutical composition can be a medicament.

[0065] Pharmaceutical compositions can be prepared by combining a compound, e.g., an oligopeptide or a peptidomimetic compound with a pharmaceutically acceptable carrier and, optionally, one or more additional therapeutic agents.

[0066] The term “subject” as used herein refers to a human. An “effective amount” refers to any amount that is sufficient to achieve a desired biological effect. Combined with the teachings provided herein, by choosing among the various active compounds and weighing factors such as potency, relative bioavailability, patient body weight, severity of adverse side-effects and mode of administration, an effective prophylactic (i.e., preventative) or therapeutic treatment regimen can be planned which does not cause substantial unwanted toxicity and yet is effective to remedy the condition or disease of a particular subject. The effective amount for any particular indication can vary depending on such factors as the disease or condition being treated, the particular compound of the present application being administered, the size of the subject, or the severity of the disease or condition. The effective amount may be determined during pre-clinical trials and clinical trials by methods familiar to physicians and clinicians. One of ordinary skill in the art can empirically determine the effective amount of a particular compound of the present application and / or other therapeutic agent without necessitating undue experimentation. A maximum dose may be used, that is, the highest safe dose according to some medical judgment. Appropriate systemic levels can be determined by, for example, measurement of the patient’s peak or sustained plasma level of the drug. Locally delivered doses can be determined by, for example, the level in a particular compartment of the body, such as the vitreous humor of the eye. “Dose” and “dosage” are used interchangeably herein. A dose can be administered by oneself, by another, or by way of a device (e.g., a pump or syringe). As a non-limiting example, an “effective amount” of a composition described herein may refer to the amount sufficient to improve, increase, enhance, or augment neurocognitive function in a subject.

[0067] Compounds for use in, e.g., the methods described herein can be tested in suitable animal model systems. Similarly, for in vivo testing, any of the animal model system known in the art can be used prior to administration to human subjects. Suitable animal model systems include, but are not limited to, rats, mice, chicken, cows, monkeys, rabbits, and the like, prior to testing in human subjects.

[0068] A therapeutic compound and optionally other therapeutic agents may be administered in freebase equivalent form or in the form of a pharmaceutically acceptable salt. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof. The term “pharmaceutically acceptable salt” as used herein includes salts derived from inorganic or organic acids including, for example, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, phosphoric, formic, acetic, lactic, maleic, fumaric, succinic, tartaric, glycolic, salicylic, citric, methanesulfonic, benzenesulfonic, benzoic, malonic, trifluoroacetic, trichloroacetic, naphthalene-2-sulfonic, and other acids. Pharmaceutically acceptable salt forms can include forms wherein the ratio of molecules comprising the salt is not 1:1. For example, the salt may comprise more than one inorganic or organic acid molecule per molecule of base, such as two hydrochloric acid molecules per molecule of compound. As another example, the salt may comprise less than one inorganic or organic acid molecule per molecule of base, such as two molecules of compound per molecule of tartaric acid. As another example, the salt may comprise two different inorganic or organic acid molecules per molecule of base such as one molecule of compound with one sodium and one potassium molecule.

[0069] Pharmaceutical compositions of the present application contain an effective amount of a therapeutic compound as described herein and may optionally be disbursed in a pharmaceutically acceptable carrier. The components of the pharmaceutical compositions also are capable of being commingled with the compounds of the present application, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency. For example, the pharmaceutically acceptable carrier(s) may be or may comprise cyclodextrins or extracellular vesicles.

[0070] Dosage, toxicity and therapeutic efficacy of any therapeutic compounds, compositions (e.g., formulations or medicaments), other therapeutic agents, or mixtures thereof can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Compounds that exhibit high therapeutic indices are advantageous.

[0071] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds may be within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms). Such information can be used to determine useful doses in humans accurately. Levels of the active pharmaceutical ingredient / therapeutic compound (e.g., the oligopeptide or the peptidomimetic) in plasma of an animal or human may be measured, for example, by high performance liquid chromatography.

[0072] An exemplary treatment regime can entail acute or sub-chronic administration (e.g., as needed or PRN), or administration once a week to once a month, or once a month to once a year, or once a month to once every six months, or once a month to once every three months, once every three months to once every six months, once every six months to once nine months, or once every nine months to once per year. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, the desired health or performance effect is achieved, or until the subject shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regimen.

[0073] EXAMPLES

[0074] The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

[0075] Example 1: Evaluation of TFF (Tangential Flow Filtration or Ultrafiltration) for biopterside product refinement

[0076] 500 grams of Spirulina-02 (BHAG-AB, 499.54 g, stored at ambient) was weighed out directly into a 15 kg Eagle Stainless steel pot. In a separate 10 L hdpe jug was weighed out 101.19 g 5N HO and 4901.02 g deionized water (Labchem, ASTM Type II, exp. 07 / 2023). The aqueous acid mixture (0.1N HC1) was swirled to mix and then combined with the spirulina. A 400 mm Ellipso-Spoon spatula (Chemglass, p / n: CG- 1987-04) was used to ensure all of the spirulina was wetted and formed a homogeneous mixture. The mixture was agitated with a 4- blade, propeller type stainless steel stir- shaft connected to a l / 9th horsepower stir motor (Arrow Engineering, 2000 electric, p / n: 2000) for exactly 60 minutes. After the extraction was complete the stir shaft was removed, and the stir shaft was rinsed with approximately 10 mL of deionized water.

[0077] In a separate 4-L Erlenmeyer flask, Celite 545 was added (504.98 g) and deionized water (1790.91g) were swirled until homogeneous, then poured into a 3-L sintered glass filter funnel fitted onto a 2-L side-arm elution flask (custom glass) with a piece of 4.5-inch diameter Whatman #1 filter paper while under vacuum. After the cake was formed the vacuum was removed and the filtrate was poured back into the 4-L Erlenmeyer flask. The Buchner funnel was then returned to the side-arm elution flask and placed back under vacuum. The water in the 4-L flask was then poured over the cake a second time and vacuum filtered into the side-arm elution flask. 1696.52 g of the Celite cake water was recovered then discarded.

[0078] The crude spirulina extract was then poured onto the Celite 545 cake while under vacuum, using a silicon cake spatula to empty the contents of the stainless steel pot. After the paste cake stopped dripping (approximately 17 hours) the golden, clarified crude extract (3504.91 g, pH=3.49 @ 21.2 °C, 0.5 mL aliquot taken for HPLC analysis: Cl-clarified crude portion #3 1062.68 g, no pH adjustment, assay value: 61.89 pg / mL xyloside; 14.27 pg / mL glucoside) was split into 3 portions for pH adjustment. Portion #1 (1504.92 g, pH 7.02 @ 23.4 °C, pH adjusted with 0.2M KOH, 0.5 mL aliquot taken named Al clarified crude; assay value: 58.00 pg / mL biopterin xyloside; 16.07 pg / mL biopterin glucoside). Portion #2 (1502.08 g, pH = 7.03 @ 23.5 °C, pH adjusted with 0.2M NaOH, 0.5 mL aliquot taken named Bl clarified crude; assay value: 58.40 pg / mL biopterin xyloside; 15.23 pg / mL biopterin glucoside). The remaining wet paste / filter paper / Celite cake was discarded. Back calculation shows 266.9 mg total bioptersides in the crude extract, indicating a total biopterside content in the spirulina of 0.53 mg / g.

[0079] Portion #1 ultrafiltration experiment

[0080] Size 16# silicon tubing was attached to the inlet of the ultrafilter (Repligen MidiKros Hollow Fiber Filter 10 kD 234 cm241.5 cm D04-E010-05-N) by Luer fitting then taped into place to prevent pulling out of the reservoir. Size 16# tubing was also connected to the outlet by Luer fitting and recycled back into the retentate beaker. Size 16# tubing was connected by Luer fitting to the permeate outlet and collected in a 2000 mL beaker. The inlet tubing was clamped into the peristaltic pump head. Magnetic stir rate set to 500 rpm. Pumping speed was set to a flow-rate of 200 mL / min. Prior to filtration the system was flushed with approximately 5 mL of deionized water to ensure all air was purged from the system. Permeate was collected for 183 minutes. After about 50% (739.47 g permeate) had been filtered an aliquot was taken for HPLC analysis: A2_50%UF KC1, assay value 57.59 pg / mL xyloside; 19.60 pg / mL glucoside. After about 90% had been filtered the light yellow permeate (1305.41 g, pH=3.53 @ 23.3 °C, 0.5 mL aliquot taken for HPLC analysis: A2_90% UF KC1, assay value 57.64 pg / mL biopterin xyloside; 15.59 pg / mL biopterin glucoside). The brown / orange retentate was turbid (193.6 g, pH=3.49 @23.2 °C, A4_10%_retentate assay value: 58.39 pg / mL biopterin xyloside; 15.14 pg / mL biopterin glucoside).

[0081] The ultrafiltrate A2 was split into 2 portions: A5 = 652.08 g and A6 = 652.22g. A5 was transferred to a 2-L single neck round bottom flask. The flask was placed onto the rotovap set at 100 rpm, bath temperature 55 °C, and pressure <700 mmHg. Water was stripped until approximately 100 mL slurry was left (approximately 5 hours). The contents were transferred to a 250 mL round bottom flask and placed back onto the rotovap. And stripped to approximately 10 grams of slurry. The contents were transferred to a 20-gram scintillation vial and placed back on the rotovap for 6 hours until 2.3709 g of a yellow dry, free-flowing powder was obtained. 8.0 mg of the homogenized dry powder was dissolved in 250 mL of mobile phase A (0.2%TFA in deionized water).

[0082] Portion A6 was transferred to a 1-L media bottle and placed into a 4 °C refrigerator for 24 hours to see if bioptersides could be crystallized. No visible precipitation observed. The same process was used for portions #2 and #3

[0083] Example 2: Evaluation of microporous resins for extract concentration and refinement

[0084] 50 grams of Spirulina-02 (B HAG- AB, 50.54 g, stored at ambient) was weighed out directly into a 1000 mL Pyrex beaker (Quark Glass, p / n: QB-1-11). In a separate 1000 mL Pyrex beaker was weighed out 10.03 g 5N HC1 and 500.07 g deionized water (Labchem, ASTM Type II, exp. 07 / 2023). The aqueous acid mixture (0.1N HC1) was swirled to mix and then combined with the spirulina. A spatula was used to ensure all of the spirulina was wetted and formed a homogeneous mixture. The mixture was agitated with a 4-blade, propeller type stainless steel stir-shaft connected to a l / 25th horsepower stir motor (Arrow Engineering, JR4000 electric, p / n: JR4000) for exactly 60 minutes. After the extraction was complete the stir shaft was removed, and the stir shaft was rinsed with approximately 10 mL of deionized water.

[0085] In a separate 2-L Erlenmeyer flask, Celite 545 was added (25.05g) and deionized water (213.77g) were swirled until homogeneous, then poured into a ceramic Buchner funnel fitted onto a 2-L side-arm flask with a piece of 5 -inch diameter Whatman #1 filter paper while under vacuum. After the cake was formed the vacuum was removed and the filtrate was poured back into the 2-L Erlenmeyer flask. The Buchner funnel was then returned to the side-arm flask and placed back under vacuum. The water in the 2-L flask was then poured over the cake a second time and vacuum filtered into the side-arm flask. 177.74 g of the Celite cake water was recovered then discarded.

[0086] The crude spirulina extract was then poured onto the Celite 545 cake while under vacuum, using a silicon cake spatula to empty the contents of the 2-L beaker. After the paste cake stopped dripping (54 minutes) the golden, clarified crude extract (372.72 g, pH=3.37 @ 24.0 °C, 0.5 mL aliquot taken for HPLC analysis: l_crude extract, assay value: I l 8.57pg / mL xyloside; 11.44 pg / mL glucoside) was transferred to a 1-L beaker fitted with a 1.5-inch magnetic stir-bar and measured for pH and weighed. The beaker was then used as the retentate reservoir for the next step. The remaining wet paste / filter paper / Celite cake was discarded.

[0087] Size 16# silicon tubing was attached to the inlet of the ultrafilter (Repligen MidiKros Hollow Fiber Filter 10 kD 234 cm241.5 cm D04-E010-05-N) by Luer fitting then taped into place to prevent pulling out of the reservoir. Size 16# tubing was also connected to the outlet by Luer fitting and recycled back into the retentate beaker. Size 16# tubing was connected by Luer fitting to the permeate outlet and collected in a 1000 mL beaker. The inlet tubing was clamped into the peristaltic pump head. Pumping speed was set to a flow-rate of 200 mL / min. Prior to filtration the system was flushed with approximately 5 mL of deionized water to ensure all air was purged from the system. Permeate was collected for 79 minutes. The light yellow permeate (311.32 g, pH=3.37 @ 23.4 °C, 0.5 mL aliquot taken for HPLC analysis: 2_permeate from UF non pH adjust, assay value 117.7 pg / mL xyloside; 9.58 pg / mL glucoside.) The brown / orange retentate was turbid (39.62g, pH=3.46 @23.3 °C).

[0088] The permeate was split into two portions: 2_permeate from UF non pH adjust 193.68g and 3_permeate from uf pH 8_89 which was pH adjusted by dropwise addition of 0.2M KOH to pH = 8.89 @ 23.5 °C. Some turbidity was observed in 3; assay value: 115.69 pg / mL xyloside and 10.06 pg / mL glucoside.

[0089] Both resins in this study were prepared by soaking 1 gram portions in deionized water overnight prior to packing the solid phase extraction columns. Columns were the barrel portion of disposable 5 mL plastic syringes with Luer lock tips. 2.5 inch silicon tubing were attached to each column. A small plug of cotton was inserted into the syringe to retain the resins. Each resin was transferred to the individual column, washing down the sides to ensure all the resin beads were accounted for. The plug was gently tamped down with the bulb end of a disposable pipette followed by another plug of cotton to prevent column cracking or unwanted pockets of solvent. Each column was zip-tied to a crossbar, clamped onto a ring stand. Each column was then rinsed with 10 column volumes of deionized water, ensuring the tubings were completely drained prior to loading.

[0090] Approximately 3.5 g of extract at both pH values were loaded onto each type of resin shown in the table below. Loading consisted of adding the extract with a pre-weighed pipette by a very slow dropwise addition of approximately 10 minutes. The two columns loaded with pH 3.5 ultrafiltrate did not change color. The two columns loaded with pH 9.0 extract turned a dark burgundy color. The unretained fractions were collected and assayed. The columns were then rinsed with an equivalent volume of deionized water to the amount of extract added in the same slow dropwise fashion. The washings were collected and assayed. Finally, the columns were eluted with a 50 / 50 mixture of ethanol and water using approximately twice the loading volume. The eluents were collected and assayed. The eluents were stripped of solvent in the spray drying method. Each solid was a yellow film.

[0091] After the elution solvent passed through the columns it was noted that both resins loaded with pH 9.0 ultrafiltrate were still a dark burgundy color. Each of the four columns were then rinsed with about 3.0 g of absolute ethanol. The ethanol rinse was collected and assayed. No biopterins were found in any sample. See Table 5.1.

[0092] A 0.1 M solution of ascorbic acid was prepared then added to the two basic columns. 3.88 g 0.1M ascorbic acid was added to column IB and 3.96 g 0.1M ascorbic acid was added to column 2B. The original color of the resins was restored. The elution was collected and assayed. No biopterins were observed in either sample, however the ascorbic acid peak in the HPLC was quite large.

[0093] The next set of resin experiments used the same ultrafiltrate that was pH adjusted to 8.98 and pre-swelled SP700 resin. Three columns were prepared as described above. The elution solvents tested were Elution Solvent 1: 5% 0.01N HO in 95% Ethanol; Elution Solvent 2: 50% 0.01N HC1 in ethanol; and Elution Solvent 3: 100% 0.01N HC1. See Table 5.2.

[0094] The final resin test was designed to determine if rinsing the column with base prior to loading helped with biopterside retention. A side-by-side comparison using the same ultrafiltrate pH adjusted to 8.98 and SP700 resin that has been pre- weighed and pre-swelled overnight. See Table 5.3.

[0095] Example 3: Biopterside stability in crude spirulina and Biopterside stability and recovery from aqueous extracts

[0096] Example Extraction 1A

[0097] 50 grams of Spirulina-02 (B HAG- AB, 50.03g, stored at ambient) was weighed out directly into a 1000 mL Pyrex beaker (Quark Glass, p / n: QB-1-11). In a separate 1000 mL Pyrex beaker was weighed out 10.03 g 5N HC1 and 501.03 g deionized water (Labchem, ASTM Type II, exp. 07 / 2023). The aqueous acid mixture (0.1N HC1) was swirled to mix and then combined with the Spirulina. A spatula was used to ensure all of the spirulina was wetted and formed a homogeneous mixture. The mixture was agitated with a 4-blade, propeller type stainless steel stir-shaft connected to a l / 25th horsepower stir motor (Arrow Engineering, JR4000 electric, p / n: JR4000) for exactly 60 minutes. After the extraction was complete the stir shaft was removed, and the stir shaft was rinsed with approximately 10 mL of deionized water.

[0098] In a separate 2-L Erlenmeyer flask, Celite 545 was added (299.86 g) and deionized water (551.77g) were swirled until homogeneous, then poured into a ceramic Buchner funnel fitted onto a 2-L side-arm flask with a piece of 5 -inch diameter Whatman #1 filter paper while under vacuum. After the cake was formed the vacuum was removed and the filtrate was poured back into the 2-L Erlenmeyer flask. The Buchner funnel was then returned to the side-arm flask and placed back under vacuum. The water in the 2-L flask was then poured over the cake a second time and vacuum filtered into the side-arm flask. 416.24g of the Celite cake water was recovered then discarded.

[0099] The crude spirulina extract was then poured onto the Celite 545 cake while under vacuum, using a silicon cake spatula to empty the contents of the 2-L beaker. After the paste cake stopped dripping (72 minutes) the golden, clarified crude extract (442.46 g, pH=3.44 @ 23.3 °C, 0.5 mL aliquot taken for HPLC analysis: 5A1, assay value: 80.30 pg / mL xyloside; 9.13 pg / mL glucoside) was transferred to a 1-L beaker fitted with a 1.5 -inch magnetic stir-bar and measured for pH and weighed. The beaker was then used as the retentate reservoir for the next step. The remaining wet paste / filter paper / Celite cake (548.07 g) was discarded.

[0100] Size 16# silicon tubing was attached to the inlet of the ultrafilter (Repligen MidiKros Hollow Fiber Filter 10 kD 234 cm241.5 cm D04-E010-05-N) by Luer fitting then taped into place to prevent pulling out of the reservoir. Size 16# tubing was also connected to the outlet by Luer fitting and recycled back into the retentate beaker. Size 16# tubing was connected by Luer fitting to the permeate outlet and collected in a 1000 mL beaker. The inlet tubing was clamped into the peristaltic pump head. Pumping speed was set to a flow-rate of 200 mL / min. Prior to filtration the system was flushed with approximately 5 mL of deionized water to ensure all air was purged from the system. Permeate was collected for 150 minutes. The light yellow permeate (404.85 g, pH=3.48 @ 23.1 °C, 0.5 mL aliquot taken for HPLC analysis: 5A2, assay value 81.51 pg / mL xyloside; 9.17 pg / mL glucoside). The brown / orange retentate was turbid (39.62g, pH=3.46 @23.3 °C).

[0101] A small portion of the permeate (3.4065 g) was transferred to a 20 g, pre-weighed scintillation vial fitted with a 3 mm magnetic stir bar (tare with stir bar 15.7040 g). Vial was placed onto a stir / hot plate and set to stir at a rate of 650 rpm. Hot plate surface temperature was set to 125 °C. An Argon stream was blown directly into the vial at a flow rate of 15 CFH. Water was evaporated for 24 minutes to dryness. The light yellow solid was recovered (19.6 mg). The solid was readily dissolved in deionized water (3.3152 g, 0.5 mL aliquot was taken for HPLC analysis: 6A, assay value 93.06 pg / mL xyloside; 9.21 pg / mL glucoside; dry wt value: 15.7397 mg biopterin xyloside per g of dried extract; 1.5577 mg biopterin glucoside per g of dried extract; Total biopterin content: 17.2974 mg biopterins per gram of dried extract; %wt of total biopterins in dried extract: 1.73%; total biopterins in spirulina sample: 0.90 mg biopterins per gram of spirulina)

[0102] Example Extraction IB

[0103] 10 grams of Spirulina (BHAG-AB, 10.0014g) was weighed out directly into a 250 mL Pyrex beaker (Quark Glass, p / n: QB- 1-7). In a separate 250 mL Pyrex beaker was weighed out 1.67 g 6N HC1 (Supelco, reagent grade, Z0750207132, exp 10 / 2024) and 98.3 g deionized water (Labchem, ASTM Type II, exp. 01 / 2022). The aqueous acid mixture (0.1N HC1) was swirled to mix and then combined with the Spirulina and a 1-inch PTFE coated magnetic stir-bar (Chemglass, CG-2001-50) was added. The beaker was placed onto a magnetic stir / hot plate (Thermo Scientific, p / n: SP88857100, s / n: C3710017101918196). The solution was stirred at 1000 rpm for 60 minutes (Casio G Shock, p / n: DW-5600E, s / n: unavailable) at an ambient temperature of 19.1 °C. When the extraction was complete the pH and temperature were measured (digital pH meter / thermometer, Yoko, p / n: PH-838, s / n: 05227127) at pH=6.88 at 18.8 °C. A 5 cm ceramic Buchner funnel (Amazon) fitted with filter paper (Whatman #1, cut to size) was prepared by addition of approximately 10 g Celite 545 (Sigma Aldrich, p / n: 22140-1KG-F). The funnel was placed atop a 250 mL side arm vacuum flask (Chemglass, p / n: CG-8514-250) fitted with the appropriately sized neoprene collar (Chemglass, p / n: CG-1401-01). Vacuum was applied (Welsh, Dryfast p / n: 2044, s / n not legible on pump) and the extract was poured slowly over the Celite bed. The filtration proceeded until the cake was visibly dry (approximately 35 minutes). The filtrate was light yellow / orange in color with no visibly suspended solids. The filtrate (94.8733 g extract recovered) was passed through a 0.22 pm nylon syringe filter and analyzed neat by HPLC (Liquid extract assay value for biopterin xyloside: 390.5629 pg / mL; biopterin glucoside: 21.04639 pg / mL; calculated recoveries in mg per gram of spirulina for biopterin xyloside: 3.46044 mg biopterin xyloside per gram of BHAG-AB Spirulina; biopterin glucoside: 0.2105 mg biopterin glucoside per gram of BHAG-AB Spirulina).

[0104] Example Extraction 2A

[0105] 25 grams of Spirulina (QC Lot 18612, 25.28 g, stored at -20 °C) was weighed out directly into a 400 mL Pyrex beaker (Quark Glass, p / n: QB- 1-8). In a separate 400 mL Pyrex beaker was weighed out 5.07 g 5N HC1 and 253.27 g deionized water (Labchem, ASTM Type II, exp. 07 / 2023). The aqueous acid mixture (0.1N HC1) was swirled to mix and then combined with the Spirulina. A spatula was used to ensure all of the spirulina was wetted and formed a homogeneous mixture. The mixture was agitated with a 4-blade, propeller type stainless steel stir-shaft connected to a l / 25th horsepower stir motor (Arrow Engineering, JR4000 electric, p / n: JR4000) for exactly 60 minutes. After the extraction was complete the stir shaft was removed, and the stir shaft was rinsed with approximately 10 mL of deionized water.

[0106] In a separate 2-L Erlenmeyer flask, Celite 545 was added (301.60 g) and deionized water (613.88 g) were swirled until homogeneous, then poured into a ceramic Buchner funnel fitted onto a 2-L side-arm flask with a piece of 5 -inch diameter Whatman #1 filter paper while under vacuum. After the cake was formed the vacuum was removed and the filtrate was poured back into the 2-L Erlenmeyer flask. The Buchner funnel was then returned to the side-arm flask and placed back under vacuum. The water in the 2-L flask was then poured over the cake a second time and vacuum filtered into the side-arm flask. 555.92 g of the Celite cake water was recovered then discarded.

[0107] The crude spirulina extract was then poured onto the Celite 545 cake while under vacuum, using a silicon cake spatula to empty the contents of the 400 mL beaker. After the paste cake stopped dripping (16 minutes) the golden, clarified crude extract (231.60 g, pH=3.60 @ 23.0 °C, 0.5 mL aliquot taken for HPLC analysis: 7A1, assay value: 67.60 pg / mL xyloside; 4.89 pg / mL glucoside)) was transferred to a 400 mL beaker fitted with a 1.5-inch magnetic stir-bar and measured for pH and weighed. The beaker was then used as the retentate reservoir for the next step. The remaining wet paste / filter paper / Celite cake (425.13 g) was discarded.

[0108] A second aliquot was transferred from the crude, clarified extract to a pre- weighed scintillation vial fitted with a 3 mm magnetic stir bar (4.2022 g). The vial was placed onto a heating / stir plate with a stir set to 650 rpm and a surface temperature set to 125 °C. Argon gas was blown directly into the vial at a rate of 15 CFH for 19 minutes to provide a yellow solid (28.3 mg) that was readily soluble in deionized water (4.4700 g, a 0.5 mL aliquot was taken for HPLC analysis: 7A2, assay value 56.61 pg / mL xyloside; 4.24 pg / mL glucoside; Biopterin content: 8.9410 mg biopterin xyloside per gram of dried extract, 0.6690 mg biopterin glucoside per gram of dried extract; Total biopterin content: 9.6109 mg biopterins per gram of dried extract; %wt biopterins in dried extract: 0.96%. Size 16# silicon tubing was attached to the inlet of the ultrafilter (Repligen MidiKros Hollow Fiber Filter 10 kD 234 cm241.5 cm D04-E010-05-N) by Luer fitting then taped into place to prevent pulling out of the reservoir. Size 16# tubing was also connected to the outlet by Luer fitting and recycled back into the retentate beaker. Size 16# tubing was connected by Luer fitting to the permeate outlet and collected in a 400 mL beaker. The inlet tubing was clamped into the peristaltic pump head. Pumping speed was set to a flow-rate of 200 mL / min. Prior to filtration the system was flushed with approximately 5 mL of deionized water to ensure all air was purged from the system. Permeate was collected for 111 minutes. The light yellow permeate (153.3 g, pH=3.55 @ 23.6 °C, 0.5 mL aliquot taken for HPLC analysis: 7A3, assay value 72.38 pg / mL xyloside; 5.45 pg / mL glucoside). The brown / orange retentate was turbid (72.15g, pH=3.46 @23.3 °C).

[0109] A small portion of the permeate (3.4893 g) was transferred to a 20 g, pre-weighed scintillation vial fitted with a 3 mm magnetic stir bar (tare with stir bar 15.6840 g). Vial was placed onto a stir / hot plate and set to stir at a rate of 650 rpm. Hot plate surface temperature was set to 125 °C. An Argon stream was blown directly into the vial at a flow rate of 15 CFH. Water was evaporated for 26 minutes to dryness. The light yellow solid was recovered (23.9 mg). The solid was readily dissolved in deionized water (4.3065 g, 0.5 mL aliquot was taken for HPLC analysis: 7A4, assay value 58.60 pg / mL xyloside; 4.74 pg / mL glucoside; Biopterin content of dried extract: 10.5584 mg biopterin xyloside per gram of dried extract, 0.8537 mg biopterin glucoside per gram of dried extract; Total biopterin content: 11.4121 mg biopterins per gram of dried extract; %wt biopterins in dried extract: 1.14 %; biopterins in spirulina sample: 0.56 mg biopterins per gram of spirulina)

[0110] A small portion of the retentate (3.727 g) was transferred to a 20 g, pre-weighed scintillation vial fitted with a 3 mm magnetic stir bar (tare with stir bar 16.2156 g). Vial was placed onto a stir / hot plate and set to stir at a rate of 650 rpm. Hot plate surface temperature was set to 125 °C. An Argon stream was blown directly into the vial at a flow rate of 15 CFH. Water was evaporated for 33 minutes to dryness. The dark yellow solid was recovered (31.3 mg).

[0111] Example Extraction 2B

[0112] 5 grams of Spirulina (QC Lot: 18612 5.0253 g) was weighed out directly into a 250 mL Pyrex beaker (Quark Glass, p / n: QB-1-7). The aqueous acid mixture (0.1N HC1, 50.0071g) was combined with the Spirulina and a 1-inch PTFE coated magnetic stir-bar (Chemglass, CG-2001- 50) was added. The beaker was placed onto a magnetic stir / hot plate (Thermo Scientific, p / n: SP88857100, s / n: C3710017101918196). The solution was stirred at 1000 rpm for 60 minutes (Casio G Shock, p / n: DW-5600E, s / n: unavailable) at an ambient temperature of 19.5 °C. When the extraction was complete the pH and temperature were measured (digital pH meter / thermometer, Yoko, p / n: PH-838, s / n: 05227127) at pH=7.34 at 19.5 °C. A 5 cm ceramic Buchner funnel (Amazon) fitted with filter paper (Whatman #1, cut to size) was prepared by addition of approximately 10 g Celite 545 (Sigma Aldrich, p / n: 22140-1KG-F). The funnel was placed atop a 250 mL side arm vacuum flask (Chemglass, p / n: CG-8514-250) fitted with the appropriately sized neoprene collar (Chemglass, p / n: CG-1401-01). Vacuum was applied (Welsh, Dryfast p / n: 2044, s / n not legible on pump) and the extract was poured slowly over the Celite bed. The filtration proceeded until the cake was visibly dry (approximately 30 minutes). The filtrate was light yellow in color with no visibly suspended solids. The filtrate was passed through a 0.22 pm nylon syringe filter and analyzed neat by HPLC (Liquid extract assay value for biopterin xyloside: 184.5502 pg / mL; biopterin glucoside: 6.718811 pg / mL; calculated recoveries in mg per gram of spirulina for biopterin xyloside: 0.96 mg biopterin xyloside per gram of BHAG-AB Spirulina; biopterin glucoside: 0.07 mg biopterin glucoside per gram of BHAG-AB Spirulina). 91.30 g liquid extract recovered.

[0113] Example 4: Evaluation of TFF (Tangential Flow Filtration or Ultrafiltration) for biopterside product refinement

[0114] Table 4.1: Biopterside recovery for exemplary extractions A, B, and C. biopterin concentration appears highest at an acidic pH, presumably due to precipitation at a higher pH.

[0115] Table 4.2: Process mass balance for exemplary extractions A, B, and C. ote: no biopterins recovered from rotovap step.

[0116] Example 5 : Evaluation of exemplary macroporous resins for extract concentration and refinement

[0117] Table 5.1: Results of initial evaluation of biopterside retention on Mitsubishi SP700 and HP2MGL resins. the fully protonated biopterins, and the SP700 appeared to be stickier than the HP2ME resin. It also appears that this is an acid / base controlled process.

[0118] Table 5.2: Evaluation of biopterside retention on SP700 at pH 9

[0119] Mote: It appears that some amount of biopterins are sticking to the column and the 100% 0.01N

[0120] HC1 is a suitable elution solvent. Column B2 appears to indicate that some fractionation may be possible with the correct choice of resin.

[0121] Table 5.3: Evaluation of biopterside retention on SP700 pre-equilibrated at pH 9 ote: It appears that pretreatment of the column with base slowed the passage through of biopterin xyloside while the opposite appears to be true for biopterin glucoside. The big take away here is that for the most part the biopterins were not retained well enough for concentration or purification purposes with this particular resin.

[0122] Example 6: Biopterside stability in crude spirulina

[0123] Table 6.1: Effect of ambient temperature storage of Spirulina powder on Biopterside content. Table 6.2: Effect of -20 °C storage of Spirulina powder on Biopterside content.

[0124] Table 6.3: Biopterside recovery and purity from Spirulina through spray-dried solid.

[0125] Note: Table 6.3 shows the comparison of two different specimens, BHAG-AB stored at ambient temperature and humidity over 18 months between extractions and QC Lot 18612 stored at -20 °C over 18 months between extractions.

[0126] Example 7: Investigation of Effects of Varying Doses of Biopterside Compositions on Nitric Oxide Synthase (eNOS) in Human Endothelial Cells

[0127] An In vitro randomized controlled trial was conducted to assess the vasodilation-inducing eNOS and NOx effects of the a) total Biopterside dose (low vs high), b) concentration of specific, highly purified (>80% w / w) Biopterside standards present within the total Biopterside dose, and c) as compared against two control conditions - unprocessed source material (Spirulina) and untreated cells (Control). The study, titled Dose-seeking effects of Bioptersides and Spirulina on acute activation of vasodilation-inducing nitric oxide synthase (eNOS) in human endothelial cells, was conducted at the Molecular & Applied Sciences Laboratory, Auburn University (Auburn, AL, USA).

[0128] Cell Type

[0129] Human umbilical vein endothelial cells (HUVEC)

[0130] Methods

[0131] 6-well plates / treatment; 10,000 HUVEC cells / well; R&D Systems HUVEC specific cell growth base medium w / endothelial growth supplement; treatments added to HUVEC media once 80% confluency (cell concentration per well) was achieved; 3-hr treatment time (i.e., acute response); SDS-PAGE Western blotting analysis using phosphor-eNos (Seri 177; 133 kD) antibody; t-tests vs control (p<0.05). Cell Culturing

[0132] Two, six-well plates were seeded, per treatment group, with 10,000 HUVEC cells per well. R&D Systems’ Endothelial Cell Growth Base Media (#390598) with Endothelial Cell Growth Supplement (#390599) were used for the expansion of HUVECs according to the suppliers’ procedures. Cultures were monitored daily and culture media changed every other day until 80% confluency was achieved per well.

[0133] SDS-PAGE Western Blotting Analysis

[0134] After 3-hr treatments all cells were lysed using ice-cold lysis buffer (cell signaling)

[0135] Cell lysates were then prepared for Western blotting; SDS-PAGE Western Blotting ensued and a Abeam Anti-eNOS (phosphor SI 177) antibody abl84154 was used for interrogation.

[0136] Treatments (8)

[0137] Growth Supplement Only (CTL)

[0138] CTL + 10.9 ug / mL Spirulina (SPI)

[0139] CTL + 10.9 pg / mL Biopterside A (BIO-ALO)

[0140] CTL + 86.8 pg / mL Biopterside A (BIO-AHI)

[0141] CTL + 10.9 pg / mL Biopterside B (BIO-BLO)

[0142] CTL + 86.8 pg / mL Biopterside B (BIO-BHI)

[0143] CTL + 10.9 pg / mL Biopterside A+B (BIO-ABLO)

[0144] CTL + 86.8 pg / mL Biopterside A+B (BIO-ABHI)

[0145] Dependent Variables (DVs)

[0146] Phosphorylation (activation) of Endothelial Nitric Oxide Synthase [p-eNOS (Seri 177)]

[0147] Statistical Analysis

[0148] Treatment responses were normalized across plates to account for gel covariance and then compared against CTL and SPI using repeated two-way t-tests (p<0.05). Results

[0149] Phosphorylation (activation) of Endothelial Nitric Oxide Synthase [p-eNOS (Serll77)]:

[0150] The unique combinations of Bioptersides, but not native Spirulina (SPI) alone or highly purified isolates of specific Biopterside fractions alone (BIO-A or BIO-B), resulted in a significant increase in acute (3-hr) eNOS activation within human endothelial cells. Generally, eNOS activation may be positively correlated with the unique combination of Biopterside fractions and possibly affected by total Biopterside dose.

[0151] Table 7.1. Activation of human endothelial nitric oxide synthase (p-eNOS) by treatment

[0152] Note: CTL = Growth Supplement Only Control Treatment; SPI = CTL + 10.9 pg / mL Spirulina Treatment; BIO-ALO = CTL + 10.9 pg / mL Biopterside A Treatment; BIO- AHI = CTL + 86.8 pg / mL Biopterside A Treatment; BIO-BLO = CTL + 10.9 pg / mL Biopterside B Treatment; BIO-BHI = CTL + 86.8 pg / mL Biopterside B Treatment; BIO-ABLO = CTL + 10.9 pg / mL Biopterside A+B Treatment; BIO-ABHI = CTL + 86.8 pg / mL Biopterside A+B Treatment. All treatments were compared to CTL and SPI. * = significant up-regulation compared to CTL (p<0.05); f = significant up-regulation compared to SPI (p<0.05). *BIO- ABLO vs CT (p=0.0259); *BIO-ABHI vs CTL (p=0.0075); fBIO-ABLO vs SPI (p=0.0010); tBIO-ABHI vs SPI (p=0.0059).

[0153] Example 8: Investigation of Effects of Bioptersides on Nitric Oxide Synthase (eNOS) and NOx (Nitrite + Nitrate) concentrations in human endothelial cells (HUVECs). An in vitro randomized controlled trial was conducted to assess the vasodilation-inducing eNOS and NOx effects of the a) Biopterside source, b) total Biopterside dose (low vs high), c) concentration of specific Bioptersides present within the total Biopterside dose, and d) as compared against a positive control (L-Citrulline) and two additional control conditions - unprocessed source material (Spirulina) and untreated cells (Control). The study, titled Effects of Bioptersides v.y L-Citrulline and Spirulina on acute activation of vasodilation-inducing nitric oxide synthase (eNOS) and NOx (Nitrite + Nitrate) concentrations in human endothelial cells (HUVECs), was conducted at the Molecular & Applied Sciences Laboratory, Auburn University (Auburn, AL, USA).

[0154] Study Design

[0155] In vitro randomized controlled trial; acute response

[0156] Cell Type

[0157] Human umbilical vein endothelial cells (HUVECs)

[0158] Methods

[0159] 6-well plates / treatment; 30,000 HUVEC cells / well; R&D Systems HUVEC specific cell growth base medium w / endothelial growth supplement; treatments added to HUVEC media once ~80%-90% confluency (cell concentration per well) was achieved; 3-hr treatment time (i.e., acute response); Fluorometric (NOx) and SDS-PAGE Western blotting analysis using phosphor- eNos (Seri 177; 133 kD) antibody; t-tests vs Control, L-Citrulline, and Spirulina (p<0.05).

[0160] Cell Culturing

[0161] HUVECs (passage 5) were grown on 150 mm culture plates using endothelial cell culture media and accompanying growth factor to enhance the growth of HUVECs. Cells were subcultured on 23 treatment plates (6 wells per condition), and evenly seeded at 30,000 HUVEC cells per well. R&D Systems’ Endothelial Cell Growth Base Media (#390598) with Endothelial Cell Growth Supplement (#390599) were used for the expansion of HUVECs according to the suppliers’ procedures. Cultures were monitored daily and culture media changed every other day until ~80%-90% confluency was achieved per well (~1.5 weeks after seeding).

[0162] Treatments (n=23) Treatment media contained endothelial cell culture media and accompanying growth factors to enhance the growth of HUVECs as well as the designated treatment quantities per 20 mL stock solution that were pre-calculated for 22 ingredients (SPIRULINA HI had to be excluded due to solubility issues). A final concentration of 0.1% DMSO was used to help dissolve the constituents as a vehicle and, thus, was also included in all the treatments along with the CONTROL (untreated) cells.

[0163] 1. Growth Supplement Only (CONTROL)

[0164] 2. CONTROL + 52.557 pg / mL L-Citrulhne USP (CITRULLINE). See Tsuboi T et al. PLoS One 2018;13(2):e0192252 (PMID: 29415069; DOI: 10.1371 / journal.pone.0192252)

[0165] 3. CONTROL + 55309.522 pg / mL Spirulina (SPIRULINA LO)

[0166] 4. CONTROL + 94.325 pg / mL Biopterside A2 (BIOPTERSIDE-A2 HI)

[0167] 5. CONTROL + 11.791 pg / mL Biopterside A2 (BIOPTERSIDE- A2 LO)

[0168] 6. CONTROL + 84.270 pg / mL Biopterside B2 (BIOPTERSIDE-B2 HI)

[0169] 7. CONTROL + 10.534 pg / mL Biopterside B2 (BIOPTERSIDE-B2 LO)

[0170] 8. CONTROL + 89.298 pg / mL Biopterside A2 + B2 (BIOPTERSIDE-A2+B2 HI)

[0171] 9. CONTROL + 11.162 pg / mL Biopterside A2 + B2 (BIOPTERSIDE- A2+B2 LO)

[0172] 10. CONTROL + 3125.152 pg / mL Biopterside 01 (BIOPTERSIDE-01 20x HI)

[0173] 11. CONTROL + 390.644 pg / mL Biopterside 01 (BIOPTERS IDE-01 20x LO)

[0174] 12. CONTROL + 6273.919 pg / mL Biopterside 03.1 (BIOPTERSIDE-03.1 20x HI)

[0175] 13. CONTROL + 784.240 pg / mL Biopterside 03.1 (BIOPTERSIDE-03.1 20x LO)

[0176] 14. CONTROL + 3075.403 pg / mL Biopterside 04 (BIOPTERSIDE-04 20x HI)

[0177] 15. CONTROL + 384.425 pg / mL Biopterside 04 (BIOPTERS IDE-0420x LO)

[0178] 16. CONTROL + 2721.294 pg / mL Biopterside 05.1 (BIOPTERSIDE-05.1 20x HI)

[0179] 17. CONTROL + 340.162 pg / mL Biopterside 05.1 (BIOPTERSIDE-05.1 20x LO) 18. CONTROL + 3272.089 pg / mL Biopterside 07 (BIOPTERSIDE-07 20x HI)

[0180] 19. CONTROL + 409.011 pg / mL Biopterside 07 (BIOPTERSIDE-07 20x LO)

[0181] 20. CONTROL + 21673.833 pg / mL Biopterside 09.2 (BIOPTERSIDE-09.2 20x HI)

[0182] 21. CONTROL + 2709.229 pg / mL Biopterside 09.2 (BIOPTERSIDE-09.220x LO)

[0183] 22. CONTROL + 3404.877 pg / mL Biopterside 10 (BIOPTERSIDE- 1020x HI)

[0184] 23. CONTROL + 425.610 pg / mL Biopterside 10 (BIOPTERSIDE- 1020x LO)

[0185] SPIRULINA-LO and all BIOPTERSIDE Treatments standardized for Total Bioptersides ([BHn]). Dosing based upon: 1) 90% of orally consumed Biopterin and Dihydrobiopterin is absorbed and raises plasma BH4; 2) Oral Biopterin = 0.02948 ug / mL per mg / kg change in plasma BH4 response; and, 3) Minimal effective dose of 6R-BH4-2HC1 (77% BH4) = 5 mg / kg (or, 3.85 mg / kg BH4). Therefore, A) LO = 5 mg / kg * 0.77 = 3.85 mg / kg / 0.90 = 4.28 mg / kg * 0.02948 = 0.1261 ug / mL * 83.5 kg BM = 10.534 ug / mL [BHn]; B) HI = 40 mg / kg * 0.77 = 30.8 mg / kg / 0.90 = 34.22 mg / kg * 0.02948 = 1.0089 ug / mL * 83.5 kg BM = 84.241 ug / mL [BHn].

[0186] Dependent Variables (DVs)

[0187] Phosphorylation (activation) of Endothelial Nitric Oxide Synthase [p-eNOS (Seri 177)], and Total NOx (Nitrite + Nitrate) Concentration in Extracellular (background NOx in media + secreted NOx by HUVECs) and Intracellular (NOx within HUVECs).

[0188] Fluorometric and SDS-PAGE Western Blotting Analysis

[0189] After 3-hr treatments, media was removed from cells and banked at -80C for NOx analysis using fluorometric reagents (two-day procedure).

[0190] Cells were washed in lx phosphate buffered saline and lysed using general cell lysis buffer. Lysates were collected and banked at -80C for phosphor-eNOS analysis via Western blotting (four-day procedure). SDS-PAGE Western Blotting ensued and a Abeam Anti-eNOS (phosphor SI 177) antibody ab 184154 was used for interrogation.

[0191] Statistical Analysis All conditions were compared to growth supplement only (CONTROL), which only received HUVEC media and 0.1% DMSO. All conditions were also compared to L- CITRULLINE and native SPIRULINA (SPIRULINA-LO). Treatment responses were normalized across plates to account for gel covariance and then compared against CONTROL, L-CITRULLINE, and SPIRULINA-LO using repeated measures (paired or dependent samples) t-tests ( / ?<0.05).

[0192] Results

[0193] Effect on Phosphorylation (activation) of Endothelial Nitric Oxide Synthase [p- eNOS (Serll77)]

[0194] Many of the Biopterside compositions, but not native Spirulina (SPI) alone or L- Citrulline (L-CIT), resulted in a significant increase in acute (3-hr) eNOS activation within human endothelial cells. Efficacious dose threshold notwithstanding, the specific Biopterside composition appears to be more important than total Biopterside dose alone. Biopterside 05.1 LO elicited the most significant activation of eNOS vs CTL (+76.02%, / ?=0.020), vs L-CIT (+68.28%, / ?=0.018), and vs SPI (+83.00%, / ?=0.007); Biopterside 03.1 LO elicited the lowest variability (most consistent) activation of eNOS vs CTL (+38.44%, / ?=0.001), vs L-CIT (+32.36%, / ?=0.000), and vs SPI (+43.94%, / ?=0.001); and, Biopterside 09.2 appears to significantly decrease acute eNOS activation within HUVECs.

[0195] Table 8.1a. Activation of human endothelial nitric oxide synthase (p-eNOS) relative to total eNOS

[0196] Table 8.1b. Activation of human endothelial nitric oxide synthase (p-eNOS) relative to total eNOS

[0197] Note: All treatments were compared to CONTROL (CTL), CITRULLINE (L-CIT), and SPIRULINA LO (SPI). p<0.05 = significant up-regulation or down-regulation (bold values). / ?<(). 10 = trend toward significant up-regulation or down-regulation.

[0198] Effect on Total NOx (Nitrite + Nitrate) Concentration in Extracellular (background NOx in media + secreted NOx by HUVECs) and Intracellular (NOx within HUVECs)

[0199] Many of the Biopterside compositions, as well as native Spirulina (SPI) and L-Citrulline (L-CIT), resulted in a significant increase in acute (3-hr) Total NOx production within human endothelial cells (HUVECs) and secreted NOx by HUVECs.

[0200] For all Biopterside treatments, a positive dose-dependent response was observed; suggesting, that the greater the dose of total Bioptersides the greater the increase in Total NOx response.

[0201] Of the varied Bioptersides compositions, Biopterside 01 HI, 03.1 HI, and 04 HI were significantly more effective than CTL, L-CIT, and SPI [01 HI vs CTL (+3775.07%, / ?=0.000), vs L-CIT (+2484.13%, ^=0.000), vs SPI (+368.65%, =0.000); 03.1 HI vs CTL (+2398.43%, / 2=0.000), vs L-CIT (+1566.10%, 72=0.000), vs SPI (+202.16%, 72=0.000); 04 HI vs CTL (+2138.01%, 72=0.000), vs L-CIT (+1392.44%, 72=0.000), vs SPI (+170.67%, 72=0.000)]. The greatest percentage increase in intracellular NOx occurred in response to Biopterside 09.2 LO (59.15% of Total ANOx), 03.1 LO (26.04% of Total ANOx), 01 LO (25.90% of Total ANOx), 07 LO (21.81% of Total ANOx), 10 HI (21.16% of Total ANOx), and 05.1 HI (21.04% of Total ANOx).

[0202] Interestingly, L-CIT only appeared to affect extracellular NOx production. The lack of observed intracellular NOx response from L-CIT may be the result of the dose used or response timing (i.e., L-CIT may elicit a faster or delayed intracellular NOx response). The latter is the most probable explanation since the L-CIT dose used within the present investigation has previously been observed to affect NOx.

[0203] Table 8.2a. Total NOx (Nitrate + Nitrite) Response in Media + Cells

[0204] Table 8.2a. Total NOx (Nitrate + Nitrite) Response in Media + Cells

[0205] Note: All treatments were compared to CONTROL (CTL), CITRULLINE (L-CIT), and SPIRULINA LO (SPI). p<0.05 = significant up-regulation or down-regulation (bold values). / ?<(). 10 = trend toward significant up-regulation or down-regulation..

[0206] Example 9: Acute Effects of Biopterside Compositions on Resting and Exercise Metabolism and Performance in Healthy, Physically Active Adults

[0207] An in vivo randomized controlled, crossover trial; acute response pilot study to assess the acute effects of 2.0 grams of a novel Spirulina (Arthrospira platensis or Blue-Green Algae) extract (standardized to 1% Total Bioptersides) on markers of resting and exercise metabolism and performance when consumed by physically active, trained adults. Specifically, to determine if acute supplementation with a commercial energy drink [C4 Energy® (Woodbolt Distribution LLC, Austin, TX, USA); C4] or C4 Energy® + 20 mg Bioptersides [Biopterside™ (Dr Chris Lockwood LLC, Casper, WY, USA); C4+BHN] affects:

[0208] • Resting Energy Expenditure

[0209] • Fuel Oxidation

[0210] • Response to a Graded Exercise Test

[0211] Study Design

[0212] In vivo randomized controlled, crossover trial; acute response pilot study. Subjects

[0213] Adult (20-65 yrs), physically active (>3 d / wk of strenuous resistance and / or endurance training for the prior >6 months), men and women who regularly consume an average of >1 serving per day, but not >21 servings per week of caffeine-containing beverages, and are otherwise healthy and free of disease, illness, or injury, nor use medications that may adversely affect or be affected by the intervention or strenuous physical activity. Subjects must also not regularly use nicotine or cannabis-containing products, have a history of chronically abusing illicit drugs or alcohol, or misusing prescription or recreational drugs, not have been clinically diagnosed by a physician as having an allergy to any ingredients present within any of the study treatments, not currently a competitive athlete, and not be pregnant, suspect they may become pregnant, recently postpartum and still breastfeeding, or have reason believe they may become pregnant over the course of participation within the study. All subjects must be willing to comply with all of the controls and conditions for participation within the study, and sign and date a written Informed Consent prior to participation in the study.

[0214] Methods

[0215] This was a randomized, positively controlled, acute crossover design study, with repeated measures (Figure 7).

[0216] After completing an Informed Consent and a familiarization session, subjects reported to the laboratory for two (2) testing sessions; each session separated by 2 (±1) days.

[0217] During each of the two (2) testing sessions, subjects arrived to the laboratory under semifasted conditions (5-7 hrs fasted; water only) and after 24-hr abstinence from caffeine, other forms of stimulants, and having participated in no strenuous physical activity. Upon arrival to the laboratory, subjects were weighed to the nearest 0.01 kg on a digital clinical scale (DETECTO Apex® Digital Clinical Scale with Height Rod; Webb City, MO, USA), and hydration status validated using bioimpedance spectroscopy (SOZO® BIS; ImpediMed Inc., Carlsbad, CA, USA) and following the manufacturer’s operating instructions. [Lukaski HC et al. Nutrients 2019 ; 11(4): DOI: 10.3390 / nul 1040809; PMID: 30974817; Norman K et al. Clin Nutr 2012;31(6):854-61 DOI: 10.1016 / j.clnu.2012.05.008; PMID: 22698802; Tinsley GM et al. J Electr Bioimpedance 2023; 14(1):3-12 DOI: 10.2478 / joeb-2023-0002; PMID: 37416523] After hydration was verified, subjects consumed one (1) of two (2) treatment conditions in randomly assigned order: A) 16 fl oz commercial energy drink [C4; C4 Energy® (Woodbolt Distribution LLC; Austin, TX, USA)] or, B) C4 + 20 mg Biopterside™ [C4+BHN; as 1% Spirulina extract (Dr Chris Lockwood LLC; Casper, WY, USA)]. Subjects were allowed five (5) minutes to consume the treatment and then were required to remain sedentary for 30 minutes before beginning testing.

[0218] After the 30-minute latent period, subjects were fitted with a heartrate monitor (Polar H10, Polar Electro Oy; Kempele, Finland), headgear, and a one-way ventilated face mask connected to a metabolic cart through a sampling line (COSMED Quark QRMR, COSMED Sri; Rome, Italy). Subjects were instructed to sit comfortably in a chair with feet flat on the ground, breath normally while wearing the face mask, and to remain quiet and relaxed for 25 minutes, without falling asleep. Data collection consisted of a 5-minute calibration period and 20 minutes of active data collection under resting conditions. Breath-by -breath data was analyzed using 10- second rolling averaging. The 5-minute interval with the lowest variability (“best”), within the 20-minute active data collection period, was used for further analysis. The dependent variables (DVs) used for comparison were: Resting Energy Expenditure (REE; kcal / d); Respiratory Quotient (RQ; #); Oxygen Consumption (VO2; mL / min); Carbon Dioxide Expiration (VCO2; mL / min); Minute Ventilation (VE; L / min); Respiratory Frequency (Rf; breaths / min); Fat Oxidation Rate (FAT; kcal / d); Fat Oxidation for Total Energy Needs (FAT%; % of total kcals); Carbohydrate Oxidation Rate (CHO; kcal / d); and, Carbohydrate Oxidation for Total Energy Needs (CHO%; % of total kcals).

[0219] Immediately after REE testing, subjects were positioned on an electronically braked cycle ergometer (Monark LC7 TT novo, Monark Exercise AB, Vansbro, Sweden) while continuing to wear the heartrate monitor (Polar H10, Polar Electro Oy, Kempele, Finland), headgear, and oneway ventilated face mask connected to a metabolic cart through a sampling line. Subjects were instructed to pedal at a cadence of 70 ±10 RPMs. The test began with a 5-minute warmup at 50 W. Thereafter, the power increased by 30 W every one ( 1 ) minute ( 1 W every 2 seconds) until the participant could no longer maintain a cadence of >60 RPMs, the participant chose to stop the test, or all three (3) indicators of attaining a true maximum were observed by the investigator. Specifically, 1) 02 consumption (VO2) plateaus despite increasing workloads (VO2 increases by <150 mL O2*min-1 despite increasing workloads), 2) HR fails to rise with increasing workloads, and 3) respiratory exchange ratio (RER) > 1.15.[Liguori G (2021). ACSM’s Guidelines for Exercise Testing and Prescription (11th Ed.). Wolters Kluwer] All gas exchange data was collected using a COSMED Quark QRMR with OMNIA CPET Module open circuit spirometry (COSMED Sri, Rome, Italy). Twenty-second rolling average filtering was applied to all raw HR and breath-by-breath gas analysis of oxygen (02) and carbon dioxide (CO2), and then used to determine the highest absolute (mL / min) and relative (ml«kg-l«min-l) oxygen uptake (VO2peak) and other exercise-specific DVs. Time to exhaustion (TTE) was determined as the total exercise time, ending when the subject’s cadence fell below 60 RPMs. Maximal fat oxidation rate (MFO; g / min) will be calculated as the highest fat oxidation rate achieved during the GXT. To determine MFO, fat oxidation rate will be calculated using the stoichiometric equation (1.695 x VO2 (L / min)) - (1.701 x VCO2 (L / min)). Only data between the start of exercise to the point at which participants achieve 80% of their VO2peak will be used to determine subjects’ total fat oxidation (MFO; g). In addition, aerobic threshold (VT1) and ventilatory threshold (VT2) were determined. For VT1, the V-slope method was used where the VT1 is defined as the VO2 corresponding to the point of intersection of two separately derived regression line plots of the VO2 (L / min) and VCO2 (L / min). [Beaver WL et al. J Appl Physiol 1986;60(6):2020-7 DOI: 10.1152 / jappl.l986.60.6.2020; PMID: 3087938] VT2 was autodetected using the manufacturer’s software and determined to correspond to where 1) minute ventilation rate (VE) demonstrates inflection relative to VO2, and 2) a rise in VE / VO2 is observed without a concomitant increase in the subject’s VE / VCO2. The VO2 data points at which VT1 and VT2 are identified were recorded as VO2@VT1 and VO2@VT2. [Simon J et al. J Appl Physiol 1986;60(3):777-81 DOI: 10.1152 / jappl.l986.60.3.777; PMID: 3957830; Sued PJ et al. Int J Exerc Sci 2020;13(2):455-69 PMID: 32509109] Other DVs, include: Peak Power (W); Oxygen Uptake Efficiency Slope (OUES; mL / min / L / min), Oxygen Consumption per Work Rate (V02 / WR; mL / min / W); Tidal Volume (VT; L / breath); Respiratory Frequency (Rf; breaths / min); Peak Energy Expenditure (Peak EE; kcal / min); and duration of time maintained within VT1 and VT2 before phase shifts or termination of exercise, respectively.

[0220] At the end of each experimental visit subjects completed an Adverse Events (AEs) Questionnaire that included a list of nine (9) common AEs and an “Other” field. Subjects were asked to answer “YES” or “NO” if, during their visit, they experienced or were experiencing each of the AEs listed. For all “YES” responses, subjects were instructed to indicate how likely - “Possible”, “Likely”, or “Very Likely” - they believed the AE effects were due to the treatment that was consumed.

[0221] Treatments (n=2)

[0222] Treatment conditions were randomly assigned and consisted of a positive control (C4) and the positive control in combination with a novel dietary supplement ingredient (C4+BHN). Subjects consumed both treatment conditions over the duration of their involvement in the study. The two (2) possible orders for which treatment conditions were assigned for visits 2 and 3, were: AB or BA.

[0223] 1. 16 fl ozs C4 Energy (C4; or POSITIVE CONTROL) a. < 5 kcals, 0 g Total Carbohydrate (0 g Added Sugars), and contains the following active ingredients, in order of inclusion (by weight): Beta- Alanine (CarnoSyn®, Natural Alternatives International, Inc.; Carlsbad, CA, USA), L-Citrulline, Betaine (as anhydrous BetaPower®, International Flavors & Fragrance, Inc; New York, NY, USA), 200 mg Caffeine (as anhydrous), Tyrosine (as N-Acetyl-L-Tyrosine), 30 mg Niacin (as Niacinamide), and 6 mcg Vitamin B 12 (as Cyanocobalamin). Banks NF et al. (J Int Soc Sports Nutr 2024;21 : 1 DOI: 10.1080 / 15502783.2023.2297988; PMID: 38197606; ClinicalTrials.gov ID: NCT05559372) recently reported on the acute efficacy and cardiac safety response of C4 Energy versus Monster Energy® (Monster Energy Company; Corona, CA, USA) versus placebo within a similar population, utilizing a crossover design, and similar graded exercise testing methods as implemented herein.

[0224] 2. C4 + 20 mg Biopterside (C4+BHN) a. Biopterside was supplied to subjects as six (6) gelatin capsules, containing a total of 2.0 g Spirulina (Arthrospira platensis or Blue-Green Algae) extract (standardized to 1% Total Bioptersides) (Dr Chris Lockwood LLC; Casper, WY, USA). Results

[0225] Effect on Resting Energy Expenditure and Substrate Oxidation

[0226] Addition of 20 mg Biopterside to a commercial energy drink (C4+BHN) resulted in a + 12.1% increase in resting energy expenditure (REE) versus commercial energy drink alone (C4), which supporting evidence suggests was likely due to a rise in stimulatory catecholamines (epinephrine and norepinephrine). Specifically, in response to C4+BHN, fat oxidation increased +82.4%, carbohydrate oxidation decreased -28.9%, and respiratory frequency (Rf), which is generally regarded as being affected mostly by central factors, increased by +60.2% versus C4. Also, oxygen utilization (VO2) increased +13.9% and minute ventilation (VE) increased by + 18.0% in response to C4+BHN versus C4; however, carbon dioxide expiration (VCO2) only increased by +5.1% in response to C4+BHN versus C4, which suggests improved oxygen uptake and / or utilization efficiency, and increased utilization of lipids for fuel under resting conditions - improved mitochondria efficiency (FIG. 8).

[0227] Effect on Graded Exercise Testing (GXT; i.e., VO2max Test) Response

[0228] Acute supplementation with 20 mg Biopterside added to a commercial energy drink (C4+BHN) did not appreciably affect time to exhaustion (TTE), peak power output, peak oxygen utilization (VO2peak), peak energy expenditure (EEpeak), or either the aerobic energy threshold (VT1) or ventilatory threshold (VT2) when compared to supplementation with the commercial energy drink alone (C4). However, evidence suggests C4+BHN elicited a positive shift in metabolic efficiency during moderate-to-high intensity exercise; which could be explained by an increase in catecholaminergic signaling and Nitric Oxide (NO) mediated vasodilation. Also, subjects subjectively reported a greater “pumped” feeling during later stages of exhaustive exercise (high-to-supramaximal intensity), and immediately following cessation of exercise and during recovery in response to C4+BHN versus C4 alone (FIG. 9).

[0229] INCORPORATION BY REFERENCE

[0230] All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control. EQUIVALENTS

[0231] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect of the invention and other functionally equivalent embodiments are within the scope of the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention.

Claims

CLAIMSWe claim:

1. A composition, comprising about 0.5% to about 10% total bioptersides (BHn), wherein the total bioptersides (BHn) comprises a first biopterin (Bl) selected from biopterin xyloside (BX), biopterin glucoside (BGlc), and biopterin arabinoside (BA), or pharmaceutically acceptable salts thereof.

2. The composition of claim 1, wherein the first biopterin (B 1) is biopterin xyloside.

3. The composition of claim 1 , wherein the first biopterin (B 1) is biopterin arabinoside.

4. The composition of claim 1, wherein the first biopterin (B 1) is biopterin glucoside.

5. The composition of any one of claims 1-4, wherein the composition does not comprise additional biopterin glycosides (BG), or pharmaceutically acceptable salts thereof.

6. The composition of any one of claims 1-4, further comprising one or more additional biopterin glycosides (BG), or pharmaceutically acceptable salts thereof.

7. The composition of claim 6, wherein the ratio of B 1 :BG is about 12: 1 to about 6: 1.

8. The composition of claim 6, wherein the ratio of B1:BG is about 10: 1 to about 8:1.

9. The composition of claim 6, wherein the ratio of B 1 :BG is about 10:1.

10. The composition of claim 6, wherein the ratio of B1:BG is about 9.5:1.

11. The composition of claim 6, wherein the ratio of B 1 :BG is about 9: 1.

12. The composition of claim 6, wherein the ratio of B1:BG is about 8.5:1.

13. The composition of claim 6, wherein the ratio of B 1 :BG is about 8: 1.

14. The composition of any one of claims 1-13, further comprising one or more pharmaceutically acceptable excipients.

15. The composition of any one of claims 1-14, wherein the total bioptersides (BHn), biopterin xyloside (BX), biopterin glucoside (BGlc), biopterin arabinoside (BA), and, when present, the one or more additional biopterin glycosides (BG) were not derived from petroleum.

16. The composition of any one of claims 1-15, further comprising one or more additional components selected from the group consisting of folate, folic acid, 5-methyltetrahydrofolate, a compound derived from folate, folic acid, or 5-methyltetrahydrofolate, iron, one or more antioxidant compounds (e.g., Vitamin C, N-acetylcysteine (NAC), glutathione, and polyphenols), one or more dopaminergic compounds [e.g., tyrosine, phenylalanine, L-DOPA, Mucuna pruriens, Musa spp. (e.g., bananas and plantains), Persea americana (e.g., avocado), Citrus spp., eggs, dairy, animal organ tissue], caffeine, arginine, citrulline, ornithine, nitrates, vitamin B6, vitamin Bl 2, zinc, and colostrum.

17. The composition of claim 16, wherein the one or more additional components are selected from folate, folic acid, 5-methyltetrahydrofolate, and a compound derived from folate, folic acid, or 5-methyltetrahydrofolate.

18. The composition of claim 16, wherein the one or more additional components comprise iron.

19. The composition of claim 16, wherein the one or more additional components comprise one or more antioxidant compounds (e.g., Vitamin C, N-acetylcysteine (NAC), glutathione, and polyphenols).

20. The composition of claim 16, wherein the one or more additional components comprise one or more dopaminergic compounds [e.g., tyrosine, phenylalanine, L-DOPA, Mucuna pruriens, Musa spp. (e.g., bananas and plantains), Persea americana (e.g., avocado), Citrus spp., eggs, dairy, animal organ tissue].

21. The composition of claim 16, wherein the one or more additional components comprise caffeine.

22. The composition of claim 16, wherein the one or more additional components comprise arginine, citrulline, ornithine, or nitrates, or a combination thereof.

23. The composition of claim 16, wherein the one or more additional components comprise Vitamin B6.

24. The composition of claim 16, wherein the one or more additional components comprise Vitamin B12.

25. The composition of claim 16, wherein the one or more additional components comprise zinc.

26. The composition of claim 16, wherein the one or more additional components comprise colostrum.

27. A method of improving, increasing, enhancing, or augmenting: weight loss, fat metabolism, physical energy, cognitive energy (e.g., mental energy or working memory), circulation, blood flow, lean mass, mitochondrial function or health, endothelial function (e.g., vascular homeostasis), neurocognitive function, cardiovascular function, muscle recovery, recovery from physiological stress, muscle performance, exercise capacity, insulin sensitivity, metabolism, blood glucose control, fat loss, weight management, oxidative stress, thermogenesis, reaction times, neuromuscular function, nitric oxide biosynthesis, L-DOPA biosynthesis, serotonin biosynthesis, neurohormone biosynthesis, sexual performance, male erectile dysfunction; tyrosine, phenylalanine, arginine, citrulline, and / or tryptophan metabolism; endothelial nitric oxide synthase phosphorylation, mood disorders, or the attenuation or reduction of reactive oxygen species (ROS) and oxidative tissue damage (e.g., effects of aging or other forms of physiological stress), comprising administering to a subject in need thereof an effective amount of a composition, comprising an amount of total bioptersides (BHn), wherein the total bioptersides (BHn) comprises a first biopterin (Bl) selected from biopterin xyloside (BX), biopterin glucoside (BGlc), and biopterin arabinoside (BA), or pharmaceutically acceptable salts thereof.

28. The method of claim 27, wherein the method improves, enhances, or augments neurocognitive function.

29. The method of claim 27, wherein the method improves, enhances, or augments cardiovascular function.

30. The method of claim 29, wherein improving, enhancing, or augmenting cardiovascular function comprises increasing blood flow.

31. The method of claim 29, wherein improving, enhancing, or augmenting cardiovascular function comprises inducing vasodilation.

32. The method of claim 27, wherein the method improves, enhances, or augments muscle recovery.

33. The method of claim 27, wherein the method improves, enhances, or augments recovery from physiological stress.

34. The method of claim 27, wherein the method improves, enhances, or augments muscle performance.

35. The method of claim 27, wherein the method improves, enhances, or augments exercise capacity.

36. The method of claim 27, wherein the method improves, enhances, or augments insulin sensitivity.

37. The method of claim 27, wherein the method improves, enhances, or augments metabolism.

38. The method of claim 27, wherein the method improves, enhances, or augments blood glucose control.

39. The method of claim 27, wherein the method improves, enhances, or augments fat loss.

40. The method of claim 27, wherein the method improves, enhances, or augments weight management.

41. The method of claim 27, wherein the method reduces oxidative stress.

42. The method of claim 27, wherein the method improves, enhances, or augments thermogenesis.

43. The method of claim 27, wherein the method improves, enhances, or augments reaction times.

44. The method of claim 27, wherein the method improves, enhances, or augments neuromuscular function.

45. The method of claim 27, wherein the method improves, enhances, or augments nitric oxide biosynthesis.

46. The method of claim 27, wherein the method improves, enhances, or augments L-DOPA biosynthesis.

47. The method of claim 27, wherein the method improves, enhances, or augments serotonin biosynthesis.

48. The method of claim 27, wherein the method improves, enhances, or augments neurohormone biosynthesis.

49. The method of claim 27, wherein the method improves, enhances, or augments sexual performance.

50. The method of claim 27, wherein the method mitigates male erectile dysfunction.

51. The method of claim 27, wherein the method improves, enhances, or augments metabolism of one or more amino acids selected from tyrosine, phenylalanine, arginine, citrulline, and tryptophan.

52. The method of claim 27, wherein the method improves, enhances, or augments endothelial nitric oxide synthase phosphorylation.

53. The method of claim 27, wherein the method attenuates or reduces reactive oxygen species (ROS) and oxidative tissue damage (e.g., effects of aging or other forms of physiological stress).

54. The method of claim 27, wherein the method improves, enhances, or augments mood.

55. The method of any one of claims 27-54, wherein the composition comprises about 0.5% to about 10% total bioptersides (BHn), wherein the total bioptersides (BHn) comprises a first biopterin (Bl) selected from biopterin xyloside (BX), biopterin glucoside (BGlc), and biopterin arabinoside (BA), or pharmaceutically acceptable salts thereof.

56. The method of claim 55, wherein the composition further comprises one or more additional biopterin glycosides (BG), or pharmaceutically acceptable salts thereof.

57. The method of claim 56, wherein the B 1 :BG ratio is about 12: 1 to about 6: 1.

58. The method of any one of claims 27-57, wherein the composition is a composition of any one of claims 1-26.

59. A method of preparing the composition of any one of claims 1-26, comprising the steps of: contacting a precursor with an acid aqueous solution to form a mixture; agitating the mixture; filtering the mixture to obtain a filter cake and a filtrate solution; optionally adjusting the pH of the filtrate solution; and isolating the composition from the filtrate solution; wherein the precursor is selected from macroalgae, microalgae (e.g., cyanobacteria), blue-green algae, (Arthrospira) Spirulina (e.g., S. plantensis, Aphanizomenon flosaquae, Arthrospira maxima), Chlorella e.g., C. vulgaris, C. pyrenoidosa, C. ellpsiodea, C. stigmatophora, C. zofingiensis), Haematococcus (e.g., H. pluvialis) Tetraselmis / Tetrasellimis (e.g., T. chuii, T. suecica), Isochryisis (e.g., I. galbana), Nanochloropsis (e.g., N. gaditana, N. oculata, N. salina), Euglena (e.g., E. gracilis), Schizachyrium, Crypthecodinium, Chlamydomonas (e.g., C. reinhardtii), Dunaliella (e.g., D. salina), Scenedesmus, Phaeodactylum, Ochromonas, Prymnesium, Coccomyxa, Skeletonema, Arthronema, Porphyra, Chlorococcum, Fucus, Microcystis, Glaucocystophyte, Lyngbya, Phaeodactylum, Haslea, Pavlova, Thalassiosira, Chaetoceros, Amphora, Annochloropsis, Pelagophyceae, Lyngbya, Anabaena, Porphyridium, Odentella, Undaria, Synechocystis, Synechococcus, Nostoc, Oscillatoria, Cylindrotheca, royal jelly, animal liver (e.g., beef liver), spinach, velvet bean (mucuna pruriens), fava bean (viciafaba), english broom (cytisus scoparius), banana, commonpurslane, Mexican rose, black caraway, black bean, soybean, watercress, water lotus, Buffalo gourd, pumpkin, acorn squash, sunflower, mung bean, black mustard, carrot, Acetobacter xyliunum, Achromobacter liquidiim, Aerobacter aerogenes, Aeromonas hydrophilia, Bacillus spp. (e.g., B. alvei, B. cereus, B. coagulans, B. roseus, B. sphaericus), Brevibacterium acetylicum, Corynebacterium equi, Crithidia fasciculata, Erwinia carotovora, Eschericia coli, Flavobacterium aquatile, Gluconobacter spp (e.g., G. melanogenus, G. suboxydans), Lactobacillus casei, Micrococcus spp. e.g., M.flavus, M. luteus, M. lysodeikiticus, M. ureae), Pseudomonas aeruginosa, P. aureofaciens, P. convexa, P. fluorescens, P. putrefaciens, Serratia spp. (e.g., S. indica, S. marcescens), Streptococcus faecalis, insects (e.g., fruit fly, silkworm, honey bee), insect cocoons, and a combination of any of them.

60. The method of claim 59, wherein the precursor is a cyanobacterium.

61. The method of claim 60, wherein the cyanobacterium is (Arthro spiro) Spirulina.

62. The method of any one of claims 59-61, wherein the precursor is not derived from petroleum.

63. The method of any one of claims 59-62, wherein the acid aqueous solution comprises a Bronsted acid (e.g., HC1, HBr) and deionized water.

64. The method of claim 63, wherein the acid aqueous solution is about 0.1 N to about 2 N HC1.

65. The method of any one of claims 59-64, wherein the mixture has a pH of about 1.5 to about 6.9.

66. The method of any one of claims 59-65, wherein agitating the mixture comprises mechanical stirring.

67. The method of any one of claims 59-66, wherein agitating the mixture comprises sonic agitation.

68. The method of any one of claims 59-67, further comprises heating the mixture while agitating the mixture.

69. The method of any one of claims 59-67, wherein the mixture is agitated at about ambient temperature.

70. The method of any one of claims 59-69, comprising the step of adjusting the pH of the filtrate solution.

71. The method of claim 70, wherein the pH of the filtrate solution is adjusted to about 4.0 to about 8.0.

72. The method of claim 71, wherein the pH of the filtrate solution is adjusted to about 6.

73. The method of claim 71, wherein the pH of the filtrate solution is adjusted to about 7.

74. The method of claim 71, wherein the pH of the filtrate solution is adjusted to about 8.

75. The method of any one of claims 59-74, wherein isolating the composition from the filtrate solution comprises adding a second solvent to the filtrate solution.

76. The method of claim 75, wherein the second solvent is a food grade solvent selected from ethyl acetate, methanol, ethanol, and 2-propanol.

77. The method of any one of claims 59-76, wherein isolating the composition comprises recrystallization, reverse osmosis, lyophilization, spray drying, removing solvent from the filtrate solution, or any combination thereof.

78. The method of any one of claims 59-77, wherein isolating the composition comprises removing solvent from the filtrate solution.

79. The method of any one of claims 59-78, wherein isolating the composition comprises heating the filtrate solution under reduced pressure.

80. The method of any one of claims 59-79, wherein filtering the mixture comprises tangential flow filtration, dead-end filtration, nanofiltration, reverse osmosis, or a combination thereof.

81. The method of any one of claims 59-80, wherein filtering the mixture comprises ultrafiltration.

82. The method of claim 81 , wherein the ultrafiltration comprises passing the mixture through a 1 kDa to 100 kDa ultrafiltration membrane.

83. The method of any one of claims 59-82, wherein isolating the composition further comprises contacting the mixture with a chromatography medium (e.g., an ion exchange resin, ion exchange membrane, or ion exchange column).

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