Chlorella and corn leaf extract compositions, methods of their production and use

Abstract

Compositions are disclosed that include a plant extract standardized for an amount of benzoxazinoid, and further include chlorella. In contemplated embodiments, these compositions are provided in an amount effective to reduce sleep onset latency, improve sleep quality and efficiency, and prevent and treat sleep disorders.

Description

[Technical Field]

[0001] The subject matter disclosed herein includes chlorella and corn leaf extract compositions, along with methods of their production and use, including their use to reduce sleep latency and improve sleep quality. [Background technology]

[0002] Poor sleep quality, manifested in the form of increased sleep onset latency, decreased deep sleep duration, decreased deep sleep time and total sleep time, fragmented nighttime sleep, frequent nighttime awakenings, and increased wakefulness time after sleep onset, are important factors for adverse effects on psychological state and mental health. Clinically validated dietary supplements with known modes of action can be considered as a preferable alternative to conventional pharmaceuticals due to their safety.

[0003] Chlorella is a type of green algae that has gained popularity as a dietary supplement due to its rich nutrient content. It is primarily known for its potential benefits for overall health and has been implicated in sleep support, but there is no substantial scientific evidence specifically linking chlorella to improvements in any particular sleep latency, sleep duration, or sleep quality.

[0004] On the other hand, Zea mays (corn) leaf extract standardized with 6-MBOA is known to increase deep sleep time and total sleep time in humans, and Zea mays (corn) leaf extract increases the incidence of sleep onset and sleep time in animals, but is not known to shorten sleep latency in either case.

[0005] Melatonin, known to shorten sleep latency, is synthesized from tryptophan. Therefore, the amount of melatonin synthesized depends on the amount of tryptophan present. 6-MBOA is known to activate AANAT, an enzyme involved in melatonin synthesis. Therefore, when tryptophan is depleted, the melatonin synthesis pathway is halted, resulting in a decreased level of sleepiness. Therefore, increasing tryptophan consumption prolongs the duration of melatonin's effects. Chlorella's dry weight is made up of more than 60% protein, which is rich in essential amino acids, with a tryptophan content of 1.1%. Furthermore, it is rich in micronutrients that play a role in sleep. For example, vitamin B12, a regulator of circadian rhythms, folic acid, a coenzyme in the melatonin synthesis pathway, and carotenoids and PUFAs, which regulate sleep, are listed (Non-Patent Documents 1 and 2). In particular, vitamin B12 is lacking in plant foods, and chlorella is known as a plant ingredient rich in vitamin B12. Due to the nutritional profile of chlorella, consuming chlorella may be complementary or synergistic with getting adequate sleep. Summary of the Invention [Problem to be solved by the invention]

[0006] To the inventors' knowledge, this is the first time that chlorella alone (the contemplated material described herein) or in combination with Zea mays (corn) leaf extract standardized with 6-MBOA has been investigated to demonstrate scientific evidence of a sleep support effect. [Means for solving the problem]

[0007] (Summary of the Invention) Disclosed and discussed herein are compositions comprising plant extracts standardized for the amount of benzoxazinoids, the compositions further comprising chlorella.

[0008] In contemplated embodiments, these compositions are provided in amounts effective to reduce sleep onset latency, improve sleep quality and efficiency, and prevent and treat sleep disorders.

[0009] In other contemplated embodiments, the plant extract standardized for the amount of benzoxazinoids is selected from the group consisting of Zea mays, Chlorella sorokiniana, Chlorella vulgaris, Oryza species, Oryza sativa, Oryz glaberrima, Oryz australiensis, Oryz brachyantha, Secale cereale, Acanthus arboreus, Acanthus ebracteatus, Acanthus illicifolius, Acanthus mollis, Avena sativa, and the like. sativa, Avena abyssinica, Avena byzantine, Avena nuda, Avena strigosa, Hordeum vulgare, Coix lachryma-jobi, Triticum aestivum, Triticum compactum, Triticum sphaerococcum, Triticum turanicum, Sorghum bicolor, Agropyron repens, Blepharis edulis edulis), Balsamocitrus paniculate; Peristrophe roxburghiana; Strobilanthes cusia;Extracted, concentrated, and standardized from a plant species selected from the group consisting of Lamium galeobdolon, Lobelia chinensis, Leymus chinensis, Aphelandra spp., Scoparia dulcis, Capparis sikkimensis ssp., or a combination thereof; [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 shows a contemplated tablet containing chlorella and zea mays (corn) leaf extract, as described in Example 14. DETAILED DESCRIPTION OF THE INVENTION

[0011] Disclosed and discussed herein are compositions comprising plant extracts standardized for amounts of benzoxazinoids, which further comprise chlorella. In contemplated embodiments, these compositions are provided in amounts effective to reduce sleep onset latency, improve sleep quality and efficiency, and prevent and treat sleep disorders.

[0012] In other contemplated embodiments, the plant extract standardized for the amount of benzoxazinoids is selected from the group consisting of Zea mays, Chlorella sorokiniana, Chlorella vulgaris, Oryza spp., Oryza sativa, Oryza glaberrima, Oryza australiensis, Oryza brachyantha, Secare cereale, Acanthus arboreus, Acanthus ebractatus, Acanthus iliquifolius, Acanthus mollis, Avena sativa, Avena abyssinica, Avena byzantine, Avena nudum, Avena strigosa, Hordeum vulgare, Coix lacrima-yobis , Triticum aestium, Triticum compactum, Triticum sphaerococcum, Triticum tulanicum, Sorghum bicolor, Agropyron repens, Blepharis edulis, Balsamocitrus paniculate; Peristrophe roxburghiana; Strobilanthes custardii; Ramium galeobdron, Lobelia chinensis, Rhamus chinensis, Aphelandra spp., Scoparia dulcis, Capparis sikkimensis spp., or combinations thereof, which are extracted, concentrated, and standardized from plant species selected from the group consisting of Triticum aestium, Triticum compactum, Triticum sphaerococcum, Triticum tulanicum, Sorghum bicolor, Agropyron repens, Blepharis edulis, Balsamocitrus paniculate; Peristrophe roxburghiana; Strobilanthes custardii; Ramium galeobdron, Lobelia chinensis, Rhamus chinensis, Aphelandra spp., Scoparia dulcis, Capparis sikkimensis spp., or combinations thereof.

[0013] Specifically, as disclosed herein, compositions containing zea mays leaf extract and the green algae chlorella have a synergistic effect in improving sleep latency and sleep quality. In a contemplated embodiment and as disclosed herein, a combination of chlorella and zea mays (corn) leaf extract standardized with 0.2% or more 6-MBOA demonstrated a statistically significant reduction in sleep onset latency, demonstrating the unexpected synergistic effect of combining these two ingredients. Sleep onset latency was reduced to 33.0 + / - 8.9 minutes when combined with 6-MBOA-standardized zea mays (corn) leaf extract and chlorella, compared to 44.0 + / - 2.1 minutes and 35.4 + / - 16.0 minutes when administered alone. This combination demonstrated a 25% and 6.8% reduction in the time it took to fall asleep compared to 6-MBOA-standardized zea mays (corn) leaf extract or chlorella administered alone, respectively. These complementary effects of 6-MBOA-standardized corn leaf extract and chlorella were also demonstrated by a 44.4% reduction in standard deviation, minimizing inter-individual benefit variability, when the combination was administered compared to either 6-MBOA-standardized corn leaf extract or chlorella alone. The reduction in sleep onset latency was also compared to melatonin as a positive control, and the combination of zea mays (corn) leaf extract standardized with 6-MBOA (250 mg / kg) and chlorella (5000 mg / kg) was found to result in a statistically significant reduction in sleep onset latency when compared to melatonin.

[0014] An unexpected synergistic effect was observed when zea mays (corn) leaf extract standardized with 0.2% or more 6-MBOA was combined with chlorella, increasing the incidence of sleep onset. Administration of chlorella alone at 1000 mg / kg to mice resulted in a statistically insignificant 47% sleep incidence. Meanwhile, adding 250 mg / kg of 6-MBOA-standardized corn leaf extract to chlorella at this dose resulted in a statistically significant 53% sleep incidence. At the highest dose tested, sleep incidence was 60% with chlorella and 53% with 6-MBOA-standardized zea mays (corn) leaf extract. This percentage increased to 73% with the addition of 6-MBOA-standardized zea mays (corn) leaf extract, highlighting the unexpected synergistic effect of these ingredients. Zea mays (corn) leaf extract standardized with 6-MBOA and chlorella increased sleep incidence by 20% and 13%, respectively, compared with their combination. The sleep incidence response for the chlorella group was not dose-related when administered alone at 1000, 2000, or 5000 mg / kg. The highest sleep incidence (67%) was observed at the mid-dose (2000 mg / kg). Addition of zea mays (corn) leaf extract standardized with 6-MBOA at 250 mg / kg resulted in dose-related sleep incidence responses of 53%, 60%, and 73%.

[0015] An inverse correlation between sleep duration and chlorella dose was observed. The longest sleep duration was observed with the low dose of chlorella. The addition of 6-MBOA-standardized zea mays (corn) leaf extract did not further increase sleep duration. A range of 59.6 to 62.6 minutes of sleep duration was observed with the combination of chlorella and 6-MBOA-standardized zea mays (corn) leaf extract. These findings were within the range observed with the high dose of chlorella (5000 mg / kg) or 6-MBOA-standardized zea mays (corn) leaf extract (250 mg / kg) administered alone, which were 61.6 ± 16.3 minutes and 63.5 ± 10.8 minutes, respectively. Adding 6-MBOA-standardized corn leaf extract to chlorella resulted in sleep durations equivalent to those observed in melatonin-treated mice at all doses. Zea mays (corn) leaf extract standardized with 6-MBOA produced similar sleep duration as melatonin by itself. Zea mays (corn) leaf extract standardized with 6-MBOA has been clinically proven to improve sleep quality. Although sleep stages were not determined in this study, the effect of zea mays (corn) leaf extract standardized with 6-MBOA is more on sleep quality than quantity.

[0016] Chlorella has been reported to have anecdotal sleep-supporting properties. This effect may possibly be related to tryptophan levels. The lowest dose of tryptophan as a dietary supplement reported to affect sleep was 250 mg / day in humans (Sutanto et al., 2022). According to FDA guidelines, this equates to a human dose of 4.2 mg / kg and a mouse dose of 50 mg / kg (Guidance for Industry: Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers; Nair and Jacob 2016). Therefore, for a 30 g mouse, 1.5 mg of tryptophan would be required to observe sleep-related benefits. In the current study, the tryptophan level in chlorella is 1.1 g / 100 g (i.e., 1.1%). Therefore, only the highest dose tested (5000 mg / kg) is expected to produce positive results on sleep (5000 mg / kg x 0.03 kg = 150 mg chlorella or 1.5 mg tryptophan). However, the addition of zea mays (corn) leaf extract standardized with 6-MBOA significantly improved the effects of lower doses, i.e., 1000 mg / kg and 2000 mg / kg chlorella. This is due to the unique MOA of zea mays (corn) leaf extract standardized with 6-MBOA, which is known to reduce the activity of the TDO enzyme, liberating more starting material (tryptophan) and increasing the activity of the rate-limiting enzyme TH in melatonin biosynthesis. Even at the highest dose (5000 mg / day for human use) in current preparations of chlorella, tryptophan levels are only 50 mg / day. This is significantly (5-fold) lower than the expected tryptophan level of 250 mg that would have any effect on physiological sleep in humans ( Sutanto et al., 2022 ).The addition of Zea mays (corn) leaf extract standardized to 0.2% or more 6-MBOA boosted the efficacy of Chlorella by preserving available tryptophan as a starting material and facilitating melatonin biosynthesis.

[0017] The unexpected synergistic effect of combining Chlorella and Zea mays (corn) leaf extract standardized with 6-MBOA in enhancing sleep parameters also stems from the 6-MBOA-standardized Zea mays (corn) leaf extract's ability to increase tryptophan levels through a reduction in cortisol. Glucocorticoids, such as cortisol, are known to induce the TDO enzyme, leading to hepatic degradation of tryptophan and reducing the amount of tryptophan available for melatonin conversion (Altman and Greengard, 1966). Clinically, 6-MBOA-standardized Zea mays (corn) leaf extract has been shown to significantly reduce early morning salivary cortisol levels. Therefore, 6-MBOA-standardized Zea mays (corn) leaf extract can directly inhibit the TDO enzyme and indirectly inhibit it through a reduction in cortisol, with the net result of increased tryptophan availability from Chlorella consumption for serotonin and melatonin biosynthesis.

[0018] Zea mays (corn) leaf extract standardized for 6-MBOA content of 0.2% or greater has previously been shown to improve sleep quality by improving deep sleep duration and reducing the stress hormone cortisol. Zea mays (corn) leaf extract did not demonstrate a reduction in sleep onset latency in either preclinical in vivo studies or human clinical trials. Chlorella, a rich source of nutrients and micronutrients, has been suggested for use in sleep support, although this has not been demonstrated. As disclosed herein, chlorella and zea mays (corn) leaf extract standardized for 6-MBOA were combined in different ratios and tested in a pentobarbital-induced sleep test in mice. The combination of Chlorella and Zea mays (corn) leaf extract standardized with 6-MBOA demonstrated a) a statistically significant reduction in sleep onset latency when compared to the individual components; b) a reduction in the standard deviation of sleep latency by 44.4%, minimizing inter-individual variability in benefit; c) a statistically significant reduction in sleep onset latency when compared to melatonin (used as a positive control compound); and d) an unexpected synergistic effect in increasing the incidence of sleep onset, demonstrating the synergistic benefit of combining these two ingredients. The Chlorella and Zea mays (corn) leaf extract standardized with 6-MBOA composition also increased the incidence of sleep and total sleep time.

[0019] Chlorella is a genus of eukaryotic, unicellular green algae found in freshwater, marine, and soil habitats (Bock, Krienitz, and Proschold 2011). Identification of species within this genus has historically relied on morphology and nutritional requirements, but more precise identification using sequencing techniques is preferred (Huss et al. 1999).

[0020] C. sorokiniana was first isolated from a stream in Austin, Texas, in 1951 (Bock, Krienitz, and Proschold 2011). Although C. vulgaris and C. sorokiniana are morphologically identical, C. sorokiniana can be separated from C. vulgaris by hydrogenase activity, preference for warmer growth temperatures, and rRNA gene sequencing (Huss et al. 1999).

[0021] Viable C. sorokiniana CK-22 cells are spherical to slightly ovoid, 3-6 μm in diameter, and have thin cell walls. C. sorokiniana CK-22 possesses cup-shaped chloroplasts with pyrenoids coated by starch granules. These morphological characteristics also correspond to those of C. vulgaris. C. sorokiniana CK-22 was originally identified as C. vulgaris CK-22 by morphology, but after 18S rRNA sequencing, it has been redesignated as C. sorokiniana.

[0022] As used herein, Chlorella powder is spray-dried whole cell biomass of C. sorokiniana CK-22, as used in contemplated embodiments with its production-scale cultivation disclosed in Examples 5 and 6 and characterization disclosed in Example 7.

[0023] In a contemplated embodiment, chlorella serves as a source of tryptophan, which benefits the biosynthesis of melatonin. Chlorella is a type of green algae that is often consumed as a dietary supplement, known for its rich nutritional profile, including high levels of protein (Example 7, Table 5). Tryptophan is one of the essential amino acids found in chlorella protein (Example 7, Table 6). As an essential amino acid, tryptophan cannot be synthesized by the human body and must be obtained through diet (e.g., chlorella supplements). Tryptophan serves as a precursor for the synthesis of serotonin and melatonin, and plays a role in mood regulation and the sleep-wake cycle.

[0024] The use of physiological agents as sleep aids has been reported as an alternative to traditional drugs. Because natural metabolic pathways already exist for these sleep-regulating physiological agents, their use may reduce the risk of unwanted side effects and toxicity. Tryptophan is one such physiological agent and can be obtained from protein-rich foods and supplements, such as chlorella. However, the effects of tryptophan on sleep are mixed; one major concern regarding tryptophan's efficacy is the high daily dosage required. While 250 mg of tryptophan can improve sleep, daily doses of 1–5 g of tryptophan have more consistent and positive effects on sleep (Sutanto et al., 2022).

[0025] After ingestion, tryptophan undergoes several chemical reactions before its conversion to serotonin and melatonin. Tryptophan dioxygenase (TDO) is an enzyme that catalyzes the first and rate-limiting step of the kynurenine pathway, the major pathway for tryptophan metabolism (Davis and Liu 2015). TDO is primarily found in the liver and regulates tryptophan levels in the body. The activity of TDO is influenced by factors such as tryptophan levels (chlorella supplementation increases tryptophan levels) and hormones, including glucocorticoids such as cortisol (cortisol is known to induce TDO; therefore, when cortisol levels decrease, tryptophan levels increase) (Jovanovic et al. 2023). Therefore, inhibiting the activity of this rate-limiting enzyme generates excess tryptophan as a starting material for the biosynthesis of serotonin and melatonin. Indeed, zea mays (corn) leaf extract standardized with 6-MBOA is known to inhibit TDO both directly and through a reduction in the stress hormone cortisol. These coordinated effects of zea mays (corn) leaf extract standardized with 6-MBOA and chlorella are important mechanisms working together for contemplated embodiments to result in reduced sleep onset latency, sleep promotion, and improved sleep quality.

[0026] A previously filed and commonly owned U.S. patent application, US 2022 / 0387366 A1, entitled "Composition for regulating homeostasis of cortisol and improving sleep quality and method of use and production thereof," is incorporated herein by reference. The 6-MBOA-standardized Zea mays (corn) leaf extract disclosed in Examples 1 and 2 herein is enriched and standardized for one or more benzoxazinoids, 6-MBOA, as contemplated herein. The contemplated benzoxazinoids isolated from corn shoot or immature corn leaf powder are extracted with any suitable solvent, including water, methanol, ethanol, acetone, alcohol, solvents mixed with water, or combinations thereof, or with supercritical fluids. In contemplated embodiments, the corn shoot or immature corn leaf extract contains from about 0.01% to about 99.9% benzoxazinoids.

[0027] Benzoxazinoids, such as 6-MBOA, can be used to treat various plant diseases, including but not limited to: maize, wheat, rye, rice, barley, oats, cereals, Job's tears, Sorghum and other plants, such as Zea mays, Oryza spp., Oryza sativa, Oryza glaberrima, Oryza australiensis, Oryza brachyantha, Secale cereale, Acanthus arboreus, Acanthus iliquifolius, Avena sativa, Avena abyssinica, Avena byzantine, Avena nudum, Avena strigosa, Hordeum vulgare, and others, as demonstrated in Examples 3 and 4. It is contemplated that the plant may be derived, obtained or selected from the seedlings and all plant parts of at least one of Um vulgare, Coix lacrima-jobi, Triticum aestium, Triticum compactum, Triticum sphaerococcum, Triticum tulanicum, Sorghum bicolor and Balsamocitrus paniculate; Peristrophe roxyburgiana; Strobilanthes custia; Scoparia dulcis, Lobelia chinensis, Reims chinensis, the marine sponge Oceanapia genus, alone or in combination with each other.

[0028] Compositions enriched with one or more benzoxazoles may be applied to, but are not limited to, seedlings, shoots, germination from plant seeds, germinated seed sprouts, immature leaves, mature leaves, whole plants, roots, seeds, flowers, stems, stem bark, root bark, silk, grains, germinated grain root hairs or fine roots, stems, cell cultures or tissue cultures or any combination thereof, corn, wheat, rye, rice, barley, oats, cereals, Job's tears, Sorghum plants, and other plants and, but not limited to, Zea mays, Oryza spp., Oryza sativa, Oryza glaberrima, Oryza australiensis, Oryza brachyantha, Secale cereale, Acanthus arboreus; Acanthus ebractatus, Acanthus iliquifolius , Acanthus mollis, Avena sativa, Avena abyssinica, Avena byzantine, Avena nudda, Avena strigosa, Hordeum vulgare, Coix lacrima-jobi, Triticum aestium, Triticum compactum, Triticum sphaerococcum, Triticum tulanicum, Sorghum bicolor, Agropyron repens, Blepharis edulis, Balsamocitrus paniculate; Peristrophe roxburghiana; Strobilanthes cuscia; Lamium galeobdron, Lobelia chinensis, Reims chinensis, Aphelandra species, Scoparia dulcis, Capparis sikkimensis species, fungal species Monocillium species (Monocillium The present invention may be obtained, derived or extracted from any suitable source or sources, including the marine sponge Oceanapia sp., or a combination thereof.

[0029] Contemplated compositions enriched with one or more benzoxazoles can be synthesized, metabolized, biodegraded, biotransformed, biotransformed, or biosynthesized from small carbon units by transgenic microorganisms, by P450 enzymes, by glycosyltransferases or combinations of enzymes, or by microbacteria.

[0030] Contemplated compositions include one or more benzoxazoles in the composition in combination with chlorella that improve sleep quality by decreasing sleep latency, increasing sleep incidence, enhancing deep sleep stages of sleep, increasing total sleep time and deep sleep time, improving REM and NREM sleep, improving overall mental well-being as measured by the Pittsburgh Sleep Quality Index (PSQI) and Profile of Mood States (POMS), providing positive mood support, enhancing emotional well-being; and maintaining homeostasis of biomarkers such as serotonin, melatonin, and GABA in mammals.

[0031] Contemplated compositions include a standardized benzoxazole extracted from Zea mays leaf, present in the composition in combination with chlorella, for preventing and treating sleep disorders, including, but not limited to, insomnia, hypersomnia, jet lag, circadian rhythm disorders, shift work sleep disorder, non-24-hour sleep-wake disorder, periodic limb movement disorder, restless legs syndrome (RLS), sleep apnea, narcolepsy, parasomnia, night terrors, sleepwalking, nightmares, sleep-related eating disorder, sleep hallucinations, sleep paralysis, sleep talking, and REM sleep behavior disorder.

[0032] Contemplated compositions, wherein one or more benzoxazoles in the composition in combination with chlorella establish and regulate homeostasis of the host stress hormone cortisol and improve symptoms of chronically high cortisol, including, but not limited to, anxiety, depression, fatigue, digestive upset such as constipation, bloating, or diarrhea, headaches, heart disease, high blood pressure, irritability, memory and concentration problems, decreased libido, erectile dysfunction or reproductive issues such as irregular menstruation and ovulation, difficulty sleeping, delayed recovery from exercise, eating disorders and weight gain, hot flashes, and other menopause-related mental and health conditions.

[0033] In the above and following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments of the present disclosure. However, those skilled in the art will understand that the contemplated embodiments may be practiced without these details.

[0034] In this description, any concentration range, percentage range, ratio range, or integer range is understood to include any integer and fraction thereof (e.g., tenths and hundredths of integers, etc.) within the recited range, unless otherwise indicated. Also, any numerical range recited herein for any physical characteristic, such as polymer subunits, size, or thickness, is understood to include any integer within the recited range, unless otherwise indicated. As used herein, the terms "about" and "consist essentially of" mean ±20% of the recited range, value, or structure, unless otherwise indicated. As used herein, the terms "a" and "an" should be understood to refer to "one or more" of the recited components. The use of alternatives (e.g., "and / or") should be understood to mean either one of the alternatives, both, or any combination thereof. Unless the context requires otherwise, throughout this specification and claims, the word "comprise" and variations thereof, such as "comprises" and "comprising," and cognate words such as "include" and "have," and variations thereof, are to be interpreted in an open and inclusive sense; that is, as meaning "including, but not limited to."

[0035] References throughout this specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one of the embodiments contemplated. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment.

[0036] "Stable compound" and "stable structure" are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.

[0037] "Biomarker" or "marker" constituent or compound is meant to refer to one or more specific chemical constituents or compounds in the disclosed plants, plant extracts, or combination compositions with two or three plant extracts that are utilized to control the quality, consistency, integrity, stability, and / or biological function of the compositions of the present invention. Quality marker compounds may not be bioactive compounds directly related to the intended method of use.

[0038] "Mammal" includes humans as well as domestic animals, such as laboratory animals or household pets (e.g., cats, dogs, pigs, cows, sheep, goats, horses, rabbits) and non-domestic animals, such as wildlife.

[0039] "Optional" or "optionally" means that the subsequently described element, component, event, or circumstance may or may not occur, and that the description includes instances where the element, component, event, or circumstance occurs and instances where it does not occur. For example, "optionally substituted aryl" means that the aryl group may be substituted or unsubstituted, and that the description includes both substituted aryl groups and aryl groups having no substituents.

[0040] A "pharmaceutically or nutritionally or nutraceutical acceptable carrier, diluent or excipient" includes any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent or emulsifier approved by the U.S. Food and Drug Administration as acceptable for human or veterinary use.

[0041] "Pharmaceutically, nutritionally, or nutraceutical acceptable salts" includes both acid addition salts and base addition salts. "Pharmaceutically, nutritionally, or nutraceutical acceptable acid addition salts" refers to salts that retain the biological effectiveness and properties of the free base and are not biologically or otherwise undesirable, as well as salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and organic acids such as acetic acid, ascorbic acid, aspartic acid, cinnamic acid, citric acid, formic acid, fumaric acid, lactic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, and the like.

[0042] "Pharmaceutically, nutritionally, or nutraceutical acceptable base addition salts" refer to salts that retain the biological effectiveness and properties of the free acids and are not biologically or otherwise undesirable. These salts are prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. In certain embodiments, the inorganic salt is an ammonium, sodium, potassium, calcium, or magnesium salt. Salts derived from organic bases include salts of primary, secondary, and tertiary amines, salts of substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Particularly useful organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0043] Crystallization often produces solvates of the compounds of the present disclosure.As used herein, the term "solvate" refers to an aggregate comprising one or more molecules of the compounds of the present disclosure together with one or more molecules of solvent.The solvent may be water, in which case the solvate may be a hydrate.Alternatively, the solvent may be an organic solvent.Therefore, the compounds of the contemplated embodiments may exist as hydrates, including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, etc., as well as corresponding solvated forms.The compounds of the present disclosure may be true solvates, but in other cases, the compounds of the present disclosure may simply retain adventitious water or may be a mixture of water and some adventitious solvent.

[0044] A "pharmaceutical composition" or "nutraceutical composition" or "functional food composition" refers to a formulation of a compound of the present disclosure and a biologically active compound in a vehicle generally accepted in the art for delivery to a mammal, e.g., a human. For example, a pharmaceutical composition of the present disclosure can be formulated or used as a stand-alone composition or as a prescription drug, over-the-counter drug (OTC), botanical, herbal medicine, natural medicine, homeopathic medicine, or any other form of health care product reviewed and approved by a government agency. An exemplary nutraceutical or functional food composition of the present disclosure can be formulated or used as a stand-alone composition or as a nutritional or bioactive ingredient in a food, functional food, beverage, bar, RTD, sachet, food flavoring, medical food, dietary supplement, or herbal product. A vehicle generally accepted in the art includes any carrier, diluent, or excipient that is pharmaceutically, nutritionally, or functionally acceptable.

[0045] As used herein, "enriched with" refers to a plant extract or other preparation that increases the amount of one or more active compounds by at least 2-fold and up to about 1000-fold compared to the amount of one or more active compounds found in the weight of the plant material or other source prior to extraction or other preparation. In certain embodiments, the weight of the plant material or other source prior to extraction or other preparation can be a dry weight, a wet weight, or a combination thereof.

[0046] As used herein, "major active ingredient" or "major active component" refers to one or more active compounds found in or concentrated in a plant extract or other preparation that provide at least one biological activity. In certain embodiments, the major active ingredient of a concentrated extract is the one or more active compounds concentrated in the extract. Generally, the one or more major active ingredients directly or indirectly impart a majority (i.e., 50% or more, or 20% or more, or 10% or more, or 1% or more, or 0.2% or more, or 0.05% or more, or 0.01% or more) of one or more measurable biological activities or effects relative to other extract components. In certain embodiments, a major active ingredient may be a minor component by weight of the extract (e.g., less than 50%, less than 25%, less than 10%, less than 5%, less than 1%, less than 0.2%, less than 0.05%, or less than 0.01% of the components contained in the extract), yet still provide a majority of the desired biological activity. Any composition of the present disclosure that contains a primary active ingredient may also contain minor amounts of active ingredients that may or may not contribute to the pharmaceutical or nutraceutical activity of the concentrated composition, but will not be at the level of the primary active ingredient, and the minor amount of the active ingredient alone may not be effective in the absence of the primary active ingredient.

[0047] An "effective amount" or "therapeutically effective amount" refers to the amount of a compound, extract, algea powder, or composition of the present disclosure that, when administered to a mammal, such as a human, is sufficient to improve sleep latency, reduce sleep disturbance and fragmentation, and improve sleep quality and efficiency through any one or a combination of pathways such as: 1) reducing nighttime and / or daytime plasma, urinary, or salivary cortisol levels; 2) binding to melatonin receptors and increasing melatonin synthesis; 3) modulating the hypothalamic-pituitary-adrenal axis; 4) affecting non-rapid eye movement stages of sleep, e.g., deep slow-wave sleep; 5) activation of rapid eye movement stages of sleep; and 6) reducing nighttime awakenings.

[0048] The amount of a compound, extract, or composition of the present disclosure that constitutes a "therapeutically effective amount" will vary depending on the bioactive compound or standardized extract or ethanolic extract or biomarker for the condition being treated and its severity, the mode of administration, the duration of treatment, or the age of the subject being treated, but can be routinely determined by one of ordinary skill in the art in view of their own knowledge and this disclosure. In certain embodiments, an "effective amount" or "therapeutically effective amount" can be expressed as the amount of substance of the bioactive compound or extract relative to the body weight of a mammal (i.e., 0.001 mg / kg, 0.005 mg / kg, 0.01 mg / kg, or 0.1 mg / kg, or 1 mg / kg, or 5 mg / kg, or 10 mg / kg, or 20 mg / kg, or 50 mg / kg, or 100 mg / kg, or 200 mg / kg, or 500 mg / kg, or 1,000 mg / kg, or 2,000 mg / kg, or 5,000 mg / kg). The human-equivalent daily dose can be extrapolated from the "effective amount" or "therapeutically effective amount" in animal studies using FDA guidelines, taking into account differences in total body area and weight between animals and humans.

[0049] As used herein, a "dietary supplement" or "functional food" is a product that improves, promotes, increases, manages, controls, maintains, optimizes, modifies, reduces, inhibits, or balances a specific condition related to natural states or biological processes or structural and functional integrity, imbalanced or impaired states, or suppressed or impaired or overstimulated states of biological function or phenotype (i.e., not used to diagnose, treat, mitigate, cure, or prevent disease). For example, with respect to sleep, dietary supplements or functional foods can be used to regulate, maintain, manage, balance, inhibit, or stimulate any component of the sleep and neuroendocrine systems, resulting in reduced sleep latency, increased deep sleep time or total sleep time, improved REM or NREM sleep, improved quality, efficiency, and duration of sleep by correcting factors that may contribute to sleep disturbances, fragmentation, wakefulness, hyperactivity, and / or HPA axis disruptions, and increased serotonin and melatonin levels and decreased cortisol levels. In certain embodiments, dietary supplements and functional foods are a special category of foods, beverages, RTDs, food bars, sachets, powders, tinctures, gummies, and medical foods, and are not drugs.

[0050] As used herein, "treating" or "treatment" means the treatment of a disease or condition of interest in a mammal, such as a human, having the disease or condition of interest, and includes (i) preventing the disease or condition from occurring in a mammal, particularly if the mammal is predisposed to the condition but has not yet been diagnosed with the condition; (ii) inhibiting the disease or condition, i.e., halting its development; (iii) alleviating or modifying the disease or condition, i.e., causing the disease or condition to regress; or (iv) alleviating symptoms resulting from the disease or condition without addressing the underlying disease or condition (e.g., reducing sleep latency, improving sleep quality and efficiency in patients diagnosed with a sleep disorder, such as insomnia); or (v) balancing the regulation of HPA axis homeostasis or altering the phenotype of the disease or condition.

[0051] As used herein, the terms "disease" and "condition" can be used interchangeably or can differ in that a particular disease or condition may not have a known causative agent (and thus the etiology has not yet been elucidated) and therefore is not yet recognized as a disease, but merely as an undesirable state or syndrome in which a more or less specific set of symptoms has been identified by clinicians. A disease or condition may be acute, such as insomnia; or chronic, such as sleep disorders caused by aging. Impaired function of the hypothalamic-pituitary-adrenal (HPA) axis due to an imbalance in homeostasis may cause a disease or condition or predispose a mammal to neurological disorders or may result in more acute or chronic elevations of cortisol that are directly or indirectly related to sleep disorders.

[0052] As used herein, "statistical significance" refers to a p-value of 0.05 or less, as calculated using Student's t-test, indicating that a particular event or result being measured is unlikely to have occurred by chance.

[0053] For purposes of administration, contemplated compositions and compounds may be administered as drug substances or formulated as pharmaceutical or nutraceutical compositions. Contemplated pharmaceutical or nutraceutical compositions include a compound of the contemplated structure described herein and a pharmaceutically or nutritionally acceptable carrier, diluent, or excipient. The compound of the structure described herein is present in the composition in an amount effective to treat a particular disease or condition of interest, i.e., in an amount sufficient to promote good sleep quality and efficiency, and general HPA axis homeostasis, or any of the other related indications described herein, and with toxicity generally acceptable to patients.

[0054] Administration of the compounds or compositions of the present disclosure, or their pharmaceutically or nutritionally acceptable salts, in pure form or in suitable pharmaceutical or nutritional compositions can be carried out via any of the accepted modes of administration of drugs to fulfill similar utilities. Pharmaceutical, nutraceutical, or nutraceutical compositions of the present disclosure can be prepared by combining the compositions of the present disclosure with suitable pharmaceutically, nutritionally, or nutraceutical-acceptable carriers, diluents, or excipients, and can be formulated into preparations in solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, powders, gummies, granules, ointments, solutions, beverages, suppositories, injections, inhalants, gels, creams, lotions, tinctures, sachets, ready-to-drink drinks, masks, microspheres, and aerosols. Typical routes of administration of such pharmaceutical or nutraceutical compositions include oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, or nasal. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.

[0055] The pharmaceutical or dietary supplement compositions of the present disclosure are formulated so that the active ingredients contained therein are bioavailable after administration of the composition to a patient. The composition administered to a subject, patient, or mammal may take the form of one or more dosage units; for example, a tablet may be a single dosage unit, and a container of a composition of the disclosed compounds or extracts or two or three plant extracts in aerosol form may hold multiple dosage units. Actual methods for preparing such dosage forms are known or apparent to those skilled in the art; see, for example, Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). The composition to be administered will, in any event, contain a therapeutically effective amount of the disclosed compounds or pharmaceutically or nutritionally acceptable salts thereof for treatment of the disease or condition of interest, in accordance with the teachings of the present subject matter.

[0056] The pharmaceutical or nutraceutical compositions of the present disclosure can be in solid or liquid form. In one aspect, the carrier is particulate, so that the composition is, for example, in tablet or powder form. The carrier may also be liquid, so that the composition is, for example, an oral syrup, an injectable liquid, or an aerosol that is useful, for example, for inhalation administration.

[0057] When intended for oral administration, the pharmaceutical or nutraceutical composition may be in either solid or liquid form, with semi-solid, semi-liquid, suspension and gel forms being included within the scope of forms considered herein as either solid or liquid.

[0058] As a solid composition for oral administration, the pharmaceutical or nutraceutical composition can be formulated into the form of powder, granules, compressed tablets, pills, capsules, gummies, chewing gum, sachets, wafers, bars, etc. Such solid compositions usually contain one or more inert diluents or edible carriers. Additionally, one or more of the following may be present: binders such as carboxymethylcellulose, ethylcellulose, cyclodextrin, microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch, lactose, or dextrin; disintegrants such as alginic acid, sodium alginate, Primogel, corn starch, etc.; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; flavorings such as peppermint, methyl salicylate, or orange flavor; and coloring agents.

[0059] When the pharmaceutical or nutraceutical composition is in the form of a capsule, for example, a gelatin capsule, it can contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil.

[0060] Pharmaceutical or dietary supplement compositions may be in the form of a liquid, such as an elixir, tincture, syrup, solution, emulsion, or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, useful compositions contain, in addition to the compound, one or more of a sweetener, a preservative, a dye / colorant, and a flavor enhancer. Compositions intended for administration by injection may contain one or more of a surfactant, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer, and an isotonic agent.

[0061] The liquid pharmaceutical, nutraceutical, or functional food compositions of the present disclosure, whether in solution, suspension, or other similar form, may contain one or more of the following adjuvants: sterile diluents, such as water for injection, physiological saline solutions, e.g., saline, Ringer's solution, isotonic sodium chloride, fixed oils, such as synthetic mono- or diglycerides, polyethylene glycol, glycerin, propylene glycol, or other solvents that can act as solvents or suspending media; antibacterial agents, such as benzyl alcohol or methylparabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetic acid, citric acid, or phosphate, and agents for adjusting tonicity, such as sodium chloride or dextrose. Parenteral preparations can be sealed in ampoules, disposable syringes, or glass or plastic multi-dose vials. Physiological saline is typically a useful adjuvant. The injectable pharmaceutical or nutraceutical composition is sterile.

[0062] A liquid pharmaceutical or nutraceutical composition of the present disclosure intended for either parenteral or oral administration should contain an amount of a compound of the present disclosure such that a suitable dosage will be obtained.

[0063] The pharmaceutical or nutraceutical compositions of the present disclosure may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, cream, lotion, ointment, gel base, or patch. Bases may include, for example, one or more of the following: petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickeners may also be present in pharmaceutical or nutraceutical compositions for topical administration. When intended for transdermal administration, the compositions may include transdermal patches or iontophoresis devices.

[0064] The pharmaceutical or nutraceutical compositions of the present disclosure can be intended for rectal administration, for example, in the form of suppositories that melt in the rectum and release the drug. Rectal administration compositions can contain an oily base as a suitable non-irritating excipient. Such bases include lanolin, cocoa butter, and polyethylene glycol.

[0065] The pharmaceutical or dietary supplement compositions of the present disclosure can contain various materials that modify the physical form of the solid or liquid dosage unit.For example, the compositions can contain materials that form a coating shell around the active ingredient.The coating shell material is generally inert and can be selected from, for example, sugar, shellac, and other enteric coatings.Alternatively, the active ingredient can be placed in a gelatin capsule.

[0066] The pharmaceutical or nutraceutical compositions of the present disclosure, in solid or liquid form, can include an agent that binds to the compounds of the present disclosure and thereby assists in the delivery of the compounds. Suitable agents that can act in this capacity include monoclonal or polyclonal antibodies, proteins, or liposomes.

[0067] Pharmaceutical or nutraceutical compositions of the present disclosure, in solid or liquid form, may include particle size reduction to improve, for example, bioavailability. The size of powders, granules, particles, microspheres, etc. in the compositions, with or without excipients, may be macro (e.g., visible or at least 100 μm in size), micro (e.g., ranging from about 100 μm to about 100 nm in size), nano (e.g., 100 nm or less in size), and any size in between, or any combination thereof to improve size and bulk density.

[0068] The pharmaceutical or nutraceutical compositions of the present disclosure may be comprised of dosage units that can be administered as an aerosol. The term aerosol is used to describe a variety of systems, from colloidal systems to systems consisting of pressurized packages. Delivery can be by liquefied or compressed gas or by a suitable pump system that dispenses the active ingredient. Aerosols of the compounds of the present disclosure can be delivered in single-phase, two-phase, or three-phase systems to deliver the active ingredient. Aerosol delivery includes the necessary container, activator, valve, and subcontainer, which can be combined to form a kit. One skilled in the art can determine the most suitable aerosol without undue experimentation.

[0069] The pharmaceutical or nutraceutical compositions of the present disclosure can be prepared by the methodology well known in the field of medicine or nutraceuticals.For example, the pharmaceutical or nutraceutical compositions intended to be administered by injection can be prepared by combining the compound of the present disclosure with sterile distilled deionized water to form a solution.Surfactants can be added to facilitate the formation of a homogeneous solution or suspension.Surfactants are compounds that interact non-covalently with the compound of the present disclosure to facilitate the dissolution or homogeneous suspension of the compound in aqueous delivery systems.

[0070] The compounds of the present disclosure, or their pharmaceutically or nutritionally acceptable salts, are administered in a therapeutically effective amount, which will vary depending on a variety of factors, including the activity of the particular compound used; the metabolic stability and length of action of the compound; the age, weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject receiving the therapy.

[0071] The compounds of the present disclosure, or pharmaceutically or nutritionally acceptable derivatives thereof, can also be administered simultaneously with, before, or after the administration of food, water, and one or more other therapeutic agents. Such combination therapy includes administration of a single pharmaceutical or dietary supplement dosage formulation containing a compound or extract of the present disclosure, or a composition having two or three plant extracts, and one or more additional active agents, as well as administration of a composition having a compound or extract or two or three plant extracts of the present disclosure, and each active agent in its own separate pharmaceutical or dietary supplement dosage formulation. For example, a composition containing a compound or extract of the present disclosure, or two or three plant extracts, and another active agent can be administered to a patient together in a single oral dosage composition, such as a tablet or capsule, or each agent can be administered in a separate oral dosage formulation. When separate dosage formulations are used, the compound of the present disclosure and one or more additional active agents can be administered essentially simultaneously, i.e., simultaneously, or separately, i.e., sequentially; combination therapy is understood to include all of these regimens.

[0072] It is understood that in this description, combinations of substituents or variables of the depicted formulae are permissible only if such combinations result in stable compounds.

[0073] Those skilled in the art will also appreciate that such protected derivatives of the compounds included as contemplated embodiments may not themselves have pharmacological activity, but may be administered to a mammal and then metabolized in the body to form a pharmacologically active compound of the present disclosure. Such derivatives may therefore be described as "prodrugs." All prodrugs of the compounds included as contemplated embodiments are included within the scope of the present disclosure.

[0074] Furthermore, all compounds or extracts of the present disclosure that exist in free base or acid form can be converted to their pharmaceutically or nutritionally acceptable salts by treatment with an appropriate inorganic or organic base or acid by methods known to those skilled in the art. Salts of the compounds of the present disclosure can be converted to their free base or acid form by standard techniques.

[0075] Contemplated compounds, medicinal compositions and compositions may include, additionally include, or consist of at least one active ingredient. In some embodiments, the at least one bioactive ingredient may include or consist of a plant powder, plant extract, or the like.

[0076] In any of the foregoing embodiments, a standardized extract comprising a mixture of water, alcohol, or supercritical fluid extracts of a composition derived from a concentrate of one or more benzoxazoles is mixed with Chlorella in a particular weight ratio. In certain embodiments, the ratio (by weight) of at least one or more benzoxazole extracts mixed with Chlorella ranges from about 0.05:99.95 to about 99.95:0.05. Similar ranges apply when two or more extracts or compounds (e.g., three, four, or five) are used. Exemplary ratios of benzoxazole extract:Chlorella include, but are not limited to, 0.05:99.95, 0.1:99.9, 0.15:99.85, 0.2:99.8, 0.25:99.75, 0.3:99.7, 0.4:99.6, 0.5:99.5, 0.6:99.4, 1:99, 2:98, and 3:97. , 4:96, 5:95, 6:94, 7:93, 8:92, 9:91, 10:90, 15:85, 20:80, 25:75, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 99:1, 99.5:0.5, 99.9:0.1, and 99.95:0.05. In a further embodiment, the disclosed standardized extract of a composition derived from a concentrate of one or more benzoxazoles coded as UP165 from Zea mays leaves in combination with Chlorella is combined in a composition enriched to or standardized at 0.2% of the quality marker compound 6-MBOA, for example, but not limited to: In a further embodiment, such enriched or standardized Zea mays extract is combined with chlorella in combination with one or more benzoxazoles, either naturally isolated or artificially synthesized with natural compounds of comparable chemical structure.

[0077] In further embodiments, such enriched or standardized extracts of Zea mays combined with Chlorella are evaluated in in vitro and / or ex vivo and / or in vivo models for perceived biological benefits / detriments and unexpected synergistic / antagonistic effects, such as reduced sleep onset latency, sleep incidence, total sleep time, deep sleep time, RAM sleep time, NREM sleep time, and effective modulation of neuroendocrine homeostasis, increased serotonin, melatonin, GABA, DHEA, and decreased cortisol, resulting in improved sleep quality and improved mood states in human clinical trials. The optimal standardization with specific ratios of individual compounds is selected based on the unexpected synergistic effects measured in in vitro and / or ex vivo and / or in vivo models and the potential enhancement of ADME of these compounds to maximize biological output.

[0078] In certain examples, the compositions of the present disclosure can be formulated to further include a pharmaceutically or nutritionally acceptable carrier, diluent, or excipient, and the pharmaceutical or nutraceutical formulation comprises about 0.01 or 0.05 weight percent (wt %), or 0.2 weight percent, or 0.5 weight percent (wt %), or 5 weight percent, or 25 weight percent to about 95 weight percent of the active compound or primary active compound or biomarker compound of the extract mixture. In any of the foregoing formulations, the compositions of the present disclosure are formulated as tablets, hard capsules, softgel capsules, powders, gummies, RTDs, or granules.

[0079] Also contemplated herein are pharmaceutical preparations of the disclosed compounds. Such products may result, for example, from oxidation, reduction, hydrolysis, amidation, esterification, etc., of the administered compound, primarily due to enzymatic processes. Thus, contemplated compounds are those produced by a process comprising administering a contemplated compound or composition to a mammal for a period of time sufficient to produce a metabolic product thereof. Such products are typically identified by administering a detectable dose of a radiolabeled or non-radiolabeled compound of the present disclosure to an animal, such as a rat, mouse, guinea pig, dog, cat, pig, sheep, horse, monkey, or human, allowing sufficient time for metabolism to occur, and then isolating the conversion product from urine, blood, or other biological sample.

[0080] The contemplated compounds, medical compositions, and compositions can include, or can additionally include, or consist of, at least one pharmaceutically, nutritionally, or cosmetically acceptable carrier, diluent, or excipient. As used herein, the phrase "pharmaceutically, nutritionally, or cosmetically acceptable carrier, diluent, or excipient" includes any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonicity agent, solvent, or emulsifier approved by the U.S. Food and Drug Administration as acceptable for use in humans or veterinary animals. The contemplated compounds, medical compositions, and compositions can include, or can additionally include, or consist of at least one pharmaceutically, nutritionally, or cosmetically acceptable salt. As used herein, the phrase "pharmaceutically, nutritionally, or cosmetically acceptable salt" includes both acid addition salts and base addition salts.

[0081] In any of the foregoing embodiments, the composition includes an enriched or standardized corn leaf extract in combination with chlorella and one or more bioactive extracts or compounds to complement or boost the effects for reducing sleep onset latency, regulating cortisol homeostasis, and improving sleep quality, which may be present at certain percentage levels or ratios. In certain contemplated embodiments, the composition may include a composition containing chlorella and corn leaf extract or standardized corn leaf extract enriched with one or more benzoxazoles as biomarker compounds and / or about 0.01% to about 99.9% benzoxazoles and / or their congeners, or derivatives or precursors that may be isolated from natural sources or synthesized.

[0082] The natural bioactive compounds disclosed in presently contemplated embodiments, chlorella or extracts in combination with concentrated or standardized benzoxazoles of Zea mays (corn) leaf or corn sprout extracts, contain molecules that shorten sleep onset latency, enhance serotonin and melatonin biosynthesis, regulate the HPA axis, and normalize cortisol levels for homeostatic feedback leading to improved sleep quality and efficiency. Those naturally occurring compounds to be further combined with presently contemplated embodiments of the disclosed compositions containing benzoxazole in dietary supplements or functional foods include, but are not limited to, melatonin, magnesium, gamma aminobutyric acid (GABA), CBD, vitamins B1, B2, B3, B6, B12, pyridoxine, methylcobalamin, niacinamide, folic acid, ascorbic acid, vitamin C, vitamins D and E, zinc, omega-3 fatty acids, glycine, glutamine, arginine, tryptophan, L-theanine, 5-hydroxytryptophan (5-HTP), SAMe, magnolia, etc. These include hydroxybenzoates, honokiol, taurine, boron, branched-chain amino acids (BCAAs), phospholipids, phosphatidylserine, phosphatidic acid, theaflavin, rosmarinic acid, catechin, epicatechin, conjugated catechins such as EGCG, ECG, and epigallocatechin, baicalein, baicalin, oroxylin, wogonin, kaempferol, genistein, quercetin, butein, betaine, luteolin, chrysin, apigenin, curcumin, resveratrol, glomelatose A, 6-shogaol, gingerol, berberine, piperine, carnosol, and carnosic acid.

[0083] Plant species that may be combined with contemplated embodiments containing chlorella and benzoxazole-standardized zea mays leaf extract in dietary supplements and functional foods to shorten sleep onset latency, enhance serotonin and melatonin biosynthesis, regulate the HPA axis, and normalize cortisol levels for homeostatic feedback leading to shortened sleep latency, improved sleep quality, and efficiency include, but are not limited to, valerian root, Valeriana officinalis, Ginkgo biloba, Kava kava, lavender, passion flower (Passiflora incarnata or Passiflora incarnata), chamomile flower, hops, Humulus lupulus, Hibiscus sabdariffa, and others. sabdariffa, St. John's Wort, Griffonia simplicifolia, Fermented Milk, Fish Oil, Rhodiola rosea, Lotus Seed, Lotus Seed Germ, Oryza sativa, Zea mays, Ziziphus jujuba, Schisandra chinensis, Magnolia officinalis, Astragalus membranaceus, Ganoderma lucidum, Echinacea purpurea, Echinacea angustifolia, Poria cocos wolffii Wolf, Wolfporia extensa, Ashwagandha, Withania somnifera, Crocus sativus (Saffron), Bupleurum falcatum, Glycyrrhiza spp., Panax quinquefoliumquinquefolium, Panax ginseng CA Meyer, Korean red ginseng, Eurycoma longifolia (Malaysian ginseng), Lentinula edodes (shiitake mushroom), and Inonotus obliquus (Chaga mushroom).

[0084] The above-mentioned plant species that can be combined with contemplated embodiments containing chlorella and benzoxazole-standardized Zea mays leaf extract in dietary supplements and functional foods can be isolated from botanical and / or marine sources included in the Examples and elsewhere in this application. Plant parts suitable for extraction with and combination with contemplated embodiments containing chlorella and benzoxazole-standardized Zea mays leaf extract include shoots, shoots, leaves, young leaves, bark, trunks, trunk bark, stems, stem bark, twigs, tubers, roots, rootlets, rhizomes, root bark, bark surfaces, young shoots, seeds, fruits, seedlings, root hairs, stamens, pistils, calyxes, stamens, petals, sepals, carpels (pistils), flowers, or any combination thereof. In some related embodiments, the above-mentioned plant species can be combined with contemplated embodiments containing chlorella and benzoxazole-standardized Zea mays leaf extract in dietary supplements and functional foods. The plant species listed above can be extracted from plant sources and synthetically produced or modified to contain any of the recited substituents. In this regard, synthetic modification of compounds isolated from plants can be accomplished using any number of techniques known in the art and within the knowledge of one of ordinary skill in the art, including biosynthesis, biotechnology, fermentation, and enzymatic conversion. [Example]

[0085] [Example 1] Preparation of organic extracts obtained from corn leaves (Zea mays) Dry-milled immature corn leaf powder (10 g) was loaded into two 100 ml stainless steel tubes and extracted twice with different organic solvents, including dichloromethane, methanol, ethanol, acetone, petroleum, and ethyl acetate, using an ASE 300 automated extractor at 80°C and 1500 psi. The extracts were automatically filtered and collected. The organic extracts were evaporated on a rotary evaporator to obtain the crude organic extracts shown in Table 1.

[0086] [Table 1]

[0087] A composition (UP165) derived from a concentrate of one or more phenylpropanoid acids and benzoxazinoids was produced as a 70% ethanol / 30% water extract of immature corn leaf powder ground at 70-90°C and standardized with at least 0.2% 6-MBOA isolated from natural sources or synthesized.

[0088] Similar results were obtained using the same procedure, but replacing the organic solvent with methanol or ethanol to produce methanol extract (ME) or ethanol extract (EE), methanol:HO (7:3) extract, methanol:HO (1:1) extract, methanol:HO (3:7) extract, ethanol:HO (7:3) extract, ethanol:HO (1:1) extract, ethanol:HO (3:7) extract, and water extract, respectively.

[0089] [Example 2] 6-MBOA quantification in corn leaf extracts Immature corn (Zea mays) leaf extracts (10 mg / mL) were analyzed using a Hitachi HPLC / PDA system equipped with a C18 reverse-phase column (Phenomenex, Luna 5 μm, 150 mm x 4.6 mm) eluted with a solvent system of 0.2% formic acid in HO and acetonitrile at a flow rate of 1 mL / min. UV detection was performed at 286 nm using a 10 μL injection volume against pure 6-MBOA (Product No. 543551, Sigma-Aldrich) as an external reference standard. The 6-MBOA content in the plant extracts was measured in the range of 0.09–0.3% in extracts obtained with different solvents, including, but not limited to, methanol, ethanol, dichloromethane (DCM), acetone, and ethyl acetate.

[0090] [Table 2]

[0091] [Example 3] Preparation of organic extracts obtained from Scoparia dulcis Dried and ground Scoparia dulcis whole plant powder (20 g) was packed into a 100 ml stainless steel tube and extracted twice with an organic solvent mixture (methylene chloride / methanol, 1:1 ratio) using an ASE 300 automated extractor at 80 °C and 1500 psi. The extract was automatically filtered and collected. The organic extract was evaporated on a rotary evaporator to obtain a crude organic extract (OE, 2.64 g, 13.2%), which was determined to have a 6-MBOA content of 0.58% by the HPLC method described in Example 2.

[0092] [Example 4] Evaluation of 6-MBOA content and composition profiles enriched with one or more benzoxazinoids in different germinated crops Plant seeds of six crop species were planted in prepared soil and grown in early spring using standard agricultural practices in Texas. Whole plant shoots were harvested 10 days after germination. Crushed and dried plant shoot powder was extracted with methanol to obtain a per-plant methanol extract. Extracts were prepared at a concentration of 10 mg / mL and analyzed using an ACQUITY UPLC-I-class Xevo G2-XS-QTof system for compositional profiles based on enrichment of one or more phenylpropanoid acids and benzoxazinoids. 6-MBOA was further quantified at 0.66% in corn shoot extract, 0.12% in corn shoot powder, 0.19% in wheat shoot extract, 0.03% in wheat grass powder, 0.0081% in rye shoot extract, and 0.0018% in rye grass powder. 6-MBOA was not detected in barley, oat, or buckwheat extracts in this study.

[0093] The ground and dried corn stalk and wheat grass powder were further extracted with water and water containing 2% acetic acid. The extracts were prepared at a concentration of 10 mg / mL and analyzed using an ACQUITY UPLC-I-class Xevo G2-XS-QTof system to determine the concentration profiles of one or more phenylpropanoid acids and benzoxazinoids, as well as the content of 6-MBOA.

[0094] Coix lacrima L. seeds were directly extracted with methanol and analyzed at a concentration of 50 mg / mL using an ACQUITY UPLC-I-class Xevo G2-XS-QTof system. The concentrations of one or more phenylpropanoid acids and benzoxazinoids, as well as the profile of 6-MBOA, were determined at 0.00226% in Coix seed powder.

[0095] [Table 3]

[0096] [Table 4]

[0097] [Example 5] Chlorella CK-22 culture CK-22 is cultured and expanded in the following steps: slant culture, flask culture, jar culture, seed culture, tank culture, and finally outdoor pool culture.

[0098] 1) Slope culture CK-22 stocks are maintained in slant cultures. Slant cultures are initiated monthly from the original frozen stock. Agar slants are inoculated using a sterile loop. These slants are maintained by continuous subculture at Chlorella Kogyo Co., Ltd. Slant cultures are considered successful if the CK-22 culture is dark green and grows within 1-2 weeks under constant fluorescent lighting. These slants are then used to inoculate flask cultures.

[0099] 2) Flask culture All culture vessels and cultures for the laboratory cultivation steps are sterilized before inoculation at each step. Flask cultures are inoculated under sterile conditions and grown for 1-2 weeks in the presence of glucose, with constant exposure to a light source and constant mechanical shaking. A portion of the flask culture is then expanded into a jar culture. The remaining culture is discarded.

[0100] 3) Wide-mouth bottle culture The jar culture is grown in ambient light with shaking. A transfer line for seed culture inoculation is connected to the jar culture and the entire biomass of the jar culture is transferred when cell growth plateaus.

[0101] 4) Seed culture After inoculation from the jar culture, the CK-22 culture is grown under temperature and pH control with constant shaking. The seed culture is cultivated in the presence of glucose and in the absence of light. A portion of the seed culture is collected and used for the next step, tank cultivation.

[0102] 5) Tank culture A portion of the seed culture is transferred to a tank culture vessel containing sterile medium in the presence of glucose and in the absence of light with constant shaking. Temperature and pH are controlled during the tank culture. After the biomass reaches an appropriate chlorophyll content, the entire tank culture is transferred by pipeline to an outdoor pool culture.

[0103] 6) Outdoor pool culture The tank culture is then expanded via pipeline to a shallow outdoor pool, where the culture is incubated with shaking in the presence of ambient light and temperature with agitation. The nitrogen concentration and pH of the medium are monitored during outdoor pool incubation as a functional readout of the culture's health. Thus, the agitation rate can be increased to maintain culture health. The culture is further monitored by microscopic examination of cell shape, lack of clumping, and for potential microbial contamination. In the event of microbial contamination, the culture is discarded. After reaching an appropriate chlorophyll content, the culture is transferred via pipeline and harvested for processing.

[0104] [Example 6] Processing and production of chlorella powder Processing and production of chlorella powder obtained from outdoor cultivation involves washing, filtering and sterilizing the algae.

[0105] After the cultivation process is completed as described in Example 5, the CK-22 biomass is harvested by filtering the culture through 800 μm and 550 μm filters. The culture is then washed and concentrated by three separate centrifugation steps to remove the medium. Each wash is performed with sterile water, and the culture is passed through a final 350 μm filter. The final slurry is passed through a magnetic strainer, cooled to 2-5°C, and stored for up to 24 hours, followed by heat inactivation at 100°C for 3 minutes.

[0106] The heat-sterilized CK-22 is then spray-dried. The spray-dried intermediate product is sieved through a magnetic strainer, passed through a 20-mesh strainer, and then packaged in 100 kg bags. One batch of seed culture corresponds to one lot of intermediate product.

[0107] The final product consists of multiple lots of intermediate product that are mixed to produce one final lot of 1000-1500 kg. After mixing, the product is passed through a magnetic strainer. The particle distribution of the chlorella powder is 19 μm (10th percentile), 60 μm (50th percentile), and 134 μm (90th percentile).

[0108] [Example 7] Chlorella CK-22 Powder Specifications The characteristics of the Chlorella described in Example 6 are summarized in Tables 5 and 6.

[0109] [Table 5]

[0110] [Table 6]

[0111] [Example 8] Experimental Design Used to Evaluate the Synergistic Effects of Zea Mays (Corn) Leaf Extract Standardized with 6-MBOA and Chlorella Purpose-bred BALB / C mice (n=405, male, 20-22g) were purchased from Beijing Hua Fukang Biological Technologies Inc. (Beijing, China). This study was conducted at the Testing Center for the Functions of Health Foods, College of Arts and Sciences of Beijing Union University (Beijing, China). Mice were housed in a specific pathogen-free, temperature-controlled room and fed rodent chow purchased from Beijing Hua Fukang Biological Technologies Inc. (Beijing, China). Three consecutive studies were conducted. Mice were randomly assigned to nine groups per experiment. Fifteen mice were assigned to each group. The first experiment was designed to determine the sleep incidence effect of test materials. The second experiment was designed to detect the total sleep time under a subthreshold dose of pentobarbital sodium (Lot#: 34E10320, Beijing Dingguo Changsheng Biotechnology Co. Ltd.). A third experiment was designed to detect sleep latency induced by sodium barbital (Lot#: 527B051, Merck).

[0112] [Table 7]

[0113] Zea mays (corn) leaf extract (lot #: FP210105-01) standardized to ≥0.2% 6-MBOA and chlorella powder (lot #: 222A) were suspended in sterile water. Melatonin purchased from By-Health Co. Ltd. (Beijing, China) was used as the reference compound in each study. All mice were orally administered the test materials at a volume of 20 mL / kg BW / day. The study lasted for 31 days.

[0114] SPSS software (Statistical Package for the Social Sciences, advanced statistical analysis software) for Windows was used for statistical analysis. The number of sleeping mice in the direct sleep test experiment and the effect of pentobarbital sodium on the subthreshold sleep time of hypnotism were analyzed using the chi-square test. Homogeneity between test groups was determined using analysis of variance. If the variances were not homogeneous or the data showed an abnormal distribution, appropriate variable transformations were performed to meet the requirements for homogeneity or normal distribution. If variable transformation or homogeneity was not achieved even after appropriate variable transformations, the statistical method of rank tests was used. The statistical method of t-tests was used to analyze the changes in efficacy between melatonin and Zea mays (corn) leaf extract standardized with 6-MBOA.

[0115] [Example 9] The test materials did not exhibit any sleep-inducing effects as measured by a 60-minute direct effects test. In the direct effect test, mice were considered asleep and were indexed as asleep when they lost their reflex to roll to the right side. Testing was performed 1 hour after administration of the test material. The principles of the test dictate that recovery of rolling over is defined as wakefulness, and the period between loss of the reflex to roll to the right side and its recovery is defined as sleep time. Sleeping time and the number of mice exhibiting these characteristics were recorded and compared. Differences in sleeping time or number of sleeping mice between the test substance and vehicle control groups were considered positive.

[0116] [Table 8]

[0117] As shown in Table 8, in the current study, all mice in each test material group immediately turned to their right side when placed in the supine position. This indicates that the test materials do not have direct sleep-inducing activity. Oral administration of 6-MBOA-standardized zea mays (corn) leaf extract, chlorella, and 6-MBOA-standardized zea mays (corn) leaf extract + chlorella to mice did not produce a sleep-inducing effect in the absence of pentobarbital.

[0118] [Example 10] Effects of materials on sleep latency Fifteen minutes after the final administration of the test material, the mice were intraperitoneally injected with 240 mg / kg sodium barbital (0.2 mL / 20 g body weight). The sleep latency was recorded, and the difference in sleep time between the test material group and the vehicle control was determined. If there was a difference in sleep onset time between the test material group and the vehicle control, the test material was considered positive (shortened sleep latency).

[0119] As shown in Table 9, sleep latency was significantly reduced in mice treated with 1 mg / kg melatonin. Similarly, mice receiving an oral dose of 5,000 mg / kg chlorella demonstrated a statistically significant decrease in sleep onset latency (p=0.024). Adding 250 mg / kg zea mays (corn) leaf extract standardized with 6-MBOA to 5,000 mg / kg chlorella further reduced sleep latency (p=0.001), demonstrating a synergistic effect of these combinations. Additional analyses were performed to evaluate the decrease in sleep onset latency observed for the melatonin (1 mg / kg) group compared to chlorella (5,000 mg / kg) alone and the combination of 250 mg / kg zea mays (corn) leaf extract standardized with 6-MBOA and chlorella (250 + 5,000 mg / kg). This was the only composition of zea mays (corn) leaf extract and chlorella standardized with 6-MBOA that achieved statistical significance. This reduction in sleep latency further strengthens the additive benefit of the combination of zea mays (corn) leaf extract and chlorella standardized with 6-MBOA.

[0120] [Table 9]

[0121] [Example 11] Increased sleep incidence with the combination of 6-mboa standardized corn (Zea mays) leaf extract with chlorella Fifteen minutes after the final administration of test material, mice were intraperitoneally injected with a subthreshold dose of pentobarbital (32 mg / kg) to determine the incidence of sleep as a result of treatment effect.One minute after pentobarbital injection, the number of mice that lost the ability to turn to the right side was recorded.The incidence of sleep in mice was analyzed, and the difference between the test substance administration group and the vehicle control group was compared.All experiments were conducted at night.

[0122] [Table 10]

[0123] Adding 6-MBOA-standardized zea mays (corn) leaf extract to chlorella improved the incidence of sleep onset in mice in a dose-related manner. When mice were administered 1000 mg / kg of chlorella alone, the incidence of sleep onset was not statistically significant compared to vehicle. However, when 250 mg / kg of 6-MBOA-standardized zea mays (corn) leaf extract was combined with 1000 mg / kg of chlorella, the incidence of sleep was significantly improved compared to vehicle-treated controls. Demonstrating these findings, a combination of 250 mg / kg of 6-MBOA-standardized zea mays (corn) leaf extract and 5000 mg / kg of chlorella increased the duration of sleep onset by 60% (over the incidence of sleep observed when 5000 mg / kg of chlorella was administered alone) to 73%. These data demonstrate the complementary effects of 6-MBOA-standardized zea mays (corn) leaf extract and chlorella. In fact, the 73% sleep incidence rate of the combination of Zea mays (corn) leaf extract standardized with 6-MBOA (250 mg / kg) and chlorella (5000 mg / kg) was better than the 60% sleep incidence rate observed with melatonin.

[0124] [Example 12] Effects of a combination of zea mays (corn) leaf extract standardized with 6-MBOA and chlorella on sleep duration Time spent sleeping was determined by intraperitoneally injecting mice with 46 mg / kg of sodium pentobarbital 15 minutes after administration of the final dose of test material. Sleep time for each mouse was recorded, and the difference in sleep time between the test material and vehicle control was analyzed. As shown in Table 11, an inverse correlation between sleep time and dose was observed in mice treated with chlorella alone. The highest sleep time was observed in mice treated with a low dose of chlorella (1,000 mg / kg) followed by a medium dose (2,000 mg / kg). As the dose of chlorella increased, the duration of sleep decreased. Adding zea mays (corn) leaf extract standardized with 6-MBOA to chlorella resulted in sleep time equivalent to that of mice treated with melatonin at all doses. Zea mays (corn) leaf extract standardized with 6-MBOA by itself produced sleep time similar to that of melatonin.

[0125] [Table 11]

[0126] [Example 13] Preparation of a sachet powder formulation combining chlorella and Zea mays (corn) leaf extract standardized with 6-MBOA Chlorella and zea mays (corn) leaf extract standardized with 6-MBOA were blended in a 9:1 ratio with glucose powder, chicory root extract powder, skim milk powder, vegetable cream powder, and sucralose. Granules were produced by wet granulation with purified water as a binder using an automated system with an inlet temperature of 55-60°C, an outlet temperature of 35-40°C, and an air injection pressure of 26-30 rpm. The granulated powder was further mixed with milk flavor powder and silicon dioxide to obtain a final 50% by weight ratio of chlorella to zea mays (corn) leaf extract standardized with 6-MBOA.

[0127] [Table 12]

[0128] [Example 14] Preparation of a tablet formulation with a combination of Chlorella and Zea mays (corn) leaf extract standardized with 6-MBOA.

[0129] A first example tablet formulation is described below, and a contemplated tablet is shown in Figure 1. The tablet shown in Figure 1 is designed to contain 2 g of chlorella and 250 mg of zea mays (corn) leaf extract standardized with 6-MBOA per day. Each tablet is designed to contain 333.333 mg of chlorella and 41.667 mg of zea mays (corn) leaf extract standardized with 6-MBOA. The tablet formulation is designed to contain six tablets per day. The tablet is not designed to contain excipients, such as diluents, although excipients can be added as needed.

[0130] The weight of the above tablets was designed to be 375 mg per tablet, and the blended amount of the prototype was 2000 kg. Prototype production and testing were outsourced to Nippon Tablet Co., Ltd. (149-1 Megawa, Makishima-cho, Uji City, Kyoto Prefecture). The trial production process consisted of weighing, mixing, and tablet preparation. The prototype results are described below. The thickness of the above tablets was 5.82 mm. The diameter of the above tablets was 9.10 mm. The tablet hardness was 8.03 kgf. The tablet weight was 375 mg. The disintegration time of the above tablets was 16-18 minutes. The tablet weight deviation mentioned above was 0.41%. The moisture content of the above tablets was 5.9%. The friability of the above tablets was 0.15%, 7.486 g before testing and 7.475 g after testing. The results of the impact test were within the acceptable range.

[0131] Pilot-scale tableting was carried out at a prototype production scale with mass production in mind, and it was confirmed that there were no problems with tablet manufacturing at a production scale.

[0132] A second example tablet formulation is described below. The tablets are designed for daily administration of 5 g of chlorella and 250 mg of zea mays (corn) leaf extract standardized with 6-MBOA. Each tablet is designed for 333.333 mg of chlorella and 16.667 mg of zea mays (corn) leaf extract standardized with 6-MBOA. The tablet formulation is designed for daily administration of 15 tablets. The tablets are not designed to include excipients, such as diluents, although excipients can be added as needed. The tablet weight is designed to be 350 mg per tablet, and the blended amount of the original was 2000 kg.

[0133] Prototype production and testing was outsourced to Nippon Tablet Co., Ltd. (149-1 Megawa, Makishima-cho, Uji City, Kyoto Prefecture). The prototyping process included weighing, mixing, and tablet production. The prototype results are described below. The thickness of the above tablets was 5.57 mm. The diameter of the above tablets was 9.11 mm. The tablet hardness was 5.9 kgf. The tablet weight was 351 mg. The disintegration time of the above tablets was 13-15 minutes. The tablet weight deviation mentioned above was 0.44%. The moisture content of the above tablets was 6%. The friability of the above tablets was 0.01%, 7.027g before testing and 7.026g after testing. The results of the impact test were within the acceptable range.

[0134] Pilot-scale tableting was carried out at a prototype production scale with mass production in mind, and it was confirmed that there were no problems with tablet manufacturing at a production scale.

[0135] [Example 15] Human clinical trial of a combination of chlorella and 6-mbOa standardized Zea mays (corn) leaf extract Human clinical trials may be initiated with larger study populations and additional biomarkers to validate the clinical trials shown in these examples. The clinical trial protocol will be provided in its entirety by the CRO.

[0136] Clinical Design: A randomized, triple-blind, placebo-controlled, parallel clinical trial investigating the safety and efficacy of an investigational drug on sleep quality in a population of healthy individuals who have difficulty falling asleep or staying asleep.

[0137] Population: Healthy adult men and women

[0138] Sample size: The sample size is 10-100 participants per group.

[0139] Clinical Trial Design Outcomes Pittsburgh Sleep Quality Index (PSQI) Profile of Mood States (POMS) Perceived Stress Scale (PSS) EEG or wearable devices Actigraph readings: sleep time, sleep onset, wake time, circadian consistency (0-100), sleep latency, sleep efficiency, sleep stages (awake, light sleep, deep sleep), wakefulness / interruptions, exercise (with intensity classification), snoring (via mobile device microphone with intensity classification), biometric trends: HR, HRV, SpO2, respiratory rate, arterial elasticity, sleep score (0-100), recovery score (0-100) Blood markers: serotonin, melatonin, GABA Salivary marker: Cortisol Questionnaire on the impact of COVID-19 on quality of life (QoL)

[0140] safety As assessed by clinical chemistry, hematology, vital signs and adverse events

[0141] Inclusion criteria Men and women between the ages of 18 and 65 Women of childbearing potential must agree to use a medically approved method of childbirth. · Control and have a negative urine pregnancy test result. Inability to fall asleep or stay asleep (waking up more than twice in seven days). Agree to maintain your current sleep schedule during the exam I agree to remain in my current time zone for the duration of the exam. ·Agree to refrain from over-the-counter (OTC) medications that aid sleep. Willingness to complete all study-related questionnaires, records, and diaries and to complete all visits Provide voluntary, written, informed consent to participate in this study.

[0142] Exclusion criteria Women who are pregnant, breastfeeding, or planning to become pregnant during the study Alcohol or drug abuse within the past year Have you ever been previously diagnosed with a sleep disorder? Currently working in a job that requires shift work Currently experiencing nightmares and / or sleepwalking Subject has a known allergy to the active or inactive ingredients of the test material Subjects with unstable medical conditions Clinically significant abnormal test results at screening Participation in a clinical trial within 30 days prior to randomization People who are cognitively impaired and / or unable to give informed consent Any other condition that, in the opinion of the investigator, may adversely affect the subject. · May pose a significant risk to the subject or their ability to complete the Study or its procedures.

[0143] treatment Investigational Drug - 500 mg of a combination of chlorella and Zea mays (corn) leaf extract standardized with 6-MBOA - approximately 5000 mg per day ·placebo

[0144] Visit 1 (Screening): Eligibility will be assessed and determined based on the inclusion and exclusion criteria. A urine pregnancy test will be performed (if applicable). Medical history and concomitant medications will be reviewed, and heart rate and blood pressure will be measured. Peripheral blood will be obtained to determine CBC, electrolytes (Na, K, Cl), HbA1c, glucose, eGFR, creatinine, AST, ALT, ALP, and bilirubin. Subjects will begin a 14-day run-in period and complete a sleep diary each morning prior to the baseline visit.

[0145] Visit 2 (Baseline - Day 0): Eligible participants return to the clinic. Sleep diaries are collected and reviewed. Heart rate and blood pressure are measured; concomitant medications are reviewed. Subjects are randomized to treatment groups. The Pittsburgh Sleep Quality Index (PSQI), Perceived Stress Scale (PSS), COVID-19 Impact on Quality of Life (QoL) Questionnaire, and Profile of Mood States (POMS) are completed. Blood samples are collected for analysis of serotonin, melatonin, and GABA. Saliva samples are collected to measure cortisol levels. Participants are provided with a wrist-worn actigraphy device to monitor nighttime sleep patterns and instructed to wear it every night when they go to bed. Participants are also provided with an EEG device or wearable device and trained to use it at home. The study drug and subject's treatment diary are distributed and subjects are instructed on how to use it. The subject's treatment diary is used to record daily product use, changes in concomitant medications, and any adverse events and symptoms during the study.

[0146] Visit 3 (Day 14): Heart rate and blood pressure will be measured. Study drug and subject treatment diaries will be returned and compliance will be calculated. Concomitant medications and adverse events will be reviewed. Sleep data will be collected from actigraphy and EEG devices. PSQI, PSS, COVID-19 Impact on QoL questionnaire and POMS will be completed. Blood samples will be taken and analysed for serotonin, melatonin and GABA. Saliva samples will be taken and cortisol levels will be measured. EEG devices or wearable devices will be redistributed. Study drug and subject treatment diaries will also be redistributed.

[0147] Visit 4 (Day 28 - End of Study): Heart rate and blood pressure will be measured. Study drug and subject treatment diaries will be returned and compliance will be calculated. Concomitant medications and adverse events will be reviewed. Sleep data will be collected from actigraphy and EEG devices. PSQI, PSS, COVID-19 Impact on QoL Questionnaire and POMS will be completed. Blood samples will be taken to analyze serotonin, melatonin and GABA. Saliva samples will be taken to measure cortisol levels. Blood samples will also be taken to determine CBC, electrolytes (Na, K, Cl), glucose, eGFR, creatinine, AST, ALT, ALP and bilirubin.

[0148] [Table 13]

Claims

1. 1. A composition comprising a plant extract standardized for an amount of benzoxazinoid, the composition further comprising chlorella.

2. 10. The composition of claim 1 provided in an amount effective to reduce sleep onset latency, improve sleep quality and efficiency, and prevent and treat sleep disorders.

3. Plant extracts standardized for benzoxazinoid content were obtained from Zea mays, Chlorella sorokiniana, Chlorella vulgaris, Oryza spp., Oryza sativa, Oryza glaberrima, Oryza australiensis, Oryza brachyantha, Secale cereale, Acanthus arboreus; Acanthus ebractatus, Acanthus iliquifolius, Acanthus mollis, Avena sativa, Avena abyssinica, Avena byzantine, Avena nudum, Avena strigosa, Hordeum vulgare, Coix lacrima-jobi, Triticum aestivum.

10. The composition of claim 1, wherein the extract is extracted, concentrated, and standardized from a plant species selected from the group consisting of: Sorghum bicolor, Triticum compactum, Triticum sphaerococcum, Triticum tulanicum, Sorghum bicolor, Agropyron repens, Blepharis edulis, Balsamocitrus paniculate; Peristrophe roxyburgiana; Strobilanthes custardii; Lamium galeobdron, Lobelia chinensis, Rhamus chinensis, Aphelandra spp., Scoparia dulcis, Capparis sikkimensis spp., or combinations thereof.

4. 2. The composition of claim 1, wherein the plant extract standardized for the amount of benzoxazinoids is extracted, concentrated, and standardized from a plant part selected from the group including shoots, sprouts from plant seeds, sprouts of germinated seeds, immature leaves, mature leaves, whole plants, roots, seeds, flowers, stems, stem bark, root bark, silk, seeds, hairy roots of germinated seeds, stem cells, cell culture tissue, or any combination thereof.

5. 2. The composition of claim 1, wherein the benzoxazinoid is one or more of 6-methoxy-2-benzoxazolol (6-MBOA); 2-benzoxazolol (2-BOA); 4-methylbenzoxazole; 2,4-dimethylbenzoxazole; 2,6-dimethylbenzoxazole; 2,6-benzoxazolediol; 2,4-benzoxazolediol; 4-acetyl-2(3H)-benzoxazolone; 6-methoxy-N-methyl-2(3H)-benzoxazolone; 3-hydroxy-6-methoxy-2-benzoxazolin-2(3H)-one; 2-hydroxy-6,7-dimethoxybenzoxazole; 5,6-dimethoxy-2-benzoxazolinone; 3,6-dimethoxybenzoxazolin-2(3H)-one; 5-chloro-6-methoxy-2-benzoxazolinone; trehalamine, or any combination thereof, and a benzoxazinoid glycoside.

6. 10. The composition of claim 1, wherein the plant extract standardized for the amount of benzoxazinoid is extracted with any suitable solvent, including supercritical fluid CO2, water, acidic water, basic water, acetone, methanol, ethanol, propenol, butanol, alcohol mixed with water, mixed organic solvents, or combinations thereof.

7. 10. The composition of claim 1, wherein the benzoxazole is synthesized, metabolized, biodegraded, biotransformed, biotransformed, or biosynthesized from small carbon units by a transgenic microorganism, by a P450 enzyme, by a glycosyltransferase or a combination of enzymes, or by a microbacterium.

8. 10. The composition of claim 1, wherein the plant extract, standardized for the amount of benzoxazinoid, is concentrated by solvent precipitation, neutralization, solvent partitioning, ultrafiltration, enzymatic digestion, column chromatography using silica gel, XAD, HP20, LH20, C-18, alumina oxide, polyamide, ion exchange, and CG161 resins, individually or in combination.

9. 10. The composition of claim 1, wherein the plant extract standardized for the amount of benzoxazinoid is extracted, concentrated, and standardized from shoots and immature leaves of Zea mays containing 0.2% or more 6-MBOA.

10. The composition of claim 1, wherein the Chlorella is CK-22, which is cultured and expanded sequentially by slant culture, flask culture, wide-mouth jar culture, seed culture, tank culture, and finally outdoor pool culture.

11. 2. The composition of claim 1, wherein the chlorella is CK-22 grown in an outdoor pool for processing by washing, filtering, sterilizing and spray drying.

12. 2. The composition of claim 1, wherein the chlorella particle distribution of the chlorella powder is in the range of 1 μm to 500 μm.

13. 2. The composition of claim 1, wherein the chlorella contains 4 mg / 100 g or more of vitamin B2, 55% or more of protein, 1 g / 100 g or more of tryptophan, and 40 mg / 100 g or more of iron.

14. 10. The composition of claim 1, wherein the plant extract standardized for the amount of benzoxazinoid is combined with chlorella in a combination ratio ranging from 0.05%:99.95% to 99.95%:0.05% by weight.

15. 10. The composition of claim 1, further comprising a pharmaceutically or nutritionally or nutraceutical acceptable active agent, adjuvant, carrier, diluent or excipient, wherein the pharmaceutical or nutraceutical formulation comprises from about 0.1 weight percent (wt %) to about 99.9 wt % of the composition in concentrated one or more benzoxazinoids.

16. The active agent, adjuvant, excipient or carrier may be selected from the group consisting of cannabis sativa oil or CBD / THC, turmeric extract or curcumin, Terminalia extract, aloe vera leaf gel powder, valerian root, Valeriana officinalis, Ginkgo biloba, kava kava, lavender, passion flower (Passiflora incarnata or Passiflora incarnata), chamomile flower, hops, humulus lupulus, hibiscus sabdariffa, St. John's wort, cauliflower,Griffonia simplicifolia, fermented milk, fish oil, Rhodiola rosea, lotus seed, lotus seed germ, Oryza sativa, Zea mays, Chlorella sorokiniana, Chlorella vulgaris, other Chlorella species, Ziziphus jujuba, Schisandra sinensis, Magnolia officinalis, Astragalus membranaceus, Ganoderma lucidum, Echinacea purpurea, Echinacea angustifolia, Poria cocos wolff, Wolfiporia extensa, Withania somnifera, Bupleurum falcatum, Glycyrrhiza species, Panax quinquefolium, Panax ginseng C.A. Mayer, Red Ginseng, Eurycoma Longifolia (Malaysian Ginseng), Lentinula Edulis (Shiitake Mushroom), Inonotus Obliquus (Chaga Mushroom), Melatonin, Magnesium, Gamma Aminobutyric Acid (GABA), Vitamins B1, B2, B3, B6, and B12, Pyridoxine, Methylcobalamin, Niacinamide, Folic Acid, Ascorbic Acid, Vitamin C, Vitamins D and E, Zinc, Omega-3 Fatty Acids, Glycine, Glutamine, Arginine, Tryptophan, L-Theanine, 5-Hydroxytryptophan (5-HTP), SAMe, Magnolol, Honokio 16. The composition of claim 15, wherein the catechin is selected from one or more of the following: methylparaben, taurine, boron, branched-chain amino acid (BCAA) phospholipids, phosphatidylserine, phosphatidic acid, theaflavin, rosmarinic acid, catechin, epicatechin, conjugated catechins such as EGCG, ECG, and epigallocatechin, baicalein, baicalin, oroxylin, wogonin, kaempferol, genistein, quercetin, butein, betaine, luteolin, chrysin, apigenin, curcumin, resveratrol, glomelatose A, 6-shogaol, gingerol, berberine, and piperine.

17. 10. The composition of claim 1 formulated as a tablet, hard capsule, soft gel capsule, powder or granules, compressed tablet, pill, gummy, chewing gum, sachet, wafer, bar or liquid form, tincture, aerial spray, semi-solid, semi-liquid, solution, emulsion, cream, lotion, ointment, gel base or similar form.

18. 10. The method of claim 1, wherein the route of administration is selected from the group comprising oral, topical, suppository, intravenous, intradermal, intragastric, intramuscular, intraperitoneal and intravenous.

19. 10. The composition of claim 1, wherein the plant extract standardized for the amount of benzoxazinoids in combination with chlorella prevents and treats sleep disorders.

20. 20. The composition of claim 19, wherein the sleep disorder comprises insomnia, hypersomnia, circadian rhythm disorder, shift work sleep disorder, non-24-hour sleep-wake disorder, periodic limb movement disorder, restless legs syndrome (RLS), sleep apnea, narcolepsy, parasomnia, night terrors, sleepwalking, nightmares, sleep-related eating disorder, sleep hallucinations, sleep paralysis, sleep talking, and REM sleep behavior disorder.

21. 10. The composition of claim 1, wherein the plant extract standardized for the amount of benzoxazinoids in combination with chlorella reduces sleep onset latency, improves sleep quality and efficiency, increases deep sleep time, increases total sleep time, and improves REM and NREM sleep.

22. 10. The composition of claim 1, wherein the plant extract standardized for the amount of benzoxazinoids in combination with chlorella maintains cortisol homeostasis leading to improvement of symptoms of chronically elevated cortisol.

23. 23. The composition of claim 22, wherein symptoms of chronically high cortisol include anxiety, depression, fatigue, gastrointestinal upset such as constipation, bloating or diarrhea, headaches, heart disease, high blood pressure, irritability, memory and concentration problems, decreased libido, reproductive problems such as erectile dysfunction or irregular menstruation and ovulation, difficulty sleeping, slow recovery from exercise, eating disorders and weight gain, hair loss, chronic inflammation, susceptibility to infection and skin blemishes.

24. 10. The composition of claim 1, wherein the plant extract standardized for the amount of benzoxazinoid in combination with chlorella is administered in an effective amount of 0.01 mg to 5,000 mg per kg of mammalian body weight.

25. 25. The composition of claim 24, wherein the mammal is a human; a companion animal, a domestic animal, or a wild animal, including a cat, a dog, a pig, a cow, a sheep, a goat, a horse, and a rabbit.

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