Pistachio (pistachio) composition rich in melatonin and preparation method of pistachio (pistachio) composition

Abstract

Provided herein are methods and compositions of a Pistachyos (Pistachyos) composition, which is rich in melatonin, and methods and compositions of the Pistachyos (Pistachyos) composition are provided.

Description

Background Technology

[0001] This invention relates to the selection, preparation, and extraction of pistachio seed kernels to obtain standardized levels of melatonin from natural sources.

[0002] Melatonin has been widely used as a dietary supplement ingredient, primarily for supporting and regulating healthy sleep patterns, overcoming jet lag, and improving mild sleep disorders.

[0003] Melatonin was once commercially produced from the pineal glands of cattle and pigs, making it animal-derived. As concerns about animal-derived sources have grown, particularly with the emergence of viruses such as mad cow disease and foot-and-mouth disease, people have begun seeking alternative sources of melatonin. Most commercially available melatonin dietary supplements utilize synthetically produced melatonin.

[0004] As more people shift towards sustainable, plant-based diets, the demand for plant-based ingredients such as melatonin is growing. It has also been learned that the quality of synthetic melatonin varies significantly, both in the ingredient itself and in dietary supplements / food products containing it.

[0005] Sleep is a finely regulated process driven by complex neurochemical interactions within the brain's sleep-promoting and wakefulness centers. Melatonin is central to this regulation, influencing various bodily systems and physiological processes, triggering immediate physiological responses such as fatigue, and contributing to long-term health outcomes due to its powerful antioxidant properties. Melatonin exerts this effect by activating highly sensitive receptors MT1 and MT2, which respond to changes in the light-dark cycle and directly influence circadian rhythms, sleep-wake cycles, and the synchronicity of experienced sleep duration and stages. While melatonin can activate both receptors, they have been reported to elicit different physiological responses due to their unique expression patterns, signaling pathways, and pharmacological properties in different organs. Activation of the MT1 receptor reduces neuronal activity in the suprachiasmatic nucleus (SCN), promoting sleep initiation; while activation of the MT2 receptor regulates circadian rhythm synchronicity in response to environmental cues such as the light-dark cycle. The synergistic effect of these receptors in regulating sleep duration, duration, and circadian rhythms highlights their value as potential pharmacological targets for optimizing sleep management and quality.

[0006] Clinical studies have extensively explored the routes of melatonin administration, primarily focusing on oral and intravenous administration in healthy volunteers. However, results regarding its pharmacokinetic properties are inconsistent.

[0007] Studies have revealed that oral melatonin follows first-order kinetics, with an average bioavailability of approximately 15%, but significant individual variability exists. Multiple factors, including age, caffeine intake, smoking, oral contraceptive use, food intake, and concurrent drug intake (such as fluvoxamine), can affect its pharmacokinetics. Nevertheless, melatonin appears to maintain a constant half-life of approximately 45 minutes, and peak melatonin concentrations in the bloodstream have been reported approximately 50 minutes after oral administration.

[0008] This invention attempts to solve these problems, as well as others. Summary of the Invention

[0009] This article provides methods and compositions for the preparation of melatonin-rich Pistacia vera (pistachio) compositions.

[0010] The methods and compositions described herein are set forth in part in the following description and will be apparent in part from the description, or may be learned by practice of the methods and compositions. The advantages of the methods and compositions will be realized and obtained by means of the elements and combinations particularly pointed out in the appended claims. It should be understood that the foregoing general description and the following detailed description are exemplary and illustrative only and do not limit the claimed methods and compositions.

[0011] Therefore, the purpose of this invention is not to cover any previously known products, processes for manufacturing products, or methods of using products, thereby reserving the rights of the applicant and disclosing a disclaimer herein regarding any previously known products, processes, or methods. It should also be noted that this invention is not intended to address products that do not meet USPTO (35 USC) requirements. 112, Paragraph 1) or any product, process, or method of making or using the product as described in writing and required by the EPO (Article 83 of the EPC) is covered within the scope of this invention, thereby reserving the rights of the applicant and hereby disclosing any previously known product, process of making the product, or method of using the product. In the practice of this invention, compliance with Article 53(c) of the EPC and Rules 28(b) and (c) of the EPC may be advantageous. It is expressly reserved that all rights to embodiments of the subject matter of any one or more of the applicant's granted patents in any series or any other series or any previously filed application of any third party are expressly excluded. Nothing herein shall be construed as a commitment. Attached Figure Description

[0012] In the accompanying drawings, similar elements are identified by similar reference numerals in several preferred embodiments of the invention.

[0013] Figure 1AThis is a flowchart illustrating a method for preparing a melatonin-rich pistachio seed / kernel extract according to one embodiment.

[0014] Figure 1B This is a flowchart illustrating a method for preparing a melatonin-rich pistachio seed / kernel extract according to one embodiment.

[0015] Figure 2A It is a chart showing the chromatograms of examples of standard substances; Figure 2B It is a graph showing the chromatograms of sample examples; and Figure 2C This is a graph showing the chromatogram of a blank example.

[0016] Figure 3A It is a chart showing melatonin absorption over 2 hours, and Figure 3B This is a chart showing melatonin absorption over 8 hours. Figures 3A-3B The data is created by recording any negative value as zero and using it as the group average for each specific time point. The data is expressed as mean ± SEM. Detailed Implementation

[0017] The foregoing and other features and advantages of the invention will become apparent from the following detailed description of exemplary embodiments, which are read in conjunction with the accompanying drawings. The detailed description and drawings are merely illustrative and not limiting of the invention, the scope of which is defined by the appended claims and their equivalents.

[0018] Embodiments of the invention will now be described with reference to the accompanying drawings, wherein like reference numerals always denote like elements. The terminology used in the description herein is not intended to be limiting or restrictive in any way, simply because it is used in conjunction with a detailed description of certain specific embodiments of the invention. Furthermore, embodiments of the invention may include several novel features, none of which is solely responsible for its desired properties or is essential for carrying out the invention described herein.

[0019] Unless otherwise stated herein or clearly contradicted by the context, the terms “a / an” and “the” and similar designations used in the context of describing the invention should be interpreted as encompassing both the singular and plural. It will also be understood that the terms “comprises,” “comprising,” “includes,” and / or “including” as used herein specify the presence of said features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0020] Unless otherwise stated herein, the descriptions of numerical ranges herein are intended only as a shorthand method for individually referring to each individual value falling within the range, and each individual value is incorporated into the specification as if it were described separately herein. When accompanied by numerical values, the term “about” should be interpreted as indicating a deviation from the indicated value of up to and including 10%. Unless otherwise stated, the use of any and all instances or exemplary language (“e.g.” or “such as”) provided herein is intended only to better illustrate the invention and does not constitute a limitation on the scope of the invention. Nothing in this specification should be construed as indicating that any unclaimed element is necessary for the practice of the invention.

[0021] References to "one embodiment," "implementation," "exemplary embodiment," "various embodiments," etc., may indicate that one or more embodiments of the invention described herein may include a particular feature, structure, or characteristic, but not every embodiment must include that particular feature, structure, or characteristic. Furthermore, repeated use of the phrases "in one embodiment" or "in an exemplary embodiment" does not necessarily refer to the same embodiment, although they may refer to the same embodiment.

[0022] As used herein, the term "method" refers to the manner, means, technique, and procedure for accomplishing a given task, including, but not limited to, those known to or readily developed by those skilled in the art of chemistry, pharmacology, biology, biochemistry, and manufacturing, from known methods, means, techniques, and procedures. Unless expressly stated otherwise, no method or aspect set forth herein is intended to be construed as requiring its steps to be performed in a particular order. Therefore, when a method claim does not specifically state in the claim or description that the steps are limited to a particular order, no order is implied in any respect. This applies to any possible non-explicit basis of interpretation, including logical questions about the arrangement of steps or operational procedures, simple meanings derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0023] Description of the implementation method Generally, methods for preparing melatonin from pistachios provide a more effective dose / serving than synthetically derived melatonin, enabling sleep regulation in an easily accessible form and consumption in dietary supplements or food products. Pistachios are a widely consumed starting material worldwide and are widely accepted as a health food. However, the fact that pistachios are a rich source of melatonin is not widely known. This method maximizes the melatonin content in the final extract by enriching the starting material, allowing an appropriate amount of melatonin composition to be consumed in the form of dietary supplements or food products to provide meaningful effects. This is the first time that a melatonin-rich product has been extracted from pistachios and presented in a form suitable for pharmaceutical and / or food delivery.

[0024] Some pistachio varieties contain high levels of melatonin, exceeding 12,000 pg / g. Furthermore, compared to other nuts, pistachios contain significantly higher levels of phytomelatonin, with some varieties reaching up to 223 µg / g. These findings highlight pistachio-derived melatonin-rich foods as a potential therapeutic option. The following examples evaluate the efficacy of naturally sourced melatonin gummies versus synthetic melatonin gummies in terms of melatonin absorption and sleep quality.

[0025] In one embodiment, the melatonin composition is bound to a complex herbal matrix comprising complementary compounds such as arginine, tryptophan, lutein, and resveratrol, and possessing high ORAC antioxidant capacity. Compared to synthetic melatonin, the melatonin composition bound to the complex herbal matrix increases the bioavailability, absorption, and efficacy of melatonin. This melatonin extract exhibits bioactivity against MT1 and MT2 receptors in terms of binding and signal transduction. The melatonin extract increases the activity of endogenous melatonin and enhances exogenous melatonin, thereby having a synergistic effect in treating sleep disorders. In one embodiment, treating sleep disorders comprises administering a small amount of the melatonin extract, in an amount of about 0.03 g to about 0.5 g, equivalent to about 300 mcg to about 5 mg of natural melatonin, to induce physiological effects in the human body.

[0026] This melatonin composition includes melatonin-rich pistachio seed / kernel extract, designed to meet the demand for plant-based melatonin that can provide a meaningful and effective dose of melatonin at a convenient dosage / serving.

[0027] like Figure 1AAs shown, a method 100 for preparing a melatonin-rich pistachio seed / kernel extract includes the following steps: Step 130, supercritical CO2 extraction to remove fatty acids and oil fractions from the seeds / kernels, thereby generating a melatonin extract; Step 140, washing the melatonin extract with water; Alternatively, Step 142, fermenting the milled seeds / kernels using yeast / culture medium; Step 150, extracting melatonin with a suitable solvent, which may include ethanol, methanol, water, acetone, hexane, butanol, and / or other suitable solvent mixtures; Step 160, repeating the extraction until process testing confirms that the melatonin determination meets the acceptance criteria; Step 170, concentrating the liquid extract solution. In one embodiment, the acceptance criterion is: not less than about 1.0% (NLT), as determined by high performance liquid chromatography (HPLC).

[0028] like Figure 1B As shown, a method 100 for preparing melatonin-rich pistachio (pistachio) seed / kernel extract includes: step 110, selecting crude pistachio (pistachio) seed / kernel raw material with an endogenous natural melatonin content between about 0.02% and about 0.04%; step 120, crushing and removing the outer shell; step 130, supercritical CO2 fluid extraction to remove fatty acids and oil fractions from the seeds / kernels; step 140, washing with water; and step 150, extraction with a suitable solvent, which may include ethanol, methanol, water, acetone, hexane, butanol, and / or others. A mixture of suitable solvents; step 160, repeating the extraction until process testing confirms that the melatonin determination meets acceptance criteria; step 170, concentrating the liquid extract solution; step 180, drying by a suitable method; step 190, standardizing until the final composition contains at least 1% melatonin; and step 200, applying quality control measures including herbal identification, melatonin potency determination, radiocarbon dating to ensure avoidance of synthetic additives, and testing for general contaminants (i.e., solvent residues, pesticide residues, aflatoxin, microorganisms, heavy metals, pyrrolizidine alkaloids, and polycyclic aromatic hydrocarbons). Standardization can be achieved by sub-batch blending or by using a suitable carrier / excipient. In one embodiment, the ratio of melatonin extract to natural extract is from about 16:1 to about 20:1, and in another embodiment, the ratio of melatonin extract to natural extract is from about 15:1. In one embodiment, the solvent is ethanol and water in a ratio of about 30:70.

[0029] A process overview for implementing this disclosure is provided in the Manufacturing Flow Chart, document number NNPVE151-MFC (valid version).

[0030] melatonin structure The chemical structure of melatonin is described by the United States National Institutes of Health National Library of Medicine - https: / / pubchem.ncbi.nlm.nih.gov / compound / Melatonin.

[0031] The chemical structure of the synthesized melatonin is identical to that found in this disclosure.

[0032] dose Melatonin compositions are available in capsule form for oral consumption. The melatonin compositions are free from any contaminants, including foreign matter, pesticides, bacterial pathogens, mold, residual solvents, and heavy metals. A Certificate of Analysis (COA) documents the analytical tests performed on the melatonin compositions.

[0033] The methods described herein may include administering an effective dose of a melatonin composition daily, every other day, or weekly. In embodiments, according to the invention, the melatonin composition may be administered daily for 1-2 weeks, 1-3 weeks, 1-12 weeks, 4-8 weeks, 8-12 weeks, 1-12 weeks, 4-12 weeks, 8-12 weeks, or 12-15 weeks or longer, or other time periods.

[0034] The melatonin composition of the present invention can be administered in combination with one or more nutritionally acceptable carriers and / or other excipients. The active ingredient in such a formulation may comprise 1% to 99% by weight, or alternatively 0.1% to 99.9% by weight. "Nutritionally acceptable carriers and / or other excipients" means any carrier, diluent, or excipient that is compatible with other components of the melatonin composition and is harmless to the user. According to one embodiment, suitable nutritionally acceptable carriers may include oils, vegetable oils, medium-chain triglyceride (MCT) oils, coconut oil, palm kernel oil, hemp seed oil, olive oil, avocado oil, and combinations thereof. Carriers may include dicalcium phosphate, rice flour, maltodextrin, microcrystalline cellulose, etc. Other excipients may include binders (such as hydroxypropyl cellulose, hydroxypropyl methyl cellulose, various gums, etc.), flow agents / anti-caking agents (such as silica, etc.), disintegrants (such as croscarmellose sodium, etc.), lubricants / release agents (such as magnesium stearate, calcium laurate, etc.), flavoring agents / sweeteners, coloring agents, or other excipients required for other dosage forms.

[0035] In some embodiments, the dosage form is formulated as granules, pills, microparticles, tablets, or hard-shell capsules suspended in a liquid, syrup, or enema. In some embodiments, the dosage form is formulated for oral or mucosal delivery. In some embodiments, the dosage form is formulated as lozenges, candies, gummies, chocolates, or cookies. In some embodiments, the tablets or pills are immediate-release, sustained-release, or controlled-release dosage forms. In some embodiments, the tablets are enteric-coated, or melt or dissolve in the mouth, or are mucosal adhesive dosage forms.

[0036] In some embodiments, the unit dosage form is a unit particle, such as a tablet, capsule, granule, pill, microparticle, or membrane, which is enterically coated or colonically coated to protect the unit dose from degradation in the acidic gastric pH and to allow timely swelling in a pH-controlled manner or both, to release the melatonin composition in the distal intestine.

[0037] In some embodiments, the melatonin composition is formulated as a semi-solid or liquid dosage form, such as a cream, lotion, ointment, dispersant, suspension, gel, foam, spray, syrup, liquid, eye drops, ear drops, enema, or oral dosage form, or topical dosage form, or topical ophthalmic or optical, or oral, vaginal, rectal, or uterine dosage form. Liquid formulations include solutions, suspensions, and emulsions, such as water or water-propylene glycol solutions. For example, parenteral liquid formulations can be formulated as solutions in aqueous polyethylene glycol solutions. Thus, compounds according to the invention can be formulated for parenteral administration (e.g., by injection, such as bolus injection or continuous infusion) and can be present in unit doses in ampoules, pre-filled syringes, small-volume infusion sets, or in multi-dose containers with added preservatives. The composition can be in the form of a suspension, solution, or emulsion in an oily or aqueous load and may contain formulations such as suspending agents, stabilizers, and / or dispersants. Alternatively, the active ingredient may be in powder form, obtained by aseptic separation of sterile solids or lyophilization from solution, for reconstitution with a suitable carrier, such as sterile, pyrogen-free water, prior to use.

[0038] In some embodiments, any of the above compositions or any of the above dosage forms are used in methods for treating insomnia or sleep disorders.

[0039] Preferred dosage forms include, but are not limited to, any liquid, semi-solid, or solid dosage form. Melatonin compositions can be formulated into pharmaceuticals by preparing topical, mucosal, or oral delivery systems. Topical delivery systems may be in the form of eye drops, suspensions, ointments, creams, foams, sprays, or topical patches. Oral delivery systems may be tablets, capsules, soft capsules, sachets, granules, or syrups. Mucosal delivery systems may be gels, vaginal suppositories, enemas, douches, lotions, foams, mucosal adhesive gels, or tablets for immediate release or sustained or controlled release. The loading medium may include any acceptable solvent and inactive ingredients, as well as preservatives, antioxidants, and colorants. Delivery forms may be single-dose or multi-dose and may include microparticles, nanoparticles, microcapsules, liposomes, micelles, etc., known in the pharmaceutical, cosmetic, veterinary, and formulation fields. Further details on suitable dosage forms are available from any standard reference work in the field, including, for example: Remington's Pharmaceutical Sciences, Mack Publishing Co, Easton, Pa, USA (1980).

[0040] Therefore, in some embodiments of the invention, the source of the melatonin composition further includes one or more excipients selected from the group consisting of solvents, stabilizers, suspending agents, emulsifiers, release modifiers, targeting agents, and viscosity agents, and combinations thereof. In one embodiment, the excipient is about 10% to about 25% maltodextrin.

[0041] Treatment The terms “treatment” or “enhancement” are used interchangeably herein. These terms can refer to methods of achieving beneficial or desired outcomes, including but not limited to therapeutic benefits and / or preventive benefits. A therapeutic benefit can refer to the eradication or improvement of the underlying disease being treated. Furthermore, a therapeutic benefit can be achieved by eradicating or improving one or more physical symptoms associated with the underlying disease, resulting in an improvement observed in the subject, although the subject may still suffer from the underlying disease. Preventive effects include delaying, preventing, or eliminating the onset of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For preventive benefits, subjects at risk of developing a specific disease, or subjects reported to have one or more physical symptoms of a disease, may receive treatment even if they have not yet been diagnosed with that disease.

[0042] As used herein, the term "synergistic" refers to the phenomenon where the cumulative pharmacological effect of two or more components used in combination is greater than the sum of the effects tested individually. As used herein, the term "enhancing" refers to the phenomenon where the efficacy of an active ingredient is significantly enhanced when combined with a second ingredient, wherein the second ingredient itself does not show any efficacy in the same pharmacological test. In some cases of enhancing effects, the second ingredient, when measured alone, may not only fail to produce a pharmacological effect but may even cause the opposite effect. An example of this is as follows: ingredient A has an anxiolytic effect; ingredient B has an anxiolytic effect; when A and B are combined, the combined anxiolytic effect is greater than that of A alone. In the context of this invention, enhancing effects are considered a special case of synergistic effects. Therefore, the term "synergistic effect" (or synergistic, etc.) in defining the properties of the compositions of this invention also includes, within its meaning, the enhancing effects just described above.

[0043] As used herein, the term "composition" refers to a pharmaceutically acceptable composition and means compounds, materials, compositions, and / or dosage forms that, within reasonable medical judgment, are suitable for contact with the tissues of mammals (especially humans) without excessive toxicity, irritation, allergic responses, or other problematic complications commensurate with a reasonable benefit / risk ratio. Formulations include configurational isomers (such as cis and trans isomers) and all optical isomers (such as enantiomers, diastereomers), racemic isomers, diastereomers, and other mixtures of these isomers, as well as solvates, hydrates, isomers, polymorphs, tautomers, esters, salt forms, and prodrugs. The term "prodrug" refers to a compound that serves as a drug precursor, releasing the drug in vivo through some chemical or physiological process after administration (e.g., the prodrug is converted to the desired drug form upon reaching physiological pH or by enzymatic action). Exemplary prodrugs release the corresponding free acid and hydrolyzable esterifying residues of the compounds of the present invention upon cleavage.

[0044] In this disclosure, the “effective amount” or “effective dose” of a melatonin extract or composition refers to the amount of melatonin extract extracted from pistachios that, once administered to a subject, would reach the subject’s bloodstream, intestines and / or body tissues.

[0045] In one embodiment, the method includes administering or ingesting a melatonin composition as a single formulation to enhance the synergistic effect between exogenous and endogenous melatonin. The method also includes combining the melatonin composition with an herbal matrix to form a formulation to enhance the synergistic effect in treating sleep disorders or improving sleep quality.

[0046] The methods described herein may include administering an effective dose of a melatonin composition having an herbal base daily, every other day, or weekly. In embodiments, according to the invention, daily administration of a melatonin composition having an herbal base may be continued for 1-2 weeks, 1-3 weeks, 1-12 weeks, 4-8 weeks, 8-12 weeks, 1-12 weeks, 4-12 weeks, 8-12 weeks, or 12-15 weeks or longer, or other time periods.

[0047] Treatment of sleep disorders involves administering an effective amount of pistachio melatonin extract and an effective amount of herbal matrix. "Sleep disorder" is any condition that causes difficulty in falling asleep and maintaining sleep, such as insomnia, and includes conditions related to: difficulty falling asleep due to anxiety, stress, and depression; difficulty falling asleep leading to poor concentration; difficulty falling asleep due to age-related memory loss, dementia, or other cognitive impairment; difficulty falling asleep due to substance abuse, mental illness, respiratory disorders, or other sleep disorders such as periodic limb movement disorder; and difficulty falling asleep or maintaining sleep due to other sleep-related problems such as poor sleep hygiene, including alcohol consumption. Consuming caffeinated beverages, eating large meals, or engaging in physical or mentally stimulating activities shortly before bedtime; poor sleep hygiene including extremely irregular bedtimes, or unsuitable temperature, poor ventilation, noise, or light in the sleep environment; including narcolepsy and rapid eye movement (REM) sleep behavior disorders; disorders associated with neurodegenerative diseases such as Alzheimer's, Parkinson's, and multiple sclerosis; REM sleep disorders associated with drug withdrawal (especially alcohol or sedative-hypnotic drug withdrawal); and circadian rhythm disorders associated with sleep apnea, shift work, and jet lag.

[0048] Complex herbal matrix The complex herbal matrix includes complementary compounds such as arginine, tryptophan, lutein, and resveratrol, and has high ORAC antioxidant capacity.

[0049] Arginine is an amino acid with the formula (H2N)(HN)CN(H)(CH2)3CH(NH2)CO2H. L-arginine is considered safe (GRAS status) when the daily intake is up to 20 grams. L-arginine (0.15-0.60 μmol) is a nitric oxide precursor and can regulate sleep, such as increasing slow-wave sleep and reducing wakefulness.

[0050] Tryptophan is an α-amino acid used in protein biosynthesis. It comprises an α-amino group, an α-carboxyl group, and an indole side chain, making it a polar molecule with nonpolar aromatic β-carbon substituents. Tryptophan is also a precursor to the neurotransmitter serotonin, the hormone melatonin, and vitamin B3. Tryptophan contributes to good sleep because the body uses it to synthesize serotonin (a neurotransmitter). Serotonin helps regulate sleep and is used in the synthesis of melatonin (a hormone that promotes sleep).

[0051] Lutein is a xanthophyll compound and one of the 600 known naturally occurring carotenoids. It has been shown to reduce the duration of sleep deprivation, i.e., less than 7 hours of sleep per day. Lutein and zeaxanthin are isomers, differing only in the position of a single double bond. Lutein and zeaxanthin can interconvert in vivo via an intermediate called meso-zeaxanthin. 100 g of pistachios contain 1,205 mcg of lutein.

[0052] Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a stilbene, belonging to the class of natural phenols or polyphenols and phytoprotective agents. Resveratrol exists in two geometric isomers: cis (Z) and trans (E), with the trans isomer shown in the image above. Resveratrol is usually conjugated with glucose. Resveratrol is known to induce sleep and reduce active-wake time and abnormal sleep patterns. Sleep observations and electroencephalogram (EEG) measurements indicate that resveratrol significantly improves non-REM sleep, which is considered the most important aspect of normal sleep.

[0053] Example The following embodiments are provided to provide those skilled in the art with a complete disclosure and description of how to prepare and evaluate the compounds, compositions, articles, apparatus, and / or methods claimed herein, and these embodiments are intended purely as examples of the invention and not to limit the scope of what the inventors consider to be their invention. However, based on this disclosure, those skilled in the art should understand that many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of the invention, and still obtain the same or similar results.

[0054] While efforts have been made to ensure the accuracy of figures (e.g., quantities, temperatures, etc.), some errors and deviations should be taken into account. Unless otherwise stated, parts are parts by weight, temperatures are °C or ambient temperature, and pressures are atmospheric pressure or close to atmospheric pressure.

[0055] Example 1: HPLC Analysis Method Objective: To describe a procedure for determining melatonin content in pistachios by UPLC-MS.

[0056] Table 1a

[0057] Figure 2A It is a chart showing the chromatograms of examples of standard substances; Figure 2B It is a graph showing the chromatograms of sample examples; and Figure 2C This is a graph showing the chromatogram of a blank example.

[0058] Melatonin extracts and / or herbal matrices exhibit bioactivity against MT1 and MT2 receptors in terms of binding and signal transduction. These melatonin extracts increase the activity of endogenous melatonin and enhance exogenous melatonin, thereby having a synergistic effect in treating sleep disorders. In one embodiment, treating sleep disorders comprises administering a small amount of melatonin extract, in an amount of about 0.03 g to about 0.5 g, equivalent to about 300 mcg to about 5 mg of natural melatonin, to induce a physiological effect in the human body.

[0059] Example 2: The effect of melatonin extract on melatonin absorption and sleep-inducing efficacy compared with the comparative substance. Effect—Randomized, Double-Blind, Crossover Study This study aims to evaluate, based on the above... Figure 1B The steps outlined in this study demonstrate the efficacy of naturally sourced melatonin (Prosomnial™) gummies prepared using these methods in terms of melatonin absorption and sleep quality compared to synthetic melatonin gummies. The assumption is that the naturally sourced melatonin prepared using these steps is equivalent to comparable synthetic melatonin in terms of absorption and its impact on sleep quality.

[0060] Research Products Table 1b: Research Products (IP).

[0061]

[0062] Participants took one gummy at each PK visit (Visits 2 and 3) and one gummy nightly for seven nights during each test product. There was a two-week washout period between each test product.

[0063] method Participants: This trial recruited 27 participants from Brisbane, Queensland who met the following screening criteria.

[0064] Inclusion criteria: Individuals aged 18 years and older; general health; BMI between 18.5 and 29.9 kg / m²; able to provide informed consent; experiencing difficulty falling asleep (>20 minutes); agreeing not to participate in other clinical trials during recruitment; agreeing not to change their current diet, frequency or intensity of exercise throughout the study; agreeing to avoid medications, supplements or foods for sleep, stress or anxiety, except for the product being tested, during the study; and women currently using prescription contraception (e.g., oral contraceptives).

[0065] Exclusion criteria: Use of prescription drugs, over-the-counter medications, or herbal supplements for sleep, stress, or anxiety; use of CBD or aromatherapy to manage sleep, stress, or anxiety; diagnosed chronic sleep disorders (e.g., insomnia, restless legs syndrome, sleep apnea); serious medical conditions. (For example, mood disorders such as depression, anxiety, or bipolar disorder; neurological disorders such as MS; kidney, liver, or heart conditions); unstable illnesses. (e.g., diabetes, thyroid dysfunction); currently diagnosed with malignant tumors (excluding basal cell carcinoma) or treated with chemotherapy / radiation within the past 2 years; using anticoagulants (e.g., coumarin / warfarin, heparin, dalteparin, enoxaparin, or low-dose aspirin); active smokers, nicotine users, or individuals who abuse medications (prescription or illegal); past or present long-term alcohol consumption (>14 drinks per week); pregnant or lactating women; allergy to any component of the active or comparative formulation; currently or recently (within the past month) participating in other clinical trials; any circumstances deemed unsuitable for participation by the investigator.

[0066] Serious illnesses are those that may endanger life, affect quality of life, or cause significant symptoms or treatment burden.

[0067] Unstable diseases are those that cannot be controlled by a stable drug dosage or whose severity fluctuates.

[0068] Recruitment and Screening: During the screening process, potential participants who do not meet the inclusion / exclusion criteria will be informed that they are not eligible. Interested individuals will review the study summary page and complete a short eligibility questionnaire. Participants deemed eligible will be provided with an electronic Participant Information and Consent Form (PICF) for review.

[0069] At least 24 hours after receiving PICT, participants will have a telemedicine consultation with the trial coordinator. During this process: participants will be screened according to inclusion / exclusion criteria; a detailed explanation and requirements of the trial procedures will be provided; and participants will be encouraged to ask questions, request clarification, and consult family or friends before providing informed consent.

[0070] Eligible participants who agree to participate sign an electronic informed consent form to confirm their understanding and willingness to participate in the recruitment.

[0071] Trial Procedure: Selected participants will be recruited and assigned a research product (IP) in a container labeled with a unique product number (based on a random code). Participants will be unaware of the product they are assigned to.

[0072] Study timeline: During the study period, participants were required to visit the RDC clinic three times: 1. Screening Phase (Remote): Telemedicine Screening and Informed Consent Form 2. Visit 1: Screening and Informed Consent: Conducted within 4 weeks prior to trial initiation. 3. Baseline 1: Sleep records were taken for 3 days prior to visit 2. 4. Visit 2: PK 1 Participants must arrive at the clinic on an empty stomach (no food after 10 PM the previous night).

[0073] A cannula is inserted into the antecubital vein to collect baseline blood samples.

[0074] Participants took the assigned IP with 250 mL of water.

[0075] Blood samples were collected at 10, 20, 30, 45, 60, 75, 90, 120, and 150 minutes, as well as at 3, 4, 6, and 8 hours after ingestion.

[0076] After all samples were collected, the cannula was removed.

[0077] Meals will be provided as follows: Breakfast: after taking IP; Lunch: 4 hours after blood sample collection; and snacks: in the afternoon (all food intake will be recorded by the trial coordinator).

[0078] Drinking water is unrestricted (you can drink as much as you want).

[0079] 5. Supplementation Phase 1: Take the assigned IP at home for 7 days. 6. Cleaning period: Two weeks between replenishment stages. 7. Baseline 2: Sleep records were taken for 3 days prior to visit 3. 8. Visit 3: PK 2: The procedure is the same as Visit 2. 9. Supplementation Phase 2: Take the assigned IP at home for 7 days.

[0080] 10. Final interview: conducted by telephone.

[0081] Participant Requirements: Participants were required to complete a 3-day baseline sleep diary before both Visit 2 and Visit 3 to establish a stable sleep pattern. Daily online sleep diaries were also required during each 7-day supplementary period. Unused IPs after Visit 2 were returned at Visit 3. Unused IPs after Visit 3 were destroyed at the end of the study.

[0082] Blood Sampling and Measurement: During the PK visit, blood samples were collected over 8 hours using the method described above. After the last sample was collected, the cannula was removed, and participants were monitored throughout the process. All dietary and fluid intake was controlled and recorded during the PK visit.

[0083] A stable 3-day sleep record was taken before each PK visit to ensure a baseline sleep pattern for comparison during the supplementary phase.

[0084] Table 2. Activity Schedule

[0085] 1 The height and weight reported during the telephone screening will be confirmed during the first visit. 2 Excluding height, 3 After sitting quietly for 5 minutes Result measurement Main results: Total melatonin uptake was measured via AUC0-8 in blood samples.

[0086] Secondary results: C via blood samples 最大 T via blood sample 最大 The study included changes in sleep quality as assessed by a sleep diary (T1 / 2 via blood sample), changes in sleep attack time as assessed by a consensus sleep diary (CSD-M), changes in sleep disturbances as assessed by a sleep diary, and the number and severity of adverse events as monitored by adverse events (AEs). result Demographic data This study recruited twenty-six participants, of whom twenty-four completed the pharmacokinetic (PK) component of Product 1 (Prosomnial) and twenty-three completed the PK component of Product 2 (Comparative). Among the participants who completed the PK component, 21 completed a 7-day sleep diary for both Product 1 (Prosomnial) and Product 2 (Comparative).

[0087] Because this study was a crossover design, there were no significant differences in characteristics between the groups at any point (Tables 3 and 4).

[0088] Table 3: Demographic data of participants.

[0089]

[0090] Table 4: Vital signs of participants at each visit.

[0091]

[0092] plasma melatonin There were no significant differences between groups when assessing absorption in the first 2 hours (p=0.4). Figure 3B ).

[0093] Table 6: Melatonin absorption data within 2 hours. Values ​​are based on each participant's specific AUC and T. 最大 and C 最大 Calculated Data is presented as an average.

[0094]

[0095] Sleep Diary There were no significant differences between the groups at baseline (Table 7). When considering the relative changes of each group relative to baseline, there were no significant differences between the groups for any measurement. Compared with baseline, the Prosomnial group showed significant changes in sleep latency, total sleep time, sleep quality, and perceived rest, while the comparison substance melatonin showed significant changes in perceived wakefulness and rest compared with baseline.

[0096] Table 7: Sleep diary data.

[0097]

[0098] Significant difference compared to baseline In summary, 100 mg of Prosomnial™ (containing 1 mg of naturally derived melatonin, delivered in the form of gummies / tablets) showed statistically significant improvements compared to baseline in sleep latency (time to fall asleep), total sleep time, sleep quality, and feelings of rest and refreshment.

[0099] All publications and patent applications mentioned in this specification are incorporated herein by reference to the same extent that each individual publication or patent application is specifically and individually indicated to be incorporated by reference.

[0100] While the invention has been described in conjunction with various embodiments, it should be understood that the invention is capable of further modifications. This application is intended to cover any variations, uses, or modifications of the invention that generally follow the principles of the invention and include deviations from this disclosure within the scope of practice known and customary in the art to which this invention pertains.

Claims

1. A method for preparing a melatonin-rich Pistacia vera (pistachio) seed / kernel extract, comprising: Melatonin extract was obtained by removing fatty acids and oil fractions from seeds / kernels using supercritical fluids. Wash the melatonin extract with water; Seeds / kernels fermented with yeast / culture medium; Melatonin was extracted using multiple solvents; extraction was repeated until process testing confirmed that the melatonin determination met the acceptance criteria; and the liquid extract solution was concentrated.

2. The method according to claim 1, wherein, The solvents are selected from the group consisting of ethanol, methanol, water, acetone, hexane, butanol and / or other suitable solvents.

3. The method according to claim 2, wherein, The supercritical fluid extraction is performed using CO2.

4. A method for preparing a melatonin-rich pistachio seed / kernel extract, comprising: Select pistachio seeds / kernels as crude raw material with an endogenous natural melatonin content between about 0.02% and about 0.04%; crush and remove the outer shell of the pistachios; use supercritical fluid extraction to remove fatty acids and oil fractions from the seeds / kernels; wash the melatonin extract with water; Melatonin was extracted using multiple solvents; extraction was repeated until process testing confirmed that the melatonin determination met acceptance criteria; the liquid extract was concentrated; the solution was dried using a suitable method; and the final composition was standardized to contain at least 1% melatonin. And apply quality control measures, including herbal identification, melatonin potency determination, radiocarbon testing to ensure the avoidance of synthetic additives, and testing for general contaminants.

5. The method according to claim 4, wherein, The quality control measures include solvent residues, pesticide residues, aflatoxin, microorganisms, heavy metals, pyrrolizidine alkaloids, and polycyclic aromatic hydrocarbons.

6. The method according to claim 5, wherein, Standardization steps are achieved through sub-batch blending or by using a suitable carrier / excipient.

7. A method for increasing sleep efficacy in subjects, comprising: Apply an effective amount of melatonin extract from pistachios; Furthermore, compared to synthetic melatonin extracts, melatonin extract from pistachios increased sleep efficacy in subjects.

8. The method of claim 7, further comprising applying an effective amount of an herbal matrix, said herbal matrix comprising arginine, lutein, resveratrol and tryptophan.

9. The method according to claim 8, wherein, The melatonin extract comprises 1 mg in the form of gummies; the melatonin extract was administered for a period of 7 days; a baseline sleep was measured without the administration of the melatonin extract; and the melatonin extract showed statistically significant improvements beyond baseline in sleep latency, total sleep time, sleep quality, and feelings of rest and refreshment; wherein sleep latency is the time required to fall asleep.

Images