Compositions and methods for promoting mental health

Abstract

This disclosure generally relates to compositions for promoting mental health, particularly compositions derived from plant extracts for use in sedating, invigorating, or promoting feelings of well-being or euphoria in an individual. The disclosed methods include promoting mental health by administering plant-derived extracts, such as extracts derived from Labisia pumila or Lignosus rhinosaras, or combinations of extracts derived from the plants Labisia pumila and Lignosus rhinosaras. Such methods are non-therapeutic. The disclosed compositions include extracts derived from Labisia pumila or Lignosus rhinosaras for use in promoting mental health. Systems and methods for testing whether a substance promotes mental health are also disclosed.

Description

Technical Field

[0001]

[0002] The present disclosure generally relates to compositions and methods for promoting mental health, particularly compositions derived from plant extracts or fungal extracts for use in calming, energizing or enhancing feelings of well-being or happiness in an individual.

Background Art

[0002]

[0003] The following discussion of the background of the invention is for the sole purpose of facilitating an understanding of the invention. It is to be recognized that no admission is made that any of the material referred to was published, known or part of the common general knowledge in any jurisdiction as of the priority date of the invention.

[0003]

[0004] Modern life can be highly stressful and extremely fatiguing. It is difficult for an individual to find ways to relax, unwind, energize and feel more well-being or happiness. Some individuals may use drugs or alcohol to achieve such feelings to some extent, but these can have an impact on the individual's health in the long term or are not legal in some cases.

[0004]

[0005] The recent COVID-19 pandemic has had a significant impact on people worldwide, not only physically but also in terms of their mental health. Due to the spread of the coronavirus, people were often unable to go out and were forced to stay at home for over two years. This restrictive lifestyle has raised concerns about its impact on individual mental health, including stress accumulation, anxiety, and depressive symptoms. The World Health Organization (WHO) reported a 25% increase in global prevalence of anxiety and depression in the first year of the COVID-19 pandemic. Numerous studies have investigated the psychological impact of COVID-19 on healthcare workers and the general public, reporting high levels of problems in these areas. The decline in mental health was found to be more severe among the general population than among healthcare workers and students. A meta-analysis of these studies reported pooled prevalence rates of 33% for anxiety and 28% for depression. Anxiety levels among university students were investigated in Malaysia (Irfan et al., The Psychological Impact of Coronavirus on University Students and its Socio-Economic Determinants in Malaysia. Inquiry. 2021; 58:469580211056217), and the results showed that 12.3% of students were normal, 30.5% experienced mild anxiety, 31.1% experienced moderate anxiety, and 26.1% experienced severe anxiety. A similar experiment was conducted in Thailand, where the COVID-19 pandemic negatively impacted anxiety symptoms among Thai students (Tiaprapong et al., PLoS One. 2021; 16(6): 2021). The study strongly supports evidence that Thai healthcare student workers were similar to students in other countries who exhibited significant sedentary behavior, weight gain, and decreased mental health.

[0005]

[0006] Mental health issues among healthcare workers include excessive stress and mental stress, long working hours, separation from family, lack of appropriate protective equipment in the work environment, and lack of effective treatment. Burnout levels and factors associated with distress among healthcare workers engaged in COVID-19 management in India were reported (Menon et al., PLoS One 2022;17(3)2022). The report found that 52.9% of those involved experienced distress. One study found that among seven middle-income Asian countries (China, Iran, Malaysia, Pakistan, the Philippines, Thailand, and Vietnam), Thailand reported the highest anxiety, depression, and stress scores (Wang et al., PLoS One 2021;16(2)2021). In contrast, the same report showed that Vietnam had the lowest average scores on the anxiety, depression, and stress scales.

[0006]

[0007] Extracts from Labisia pumila, also known as Kacipfatimah, are thought to be effective for female reproductive disorders (Chua et al., Fitoterapia, 83(8), pp. 1322-135, 2012; Zakaria et al., BioMed Res. International, 2021, 9928199). Lignosus rhinoceros, or tiger milk mushroom, is a medicinal fungus. Extracts from Scerotia have been used to treat asthma, cough, fever, liver-related diseases, and gastric ulcers (Chang and Lee 2004, Fungal diversity 15: pp. 15-22). Lignosus rhinosalas extract has been shown to possess antioxidant properties and neuroprotective properties in nematodes (C. elegans), which have led to its potential use in cancer treatment (Kittimonkolsuk et al., Pharmaceuticals 27;14(2):93, 2021).

[0007]

[0008] There is a need to find alternative compounds or compositions that promote mental health and / or alleviate at least one of the aforementioned problems. [Overview of the project]

[0008]

[0009] Compositions and methods for promoting mental health using the same substance are conceivable.

[0009]

[0010] Accordingly, aspects of the present invention refer to a method for promoting mental health, comprising delivering a composition comprising a powder extract, optionally soluble in a solvent, which is (a) extracted from the plant Labisia pumila or the fungus Lignosus rhinosalas, and (b) capable of inhibiting cyclooxygenase receptors by at least 30%.

[0010]

[0011] According to another aspect of the present invention, there is a composition comprising a powder extracted from the plant Labisia pumila or the fungus Lignosus rhinosala, which can inhibit cyclooxygenase receptors by at least 30% and promote mental health, and which can be formulated as a food preparation, nutritional supplement, or beverage.

[0011]

[0012] According to another aspect of the present invention, there is a food preparation comprising the composition described herein for use in promoting mental health.

[0012]

[0013] According to another aspect of the present invention, a system for testing the effect of a composition comprising a powder extract derived from a living organism on promoting mental health, A home cage for feeding, a) An open field arena having red / near-infrared light that can illuminate the arena using a computer video tracking system that uses a near-infrared sensing camera capable of recording the arena; b) A chamber having a light region and a dark region, with a door between the light region and the dark region, and a sensor on the door between the light region and the dark region; c) A maze having open areas, enclosed areas surrounded by side walls, and elevated areas; a chamber equipped with a near-infrared sensing camera capable of recording each of the open areas, enclosed areas, and elevated areas; d) Two small ceramic cups placed at both ends of the home cage; e) A Y-shaped maze having three arms angled 120° to each other, equipped with a computer video tracking system capable of recording the movement of each arm; f) A component located above the sensing range, a fixing device connected to the component for securing the animal's tail; and a base equipped with a computer video tracking system capable of recording movement within the sensing range; and g) A transparent plexiglass cylinder having a base into which water can be placed, and comprising a computer video tracking system capable of recording movement within the transparent plexiglass cylinder. There is a system that includes a series of test containers.

[0013]

[0014] According to another aspect of the present invention, a method for testing the effect of a composition on mental health, a) Step of extracting a powder extract from an organism; b) The step of preparing the powdered extract as a food preparation, nutritional supplement, or beverage; c) A step in which a food preparation, nutritional supplement, or beverage is fed to a test subject in a home cage, and a similar food preparation, nutritional supplement, or beverage that does not contain the powder extract is fed to a control subject; d) A step of placing the test subject and control subject in an open field arena, which is illuminated with red / near-infrared light that provides illumination of less than 20 lux in the arena, and recording the movements of the test subject and control subject; e) A step of administering the test subject and control subject for at least two days to allow them to recover and to provide a handling interaction between the test subject and the control subject; f) A step in which the test subject and the control subject are placed in a chamber having a light area and a dark area, with a door between the light areas, and the movement of the test subject and the control subject between the light area and the dark area is measured; g) A step of placing the test subject and control subject in a chamber equipped with a maze having an open area, a closed area enclosed by side walls, and an elevated area, and recording the movement of the test subject and control subject in each of the open area, closed area, and elevated area; h) Place two small ceramic cups at opposite ends of the home cage, filling the first small ceramic cup with a food preparation, nutritional supplement, or beverage, and the second small ceramic cup with a food preparation, nutritional supplement, or beverage that does not contain powder extracts, and measure which food preparation, nutritional supplement, or beverage the test subject and control subject prefer; i) A step in which the test subject and control subject are placed in a Y-shaped maze having three arms at 120° angles to each other, and the movement of the test subject and control subject is recorded in each arm; j) The tails of the test subject and the control subject are fixed to a stationary device connected to a component located above the sensing range, the test subject and the control subject are suspended at least 25 cm above the base, and the movement of the test subject and the control subject within the sensing range is recorded; k) A step of providing the test subject and control subject with at least two days to allow them to recover and to provide a handling interaction between the test subject and the control subject; and l) Place the test subject and control subject inside a transparent plexiglass cylinder having a base partially filled with water, and record the movement of the test subject and control subject in the transparent plexiglass cylinder underwater. Includes, Between each of steps d) to l), the test subjects and control subjects are returned to their home cages, and the test subjects are fed a food preparation, nutritional supplement, or beverage, while the control subjects are fed a similar food preparation, nutritional supplement, or beverage that does not contain the powder extract.

[0014]

[0015] Other aspects and features of the present invention will become apparent to those skilled in the art by examining the following description of a particular embodiment of the invention in conjunction with the accompanying drawings.

[0015]

[0016] The drawings illustrate embodiments of the present invention by non-limiting embodiments only.

Brief Description of Drawings

[0016] [Figure 1] Cell viability of R-hth-507 cells in the first screening. R-hth-507 cells were treated with 50 μg / mL aqueous extracts of each sample. Cell viability after 24-hour treatment was measured by MTT assay. Control; MQ, *; p < 0.05, **: p < 0.01. Mean ± SD, n = 3. [Figure 2] Cell viability of R-hth-507 cells in the second screening. R-hth-507 cells were treated with aqueous extracts of different concentrations of each sample. Cell viability after 24-hour treatment was measured by MTT assay. Control; MQ, *; p < 0.05, **: p < 0.01. Mean ± SD, n = 3. [Figure 3A] Expression level of proopiomelanocortin gene in R-hth-507 cells. Relative values of POMS gene expression were estimated by real-time PCR. Control; MQ, *; p < 0.05, **: p < 0.01. Mean ± SD, n = 3. [Figure 3B] The functional evaluation test was conducted on 20.0 μg / ml extract samples of Rabicia pumila or Lignosus rhinocerus. The targeting inhibitory activity against human cyclooxygenase (COX1(h)) is listed as a percentage of the control. [Figure 4] Schematic flowchart of the steps to make plant extracts from Rabicia pumila or Lignosus rhinocerus. [Figure 5] HPLC results for leaf and stem extracts of Rabicia pumila. [Figure 6] Diagrams of relaxation tests (A) light-dark box test and (B) elevated zero maze. [Figure 7]Panel A. Average time spent in the light box. Control group (c), 0.25% Lavandula pumila leaf extract group (KP25), 0.5% Lavandula pumila leaf extract group (KP50), 0.25% Lignosus rhinocerus sclerotium extract group (TMM25), 0.5% Lignosus rhinocerus sclerotium extract group (TMM50), Panel B. Average time spent in the open arms. There was no significant difference in the time spent in the open arms between the herb diet group and the control. For all extract diet groups, N = 10 (5 males and 5 females). For the control group, N = 20 (10 males and 10 females). [Figure 8] Schematic diagrams of the (A) open field test and (B) Y - maze for the arousal test. [Figure 9] Panel A. Distance traveled in the dark open field test (OFT). Control group (c), 0.25% Lavandula pumila leaf extract group (KP25), 0.5% Lavandula pumila leaf extract group (KP50), 0.25% Lignosus rhinocerus sclerotium extract group (TMM25), 0.5% Lignosus rhinocerus sclerotium extract group (TMM50), Panel B. Percent alternation in the maze. There was no significant difference between the herb diet group and the control. For all extract diet groups, N = 10 (5 males and 5 females). For the control group, N = 20 (10 males and 10 females). [Figure 10] Schematic diagrams of the happiness tests: (A) tail suspension test, (B) forced swim test and (C) diet preference test. [Figure 11] Panel A. Average percentage of herb diet consumed relative to total diet consumed. Panel B. Average percent time spent immobile in the forced swim test. Panel C. Average percent time spent immobile in the tail suspension test. Control group (c), 0.25% Lavandula pumila leaf extract group (KP25), 0.5% Lavandula pumila leaf extract group (KP50), 0.25% Lignosus rhinocerus sclerotium extract group (TMM25), 0.5% Lignosus rhinocerus sclerotium extract group (TMM50). For all plant extract diet groups, N = 10 (5 males and 5 females). For the control group, N = 20 (10 males and 10 females). [Figure 12] Schematic diagram of the collection of serum and brain tissues for the detection and analysis of dopamine, serotonin and corticosteron. [Figure 13] Mean tissue levels of dopamine. Control group (c), 0.25% Labisia pumila leaf extract group (KP25), 0.5% Labisia pumila leaf extract group (KP50), 0.25% Lignosus rhinosala sclerotia extract group (TMM25), 0.5% Lignosus rhinosala sclerotia extract group (TMM50). N=6-8 (balanced males and females) in all plant extract diet groups. N=10 (5 males and 5 females) in the control group. [Figure 14] Mean serum serotonin levels. Control group (c), 0.25% Labisia pumila leaf extract group (KP25), 0.5% Labisia pumila leaf extract group (KP50), 0.25% Lignosus rhinosala sclerotia extract group (TMM25), 0.5% Lignosus rhinosala sclerotia extract group (TMM50). N=4-6 (balanced males and females) in all extract diet groups. N=8 (4 males and 4 females) in the control group. [Figure 15] Serum corticosterone concentration. N=6 (3 males and 3 females) in all plant extract diet groups. Control group (c), 0.25% Labisia pumila leaf extract group (KP25), 0.5% Labisia pumila leaf extract group (KP50), 0.25% Lignosus rhinosala sclerotia extract group (TMM25), 0.5% Lignosus rhinosala sclerotia extract group (TMM50). N=8 (4 males and 4 females) in the control group. [Modes for carrying out the invention]

[0017]

[0032] Throughout this document, unless otherwise indicated, terms such as “comprising,” “consisting of,” “having,” and similar terms should be interpreted as not exhaustive, or in other words, “including, but not limited to.”

[0018]

[0033] Furthermore, throughout the document, unless the context requires a different interpretation, variations of the word "include," "includes," or "including" are understood to mean the inclusion of the integer or group of integers mentioned, but not the exclusion of any other integer or group of integers.

[0019]

[0034] In various embodiments, a method for promoting mental health includes administering an extract selected from Labisia pumila and Lignosus rhinosalas, and the method is a non-therapeutic method.

[0020]

[0035] As used herein, the term “enhancing mental health” relates to elevating and improving a person’s mood in a non-therapeutic way. In various embodiments, enhancing mental health includes making a person who takes or ingests the extract feel relaxed. In various embodiments, enhancing mental health includes making a person who takes or ingests the extract feel energetic. In various embodiments, enhancing mental health includes making a person who takes or ingests the extract feel happy. In various embodiments, enhancing mental health includes making a person who takes or ingests the extract feel relaxed and happy. In various embodiments, enhancing mental health includes making a person who takes or ingests the extract feel energetic and happy. In various embodiments, enhancing mental health includes reducing or lowering dopamine levels in a person who takes or ingests the extract. In various embodiments, enhancing mental health includes reducing or lowering serotonin or corticosterone levels in the serum of a person who takes or ingests the extract. In various embodiments, enhancing mental health includes increasing dopamine levels in a person who takes or ingests the extract. In various embodiments, promoting mental health includes increasing serotonin or corticosterone levels in the serum of a person taking or ingesting the extract. In various embodiments, promoting mental health includes anxiety-inducing effects that cause a person taking or ingesting the extract to feel anxious or vigilant. In various embodiments, promoting mental health is selected from the group consisting of anxiety-inducing effects, vigilance, joy, excitement, invigoration, relaxation, and well-being.

[0021]

[0036] In various embodiments, the composition is extracted from either Labisia pumila or Lignosus rhinosaras for use in promoting mental health.

[0022]

[0037] In various embodiments, a method for promoting mental health comprises the step of delivering a composition comprising a powder extract, optionally dissolved in a solvent, wherein the composition is (a) extracted from the plant Labisia pumila or the fungus Lignosus rhinosalas, and (b) capable of inhibiting cyclooxygenase receptors by at least 30%.

[0023]

[0038] As used herein, the term “extract” refers to any preparation obtained from the plant Labisia pumila or the fungus Lignosus rhinosalas. In various embodiments, the extract may be obtained using the process outlined in [Figure 4] or a similar extraction method that produces a dry powder extract. In various embodiments, the average particle size of the powder extract includes at least 70 μm, or at least 71 μm, or at least 72 μm, or at least 73 μm, or at least 74 μm, or at least 75 μm. In various embodiments, the average particle size of the powder extract includes 70–100 μm, or 71–100 μm, or 72–100 μm, or 73–100 μm, or 74–100 μm, or 75–100 μm.

[0024]

[0039] In various embodiments, the solvent is water. In various embodiments, the solvent is ethanol or a mixture of ethanol and water.

[0025]

[0040] In various embodiments, the composition can increase the viability of PC-12 cells in vitro by at least 130%. Increasing the viability of PC-12 cells in vitro is a source of pleasure. In various embodiments, increasing the viability of PC-12 cells includes increasing the viability of PC-12 cells in vitro by 130% to 150%, 135% to 150%, 138% to 148%, or at least 139% to 147%.

[0026]

[0041] In various embodiments, the compositions can inhibit the cyclooxygenase receptor by at least 35%, or at least 36%, or at least 37%, or at least 38%, or at least 39%, or at least 40%, or at least 45%, or at least 50%, or at least 55%. Cyclooxygenase inhibition can be determined by measuring oxygen consumption, peroxidase co-substrate oxidation, prostaglandin (PG) detection, or by quantifying PGE2 produced by the enzymatic conversion of arachidonic acid in or without a potential inhibitor, or by any other method known in the art.

[0027]

[0042] In various embodiments, the method includes extracting the composition from the leaves and / or stems of Labisia pumila. In various embodiments, the method for extracting the composition from the leaves and / or stems of Labisia pumila may follow the method outlined in Figure 4.

[0028]

[0043] In various embodiments, the method includes determining that 0.1% to 0.2% gallic acid is present in the extract prior to delivery of the composition. In various embodiments, gallic acid may be determined by any known method, including spectroscopy, chromatography, and capillary electrophoresis, or by any other method developed to identify and quantify gallic acid in most biological matrices. In various embodiments, gallic acid may be determined by high-performance liquid chromatography (HPLC). In various embodiments, determining the amount of gallic acid in the extract is only necessary if it is necessary to ensure that the powder extract contains a composition that can promote mental health.

[0029]

[0044] In various embodiments, the method involves ingesting the described Labisia pumila extract to enhance mental health, and the enhancement of mental health includes an anxiety-inducing effect that results in a feeling of anxiety. Such a feeling has the benefit of increasing or stimulating the individual's vigilance.

[0030]

[0045] In various embodiments, the method involves extracting the composition from the sclerotia of Lignosus rhinosarasu. In various embodiments, the composition is extracted only from Lignosus rhinosarasu sclerotia having a pore size of 6-7 pores / mm. This allows for accurate classification of Lignosus rhinosarasu prior to extraction. In various embodiments, the method for extracting the composition from the sclerotia of Lignosus rhinosarasu may follow the method outlined in [Figure 4].

[0031]

[0046] In various embodiments, the method includes determining the presence of SEQ ID NO: 1 in the sclerotia of *Lignosus rhinosaras* prior to extraction of the composition. The nucleotide sequence shown in SEQ ID NO: 1 includes a unique nucleotide sequence in the internal transcription spacer (ITS) ITS1 region located between rRNA structural genes (bases 149-168 in the nucleic acid sequence shown in SEQ ID NO: 1). This allows *Lignosus rhinosaras* to be accurately classified prior to extraction. In various embodiments, the presence of SEQ ID NO: 1 may be determined using PCR primers (5'-GTTTTCAGCCGGCGTTTGC-3' shown in SEQ ID NO: 2) and (5'-TCCTCCGCTTATTGATATGC-3' shown in SEQ ID NO: 3). In various embodiments, determining the presence of SEQ ID NO: 1 in the extract is only necessary if it is necessary to ensure that the powder extract contains a composition that can promote mental health.

[0032]

[0047] In various embodiments, the method comprises taking or ingesting the described Lignosus rhinosalas extract, and enhancing mental health includes experiencing pleasure from increased levels of dopamine. In various embodiments, the method comprises measuring dopamine levels.

[0033]

[0048] In various embodiments, the method comprises taking or ingesting the described Lignosus rhinosarasu extract, and the enhancement of mental health comprises feeling excited from increased levels of serum corticosterone. In various embodiments, the method comprises measuring serum corticosterone levels before and after taking or ingesting the described Lignosus rhinosarasu extract to confirm that it has enhanced mental health in the individual taking or ingesting the described Lignosus rhinosarasu extract.

[0034]

[0049] In various embodiments, the method includes the step of preparing the composition as a food preparation, nutritional supplement, or beverage.

[0035]

[0050] In various embodiments, the term "food preparation" generally refers to a material of plant or animal origin, or a synthetic source, that contains essential nutrients, carbohydrates, proteins, fats, vitamins, minerals, and other elements that can be consumed or ingested by living organisms such as individuals in order to sustain growth, recovery, life-sustaining processes, or to provide energy.

[0036]

[0051] In various embodiments, the term “nutritional supplement” generally refers to a product containing substances such as vitamins, minerals, nutrients, botanicals, and amino acids that are intended to complement the food and / or beverages consumed or ingested by organisms such as individuals in order to sustain growth, recovery, life-sustaining processes, or to supply energy.

[0037]

[0052] In various embodiments, the term "beverage" generally refers to a liquid intended for consumption. In various embodiments, the liquid may be water, flavored water, soft drinks, alcoholic beverages, health drinks, concentrated beverages, dairy beverages such as milk or yogurt, fruit juices, vegetable juices, or any combination thereof, which can be consumed or ingested by living organisms such as individuals.

[0038]

[0053] In various embodiments, the method includes delivering the composition orally. In various embodiments, the method includes delivering the composition nasally. In various embodiments, the method includes delivering the composition as a gas or aerosol. In various embodiments, the method includes delivering the composition intravenously. In various embodiments, the composition is delivered orally. In various embodiments, other known delivery methods may be used to deliver the dose that produces enhanced mental health or mood.

[0039]

[0054] In various embodiments, the composition comprises a powder extracted from the plant Labisia pumila or the fungus Lignosus rhinosalas, and the composition can inhibit cyclooxygenase receptors by at least 30%, promote mental health, and the composition is formulated as a food preparation, nutritional supplement, or beverage.

[0040]

[0055] In various embodiments, the extracted powder is extracted from the leaves and / or stems of Labisia pumila and contains 0.1% to 0.2% gallic acid of the total powder extract. In various embodiments, the amount of gallic acid in the extract is detected solely to ensure that the powder extract contains a composition that can promote mental health.

[0041]

[0056] In various embodiments, the extract derived from Labisia pumila, when eluted with 50% water:50% pure methanol at a concentration of 10 mg / ml of extract, includes one or more HPLC peaks containing gallic acid with a retention time of 4–5 minutes and a second peak with a retention time of 9–11 minutes. In various embodiments, the extract derived from Labisia pumila, when eluted with 50% water:50% pure methanol at a concentration of 10 mg / ml of extract, includes an HPLC peak of gallic acid with a retention time of 4–5 minutes and one or more additional peaks having a pattern substantially similar to the peak pattern depicted in [Figure 5].

[0042]

[0057] In various embodiments, the composition is formulated as a food preparation, nutritional supplement, or beverage as described herein.

[0043]

[0058] In various embodiments, the powder extracted from the Labisia pumila plant or the Lignosus rhinosala fungus contains at least 25% by weight of a food preparation, nutritional supplement, or beverage. In various embodiments, at least 25% by weight represents the weight of the food preparation, nutritional supplement, or beverage per unit of total weight. In various embodiments, at least 25% by weight represents the weight of the food preparation, nutritional supplement, or beverage per unit of total volume. In various embodiments, at least 25% by weight represents 25% to 51% by weight of the food preparation, nutritional supplement, or beverage per unit of total weight. In various embodiments, at least 25% by weight represents 25% to 51% by weight of the food preparation, nutritional supplement, or beverage per unit of total volume.

[0044]

[0059] In various embodiments, the food preparation includes compositions described herein for use in promoting mental health.

[0045]

[0060] In various embodiments, the powder extracted from the Labisia pumila plant or the Lignosus rhinosala fungus contains at least 25% by weight of a food preparation. In various embodiments, at least 25% by weight includes the weight of the food preparation per unit of total weight. In various embodiments, at least 25% by weight includes the weight of the food preparation per unit of total volume. In various embodiments, at least 25% by weight includes 25% to 51% by weight of the food preparation per unit of total weight. In various embodiments, at least 25% by weight includes 25% to 51% by weight of the food preparation per unit of total volume.

[0046]

[0061] According to various embodiments, a system for testing the effect of a composition containing a powder extract derived from an organism on promoting mental health, comprising a home cage for feeding, h) An open field arena having red / near-infrared light that can illuminate the arena using a computer video tracking system that uses a near-infrared sensing camera capable of recording the arena; i) A chamber having a light region and a dark region, with a door between the light region and the dark region, and a sensor on the door between the light region and the dark region; j) A maze having open areas, enclosed areas surrounded by side walls, and elevated areas; a chamber equipped with a near-infrared sensing camera capable of recording each of the open areas, enclosed areas, and elevated areas; k) Two small ceramic cups to be placed at both ends of the home cage; l) A Y-shaped maze having three arms angled 120° to each other, equipped with a computer video tracking system capable of recording movement in each arm; m) A component located above the sensing range, a fixing device connected to the component for securing the animal's tail; and a base equipped with a computer video tracking system capable of recording movement within the sensing range; and n) A transparent plexiglass cylinder having a base into which water can be placed, and comprising a computer video tracking system capable of recording movement inside the transparent plexiglass cylinder. A system comprising a series of test containers, including a set of test containers.

[0047]

[0062] According to various embodiments, the test container is the same as the test container depicted in Figures 6A and B, Figures 8A and B, and Figures 10A, B, and C.

[0048]

[0063] According to various embodiments, a method for testing the effects of a composition on mental health, a) Step of extracting a powder extract from an organism; b) The step of preparing the powdered extract as a food preparation, nutritional supplement, or beverage; c) A step in which a food preparation, nutritional supplement, or beverage is fed to a test subject in a home cage, and a similar food preparation, nutritional supplement, or beverage that does not contain the powder extract is fed to a control subject; d) A step of placing the test subject and control subject in an open field arena, which is illuminated with red / near-infrared light that provides illumination of less than 20 lux in the arena, and recording the movements of the test subject and control subject; e) A step of administering the test subject and control subject for at least two days to allow them to recover and to provide a handling interaction between the test subject and the control subject; f) A step in which the test subject and the control subject are placed in a chamber having a light area and a dark area, with a door between the light areas, and the movement of the test subject and the control subject between the light area and the dark area is measured; g) A step of placing the test subject and control subject in a chamber equipped with a maze having an open area, a closed area enclosed by side walls, and an elevated area, and recording the movement of the test subject and control subject in each of the open area, closed area, and elevated area; h) Place two small ceramic cups at opposite ends of the home cage, filling the first small ceramic cup with a food preparation, nutritional supplement, or beverage, and the second small ceramic cup with a food preparation, nutritional supplement, or beverage that does not contain powder extracts, and measure which food preparation, nutritional supplement, or beverage the test subject and control subject prefer; i) A step in which the test subject and control subject are placed in a Y-shaped maze having three arms at 120° angles to each other, and the movement of the test subject and control subject is recorded in each arm; j) The tails of the test subject and the control subject are fixed to a stationary device connected to a component located above the sensing range, the test subject and the control subject are suspended at least 25 cm above the base, and the movement of the test subject and the control subject within the sensing range is recorded; k) A step of providing the test subject and control subject with at least two days to allow them to recover and to provide a handling interaction between the test subject and the control subject; and l) Place the test subject and control subject inside a transparent plexiglass cylinder having a base partially filled with water, and record the movement of the test subject and control subject in the transparent plexiglass cylinder underwater. Includes, A method in which, between each step of d) to l), the test subjects and control subjects are returned to their home cages, the test subjects are fed a food preparation, nutritional supplement, or beverage, and the control subjects are fed a similar food preparation, nutritional supplement, or beverage that does not contain the powder extract.

[0049]

[0064] In various embodiments, the tests should be performed sequentially from a) to l) and / or at least from d) to l), providing the tests in a specific order listed in steps d) to l) or a) to l), allowing the test subjects and control subjects to be gradually exposed to stressful tests. This has the advantage of minimizing potential interactions between tests, as stress levels from a preceding test do not adversely affect subsequent tests.

[0050]

[0065] In various embodiments, steps d) and i) are suitable for testing the invigorating effect of the test composition. In various embodiments, steps f) and g) are suitable for testing the relaxing and sedative effect of the test composition. In various embodiments, steps h), j) and l) are suitable for testing the well-being and invigorating effect of the test composition.

[0051]

[0066] In various embodiments, the organism from which the test composition is extracted may be a plant, a fungus, or an animal. In various embodiments, the test composition may include a powder extracted from the Labisia pumila plant or the Lignosus rhinosaras fungus described herein.

[0052]

[0067] In various embodiments, the test subject is a mouse or rat, and the control subject is the same species or variant of the mouse or rat used in the test subject. In various embodiments, the test composition or powder extract is incorporated into the diet so that the intake of each animal is expected to be 0.25% or 0.5% of the dietary composition. Sequence ID 1, Internal transfer spacer of Lignosus rhinosarasu: . Sequence ID 2, Forward primer for internal transfer spacer of Lignosus rhinosaras: 5'-GTTTTCAGCCGGCGTTTGC-3' Sequence ID 3, Reverse primer for internal transfer spacer of Lignosus rhinosaras: 5'-TCCTCCGCTTATTGATATGC-3' Sequence ID 4, Forward primer for POMC: 5'-CCTCACCACGGAAAGCA-3' Sequence ID 5, Reverse primer for POMC: 5'-TCAAGGGCTGTTCATCTCC-3' Sequence ID 6, Forward primer for housekeeping gene GAPDH: 5'-TTGTGCAGTGCCAGCCTC-3' Sequence ID 7, Reverse primer for housekeeping gene GAPDH: 5'-CCAATACGGCCAAATCCG-3'

[0053]

[0068] The following are non-limiting embodiments of the model. [Examples]

[0054]

[0069]

[0070] Example 1 Screening of plant extracts for neuronal cytotoxicity

[0071] This study screened natural products and nutritional supplements from Asia for compositions that may improve human mood and mental health. Among 23 tested samples, three showed significantly higher cell viability against rat hypothalamic nerve (R-hth-507) cells. Real-time PCR experiments showed that seven samples exhibited significant β-endorphin synthesis activity. These results suggest that the extracts have potential as candidate mental health drugs. Two of the most promising plant extracts were further studied.

[0055]

[0072] Sample preparation

[0073] Twenty-three natural product samples were ground and dissolved in MQ water at a concentration of 50 mg / mL for the first screening. The dissolved samples were thoroughly mixed by vortexing and sonicated for 5 minutes. This step was repeated six times. For the second screening and real-time PCR experiments, each sample was prepared at the different concentrations shown in [Table 1].

[0056]

[0074] First screening

[0075] To investigate the anti-stress activity derived from plant extracts, their activating activity against rat hypothalamic neurons (R-hth-507) was examined.

[0057]

[0076] cell culture

[0077] Rat hypothalamic neurons (R-hth-507) were purchased from Lonza Bioscience (Basel, Switzerland) and cultured in primary neuronal growth medium (PNGM) supplemented with Singlequots, which contains all the growth factors and nutritional supplements necessary for successful culture of rat and mouse neurons.

[0058]

[0078] Cell viability assay

[0079] The cytotoxicity of R-hth-507 was measured in vitro by a colorimetric method that measures the mitochondrial response to the dye 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT), purchased from Tokyo Chemical Industry (Tokyo, Japan). Generally, the reduction of MTT occurs in metabolically active cells. Therefore, the level of activity is measured spectrophotometrically. The cells were 1 × 10⁻⁶ 5 Cells were cultured in a 96-well plate at a density of cells / mL for 24 hours. Next, R-hth-507 cells were treated with a sample and lysed in MQ. After 24 hours, cell viability was determined using MTT reagent as follows: 10 mL of MTT solution (5 mg / mL PBS) was added to each well, followed by incubation at 37°C in 5% CO2 for 4 hours. The supernatant was discarded, and 40 mM HCl-isopropanol (100 mL) was added to dissolve the formazan crystals. Absorbance was measured at 570 nm using a Microplate Reader 310-Lab (Corona Electric Company, Ibaraki, Japan).

[0059]

[0080] The cytotoxicity test results showed that only one sample (number 7) exhibited a higher cell viability compared to the water control among the 23 samples tested. On the other hand, 13 samples showed cytotoxicity to R-hth-507 cells.

[0060]

[0081] Areca catechu (Sample 7), also known as betel nut, is widely used as chewing tobacco. Chewing betel nut produces mental stimulating effects such as stress reduction, euphoria, and happiness. Our results also support the possibility that betel nut can protect nerve cells that can alter a person's mood. However, such extracts have been reported to increase the risk of oral and pharyngeal cancers from which the plant is chewed (Athukorala et al., J. Addict. 2021; 2021:9967097).

[0061]

[0082] Cell death can occur due to the high concentrations used in each sample. While natural plant extracts can be safe and beneficial to health and well-being, it is important to note that even if a natural plant extract has a positive effect on an individual, it can be harmful to our bodies at different concentrations. Therefore, it is crucial to identify the appropriate amount to obtain the functional effects of natural products while reducing the risk of side effects.

[0062]

[0083] Second screening: Optimizing the concentration

[0084] In the second screening, each sample was prepared at different concentrations as shown in [Table 1]. Samples 6 and 10 showed significantly higher cell viability of R-hth-507 cells than cells treated with MQ in the second screening. The mean cell viability of samples 4, 5, 7, and 9 was slightly higher than that of the water control. This is because the cells used in this study were rat brain hypothalamic neurons, and the high cell viability of these samples against R-hth-507 is consistent with previous studies. In Figure 2, it can be seen that sample 9 (Lignosus rhinosalas) at 10 μg / mL was not cytotoxic to R-hth-507 cells.

[0063]

[0085] [Table 1]

[0064]

[0086] Eight samples showed cytotoxicity to the tested cells, and these samples showed a similar trend in the first screening (sample numbers 11-14 and 17-20). Conversely, based on the results of the second screening of the second sample concentrations, eight samples that showed the highest neuroprotection and no cytotoxicity to R-hth-507 cells (sample numbers 2, 4, 5, 6, 7, 9, 10, and 23) were selected for use in real-time PCR testing. Further experiments were conducted with these better-performing extracts.

[0065]

[0087] Real-time PCR

[0088] The gene expression of proopiomelanocortin (POMC), which is involved in β-endorphin synthesis, was investigated by real-time PCR. Sample concentrations in this experiment [Table 1] were determined based on the first and second screenings. After synthesis in the brain, POMC is broken down into endorphins and corticosteroids by enzymatic action. Therefore, increased POMC gene expression is suggested to increase endorphin production.

[0066]

[0089] Real-time PCR

[0090] R-hth-507 cells were placed in a 100 mm dish at a rate of 0.4 × 10⁶ cells per dish. 5Cells were seeded. After 24 hours of incubation, water-extracted samples or MQ as a control were added to the R-hth-507 cells. The R-hth-507 cells were then cultured for 96 hours. Total RNA was purified from R-hth-507 cells cultured using ISOGENE II (Nippon Gene, Tokyo, Japan). cDNA strands were synthesized from 500 ng of total RNA using ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan). Quantitative real-time PCR was performed in a total reaction volume of 25 μL using SYBR Premix Ex Taq II (TaKaRa Bio, Shiga, Japan) and 20 ng of cDNA per reaction. Real-time PCR was performed using a Thermal Cycler Dice Real Time System Lite TP700 (TakaRa, Japan) under the following conditions: 30 seconds at 95°C, followed by 5 seconds at 95°C and 30 seconds at 60°C, each for 40 cycles. The primers used for amplification were as follows: CCTCACCACGGAAAGCA and TCAAGGGCTGTTCATCTCC for POMC, and TTGTGCAGTGCCAGCCTC and CCAATACGGCCAAATCCG for GAPDH.

[0067]

[0091] As a result, POMC gene expression was confirmed to increase with the addition of most of the tested plant extracts compared to the control, with the exception of sample 2. Samples 5 and 7 showed more than three times the POMC gene expression of water-treated cells, and sample 9 (Lignosus rhinosaras) induced more than two times the expression of POMC genes, as can be seen in Figures 3A and 3B. Each of these results suggests that such substances or extracts may induce euphoric feelings in individuals who ingest them. A water extract of Centella asiatica, a plant similar to sample 5, has previously been found to have cognitive-enhancing effects in animal models of aging and Alzheimer's disease. The mechanism of Centella asiatica's cognitive-enhancing effect is hypothesized to involve the upregulation of nicotinamide adenine dinucleotide and the regulation of brain-derived neurotrophic factor in the brain (Speers et al., Front Pharmacol. 2021;12:788312). The cells used in the current study are nerve cells, and the regulation of neurotrophic factors can affect R-hth-507 cells. C. pluricaulis, a plant similar to sample 4, has been previously reported to have antidepressant-like effects on neuroinflammation-associated depressive behavior induced by chronic, unpredictable mild stress in rats (Gupta & Fernandes Biomed Pharmacother. 2019; 109: pp. 1698-1708). The current results also support the possibility that samples 4, 5, and 9 possess neuroprotective systems that promote calmness and reduce stress and anger.

[0068]

[0092] Sample 9 (Lignosus rhinosaras), commonly known as tiger milk mushroom, also exhibited high POMC gene expression activity. L. rhinosaras possesses functional activities such as antioxidant, immunomodulatory, and skin conditioning effects (Kittimonkolsuk et al., Pharmaceuticals 27;14(2):93 2021). To our knowledge, this is the first report that L. rhinosaras may have an impact on mental health. Samples 23 and 10 activated POMC genes approximately twice as much as the control. POMC gene expression activity of sample 10 was observed at 10 μg / mL, a concentration far below the reported toxic concentration; therefore, if an aqueous extract of sample 10 is to be used as a medicine or dietary supplement, it must be at a low and safe concentration.

[0069]

[0093] Endorphins are neurotransmitters that function in the brain and are thought to be involved in the endogenous analgesic system, producing feelings of euphoria. The seven plant extracts tested were able to increase POMC production, which may produce varying degrees of euphoria.

[0070]

[0094] Cells are selected based on their ability to produce certain hormones related to mood, such as: The cell line SH-SY5Y may be associated with the gene expression of arylalkylamine N-acetyltransferases, which are involved in melatonin and sleep. The Neuro2A cell line may be associated with the gene expression of serotonin / melatonin and tryptophan hydroxylase, which is involved in healing or happiness. The cell line R-HTH 507 may be associated with the gene expression of β-endorphin and the β-endorphin POMC gene, which is involved in euphoria; The cell line PC12 may be associated with the gene expression of dopamine, NE or epinephrine, and tyrosine hydroxylase, which is involved in pleasure; and The cell line CNI-H295R may be associated with the gene expression of cortisol and cortisol 11-beta-hydroxysteroid dehydrogenase.

[0071]

[0095] The initial aqueous extracts of Labisia pumila and Lignosus rhinosalas were prepared and tested in the aforementioned cells, and the expression of genes involved in cell activation and hormone production was identified, based on this screening step.

[0072]

[0096] Further studies in the PC12 cell line demonstrated that extracts of either the Lavisia pumila plant or the Lignosus rhinosala fungus, dissolved in ethanol, both exhibited positive cell activity activation. Lignosus rhinosala extracts at concentrations of 50 μg / mL and 100 μg / mL in 50% ethanol improved PC-12 cell viability by 147% and 139%, respectively. Similarly, Lavisia pumila extract at 50 μg / mL improved PC-12 cell viability by 143%.

[0073]

[0097] Example 2 Functional evaluation tests targeting genes and enzymes related to mental function.

[0098] Hot water extracts were prepared from two promising plant samples (i.e., Sample 13 and Sample 9). Sample 1 was newly named as it was an extract from Lignosus rhinosalas. Sample 2 was an extract from Labisia pumila. These samples were stored in a 20 mg / mL DMSO solution.

[0074]

[0099] Functional evaluation tests [Table 2] were performed on two extract samples prepared as described above, targeting genes and enzymes related to mental state-related functionality. The compounds were tested at 20.0 μg / ml.

[0075] [Table 2]

[0076]

[0100] Compound binding was calculated as the percentage inhibition of binding to the radiolabeled ligand specific to each target. Compound enzyme inhibitory effect was calculated as the percentage inhibition of control enzyme activity. Cellular agonist effect was calculated as the percentage of the control response to a known reference agonist for each target, and cellular antagonist effect was calculated as the percentage inhibition of the control reference agonist response to each target. Results showing inhibition or stimulation higher than 50% were considered to represent a significant effect of the test compound sample, while samples with 30% to 50% activity were considered "probably active."

[0077]

[0101] The results of the functional evaluation study revealed that the extract exhibited activity at six different receptors targeting cannabinoid receptor 1, dopamine D2 receptor, μ (mu) opioid receptor, δ (delta) opioid receptor, cyclooxygenase, and GABAA receptor. In six studies targeting cannabinoid receptor 1, dopamine D2 receptor, GABAA receptor, μ opioid receptor, delta opioid receptor, and cyclooxygenase, both samples (Lignosus rhinosaras and Labisia pumila) showed activity.

[0078]

[0102] In vitro pharmacology: Cellular and nuclear receptor function assays. Agonist effects: Results of test compounds.

[0079]

[0103] Agonist activity against the human opioid receptor mu-1 (mu(MOP)(h)) was observed in extracts from Lignosus rhinosalas (23%) and Labisia pumila (13%). The expected function of these materials is euphoria.

[0080]

[0104] In vitro pharmacology: Enzyme and uptake assays

[0105] Inhibitory activity against human cyclooxygenase (COX1(h)) was found in Labisia pumila extract at 55.9%. Potential activity was observed in Lignosus rhinosalas (40.2%). The expected functionality of these materials is euphoria or well-being. Inhibition of the Cox1 pathway is also known to reduce inflammation and, therefore, lower body temperature and alleviate pain, and is an established mechanism for drugs such as aspirin and ibuprofen, as well as other pharmaceuticals. While speculation on how a reduction in body temperature or pain can deliver euphoria is still uncertain, it is thought that such a reduction in inflammation could reduce systemic stress, and therefore, anti-stress, relaxation, and calming effects can be explored.

[0081]

[0106] Example 3: Gene expression analysis of cells

[0107] Increased expression of three genes was observed in cells exposed to Lignosus rhinosalas extract: APH1B (aph1 homolog B) and the gamma-secretase subunit, one of the subunits of gamma-secretase, an enzyme that produces amyloid-beta, a cause of dementia. Gamma-secretase, an enzyme that produces amyloid-beta, was downregulated by the plant extract.

[0082]

[0108] OPTN optineurin is one of the autophagy functions involved in cellular quality control and is responsible for the normal functioning of cells. OPTN optineurin also plays a role in maintaining normal cellular function. Abnormalities in this gene can cause neurodegeneration. It is expected that increased expression of this gene will suppress cell deterioration. However, the current state of this gene is unclear. Currently, there is no data regarding the suppression of cell deterioration by increasing the expression of this gene.

[0083]

[0109] VGF is known to play a role in neuroprotection by activating cell survival signals. VGF has been shown to promote nerve survival and dendritic growth, protect against retinal nerve damage, repair and grow hippocampal neurons, and act as an antidepressant.

[0084]

[0110] This gene has been shown to promote nerve survival and dendritic growth, protect against retinal nerve damage, repair and grow hippocampal neurons, and exhibit antidepressant effects.

[0085]

[0111] Furthermore, the overall results of the evaluation of the Lignosus rhinosalas extract include gene expression patterns in neuronal cells treated with the plant extract, as shown in [Table 3]. SH-SY5Y cells were treated with a concentration of 10 mg / ml Lignosus rhinosalas extract, and RNA was harvested after overnight incubation. Treatment with Lignosus rhinosalas extract showed a decrease in gene expression of Aph1B, one of the enzymes that produce proteins that cause Alzheimer's disease. Optinulin was an upregulated gene in Lignosus rhinosalas extract-treated samples, and is involved in the autophagy process, suggesting that its extract may maintain cell health. In addition, VGF was also upregulated in Lignosus rhinosalas extract-treated samples. VGF is a gene that induces nerve growth and activates dendritic survival signals, suggesting its role in neuroprotection overall [Table 3]. In summary, based on its use in neuronal cell types such as SH-SY5Y, Lignosus rhinosalus extract can protect nerve cells, induce their growth, and activate genes that maintain nerve cell health.

[0086] [Table 3]

[0087]

[0112] Based on the in vitro results, two plant extracts, an extract from Lignosus rhinosaras and an extract from Labisia pumila, were selected for further detailed analysis and characterization.

[0088]

[0113] Example 4: Extraction process and characterization of Labisia pumila

[0114] The leaves and / or stems of Labisia pumila were prepared as outlined in [Figure 4]. First, the raw material leaves or leaves and stems are collected [Step 1]. Next, these raw materials are dried in an oven at 35-50°C for 2-4 days. Alternatively, these raw materials may be freeze-dried or vacuum freeze-dried. After drying, the stems and leaves are ground to a powder, and the powder (100-200 kg) is then fortified by dissolving the powder in water (500-1000 L) at a temperature of 70-90°C for several hours with constant stirring [Step 2]. The fortification step may be repeated, and filtration [Step 3] may be performed after the fortification step(s) and / or between these fortifications if it is repeated. Next, the fortified liquid is concentrated or evaporated by any known conventional means [Step 4]. The concentrate is pasteurized at a temperature of 70-90°C [Step 5], then filtered [Step 6], followed by spray drying [Step 7], and separated using a metal separator [Step 8], leaving a purified extract powder. The purified extract is a brown powder containing 0.1-0.2% gallic acid.

[0089]

[0115] Characterization of Labisia pumila plant extract

[0116] Prior to characterizing the purified Labisia pumila extract by HPLC, the extract was dissolved in a solvent (water) in a weight ratio of 4:1 (water to extract) or 5000 μl of 10 mg / ml was injected into a 1290LC01 HPLC in a 50% pure methanol to water solvent ratio. As seen in [Figure 5], a peak conforming to the gallic acid reference standard was observed at a retention time of 4.301 minutes, which was calculated from the standard reference to be approximately 0.191% of the extract composition, and an unidentified peak was observed at a retention time of 9.978 minutes, with a first series of smaller unidentified peaks observed at retention times of 1–2 minutes, and a second series of even smaller unidentified peaks observed at retention times of 10–13.5 minutes.

[0090]

[0117] Example 5: Extraction process and characterization of Lignosus rhinosaras

[0118] Lignosus rhinosalas is a polyporous or fungal species with large fruiting bodies that have pores or tubes on the underside. Only Lignosus rhinosalas with a pore size of 6-7 pores / mm were selected. Selection can also be made based on the unique nucleotide sequence of the internal transcription spacer (ITS) ITS1 region (bases 149-168) located between the rRNA structural genes, which is listed here as Sequence ID No. 1, using PCR primers (5'-GTTTTCAGCCGGCGTTTGC-3') and (5'-TCCTCCGCTTATTGATATGC-3'). Using subterranean sclerotia, extracts were prepared according to the procedure in [Step 1] [Figure 4], in which subterranean sclerotia are first collected. These sclerotia were then dried in an oven at 35-50°C for 2-4 days, or alternatively, these sclerotia may be freeze-dried or vacuum freeze-dried. The stems and leaves are dried and then ground into a powder (100-200 kg), which can then be fortified by dissolving the powder in water (500-1000 L) at a temperature of 70-90°C for several hours with constant stirring [Step 2]. The fortification step may be omitted in some cases, and filtration [Step 3] may be performed after the fortification step or immediately after the grinding step or extraction. The filtered product is then concentrated or evaporated by any known conventional means [Step 4]. The concentrated product is pasteurized at a temperature of 70-90°C [Step 5], then filtered [Step 6], then spray-dried [Step 7], separated using a metal separator [Step 8], leaving a purified extract powder. The purified extract is a fine, light brown powder.

[0091]

[0119] Lignosus rhinosalas extract was analyzed and found to contain 1% fat, 28-30% carbohydrates, 28% protein (of which over 10% is glycoprotein), 2% calcium, 4% potassium, and 35% magnesium.

[0092]

[0120] Example 6: In vivo study in mice

[0121] A study design for a preliminary evaluation of the relaxation / sedation, invigorating, and happiness / euphoria effects of eight extract samples at two different doses in mice. The animal studies were designed to gradually expose animals to increasingly stressful studies, minimizing potential interactions between studies. This ensured that stress levels from previous studies would not affect subsequent studies. The study schedule is outlined in [Table 4].

[0093] [Table 4]

[0094]

[0122] Behavioral assessments of relaxation / sedation, invigoration, and well-being effects were conducted. In each behavioral test, control mice fed a standard laboratory animal solid diet were randomly included in a series of animals tested for comparison with mice fed an extract-supplemented diet. The application of the extract incorporated into the diet was designed so that the uptake by each animal was expected to be 0.25% and 0.5% of the diet composition. On average, one animal consumed 3–5 g of diet per day, and therefore, each animal was expected to consume approximately 10 mg (0.25%)–20 mg (0.50%) daily.

[0095]

[0123] Each study consists of three groups: 1) a control group, 2) a 0.25% diet group, and 3) a 0.50% diet group. Each group contains 10 mice.

[0096]

[0124] Food pellet preparation

[0125] Plant extract samples were custom-formulated into standard mouse solid feed pellets for oral administration in the diet. Mice were freely given the custom-formulated diet. Control animals were freely given the same standard diet without any extracts. All diets were gamma-irradiated to ensure sterility.

[0097]

[0126] To test the acute effects of dietary consumption of the extracted substance, mice were fed a custom-formulated diet daily from the day before the test until the end of the test. Since most mice eat at night, the delivery of the custom-formulated diet containing the extracted substance from the day before the test ensured that the mice received their diet overnight. Following this schedule, each group of mice needed to be fed the custom-formulated diet for 10 days.

[0098]

[0127] On average, mice consume 3g to 5g of experimental food per day. However, some food is lost during feeding and gnawing (approximately 10% loss), and pellets remain uneaten in the hopper (approximately 20% loss). Therefore, each group of 10 animals fed for 10 days used at least 660g of food, taking into account maximum food consumption and potential losses.

[0099]

[0128] The limiting factor for the amount of each plant extract required is the production scale of the custom-formulated feed. The formulation, mixing, and pelletizing processes are limited by the scale of the equipment used by the feed manufacturer. Therefore, the minimum amount that can actually be custom-formulated is approximately 2.65 kg, yielding approximately 1.9 kg to 2 kg per batch.

[0100]

[0129] Therefore, 20g of each plant extract was required to obtain approximately 2kg of 0.25% custom-formulated diet and approximately 2kg of 0.5% custom-formulated diet.

[0101]

[0130] The following are plant extracts: • Lavisia pumila 0.25%; • Lavisia pumila 0.5% • Lignosus rhinosaras 0.25%; and • Lignosus rhinosaras 0.5% Includes.

[0102]

[0131] Behavioral evaluation in mice

[0132] Behavioral assessments of relaxation / sedation, invigoration, and happiness / euphoria effects will be conducted. In each behavioral test, control mice fed a standard laboratory animal solid diet will be randomly included in a series of animals tested for comparison with mice fed a diet supplemented with plant extract samples.

[0103]

[0133] Relaxation / Sedation

[0134] Relaxation and sedation effects were assessed through a standard behavioral paradigm to detect anxiety and anxiolytic-like effects. These tests were the light-dark box paradigm [Figure 6A] and the elevated zero maze [Figure 6B]. The elevated zero maze is an improved version of the conventional elevated cruciform maze that overcomes the problem of an uncertain central zone in the cruciform maze.

[0104]

[0135] Light and dark box

[0136] Mice were tested in chambers with light and dark zones. Animals were individually positioned in the center of the light arena. The duration of time spent in the light and dark arenas and the number of times they passed between the two arenas were recorded. Sensors on the doors between the light and dark arenas allowed for unbiased measurement of time spent in each arena and the number of times they passed between them. Mice have an innate motivation to explore as wide as possible to find potential food sources. However, this motivation to explore in the light chamber conflicts with the mouse's natural behavioral response of preferring dark areas to light areas to avoid being discovered by potential predators. Therefore, the increased time spent exploring in the light chamber becomes a measure of relaxation or sedation.

[0105]

[0137] In the light-dark box test, anxiety levels are assessed by the amount of time spent in the light box. If the test group spends longer in the light box than the control group, the animals in the test group are considered to have reduced anxiety-like behavior. Conversely, if the time spent in the light box is shorter than that spent in the control group, it suggests that anxiety-like behavior is increased. From [Figure 7A], mice in the 0.5% groups of both Labisia pumila and Lignosus rhinosaras spent more time in the light box than the control group mice, suggesting that these two plant extract diets may have an anxiolytic effect, although this is not statistically significant.

[0106]

[0138] Elevated Zero Maze

[0139] Mice are individually allowed to freely explore a zero-type maze consisting of open areas and enclosed areas surrounded by side walls. The maze is elevated approximately 50 cm. Subjects are allowed to explore the maze for 5 minutes. The number of times they pass between the open and enclosed areas, the waiting time to enter the open area, the amount of time spent in the open area, and the number of times they enter it are recorded. A computer video tracking system using near-infrared illumination, which allows tracking of the mice even in the enclosed areas, is used for unbiased recording of their behavior. Mice have an innate motivation to explore, but this is in the open area of ​​the zero-type maze, which conflicts with exposure to potential predators and an innate fear of the maze's elevated height. Therefore, an increase in the time spent exploring the open area is a measure of relaxation or sedation.

[0107]

[0140] In the zero maze, anxiety levels are assessed by the amount of time spent in the open arm. Longer periods of time spent indicate a reduction in anxiety-like behavior. While there was no significant difference between the extract diet group and the control diet group, both the 0.5% groups of Labisia pumila and Lignosus rhinosaras showed a longer average time spent in the open arm than the control group.

[0108]

[0141] Cheer up

[0142] The mental function-enhancing effect was assessed through the measurement of spontaneous exploratory behavior in a dark, open field [Figure 8A] and through the measurement of spontaneous working memory alternation behavior in a Y-shaped maze [Figure 8B]. These behavioral paradigms utilize the normal behavior of mice to measure mental activation.

[0109]

[0143] Exploring open fields in the dark

[0144] Mice were individually placed in an open-field arena in darkness (less than 20 lux) illuminated only by red / near-infrared light. Mouse movements were tracked using a computer video tracking system employing a near-infrared sensing camera. Because mice have limited vision for red light, the room appeared dark from their perspective, and they were not inhibited from exploring the open field by their innate fear of exposure to bright light. Under these conditions, the open-field paradigm measures spontaneous exploratory behavior. Increased exploratory behavior indicates mental activation.

[0110]

[0145] A dark, open field measures spontaneous exploratory behavior. Increased exploratory behavior indicates mental activation. Among the groups fed a plant extract diet, mice fed a 0.5% Labisia pumila extract diet showed increased exploration in a dark, open field compared to the control group [Figure 9A].

[0111]

[0146] Spontaneous alternating work memory

[0147] Mice are individually positioned in the center of a Y-shaped maze with three arms at 120° angles to each other. The animals are allowed to freely explore all three arms. The animals are tracked using a computer video tracking system. If a mouse possesses intact spatial working memory, it is less likely to enter the most recently visited arm. The number of arm entries is scored to determine the number of alternations. A mouse is considered to have entered an arm if all four limbs are within that arm. An increase in spontaneous alternation behavior indicates mental activation.

[0112]

[0148] In the spontaneous alternation working memory test, animals freely explore all three arms of a Y-shaped maze. If a mouse has intact spatial working memory, it will have a reduced tendency to enter the most recently visited arm. Arm entries are scored to determine the number of alternations. Increased spontaneous alternation behavior indicates mental activation. Results of spontaneous alternation in the Y-maze compared to a control as an indicator of working memory [Figure 9B].

[0113]

[0149] Happiness / Euphoria

[0150] Euphoria and euphoric effects were measured using a dietary preference test [Figure 10C] and two conventional paradigms used to detect antidepressant-like activity. The two paradigms for testing antidepressant-like effects are the tail suspension test [Figure 10A] and the forced swim test [Figure 10B]. The two tests are performed because they have different sensitivities. The tail suspension test yields clearer results with less individual variability between mice, but has lower sensitivity. Therefore, it is also helpful to include the forced swim test, which has better sensitivity.

[0114]

[0151] Feeding palatability

[0152] Mice are presented with conventional laboratory animal solid feed individually, while diets containing herbaceous samples are placed separately in two small ceramic cups located at opposite ends of their home cage. The amount of food consumed is weighed. The longer the time spent eating from the cups containing the supplemented diet, the greater the pleasure or well-being effect of the diet.

[0115]

[0153] In the dietary preference test, mice were individually presented with conventional laboratory animal solid feed, while diets containing herbaceous samples were placed separately in two small ceramic cups located at opposite ends of their home cages. The amount of food consumed was weighed. The longer the time spent eating from the cups containing the supplemented diet, the greater the pleasure or well-being effect of the diet. Among the herbaceous diet groups, mice fed with Lignosus rhinosaras extract showed the same preference for both the herbaceous diet and the control diet [Figure 11A]. In contrast, mice fed with Labisia pumila extract preferred the control diet to the plant extract diet.

[0116]

[0154] Compulsory swimming test

[0155] Mice are individually placed in cylinders filled with water. Their movements are monitored. Immobility is considered to mimic a state of emotional depression. An increase in the time it takes to become immobile indicates an antidepressant-like euphoric effect.

[0117]

[0156] In the compulsory swimming test, a reduction in immobility time compared to the control group indicates an antidepressant-like euphoric effect. In the extract diet group, little difference was observed compared to the control group, suggesting that these extracts may not have an antidepressant-like euphoric effect [Figure 11B].

[0118]

[0157] Tail suspension test

[0158] Mice are suspended by their tails for six minutes. Movement is monitored, and a decrease in movement is considered to mimic emotional depression. The animals are suspended at a fixed distance from the base of their tails on a standard behavioral apparatus widely used for behavioral testing, so there is no risk of tail injury or slippage. An increasing time until the animal becomes still indicates an antidepressant-like euphoric effect.

[0119]

[0159] In the tail suspension test, a reduction in the length of time spent motionless compared to the control group indicates an antidepressant-like well-being effect. There were no significant differences between the groups in the tail suspension test [Figure 11C].

[0120]

[0160] Example 7: Measurement of hormones and neurotransmitters

[0161] At the end of the in vivo experiment, brain tissue and plasma derived from core blood samples were assayed for corticosterone, dopamine, and 5-HT (serotonin) [Figure 12]. The assays were performed using ELISA kits. Each ELISA kit can be run in a 96-well plate. In each plate, samples from mice fed a control diet were run alongside samples from mice fed a herbaceous supplement diet.

[0121]

[0162] Brain tissue was homogenized according to the instructions provided with the Enzo Lifesciences kit. Mice consuming 0.25% and 0.5% Labisia pumila had significantly lower tissue dopamine levels compared to the control group [Figure 13]. Mice consuming 0.5% Lignosus rhinosala had higher tissue dopamine levels compared to the control. Mice consuming 0.25% Lignosus rhinosala had significantly higher tissue dopamine levels than the control.

[0122]

[0163] Whole blood was collected when the mice were euthanized and the serum was centrifuged to isolate it. The serum samples were then run through the Biovision Serotonin Kit. Serotonin levels were not significantly different in any group compared to the control [Figure 14].

[0123]

[0164] Whole blood was collected when the mice were euthanized and the serum was centrifuged to isolate it. The serum samples were then run through the Enzo Lifesciences corticosterone kit with some modifications to the protocol. The serum samples were incubated with the steroid replacement hormone before being added to the wells. Standards and serum samples were incubated in the wells for 1 hour before the addition of conjugates and antibodies.

[0124]

[0165] The 0.5% Lignosus rhinosalas group showed higher corticosterone levels than the control group [Figure 15].

[0125] [Table 5]

[0126]

[0166] Here, we observed that the Labisia pumila group showed decreased activity in a light box and increased activity in a dark open field, indicating an increase in anxiety-like behavior. Biochemically, this group showed a significant decrease in dopamine levels in brain tissue compared to the control group, and serum levels of serotonin and corticosterone appeared to be decreased, which may underlie the observed anxiety-inducing effect.

[0127]

[0167] In vivo, brain tissue from mice fed a 0.5% Lignosus rhinosarasu diet showed higher dopamine levels compared to controls [Figure 13]. The results suggest that these mice may have experienced feelings of happiness or euphoria. In vivo, serum samples from mice fed a 0.5% Lignosus rhinosarasu diet showed higher corticosterone levels than controls (Figure 15). Higher corticosterone levels are thought to be associated with stress. The results suggest that these mice may have been excited. Here, the behavioral changes in the Lignosus rhinosarasu diet group were not statistically significant. However, dopamine levels were increased in brain tissue, and serum serotonin and corticosterone levels were elevated.

[0128]

[0168] Those skilled in the art will further recognize that variations and combinations of the above features may not be substitutes or replacements, but rather may combine to form further embodiments that fall within the intended scope of the present invention.

[0129]

[0169] As those skilled in the art will understand, each embodiment can be used in combination with other embodiments or with several embodiments.

[0130] [Reference to related applications]

[0001] This application claims priority to Singapore Patent Application No. 10202251560, filed on 31 October 2023, the contents of which the contents of the said patent are incorporated herein by reference.

Claims

1. A method for promoting mental health, comprising the step of delivering a composition comprising a powder extract, optionally wherein the powder extract is dissolved in a solvent, and the composition is (a) Extracted from the sclerotia of the fungus Lignosus rhinoceros, which has a pore size of approximately 7 pores / mm. (b) Increase dopamine levels, (c) Cyclooxygenase can be inhibited by at least 30% in vitro, (d) A method for more than doubling the expression of the proopiomelanocortin (POMC) gene.

2. The method according to claim 1, wherein the composition can increase the viability of PC-12 cells by at least 130% in vitro.

3. The method according to claim 1 or 2, comprising the step of determining that Sequence ID No. 1 is present in the sclerotia of Lignosus rhinosaras prior to extracting the composition.

4. The method according to any one of claims 1 to 3, wherein the enhancement of mental health includes experiencing pleasure from increased levels of dopamine.

5. The method according to any one of claims 1 to 4, wherein promoting mental health includes feeling excited from increased levels of serum corticosterone.

6. The method according to any one of claims 1 to 5, comprising the step of preparing the composition as a food preparation, nutritional supplement, beverage, or aerosol.

7. The method according to any one of claims 1 to 6, wherein the composition is delivered orally.

8. The method according to any one of claims 1 to 6, wherein the composition is delivered transnasally.

9. The method according to any one of claims 1 to 6, wherein the composition is delivered intravenously.

10. A composition comprising a powder extracted from the sclerotia of the fungus Lignosus rhinosalas having a pore size of approximately 7 pores / mm, which can increase dopamine levels, inhibit cyclooxygenase by at least 30%, induce more than twofold expression of POMC genes, and promote mental health, and which can be formulated as a food preparation, nutritional supplement, beverage, or aerosol.

11. The composition according to claim 10, wherein the powder extract derived from the sclerotia of the fungus Lignosus rhinosalas having a pore size of approximately 7 pores / mm comprises 1% fat, 28-30% carbohydrates, 28% protein, of which more than 10% is glycoprotein, 2% calcium, 4% potassium, and 35% magnesium.

12. A food, beverage or nutritional supplement formulation comprising the composition according to claim 10 or 11 for use in promoting mental health, wherein the composition can increase dopamine levels, inhibit cyclooxygenase by at least 30%, and induce more than twofold expression of the POMC gene.

13. An aerosol comprising the composition according to claim 10 or 11 for use in promoting mental health, wherein the composition can increase dopamine levels, inhibit cyclooxygenase by at least 30%, and induce more than twofold expression of the POMC gene.

14. A method for promoting mental health, (a) A step of extracting a powder extract from the leaves and / or stems of Labisia pumila, (b) The step of determining that 0.1% to 0.2% gallic acid is present in the extract, (c) A step of delivering an anxiety-inducing composition comprising a purified extract powder, wherein the purified extract powder is dissolved in a solvent and the composition can inhibit cyclooxygenase by at least 30%. Includes, Methods that promote mental health, including those that induce anxiety.

15. The method according to claim 14, wherein the composition can increase the viability of PC-12 cells by at least 130% in vitro.

16. The composition is prepared as a food preparation, nutritional supplement, beverage, or aerosol. The powder extract derived from the leaves and / or stems of Labisia pumila contains 0.1% to 0.2% gallic acid in the total powder extract. The method according to claim 14 or 15, wherein the powder extract derived from Labisia pumila contains a gallic acid HPLC peak with a retention time of 4 to 5 minutes and a second peak with a retention time of 9 to 11 minutes when eluted at a concentration of 10 mg / ml of the powder extract with 50% water:50% methanol.

17. The method according to any one of claims 14 to 16, wherein the composition is delivered orally.

18. The method according to any one of claims 14 to 16, comprising delivering the composition via the nose.

19. The method according to any one of claims 14 to 16, wherein the composition is delivered intravenously.

20. An aerosol composition having an anxiety-inducing effect, comprising a powder extracted from the leaves and / or stems of the Lavisia pumila plant, The composition can inhibit cyclooxygenase receptors by at least 30% and promote mental health. The powder extract derived from the leaves and / or stems of Labisia pumila contains 0.1% to 0.2% gallic acid in the total powder extract. An aerosol composition comprising the powder extract derived from Labisia pumila, wherein when 10 mg / ml of the powder extract is eluted with 50% water:50% methanol, it contains an HPLC peak of gallic acid with a retention time of 4 to 5 minutes and a second peak with a retention time of 9 to 11 minutes.

21. A system for testing the effects of a composition containing a powder extract derived from an organism on promoting mental health, comprising a home cage for feeding, a) An open field arena having red / near-infrared light that can illuminate the arena using a computer video tracking system that uses a near-infrared sensing camera capable of recording the arena. b) A chamber having a light region and a dark region, a door between the light region and the dark region, and a sensor on the door between the light region and the dark region, c) A maze having open areas, enclosed areas surrounded by side walls, and elevated areas; a chamber equipped with a near-infrared sensing camera capable of recording each of the open areas, enclosed areas, and elevated areas; d) Two small ceramic cups placed at both ends of the home cage, e) A Y-shaped maze having three arms at 120° angles to each other, and equipped with a computer video tracking system capable of recording the movement of each arm, f) A component located above the sensing range, a fixing device connected to the component for fixing the tail of an animal; and a base comprising a computer video tracking system capable of recording movement within the sensing range, and g) A transparent plexiglass cylinder having a base into which water can be placed, and comprising a computer video tracking system capable of recording movement within the transparent plexiglass cylinder. A system comprising a series of test containers, including a set of test containers.

22. A method for testing the effects of a composition on mental health, a) Step of extracting a powder extract from an organism, b) A step of preparing the powder extract as a food preparation, nutritional supplement, or beverage. c) Feeding the test subject the food preparation, nutritional supplement, or beverage in a home cage, and feeding the control subject a similar food preparation, nutritional supplement, or beverage that does not contain the powder extract, d) A step of placing the test subject and the control subject in an open field arena, which is illuminated with red / near-infrared light that provides illumination of less than 20 lux in the arena, and recording the movements of the test subject and the control subject. e) A step of providing the test subject and the control subject with a handling interaction for at least two days to allow them to recover, f) A step of placing the test subject and the control subject in a chamber having a light area and a dark area, with a door between the light areas, and measuring the movement of the test subject and the control subject between the light area and the dark area. g) A step of placing the test subject and the control subject in a chamber having a maze with an open area, a closed area enclosed by side walls, and an elevated area, and recording the movement of the test subject and the control subject in each of the open area, closed area, and elevated area, h) A step of placing two small ceramic cups at both ends of a home cage, filling the first small ceramic cup with a food preparation, nutritional supplement, or beverage, and filling the second small ceramic cup with the food preparation, nutritional supplement, or beverage that does not contain the powder extract, and measuring which food preparation, nutritional supplement, or beverage the test subject and the control subject prefer. i) The steps of placing the test subject and the control subject in a Y-shaped maze having three arms at an angle of 120° to each other, and recording the movement of the test subject and the control subject at each arm; j) The tails of the test subject and the control subject are fixed to a fixing device connected to a component located above the sensing range, the test subject and the control subject are suspended at least 25 cm above the base, and the movement of the test subject and the control subject within the sensing range is recorded. k) A step of providing the test subject and the control subject with a solution for at least two days to allow them to recover and to provide a handling interaction between the test subject and the control subject, l) A step of placing the test subject and the control subject inside a transparent plexiglass cylinder having a base partially filled with water, and recording the movement of the test subject and the control subject in the water inside the transparent plexiglass cylinder. Includes, A method wherein, between each of steps d) to l), the test subject and the control subject are returned to their home cages, the test subject is fed the food preparation, nutritional supplement, or beverage, and the control subject is fed a similar food preparation, nutritional supplement, or beverage that does not contain the powder extract.

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