WO2024096818A1

WO2024096818A1 - Compositions and methods for enhancing mental wellbeing

Info

Publication number: WO2024096818A1
Application number: PCT/SG2023/050724
Authority: WO
Inventors: Yutaka KUROKI
Original assignee: Delightex Pte. Ltd.
Priority date: 2022-10-31
Filing date: 2023-10-31
Publication date: 2024-05-10

Abstract

The present disclosure relates generally to compositions for enhancing mental wellbeing, particularly compositions from plant extracts for use in calming energizing or enhancing happiness or euphoria in individuals. Methods disclosed include enhancing mental wellbeing by administering an extract from plants such as extracts from labisia pumila; or Lignosus rhinoceros or a combination of extracts form Iabisia pumila and Lignosus rhinoceros plants. Such methods are non-therapeutic methods. Compositions disclosed include extracts from labisia pumila or Lignosus rhinoceros for use in enhancing mental wellbeing. Systems and methods for testing whether a substance enhances mental wellbeing are also disclosed.

Description

DESCRIPTION

TITLE OF INVENTION: COMPOSITIONS AND METHODS FOR ENHANCING MENTAL WELLBEING

REFERENCE TO RELATED APPLICATIONS

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

TECHNICAL FIELD

[0002] The present disclosure relates generally to compositions and methods for enhancing mental wellbeing, particularly compositions from plant extracts or fungi extracts for use in calming energizing or enhancing happiness or euphoria in individuals.

BACKGROUND

[0003] The following discussion of the background to the invention is intended to facilitate an understanding of the present invention only. It should be appreciated that the discussion is not an acknowledgement or admission that any of the material referred to was published, known or part of the common general knowledge of the person skilled in the art in any jurisdiction as at the priority date of the invention.

[0004] Modern life can be stressful and exhausting. It is difficult for individuals to find ways to relax, calm down, reenergize themselves and feel happier or more euphoric. While some individuals may use drugs or alcohol for some of achieve such feelings these can affect the individuals health in the long run or in some cases are not legal.

[0005] The recent pandemic of COVID-19 had great influence on people all overthe world not only physically but also in relation to their mental wellbeing. Due to the spread of the coronavirus, people were often unable to go out and were forced to spend their time at home for more than two years. Because of this restricted lifestyle, there were concerns regarding the effects on individuals’ mental wellbeing, such as stress build up, anxiety and depressionlike symptoms. World Health Organization (WHO) reported that, global prevalence of anxiety and depression increased by 25% in the first year of the COVID-19 pandemic. Many studies have investigated the psychological effects of COVID-19 on health care workers and the general public and reported high levels of problems in these aspects. Decline in mental wellbeing was found to be more profound among the general population compared to healthcare workers and students. Meta analysis of these studies reported a pooled prevalence of 33% for anxiety and 28% for depression. Anxiety levels of university students' were examined in Malaysia (Irfan et al. The Psychological Impact of Coronavirus on University Students and its Socio-Economic Determinants in Malaysia. Inquiry. 2021 ;58:469580211056217), as a result 12.3% of students were normal, whereas 30.5% were experiencing mild anxiety, 31 .1 % moderate anxiety, and 26.1 % severe anxiety. Similar experiments were conducted in Thailand, COVID-19 pandemic had negative influence on anxiety symptoms to students in Thailand (Tiaprapong el al. PLoS One. 2021 ;16(6):2021). The study strongly supports evidence that, Thai health professional students were similar to students in other countries in which sedentary behavior, weight gain, and reduction of mental wellbeing had been prominent.

[0006] For the mental wellbeing of healthcare professionals, such as excessive stress and mental stress, long working hours, being away from the family, lacking proper protective equipment in the working environment, and no effective treatment. Burnout levels and factors associated with distress among healthcare workers engaged in the management of COVID 19 in India was reported (Menon et al. PLoS One 2022; 17 (3) 2022). In the report, 52.9% of the participants had distress. According to one study, among seven middle income countries in Asia (China, Iran, Malaysia, Pakistan, Philippines, Thailand, and Vietnam), Thai reported the highest anxiety, depression and stress scores (Wang et. al. PLoS One 2021 ; 16(2) 2021). In contrast, in the same report Vietnam exhibited the lowest mean scores in anxiety, depression and stress scales.

[0007] Labisia pumila extract also known as Kacip Fatimah, is believed to have effects on female reproductive diseases (Chua et al., Fitoterapia, 83(8), 1322-135, 2012; Zakaria et al., BioMed Res. International. 2021 , 9928199). Lignosus rhinoceros or tiger milk mushrooms are a medicinal fungus. Extracts from the scerotia have been used for treatment of asthma, cough, fever, liver related illnesses and gastric ulcer (Chang and Lee 2004, Fungal diversity 15: 15-22). Lignosus rhinoceros extracts have been shown to have antioxidant properties that have been postulated for use in cancer treatment and neuroprotective properties in C. elegans (Kittimonkolsuk et al., Pharmaceuticals 27;14(2):93 2021).

[0008] There exists a need to find alternative compounds or compositions for enhancing mental wellbeing and/or alleviate at least one of the aforementioned problems.

SUMMARY [0009] Compositions and methods of using the same to enhance mental wellbeing are envisaged.

[0010] Accordingly, an aspect of the invention refers to a method of enhancing mental wellbeing comprising delivering a composition comprising a powdered extract, optionally dissolved in a solvent, wherein the composition is (a) extracted from a labisia pumila plant, or Lignosus rhinoceros fungus; and (b) capable of inhibiting cyclooxygenase receptor by at least 30%.

[0011] According to another aspect of the invention there is a composition comprising a powder extracted from a labisia pumila plant; or a Lignosus rhinoceros fungus wherein the composition is capable of: inhibiting cyclooxygenase receptor by at least 30%; and enhancing mental wellbeing, and wherein the composition is formulated as a food preparation, a supplement, or a beverage.

[0012] According to another aspect of the invention there is a food formulation comprising a composition as described herein above for use in enhancing mental wellbeing.

[0013] According to another aspect of the invention there is a system fortesting the effect of a composition comprising a powdered extract extracted from a biological organism on enhancing mental wellbeing, having a home cage for feeding and a series of test containers comprising: a) an open field arena having a red/near-infrared light capable of illuminating the arena with a computer video tracking system using a near-infrared sensitive camera capable of recording the arena; b) a chamber with a light zone and a dark zone with a door between the light zone and the dark zone and a sensor on the door between the light zone and the dark zone; c) a chamber comprising a maze with open segments, closed segments enclosed with side walls, and elevated segments, a near-infrared sensitive camera capable of recording each of the open segments, closed segments, and elevated segments; d) two small ceramic cups to be placed at opposite ends of the home cage; e) a Y-shaped maze with three arms at a 120° angle from each other with a computer video tracking system capable of recording movement in each arm; f) a piece located above a sensing range, a fixing device connected with said piece for fixing a tail of an animal; and a base with a computer video tracking system capable of recording movement within the sensing range; and g) a transparent Plexiglas cylinder with a base capable of containing water with a computer video tracking system capable of recording movement within the transparent Plexiglas cylinder.

[0014] According to another aspect of the invention there is a method fortesting the effect of a composition on mental wellbeing comprising: a) extracting a powdered extract from a biological organism; b) formulating the powdered extract as a food preparation, a supplement, or a beverage; c) feeding the food preparation, supplement, or beverage to a test subject and feeding a similar food preparation, supplement, or beverage that does not contain the powdered extract to a control subject in a home cage; d) placing the test subject and the control subject in an open field arena illuminated by a red/near-infrared light providing less than 20 lux of illumination in the arena and recording the movement of the test subject and the control subject; e) giving the test subject and the control subject at least two days to recuperate and providing handling interaction of the test subject and the control subject; f) placing the test subject and the control subject in a chamber with a light zone and a dark zone with a door between the light zone and measuring the movement of the test subject and the control subject between the light zone and the dark zone; g) placing the test subject and the control subject in a chamber comprising a maze with open segments, closed segments enclosed with side walls, and elevated segments, recording the movement of the test subject and the control subject in each of the open segments, closed segments, and elevated segments; h) placing two small ceramic cups at opposite ends of the home cage a first small ceramic cup filled with the food preparation, supplement, or beverage and a second small ceramic cup filled with the food preparation, supplement, or beverage that does not contain the powdered extract and measuring which food preparation, supplement, or beverage the test subject and the control subject prefer; i) placing the test subject and the control subject in a Y-shaped maze with three arms at a 120° angle from each other and recording movement of the test subject and the control subject in each arm; j) fixing a tail of the test subject and the control subject to a fixing device connected with a piece located above a sensing range, suspending the test subject and the control subject at least 25cm above a base wand recording movement of the test subject and the control subject within the sensing range; k) giving the test subject and the control subject at least two days to recuperate and providing handling interaction of the test subject and the control subject; and l) placing the test subject and the control subject in a transparent Plexiglas cylinder with a base partially containing water and recording movement of the test subject and the control subject within the water of the transparent Plexiglas cylinder; wherein between each step of d) to I) the test subject and the control subject are returned to the home cage where the test subject is fed the food preparation, supplement, or beverage and the control subject is fed the similar food preparation, supplement, or beverage that does not contain the powdered extract.

[0015] Other aspects and features of the present invention will become apparent to those of ordinary skill in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In the figures, which illustrate, by way of non-limiting examples only, embodiments of the present invention,

[0017] [Fig. 1]: Cell viability of R-hth-507 cells at first screening. R-hth-507 cells were treated with 50 pg/mL water extract of each sample. The cell viability after 24 hours of treatment were measured by MTT assay. Control; MQ, *; p<0.05, **; p<0.01 . average ± SD, n=3.

[0018] [Fig. 2]: Cell viability of R-hth-507 cells at second screening. R-hth-507 cells were treated with different concentration of water extract of each sample. The cell viability after 24 hours of treatment were measured by MTT assay. Control; MQ, *; p<0.05, **; p<0.01 . average ± SD, n=3. [0019] [Fig. 3]: (A) Expression level of proopiomelanocortin gene in R-hth-507 cells. Relative value of POMS gene expression was estimated by real-time PCR. Control; MQ, *; p<0.05, **; p<0.01 . average ± SD, n=3. (B) Functional evaluation test were conducted on the 20.0 pg/ml extract samples of Labisia pumila or Lignosus rhinoceros targeting inhibitory activity against Human Cyclooxygenase (COX1 (h)) are listed as a % of the control.

[0020] [Fig. 4]: A schematic flow chart of the steps to make plant extracts from Labisia pumila or Lignosus rhinoceros.

[0021] [Fig. 5]: HPLC results for Labisia pumila leaf and stem extracts.

[0022] [Fig. 6]: A schematic of the relaxation tests (A) light dark box test and (B) elevated zero mase.

[0023] [Fig. 7]: Panel A. Average time spent in the light box. The Control group (c), 0.25% Labisia pumila Leaf extract group (KP25), 0.5% Labisia pumila leaf extract group (KP50), 0.25% Lignosus rhinoceros sclerotia extract group (TMM25), 0.5% Lignosus rhinoceros sclerotia extract group (TMM50), Panel B. Average time spent in open arms. There were no significant differences in time spent in open arms between the herbal diet groups and control. N=10 (5 males and 5 females) for all extract diet groups. N=20 (10 males and 10 females) for control group.

[0024] [Fig. 8]: A schematic of the energizing tests (A) open field test and (B) Y mase.

[0025] [Fig. 9]: Panel A. Distance travelled in the dark open field test (OFT). The Control group (c), 0.25% Labisia pumila Leaf extract group (KP25), 0.5% Labisia pumila leaf extract group (KP50), 0.25% Lignosus rhinoceros sclerotia extract group (TMM25), 0.5% Lignosus rhinoceros sclerotia extract group (TMM50), Panel B percent of alternation in the mase. There were no significant differences between the herbal diet groups and control. N=10 (5 males and 5 females) for all extract diet groups. N=20 (10 males and 10 females) for control group.

[0026] [Fig. 10]: A schematic of the happiness tests (A) Tail suspension test, (B) Forced Swim test and (C) Feed preference test.

[0027] [Fig. 11]: Panel A. Average proportion of herbal diet consumed over 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. The Control group (c), 0.25% Labisia pumila Leaf extract group (KP25), 0.5% Labisia pumila leaf extract group (KP50), 0.25% Lignosus rhinoceros sclerotia extract group (TMM25), 0.5% Lignosus rhinoceros sclerotia extract group (TMM50). N=10 (5 males and 5 females) for all plant extract diet groups. N=20 (10 males and 10 females) for control group.

[0028] [Fig. 12]: A schematic of the collection of serum and brain tissue for detection and analysis of dopamine, Serotonin and Corticosterone

[0029] [Fig.13]: Average tissue levels of dopamine. The Control group (c), 0.25% Labisia pumila Leaf extract group (KP25), 0.5% Labisia pumila leaf extract group (KP50), 0.25% Lignosus rhinoceros sclerotia extract group (TMM25), 0.5% Lignosus rhinoceros sclerotia extract group (TMM50). N=6 to 8 (balanced males and females) for all plant extract diet groups. N=10 (5 males and 5 females) for control groups.

[0030] [Fig. 14]: Average serum levels of serotonin. The Control group (c), 0.25% Labisia pumila Leaf extract group (KP25), 0.5% Labisia pumila leaf extract group (KP50), 0.25% Lignosus rhinoceros sclerotia extract group (TMM25), 0.5% Lignosus rhinoceros sclerotia extract group (TMM50). N=4 to 6 (balanced males and females) for all extract diet groups. N=8 (4 males and 4 females) for control group.

[0031] [Fig. 15]: Concentration of corticosterone in serum. N=6 (3 males and 3 females) for all plant extract diet groups. The Control group (c), 0.25% Labisia pumila Leaf extract group (KP25), 0.5% Labisia pumila leaf extract group (KP50), 0.25% Lignosus rhinoceros sclerotia extract group (TMM25), 0.5% Lignosus rhinoceros sclerotia extract group (TMM50). N=8 (4 males and 4 females) for control group.

DETAILED DESCRIPTION

[0032] Throughout this document, unless otherwise indicated to the contrary, the terms “comprising”, “consisting of’, “having” and the like, are to be construed as non-exhaustive, or in other words, as meaning “including, but not limited to”.

[0033] Furthermore, throughout the document, unless the context requires otherwise, the word “include” or variations such as “includes” or “including” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. [0034] In various embodiments, a method of enhancing mental wellbeing comprises administering an extract selected from Labisia pumila; and Lignosus rhinoceros wherein the method is a non-therapeutic method.

[0035] As used herein the term “enhancing mental wellbeing” is related to lifting a person’s mood and making them feel good in a non-therapeutic way. In various embodiments, enhancing mental wellbeing comprises making the person taking or ingesting the extract feel relaxed. In various embodiments, enhancing mental wellbeing comprises making the person taking or ingesting the extract feel energized. In various embodiments, enhancing mental wellbeing comprises making the person taking or ingesting the extract feel happy. In various embodiments, enhancing mental wellbeing comprises making the person taking or ingesting the extract feel relaxed and happy. In various embodiments, enhancing mental wellbeing comprises making the person taking or ingesting the extract feel energized and happy. In various embodiments, enhancing mental wellbeing comprises decreasing or reducing the dopamine levels in a person taking or ingesting the extract. In various embodiments, enhancing mental wellbeing comprises decreasing or reducing the serotonin or corticosterone levels in the serum of a person taking or ingesting the extract. In various embodiments, enhancing mental wellbeing comprises increasing the dopamine levels in a person taking or ingesting the extract. In various embodiments, enhancing mental wellbeing comprises increasing the serotonin or corticosterone levels in the serum of a person taking or ingesting the extract. In various embodiments, enhancing mental wellbeing comprises an anxiogenic effect making the person taking or ingesting the extract feel anxious or alert. In various embodiments, enhancing mental wellbeing is selected from the group consisting of anxiogenic effect, alertness, pleasure, excitement, energized, relaxed, and happiness.

[0036] In various embodiments, a composition extracted from any one of Labisia pumila; or Lignosus rhinoceros for use in enhancing mental wellbeing.

[0037] In various embodiments, a method of enhancing mental wellbeing comprising delivering a composition comprising a powdered extract, optionally dissolved in a solvent, wherein the composition is (a) extracted from; a Labisia pumila plant or a Lignosus rhinoceros fungus; and (b) capable of inhibiting cyclooxygenase receptor by at least 30%.

[0038] As used herein the term “extract” is related to any preparation obtained from the plant Labisia pumila or the fungus Lignosus rhinoceros. In various embodiments the extract may be obtained using the process outlined in [Fig. 4] or similar extraction methods that result in a dry powdered extract. In various embodiments the average particle size of the powdered extract comprises at least 70pm, or at least 71 m, or at least 72pm, or at least 73|jm, or at least 74|jm, or at least 75pm. In various embodiments the average particle size of the powdered extract comprises 70-100pm, or 71 -100pm, or 72-100pm, or 73-100pm, or 74-100pm, or 75-100pm.

[0039] In various embodiments the solvent is water. In various embodiments the solvent is ethanol or ethanol and water mix.

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

[0041] In various embodiments, the composition is capable of inhibiting 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 may be determined by measurement of oxygen consumption, peroxidase co-substrate oxidation, prostaglandin (PG) detection or by quantifying PGE2 produced by enzymatic conversion of arachidonic acid, in the presence or absence of potential inhibitors or by any other method known in the art.

[0042] In various embodiments, the method comprises extracting the composition from leaves and/or stems of Labisia pumila. In various embodiments, the method of extracting the composition from leaves and/or stems of Labisia pumila may be according to the method outlined in [Fig. 4],

[0043] In various embodiments, the method comprises determining that 0.1 % to 0.2% of gallic acid is present in the extract prior to delivering the composition. In various embodiments, gallic acid may be determined by any of the known methods including spectrometry, 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 where there is a need to confirm the powdered extract includes the composition capable of enhancing mental wellbeing.

[0044] In various embodiments, the method comprises ingesting the described Labisia pumila extracts to enhance mental wellbeing comprising an anxiogenic effect wherein enhancing mental wellbeing results in feeling anxious. Such feelings have the advantage of increasing or stimulating the alertness of an individual.

[0045] In various embodiments, the method comprises extracting the composition from sclerotia of Lignosus rhinoceros. In various embodiments, the composition is extracted only from Lignosus rhinocerus sclerotia with pore size of 6-7 pore/mm. This allows the Lignosus rhinoceros to be accurately classified prior to the extraction. In various embodiments, the method of extracting the composition from sclerotia of Lignosus rhinoceros may be according to the method outlined in [Fig. 4],

[0046] In various embodiments, the method comprises determining that SEQ ID NO. 1 is present in the sclerotia of Lignosus rhinoceros prior to extracting the composition. The nucleotide sequence set forth in SEQ ID NO. 1 comprises a unique nucleotide sequence of the internal transcribed spacer (ITS) ITS1 region located between rRNA structural genes (bases 149-168 in nucleic acid sequence set forth in SEQ ID NO. 1). This allows the Lignosus rhinoceros to be accurately classified prior to the extraction. In various embodiments, the presence of SEQ ID NO. 1 may be determined using PCR primers (5’-

GTTTTCAGCCGGCGTTTGC-3’ set forth in SEQ ID NO. 2) and (5’-

TCCTCCGCTTATTGATATGC-3’ set forth in SEQ ID NO. 3). In various embodiments determining the presence of SEQ ID NO. 1 in the extract is only necessary where there is a need to confirm the powdered extract includes the composition capable of enhancing mental wellbeing.

[0047] In various embodiments, the method comprises taking or ingesting the described Lignosus rhinoceros extract wherein enhancing mental wellbeing comprises feeling pleasure from an increased level of dopamine. In various embodiments, the method comprises measuring the dopamine level.

[0048] In various embodiments, the method comprises taking or ingesting the described Lignosus rhinoceros extract wherein enhancing mental wellbeing comprises feeling excited from an increased level of serum corticosterone. In various embodiments, the method comprises measuring the serum corticosterone level before and after taking or ingesting the described Lignosus rhinoceros extract to confirm it has enhanced the mental wellbeing in the individual taking or ingesting the described Lignosus rhinoceros extract.

[0049] In various embodiments, the method comprises formulating the composition as a food preparation, a supplement, or a beverage. [0050] In various embodiments, the term “food preparation” refers generally to material of either plant or animal origin, or of synthetic sources, that contain elements such as essential nutrients, carbohydrates, protein, fat, vitamin, mineral that may be consumed or ingested by an organism such as an individual to sustain growth, repair, vital processes or provide energy.

[0051] In various embodiments, the term “supplement” refers generally to a product that contains substances like vitamins, minerals, nutrients, botanicals, amino acids that are intended to supplement food and/or drink consumed or ingested by an organism such as an individual to sustain growth, repair, vital processes or provide energy.

[0052] In various embodiments, the term “beverage” refers generally to a liquid for drinking. In various embodiments the liquid may be water, flavoured water, soft drinks, alcoholic drinks, health drinks, an enriched drink, a diary-based drink such as milk or yogurt a fruit juice, a vegetable juice or any combination of these that may be consumed or ingested by an organism such as an individual.

[0053] In various embodiments, the method comprises delivering the composition orally. In various embodiments, the method comprises delivering the composition nasally. In various embodiments, the method comprises delivering the composition as a gas or an aerosol. In various embodiments, the method comprises delivering the composition intravenously. In various embodiments, the composition is delivered orally. In various embodiments, other known delivery methods may also be used to deliver a dosage that results in enhanced mental wellbeing or mood.

[0054] In various embodiments, a composition comprises a powder extracted from a Labisia pumila plant or Lignosus rhinoceros fungus wherein the composition is capable of: inhibiting cyclooxygenase receptor by at least 30%; and enhance mental wellbeing, and wherein the composition is formulated as a food preparation, a supplement, or a beverage.

[0055] In various embodiments, the powder extracted is extracted from leaves and/or stems of Labisia pumila and comprises 0.1 % to 0.2% gallic acid of the total powder extract. In various embodiments the amount of gallic acid in the extract is only detected to confirm the powdered extract includes the composition capable of enhancing mental wellbeing.

[0056] In various embodiments, the extract from Labisia pumila comprises one or more HPLC peak including gallic acid having a retention time between 4 and 5 minutes and a second peak between 9 and 1 1 minutes when 10 mg/ml of the extract is eluted in 50% water: 50% pure methanol. In various embodiments, the extract from Labisia pumila comprises a HPLC peak for gallic acid having a retention time between 4 and 5 minutes and one or more additional peak/s with a pattern substantially similar to the peak pattern depicted in [Fig. 5] when 10 mg/ml of the extract is eluted in 50% water: 50% pure methanol.

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

[0058] In various embodiments, the powder extracted from the Labisia pumila plant or Lignosus rhinoceros fungus comprises at least 25% weight of the food preparation, supplement, or beverage. In various embodiments, the at least 25% weight comprises weight per total weight of the food preparation or supplement or beverage. In various embodiments, the at least 25% weight comprises weight per total volume of the food preparation or supplement or beverage. In various embodiments, the at least 25% weight comprises 25% to 51 % weight per total weight of the food preparation or supplement or beverage. In various embodiments, the at least 25% weight comprises 25 to 51 % weight per total volume of the food preparation or supplement or beverage.

[0059] In various embodiments, a food formulation comprising a composition as described herein above for use in enhancing mental wellbeing.

[0060] In various embodiments, the powder extracted from the Labisia pumila plant or Lignosus rhinoceros fungus comprises at least 25% weight of the food formulation. In various embodiments, the at least 25% weight comprises weight per total weight of the food formulation. In various embodiments, the at least 25% weight comprises weight per total volume of the food formulation. In various embodiments, the at least 25% weight comprises 25% to 51 % weight per total weight of the food formulation. In various embodiments, the at least 25% weight comprises 25 to 51 % weight per total volume of the food formulation.

[0061] According to various embodiments a system fortesting the effect of a composition comprising a powdered extract extracted from a biological organism on enhancing mental wellbeing, the system having a home cage for feeding and a series of test containers comprising: h) an open field arena having a red/near-infrared light capable of illuminating the arena with a computer video tracking system using a near-infrared sensitive camera capable of recording the arena; i) a chamber with a light zone and a dark zone with a door between the light zone and the dark zone and a sensor on the door between the light zone and the dark zone; j) a chamber comprising a maze with open segments, closed segments enclosed with side walls, and elevated segments, a near-infrared sensitive camera capable of recording each of the open segments, closed segments, and elevated segments; k) two small ceramic cups to be placed at opposite ends of the home cage; l) a Y-shaped maze with three arms at a 120° angle from each other with a computer video tracking system capable of recording movement in each arm; m) a piece located above a sensing range, a fixing device connected with said piece for fixing a tail of an animal; and a base with a computer video tracking system capable of recording movement within the sensing range; and n) a transparent Plexiglas cylinder with a base capable of containing water with a computer video tracking system capable of recording movement within the transparent Plexiglas cylinder.

[0062] In various embodiments the test containers are the same as those depicted in Fig, 6, A and B, Fig, 8 A and B, and Fig. 10 A, B and C.

[0063] According to various embodiments a method for testing the effect of a composition on mental wellbeing comprising: a) extracting a powdered extract from a biological organism; b) formulating the powdered extract as a food preparation, a supplement, or a beverage; c) feeding the food preparation, supplement, or beverage to a test subject and feeding a similar food preparation, supplement, or beverage that does not contain the powdered extract to a control subject in a home cage; d) placing the test subject and the control subject in an open field arena illuminated by a red/near-infrared light providing less than 20 lux of illumination in the arena and recording the movement of the test subject and the control subject; e) giving the test subject and the control subject at least two days to recuperate and providing handling interaction of the test subject and the control subject; f) placing the test subject and the control subject in a chamber with a light zone and a dark zone with a door between the light zone and measuring the movement of the test subject and the control subject between the light zone and the dark zone; g) placing the test subject and the control subject in a chamber comprising a maze with open segments, closed segments enclosed with side walls, and elevated segments, recording the movement of the test subject and the control subject in each of the open segments, closed segments, and elevated segments; h) placing two small ceramic cups at opposite ends of the home cage a first small ceramic cup filled with the food preparation, supplement, or beverage and a second small ceramic cup filled with the food preparation, supplement, or beverage that does not contain the powdered extract and measuring which food preparation, supplement, or beverage the test subject and the control subject prefer; i) placing the test subject and the control subject in a Y-shaped maze with three arms at a 120° angle from each other and recording movement of the test subject and the control subject in each arm; j) fixing a tail of the test subject and the control subject to a fixing device connected with a piece located above a sensing range, suspending the test subject and the control subject at least 25cm above a base wand recording movement of the test subject and the control subject within the sensing range; k) giving the test subject and the control subject at least two days to recuperate and providing handling interaction of the test subject and the control subject; and l) placing the test subject and the control subject in a transparent Plexiglas cylinder with a base partially containing water and recording movement of the test subject and the control subject within the water of the transparent Plexiglas cylinder; wherein between each step of d) to I) the test subject and the control subject are returned to the home cage where the test subject is fed the food preparation, supplement, or beverage and the control subject is fed the similar food preparation, supplement, or beverage that does not contain the powdered extract.

[0064] In various embodiments, the tests should be carried out sequentially from a) to I) and/or at least d) to I) providing the tests in the specific order as listed from step d) to I) or a) to I) allows the test subject and control subject to gradually be exposed to stressful tests. This has the advantage of minimizing potential interaction between the tests as the stress level from the prior test does not negatively affect the next test.

[0065] In various embodiments steps d) and i) are suitable fortesting energizing effect of the test composition. In various embodiments step f) and g) are suitable fortesting relaxation and calming effect of the test composition. In various embodiments steps h), j) and I) are suitable for testing happiness and energizing effect of the test composition.

[0066] In various embodiments the biological organism from which a test composition is extracted may be a plant, fungus, or animal. In various embodiments the test composition may comprise the powder extracted from the Labisia pumila plant or Lignosus rhinoceros fungus as described herein above.

[0067] In various embodiments the test subject are mice or rats, and the control subject is the same species or variety of mouse or rat used for the test subject. In various embodiments the test composition or powdered extracts are integrated into food so that the intake of each animal is expected to be at 0.25% or 0.5% of the diet composition.

SEQ ID NO. 1 , internal transcribed spacer of Lignosus rhinoceros: GGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAACG AGTTTTGAAATGGGTTGTAGCTGGCCGTCAAGGCATGTGCACGCCCTGCTCATCCAC TCTACACCTGTGCACTCACTGTGGGTTTTCAGGCGGCGTCGCCTCCGTGCGGCGTTG

GTTGGGGGCCTGCGTTTTTACCACAAACCACTGTCAGTATCAGAATGTCGTGTGTTGC GATGTTAACGCAAACAATGTACAACTTTCAGCAACGGATCTCTTGGCTCTCGCATCGA TGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCG AATCTTTGAACGCACCTTGCGCTCCTTGGTATTCCGAGGAGCATGCCTGTTTGAGTGT CATGAAATTCTCAACCTGCAGATTTTTGCTGCAGGCTTGGACTTGGAGGCCCCTTTGC CGGTTCTAGTCAGTCGGCTCCTCTTAAACGCATTAGCCTGTGTCCTTGCGGATCGGC CCCTCGGTGTGATAAAACTTGTCTACGCCGGTGACCGTGAAGCGTTTTTGGCTGGCTT CCAACCGTCTCGCTGGAGACAGATTTTCACTATTTGACATCTGACCTCAAATCAGGTA GGACTACCCGCTGAACTTAAGCATATCAATAAGCGGAGGA.

SEQ ID NO. 2, forward primer for internal transcribed spacer of Lignosus rhinoceros: 5’- GTTTTCAGCCGGCGTTTGC-3’

SEQ ID NO. 3, reverse primer for internal transcribed spacer of Lignosus rhinoceros: 5’- TCCTCCGCTTATTGATATGC-3’

SEQ ID NO. 4, forward primer for POMC: 5’-CCTCACCACGGAAAGCA-3’ SEQ ID NO. 5, reverse primer for POMC: 5’-TCAAGGGCTGTTCATCTCC-3’

SEQ ID NO. 6, forward primer for housekeeping gene GAPDH: 5’-

TTGTGCAGTGCCAGCCTC-3’

SEQ ID NO. 7, reverse primer for housekeeping gene GAPDH: 5’-

CCAATACGGCCAAATCCG-3’

[0068] The following are non-limiting examples of embodiments.

[0069] Examples

[0070] Example 1 : screening of plant extracts for cytotoxicity of neural cells

[0071] In this study, substances from natural products and supplements from Asia were screened, for compositions that may improve mood and mental wellbeing of humans. Among 23 tested samples, three samples showed significantly higher cell viability against Rat Hypothalamus Neurons (R-hth-507) cell. In Realtime-PCR experiment, seven samples showed p-endorphin synthesis activity, significantly. These results suggested that extracts have a potential as mental wellbeing drug candidate. The two most promising plant extracts were studied further.

[0072] Sample preparation

[0073] 23 natural product samples were ground and dissolved in MQ water at concentration of 50 mg/mL for first screening. The dissolved samples were mixed well by vortex and sonicated for 5 minutes. This step was repeated 6 times. In the second screening and Realtime PCR experiment, each sample were prepared at different concentrations as indicated in [Table 1],

[0074] First screening

[0075] To investigate anti stress activity from plant extract, activation activity for Rat Hypothalamus Neurons cell (R-hth-507) was examined.

[0076] Cell culture

[0077] Rat Hypothalamus Neurons (R-hth-507) were purchased from Lonza Bioscience (Basel, Switzerland) and cultured in Primary Neuron Growth Medium (PNGM) supplemented with Primary Neuron Growth Medium Singlequots which contains all growth factors and supplements required for successful culture of rat and mouse neurons.

[0078] Cell viability assay

[0079] The cytotoxicity of R-hth-507 was measured in vitro by a colorimetric method that measures the reaction of mitochondria to the dye 3-(4,5-dimethylthiazol-2-yl)-2,5- diphynyltrazoliumbromide (MTT) purchased from Tokyo Chemical Industry (Tokyo, Japan). Generally, reduction of MTT occurs in metabolically active cells. The level of activity is thus measured spectrophotometrically. Cells were cultured in a 96-well plate at a density of 1 x 10⁵ cells/mL for 24 h. Then, R-hth-507 cells were treated with samples, dissolved in MQ. After 24 h, 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 a 4 h incubation at 37°C in 5% CO₂. The supernatant was discarded, and 40 mM HCI-isopropanol (100 mL) was added to dissolve the formazan crystals. The absorbance was measured at 570 nm using a Microplate Reader 310-Lab (Corona Electric Company, Ibaraki, Japan).

[0080] As the result of cytotoxicity test, only one sample (No. 7) showed higher cell viability among 23 samples tested, compared to a water control. On the other hand, 13 samples demonstrated cytotoxicity to R-hth-507 cells.

[0081] Areca catechu (sample 7), also known as betel tree, is wildly used as chewable tabacco. Betel nut chewing bring mental stimulation effect such as reduce stress, feeling of euphoria, and sense of wellbeing. Our result also supported that A. catechu can protect neurons which may change people’s moods. However, such extracts are reported to increase the risk of oral and pharyngeal cancers where the plant is chewed (Athukorala et al. J. Addict. 2021 ; 2021 :9967097).

[0082] The cell death may occur because of high concentration used in each sample. While natural plant extracts may be safe and good for health and wellbeing, there is a need to be careful that even if the natural plant extracts have positive effects on individuals, they can also be harmful to our body at different concentration. Thus, it is important to identify appropriate amount to earn functional effect of natural products with reduced risks of side effects.

[0083] Second screening: optimizing concentrations

[0084] In a second screening, each sample was prepared at different concentrations as shown in [Table 1], Sample 6 and 10 showed significantly higher cell survival rate of the R- hth-507 cells than the cells treated with MQ in second screening. The average cell viability of sample 4, 5, 7 and 9 were slightly higher than water control, because the cells used in this study was rat brain hypothalamus neurons, the high cell viability of this sample against R- hth-507 is consistent to these previous studies. It can be seen in Fig. 2 that 10 pg/mL of sample 9 (Lignosus rhinoceros) have no cell cytotoxicity on R-hth-507 cells.

[0085] [Table 1]: Natural materials from plant extracts obtained in Asia.

[0086] Eight samples showed cytotoxicity against tested cells and these samples showed similar tendency in first screening as well (sample Nos. 1 1-14 and 17-20). Conversely, based on second screening results of the second sample concentrations, the 8 samples having no cell cytotoxicity on R-hth-507 cells (sample Nos. 2, 4, 5, 6, 7, 9, 10 and 23) that demonstrated the highest neuroprotection were chosen to use in real-time PCR examination. Further experiments were conducted on these more successful extracts.

[0087] Real-time PCR

[0088] The gene expression of proopiomelanocortin (POMC) involved in p-endorphin synthesis was investigated by real-time PCR. The sample concentration [Table 1] for this experiment, was decided based on first and second screening. After being synthesized in the brain, POMC is broken down into endorphins and corticosteroids by the action of enzymes. Therefore, it is suggested that increased expression of the POMC gene also increases the production of endorphins.

[0089] Realtime PCR

[0090] The R-hth-507 cells were seeded in 100 mm dishes at 0.4 x 10⁵ cells per dish. After 24 hours of incubation, water extracted samples or MQ as a control was added to the R-hth-507 cells. The R-hth-507 cells were then cultured for 96 hours. Total RNA was purified from the cultured R-hth-507 cells using ISOGENE II (Nippon Gene, Tokyo, Japan). The cDNA strand was synthesized from 500 ng of total RNA using ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan). The quantitative real-time PCR was performed in a total reaction volume of 25 pL using SYBR Premix Ex Taq II (TaKaRa Bio, Shiga, Japan) and 20 ng cDNA per reaction. Real-time PCR was performed using Thermal Cycler Dice Real Time System Lite TP700 (TakaRa, Japan) under the following conditions: 30 sec at 95°C, followed by 40 cycles each of 95°C for 5 sec, 60°C for 30 sec. Primers used for amplification were as follows: CCTCACCACGGAAAGCA and TCAAGGGCTGTTCATCTCC for POMC, TTGTGCAGTGCCAGCCTC and CCAATACGGCCAAATCCG for GAPDH.

[0091] As a result, it was confirmed that the expression of the POMC gene was increased by adding most of the plant extracts tested as compared with the control with the exception of sample 2. It can be seen in Fig. 3 that samples 5 and 7 showed more than three times higher POMC gene expression than water treated cells and sample 9 (Lignosus rhinoceros) have induced expression of POMC gene by more than 2-fold. Each of these results is an indication that such substances or extracts are likely to elicit a feeling of euphoria in individuals that ingest such substances. Water extract of C. asiatica a similar plant to sample 5, was earlier found to have cognitive-enhancing effects in animal models of aging and Alzheimer's disease. The mechanisms of cognitive-enhancing effect of C. asiatica, is postulated to be upregulation of nicotinamide adenine dinucleotide in the brain and modulation of brain-derived neurotrophic factor (Speers et al. Front Pharmacol. 2021 ; 12:788312). The cells used in the current study were neuron cells, where the modulation of neurotrophic factor can affect the R-hth-507 cells. C. pluricaulis a similar plant to sample 4, has earlier been reported to have antidepressant-like effect against neuroinflammation associated depressive behavior induced by chronic unpredictable mild stress in rat (Gupta & Fernandes Biomed Pharmacother. 2019;109:1698-1708). The current results also support that samples 4, 5 and 9 have a nerve protection system and may promote calmness, reduce stress and anger.

[0092] Sample 9 (JJgnosus rhinoceros) commonly known as tiger milk mushroom, also had high POMC gene expression effect. L. rhinoceros has functional activities, such as antioxidant, immunomodulating effect and skin conditioning (Kittimonkolsuk et al., Pharmaceuticals 27;14(2):93 2021). To the best of our knowledge this is the first report that L. rhinoceros may affect mental wellbeing. Sample 23 and Sample 10 activate POMC gene about double that of the control. The POMC gene expression activity of Sample 10 was observed at 10 pg/mL, the concentration is for far under the toxic concentration reported, thus when using of a water extract of Sample 10 as medicine or nutraceutical need to be in low and safe concentration. [0093] Endorphins are neurotransmitters that function in the brain and are thought to be involved in the endogenous analgesic system and bring about euphoria. Seven plant extracts examined were able to increase the production of POMC, which is likely to result in euphoria, to varying degrees.

[0094] Cells are selected based on its capacity to produce certain hormone related to mood as follows:

Cell line SH-SY5Y may be associated with melatonin and gene expression of Arylalkylamin N-acetyltransferase involved with sleep;

Cell line Neuro2A may be associated with serotonin/melatonin and gene expression of Tryptophan hydroxylase involved in healing or happiness;

Cell line R-HTH 507 may be associated with p-endorphin and gene expression of p- endorphin POMC gene involved in euphoria;

Cell line PC12 may be associated with dopamine, NE or epinephrine and gene expression of tyrosine hydroxylase involved in pleasure; and

Cell line CNI-H295R may be associated with cortisol and gene expression of cortisol 11-beta-hydroxysteroid dehydrogenase.

[0095] First water extract of Labisia pumila and Lignosus rhinoceros were prepared and tested on above cells and identified activation of cells and expression of gene involved in hormone production and based on this screening steps.

[0096] Further studies on PC12 cell lines demonstrated that extracts of Labisia pumila plant or Lignosus rhinoceros fungus each dissolved in ethanol were both observed to activate cell activity being indicative of pleasure. It was observed that 50 pg/mL and 100 pg/mL of Lignosus rhinoceros extract in 50% ethanol improved PC-12 cell viability by 147% and 139%, respectively. Similarly, 50 pg/mL Labisia pumila extracts improved PC-12 cell viability by 143%.

[0097] Example 2: functional evaluation tests targeting genes and enzymes related to mental functionality.

[0098] Hot water extract samples were prepared from 2 promising plant samples (i.e., samples 13 and sample 9). These were renamed as Sample 1 is an extract from Lignosus rhinoceros. Sample 2 is an extract from Labisia pumila. These samples were stored in 20 mg/mL DMSO solution.

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

[Table 2]: functional evaluation tests

[00100] Compound binding was calculated as a % inhibition of the binding of a radioactively labeled ligand specific for each target. Compound enzyme inhibition effect was calculated as a % inhibition of control enzyme activity. Cellular agonist effect was calculated as a % of control response to a known reference agonist for each target and cellular antagonist effect was calculated as a % inhibition of control reference agonist response for each target. Results showing an inhibition or stimulation higher than 50% are considered to represent significant effects of the test compounds samples with activity between 30% and 50% are considered "possibly active".

[00101] The results of the functional evaluation study showed that the extracts showed activity at six different receptors targeting cannabinoid receptor 1 , dopamine D2 receptor, p (mu) opioid receptor, delta (delta) opioid receptor, cyclooxygenase, and GABAA receptor. In six studies targeting cannabinoid receptor 1 , dopamine D2 receptor, GABAA receptor, mu opioid receptor, delta opioid receptor, and cyclooxygenase, both samples (JJgnosus rhinoceros, Labisia pumila) demonstrated activity.

[00102] In Vitro Pharmacology: Cellular and Nuclear Receptor Functional Assays.

Agonist Effect: Test Compound Results. [00103] Agonist activity against human opioid receptor mu 1 (mu (MOP) (h)) was observed in extract from Lignosus rhinoceros (23 %), and extract from Labisia pumila (13 %). The expected functionality of these materials is euphoria.

[00104] In Vitro Pharmacology: Enzyme and Uptake Assays

[00105] The inhibitory activity against Human Cyclooxygenase (COX1 (h)) was found to be 55.9 % in Labisia pumila extracts. Potential activity was observed in Lignosus rhinoceros (40.2 %). The expected functionality of these materials are happiness or euphoric feelings. Inhibition of Cox1 pathways have also been known to reduce inflammation thus lowering of body temperature, relieving of pain and have been established as a mechanism for medicine such as aspirin, ibuprofen, and other medicine. Speculation on how reducing of temperature or pain can deliver euphoria is still unclear but reduction of such inflammation can decrease stress of entire body and thus, could explore anti-stress, relaxation and clamming effects.

[00106] Example 3: Gene expression analyses of the cells

[00107] Increased expression of 3 genes was observed in cells exposed to Lignosus rhinoceros extracts. The expression of APH1 B aph1 homolog B, gamma secretase subunit, one of the subunits of y-secretase, one of the enzymes that produce amyloid-beta, a cause of dementia, is decreased, y-secretase, one of the enzymes that produce amyloid p, is downregulated by the plant extract.

[00108] OPTN optineurin is one of the autophagy functions of cell quality control and is responsible for the normal functioning of cells. It also plays a role in maintaining normal cell function. Abnormalities in this gene can lead to neurodegeneration. It is expected that increased expression of this gene will suppress cell deterioration. However, the current status of this gene is not clear. At present, however, there is no data on the suppression of cell degradation by increased expression of this gene.

[00109] VGF is known to play a role in neuronal protection by activating cell survival signals. It has been shown to promote neuronal survival and dendrite growth, to protect against retinal neuronal damage, to repair and grow hippocampal neurons, and to act as an antidepressant.

[00110] This gene has been shown to promote neuronal survival and dendrite growth, protect against retinal neuronal damage, repair and growth of hippocampal neurons, and exhibit antidepressant effects. [00111] In addition, the overall results of the evaluation of Lignosus rhinoceros extracts include gene expression pattern listed in [Table 3] upon the treatment of plants extract on neuronal cell types. SH-SY5Y cells were treated with Lignosus rhinoceros extracts at concentration 10mg/ml and the RNA was harvested after incubating overnight. Lignosus rhinoceros extracts treatment showed reduction of gene expression of Aph1 B, that is one of the enzymes produce protein cause Alzheimer’s disease. Otinuerin is the gene upregulated in Lignosus rhinoceros extracts treated samples is the gene involved in autophagy process and it suggest that the extract may maintain cellular health. In addition, VGF also upregulated in the Lignosus rhinoceros extracts treated samples. VGF is the gene that induce nerve growth and activating survival signals of dendrites and in all, suggest its role in neuronal protection [Table 3], Taking together the results of Lignosus rhinoceros extracts use on neuronal cell types, such as SH-SY5Y, can activate gene that protect and induce growth of neuron and maintain neural cellular health.

Table 3]: gene expression change in Lignosus rhinoceros extract treated SH-SY5Y cell line.

[00112] Based on the in vitro results two plant extracts, an extract from Lignosus rhinoceros and an extract from Labisia pumila were selected for more detailed analysis and characterization.

[00113] Example 4: Extraction process and characterization of Labisia pumila

[00114] The leaves and/or stems from Labisia pumila were prepared as outlined in [Fig. 4] First the raw material of either leaves or leaves and stems are collected [step 1], These are then dried in an oven at 35-50°C for 2 to 4 days alternatively they may be freeze dried or vacuum freeze dried. Once the stems and leaves are died, they are ground to a powder and the powder (100-200kg) is then intensified by dissolving the powder in water (500-1000L) at a temperature of 70-90°C for a few hours with constant stirring [Step 2], The intensification step may be repeated and the filtration [Step 3] may be after the intensification step/s and/or between these intensifications where it is repeated. The intensified liquid is then condensed or evaporated by any known conventional means [Step 4], The condensed liquid is pasteurized at a temperature of 70-90°C [Step 5] and then filtered [Step 6] before being spray dried [Step 7] and separated with a metal separator [Step 8] leaving a purified extracted powder. The purified extract is a brown-coloured powder and contains 0.1-0.2% Gallic acid.

[00115] Characterization of Labisia pumila plant extracts

[00116] Prior to characterization of the purified Labisia pumila extract via HPLC it was dissolved in a solvent (water) at a weight ratio of 4:1 (water: extract) or 10mg/ml 5000pl was injected in a 1290LC01 HPLC with 50% pure Methanol to water solvent ratio. As seen in [Fig. 5] a peak matching the Gallic acid reference standard was observed at retention time 4.301 min and from the standard reference was calculated to be about 0.191 % of the extract composition and an unidentified peak was observed at retention time 9.978 min an initial series of smaller unidentified peaks were observed at retention times between 1 to 2 minutes and a second series of smaller unidentified peaks were observed at retention times between 10 to 13.5 minutes.

[00117] Example 5: Extraction process and characterization of Lignosus rhinoceros

[00118] Lignosus rhinocerus is a polypore or a fungus with large fruiting bodies with pores or tubes on the underside. Only Lignosus rhinocerus with pore size of 6-7 pore/mm were selected. Selection can also be made based on the unique nucleotide sequence of the internal transcribed spacer (ITS) ITS1 region located between rRNA structural genes (bases 149-168) listed here as SEQ ID NO. 1 using PCR primers (5’-GTTTTCAGCCGGCGTTTGC- 3’) and (5’-TCCTCCGCTTATTGATATGC-3’) before the extraction process. The underground sclerotia were used to prepare the extract according to the process in [Fig. 4] whereby first the underground sclerotia are collected [step 1], These are then dried in an oven at 35-50°C for 2 to 4 days alternatively they may be freeze dried or vacuum freeze dried. Once the stems and leaves are died, they are ground to a powder and the powder (100-200kg) may then be intensified by dissolving the powder in water (500-1000L) at a temperature of 70-90°C for a few hours with constant stirring [Step 2], The intensification step may be removed in some cases and the filtration [Step 3] may be after the intensification step or directly after the grinding step or extraction. The filtered product is then condensed or evaporated by any known conventional means [Step 4], The condensed product is pasteurized at a temperature of 70-90°C [Step 5] and then filtered [Step 6] before being spray dried [Step 7] and separated with a metal separator [Step 8] leaving a purified extracted powder. The purified extract is a fine light brown powder. [00119] The Lignosus rhinocerus extract was analyzed and found to contain 1 % fat, 28% -30% carbohydrate, 28% protein of which more than 10% is glycoprotein, 2% Calcium, 4% potassium, 35% magnesium.

[00120] Example 6: In vivo studies in mice

[00121] Study Design for Preliminary Evaluation of Relaxation/Calming, Energising, and Happiness/Euphoria Effects of Eight extract Samples at Two Different Doses in Mice. Animal tests were designed to expose animals to gradually stressful tests and to minimize potential interaction between the tests. This is to ensure the stress level from the prior test won’t affect the next test. The test schedule is outlined in [Table 4],

[Table 4]: Animal test schedule over 12 days.

[00122] Behavioural assessments for relaxation/calming, energising, and happiness effects were performed. In each behavioural test, control mice fed normal laboratory chow were randomly included in the sequence of animals tested for comparison with the mice fed the extract sample supplemented diet. Application of extracts integrated into food was designed so that the intake of each animal is expected to be at 0.25% and 0.5% of the diet composition. On average one animal consumes 3-5g of food per day, therefore, each day each animal is supposed to consume about 10mg (0.25%) to 20mg (0.50%).

[00123] For each test there are 3 groups 1) control group, 2) 0.25% diet group and 3) 0.50% diet group. For each group, there are 10 mice.

[00124] Food Pellet Preparation

[00125] The plant extract samples were custom formulated into standard mouse chow diet pellets for oral administration in the diet. Mice were fed ad libitum with the custom formulated diet. Control animals were fed ad libitum with the same standard diet without inclusion of any extract substance. All the diets were gamma irradiated to ensure that they are sterile.

[00126] To test the acute effects of dietary consumption of the extract substances, the mice were fed with the custom formulated diets from the day before testing and every day during testing. As mice eat mostly at night, delivery of the custom formulated diet containing the extract substances from the day before testing will allow the mice to eat the diet overnight before testing. According to this schedule, each group of mice needs to be fed the custom formulated diet for 10 days.

[00127] On average, mice consume from 3g to 5g of laboratory diet per day. However, while feeding some food is also lost through dropping of food while gnawing (approximately 10 % loss) and pellets left uneaten in the hopper (approximately 20 % loss). Therefore, for each group of 10 animals fed for 10 days, allowing for maximum food consumption and potential loss, we used at least 660g of feed.

[00128] The limiting factor on the amount of each plant extract substance required becomes the scale of production of the custom formulated diet. The formulation mixing and pelleting process is limited by the scale of the equipment used by the diet manufacturing companies. The smallest amount that can be practically custom formulated is therefore about 2.65 kg to yield approximately 1 .9 kg to 2 kg per batch.

[00129] Therefore, 20 g of each plant extract substance was required to yield approximately 2 kg of 0.25% and approximately 2 kg of 0.5% custom formulated diet.

[00130] The plant extracts include the following:

• Labisia pumila 0.25%;

• Labisia pumila 0.5%;

• Lignosus rhinoceros 0.25%; and

• Lignosus rhinoceros 0.5%.

[00131] Behavioural Assessments in Mice

[00132] Behavioural assessments for relaxation/calming, energising, and happiness/euphoria effects will be performed. In each behavioural test, control mice fed normal laboratory chow will be randomly included in the sequence of animals tested for comparison with the mice fed the plant extract sample supplemented diet. [00133] Relaxation/Calming

[00134] Relaxation and calming effects were evaluated through standard behavioural paradigms for detection of anxiety and anxiolytic-like effects. Relaxation and calming effects were evaluated through standard behavioural paradigms for the detection of anxiety and anxiolytic-like effects. These tests were the light-dark box paradigm [Fig. 6A] and the elevated zero maze [Fig 6B], The elevated zero maze is an improved version of the conventional elevated plus maze that overcomes the problem of an indeterminate central zone in the plus-shaped maze.

[00135] Light-dark box

[00136] The mice were tested in a chamber with light and dark zones. Animals were placed individually in the centre of the light arena. The duration of time spent in the light and dark arenas and the number of transits between them were recorded. A sensor on the door between the light and dark arenas allows for unbiased measurement of time spent in each arena and the number of transits between the arenas. Mice have an innate motivation to explore as widely as possible to find potential food sources. However, when in the light chamber this motivation to explore conflicts with the natural behavioural response of mice to prefer dark over light to avoid detection by potential predators. Increased time spent exploring the light chamber is therefore a measure relaxation or calming effects.

[00137] In the light-dark boxtest, anxiety levels are assessed by the amount of time spent in the light box. When a test group spends a longer length of time spent in the light box compared to the control group, the animals in the test group are considered to have reduced anxiety-like behaviour. Conversely, shorter lengths of time spent in the light box, compared to the control group, suggests increased anxiety-like behaviour. From [Fig. 7A], mice in the 0.5% groups for both Labisia pumila and Lignosus rhinoceros spent more time in the light box than mice in the control group, suggesting that these two plant extract diets may have anxiolytic effects although it is not significant.

[00138] Elevated zero maze

[00139] The mice are allowed individually to freely explore a zero-shaped maze with open segments and closed segments enclosed with side walls. The maze is elevated at approximately 50 cm. The subjects are allowed to explore the maze for 5 minutes. The number of transitions between the open and closed segments, latency to enter the open segment, the amount of time spent and number of entries into the open segments are recorded. A computer video tracking system using near-infrared illumination to enable tracking of the mice even in the enclosed segments is used for unbiased recording of the behaviour. Mice have a natural motivation to explore but in the open segments of the zero maze this is in conflict innate fear of exposure to potential predators and the elevated height of the maze. Increased time spent exploring the open segments is therefore a measure relaxation or calming effects.

[00140] In the zero maze, anxiety levels are assessed by the amount of time spent in the open arms. The longer the length of time spent indicates reduced anxiety-like behaviours. Although there are no significant differences in the extract diet groups compared to the control diet group, the 0.5% groups for both Labisia pumila and Lignosus rhinoceros showed a longer average amount of time spent in the open arm than the control group.

[00141] Energising

[00142] Mental function energising effects were assessed through measurements of spontaneous exploratory behaviour in an open field in the dark [Fig. 8A] and through measurement of working memory spontaneous alternation behaviour in a Y-shape maze [Fig. 8B], These behavioural paradigms leverage on normal behaviours of mice to measure mental energisation.

[00143] Open field exploration in the dark

[00144] The mice were placed individually in an open field arena in a dark room (less than 20 lux) illuminated only with red/near-infrared light. The movement of the mice was tracked with a computer video tracking system using a near-infrared sensitive camera. Mice have limited vision for red light, so the room appears dark from the perspective of the mice and they are not inhibited from exploring the open field by their innate fear being exposed under bright light. Under these conditions, the open field paradigm measures spontaneous exploratory behaviour. An increase in exploratory behaviour will indicate mental energisation.

[00145] The dark open field measures spontaneous exploratory behaviour. An increase in exploratory behaviour will indicate mental energisation. Of the plant extract diet groups, mice on Labisia pumila extracts of 0.5% diet showed increased exploration in the dark open field, compared to the control group [Fig. 9A],

[00146] Spontaneous alternation working memory [00147] Mice are placed individually in the centre of a Y-shaped maze with three arms at a 120° angle from each other. The animal is allowed to freely explore all three arms. The animals are tracked with computer video tracking system. If the mouse has an intact spatial working memory, it will have a reduced tendency to enter the recently visited arm. The number of arm entries are scored to determine the number of alternations. A mouse is considered as having entered an arm when all four limbs are within the arm. Increased spontaneous alternation behaviour will indicate mental energisation.

[00148] In the spontaneous alternation working memory test, the animals freely explore all three arms of a Y-shaped maze. If the mouse has an intact spatial working memory, it will have a reduced tendency to enter a recently visited arm. The arm entries are scored to determine the number of alternations. An increase in spontaneous alternation behaviour will indicate mental energisation. The results in the spontaneous alternation of the Y maze compared to controls, as an indication of working memory [Fig. 9B],

[00149] Happiness/Euphoria

[00150] Happiness and euphoria effects were measured with a food preference test [Fig. 10C] and two paradigms conventionally used to detect antidepressant-like activity. Two paradigms for testing antidepressant-like effects are the tail suspension test [Fig. 10A] and the forced swim test [Fig. 10B], Two tests are preformed because they have different sensitivity. The tail suspension test produces cleaner results with less individual differences between mice but is less sensitive. It is therefore also helpful to include the forced swim test, which is more sensitive.

[00151] Feeding preference

[00152] Mice are individually presented with conventional laboratory chow and the feed containing the herbal sample placed separately in two small ceramic cups placed at opposite ends of the home cage. The amount of food consumed is weighed. Greatertime spent eating from the cup containing the food supplemented with the extract sample will indicate a pleasure or happiness effect of the food.

[00153] In the food preference test, mice were individually presented with conventional laboratory chow and the feed containing the herbal sample placed separately in two small ceramic cups placed at opposite ends of the home cage. The amount of food consumed was weighed. Greater time spent eating from the cup containing the food supplemented with the extract sample indicates a pleasure or happiness effect of the food. Of the herbal diet groups, mice on a diet of Lignosus rhinocerus extract showed the same preference for the herbal diet and the control diet [Fig. 11 A], In contrast, mice on a diet of Labisia pumila extract preferred the control diet over the plant extract diet.

[00154] Forced swim test

[00155] Mice are individually placed in a cylinder containing water. Movement is monitored. Immobility is considered to model emotional depression. An increase in the time to immobility will indicate an anti-depressant-like happiness effect.

[00156] In the forced swim test, a reduction in immobility time compared to controls will indicate an anti-depressant-like happiness effect. In the extract diet groups, little difference was observed compared to controls, suggesting that these extracts may not have an anti- depressant-like happiness effect [Fig. 11 B].

[00157] Tail suspension test

[00158] The mice are suspended by the tail for six minutes. Movement is monitored and reduced movement is considered to model emotional depression. There is no risk of tail injury or exclusion as the animal are suspended at a fixed distance from the base of the tail on a standard behavioural apparatus widely used for behavioural testing without any incidence of tail injury. An increase in the time to immobility will indicate an anti-depressant- like happiness effect.

[00159] In the tail suspension test, a reduction in the length of immobility time compared to controls indicate an anti-depressant-like happiness effect. There were no significant differences across groups in the tail suspension test [Fig. 11 C],

[00160] Example 7: Measurement of hormones and neurotransmitters

[00161] At the end of the in vivo experiments, brain tissue and plasma from core blood collection was assayed for corticosterone, dopamine, and 5-HT (serotonin) [Fig. 12]. The assays were performed with ELISA kits. Each ELISA kit allows for a 96 well plate to be run. In each plate, samples from the mice on the control feed will be run alongside samples from the mice feed on the herbal sample supplemented diets.

[00162] Brain tissues were homogenized as per instructions provided by the Enzo Lifesciences kit. Mice that consumed 0.25% and 0.5% of Labisia pumila groups had significantly lower levels of tissue dopamine compared to control groups [Fig. 13], The mice that consumed 0.5% Lignosus rhinocerus had higher tissue levels of dopamine compared to controls. Mice that consumed 0.25% Lignosus rhinoceros had close to significantly higher tissue levels of dopamine than controls.

[00163] Whole blood was collected when the mice were euthanised and spun down to isolate the serum. Serum samples were then run through the Biovision Serotonin kit. The serotonin levels were not significantly different in all groups compared to the control [Fig. 14],

[00164] Whole blood was collected when the mice were euthanised and spun down to isolate the serum. Serum samples were then run through the Enzo Lifesciences Corticosterone kit, with some modifications to the protocol. Serum samples were incubated with steroid displacement hormone before being added to the wells. Standards and serum samples were incubated in the wells for an hour before conjugate and antibody were added.

[00165] The 0.5% Lignosus rhinocerus group showed higher corticosterone levels than controls [Fig. 15],

[Table 5]: Summary of in vivo results where + indicates a statistically significant increase in the extract diet group compared to the control diet group - indicates a statistically significant decrease in the plant extract diet group compared to the control diet group and () indicates a change that is not statistically significant.

[00166] Here, we observe that the Labisia pumila group had reduced activity in the light box and increased activity in the dark open field, which is suggestive of increased anxietylike behaviour. Biochemically, they showed significantly reduced brain tissue levels of dopamine compared to the control group and seemed to have reduced serum levels of serotonin and corticosterone, which may underly the observed anxiogenic-like effects.

[00167] In the in vivo, brain tissues of mice fed by 0.5% Lignosus rhinocerus diet had higher levels of dopamine compared to controls [Fig. 13], The results seem to indicate that the mice on this diet may have been happy or euphoric. In the in vivo, serum samples of mice fed by 0.5% Lignosus rhinocerus diet showed higher corticosterone levels than controls (Figure 15). Higher corticosterone seems to be linked to stress. The results seem to indicate that the mice on this diet may have been excited. Here, in the behaviour of the Lignosus rhinocerus diet group were not statistically significant. However, there was an increase in dopamine levels in brain tissues, and increased serotonin and corticosterone levels in serum.

[00168] It should be further appreciated by the person skilled in the art that variations and combinations of features described above, not being alternatives or substitutes, may be combined to form yet further embodiments falling within the intended scope of the invention.

[00169] As would be understood by a person skilled in the art, each embodiment, may be used in combination with other embodiment or several embodiments.

Claims

1 . A method of enhancing mental wellbeing comprising delivering a composition comprising a powdered extract, optionally dissolved in a solvent, wherein the composition is:

(a) extracted from; a Labisia pumila plant or a Lignosus rhinoceros fungus; and

(b) capable of inhibiting cyclooxygenase receptor in vitro by at least 30%.

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

3. The method according to claim 1 or 2, comprising extracting the composition from leaves and/or stems of Labisia pumila.

4. The method according to claim 3, comprising determining that 0.1 % to 0.2% of gallic acid is present in the extract prior to delivering the composition.

5. The method according to claim 3 or 4, wherein enhancing mental wellbeing comprises an anxiogenic effect.

6. The method according to claim 1 or 2, comprising extracting the composition from sclerotia of Lignosus rhinoceros.

7. The method according to claim 6, comprising determining that SEQ ID NO. 1 is present in the sclerotia of Lignosus rhinoceros prior to extracting the composition.

8. The method according to claim 6 or 7, wherein enhancing mental wellbeing comprises feeling pleasure from an increased level of dopamine.

9. The method according to claim 6 or 7, wherein enhancing mental wellbeing comprises feeling excited from an increased level of serum corticosterone.

10. The method according to any one of claims 1 to 9, comprising formulating the composition as a food preparation, a supplement, or a beverage.

11. The method according to any one of claims 1 to 10, wherein the composition is delivered orally.

12. The method according to any one of claims 1 to 9, comprising wherein the composition is delivered nasally.

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

14. A composition comprising a powder extracted from a Labisia pumila plant or Lignosus rhinoceros fungus wherein the composition is capable of: inhibiting cyclooxygenase receptor by at least 30%; and enhance mental wellbeing, and wherein the composition is formulated as a food preparation, a supplement, or a beverage.

15. The composition according to claim 14, wherein the powdered extract is extracted from leaves and/or stems of Labisia pumila and comprises 0.1% to 0.2% gallic acid of the total powdered extract.

16. The composition according to claim 14 or 15, wherein the powdered extract from Labisia pumila comprises a HPLC peak for gallic acid having a retention time between 4 and 5 minutes and a second peak between 9 and 11 minutes when 10 mg/ml of the powdered extract is eluted in 50% water: 50% methanol.

17. A food formulation comprising a composition of anyone of claims 14 to 16 for use in enhancing mental wellbeing.

18. The food formulation according to claim 17, wherein the powder extracted from the Labisia pumila plant or Lignosus rhinoceros fungus comprises at least 25% weight of the food.

19. A system for testing the effect of a composition comprising a powdered extract extracted from a biological organism on enhancing mental wellbeing, having a home cage for feeding and a series of test containers comprising: o) an open field arena having a red/near-infrared light capable of illuminating the arena with a computer video tracking system using a near-infrared sensitive camera capable of recording the arena; p) a chamber with a light zone and a dark zone with a door between the light zone and the dark zone and a sensor on the door between the light zone and the dark zone; q) a chamber comprising a maze with open segments, closed segments enclosed with side walls, and elevated segments, a near-infrared sensitive camera capable of recording each of the open segments, closed segments, and elevated segments; r) two small ceramic cups to be placed at opposite ends of the home cage; s) a Y-shaped maze with three arms at a 120° angle from each other with a computer video tracking system capable of recording movement in each arm; t) a piece located above a sensing range, a fixing device connected with said piece for fixing a tail of an animal; and a base with a computer video tracking system capable of recording movement within the sensing range; and u) a transparent Plexiglas cylinder with a base capable of containing water with a computer video tracking system capable of recording movement within the transparent Plexiglas cylinder.

20. A method for testing the effect of a composition on mental wellbeing comprising: a) extracting a powdered extract from a biological organism; b) formulating the powdered extract as a food preparation, a supplement, or a beverage; c) feeding the food preparation, supplement, or beverage to a test subject and feeding a similar food preparation, supplement, or beverage that does not contain the powdered extract to a control subject in a home cage; d) placing the test subject and the control subject in an open field arena illuminated by a red/near-infrared light providing less than 20 lux of illumination in the arena and recording the movement of the test subject and the control subject; e) giving the test subject and the control subject at least two days to recuperate and providing handling interaction of the test subject and the control subject; f) placing the test subject and the control subject in a chamber with a light zone and a dark zone with a door between the light zone and measuring the movement of the test subject and the control subject between the light zone and the dark zone; g) placing the test subject and the control subject in a chamber comprising a maze with open segments, closed segments enclosed with side walls, and elevated segments, recording the movement of the test subject and the control subject in each of the open segments, closed segments, and elevated segments; h) placing two small ceramic cups at opposite ends of the home cage a first small ceramic cup filled with the food preparation, supplement, or beverage and a second small ceramic cup filled with the food preparation, supplement, or beverage that does not contain the powdered extract and measuring which food preparation, supplement, or beverage the test subject and the control subject prefer; i) placing the test subject and the control subject in a Y-shaped maze with three arms at a 120° angle from each other and recording movement of the test subject and the control subject in each arm; j) fixing a tail of the test subject and the control subject to a fixing device connected with a piece located above a sensing range, suspending the test subject and the control subject at least 25cm above a base wand recording movement of the test subject and the control subject within the sensing range; k) giving the test subject and the control subject at least two days to recuperate and providing handling interaction of the test subject and the control subject; and l) placing the test subject and the control subject in a transparent Plexiglas cylinder with a base partially containing water and recording movement of the test subject and the control subject within the water of the transparent Plexiglas cylinder; wherein between each step of d) to I) the test subject and the control subject are returned to the home cage where the test subject is fed the food preparation, supplement, or beverage and the control subject is fed the similar food preparation, supplement, or beverage that does not contain the powdered extract.