COMPOSITION OF BOTANICAL EXTRACTS FOR SLEEP AND MOOD SUPPORT

The present invention provides a composition for sleep and mood support, which comprises composition for sleep and mood support, which consists essentially of Xylaria nigripes extract, Panax notoginseng extract and Cuscuta chinensis extract, as main active components at a relative ratio to provide a synergistic effect in increasing GABA content, and GABA-related enzyme activities, and decreasing β-amyloid protein aggregation. Also provided is the use of the composition for manufacturing a formulation for treating insomnia, depression and/or various neurological conditions.

FIELD OF THE INVENTION

The invention concerns a composition of botanical extracts for sleep and mood support, which provides synergistic effects in improving sleep quality, promoting relaxation, and elevating mood.

BACKGROUND Z OF THE INVENTION

γ-Aminobutyric acid (GABA) is a non-proteinogenic amino acid that acts as an inhibitory neurotransmitter, which helps to block the neuro signals/message and thereby reduce stress, anxiety, and fear (calming effect). Hence GABA plays a crucial role in stabilizing mood (Sarasa et al., 2020). Researchers are showing immense interest in GABA and its uptake to regulate various neuropsychological issues, which are related to hyper neuronal excitability and thus useful for treating autism, schizophrenia, Tourette's syndrome, and other hyperactive neuronal disorders (Sarawagi et al., 2021; Kendell et al., 2005). GABA is produced from glutamate/glutamic acid (in GABA-ergic neuron) with the help of GABA decarboxylase (GAD) enzyme and degraded/broken down by GABA transaminase or aminotransferase (GABA-T) enzyme therefore to maintain a nominal level of GABA in the system, the above-mentioned enzymes play a pivotal role (Ngo et al., 2019). Moreover, studies have shown that GABA has a strong association with insomnia, hypertension, and Alzheimer's disease as it balances the production of various neurotransmitters/neurotropic factors and hormones (Yu et al., 2020; Solas et al., 2015). Recently, many scientists have shown interest in regulating GABA levels (nominal level) to maintain a strong healthy mental status in normal and various neurological disorders subjects using various traditional medicine, which has traditionally been used in regulating mood, sleep, and stress/depression (Yu et al., 2020; Byun et al., 2018).

It is still desirable to develop a new composition for sleep and mood support based on the study on GABA.

SUMMARY OF THE INVENTION

Accordingly, it is unexpectedly found in the invention that a composition consisting of three botanical extracts/ingredients exhibits a synergistic effect in increasing GABA content, and GABA-related enzyme activities, and decreasing β-amyloid protein aggregation in PC12 cells, so as to be a potent candidate for treating insomnia, depression and various neurological conditions.

In one aspect, the invention provides a composition for sleep and mood support, which consists essentially of comprisesXylaria nigripesextract,Panax notoginsengextract andCuscuta chinensisextract at a relative ratio to provide a synergistic effect in increasing GABA content, and GABA-related enzyme activities, and decreasing β-amyloid protein aggregation.

In one embodiment of the invention, the composition consists ofXylaria nigripesextract,Panax notoginsengextract andCuscuta chinensisextract, as main active components, at a relative ratio to provide a synergistic effect in increasing GABA content, and GABA-related enzyme activities, and decreasing β-amyloid protein aggregation, and optionally an edible carrier.

In one further embodiment of the invention, the composition consists of 50%-96% fermentedXylaria nigripesextract, 0.001%-10%Panax notoginsengextract and 4%-50%Cuscuta chinensisextract, and optionally an edible carrier.

In one preferred embodiment of the invention, the composition consists of 70%-90%Xylaria nigripesextract, 0.01%-5%Panax notoginsengextract and 10%-30%Cuscuta chinensisextract, and optionally an edible carrier.

In one more preferred embodiment of the invention, the composition consist of 80%-90% fermentedXylaria nigripesextract, 0.05%-1%Panax notoginsengextract and 10%-20%Cuscuta chinensisextract, and optionally an edible carrier.

In another aspect, the present invention provides a method for treating insomnia, depression or a neurological condition in a subject, comprising administering the subject the composition according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. It should be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting.

As used herein, the singular forms “a”, “an” and “the” include plural references unless explicitly indicated otherwise. Thus, for example, reference to “a sample” includes a plurality of such samples and their equivalents known to those skilled in the art.

As used herein, the terms “around”, “about” or “approximately” can generally mean within 20 percent, particularly within 10 percent, and more particularly within 5 percent of a given value or range. Numerical quantities given herein are approximate, meaning that the term “around”, “about” or “approximately” can be inferred if not expressly indicated.

The term “edible carrier” as used herein refers to a carrier that is compatible with the other ingredients of the composition and is not deleterious to a subject. The compositions may contain other components, and may be formulated, for example, by employing conventional solid or liquid vehicles or diluents, as well as additives of a type appropriate to the mode of oral administration (for example, excipients, binders, preservatives, stabilizers, flavours, etc.) according to techniques such as those well known in the art.

According to the invention provides a composition for sleep and mood support, which comprisesXylaria nigripesextract,Panax notoginsengextract andCuscuta chinensisextract at a relative ratio to provide a synergistic effect on GABA content, GABA-related enzyme activities, β-amyloid protein aggregation in PC12 cells.

In one embodiment of the invention, the composition for sleep and mood support consists essentially ofXylaria nigripesextract,Panax notoginsengextract andCuscuta chinensisextract at a relative ratio to provide a synergistic effect on GABA content, GABA-related enzyme activities, β-amyloid protein aggregation in PC12 cells.

In one specific embodiment of the invention, the composition for sleep and mood support consists ofXylaria nigripesextract,Panax notoginsengextract andCuscuta chinensisextract, as main active components, at a relative ratio to provide a synergistic effect on GABA content, GABA-related enzyme activities, β-amyloid protein aggregation in PC12 cells, and an edible carrier.

In one embodiment of the invention, the composition comprises/consists essentially of/consists of 50%-96%Xylaria nigripesextract, 0.001%-10%Panax notoginsengextract and 4%-50%Cuscuta chinensisextract.

In one preferred embodiment of the invention, the composition comprises/consists essentially of/consists of 70%-90%Xylaria nigripesextract, 0.01%-5%Panax notoginsengextract and 10%-30%Cuscuta chinensisextract.

In one more preferred embodiment of the invention, the composition comprises/consists essentially of/consists of 80%-90%Xylaria nigripesextract, 0.05%-1%Panax notoginsengextract and 10%-20%Cuscuta chinensisextract.

Xylaria nigripes, is an edible fungus (WulinShen) belonging to the Xylariaceae family.Xylaria nigripeshas been traditionally used for treating various ailments including insomnia, anxiety, and neurodegenerative disorders (Zhou et al., 2022). It is commonly grown on dead trees and near abandoned termite nests (Hung et al., 2015). Recently many researchers have shown thatXylaria nigripespossesses various biological functions like antioxidant, anti-inflammatory, immunomodulatory, and anti-depressive properties along with hepato-protective and neuroprotective activities (Peng et al., 2015; Song et al., 2011; Ko et al., 2011). The above-mentioned activities are due to the presence of various phytocomponents including polyphenols, adenosine, triterpenoids, and polysaccharides (Divate et al., 2017; Ma et al., 2013). Ample amount of evidence indicated thatXylaria nigripeswould improve GABA production and thus plays a key role in reducing depression and induce sleep (Zhoa et al., 2014; Wu et al., 2012). In the present invention, theXylaria nigripesextract is obtained from the mycelium ofXylaria nigripes, particularly the fermented extract ofXylaria nigripesmycelium.

Panax notoginseng(root) is a popular traditional Chinese medicine (ChineseGinseng) rich in ginsenosides (saponins). Previous studies have shown that ginsenosides fromPanax notoginsenghave considerably improved memory (cognitive function) and learning ability along with anti-depression, antioxidant and anti-inflammatory properties and thus altering the amyloid genesis process (Lin et al., 2019; Chen et al., 2019). Likewise, Zheng and his colleagues (2019) in his study confirmed that ginsenosides fromGinsengcould enhance the production of acetylcholine and GABA as well as lower β-amyloid protein accumulation and thereby attenuates Aβ-amyloid protein-induced neuronal damage. In the present invention, thePanax notoginsengis obtained from the roots ofXylaria nigripes, particularly the water extract ofPanax notoginsengroots.

Cuscuta chinensisis traditional medicine and is rich in flavonoids, phenolic acids, and polysaccharides (Donnapee et al., 2014). Previously, various scientists have proved thatCuscuta chinensisexhibits numerous pharmacological activities including antioxidant, anti-inflammatory, anti-depressant, anti-aging as well as renoprotective, hepatoprotective, and neuroprotective properties (Lin et al., 2018; Donnapee et al., 2014; Mokhtarifar et al., 2012). Zhen and others (2006), indicated thatCuscuta chinensiscould considerably improve the survival rate of PC12 cells via lowering oxidative stress and thereby exhibiting neuroprotective properties. In the present invention, thePanax notoginsengis obtained from the seeds ofXylaria nigripes, particularly the ethanol extract ofPanax notoginsengseeds.

According to the invention, Some examples of the composition for sleep and mood support are given below:

The present invention is further illustrated by the following examples, which are provided for the purpose of demonstration rather than limitation.

Examples

Methods

Preparation of Three Compositions

Three highly purified botanical extracts including XE, XPE, and XPCE extracts were tested for the study. The three compositions were provided by NuLiv Sciences USA, inc. (Brea, CA, USA): XE (Xylaria nigripesextract) rich in polysaccharides and flavonoids, XPE (Xylaria nigripesextract andPanax notoginsengextract) rich in ginsenosides and XPCE (Xylaria nigripesextract,Panax notoginsengextract andCuscuta chinensisextract) rich in polysaccharides and flavonoids.

TheXylaria nigripesextract was obtained from the mycelium ofXylaria nigripes, particularly the fermentedXylaria nigripesextract. ThePanax notoginsengextract was obtained from the roots ofPanax notoginseng, particularly the water extract ofPanax notoginsengroots. TheCuscuta chinensisextract was obtained from the seeds ofCuscuta chinensis, particularly the extract ofCuscuta chinensisseeds with a 65%-95% ethanol solution.

Culturing of PC12 Cells

The PC12 cell line was purchased from BCRC (Hsinchu, Taiwan). PC12 cells were grown under Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 5% fetal bovine serum (FBS), 10% horse serum, and 50 μg/L gentamycin in a humidified atmosphere of 5% CO2 at 37° C. Experiments were performed only when PC12 cells grown to ˜80% confluence between passages 2-6. Before three compositions (XE, XPE and XPCE) treatments at different concentrations (40-120 μg/mL), the cells were grown in FBS/horse serum and an antibiotic-free medium for 16 h. The protein content of PC12 cell lysate was estimated using the BCA protein assay kit (ab102536) provided by Abcam (Cambridge, UK).

GABA Content

GABA contents in PC12 cells treated with different concentrations of the three compositions (XE, XPE and XPCE) were assayed by BioVision Quick Detect-GABA ELISA kit (E4457) purchased from BioVision (MA, USA) according to the manufacturer's protocol.

GABA Related Enzymes

GABA-T activity in PC12 cells was detected using a GABA-T assay kit (E-134) provided by Biomedical Research Service Center and Application (NY, USA) based on the supplier's instruction.

GAD activity was assayed using BioVision Glutamate Decarboxylase Activity Assay Kit (K2021) purchased from BioVision (MA, USA) according to the manufacturer's protocol.

PC12 cells viability with and without Aβ 1-42 peptide

PC12 cells were plated in 96 well plates at a density of 30,000 cells/well and incubated with each Zylaria composition at different concentrations (30-120 μg/mL) for 24 h at 37° C. Add 10 μl of the CCK-8 solution (provided with a Cell counting kit) to each well of the plate. Incubate the plate for 24 h in the incubator and the absorbance at 450 nm was measured using a microplate reader. Also, the cell viability of PC12 cells with Aβ1-42(cytotoxic) peptide was also measured similarly as mentioned above to confirm the neuroprotective activity of the three compositions. In brief, Aβ1-42(10 μM) was diluted with serum-free culture medium and incubated with PC12 cells (30,000 cells/well) for 24 h and then treated with 3 different Zylaria compositions for another 24 h. Then, the PC12 cell viability was determined by using the Cell Counting Kit-8 (CCK-8) as mentioned above.

The process of beta-amyloid protein aggregation was monitored using the ThT assay kit. ThT can assemble with misfolded amyloid proteins and emit fluorescence as a result of this union. Initially, Aβ1-42(10 μM final concentration) peptide/protein was mixed in equal volumes with each Zylaria composition (30-120 μg/mL), and ThT (200 μM final concentration) was added on 96-well plates. The ThT fluorescence was measured using a multi-detector microplate reader fluorescence spectrophotometer set at 450 nm for excitation and 485 nm for emission wavelengths. Fluorescence emission data were recorded every 5 minutes over a 3 h period.

Statistical Analysis

All the experiments were performed in triplicate (n=3). Data are expressed as the mean±standard deviation (SD). All statistical analysis was conducted using SPSS software, version 17 (SPSS, IBM, NY, USA). All the values were evaluated using one-way ANOVA, followed by Duncan's multi-comparison test to compare the difference between the three compositions at different concentrations. Differences were deemed significant when the probability values were less than 0.05 (p<0.05).

Results

Evaluation of GABA Content in PC12 Cells after Treatment with the Three Compositions

As shown inFIG.1providing the GABA content (ng/mg protein) and Table 1 listing the relative GABA content (%) in PC12 after treatment with the three compositions, the GABA content was considerably increased in PC12 cells, which were treated with the three compositions. Particularly, the 120 μg/mL concentration of XE (138.1%), XPE (139.4%), and XPCE (180.8%) showed the highest GABA levels than other concentrations. However, XPCE compositions rich in polysaccharides, flavonoids, and saponins exhibited the best GABA enhancing property (increase GABA synthesis) as compared to XE and XPE.

GABA Transaminase (GABA-T) Activity in PC12 Cells after Treatment with the Three Compositions

The GABA-T activity in PC12 cells after treatment with the three compositions and control were shown in Table 2 (%) andFIG.2. The relative activity of GABA-T was considerably suppressed by all the three compositions (XE/XPE/XPCE) in PC12 cells in a dose-dependent manner than the control group. Here also, XPCE (120 μg/mL; 60.2%) displayed the highest (p<0.001) GABA-T inhibitory activity and subsequently improved GABA content.

GABA Decarboxylase (GAD) Activity in PC12 Cells after Treatment with the Three Compositions

As shown in Table 3 (%) andFIG.3, the GAD activity in PC12 cells were significantly increased after treatment with XE (p<0.05; p<0.01), XPE (p<0.01; p<0.001), and XPCE (p<0.001) with different concentration. XPCE displayed greater GAD enhancing activity in a dose-dependent fashion than XE and XPE.

Cells Viability with and without Aβ 1-42 Peptide in PC12 Cells after Treatment with the Three Compositions

Initial analysis indicated the cell viability of PC12 cells was significantly increased when treated with the three compositions for 24 h. The result showed that the three compositions had no cytotoxic activity against the PC-12 cell (Table 4 andFIG.4). XPCE composition exhibited a higher proliferative effect (p<0.001) on the PC-12 cells than XE and XPE compositions.

However, the cell viability of PC12 cells exposed to Aβ1-42peptides for 24 h, followed by pretreatment with the three compositions has shown a considerable increase in a dose-dependent manner (Table 5 andFIG.5). In addition, XPCE (120 μg/mL) displayed the highest proliferative and cytoprotective activity (p<0.001) as compared to other compositions (XE and XPE).

Aβ Protein Aggregation in PC12 Cells after Treatment with the Three Compositions

The PC12 cells exposed with Aβ1-42peptides and followed by the addition of the three compositions showed potent inhibitory activity of beta-amyloid protein aggregation and subsequent neuronal injury/damage. All the three compositions showed good anti-Aβ1-42peptides aggregation properties (FIG.6). Nevertheless, XPCE exhibited the highest (p<0.001) inhibitory activity of beta-amyloid protein aggregation in a dose-dependent fashion as compared with XE and XPE compositions. Table 6 shows the percentage change in inhibition of Aβ protein aggregation (%). Based on the above data, the author confirmed that XPCE composition showed better neuroprotective activity than XE and XPE compositions.

DISCUSSION

It was the first time to conduct an experiment to check the combinations of different botanical compositions (XE, XPE and XPCE) on the GABA system and Aβ peptides aggregation. Since, various researchers have hinted that many Chinese traditional medicines likeXylaria nigripes, Panax notoginseng, andCuscuta chinensiscould positively maintain GABA levels and also lower Aβ peptides deposition in neuronal cells to exhibit potent neuroprotective activity (Lin et al., 2018; Donnapee et al., 2014; Song et al., 2011). Hence, this novel combination of botanical compositions (XE, XPE and XPCE) was used to check the holistic effect on GABA production and lower beta-amyloid protein production and its aggregation. GABA plays a major role in maintaining a healthy mental status, as it regulates various neurotransmitters/neurotropic factors and hormones production and thus useful in treating insomnia and depression (Yu et al., 2020; Solas et al., 2015) as well as various neuropsychological issues especially autism, schizophrenia, and other hyperactive neuronal disorders (Sarawagi et al., 2021; Sarasa et al., 2020).

GABA shunt system play a crucial role in maintaining proper balance in GABA levels (metabolism of GABA) using various GABA metabolizing enzymes including GAD and GABA-T (Sarawagi et al., 2021). In the present study, all the three compositions treatments effectively improved the GABA production and hence the GABA content was considerably improved. The author hypothesizes that the rich content of flavonoids, saponins, and polysaccharides in the three compositions might influence the GABA synthesis. Previously, many studies have proved that (Xylaria nigripes, Panax notoginseng, Cuscuta) would trigger GABA production and thus plays a key role in reducing depression and insomnia (Forouzanfar et al., 2020; Zheng et al., 2018; Zhao et al., 2014; Wu et al., 2012). To confirm the above statement, the author would like the check the activity of two major enzymes like GAD and GABA-T, which are involved in the GABA shunt system. The activities of GAD and GABA-T were considerably improved and suppressed respectively by all three compositions (XE, XPE and XPCE) in PC12 cells in a dose-dependent manner. As the concentration of XE, XPE and XPCE increased the activity of GAD and GABA-T were significantly altered. However, the 120 μg/mL of XPCE composition showed the highest GABA synthesizing property (elevated GABA content), via increasing the activation of GAD and lowering the activity of GABA-T. Wu and other (2012), also indicated thatXylaria nigripecan increase the permeability of glutamate and thus enhance the activity of GAD, which in turn regulate the activity of GABA-T and thus maintain the nominal levels of GABA.

To cross-check the cytoprotective and proliferative activity the author decided to evaluate the PC12 Cell's viability with or without Aβ1-42peptide (cytotoxin). The PC12 cells after treatment with three compositions showed potent cytoprotective activity by increasing the PC12 cells count (proliferative activity) after 24 h of exposure to Aβ1-42peptide. The above statement proved that three compositions in particular XPCE displayed good cytoprotective and proliferative properties due to the presence of saponins and flavonoids. Likewise, Divate and his colleagues (2017), proved that the GABA-like property ofXylariahelps to protect the cells from H2O2-induced cytotoxicity. Also, ginsenosides (saponins) are reported to improve PC12 cell proliferation and could effectively prevent MMP-induced cytotoxicity (Hashimoto et al., 2012). As mentioned previously that Aβ peptide/protein is a neurotoxin, which is the major contributor to various neurodegenerative disorders especially Alzheimer's disease (AD) (Solas et al., 2015). To assess the neuroprotective property of three compositions, the PC12 cells were exposed to Aβ peptide/protein and the amount of Aβ peptide/protein aggregation/deposition was measured. The present data indicated that PC12 cells exposed to Aβ peptide/protein and followed by the addition of three compositions showed significant inhibitory activity of beta-amyloid protein aggregation/deposition. In view of the experiment results, the compositions of three botanical extracts (XPCE) exhibited a potent neuroprotective activity. In addition, the compositions of three botanical extracts (XPCE) showed the highest inhibitory activity of beta-amyloid protein aggregation than each of the three extracts or the combination of two botanical extracts. It was proved by the experimental data that the compositions of three botanical extracts (XPCE) had higher cytoprotective and neuroprotective properties than eitherXylaria nigripesextract (XE) or a composition ofXylaria nigripesextract andCuscuta chinensisextract (XCE), but lower oxidative stress (suppress free radical production) as well as inhibited Aβ protein misfolded/aggregation (altering various mitochondrial proteins), thus possibly lowering the onset of Alzheimer's Disease (AD) (Zheng et al., 2019; Shim et al., 2017; Choi et al., 2014).

CONCLUSION

The three compositions (XE, XPE and XPCE) displayed good glutamate uptake and GABA release through elevated glutamate decarboxylase activity with decreased GABA transaminase activity, but the composition of three botanical extracts is the best. Also, the three compositions showed potent neuroprotective activity by improving the cell viability of PC12 cells and significantly lowering the Aβ aggregation. However, XPCE showed the best neuroprotective and GABA releasing properties than XE and XPE owing to high contents of various phytocomponents like polysaccharides, flavonoids, and saponins (holistic activity). The above data clearly showed that the composition of three botanical extracts exhibiting an improved effect on GABA levels and exhibiting strong neuroprotective activity, which makes these novel composition (XPCE) as a potent candidate for treating insomnia, depression and various neurological conditions (e.g., Alzheimer's Disease).

The invention has been described with reference to various specific and preferred embodiments and techniques. However, one skilled in the art will understand that many variations and modifications may be made while remaining within the spirit and scope of the invention

REFERENCES