CN118201500A - Method for treating sleep disorder by exopolysaccharide - Google Patents

Method for treating sleep disorder by exopolysaccharide Download PDF

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CN118201500A
CN118201500A CN202280057975.0A CN202280057975A CN118201500A CN 118201500 A CN118201500 A CN 118201500A CN 202280057975 A CN202280057975 A CN 202280057975A CN 118201500 A CN118201500 A CN 118201500A
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fraction
eps
sleep
mole
lactic acid
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蔡英杰
吴健诚
黄瑾琳
许智捷
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Bened Biomedical Co Ltd
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Bened Biomedical Co Ltd
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Priority claimed from PCT/CN2022/114742 external-priority patent/WO2023025235A1/en
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Abstract

The present invention provides the use of exopolysaccharides of lactic acid bacteria or pharmaceutical compositions comprising the same for the manufacture of a medicament for preventing, ameliorating and/or treating sleep disorders in an individual in need thereof. The pharmaceutical composition comprises an effective amount of Exopolysaccharide (EPS) of lactic acid bacteria, and optionally an edible carrier or a pharmaceutically acceptable carrier.

Description

Method for treating sleep disorder by exopolysaccharide
Technical Field
The present disclosure relates to the use of probiotics. In particular, the present disclosure relates to methods of using lactic acid bacteria.
Background
Probiotics are living microorganisms that can provide some beneficial effect to their host. Research exploring the various functions of probiotics has progressed significantly over the past decade, benefiting from the higher commercial value of probiotics. One of the most important milestones is the proposal of "mental probiotics", which further extends the function of probiotics to behavioral and psychological wellbeing. In addition to finding new applications, many probiotic functions were found to be associated with non-viable cells, which in turn led to a proliferation of studies of functional probiotic effectors (Teame T et al ,2020.Paraprobiotics and Postbiotics of Probiotic Lactobacilli,Their Positive Effects on the Host and Action Mechanisms:A Review.Front Nutr 7:570344;Lebeer S et al ,2018.Identification of probiotic effector molecules:present state and future perspectives.Curr Opin Biotechnol 49:217-223).
In general, functional effectors produced by probiotics can be divided into two main categories: the secondary probiotics and the metazoan. A secondary probiotic is a cell component of a non-activated probiotic cell or probiotic that includes intracellular proteins, cell wall components, cell surface related molecules and Exopolysaccharides (EPS). Metazoans are non-cellular components that include secreted molecules and bacterial metabolites. Among the isolated molecules, EPS is of particular interest due to its diverse chemical nature and its role in host-microorganism interactions (Lee IC et al ,Strain-Specific Features of Extracellular Polysaccharides and Their Impact on Lactobacillus plantarum-Host Interactions.Appl Environ Microbiol 82:3959-3970).
However, new health applications need to be developed.
Disclosure of Invention
The present disclosure provides a method for preventing, ameliorating and/or treating sleep disorders in a subject in need thereof comprising administering to the subject an effective amount of exopolysaccharides of lactic acid bacteria or a pharmaceutical composition comprising the exopolysaccharides.
The present disclosure also provides the use of exopolysaccharides of lactic acid bacteria or pharmaceutical compositions comprising the exopolysaccharides for the manufacture of a medicament for preventing, ameliorating and/or treating sleep disorders in an individual in need thereof.
The present disclosure provides a method for improving sleep quality in an individual in need thereof, comprising administering to the individual an effective amount of exopolysaccharides of lactic acid bacteria or a pharmaceutical composition comprising the exopolysaccharides.
The present disclosure also provides the use of exopolysaccharides of lactic acid bacteria or pharmaceutical compositions comprising the exopolysaccharides for the manufacture of a medicament for improving sleep quality in an individual in need thereof.
The present disclosure provides a method for preventing, ameliorating and/or treating insomnia in a subject in need thereof, comprising administering to the subject an effective amount of exopolysaccharides of lactic acid bacteria or a pharmaceutical composition comprising the exopolysaccharides.
The present disclosure also provides the use of exopolysaccharides of lactic acid bacteria or pharmaceutical compositions comprising the exopolysaccharides for the manufacture of a medicament for preventing, ameliorating and/or treating insomnia in a subject in need thereof.
In some embodiments of the present disclosure, the lactic acid bacteria described herein are lactobacillus (Lactobacillus spp). In one embodiment of the present disclosure, the lactic acid bacteria are Lactobacillus fermentum (Lactobacillus fermentum). Examples of lactobacillus fermentum strains include, but are not limited to, lactobacillus fermentum Lf2, lactobacillus fermentum MTCC 25067 or lactobacillus fermentum PS150 (which was deposited according to the budapest treaty at german collection of microbial species (Deutsche Sammlung von Mikroorganismen und Zellkulturen) and assigned deposit number DSM 32323 at 6.6 of 2016).
EPS as described herein are provided in various forms. In some embodiments of the present disclosure, EPS is included in the extract.
Furthermore, in one embodiment, the EPS is obtained by a method comprising the steps of: culturing lactic acid bacteria to obtain a bacterial culture; and removing the bacterial cell bodies from the bacterial culture to obtain a mixture containing EPS.
In some embodiments of the present disclosure, the method further comprises the steps of:
Precipitating the mixture with ethanol; and
Ethanol was removed to obtain an extract containing EPS.
In some embodiments of the present disclosure, EPS is obtained by a method comprising the steps of:
culturing lactic acid bacteria to obtain a bacterial culture;
removing bacterial cell bodies from the bacterial culture to obtain a mixture containing EPS;
precipitating the mixture with ethanol;
removing the ethanol to obtain an extract containing EPS; and
Fractions having a molecular weight in the range of about 9kDa to about 2000kDa were obtained from the extract.
In one embodiment of the present disclosure, the extract comprises fraction 1 having a molecular weight in the range of about 30kDa to about 2,000kDa, and fraction 1 is separated by size exclusion chromatography.
In some embodiments of the present disclosure, fraction 1 comprises a major component having a molecular weight of 1,447 kda, and in one embodiment the major component comprises about 74% of fraction 1.
In some embodiments of the present disclosure, fraction 1 comprises trehalose, galactamine, glucosamine, galactose, glucose, and mannose. In some embodiments, fraction 1 further comprises other monosaccharides, including, but not limited to, inositol and sorbitol. In one embodiment, fraction 1 comprises about 3.7 to about 5.5 mole% trehalose, about 9.0 to about 13.5 mole% galactosamine, about 10.8 to about 16.2 mole% glucosamine, about 27.8 to about 41.7 mole% galactose, about 7.9 to about 11.8 mole% glucose, and about 20.9 to about 31.3 mole% mannose, wherein the total mole percent of monosaccharides in fraction 1 is about 100 mole%.
In another embodiment of the present disclosure, the extract comprises fraction 2 having a molecular weight in the range of about 15kDa to about 750kDa, and fraction 2 is separated by size exclusion chromatography.
In another embodiment of the present disclosure, the extract comprises fraction 3 having a molecular weight in the range of about 9kDa to about 380kDa, and fraction 3 is separated by size exclusion chromatography.
The route of administration of the pharmaceutical compositions as described herein may vary. In one embodiment, the composition is administered orally, and the composition is in a form suitable for oral administration. In another aspect, the composition is in the form of solid, semi-solid, liquid, or powder particles.
In some embodiments of the present disclosure, the method is used to reduce sleep latency.
In some embodiments of the present disclosure, the method is used to reduce the time to recover from sleep.
Brief description of the drawings
Fig. 1 shows a timeline of pentobarbital-induced sleep tests. PBS: phosphate buffered saline. This group served as a normal control. DIPH: diphenhydramine hydrochloride, a well-known antihistamine used as a sleep aid. DIPH was dissolved in PBS (20 mg/mL). This group served as a positive control. MRS Deman (De Man), luo Gesa (Rogosa), and Sharpe (Sharpe) culture fluids. The unfermented MRS broth was used as a negative control.
FIGS. 2A to 2D show the results of size exclusion chromatography of crude extracts containing PS150 exopolysaccharide. FIG. 2A shows the fine portioning of crude extracts containing PS150 exopolysaccharide. The finely divided fractions are divided into three groups (fraction 1, fraction 2 and fraction 3) according to the retention volume of the finely divided fraction. The carbohydrate content of each finely divided fraction was quantified using the phenol-sulphur method with a glucose reference. FIG. 2B shows the molecular weight distribution of fraction 1. FIG. 2C shows the molecular weight distribution of fraction 2. FIG. 2D shows the molecular weight distribution of fraction 3.
Fig. 3A to 3D show monosaccharide composition analysis of PS150 exopolysaccharide. Figure 3A shows monosaccharide standard. Fig. 3B shows monosaccharide composition of fraction 1. Fig. 3C shows monosaccharide composition of fraction 2. Figure 3D shows monosaccharide composition of fraction 3. The samples were hydrolyzed to monosaccharides and analyzed using high performance anion exchange chromatography in combination with a pulsed amperometric detector.
Fig. 4A shows the result of sleep latency in example 2. Fig. 4B shows the result of sleep duration in example 2. Fig. 4C shows the results of recovery time in example 2. Data are expressed as mean ± SEM. Different superscripts (a, b) differ significantly at p <0.05, based on one-way anova and Tukey HSD post hoc test.
Description of the embodiments
Unless defined otherwise, all technical or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein can be understood and used by those of ordinary skill in the art to practice the present disclosure.
It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Accordingly, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular.
As used herein, the term "optional/optional" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, the phrase "optionally comprising an agent" means that the agent may or may not be present.
Generally, ranges are expressed herein as from "about" one particular value, and/or to "about" another particular value. When such ranges are expressed, an embodiment includes ranges from one particular value and/or to another particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. As used herein, the term "about" refers to ± 20%, preferably ± 10% and even more preferably ± 5%.
The term "and/or" is used to refer to two things or any of the two things mentioned.
The term "preventing" is art-recognized and, when used in connection with a condition, includes administration of an agent prior to the onset of the condition to reduce the frequency or severity of a medical condition in an individual or to delay the onset of symptoms thereof relative to an individual not receiving the agent.
The term "treatment" generally refers to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease, disorder, or symptom thereof, and may be therapeutic in terms of partially or completely curing the disease, disorder, and/or symptom attributed thereto. As used herein, "treating" encompasses any treatment of a disease in a mammal (preferably a human) and includes (1) inhibiting the development of a disease, disorder, or symptom thereof in an individual, or (2) alleviating or ameliorating a disease, disorder, or symptom thereof in an individual.
As used herein, the term "disorder" may be used interchangeably with "disease" or "condition.
As used herein, the term "sleep disorder" includes conditions recognized by those of skill in the art as related to sleep disorders; for example, conditions known in the art are either proposed as sleep disorders or conditions found as sleep disorders. Sleep disorders also occur in individuals suffering from other medical conditions, diseases or injuries, or who are undergoing other medications or medical treatments, wherein the individual is therefore difficult to fall asleep and/or remain asleep, or experience unpleasant sleep, e.g., the individual experiences sleep deprivation.
As used herein, "sleep mode" or "sleep structure" refers to the time spent at each sleep stage (e.g., REM, N1, N2, and N3, or alternatively REM, stage I, stage II, stage III, and stage IV) for a given architecture, including the relative proportion of the duration of each stage to the duration of the other stages. Parameters of a "normal" sleep pattern or sleep structure for a particular population (e.g., post-menstrual female) are known in the art. See, e.g., latta et al, 2005, sleep,28:1525-1534; sahlin et al 2009, sleep Med, 10:1025-1030.
As used herein, the term "individual" is any animal that may benefit from administration of a compound or composition as disclosed herein. In some embodiments, the individual is a mammal, e.g., a human, primate, canine, feline, equine, bovine, porcine, rodent, such as a rat or mouse. Typically, the mammal is a human.
The term "probiotic" is currently recognized in the art as a microorganism that, when administered in sufficient quantity, imparts a health benefit to the host. Probiotic microorganisms must meet several requirements related to lack of toxicity, viability, adhesion and beneficial effects. These probiotic characteristics are related to the strain, even between bacteria of the same species.
The term "effective amount" of an active ingredient as provided herein means a sufficient amount of the ingredient to provide the desired modulation of the desired function. As will be noted below, the precise amount required will vary from individual to individual, depending on the individual's disease state, physical condition, age, sex, species and weight, the particular characteristics and formulation of the composition, and the like. The dosing regimen may be adjusted to induce an optimal therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the urgency of the treatment situation. Thus, it is not possible to specify an exact "effective amount". However, one of ordinary skill in the art can determine a suitable effective amount using only routine experimentation.
The term "exopolysaccharide" refers to a high molecular weight polysaccharide secreted by a microorganism.
The term "edible carrier" refers to a compound, material, composition, and/or dosage form suitable for contact with the tissue of an individual. Each carrier must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.
As used herein, the term "pharmaceutically acceptable" refers to a compound, material, composition, and/or dosage form that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of an individual (human or non-human animal) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, excipients, and the like can be found in standard pharmaceutical text.
As used herein, the term "lactic acid bacteria" refers to gram-positive microaerophilic or anaerobic bacteria that ferment sugars while producing acids (including lactic acid as the primary acid production).
The highly diverse chemical compositions of various EPS lead to excellent and useful industrial applications, i.e. emulsifiers, food additives, antioxidants, cryoprotectants and even nanoparticle stabilizers (Wang J,Salem DR,Sani RK.2019.Extremophilic exopolysaccharides:A review and new perspectives on engineering strategies and applications.Carbohydr Polym 205:8-26;Zhou Y,Cui Y and Qu X.2019.Exopolysaccharides of lactic acid bacteria:Structure,bioactivity and associations:A review.Carbohydr Polym 207:317-332)., in addition, EPS has potential in biomedical applications. One exemplary marker is EPS of Lactobacillus rhamnosus (Lactobacillus rhamnose) GG, which inhibits adipogenesis (Zhang Z et al) ,2019.Ability of prebiotic polysaccharides to activate a HIF1α-antimicrobial peptide axis determines liver injury risk in zebrafish.Commun Biol 2:274).
Accordingly, the present disclosure provides a method for preventing, ameliorating and/or treating sleep disorders (such as insomnia), improving the sleep quality of an individual in need thereof, comprising administering to the individual an effective amount of exopolysaccharides of lactic acid bacteria or a pharmaceutical composition comprising the exopolysaccharides.
The present disclosure also provides the use of exopolysaccharides of lactic acid bacteria or pharmaceutical compositions comprising the exopolysaccharides for the manufacture of a medicament for preventing, improving and/or treating sleep disorders (such as insomnia) and/or improving the sleep quality of an individual in need thereof.
The most common lactic acid bacteria are found in "Lactobacillus (Lactobacillales)", including Lactococcus (Lactococcus), streptococcus (Streptococcus), lactobacillus (Lactobacillus), candida albicans (Leuconostoc), pediococcus (Pediococcus) and Enterococcus (Enterococcus). In addition, lactic acid producing bacteria belonging to the group of strictly anaerobic bacteria Bifidobacterium (i.e., bifidobacterium) are generally included in the group of lactic acid bacteria.
As the most well studied probiotics, lactobacillus is the main object for isolating bioactive EPS. As a common species in various fermented foods, lactobacillus ferments are known for their sticky texture and ability to produce EPS, making them an ideal source for the discovery of novel EPS. The chemical structure, genetics, rheological properties and EPS production of EPS producing strains such as lactobacillus fermentum Lf2 and lactobacillus fermentum MTCC 25067 are widely studied. More importantly, there is growing evidence (yet to be preliminary) linking the probiotic function of the fermenting lactobacillus strain with EPS. Specifically, the lactic acid bacteria is Lactobacillus fermentum PS150. Lactobacillus fermentum PS150 is disclosed in WO2018129722A1 and has been deposited under the budapest treaty on the german collection of microorganisms and bacteria under deposit number DSM 32323.
Lactobacillus fermentum PS150 (PS 150) has any one of the nucleic acid sequences as set forth in SEQ ID NOs 1 to 3. The sequences of SEQ ID NOS.1 to 3 are listed below.
Typically, the exopolysaccharide comprises a polymer of monosaccharides. However, some exopolysaccharides also contain non-carbohydrate substituents (such as acetates, pyruvates, succinates, and phosphates). EPS can be classified into homoglycans and heteropolysaccharides according to monosaccharide composition. Homoglycans are composed of repeating units built up from a single type of monosaccharide. However, the repeating units of heteropolysaccharides comprise various types of monosaccharides. In addition to monosaccharide composition, factors including the number of repeating units, type of glycosidic bond, branching, sulfation, and phosphorylation may vary among EPS produced by different organisms.
As described herein, intervention of EPS may reduce sleep latency, although post-treatment sleep duration is unaffected. EPS treatment, on the other hand, significantly reduces recovery time. These results show that EPS is sufficient to produce a hypnotic effect.
EPS as described herein may be provided in purified, semi-purified, or unpurified form. In some embodiments, EPS as described herein is semi-purified and included in the extract. The extract may be prepared by a process comprising the steps of:
Culturing lactic acid bacteria to obtain a bacterial culture; and
Bacterial cell bodies were removed from the bacterial culture to obtain a mixture.
In addition, the method for preparing the extract further comprises the steps of:
Precipitating the mixture with ethanol; and
Ethanol was removed to obtain an extract containing EPS.
In addition, the method for preparing the extract further comprises the step of obtaining a fraction having a molecular weight in the range of about 9kDa to about 2,000kDa from the extract.
Furthermore, the EPS-containing extract as described herein may be further separated into several fractions. The separation means may be size exclusion chromatography. The extract contained some fractions. One of the fractions is fraction 1 having a molecular weight in the range of about 30kDa to about 2,000kDa, and fraction 1 is separated by size exclusion chromatography; one of the fractions is fraction 2 having a molecular weight in the range of about 15kDa to about 750kDa, and fraction 2 is separated by size exclusion chromatography; one of the fractions is fraction 3 having a molecular weight in the range of about 9kDa to about 380kDa, and fraction 3 is separated by size exclusion chromatography.
The term "size exclusion chromatography" means a method of separating molecules using a porous chromatography material. Size exclusion chromatography may consist of one or more different types of porous chromatography materials used in a single step or one or more different types of porous chromatography materials used in multiple separate steps.
In some embodiments of the present disclosure, fraction 1 comprises a major component having a molecular weight of about 1,447 kda. The contents of fraction 1 were further analyzed using a high performance anion exchange column chromatography system (HPAEC) with a Pulse Amperometric Detector (PAD), using a gold working electrode and an anion exchange column (Carbopac TM PA-10,4.6×250mm)(DionexTM BioLC), fraction 1 comprising about 3.7 to about 5.5 mole% trehalose, about 9.0 to about 13.5 mole% galactosamine, about 10.8 to about 16.2 mole% glucosamine, about 27.8 to about 41.7 mole% galactose, about 7.9 to about 11.8 mole% glucose, and about 20.9 to about 31.3 mole% mannose, wherein the total mole percentage of monosaccharides in fraction 1 was 100 mole%. Quantitative analysis showed that galactose and mannose were most abundant in fraction 1.
Individuals in need of improved sleep architecture or sleep quality may exhibit sleep patterns that deviate from "normal" sleep patterns in one or more parameters, may suffer from or be at risk of sleep pattern disorders, or may be diagnosed as sleep disorders. In particular embodiments, the individual has a problem falling asleep.
A variety of different parameters may be considered in determining whether a drug or treatment regimen achieves sleep architecture or sleep quality improvement. For example, one or more of reduced wake time, increased Slow Wave (SW) and/or fast eye movement (REM) sleep, increased sleep maintenance, increased sleep efficiency, reduced sleep latency, and/or normalization of the distribution of SW and REM stages during sleep may be considered an improvement in sleep quality or sleep architecture. Methods by which sleep quality improvement may be determined are known in the art and are illustrated in the examples. Providing an individual with a drug or treatment regimen that improves any one or more of these parameters would be considered to have improved the sleep quality or sleep architecture of that individual.
The compositions of the present disclosure may be in any form suitable for administration, particularly oral administration. This includes, for example, solids, semisolids, liquids, and powders.
Examples of the compositions of the present disclosure are nutritional compositions, including food products, especially dairy products.
The composition may be, for example, a capsule, lozenge, beverage, powder or dairy product. Preferably, the disclosed composition is a nutraceutical or pharmaceutical product, a nutritional supplement or a medical food.
Nutritional compositions of the present disclosure also include food supplements and functional foods. "food supplement" means a product made from compounds commonly used in foods, but in the form of lozenges, powders, capsules, drinks or any other product that is generally food independent and has beneficial effects on human health. The functional food is a food which also has beneficial effects on human health. In particular, food supplements and functional foods may exert a physiological effect on the disease-protective or therapeutic.
If the composition according to the present disclosure is a dietary supplement, it may be administered as such, may be mixed with a suitable drinkable liquid, such as water, yogurt, milk or juice, or may be mixed with a solid or liquid food. In this case, the dietary supplement may be in the form of lozenges, pills, capsules, troches, granules, powders, suspensions, sachets, tablets, dragees, strips, syrups and corresponding administration forms, typically in unit dosage form. Preferably, the dietary supplements comprising the compositions of the present disclosure are administered in the form of lozenges, buccal tablets, capsules or powders, manufactured in conventional processes for preparing dietary supplements.
The compositions described herein may be pharmaceutically acceptable compositions, which may include one or more pharmaceutically acceptable carriers, excipients, binders, diluents, or the like. The compositions of the present disclosure may be formulated for various routes of administration, such as oral administration. It may also be provided in combination with a delivery vehicle such as in some encapsulation techniques.
For oral administration, powders, suspensions, granules, lozenges, pills, capsules, gel capsules and caplets are acceptable as solid dosage forms. Such can be prepared, for example, by mixing one or more compounds disclosed herein with at least one additive, such as starch or another additive. Suitable additives are sucrose, lactose, cellulose sugar, mannitol, maltitol, dextran, starch, agar, alginate, chitin, chitosan, pectin, tragacanth, acacia, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers or glycerides. Optionally, the oral dosage form may contain other ingredients to aid in administration, such as inert diluents, or lubricants (such as magnesium stearate), or preservatives (such as parabens or sorbic acid), or antioxidants (such as ascorbic acid, tocopherol, or cysteine), disintegrants, binders, thickeners, buffers, sweeteners, flavoring or perfuming agents. The tablets and pills may be further treated with suitable coating materials known in the art.
Liquid dosage forms for oral administration may be in the form of pharmaceutically acceptable emulsions, syrups, elixirs, suspensions and solutions, which may contain inert diluents such as water. Pharmaceutical formulations and compositions may be prepared as liquid suspensions or solutions using sterile liquids, such as, but not limited to, oils, water, ethanol, and combinations thereof. Pharmaceutically suitable surfactants, suspending agents, emulsifying agents may be added for oral or parenteral administration.
It will be appreciated that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art.
Although the disclosure has been provided in detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to those skilled in the art that various changes and modifications may be practiced without departing from the spirit or scope of the disclosure. Accordingly, the foregoing description and examples should not be considered as limiting.
Examples
Purification of crude extract containing exopolysaccharide
The bacterial strain Lactobacillus fermentum PS150 was anaerobically cultured in Deman, luo Gesa and in a summer broth (MRS; criterion, hardy diagnostics, SANTA MARIA, CA, USA) at 37℃for 18 hours. The overnight culture was heated at 80 ℃ for 1 hour and bacterial cell bodies were removed using centrifugation at 7,000Xg for 30 minutes. The polysaccharide fraction was then precipitated at 4℃for 24 hours using 3X volumes of absolute ethanol. The precipitate was then collected at 7,000Xg for 30 minutes and washed with 70% ethanol. Residual ethanol was removed by evaporation, followed by dissolution of the EPS-containing extract in ddH 2 O and storage at-30 ℃ until use.
Size exclusion column chromatography
Briefly, 40mg of lyophilized EPS was dissolved in 3mL of buffer (pH 6.8) containing 150mM NaCl and 10mM NaH 2PO4 and applied to a Fractogel TM BioSec column (102 x 1.5 cm) (Merck, n.j., u.s.a.). Subsequently, 2.8 mL/tube was collected and hexoses were analyzed by the phenolsulfuric acid method (DuBois DuBois, m., gilles, k.a., hamilton, j.k., rebers, p.a., and Smith,F.Colorimetric method for determination of sugars and related substances,Anal Chem 28:350-356et al.,1956). were subjected to spectroscopic brightness analysis at a wavelength of 488 nm.
Estimation of exopolysaccharide molecular weight
The sugar-containing tube collected by the size exclusion column was divided into three fractions. The fractions were further separated using gel permeation chromatography in combination with a refractive index detector to estimate molecular weight. Calibration curves were constructed using reliable standards (dextran series, sigma-Aldrich co.) with molecular weights 670.0 x 10 3、69.8×103、40.0×103、10.5×103 and 0.18 x 10 3 Da.
Analysis of monosaccharide composition
The monosaccharide composition of EPS was analyzed using a high performance anion exchange column chromatography system (HPAEC) with a Pulsed Amperometric Detector (PAD) using a gold working electrode and an anion exchange column (Carbopac TM PA-10,4.6×250mm)(DionexTM BioLC). EPS (1 mg) was acid hydrolyzed with 1.95N trifluoroacetic acid at 80℃for 6 hours, evaporated to remove residual TFA, and dissolved in Milli-Q water. The aqueous phase was filtered through a 0.22 μm filter prior to HPAEC analysis. An isocratic NaOH (18 mM) at ambient temperature was used as a stripping agent. The flow rate was 1.0mL/min. Ten monosaccharide standards, including inositol, sorbitol, mannitol, trehalose, galactagose, glucosamine, galactose, glucose, mannose, and fructose, were purchased from Sigma-Aldrich co. (St.Louis, MO, USA). Data were collected and integrated on PRIME DAK system (HPLC Technology, ltd.
Animals
Male C57BL/6J mice (6 weeks old) were purchased from experimental animal centers. Mice were housed in experimental animal centers. The room was kept at a constant temperature (22.+ -. 1 ℃) and humidity (55-65%) for 12hr of light/dark cycle. Mice were fed ad libitum with standard diet and sterilized water.
Pentobarbital-induced sleep test
For functional verification of crude extracts containing EPS, C57BL/6J mice were orally administered with PBS, MRS broth, and EPS (0.4 mg/day) for 13 days. On day 14, PBS, DIPH (20 mg/kg), MRS medium or EPS was administered to mice 30 minutes prior to the injection of pentobarbital (50 mg/kg). After injection, mice were tested for orthostatic reflection by flipping up and down. The time between pentobarbital injection and disappearance of the eversion front is defined as sleep latency. The time required for recovery of the flip-flop is defined as sleep time, and the time taken between the flip-flop action and the autonomous movement is defined as recovery time.
The timeline of pentobarbital-induced sleep tests is shown in fig. 1.
Example 1 fine portioning and characterization of PS150 EPS
To investigate the composition of EPS, size exclusion chromatography was performed for fractionation (FIG. 2A). EPS is separated into three finely divided fractions, namely a fraction 1, a fraction 2 and a fraction 3. Subsequently, the homogeneity of the three fractions was analyzed using a gel permeation chromatography system. Interestingly, fraction 1 consisted of a major component (74%) with a molecular weight of 1,447 kDa (FIG. 2B). The chromatograms of fraction 2 and fraction 3 show a mixed molecular weight distribution and higher background noise compared to the high purity of fraction 1 (fig. 2C and 2D). Thus, we speculate that both fraction 2 and fraction 3 are mixtures of various molecules, while fraction 1 is purified EPS.
Next, monosaccharide composition analysis was performed to further characterize the chemistry of the three fractions (fig. 3A-3D). The results show that fraction 1 is a polysaccharide consisting mainly of six different monosaccharides: trehalose, galactosamine, glucosamine, galactose, glucose and mannose. Quantitative analysis showed that galactose and mannose were the most abundant components in fraction 1 (table 1). Background noise for fraction 2 and fraction 3 persisted in our monosaccharide analysis (fig. 3C and 3D), and quantitative analysis of monosaccharide composition is also shown in table 1. In summary, we conclude that the main component of EPS is a heteropolysaccharide composed of six monosaccharides, and that this EPS molecule can be an active component that mediates the hypnotic effect of PS 150.
Table 1. Monosaccharide composition of purified fractions.
EXAMPLE 2 crude extract containing EPS was sufficient for hypnotic function
To demonstrate our hypothesis that EPS of PS150 is an active effector of hypnotic function, EPS was precipitated from spent medium of PS 150. The final yield of carbohydrate content in the crude extract was about 0.4mg/ml liquid culture. The crude extract was then tested for hypnotic activity in a mouse model injected with pentobarbital, as described in pentobarbital-induced sleep test. The results show that intervention with EPS-containing extracts from PS150 spent medium can reduce sleep latency (fig. 4A), although sleep duration is unaffected after treatment (fig. 4B). EPS treatment, on the other hand, significantly reduced recovery time (fig. 4C).
While the present disclosure has been described in conjunction with the specific embodiments described above, many alternatives, modifications and variations thereof will be apparent to those skilled in the art. All such alternatives, modifications, and variations are considered to be within the scope of the present disclosure.

Claims (17)

1. Use of exopolysaccharides of lactic acid bacteria or pharmaceutical compositions comprising the same for the manufacture of a medicament for preventing, improving and/or treating sleep disorders and/or improving the sleep quality of an individual in need thereof.
2. The use of claim 1, wherein the sleep disorder is insomnia.
3. The use according to claim 1, wherein the lactic acid bacteria is lactobacillus (Lactobacillus spp).
4. The use of claim 1, wherein the lactic acid bacteria are lactobacillus fermentum (Lactobacillusfermentum).
5. The use of claim 1, wherein the lactic acid bacteria are lactobacillus fermentum Lf2, lactobacillus fermentum MTCC 25067 or lactobacillus fermentum PS150.
6. The use according to claim 1, wherein the EPS is comprised in an extract.
7. Use according to claim 1, wherein the EPS is obtained by a method comprising the steps of:
Culturing the lactic acid bacteria to obtain a bacterial culture;
removing bacterial cell bodies from the bacterial culture to obtain a mixture containing the EPS;
Precipitating the mixture with ethanol; and
Ethanol was removed to obtain extracts containing the EPS.
8. Use according to claim 1, wherein the EPS is obtained by a method comprising the steps of:
Culturing the lactic acid bacteria to obtain a bacterial culture;
removing bacterial cell bodies from the bacterial culture to obtain a mixture containing the EPS;
precipitating the mixture with ethanol;
Removing the ethanol to obtain an extract containing the EPS; and
Obtaining fractions having a molecular weight in the range of about 9kDa to about 2,000kDa from the extract.
9. The use of claim 7 or 8, wherein the extract comprises fraction 1 having a molecular weight in the range of about 30kDa to about 2,000kDa, and the fraction 1 is separated by size exclusion chromatography.
10. The use according to claim 9, wherein the fraction 1 comprises a major component having a molecular weight of 1,447 kda.
11. The use of claim 9, wherein the fraction 1 comprises trehalose, galactamine sugar, glucosamine, galactose, glucose, and mannose.
12. The use of claim 9, wherein the fraction 1 comprises from about 3.7 to about 5.5 mole% trehalose, from about 9.0 to about 13.5 mole% galactamine sugar, from about 10.8 to about 16.2 mole% glucosamine, from about 27.8 to about 41.7 mole% galactose, from about 7.9 to about 11.8 mole% glucose, and from about 20.9 to about 31.3 mole% mannose, wherein the total mole percent of monosaccharides in the fraction 1 is 100 mole%.
13. The use of claim 7 or 8, wherein the extract comprises fraction 2 having an average molecular weight in the range of about 15kDa to about 750kDa, and the fraction 2 is separated by size exclusion chromatography.
14. The use of claim 7 or 8, wherein the extract comprises fraction 3 having an average molecular weight in the range of about 9kDa to about 380kDa, and the fraction 3 is separated by size exclusion chromatography.
15. The use of claim 1, wherein the pharmaceutical composition is in a form suitable for oral administration.
16. The use of claim 1, wherein the pharmaceutical composition is in the form of solid, semi-solid, liquid or powder particles.
17. Use according to claim 1 for reducing sleep latency and/or reducing the time to recover from sleep.
CN202280057975.0A 2021-08-27 2022-08-25 Method for treating sleep disorder by exopolysaccharide Pending CN118201500A (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US63/237,964 2021-08-27
US202263267326P 2022-01-31 2022-01-31
US63/267,326 2022-01-31
PCT/CN2022/114742 WO2023025235A1 (en) 2021-08-27 2022-08-25 Method for treating sleeping disorders with exopolysaccharides

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