Use of a traditional Chinese medicine composition in serum neurotransmitters

Abstract

The application relates to the technical field of medicines, and discloses application of a traditional Chinese medicine composition in serum neurotransmitters, which comprises the following steps: selecting 6-8-week-old mice to establish normal models, chronic fatigue syndrome models and functional dyspepsia models; blood sampling is conducted on the mice of the normal models, the chronic fatigue syndrome models and the functional dyspepsia models to obtain normal blood samples, CFS blood samples and FD blood samples respectively; protein precipitation operation is conducted to obtain normal deproteinized supernatants, CFS deproteinized supernatants and FD deproteinized supernatants; separation is conducted by using a reversed-phase chromatographic column, and then mass spectrometry is conducted to obtain normal serum neurotransmitter mass spectra, CFS serum neurotransmitter mass spectra and FD serum neurotransmitter mass spectra respectively; concentration value calculation and rhythm fluctuation analysis are conducted to obtain a neurotransmitter function index and rhythm stability quantitative score respectively. Therefore, the mechanism of the traditional Chinese medicine composition in serum neurotransmitters is clearly quantitatively characterized.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of a traditional Chinese medicine composition in serum neurotransmitters. Background Technology

[0002] The application of traditional Chinese medicine (TCM) compositions in serum neurotransmitter analysis has significant practical implications, primarily in regulating nerve function, elucidating pharmacodynamic mechanisms, and assisting in the validation of disease models. In pathological states closely related to neurotransmitter disorders, such as nervous system diseases, chronic fatigue syndrome (CFS), and functional dyspepsia (FD), TCM compositions may intervene in nerve regulation and emotional behavior by influencing the concentrations of key neurotransmitters such as dopamine, serotonin, norepinephrine, γ-aminobutyric acid (GABA), or glutamate.

[0003] However, the traditional Chinese medicine (TCM) compositions used in related technologies often contain dozens or even hundreds of active compounds, such as flavonoids, saponins, polysaccharides, phenolic acids, and alkaloids. These components may exert their effects in vivo through different metabolic pathways or signal transduction pathways. Currently, the evaluation of the intervention effects of TCM compound prescriptions still largely relies on experience or single-indicator judgments, lacking a unified and reproducible quantitative indicator system. Compared with single-target drugs, the mechanism of action of TCM often manifests as systemic regulation rather than point-to-point intervention, making it difficult for traditional quantitative methods to clearly define the specific biological contribution of each component. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides an application of a traditional Chinese medicine composition in serum neurotransmitters, aiming to solve the problem of the difficulty in quantifying and characterizing the mechanism of action of traditional Chinese medicine compositions on serum neurotransmitters.

[0005] To address the above problems, this invention proposes the application of a traditional Chinese medicine composition in serum neurotransmitters, comprising the following steps:

[0006] S1. Use 6-8 week old mice to establish normal models, chronic fatigue syndrome models and functional dyspepsia models;

[0007] S2. Blood samples were collected from mice in the normal model, chronic fatigue syndrome model, and functional dyspepsia model to obtain normal blood samples, CFS blood samples, and FD blood samples, respectively. Protein precipitation was performed on the normal blood samples, CFS blood samples, and FD blood samples to obtain normal deproteinized supernatant, CFS deproteinized supernatant, and FD deproteinized supernatant, respectively.

[0008] S3. Add acidified reconstitution solution to the normal deproteinized supernatant, CFS deproteinized supernatant and FD deproteinized supernatant respectively, separate them using reversed-phase chromatography column, and then perform mass spectrometry detection to obtain normal serum neurotransmitter mass spectra, CFS serum neurotransmitter mass spectra and FD serum neurotransmitter mass spectra respectively.

[0009] S4. Concentration values ​​and rhythm fluctuation analysis were performed on the mass spectra of normal serum neurotransmitters, CFS serum neurotransmitters, and FD serum neurotransmitters to obtain neurotransmitter function index and rhythm stability quantitative score, respectively.

[0010] In some embodiments, step S1 includes:

[0011] S1.1 Select male mice aged 6-8 weeks, with a weight of 18-22g. After 7 days of acclimatization, randomly label them into normal group, chronic fatigue syndrome group and functional dyspepsia group using a random number table method, with no less than 10 mice in each group.

[0012] S1.2 Three pre-experiments were conducted on the chronic fatigue syndrome group. Each pre-experiment lasted for 7 days. The three pre-experiments corresponded to three factors, including physical fatigue factor, chronic psychological stress factor and rhythm disorder factor. During the three pre-experiments, the Chinese medicine composition was administered by gavage at a rate of 10 mL / kg per day, with the concentration converted according to body weight. The medicine was administered at a fixed time every day, at 9:00 am, to avoid affecting the diurnal rhythm and to obtain the chronic fatigue syndrome model.

[0013] S1.3 Three pre-experiments were conducted on the functional dyspepsia group. Each pre-experiment lasted for 7 days. The three pre-experiments corresponded to three stimuli, including restraint stimulation, feeding restriction stimulation, and stomach cold stimulation. During the three pre-experiments, the Chinese medicine composition was administered by gavage at a rate of 10 mL / kg per day, with the concentration converted according to body weight. The medicine was administered at a fixed time every day, at 9:00 am, to avoid affecting the circadian rhythm and to obtain the functional dyspepsia model.

[0014] S1.4. The normal group was kept in a standard diurnal cycle for 21 days, with a temperature of 22±1℃ and a relative humidity of 50~60%, and was provided with free access to food and water to obtain a normal model.

[0015] In some embodiments, step S1.2 includes:

[0016] The preliminary experiment on physical fatigue factors was conducted by performing a 10-minute forced swimming test every day, with the water temperature maintained at 25±1℃ and the water depth at 20~25cm.

[0017] The preliminary experiment for chronic psychological stress factors involved placing a 1cm layer of cold, damp cotton pad at the bottom of the cage to maintain a humidity level of 75-85%.

[0018] The preliminary experiment on the circadian rhythm disorder factor was to randomly adjust the light duration by ±2 hours each day and use an intelligent light control box to achieve daytime circadian rhythm mismatch.

[0019] In some embodiments, step S1.3 includes:

[0020] The restraint stimulation pre-experiment involved placing mice in a restraint tube for 2 hours daily.

[0021] The feeding restriction stimulus pre-experiment was conducted by providing 3 mL of semi-liquid rice cereal only at fixed times each day, avoiding free feeding.

[0022] The preliminary experiment for stomach cold stimulation was conducted by administering 4°C saline solution orally every two days at a dose of 10 mL / kg.

[0023] In some embodiments, the traditional Chinese medicine composition includes hawthorn, Shenqu (medicated leaven), malt, white peony root, codonopsis root, poria cocos, atractylodes macrocephala, costus root, amomum villosum, corydalis rhizome, and prepared licorice root.

[0024] In some embodiments, step S2:

[0025] S2.1 At the end of day 14, mice were rapidly anesthetized using an isoflurane anesthesia system. Blood samples were collected from mice in the normal group, chronic fatigue syndrome group, and functional dyspepsia group to obtain normal blood samples, CFS blood samples, and FD blood samples.

[0026] S2.2 Transfer normal blood samples, CFS blood samples and FD blood samples to anticoagulant-free polypropylene centrifuge tubes respectively, place them at room temperature for 30 minutes to allow the blood to coagulate naturally, and then centrifuge at 4℃ and 3000rpm for 15 minutes to separate the serum. Collect the supernatant to obtain normal serum, CFS serum and FD serum respectively.

[0027] S2.3 Add acidified acetonitrile methanol solution, sodium ascorbate, and sodium acetate buffer to CFS serum. The volume ratio of acidified acetonitrile-methanol solution: sodium ascorbate: sodium acetate buffer: CFS serum is 10:1:1:1. Adjust the rotation speed to 1200 rpm and stir for 15 minutes. Cool down to 4℃ and centrifuge at 15000 rpm for 10 minutes to obtain CFS deproteinized supernatant.

[0028] S2.4 Add acidified acetonitrile methanol solution, zinc acetate, and ammonium formate buffer to FD serum. The volume ratio of acidified acetonitrile methanol solution: zinc acetate: ammonium formate buffer: FD serum is 6:1:1:1. Adjust the rotation speed to 1600 rpm and stir for 10 minutes. Cool down to 8℃ and centrifuge at 15000 rpm for 10 minutes to obtain FD deproteinized supernatant.

[0029] S2.5 Add acidified acetonitrile-methanol solution to normal serum. The volume ratio of acidified acetonitrile-methanol solution to normal serum is 8:1. Adjust the rotation speed to 1400 rpm and stir for 12 minutes. Cool down to 6℃ and centrifuge at 15000 rpm for 10 minutes to obtain normal deproteinized supernatant.

[0030] S2.6 Take 100µL of each of the normal deproteinization supernatant, CFS deproteinization supernatant, and FD deproteinization supernatant, and dilute them to 200µL with acidified acetonitrile aqueous solution.

[0031] In some embodiments, step S3 includes:

[0032] S3.1 Add acidified reconstitution solution to the normal deproteinized supernatant, CFS deproteinized supernatant and FD deproteinized supernatant respectively, and set the stirring speed to 1500 rpm and shake for 1 to 3 minutes to obtain normal reconstituted sample, CFS reconstituted sample and FD reconstituted sample;

[0033] S3.2. Configure a reversed-phase column using a high-performance liquid chromatography system. Load the normal reconstituted sample, CFS reconstituted sample, and FD reconstituted sample into the autosampler. The injection volume is 2µL each time. Set the reversed-phase column temperature to 30℃. Use positive and negative ion switching mode for multiple reaction monitoring to obtain the chromatographic peaks of the target metabolites. The target metabolites include tyrosine, tryptophan, glutamine, and glutamic acid.

[0034] S3.3. Integrate the target metabolite chromatographic peaks of the normal reconstituted sample, CFS reconstituted sample, and FD reconstituted sample respectively, and perform standardization and normalization processing to obtain the normal serum neurotransmitter mass spectrum, CFS serum neurotransmitter mass spectrum, and FD serum neurotransmitter mass spectrum.

[0035] In some embodiments, the acidification complex solution includes at least one of the following: a mixture of formic acid and acetonitrile, a mixture of formic acid and methanol, a mixture of glacial acetic acid and methanol, and a mixture of formic acid and formic acid.

[0036] In some embodiments, step S4 includes:

[0037] S4.1 Calculate the target metabolite concentration values ​​in the normal serum neurotransmitter mass spectrum, CFS serum neurotransmitter mass spectrum, and FD serum neurotransmitter mass spectrum, and calculate the neurotransmitter function index data based on the target metabolite concentration values.

[0038] S4.2 Calculate the concentration difference of a single neurotransmitter concentration value within a fixed time period in the target metabolite concentration value, and obtain a quantitative score of rhythm stability after standardization.

[0039] Compared with existing technologies, the application of a traditional Chinese medicine composition in serum neurotransmitters in this invention has the following advantages:

[0040] In steps S1 to S4, a mouse model with a clearly defined physiological / pathological state is first established. Then, by administering medication and collecting blood at uniform time points, circadian rhythm deviations are eliminated, ensuring the controllability of the experimental design. Subsequently, a standard procedure of protein precipitation, acidification and reconstitution, reversed-phase chromatography, and mass spectrometry is used to obtain neurotransmitter mass spectra under different conditions. This method can accurately identify precursor neurotransmitters closely related to central nervous system regulation and perform quantitative analysis from two dimensions: concentration distribution and rhythmic fluctuations. It transforms the mechanism of action of traditional Chinese medicine (TCM) compositions into a specific, continuous, and comparable data format. This quantitative approach effectively overcomes the interpretative obstacles caused by the complexity of TCM effects, multiple targets, and unclear metabolic pathways, providing a clear quantitative characterization of the mechanism of action of serum neurotransmitters in TCM compositions. Attached Figure Description

[0041] Figure 1 This is a schematic flowchart illustrating the application of a traditional Chinese medicine composition in serum neurotransmitters according to an embodiment of the present invention. Detailed Implementation

[0042] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0043] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0044] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0045] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0046] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0047] Please refer to Figure 1 This invention proposes the application of a traditional Chinese medicine composition in serum neurotransmitters, comprising the following steps:

[0048] S1. Use 6-8 week old mice to establish normal models, chronic fatigue syndrome models and functional dyspepsia models.

[0049] Step S1 includes:

[0050] S1.1 Select male mice aged 6-8 weeks, with a weight of 18-22g. After 7 days of acclimatization, randomly label them into normal group, chronic fatigue syndrome group and functional dyspepsia group using a random number table method, with no less than 10 mice in each group.

[0051] This step, using male mice aged 6–8 weeks and weighing 18–22g, ensured the model animals were in a stable physiological state, contributing to the uniformity of subsequent behavioral and metabolic indicators. A 7-day acclimatization period allowed the animals to adapt to the experimental environment, reducing interference from non-specific stress. Using a random number table for grouping significantly reduced group bias, enhancing the scientific rigor and reproducibility of the experiment. Dividing the mice into normal, chronic fatigue syndrome, and functional dyspepsia groups provided a clear basic grouping framework for subsequent model induction and drug intervention. Female mice exhibit a distinct hormonal cycle, approximately every 4–5 days, which significantly affects neurotransmitter levels in the central nervous system. This cyclical fluctuation introduces uncontrollable sources of variation, especially when studying diurnal rhythm fluctuations and neurotransmitter ratio stability. Male mice are more stable in physiological, behavioral, and metabolic characteristics, making them particularly suitable for multi-timepoint, long-cycle rhythmic neurotransmitter analysis. Male mice exhibit clearer phenotypic and neurotransmitter abnormalities in stress models, facilitating differential amplification and mechanistic tracing. Male mice are more tolerant of experimental procedures, and their mortality and spontaneous disease rates are lower than those of female mice, which helps to ensure the success rate of experiments and the integrity of data.

[0052] S1.2. Three pre-experiments were conducted on the chronic fatigue syndrome group, each lasting 7 days. The three pre-experiments corresponded to three factors: physical fatigue factor, chronic psychological stress factor, and circadian rhythm disorder factor. During the three pre-experiments, a traditional Chinese medicine combination was administered by gavage at a rate of 10 mL / kg per day, with the concentration adjusted according to body weight. The medication was administered at a fixed time each day, at 9:00 AM to avoid affecting the diurnal rhythm, thus obtaining the chronic fatigue syndrome model. The physical fatigue factor pre-experiment involved a 10-minute forced swimming test daily, with the water temperature maintained at 25±1℃ and the water depth at 20~25cm. The chronic psychological stress factor pre-experiment involved adding a 1cm cold, wet cotton pad to the bottom of the cage to maintain a humidity of 75~85%. The circadian rhythm disorder factor pre-experiment involved randomly adjusting the light duration by ±2 hours daily, using an intelligent light control box to achieve diurnal rhythm mismatch.

[0053] This step induces a chronic fatigue syndrome model through three methods, simulating the etiology of human chronic fatigue syndrome (CFS) from three aspects: physical exertion, psychological stress, and circadian rhythm mismatch. Forced swimming (10 min / day, water temperature 25±1°C, water depth 20–25 cm) induces physical fatigue, compelling animals to exercise continuously, inducing energy metabolism disorders and neurotransmitter depletion, manifesting as behavioral depression and learning and memory impairment. The wet and cold cotton pad method (75–85% humidity, for 7 days) simulates a state of chronic psychological stress, activating the hypothalamic-pituitary-adrenal axis pathway, causing abnormal release of stress hormones, and interfering with central nervous system function. Light rhythm mismatch (±2 h / day) interferes with melatonin and neuroendocrine rhythms by regulating the daily diurnal variation of the intelligent light control box, producing a chronic phenotype similar to insomnia and mental fatigue. During the model building phase, a traditional Chinese medicine (TCM) composition was administered daily via gavage (10 mL / kg, 9:00 AM). Components such as Codonopsis pilosula and Poria cocos were used to tonify Qi and strengthen the spleen, relieving fatigue; Paeonia lactiflora and Glycyrrhiza uralensis were used to regulate the central nervous system's excitation-inhibition balance; and Corydalis yanhusuo and Aucklandia lappa were used to improve Qi stagnation and blood stasis, as well as chronic muscle aches and pains. This simultaneous drug administration design with model building is of significant innovative importance, as it can simulate the effects of TCM on the circadian rhythm-endocrine-immune axis system under early intervention, exploring its pharmacological mechanism in improving the physiological rhythm disorder of chronic fatigue syndrome (CFS).

[0054] S1.3 Three pre-experiments were conducted on the functional dyspepsia group. Each pre-experiment lasted for 7 days. The three pre-experiments corresponded to three stimuli, including restraint stimulation, feeding restriction stimulation, and stomach cold stimulation. During the three pre-experiments, the Chinese medicine composition was administered by gavage at a rate of 10 mL / kg per day, with the concentration converted according to body weight. The medicine was administered at a fixed time every day, at 9:00 am, to avoid affecting the circadian rhythm and to obtain the functional dyspepsia model.

[0055] The restraint stimulation pre-experiment involved placing mice in a restraint tube for 2 hours daily.

[0056] The feeding restriction stimulus pre-experiment was conducted by providing 3 mL of semi-liquid rice cereal only at fixed times each day, avoiding free feeding.

[0057] The preliminary experiment for stomach cold stimulation was conducted by administering 4°C saline solution orally every two days at a dose of 10 mL / kg.

[0058] The traditional Chinese medicine composition includes hawthorn, Shenqu (medicated leaven), malt, white peony root, codonopsis, poria cocos, atractylodes macrocephala, costus root, amomum villosum, corydalis rhizome, and prepared licorice root.

[0059] This step employed three methods to construct a functional dyspepsia (FD) model, simulating a multifactorial etiology including gastrointestinal motility disorders, gastrointestinal hypersensitivity, and external invasion of cold pathogens. Restraint stimulation (2 hours / day) restricted mouse movement, enhancing sympathetic nerve excitability and inducing gastrointestinal motility disorders and vagal reflex disturbances. Feeding restriction stimulation (providing only 3 mL of semi-liquid rice cereal daily for a limited time) restricted food intake and abnormal gastric emptying, interfering with the mice's feeding rhythm and energy balance. Cold stimulation (administering 4°C saline solution every other day at a dose of 10 mL / kg) directly interfered with gastrointestinal mucosal temperature and functional status, reducing gastric juice secretion and digestive enzyme activity, leading to cold-induced qi stagnation-type functional dyspepsia. During the modeling process, a traditional Chinese medicine composition (10 mL / kg, 9:00 AM) was administered simultaneously. Hawthorn, Shenqu (a type of fermented wheat), and malt promoted gastric emptying and enzyme activity, regulating gastrointestinal motility; Amomum villosum and Atractylodes macrocephala had warming and cold-dispelling effects, enhancing mucosal protection under cold conditions; and Codonopsis pilosula and Poria cocos improved spleen and stomach function. By implementing a design that combines etiological induction and intervention, the model's representativeness is enhanced, and the multi-target mechanism of action of traditional Chinese medicine on the neuro-digestive-endocrine linkage regulation can be evaluated.

[0060] S1.4. The normal group was kept in a standard diurnal cycle for 21 days, with a temperature of 22±1℃ and a relative humidity of 50~60%, and was provided with free access to food and water to obtain a normal model.

[0061] This step, by setting standardized feeding conditions, ensures that the normal group mice are at homeostatic physiological baselines under key factors such as circadian rhythm, temperature and humidity environment, and nutritional status. Specifically, maintaining a constant temperature of 22±1℃ and relative humidity of 50~60% avoids interference with the endocrine system and metabolic pathways caused by cold and heat stress and changes in humidity, ensuring that the natural fluctuations of the mice's physiological rhythms are not disrupted by the external environment; maintaining a circadian rhythm of 12h:12h light-dark cycle for 21 days helps stabilize the secretion of rhythmic hormones such as melatonin and cortisol from the pineal gland, supporting the rhythmic synchronization of the brain-gut-immune axis; free access to food and water avoids rhythmic disorders of energy metabolism and provides a stable reference for the basic expression of neurotransmitters. By optimizing the rhythmic environment and setting up free conditions, a mouse model representing normal neuroendocrine and gastrointestinal function under natural conditions was established. This model provides a real and reproducible negative reference for the differential analysis of neurotransmitter rhythm changes, anti-inflammatory and antioxidant status, and drug response between the CFS and FD model groups. It has good comparability and control value and is an indispensable basic group in rhythmic experimental design.

[0062] S2. Blood samples were collected from mice in the normal model, chronic fatigue syndrome model, and functional dyspepsia model to obtain normal blood samples, CFS blood samples, and FD blood samples, respectively. Protein precipitation was performed on the normal blood samples, CFS blood samples, and FD blood samples to obtain normal deproteinized supernatant, CFS deproteinized supernatant, and FD deproteinized supernatant, respectively.

[0063] S2.1 At the end of day 14, mice were rapidly anesthetized using an isoflurane anesthesia system. Blood samples were collected from mice in the normal group, chronic fatigue syndrome group, and functional dyspepsia group to obtain normal blood samples, CFS blood samples, and FD blood samples.

[0064] This step involves rapidly and effectively anesthetizing mice using an isoflurane anesthesia system. This avoids the increase in stress hormones such as cortisol secretion caused by stress responses, which could interfere with neurotransmitter levels and ensure the accuracy of neurotransmitter concentrations in the blood during blood collection. Isoflurane is a volatile inhaled anesthetic with rapid onset and low metabolic burden. It can induce deep anesthesia in animals in a very short time, ensuring a rapid and stable blood collection process. This effectively reduces the degradation or transformation of metabolites caused by prolonged operation time, contributing to obtaining high-quality, low-interference blood samples for subsequent neurotransmitter analysis.

[0065] S2.2 Transfer normal blood samples, CFS blood samples, and FD blood samples to anticoagulant-free polypropylene centrifuge tubes, place them at room temperature for 30 minutes to allow the blood to coagulate naturally, and then centrifuge at 4℃ and 3000 rpm for 15 minutes to separate the serum. Collect the supernatant to obtain normal serum, CFS serum, and FD serum, respectively.

[0066] This step involves allowing blood to clot naturally in anticoagulant-free polypropylene centrifuge tubes, thus avoiding the influence of exogenous chemical components on endogenous small-molecule neurotransmitters. Allowing the blood to clot fully at room temperature for 30 minutes, followed by centrifugation at a low temperature and moderate speed (4℃, 3000 rpm), effectively separates the serum and reduces interfering substances released from erythrocyte and platelet lysis, yielding a clear serum supernatant. This process helps ensure the accuracy and repeatability of neurotransmitter detection, and is particularly suitable for subsequent mass spectrometry analyses requiring high sensitivity for low-abundance molecules.

[0067] S2.3 Add acidified acetonitrile methanol solution, sodium ascorbate, and sodium acetate buffer to CFS serum. The volume ratio of acidified acetonitrile-methanol solution: sodium ascorbate: sodium acetate buffer: CFS serum is 10:1:1:1. Adjust the rotation speed to 1200 rpm and stir for 15 minutes. Cool down to 4℃ and centrifuge at 15000 rpm for 10 minutes to obtain CFS deproteinized supernatant.

[0068] CFS serum was used for protein precipitation using acidified acetonitrile-methanol solution, sodium ascorbate, and sodium acetate buffer, forming an efficient and mild protein removal system. The acidified acetonitrile-methanol solution rapidly denatures and precipitates proteins while exhibiting good polarity matching, enabling stable extraction of various neurotransmitters. Sodium ascorbate, as an antioxidant, prevents the degradation of easily oxidized neurotransmitters such as dopamine; the sodium acetate buffer maintains an acidic environment, stabilizing the ionic state of amines and improving the sensitivity of subsequent mass spectrometry detection. The mixture was first stirred at low speed to promote mixing, then cooled to 4°C and centrifuged at high speed to effectively precipitate large protein molecules, obtaining a high-purity CFS deproteinized supernatant, providing an ideal sample for neurotransmitter quantification.

[0069] S2.4 Add acidified acetonitrile methanol solution, zinc acetate, and ammonium formate buffer to FD serum. The volume ratio of acidified acetonitrile methanol solution: zinc acetate: ammonium formate buffer: FD serum is 6:1:1:1. Adjust the rotation speed to 1600 rpm and stir for 10 minutes. Cool down to 8℃ and centrifuge at 15000 rpm for 10 minutes to obtain FD deproteinized supernatant.

[0070] FD serum was treated with a combination of acidified acetonitrile-methanol solution, zinc acetate, and ammonium formate buffer to form a composite deproteinization scheme characterized by metal ion precipitation. Acidified acetonitrile-methanol also exhibits excellent protein denaturation and neurotransmitter dissolution capabilities; zinc acetate can bind to phosphoproteins or amide proteins in serum to form precipitates, making it particularly suitable for removing enzymes activated and released due to gastrointestinal inflammation in FD models; ammonium formate buffer, as a volatile buffer system, stabilizes the pH without introducing background interference during mass spectrometry detection. This deproteinization system can more efficiently adapt to the protein interference background unique to FD models, providing highly adaptable samples for subsequent separation and analysis.

[0071] S2.5 Add acidified acetonitrile-methanol solution to normal serum. The volume ratio of acidified acetonitrile-methanol solution to normal serum is 8:1. Adjust the rotation speed to 1400 rpm and stir for 12 minutes. Cool down to 6℃ and centrifuge at 15000 rpm for 10 minutes to obtain normal deproteinized supernatant.

[0072] Normal serum was deproteinized using a single acidified acetonitrile-methanol solution at a volume ratio of 8:1. This method efficiently removes most large-molecule proteins from the serum while avoiding the impact of additional compounds on the baseline levels of neurotransmitters. Because the endogenous metabolic load and oxidative stress and inflammation levels in the normal serum group are low, high-purity supernatant can be obtained without adding antioxidants or metal precipitants. High-speed centrifugation at 6°C and 1400 rpm after stirring reduces the low-temperature condensation effect, maintains neurotransmitter stability, ensures clear separation of the supernatant layers, and improves the recovery rate of the supernatant.

[0073] S2.6 Take 100µL of each of the normal deproteinization supernatant, CFS deproteinization supernatant, and FD deproteinization supernatant, and dilute them to 200µL with acidified acetonitrile aqueous solution.

[0074] This step involves diluting the three groups of deproteinized supernatants at a 1:1 volume ratio using acidified acetonitrile aqueous solution. This serves two purposes: firstly, it adjusts the solution polarity to match the initial mobile phase conditions for reversed-phase chromatography, preventing peak broadening or dissolution lag during sample loading; secondly, the acidic conditions maintain the ionization efficiency of neurotransmitters before mass spectrometry, avoiding signal drift caused by pH fluctuations. Furthermore, this dilution step uniformly processes the three sample groups, ensuring greater consistency in baseline levels and response ranges in subsequent chromatographic and mass spectrometric analyses, improving data comparability, and laying a quantitative foundation for rhythmicity and functional ratio analysis.

[0075] S3. Add acidified reconstitution solution to the normal deproteinized supernatant, CFS deproteinized supernatant and FD deproteinized supernatant respectively, separate them using reversed-phase chromatography column, and then perform mass spectrometry detection to obtain normal serum neurotransmitter mass spectra, CFS serum neurotransmitter mass spectra and FD serum neurotransmitter mass spectra respectively.

[0076] S3.1. Add acidification reconstitution solution to the normal deproteinized supernatant, CFS deproteinized supernatant, and FD deproteinized supernatant, respectively. Set the stirring speed to 1500 rpm and shake for 1-3 minutes to obtain the normal reconstituted sample, CFS reconstituted sample, and FD reconstituted sample. The acidification reconstitution solution includes at least one of the following: formic acid-acetonitrile-water mixture, formic acid-methanol-water mixture, glacial acetic acid-water-methanol mixture, and formic acid-water-formic acid-acetonitrile mixture. This step restores soluble small-molecule neurotransmitters in the sample by adding acidification reconstitution solution to the three types of deproteinized supernatant and provides a good solvent environment for subsequent chromatographic-mass spectrometric analysis. The acidification reconstitution solution used includes at least one of the following: formic acid-acetonitrile-water mixture, formic acid-methanol-water mixture, glacial acetic acid-water-methanol mixture, and formic acid-water-formic acid-acetonitrile mixture. These solutions have moderate polarity, good volatility, and an acidic environment. Acidifying agents such as formic acid or glacial acetic acid can stabilize the ionic state of amine neurotransmitters, reducing their degradation or adsorption losses during reconstitution or loading; while acetonitrile and methanol can improve the solubility efficiency of lipid-soluble and neutral small molecule metabolites. Setting the shaking speed to 1500 rpm can achieve complete dissolution in a short time, ensuring that the target neurotransmitter is in a homogeneous and detectable state in the system, thus ensuring the clarity and reproducibility of the chromatographic peaks.

[0077] S3.2. A reversed-phase column was configured using a high-performance liquid chromatography (HPLC) system. Normally reconstituted samples, CFS reconstituted samples, and FD reconstituted samples were loaded into the autosampler, with an injection volume of 2 µL each time. The reversed-phase column temperature was set to 30°C, and multiple reaction monitoring (MRM) was performed using a positive / negative ion switching mode to obtain the target metabolite chromatographic peaks. The results are shown in Table 1. L-tyrosine had the highest average abundance in the sample, followed by L-tryptophan and L-glutamine, indicating that these amino acid precursors or metabolites occupy a high proportion in the neurotransmitter metabolic network. Therefore, in this embodiment, the target metabolites include tyrosine, tryptophan, and glutamine. This step uses an HPLC system to configure a reversed-phase column, separating compounds based on the difference in hydrophobicity between the column and the sample. Reversed-phase columns often use C18 packing as the stationary phase, which has strong hydrophobicity, allowing polar small molecules such as amino acid neurotransmitters to elute sequentially according to their hydrophobicity, thus improving resolution. Samples are injected via an autosampler with a 2 μL injection volume per injection, effectively balancing detection sensitivity and peak broadening control, making it suitable for high-throughput analysis. A constant column temperature of 30°C helps improve peak shape consistency and retention time stability. Mass spectrometry employs a positive / negative ion switching mode, enabling the simultaneous acquisition of target metabolite signals in different ionized forms within a single analysis, improving analytical efficiency and accuracy. This step ensures efficient identification and separation of key neurotransmitter precursors or metabolites such as tyrosine, tryptophan, glutamine, and glutamate. These components play a central regulatory role in neurotransmitter synthesis pathways and are therefore important indicators for assessing neurological states.

[0078] Table 1:

[0079]

[0080] S3.3. Integrate the target metabolite chromatographic peaks of the normal reconstituted sample, CFS reconstituted sample, and FD reconstituted sample respectively, and perform standardization and normalization processing to obtain the normal serum neurotransmitter mass spectrum, CFS serum neurotransmitter mass spectrum, and FD serum neurotransmitter mass spectrum.

[0081] After chromatographic separation, this step integrates the chromatographic peaks of the target metabolites in various samples, extracts the peak area as the response intensity, and performs standardization and normalization to eliminate non-biological differences between samples caused by concentration differences, sample volume differences, or instrument drift. Integration accurately quantifies the relative abundance of each target neurotransmitter, forming the basis for subsequent ratio analysis and rhythm fluctuation analysis. Standardization typically uses total ion current or internal reference substances as a benchmark to unify peak areas, while normalization further converts the signal into a comparable scale, yielding a relative expression spectrum. The resulting neurotransmitter mass spectrometry data not only reveals differences in metabolic states between models but also provides precise input data for subsequent multidimensional statistical analysis and metabolic pathway reconstruction, enhancing the data interpretability and scientific value of the entire experimental system.

[0082] S4. The concentration ratios and rhythm fluctuations of normal serum neurotransmitter mass spectra, CFS serum neurotransmitter mass spectra, and FD serum neurotransmitter mass spectra were calculated and analyzed to obtain the neurotransmitter function index and rhythm stability quantitative score, respectively.

[0083] S4.1 Calculate the target metabolite concentration values ​​in the normal serum neurotransmitter mass spectrum, CFS serum neurotransmitter mass spectrum, and FD serum neurotransmitter mass spectrum, and calculate the neurotransmitter function index data based on the target metabolite concentration values.

[0084] This step, by calculating the concentration values ​​of target metabolites in normal serum neurotransmitter mass spectra, CFS serum neurotransmitter mass spectra, and FD serum neurotransmitter mass spectra, enables the functional quantitative characterization of the intervention effect of traditional Chinese medicine compositions. The target metabolites include tyrosine, tryptophan, glutamine, and glutamate. These substances are precursors or regulatory molecules of important neurotransmitters such as dopamine, serotonin, γ-aminobutyric acid, and glutamate, respectively, representing the excitatory, inhibitory, and harmonizing states of neurotransmitters. Obtaining their precise concentration values ​​through mass spectrometry, and then constructing ratio models (e.g., tyrosine / glutamate, tryptophan / glutamine, etc.), can reflect the balance or imbalance state of the body in the dimension of neurofunctional function. The introduction of this neurotransmitter functional index breaks away from the previous reliance on subjective behavioral judgment, providing an objective, repeatable, and structured data output path for the functional regulatory effects of traditional Chinese medicine compound formulas.

[0085] S4.2 Calculate the concentration difference of a single neurotransmitter concentration value within a fixed time period in the target metabolite concentration value, and obtain a quantitative score of rhythm stability after standardization.

[0086] This step calculates the difference in concentration of a single target metabolite over a fixed time period, combined with standardization methods, to obtain a quantitative score for rhythm stability, thereby achieving a quantitative evaluation of the effects of traditional Chinese medicine (TCM) at the level of biological rhythm regulation. This method is based on the physiological characteristic of neurotransmitters exhibiting significant diurnal rhythms, especially glutamine and glutamate, which show marked fluctuations during the day-night cycle. By collecting mass spectrometry data of neurotransmitters at fixed time points (e.g., morning and night) and calculating their concentration differences, the synchronicity and stability of neurotransmitter rhythms under different models can be revealed. Standardization eliminates bias caused by individual differences, making the score more comparable. This scoring index further complements the functional index, enabling a comprehensive assessment of whether the TCM composition possesses dual regulatory capabilities, i.e., intervention effects on both functional deviation and rhythm disorder. Therefore, this step provides quantitative evidence to reveal the mechanism by which TCM regulates diurnal rhythms, possessing significant theoretical and applied value.

[0087] The quantitative score for rhythm stability is calculated as follows:

[0088] ;

[0089] ;

[0090] RMSE, or root mean square error, reflects the deviation of the fitted curve from the actual measured neurotransmitter concentration, expressed in μmol / L, and can range from 0.01 to 1.5 μmol / L, depending on the specific concentration range of the neurotransmitter. The measured concentration values ​​C at different time points are then considered. i with fitted value The average of the squared differences is calculated, and the square root is taken. ΔP represents the peak / trough concentration deviation, indicating the concentration difference between the extreme points (maximum or minimum values) of the metabolic rhythm curve in the intervention group and the control group, in μmol / L. The value ranges from 0 to 5 μmol / L, depending on the type of metabolite. The peak (maximum value) and trough (minimum value) are extracted from the fitted curve, and the difference is calculated by comparing the intervention group and the control group. The metabolic rhythm curve is a dynamic trend diagram of the concentration change of a specific metabolite over a certain time period. ΔT represents the rhythm phase shift, indicating the difference in the timing of the peak / trough between the intervention group and the control group, i.e., the phase drift of the diurnal rhythm, in hours (h), in the range of 0 to 24 hours. It is calculated from the time point of the peak occurrence extracted from the fitted curve. α, β, and γ are weighting coefficients used to standardize and weight differences across different dimensions, forming a unified scoring index. The units are dimensionless, α + β + γ = 1, and each can be between 0.1 and 0.8, set according to research preferences, for example, α = 0.4, β = 0.4, γ = 0.2. rhythm The quantitative score represents the rhythm stability; a higher score indicates greater rhythm instability. When traditional Chinese medicine (TCM) compositions are introduced as an intervention, their sustained-release regulatory effect on neurotransmitter concentration and metabolic rhythm fluctuations becomes more significant. If the score tends to decrease after intervention, it indicates reduced rhythm fluctuations and a more stable rhythm, suggesting that the TCM composition has achieved metabolic rhythm modulation. This non-mutagenic intervention result is highly consistent with the explanation of the mechanism of action of TCM. Using metabolic rhythm modulation as an indicator of efficacy observation not only has mechanistic explanatory power but also expands the ways of constructing clinical models. For example, it can be used to assess chronic disease models centered on rhythm disorders, such as anxiety and depression; it can support the optimization of TCM dosage and precise setting of dosing time windows (e.g., tonifying the kidneys at night and regulating qi during the day); and it can establish a prescription database screening system based on rhythm scores to discover potential matching combinations.

Claims

1. The application of a traditional Chinese medicine composition in serum neurotransmitters, characterized in that, Includes the following steps: S1. Use 6-8 week old mice to establish normal models, chronic fatigue syndrome models and functional dyspepsia models; S2. Blood samples were collected from mice in the normal model, chronic fatigue syndrome model, and functional dyspepsia model to obtain normal blood samples, CFS blood samples, and FD blood samples, respectively. Protein precipitation was performed on the normal blood samples, CFS blood samples, and FD blood samples to obtain normal deproteinized supernatant, CFS deproteinized supernatant, and FD deproteinized supernatant, respectively. S3. Add acidified reconstitution solution to the normal deproteinized supernatant, CFS deproteinized supernatant and FD deproteinized supernatant respectively, separate them using reversed-phase chromatography column, and then perform mass spectrometry detection to obtain normal serum neurotransmitter mass spectra, CFS serum neurotransmitter mass spectra and FD serum neurotransmitter mass spectra respectively. S4. Concentration values ​​and rhythm fluctuation analysis were performed on the mass spectra of normal serum neurotransmitters, CFS serum neurotransmitters, and FD serum neurotransmitters to obtain neurotransmitter function index and rhythm stability quantitative score, respectively. Step S4 includes: S4.1 Calculate the target metabolite concentration values ​​in the normal serum neurotransmitter mass spectrum, CFS serum neurotransmitter mass spectrum, and FD serum neurotransmitter mass spectrum, and calculate the neurotransmitter function index data based on the target metabolite concentration values. S4.2 Calculate the concentration difference of a single neurotransmitter concentration value within a fixed time period in the target metabolite concentration value, and obtain a quantitative score of rhythm stability after standardization.

2. The application of the traditional Chinese medicine composition according to claim 1 in serum neurotransmitters, characterized in that, Step S1 includes: S1.1 Select male mice aged 6-8 weeks, with a weight of 18-22g. After 7 days of acclimatization, randomly label them into normal group, chronic fatigue syndrome group and functional dyspepsia group using a random number table method, with no less than 10 mice in each group. S1.2 Three pre-experiments were conducted on the chronic fatigue syndrome group. Each pre-experiment lasted for 7 days. The three pre-experiments corresponded to three factors, including physical fatigue factor, chronic psychological stress factor and rhythm disorder factor. During the three pre-experiments, the Chinese medicine composition was administered by gavage at a rate of 10 mL / kg per day, with the concentration converted according to body weight. The medicine was administered at a fixed time every day, at 9:00 am, to avoid affecting the diurnal rhythm and to obtain the chronic fatigue syndrome model. S1.3 Three pre-experiments were conducted on the functional dyspepsia group. Each pre-experiment lasted for 7 days. The three pre-experiments corresponded to three stimuli, including restraint stimulation, feeding restriction stimulation, and stomach cold stimulation. During the three pre-experiments, the Chinese medicine composition was administered by gavage at a rate of 10 mL / kg per day, with the concentration converted according to body weight. The medicine was administered at a fixed time every day, at 9:00 am, to avoid affecting the circadian rhythm and to obtain the functional dyspepsia model. S1.

4. The normal group was kept in a standard diurnal cycle for 21 days, with a temperature of 22±1℃ and a relative humidity of 50~60%, and was provided with free access to food and water to obtain a normal model.

3. The application of the traditional Chinese medicine composition according to claim 2 in serum neurotransmitters, characterized in that, Step S1.2 includes: The preliminary experiment on physical fatigue factors was conducted by performing a 10-minute forced swimming test every day, with the water temperature maintained at 25±1℃ and the water depth at 20~25cm. The preliminary experiment for chronic psychological stress factors involved placing a 1cm layer of cold, damp cotton pad at the bottom of the cage to maintain a humidity level of 75-85%. The preliminary experiment on the circadian rhythm disorder factor was to randomly adjust the light duration by ±2 hours each day and use an intelligent light control box to achieve daytime circadian rhythm mismatch.

4. The application of the traditional Chinese medicine composition according to claim 2 in serum neurotransmitters, characterized in that, Step S1.3 includes: The restraint stimulation pre-experiment involved placing mice in a restraint tube for 2 hours daily. The feeding restriction stimulus pre-experiment was conducted by providing 3 mL of semi-liquid rice cereal only at fixed times each day, avoiding free feeding. The preliminary experiment for stomach cold stimulation was conducted by administering 4°C saline solution orally every two days at a dose of 10 mL / kg.

5. The application of the traditional Chinese medicine composition according to claim 2 in serum neurotransmitters, characterized in that, The traditional Chinese medicine composition includes hawthorn, Shenqu (medicated leaven), malt, white peony root, codonopsis, poria cocos, atractylodes macrocephala, costus root, amomum villosum, corydalis rhizome, and prepared licorice root.

6. The application of the traditional Chinese medicine composition according to claim 1 in serum neurotransmitters, characterized in that, Step S2 includes: S2.1 At the end of day 14, mice were rapidly anesthetized using an isoflurane anesthesia system. Blood samples were collected from mice in the normal group, chronic fatigue syndrome group, and functional dyspepsia group to obtain normal blood samples, CFS blood samples, and FD blood samples. S2.2 Transfer normal blood samples, CFS blood samples and FD blood samples to anticoagulant-free polypropylene centrifuge tubes respectively, place them at room temperature for 30 minutes to allow the blood to coagulate naturally, and then centrifuge at 4℃ and 3000rpm for 15 minutes to separate the serum. Collect the supernatant to obtain normal serum, CFS serum and FD serum respectively. S2.3 Add acidified acetonitrile methanol solution, sodium ascorbate, and sodium acetate buffer to CFS serum. The volume ratio of acidified acetonitrile-methanol solution: sodium ascorbate: sodium acetate buffer: CFS serum is 10:1:1:

1. Adjust the rotation speed to 1200 rpm and stir for 15 minutes. Cool down to 4℃ and centrifuge at 15000 rpm for 10 minutes to obtain CFS deproteinized supernatant. S2.4 Add acidified acetonitrile methanol solution, zinc acetate, and ammonium formate buffer to FD serum. The volume ratio of acidified acetonitrile methanol solution: zinc acetate: ammonium formate buffer: FD serum is 6:1:1:

1. Adjust the rotation speed to 1600 rpm and stir for 10 minutes. Cool down to 8℃ and centrifuge at 15000 rpm for 10 minutes to obtain FD deproteinized supernatant. S2.5 Add acidified acetonitrile-methanol solution to normal serum. The volume ratio of acidified acetonitrile-methanol solution to normal serum is 8:

1. Adjust the rotation speed to 1400 rpm and stir for 12 minutes. Cool down to 6℃ and centrifuge at 15000 rpm for 10 minutes to obtain normal deproteinized supernatant. S2.6 Take 100µL of each of the normal deproteinization supernatant, CFS deproteinization supernatant, and FD deproteinization supernatant, and dilute them to 200µL with acidified acetonitrile aqueous solution.

7. The application of the traditional Chinese medicine composition according to claim 1 in serum neurotransmitters, characterized in that, Step S3 includes: S3.1 Add acidified reconstitution solution to the normal deproteinized supernatant, CFS deproteinized supernatant and FD deproteinized supernatant respectively, and set the stirring speed to 1500 rpm and shake for 1 to 3 minutes to obtain normal reconstituted sample, CFS reconstituted sample and FD reconstituted sample; S3.

2. Configure a reversed-phase column using a high-performance liquid chromatography system. Load the normal reconstituted sample, CFS reconstituted sample, and FD reconstituted sample into the autosampler. The injection volume is 2µL each time. Set the temperature of the reversed-phase column to 30℃. Use the positive and negative ion switching mode for multiple reaction monitoring to obtain the chromatographic peak of the target metabolite. S3.

3. Integrate the target metabolite chromatographic peaks of the normal reconstituted sample, CFS reconstituted sample, and FD reconstituted sample respectively, and perform standardization and normalization processing to obtain the normal serum neurotransmitter mass spectrum, CFS serum neurotransmitter mass spectrum, and FD serum neurotransmitter mass spectrum.

8. The application of the traditional Chinese medicine composition according to claim 7 in serum neurotransmitters, characterized in that, The acidified complex solution includes at least one of the following: a mixture of formic acid and acetonitrile, a mixture of formic acid and methanol, a mixture of glacial acetic acid and methanol, and a mixture of formic acid and formic acid.

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