Liver-soothing and sleep-aiding traditional Chinese medicine composition for stimulating PI3K / Akt signal pathway

Abstract

The invention discloses a liver-soothing sleep-aiding traditional Chinese medicine composition for stimulating a PI3K / Akt signal channel and a preparation method and application thereof, and the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 5-20 parts of radix bupleuri, 10-30 parts of spina date seed, 10-25 parts of poria with hostwood, 10-25 parts of tuber fleeceflower stem, 5-20 parts of radix paeoniae alba, 3-15 parts of radix curcumae and 5-20 parts of cortex albiziae. The traditional Chinese medicine composition is based on a traditional Chinese medicine formula for regulating liver and regulating qi, nourishing blood and tranquilizing, accords with the compatibility of monarch, minister, assistant and guide, and has the effects of soothing liver-qi stagnation, helping sleep and tranquilizing by combining the medicines.

Description

Technical Field

[0001] The present invention relates to the technical field of Chinese medicine compositions, and in particular to a liver-soothing and sleep-promoting Chinese medicine composition for stimulating a PI3K / Akt signaling pathway, and a preparation method and application thereof. Background Art

[0002] Insomnia is a subjective experience, which refers to difficulty falling asleep at night, easy awakening, insufficient sleep time, decreased sleep quality, accompanied by daytime weakness, fatigue, abnormal mood, etc. Epidemiological research results show that about 1 / 3 of the world's people have insomnia symptoms, accompanied by daytime dysfunction, and about 50% of patients have a chronic course of the disease. Insomnia is seriously affecting the physical and mental health of many patients and requires great attention. However, there is currently no clear treatment or drug treatment for insomnia, so there is an urgent need to find an effective, economical and safe drug solution.

[0003] In traditional Chinese medicine, insomnia is mostly described by the patient's clinical manifestations. There are also related records in the Yellow Emperor's Classic of Internal Medicine, such as "cannot lie down", "cannot sleep at night", "cannot close eyes", "cannot sleep", etc. The medical book believes that evil qi invades the internal organs, and the imbalance of yin and yang in the human body leads to insomnia. The pathological mechanism of insomnia is also relatively complex. After summarizing and sorting out, its pathogenesis is mainly yin excess and yang deficiency, yin and yang miscommunication, and internal organ disorder. The syndrome types include liver depression and qi stagnation, liver fire disturbing the heart syndrome, phlegm heat disturbing the heart syndrome, heart and spleen deficiency syndrome, heart and gallbladder qi deficiency syndrome, and heart and kidney disharmony syndrome. Zhang Biqing explored the emotional correlation insomnia based on the differentiation of internal organs, and believed that liver depression type insomnia is insomnia caused by excessive emotions. The pathogenesis is that excessive emotions hurt the liver, the liver fails to dredge, and qi is stagnant. The liver is the mother of the heart, and the heart is the master of the spirit, which is the monarch. Depression and anger hurt the liver, and the child's disease affects the mother. Therefore, liver injury can also show that the function of the heart's master spirit is damaged, resulting in sleep disorders such as difficulty falling asleep and waking from sleep. Traditional Chinese medicine has great advantages in the diagnosis and treatment of insomnia patients. Traditional Chinese medicine can regulate the body from multiple targets and has a low incidence of adverse reactions.

[0004] The invention CN116139236B discloses a traditional Chinese medicine composition for treating liver-depression type insomnia and its application. The traditional Chinese medicine composition of this invention consists of a daytime formula and a nighttime formula. The daytime formula is made from the following raw materials by weight: 3 - 15 parts of Bupleuri Radix, 12 - 24 parts of stir-fried Paeoniae Radix Alba, 20 - 40 parts of calcined Os Draconis, 1 - 5 parts of Amomi Fructus Rotundus, 3 - 15 parts of Cyperi Rhizoma Preparatum, 3 - 15 parts of Acori Tatarinowii Rhizoma, 2 - 10 parts of Cryptotympanae Periostracum, 2 - 10 parts of Angelicae Dahuricae Radix, 3 - 15 parts of Sophorae Flavescentis Radix, 3 - 15 parts of Bombyx Batryticatus, 3 - 15 parts of Chuanxiong Rhizoma, 2 - 10 parts of Glycyrrhizae Radix; The nighttime formula is made from the following raw materials by weight: 3 - 15 parts of Scutellariae Radix, 1 - 5 parts of Cinnamomi Cortex, 3 - 15 parts of Curcumae Radix, 20 - 40 parts of Salviae Miltiorrhizae Radix, 3 - 15 parts of Cyperi Rhizoma Preparatum, 3 - 15 parts of Fructus Gardeniae Carbonisatus, 5 - 25 parts of Caulis Polygoni Multiflori, 12 - 24 parts of Albizziae Cortex, 3 - 15 parts of Polygalae Radix, 5 - 25 parts of Ganoderma Lucidum, 2 - 10 parts of Glycyrrhizae Radix. This prescription has the defects of a broad action path and redundant pharmacodynamic components in the intervention of liver-depression pathogenesis, and it is necessary to distinguish between the daytime and nighttime prescriptions, with complex components, which is not conducive to the daily use of patients.

[0005] The invention CN115350256B discloses a traditional Chinese medicine composition for treating insomnia and its preparation method. The raw materials of this traditional Chinese medicine composition include: 5 - 40 parts of Polygalae Radix, 10 - 30 parts of Ziziphi Spinosae Semen, 10 - 25 parts of Pseudostellariae Radix, 20 - 40 parts of Ostreae Concha, 5 - 15 parts of Cinnamomi Ramulus, 5 - 30 parts of Chuanxiong Rhizoma, 5 - 30 parts of Citri Sarcodactylis Fructus, 1 - 20 parts of Alismatis Rhizoma, 5 - 35 parts of Atractylodis Macrocephalae Rhizoma, 1 - 30 parts of Angelicae Sinensis Radix, 1 - 20 parts of Curcumae Radix, 5 - 30 parts of Bupleuri Radix, and 5 - 30 parts of Ginseng Radix. However, the raw materials of this traditional Chinese medicine composition contain precious medicinal materials such as Pseudostellariae Radix, Ginseng Radix, and Ostreae Concha, and Ostreae Concha needs to be calcined and processed, resulting in a significant increase in the comprehensive cost of raw material procurement, processing, and quality control.

[0006] With the in-depth understanding of the pathogenesis of insomnia, a large number of studies have shown that neurotransmitter secretion is an important factor affecting sleep and is closely related to insomnia. Some studies have shown that sleep depends on inhibitory neurotransmitters, while wakefulness is related to excitatory neurotransmitters. Neurotransmitters are involved in maintaining the dynamic balance of the sleep-wake cycle and regulate the sleep process through interactions with the central nervous system. Some studies have shown that blocking the PI3K / AKT signaling pathway with the PI3K / AKT signaling pathway inhibitor LY294002 also reduces the neurotransmitters secreted by cells, indicating that PI3K / AKT, as an important signaling pathway in neuronal cells, is a key factor regulating the secretion of neurotransmitters by neuronal cells.

[0007] However, the current traditional Chinese medicine (TCM) treatment for insomnia due to liver depression faces dual technical dilemmas: at the theoretical level, although traditional prescriptions follow the compatibility principle of "soothing the liver and relieving depression, nourishing blood and calming the mind", the dynamic balance between the dispersing intensity of Bupleuri Radix and the astringent properties of Ziziphi Spinosae Semen lacks precise regulation, resulting in the failure to fully exert the synergistic effect between the monarch drug and the ministerial drug. At the practical level, the existing preparation processes generally adopt a single water extraction method, which is difficult to achieve the synchronous dissolution of active ingredients with different polarities such as saikosaponins in Bupleuri Radix and flavonoids in Albiziae Cortex. In addition, the high-temperature treatment during the conventional granulation process is likely to cause the degradation of heat-sensitive components such as paeoniflorin, directly affecting the activation efficiency of the drug on the PI3K / Akt pathway. Therefore, even with the combined use of a large number of traditional Chinese medicine components, the therapeutic effect is still very limited while the medication cost increases significantly, which is not conducive to the long-term use by a large number of patients with insomnia due to liver depression.

[0008] Therefore, how to construct a TCM prescription system that can precisely regulate the PI3K / Akt pathway, solve the key problems in the existing technology such as the lack of dynamic balance between soothing the liver and relieving depression and calming the mind, insufficient synergistic effect of multi-target active ingredients, limited synchronous dissolution of components with polarity differences, and irreversible degradation of heat-sensitive active substances during the preparation process, so as to achieve multi-dimensional precise intervention in the pathological mechanism of insomnia due to liver depression, improve the therapeutic effect while reducing the medication cost, is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the Invention

[0009] In view of the key problems in the existing technology, such as the lack of dynamic balance between soothing the liver and relieving depression and calming the mind, insufficient synergistic effect of multi-target active ingredients, limited synchronous dissolution of components with polarity differences, irreversible degradation of heat-sensitive active substances during the preparation process, and high medication cost, the present invention develops a liver-soothing and sleep-promoting traditional Chinese medicine composition that can stimulate the PI3K / Akt signaling pathway, as well as its preparation method and application.

[0010] In the first aspect, the present invention provides a liver-soothing and sleep-promoting traditional Chinese medicine composition that can stimulate the PI3K / Akt signaling pathway. Calculated by weight, the traditional Chinese medicine composition is prepared from the following raw materials:

[0011] 5 - 20 parts of Bupleuri Radix, 10 - 30 parts of Ziziphi Spinosae Semen, 10 - 25 parts of Poria, 10 - 25 parts of Polygoni Multiflori Caulis, 5 - 20 parts of Paeoniae Radix Alba, 3 - 15 parts of Curcumae Radix, 5 - 20 parts of Albiziae Cortex.

[0012] Among the above drugs:

[0013] Bupleuri Radix: Soothes the liver and relieves depression, raises yang qi - targets the pathogenesis of liver qi stagnation and regulates the qi movement ascending and descending pivot; saikosaponins can inhibit the release of inflammatory factors through the PI3K / Akt / mTOR pathway and relieve the increased nerve excitability caused by liver depression transforming into fire;

[0014] Suanzaoren (Spina Date Seed): Nourish the heart and tonify the liver, calm the mind and soothe the nerves - Nourish the liver blood to nourish the mind, improve restlessness and insomnia; Research has confirmed that its flavonoid components can inhibit neuronal apoptosis by activating the PI3K / Akt pathway, enhance the expression of γ-aminobutyric acid (GABA) receptors, thereby improving sleep;

[0015] Fushen (Poria with hostwood): Calm the mind and soothe the nerves, promote diuresis and percolate dampness - Guide the heart fire downward to communicate the heart and kidney, eliminate the drawback of phlegm-dampness disturbing the mind, with emphasis on strengthening the spleen and calming the mind;

[0016] Yejiateng (Caulis Polygoni Multiflori): Nourish the blood and calm the nerves, dispel wind and dredge collaterals - Have both the dual characteristics of nourishing and dredging, improve dreaminess and easy waking, and improve sleep quality; Modern pharmacology shows that its hypnotic effect is related to the regulation of the hypothalamic-pituitary-adrenal axis (HPA axis), and PI3K / Akt is the key pathway for the negative feedback regulation of the HPA axis;

[0017] Baishao (White Peony Root): Nourish the blood and regulate menstruation, soothe the liver and relieve pain - Alleviate headache and dizziness caused by hyperactivity of liver yang, restore the gentle nature of the liver body;

[0018] Yujin (Turmeric Root Tuber): Promote blood circulation to relieve pain, promote qi movement and relieve depression - Break stagnation in the liver meridian and clear the heart and cool the blood, relieve the pathogenesis of qi stagnation transforming into fire;

[0019] Hehuanpi (Inner Bark of Silktree Albizzia): Relieve depression and calm the nerves, promote blood circulation and reduce swelling - Bidirectionally regulate the sleep rhythm by regulating emotions and improving microcirculation;

[0020] In the traditional Chinese medicine composition for soothing the liver and promoting sleep provided by the present invention, Bupleuri Radix and Suanzaoren (Spina Date Seed) are the monarch drugs, which soothe liver depression, nourish the mind and calm the nerves, and help sleep and soothe the nerves; Fushen (Poria with hostwood) and Yejiateng (Caulis Polygoni Multiflori) are the minister drugs, which nourish the heart blood, calm the soul, and calm the mind and suppress fright; Baishao (White Peony Root) and Yujin (Turmeric Root Tuber) are the assistant drugs, which nourish the liver and level the liver, nourish the blood and astringe yin, regulate qi and strengthen the spleen; Hehuanpi (Inner Bark of Silktree Albizzia) is the guiding drug, which pacifies the five zang-organs and benefits the mind. The combination of various drugs together exerts the effects of soothing liver depression and helping sleep and soothe the nerves.

[0021] On the other hand, when Bupleuri Radix and Baishao (White Peony Root) are combined, Bupleuri Radix soothes the liver qi, and Baishao (White Peony Root) nourishes the liver, and the two cooperate to regulate the liver qi. Yujin (Turmeric Root Tuber) and Hehuanpi (Inner Bark of Silktree Albizzia) can further assist Bupleuri Radix to strengthen the effect of soothing liver depression. Aiming at the core pathogenesis of liver qi stagnation, it improves sleep disorders caused by emotional fluctuations. At the same time, Hehuanpi (Inner Bark of Silktree Albizzia) itself also has the effect of calming the nerves. Suanzaoren (Spina Date Seed), Fushen (Poria with hostwood), and Yejiateng (Caulis Polygoni Multiflori) form a combination for calming the nerves, enhancing each other's effects. Fushen (Poria with hostwood) promotes diuresis and percolates dampness, and relieves the interference of frequent nocturia on sleep by regulating the kidney AQP2 water channel protein (depending on the PI3K / Akt pathway); Yejiateng (Caulis Polygoni Multiflori) inhibits ROS from overactivating PI3K / Akt and reduces the damage of oxidative stress to the blood-brain barrier. The two synergistically improve sleep continuity from the dual pathways of 'water metabolism - oxidative stress'. The whole formula of each component synergistically strengthens the sedative and sleep-promoting effect, taking into account both the symptoms and the root causes, directly improving sleep quality from the level of the mind, reflecting the compatibility wisdom of "simultaneously soothing and tonifying".

[0022] Preferably, the traditional Chinese medicine composition is prepared from the following raw materials:

[0023] 8 - 15 parts of Bupleuri Radix, 12 - 20 parts of Semen Ziziphi Spinosae, 12 - 20 parts of Poria, 12 - 20 parts of Caulis Polygoni Multiflori, 10 - 15 parts of Radix Paeoniae Alba, 5 - 10 parts of Radix Curcumae, 10 - 15 parts of Cortex Albiziae.

[0024] Preferably, the traditional Chinese medicine composition is made from the following raw materials:

[0025] 10 parts of Bupleuri Radix, 15 parts of Semen Ziziphi Spinosae, 18 parts of Poria, 15 parts of Caulis Polygoni Multiflori, 12 parts of Radix Paeoniae Alba, 8 parts of Radix Curcumae, 12 parts of Cortex Albiziae.

[0026] Preferably, by weight, the raw materials of the traditional Chinese medicine composition further include at least one of the following:

[0027] 1) 5 - 12 parts of Atractylodis Macrocephalae Rhizoma;

[0028] 2) 8 - 12 parts of Aurantii Fructus Immaturus.

[0029] Considering that the whole prescription is slightly on the cool side and suitable for those with liver depression transforming into heat or yin deficiency constitution, if the patient has spleen - stomach deficiency - cold, Atractylodis Macrocephalae Rhizoma can be additionally added in this invention for balance.

[0030] Modern people have great work pressure, often stay up late, sit for long periods with little movement, have irregular diets and are high in fat, often presenting symptoms such as abdominal distension and fullness, belching and acid reflux. Traditional Chinese medicine says "if the stomach is not harmonious, sleep will be disturbed". Aurantii Fructus Immaturus, with a bitter, sour and slightly cold nature, mainly enters the spleen and stomach meridians, and has the effects of breaking qi to eliminate accumulation and promoting qi circulation to relieve fullness. The added Aurantii Fructus Immaturus in this invention mainly sinks (guiding qi downward), while Bupleuri Radix mainly ascends and scatters (soothing the liver qi upward), forming a combination of "one ascending and one descending" to regulate qi movement, further playing the roles of soothing the liver and regulating qi, regulating the middle - jiao, and strengthening the ascending and descending of qi. Especially for insomnia caused by liver qi stagnation combined with gastrointestinal dysfunction, it can more comprehensively improve the patient's emotional and physical symptoms.

[0031] Preferably, the dosage form of the traditional Chinese medicine composition is granule.

[0032] In the second aspect, the present invention also provides a preparation method for the traditional Chinese medicine composition for soothing the liver and promoting sleep that activates the PI3K / Akt signaling pathway as described above, including the following steps:

[0033] S1 Raw material treatment: Wash, dry and pulverize each raw material, then add water to mix evenly and soak for 1 - 5 h;

[0034] S2 Extraction and concentration: Use water extraction method and / or alcohol extraction method for extraction, and concentrate the extraction solution to obtain an extract;

[0035] S3 Granulation and drying: Mix the extract with optional excipients evenly, granulate by wet method, and dry at low temperature.

[0036] Preferably, ultrasonic - assisted treatment is applied for 0.5 - 1 h during the soaking stage to improve the soaking effect.

[0037] Preferably, in step S2, extraction is carried out by water extraction first and then alcohol extraction, including:

[0038] S2.1 Water extraction: Extract 1 - 3 times, add 5 - 20 times the amount of water each time, boil for 0.5 - 3 h, combine the water extracts, centrifuge to remove impurities, and reserve for later use;

[0039] S2.2 Alcohol extraction: Add 8 - 10 times the amount of 60 - 75 wt% ethanol aqueous solution to the medicinal residues after water extraction, reflux and extract 1 - 2 times, each time for 1 - 1.5 h, combine the alcohol extracts, centrifuge to remove impurities, and then recover ethanol under reduced pressure for later use;

[0040] S2.3 Combine the water extract obtained in step S2.1 and the alcohol extract obtained in step S2.2, and concentrate to obtain an extract with a relative density of 1.1 - 1.15.

[0041] When only water extraction is used in step S2, preferably, water extraction is carried out 2 - 3 times, the amount of water added each time is 8 - 12 times, boil strongly for 1 - 3 h for the first time, add water and boil gently for 1 - 3 h for the second time, and optionally boil gently for 0.5 - 1 h for the third time, combine the water extracts, let the filtrate stand, centrifuge to remove impurities, and concentrate to obtain an extract.

[0042] When water extraction is followed by alcohol extraction, in S2.1, preferably, water extraction is carried out 2 - 3 times, the amount of water added each time is 8 - 12 times, boil strongly for 1 - 3 h for the first time, add water and boil gently for 1 - 3 h for the second time, and optionally boil gently for 0.5 - 1 h for the third time, combine the water extracts, let the filtrate stand, centrifuge to remove impurities, and reserve for later use.

[0043] Preferably, after step S1 and before step S2, the raw materials further include at least one of the following additional treatments:

[0044] 1) Enzymatic hydrolysis treatment: Immerse the raw materials in the enzyme solution, and carry out enzymatic hydrolysis at 40 - 55 °C and pH 4.0 - 5.5 for 1 - 3 hours;

[0045] 2) Fermentation treatment: Ferment the raw materials with probiotics at 25 - 37 °C for 12 - 72 hours.

[0046] Enzymatic hydrolysis treatment can be used to decompose cell walls, promote the release of active ingredients, and improve extraction efficiency and bioavailability. Preferably, at least one of the raw materials of Bupleurum chinense, Ziziphus jujuba var. spinosa, and Paeonia lactiflora is subjected to enzymatic hydrolysis treatment.

[0047] More preferably, Bupleurum chinense, Ziziphus jujuba var. spinosa, and Paeonia lactiflora are subjected to enzymatic hydrolysis treatment, and in the enzyme solution, the mass ratio of cellulase, pectinase, and β - glucosidase is (1 - 2):(1 - 3):1.

[0048] Through the synergistic action of cellulase, pectinase and β-glucosidase, the structural polysaccharides (such as cellulose, hemicellulose and pectin, etc.) in the cell wall of medicinal materials are hydrolyzed directionally, the dense network structure of the plant cell wall is destroyed, and microporous channels are formed. This treatment can significantly increase the dissolution surface area of active ingredients such as saikosaponin and zizyphus jujuba flavonoids, convert the bound components originally wrapped by the cell wall into free forms, improve the component transfer efficiency in the water extraction / alcohol extraction stage, and at the same time reduce the risk of damage to thermosensitive substances during high-temperature extraction.

[0049] For Bupleurum chinense, β-glucosidase can hydrolyze glycosyl, destroy cell structure and promote the release of active ingredients of saikosaponin.

[0050] Zizyphus jujuba contains relatively more cell wall structures. Enzymatic hydrolysis treatment with cellulase, pectinase, etc. can promote the release of components such as saponins.

[0051] Paeonia lactiflora contains paeoniflorin. Pectinase can degrade pectin and improve the extraction efficiency of paeoniflorin.

[0052] Fermentation treatment. Through probiotic fermentation treatment, the release efficiency of active ingredients in raw materials such as Bupleurum chinense, Zizyphus jujuba and Paeonia lactiflora can be significantly improved, and the overall synergy of the "soothing the liver - calming the mind - regulating the stomach" chain can be enhanced, which is especially suitable for the long-term conditioning of sub-healthy people. For example, the acidic environment generated during fermentation can promote the stability of components such as paeoniflorin in Paeonia lactiflora, and at the same time generate precursor substances of γ-aminobutyric acid (GABA) through microbial transformation, which has a synergistic effect with the GABAergic system regulation of Zizyphus jujuba in the formula, and strengthens the targeted regulation ability of the PI3K / Akt pathway.

[0053] Preferably, the probiotics for fermentation treatment include at least one of lactic acid bacteria, yeasts, Bacillus subtilis, Bifidobacterium, and acetic acid bacteria.

[0054] For Bupleurum chinense, it can be fermented with lactic acid bacteria, etc., acid-hydrolyze saponins and improve bioavailability.

[0055] For Zizyphus jujuba, it can be fermented with lactic acid bacteria or Bifidobacterium, etc., decompose oils and saponins, generate γ-aminobutyric acid (GABA), enhance the sedative and anti-anxiety effects and sedative effects, and reduce the irritation to the gastrointestinal tract.

[0056] For Paeonia lactiflora, it can be fermented with Bacillus subtilis, etc., decompose paeoniflorin components, reduce the tannic acid content, enhance the effect of soothing the liver and regulating blood, and reduce gastric irritation.

[0057] For Albizia julibrissin, it can be fermented with yeasts, etc., degrade lignin fibers to release more triterpenoid saponin active substances, and synthesize phenyl ethanol glycoside components to better synergistically enhance the effect of relieving depression and calming the mind.

[0058] For Aurantii Fructus Immaturus, it can be fermented with acetic acid bacteria, etc., generate limonoids which is beneficial to enhancing gastrointestinal motility and soothing the liver and regulating the stomach.

[0059] Preferably, at least one of the raw materials of Bupleuri Radix, Semen Ziziphi Spinosae, Paeoniae Radix Alba, Cortex Albiziae, and Aurantii Fructus Immaturus is subjected to fermentation treatment.

[0060] More preferably, after Bupleuri Radix, Semen Ziziphi Spinosae, and Paeoniae Radix Alba are subjected to enzymatic hydrolysis treatment, Cortex Albiziae and optional Aurantii Fructus Immaturus are added to the enzymatic hydrolysis product, and fermentation treatment is carried out together.

[0061] After the physical barrier of the cell wall is broken by enzymatic hydrolysis treatment, the active ingredients exposed in the medicinal residues can provide a directional metabolic substrate for probiotic fermentation. Lactobacilli further decompose the saponin sugar side chains in the medicinal materials in the enzymatic hydrolysate, improving the polarity adaptability of lipophilic components such as bupleurum saponins, making them more easily extracted by ethanol subsequently. At the same time, organic acids such as lactic acid produced by fermentation can chelate metal ions, inhibit the oxidative polymerization of phenolic components during the extraction process, ensure the chemical stability of albizia bark flavonoids, and ultimately achieve the efficient enrichment and functional enhancement of the active ingredient group.

[0062] Thus, after step S1 and before step S2, the additional treatment is to combine enzymatic hydrolysis treatment and fermentation treatment for some raw materials, including:

[0063] A. Subject Bupleuri Radix, Semen Ziziphi Spinosae, and Paeoniae Radix Alba to enzymatic hydrolysis treatment to obtain an enzymatic hydrolysis product;

[0064] B. Add Cortex Albiziae and optional Aurantii Fructus Immaturus to the enzymatic hydrolysis product, inoculate probiotics and ferment for 24 - 36 hours, and inactivate the bacteria after fermentation.

[0065] Preferably, step S3 satisfies at least one of the following conditions:

[0066] 1) The auxiliary materials are selected from at least one of dextrin, sucrose, starch, lactose, and magnesium stearate, and the mass ratio of the extract to the auxiliary materials is 1:1 to 1:4. Preferably, the auxiliary materials are sucrose and dextrin, and the mass ratio of the extract, sucrose, and dextrin is 1:(0.5 - 1):(1 - 2);

[0067] 2) Feed the uniformly mixed soft drug material into a wet granulator and pass it through a 10 - 20 - mesh sieve to make wet granules;

[0068] 3) The temperature of low - temperature drying is 50 - 70 °C, the particle size of the dried granules is greater than 250 μm and less than 2000 μm, and the water content does not exceed 2 wt%.

[0069] Preferably, the particle size range of the dried granules meets the pharmacopoeia regulations, that is, the particle size range of the granules is: the sum of the coarse granules that cannot pass through sieve No. 1 (2000 μm) and the fine granules that pass through sieve No. 4 (250 μm) does not exceed 8.0 wt%, preferably does not exceed 5.0 wt%.

[0070] In a third aspect, the present invention provides an application of the traditional Chinese medicine composition for soothing the liver and promoting sleep by activating the PI3K / Akt signaling pathway in the preparation of a medicament for treating insomnia of liver depression type.

[0071] Preferably, the traditional Chinese medicine composition for soothing the liver and promoting sleep increases the expression level of PI3K protein in peripheral blood mononuclear cells by more than 35%, preferably more than 46%; the expression level of Akt protein is increased by more than 26%, preferably more than 37%; the expression level of p-Akt protein is increased by more than 50%, preferably more than 67%.

[0072] The present invention provides at least the following beneficial effects:

[0073] (1) Based on the theory of "liver depression causing insomnia" in traditional Chinese medicine, the present invention constructs a multi-dimensional synergistic regulation formula system. Through the monarch drug compatibility of Bupleuri Radix and Semen Ziziphi Spinosae, the dual effects of soothing the liver and relieving depression and nourishing the heart and calming the mind are innovatively and organically integrated, which can not only regulate the ascending and descending pivot of qi movement, but also nourish the liver blood to nourish the heart spirit, forming a two-way intervention on the core pathogenesis of insomnia of liver depression type. The ministerial drug combination of Poria and Caulis Polygoni Multiflori realizes the synchronous regulation of the intersection of heart and kidney and the resolution of phlegm-dampness by the synergistic effect of "guiding the heart fire downward" and "nourishing and dredging collaterals", breaking through the limitation of the single sedative effect of traditional sedative drugs. The adjuvant and guiding drugs of Paeoniae Radix Alba, Curcumae Radix and Cortex Albiziae form a compatibility of "softening the liver - promoting blood circulation - relieving depression", while harmonizing the rigid and soft characteristics of the liver body, and optimizing the sleep rhythm in two ways through improving microcirculation and emotional regulation. The whole formula realizes the targeted regulation of the PI3K / Akt signaling pathway through the synergistic effect of multi-level active ingredients, providing an innovative treatment strategy for multi-target intervention of insomnia of liver depression type.

[0074] (2) The present invention develops a stepwise extraction and biotransformation technology system based on the characteristics of active ingredients. The gradient extraction process of water extraction and alcohol extraction is adopted to selectively enrich different polar components such as saponins and flavonoids, significantly improving the extraction efficiency of active ingredients. The innovative enzymatic hydrolysis-fermentation coupling technology breaks through the plant cell wall barrier through biotransformation, promotes the release of bound components under mild conditions, and simultaneously generates new active metabolites with synergistic effects. The combination of low-temperature dynamic drying and intelligent humidity control technology effectively retains the chemical stability of thermosensitive components and ensures the complete retention of key pharmacodynamic substances in the granule. Detailed implementation manners

[0075] In order to better understand the above technical solutions, the following will describe the above technical solutions in detail in combination with specific implementation manners. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0076] The terms used in the embodiments of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The singular forms "a", "said", and "the" used in the embodiments of the present invention and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise. "Plural" generally includes at least two.

[0077] It should also be noted that the term "comprising", "including", or any other variant thereof is intended to cover non-exclusive inclusion, such that a commodity or device comprising a series of elements not only includes those elements but also includes other elements not explicitly listed, or further includes elements inherent to such commodity or device. Without further limitation, an element defined by the statement "comprising an..." does not exclude the existence of additional identical elements in the commodity or device comprising the element.

[0078] A traditional Chinese medicine composition for soothing the liver and promoting sleep by activating the PI3K / Akt signaling pathway is prepared from the following raw materials by weight:

[0079] (1) Bupleurum chinense, 5 - 20 parts, preferably 8 - 15 parts, more preferably 10 parts;

[0080] (2) Semen Ziziphi Spinosae, 10 - 30 parts, preferably 12 - 20 parts, more preferably 15 parts;

[0081] (3) Poria cum Radice Pini, 10 - 25 parts, preferably 12 - 20 parts, more preferably 18 parts;

[0082] (4) Caulis Polygoni Multiflori, 10 - 25 parts, preferably 12 - 20 parts, more preferably 15 parts;

[0083] (5) Radix Paeoniae Alba, 5 - 20 parts, preferably 10 - 15 parts, more preferably 12 parts;

[0084] (6) Radix Curcumae, 3 - 15 parts, preferably 5 - 10 parts, more preferably 8 parts;

[0085] (7) Cortex Albiziae, 5 - 20 parts, preferably 10 - 15 parts, more preferably 12 parts;

[0086] And optional raw materials:

[0087] (8) Rhizoma Atractylodis Macrocephalae, 5 - 12 parts, preferably 8 - 10 parts;

[0088] (9) Fructus Aurantii Immaturus, 8 - 12 parts, preferably 10 - 12 parts.

[0089] The preparation method of the traditional Chinese medicine composition for soothing the liver and promoting sleep by activating the PI3K / Akt signaling pathway specifically includes the following steps:

[0090] S1 Raw material treatment: After cleaning, drying, and pulverizing each raw material, add water, mix evenly, and soak for 1 - 5 h. Preferably, perform ultrasonic-assisted treatment for 0.5 - 1 h.

[0091] Preferably, after step S1, perform at least one of the following additional treatments on the soaked raw materials:

[0092] 1) Enzymatic hydrolysis treatment: Soak the raw materials in an enzyme solution and perform enzymatic hydrolysis at 40 - 55 °C and pH 4.0 - 5.5 for 1 - 3 hours.

[0093] 2) Fermentation treatment: Ferment the raw materials with probiotics at 25 - 37 °C for 12 - 72 hours. Inactivate the bacterial cells after fermentation. The dosage of probiotics is 1 - 3% of the weight of the raw materials (solid part).

[0094] Specifically, the additional treatment is to combine enzymatic hydrolysis treatment and fermentation treatment for some raw materials, including:

[0095] A. Perform enzymatic hydrolysis treatment on Bupleurum chinense, Ziziphus jujuba var. spinosa, and Paeonia lactiflora to obtain enzymatic hydrolysis products. In the enzyme solution, the mass ratio of cellulase, pectinase, and β - glucosidase is (1 - 2):(1 - 3):1.

[0096] B. Add Albizia julibrissin and optional Aurantii Fructus Immaturus to the enzymatic hydrolysis products, inoculate with probiotics, and ferment for 24 - 36 hours. Inactivate the bacterial cells after fermentation.

[0097] S2 Extraction and concentration: Perform extraction using water extraction method and / or alcohol extraction method, and concentrate the extract to obtain an extract paste.

[0098] Optionally, when only using the water extraction method, perform water extraction 2 - 3 times, add 8 - 12 times the amount of water each time. Boil vigorously for 1 - 3 h for the first time, add water and boil gently for 1 - 3 h for the second time. Optionally, boil gently for 0.5 - 1 h for the third time. Combine the water extracts, let the filtrate stand, remove impurities by centrifugation, and concentrate to obtain an extract paste.

[0099] Preferably, perform extraction by first using the water extraction method and then the alcohol extraction method, specifically including the following steps:

[0100] S2.1 Water extraction method: Perform extraction 1 - 3 times, add 5 - 20 times the amount of water each time and boil for 0.5 - 3 h. Combine the water extracts, remove impurities by centrifugation, and reserve. More preferably, perform water extraction 2 - 3 times, add 8 - 12 times the amount of water each time. Boil vigorously for 1 - 3 h for the first time, add water and boil gently for 1 - 3 h for the second time. Optionally, boil gently for 0.5 - 1 h for the third time. Combine the water extracts, let the filtrate stand, remove impurities by centrifugation, and reserve.

[0101] S2.2 Alcohol extraction method: Add 8 - 10 times the amount of 60 - 75wt% ethanol aqueous solution to the medicinal residues after water extraction, reflux and extract 1 - 2 times, each time for 1 - 1.5 h. Combine the alcohol extracts, remove impurities by centrifugation, and then recover ethanol under reduced pressure for standby.

[0102] S2.3 Combine the water extract obtained in step S2.1 and the alcohol extract obtained in step S2.2, and concentrate to obtain an extract with a relative density of 1.1 - 1.15.

[0103] S3 Granulation and drying: Mix the extract evenly with optional excipients and feed it into a wet granulator. Make wet granules through a 10 - 20 - mesh sieve, and dry at a low temperature of 50 - 70 °C. The particle size of the dried granules is greater than 250 μm and less than 2000 μm, and the water content does not exceed 2wt%. The excipients are selected from at least one of dextrin, sucrose, starch, lactose, and magnesium stearate. The mass ratio of the extract to the excipients is 1:1 to 1:4.

[0104] Example A1

[0105] In Example A1, calculated by weight, the traditional Chinese medicine composition is made from the following raw materials:

[0106] 8 parts of Bupleurum chinense, 12 parts of Ziziphus jujuba var. spinosa, 22 parts of Poria cocos, 20 parts of Polygonum multiflorum Thunb. var. thomsonii, 10 parts of Paeonia lactiflora Pall., 8 parts of Curcuma aromatica Salisb., 10 parts of Albizia julibrissin Durazz.

[0107] Example A1 only uses the water extraction method to extract and concentrate the raw materials. Its preparation steps include:

[0108] S1 Raw material treatment: Clean, dry, and pulverize each raw material, then add water and mix evenly and soak for 3 h, with ultrasonic - assisted treatment added for the first 15 minutes of each hour.

[0109] S2 Extraction and concentration: Use the water extraction method to extract 3 times. For the first time, add 8 times the amount of water and boil vigorously for 2 h; for the second time, add 12 times the amount of water and boil gently for 2 h; for the third time, add 10 times the amount of water and boil gently for 1 h. Combine the extraction solutions, let the filtrate stand, remove impurities by centrifugation, and concentrate the water extract to obtain an extract with a relative density of about 1.12.

[0110] S3 Granulation and drying: Mix the extract evenly with sucrose and dextrin in a mass ratio of 1:1:2 and feed it into a wet granulator. Make wet granules through a 15 - mesh sieve, and dry at a low temperature of 60 °C. The water content of the dried granules does not exceed 2wt%, the particle size is greater than 250 μm and less than 2000 μm, meeting the pharmacopoeia requirements.

[0111] Examples A2 - A8

[0112] The raw material ratio of Example A2 is the same as that of Example A1, and the raw material ratios of Examples A3 - A8 are different, as shown in Table 1 specifically.

[0113] Examples A2 - A8 have a different preparation method from Example A1. The preparation steps of first extracting with water and then with alcohol include:

[0114] S1 Raw material treatment: After cleaning, drying, and pulverizing each raw material, add water, mix evenly, and soak for 3 h, with ultrasonic-assisted treatment added for the first 15 min of each hour;

[0115] S2 Extraction and concentration: Extract using the method of first extracting with water and then with alcohol, and concentrate the extract to obtain an extract paste, including the following steps:

[0116] S2.1 Water extraction method: Extract 3 times. For the first time, add 8 times the amount of water and boil vigorously for 2 h; for the second time, add 12 times the amount of water and boil gently for 2 h; for the third time, add 10 times the amount of water and boil gently for 1 h. Combine the extraction solutions, let the filtrate stand, centrifuge to remove impurities, and reserve;

[0117] S2.2 Alcohol extraction method: Add 8 times the amount of 75 wt% ethanol aqueous solution to the residue after water extraction, reflux and extract 2 times, 1 h each time. Combine the alcohol extraction solutions, centrifuge to remove impurities, and then recover ethanol under reduced pressure, and reserve;

[0118] S2.3 Combine the water extraction solution obtained in step S2.1 and the alcohol extraction solution obtained in step S2.2, and concentrate to obtain an extract paste with a relative density of about 1.12;

[0119] S3 Granulation and drying: Mix the extract paste, sucrose, and dextrin evenly at a mass ratio of 1:1:2, feed them into a wet granulator, pass through a 15-mesh sieve to make wet granules, and dry them at a low temperature of 60 °C. The water content of the dried granules does not exceed 2 wt%, and the particle size is greater than 250 μm and less than 2000 μm, meeting the pharmacopoeia requirements.

[0120] The raw material ratios in Examples A2 - A8 are different as shown in Table 1:

[0121] Table 1 Raw material ingredient table of Examples A2 - A8

[0122] Bupleurum Semen Ziziphi Spinosae Poria cum Radice Pini Caulis Polygoni Multiflori White Peony Root Curcuma aromatica Salisb Cortex Albiziae Atractylodes macrocephala Koidz Fructus Aurantii Immaturus Example A2 8 12 22 20 10 8 10 - - Example A3 18 22 10 10 10 10 15 - - Example A4 10 15 18 15 12 8 12 - - Example A5 10 10 12 12 20 15 18 - - Example A6 15 15 20 22 15 3 8 - - Example A7 10 15 18 15 12 8 12 10 - Example A8 10 15 18 15 12 9 12 9 11

[0123] Example B1

[0124] Example B1 has the same raw material composition as Example A4. The difference lies in the preparation method. After step S1 and before step S2, partial raw materials are subjected to enzymatic hydrolysis treatment, including:

[0125] Soak Bupleurum chinense, Ziziphus jujuba var. spinosa, and Paeonia lactiflora in the enzyme solution, carry out enzymatic hydrolysis treatment at 45 °C and a pH value of about 5.0 for 2 h to obtain an enzymatic hydrolysis product; in the enzyme solution, the mass ratio of cellulase, pectinase, and β-glucosidase is 2:2:1;

[0126] After that, the enzymatic hydrolysate and other raw materials are fed into step S2 for extraction and concentration together.

[0127] Example B2

[0128] Example B2 has the same raw material composition as Example A8. The difference lies in the preparation method. After step S1 and before step S2, some of the raw materials are treated by combined enzymatic hydrolysis and fermentation, including:

[0129] A. Soak Bupleurum chinense, Ziziphus jujuba var. spinosa, and Paeonia lactiflora in the enzyme solution and carry out enzymatic hydrolysis at 45 °C and a pH value of about 5.0 for 2 h to obtain an enzymatic hydrolysate; in the enzyme solution, the mass ratio of cellulase, pectinase, and β-glucosidase is 2:2:1;

[0130] B. Add the soaked Albizia julibrissin and Aurantii Fructus Immaturus to the enzymatic hydrolysate, inoculate Lactobacillus (0.8 wt%), Saccharomyces cerevisiae (0.8 wt%), and Bacillus subtilis (0.6 wt%), culture and ferment for 36 hours, and inactivate at 90 °C for 10 min after fermentation.

[0131] The viable count of Lactobacillus is about (2-3)×10 9 cfu / mL, the viable count of Saccharomyces cerevisiae is about (3-4)×10 8 cfu / mL, and the viable count of Bacillus subtilis is about (8-9)×10 8 cfu / mL.

[0132] After that, the products obtained by enzymatic hydrolysis and fermentation and other raw materials are fed into step S2 for extraction and concentration together.

[0133] Table 2 Treatment methods of each raw material before extraction in Examples B1-B2

[0134]

[0135] (I) Tests and results

[0136] 1. Determination method and instrument for active ingredients

[0137] Determination method: Use the combined technology of high performance liquid chromatography (HPLC) and ultraviolet-visible spectrophotometry, and refer to the relevant standards of the 2020 edition of the Chinese Pharmacopoeia to test the granule samples of each example.

[0138] Target components:

[0139] Saikosaponin a / d: HPLC method, C18 chromatographic column, mobile phase is acetonitrile-0.1% phosphoric acid aqueous solution, gradient elution, detection wavelength 254 nm;

[0140] Spinosin in Ziziphus jujuba var. spinosa: HPLC method, amino column, mobile phase is acetonitrile-water, detection wavelength 335 nm;

[0141] Paeoniflorin: HPLC method, C18 column, mobile phase is methanol - 0.1% phosphoric acid aqueous solution, detection wavelength 230 nm;

[0142] Total flavonoids from Cortex Albiziae: UV spectrophotometry, using rutin as reference substance, detection wavelength 510 nm;

[0143] Hesperidin from Fructus Aurantii Immaturus: HPLC method, C18 column, gradient elution with methanol - 0.1% phosphoric acid water, detection wavelength 283 nm.

[0144] The contents of active ingredients in Examples A1 - A8 and Examples B1 and B2 are shown in Table 3:

[0145] Table 3

[0146]

[0147]

[0148] Comparatively, in Example A1, only water extraction method is used for extraction, and the extracted active ingredients are relatively lower than those in Example A2, and the utilization rate of raw materials is relatively insufficient. When the extraction method of first water extraction and then alcohol extraction is used, due to the differences in the component ratios of raw materials from Example A2 to A8, there are differences in the extraction amounts of various active ingredients. On this basis, the formula can be adjusted according to the actual constitution and treatment needs of patients to achieve a more targeted therapeutic effect. In addition, hesperidin, the main active ingredient of Fructus Aurantii Immaturus, is extracted after adding Fructus Aurantii Immaturus in Example A8, and it may have a positive effect on soothing the liver and promoting sleep through the synergistic effects of multiple pathways such as soothing the liver, antioxidation, regulating neurotransmitters and sedation.

[0149] On the basis of Example A4, in Example B1, some raw materials such as Bupleuri Radix are subjected to enzymatic hydrolysis treatment, resulting in an increase of about 21.9% in the contents of saikosaponin a / d, an increase of about 18.8% in the content of paeoniflorin, and an increase of about 9.4% in the content of total flavonoids from Cortex Albiziae; on the basis of Example A8, in Example B2, enzymatic hydrolysis treatment and fermentation treatment are further combined, resulting in an increase of about 32.4% in the contents of saikosaponin a / d, an increase of about 37.9% in the content of paeoniflorin, an increase of about 38.5% in the content of total flavonoids from Cortex Albiziae, and an increase of about 14.8% in the content of hesperidin from Fructus Aurantii Immaturus. It can be seen that enzymatic hydrolysis treatment and fermentation treatment have a positive effect on the extraction of corresponding active ingredients from raw materials. By increasing the extraction ratio of active ingredients, it is beneficial to improve the utilization rate of traditional Chinese medicine raw materials and reduce the medication cost of patients.

[0150] (II) Clinical tests

[0151] 1. Source of cases

[0152] A total of 150 patients with insomnia due to liver qi stagnation in Taihe Hospital of Traditional Chinese Medicine Affiliated to Anhui University of Traditional Chinese Medicine from February 2023 to February 2025 were selected. According to the random number table method, they were divided into a control group of 50 cases, and treatment groups 1 and 2 with 50 cases each. During the treatment period, 2 cases dropped out from the control group and 1 case dropped out from treatment group 2. The basic information of the control group, treatment group 1 and treatment group 2 is shown in Table 4. There were no statistically significant differences in the basic information of the three groups of patients (P>0.05), and they were comparable.

[0153] Table 4 Basic information of the three groups of patients

[0154] Group Number of people Male Female Average age / year Average course of disease / month Control group 48 25 23 46.15±9.36 11.59±2.54 Treatment group 1 50 25 25 46.58±10.14 11.85±2.65 Treatment group 2 49 24 25 45.82±10.78 12.11±1.98

[0155] 2. Diagnostic criteria

[0156] 2.1 Western medicine diagnostic criteria:

[0157] Referring to the diagnostic criteria for primary insomnia in the International Classification of Sleep Disorders (3rd Edition), and having insomnia 3 times or more per week for a duration of 1 month.

[0158] 2.2 Traditional Chinese medicine diagnostic criteria

[0159] (1) The diagnosis of insomnia is formulated according to the Clinical Practice Guidelines for Insomnia in Traditional Chinese Medicine (WHO / WPO): ① Anyone with the main clinical manifestations of difficulty falling asleep, easy waking up during sleep, or even staying awake all night can be diagnosed with insomnia; ② Often accompanied by symptoms such as fatigue, tiredness, loss of appetite, and decreased work ability due to insomnia; ③ No organic lesions are found in clinical examinations, and polysomnography (PSG) shows sleep structure disorders; combined with sleep scales and relevant biochemical examinations for confirmation;

[0160] (2) The diagnostic criteria for the syndrome of liver qi stagnation: formulated referring to the Expert Consensus on the Treatment of Adult Insomnia by Chinese Ethnic Medicine. The main clinical manifestations: difficulty falling asleep, frequent sighing, irritability, distension in both flanks, fullness and discomfort in the epigastrium after eating, abdominal distension, breast distending pain in women, and irregular menstruation. The tongue is dark red or light red, the coating is white, and the pulse is stringy.

[0161] 3. Screening criteria

[0162] 3.1 Inclusion criteria:

[0163] Meeting the above-mentioned Chinese and Western medicine diagnostic criteria, mainly including:

[0164] (1) Those who can understand the content of relevant scales, abide by the treatment plan and follow-up as required;

[0165] (2) The subjects voluntarily participate, sign the informed consent form, and pass the ethical review.

[0166] 3.2 Exclusion criteria:

[0167] (1) Patients with severe organic diseases (such as malignant tumors, patients recovering from cardiovascular and cerebrovascular events, etc.);

[0168] (2) patients with impaired cognitive function who are unable to communicate normally with researchers;

[0169] (3) Those who are allergic to the drugs used in the trial or have severe organ dysfunction.

[0170] 3.3 Elimination criteria:

[0171] (1) Those who cannot be followed up or cannot cooperate with treatment, resulting in inability to determine the efficacy;

[0172] (2) Those who take other drugs or receive other treatments during treatment that may interfere with the study results;

[0173] (3) Those who are unable to continue participating in the trial due to other diseases during the treatment process.

[0174] 4. Treatment options

[0175] The control group was given 2 mg of Estazolam tablets (Changzhou Siyao Pharmaceutical Co., Ltd.; approval number: National Medicine Standard H32020699), taken orally 30 minutes before going to bed every night, with a dose reduction of 25% per week. On the basis of the control group, the treatment group 1 was given the Shugan Zhumian Granules of Example A4, and the treatment group 2 was given the Shugan Zhumian Granules of Example A8, twice a day, 1 bag each time, 10 g per bag, taken after breakfast and half an hour before going to bed at night, all drugs were provided by the Taihe County Traditional Chinese Medicine Hospital Preparation Center, and the course of treatment was 4 weeks.

[0176] 5. Efficacy indicators

[0177] (1) PSQI scale and TCM syndrome score

[0178] The PSQI scale was used to evaluate the changes in the subjects' sleep quality before (first day) and after (fourth week) treatment. The TCM syndrome scoring scale for liver-qi stagnation-type insomnia, which was developed with reference to the "Expert Consensus on the Treatment of Adult Insomnia with Chinese Ethnic Medicine", was used to observe the improvement of TCM symptoms of liver-qi stagnation-type insomnia.

[0179] (2) Determination of inflammatory markers

[0180] Peripheral venous blood was drawn from the subjects on an empty stomach before treatment (day 1) and after treatment (week 4). The supernatant was collected after centrifugation and the changes in TNF-α and IL-1β levels in the subjects' serum were determined by enzyme-linked immunosorbent assay.

[0181] (3) Determination of protein content in subjects

[0182] Before treatment (day 1) and after treatment (week 4), fasting venous blood was drawn from the patients, and mononuclear cells were extracted by centrifugation and stored in a -80 °C refrigerator for later use. The protein immunoblotting method was used to detect the changes in the protein expression levels of PI3K, AKT, and p-AKT in peripheral blood mononuclear cells of the subjects: total protein in the cells was extracted and its concentration was measured. Concentrating gels and separating gels were prepared. After protein loading, the membrane was transferred overnight. After blocking, it was incubated at room temperature for 1 h and then washed. Then, rabbit anti-human polyclonal antibody was added and incubated at room temperature for 1.5 h and then washed. After adding rabbit secondary antibody and incubating for 1 h, it was washed again. Chemiluminescence and development were carried out by chemical method, and then the gray values of each protein were measured using ImageJ software, with β-actin as the internal standard.

[0183] 6. Efficacy evaluation criteria

[0184] The insomnia efficacy evaluation criteria were formulated with reference to the Diagnostic and Therapeutic Criteria for Traditional Chinese Medicine Diseases and Syndromes and combined with the PSQI score reduction rate (using the nimodipine method).

[0185] PSQI score reduction rate = [(total score before treatment - total score after treatment) / total score before treatment] × 100%.

[0186] Ineffective = no improvement in insomnia symptoms and sleep quality, PSQI score reduction rate < 25%

[0187] Effective = improvement in the main clinical symptoms of insomnia, longer sleep time than before, 25% ≤ PSQI score reduction rate < 50%

[0188] Markedly effective = most of the clinical symptoms of insomnia disappear, significant improvement in sleep quality, 50% ≤ PSQI score reduction rate < 75%

[0189] Clinical cure = sleep time returns to normal, sleep is deep, and the patient is energetic after waking up, accompanied by the disappearance of the main clinical symptoms, PSQI score reduction rate ≥ 75%

[0190] 8. Statistical analysis

[0191] SPSS 27.0 statistical software was used for statistical analysis of the data. Measurement data were expressed as mean ± standard deviation (x`±s). If they conformed to the normal distribution, the independent samples t-test was used for comparison of the means between groups, and the paired t-test was used for within-group comparison; if not, the non-parametric test Mann-Whitney U test was used; count data were expressed as rate or constituent ratio, the χ2 test was used for between-group comparison, and the Mann-Whitney U test was used for between-group comparison of ranked data. P < 0.05 indicated that the difference was statistically significant.

[0192] Table 5 Comparison of PSQI total scores before and after treatment

[0193]

[0194]

[0195] *Note: Compared with the control group after treatment, *P < 0.05.

[0196] Table 5 results showed that after treatment, the scores of each item and the total score of PSQI in the three groups of patients were lower than those before treatment (P < 0.05), and the reduction in Group 1-2 was more obvious than that in the control group (P < 0.05).

[0197] Comparison of curative effects in Table 6 / n(%)

[0198] Group Number of cases Cured Markedly effective Effective Invalid Total effective rate Control group 48 0(0.00) 6(12.50) 21(43.75) 21(43.75) 27(56.25) Treatment group 1 50 3(6.00) 20(40.00) 24(48.00) 3(6.00) 47(94.00) Treatment group 2 49 5(10.20) 23(46.94) 20(40.82) 1(2.04) 48(97.96)

[0199] As shown in the results of Table 6, after treatment, the total effective rates of Group 1 and Group 2 were higher than 90%, and some insomnia patients were cured, and the treatment effect was better than that of the control group.

[0200] Table 7 Changes in the expression levels of PI3K, AKT, and p-AKT proteins in the three groups of patients before and after treatment

[0201]

[0202] Note:

[0203] 1. * indicates P < 0.05 compared with before treatment, ** indicates P < 0.01 (if group comparison is involved, additional annotations are required)

[0204] 2. Calculation formula for the change rate: (value after treatment - value before treatment) / value before treatment × 100%

[0205] 3. All protein expression levels were standardized by β-actin (gray value of the target protein / gray value of the internal reference)

[0206] 4. The data conformed to a normal distribution after Shapiro-Wilk test (P > 0.05)

[0207] By comparison, after treatment, the expression levels of proteins related to the PI3K / AKT signaling pathway in peripheral blood mononuclear cells of the three groups of patients showed differential changes. Although the expression levels of the three proteins (PI3K, AKT, and p-AKT) in the control group of patients increased slightly (change rate +4.5% to +7.8%), there was no statistical significance (P>0.05). In contrast, the protein expression levels in the two treatment groups were significantly increased. After treatment, the levels of PI3K, AKT, and p-AKT in treatment group 1 increased by 35.2%, 26.4%, and 50.8% respectively (all P<0.05), and the increase in treatment group 2 was even more significant, reaching 46.3%, 37.6%, and 67.2% respectively (all P<0.01). It can be seen that the traditional Chinese medicine composition for soothing the liver and promoting sleep of the present invention can increase the expression level of PI3K protein in peripheral blood mononuclear cells by more than 35%, preferably more than 46%; increase the expression level of Akt protein by more than 26%, preferably more than 37%; and increase the expression level of p-Akt protein by more than 50%, preferably more than 67%. It is worth noting that p-AKT, as an activation marker of this pathway, showed the strongest upward trend in the treatment groups (reaching +67.2% in treatment group 2, P<0.001), suggesting that the granules for soothing the liver and promoting sleep may play a therapeutic role by activating the PI3K / AKT signaling pathway. These molecular-level improvements were consistent with the clinical efficacy indicators (decrease in PSQI score and total effective rate exceeding 90%), providing experimental evidence for the "pathway-phenotype" association mechanism of insomnia with liver qi stagnation, and at the same time revealing the potential advantages of treatment group 2 (the preparation corresponding to Example A8) in regulating the phosphorylation level of key proteins.

[0208] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the present invention is intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of the present invention. Obviously, those skilled in the art can make various changes and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the present invention and its equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A liver-soothing and sleep-inducing Chinese medicine composition that stimulates the PI3K / Akt signaling pathway, characterized in that: The Chinese medicine composition is made of the following raw materials in parts by weight: 5-20 parts of bupleurum, 10-30 parts of spinach seed, 10-25 parts of poria, 10-25 parts of polyphylla, 5-20 parts of white peony root, 3-15 parts of curcuma, and 5-20 parts of julibrissin bark.

2. The liver-soothing and sleep-inducing Chinese medicine composition according to claim 1, characterized in that: The Chinese medicine composition is made from the following raw materials: 8-15 parts of bupleurum, 12-20 parts of spinach seed, 12-20 parts of poria, 12-20 parts of polyphylla, 10-15 parts of white peony root, 5-10 parts of curcuma, and 10-15 parts of julibrissin bark.

3. The liver-soothing and sleep-inducing Chinese medicine composition according to claim 2, characterized in that: The Chinese medicine composition is made from the following raw materials: 10 parts of bupleurum, 15 parts of spinach seed, 18 parts of poria, 15 parts of polyphylla, 12 parts of white peony root, 8 parts of curcuma, and 12 parts of julibrissin bark.

4. The liver-soothing and sleep-inducing Chinese medicine composition according to any one of claims 1 to 3, characterized in that: The raw materials of the Chinese medicine composition further include at least one of the following by weight: 1) 5-12 parts of Atractylodes macrocephala; 2) 8-12 portions of Citrus aurantium.

5. A method for preparing a liver-soothing and sleep-inducing Chinese medicinal composition for stimulating the PI3K / Akt signaling pathway as claimed in any one of claims 1 to 4, characterized in that: The following steps are involved: S1 Raw material processing: After cleaning, drying and crushing the raw materials, add water to mix evenly and soak for 1-5 hours; S2 extraction and concentration: extracting by water extraction and / or alcohol extraction, and concentrating the extract to obtain an extract; S3 granulation and drying: mix the extract and optional excipients evenly, wet granulate, and dry at low temperature.

6. The preparation method according to claim 5, characterized in that: In step S2, the extraction is performed by first water extraction and then alcohol extraction, including: S2.1 Water extraction method: extract 1-3 times, add 5-20 times the amount of water each time and boil for 0.5-3h, combine the water extracts, centrifuge to remove impurities, and set aside; S2.2 Alcohol extraction method: add 8-10 times the amount of 60-75wt% ethanol aqueous solution to the drug residue after water extraction, reflux extraction 1-2 times, combine the alcohol extracts, centrifuge to remove impurities, and recover ethanol under reduced pressure for standby use; S2.3 The water extract obtained in step S2.1 and the alcohol extract obtained in step S2.2 are combined and concentrated to obtain an extract with a relative density of 1.1-1.

15.

7. The preparation method according to claim 5 or 6, characterized in that: After step S1 and before step S2, the raw material further includes at least one of the following additional treatments: 1) Enzymatic hydrolysis: soak the raw material in enzyme solution at 40-55°C, pH 4.0-5.5, and perform enzymatic hydrolysis for 1-3 hours; 2) Fermentation treatment: Ferment the raw materials and probiotics at 25-37°C for 12-72 hours.

8. The preparation method according to claim 7, characterized in that: Additional treatment is to combine enzymatic hydrolysis and fermentation treatment on some raw materials, including: A. Radix Bupleuri, Semen Ziziphi Spinosae and Radix Paeoniae Alba are subjected to enzymolysis to obtain enzymolysis products; in the enzyme solution, the mass ratio of cellulase, pectinase and β-glucosidase is (1-2): (1-3): 1; B. Add Albizzia julibrissin bark and optional Citrus aurantium to the enzymatic hydrolysate, inoculate with probiotics and ferment for 24-36 hours, and inactivate the bacteria after fermentation.

9. The preparation method according to claim 5 or 6, characterized in that: Step S3 satisfies at least one of the following conditions: 1) The auxiliary material is selected from at least one of dextrin, sucrose, starch, lactose and magnesium stearate, and the mass ratio of the extract to the auxiliary material is 1:1 to 1:4; 2) The uniformly mixed drug soft material is fed into a wet granulator and passed through a 10-20 mesh screen to form wet granules; 3) The temperature of low-temperature drying is 50-70° C. The particle size of the granules after drying is greater than 250 μm and less than 2000 μm, and the water content does not exceed 2 wt %.

10. Use of the liver-soothing and sleep-aiding Chinese medicinal composition that stimulates the PI3K / Akt signaling pathway according to any one of claims 1 to 4 in the preparation of a medicament for treating insomnia of liver depression type.

11. The use according to claim 10, characterized in that The liver-soothing and sleep-aiding Chinese medicine composition increases the expression of PI3K protein in peripheral blood mononuclear cells by more than 35%, preferably more than 46%; increases the expression of Akt protein by more than 26%, preferably more than 37; and increases the expression of p-Akt protein by more than 50%, preferably more than 67%.

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