Traditional Chinese medicine composition for treating depression and preparation method thereof

Abstract

The invention discloses a traditional Chinese medicine composition for treating depression and a preparation method thereof. The invention relates to the field of traditional Chinese medicine compositions, in particular to an edible and medicinal traditional Chinese medicine composition for preventing and treating depression as well as a preparation method and application thereof. Comprising rose flowers, peony flowers, seville orange flowers, dendrobium flowers and sanchi flowers. The traditional Chinese medicine composition provided by the invention can improve symptoms such as depression, mood disorder and schizophrenia body weight loss, pleasant sensation loss and the like, protect neurons and improve the levels of 5-HT and DA in serum, is good in safety, provides a new edible and medicinal scheme for prevention and treatment of depression, and has a relatively good application prospect.

Description

Technical Field

[0004] , , ,

[0006] , ,

[0005]

[0001] The present invention relates to the field of traditional Chinese medicine compositions, and particularly relates to a homologous medicine and food traditional Chinese medicine composition for preventing and treating depression, a preparation method and an application thereof. Background Art

[0002] The core characteristics of depression are manifested as persistent low mood, reduced interest, and a significant decline in activity ability. Depression may also lead to impaired cognitive function, such as difficulty in concentrating, memory loss, and a decline in decision-making ability. In severe cases, patients with depression may exhibit self-harm behavior and even have a risk of suicide, posing a serious threat to the patient's life safety.

[0003] The pathological mechanism of depression is complex, involving a decrease in the concentration of monoamine neurotransmitters (such as 5-HT, DA, etc.), hyperfunction of the HPA axis (abnormal elevation of ACTH and CORT levels), a neuroinflammatory cascade reaction (activation of pro-inflammatory factors such as IL-6 and TNF-α), and a synergistic disorder of the overactivation of the JAK2 / STAT3 signaling pathway and the inhibition of the BDNF / CREB neurotrophic pathway. Currently, the main treatment in Western medicine is SSRIs drugs. Although they can inhibit the reuptake of neurotransmitters, there are obvious limitations. For example, long-term use can lead to a downregulation of 5-HT receptor sensitivity, resulting in drug tolerance; about 30-40% of patients will experience gastrointestinal and central nervous system side effects such as nausea and insomnia; the incidence of withdrawal reactions after drug discontinuation is as high as 56%, etc.

[0004] Traditional Chinese medicine theory classifies depression as "depressive syndrome". Traditional compound prescriptions (such as Xiaoyao San and Chaihu Shugan San) play an antidepressant role by regulating the functions of the liver and spleen and dredging qi mechanism. However, there are deficiencies in existing research: the prescription is mostly based on empirical medication, lacking a systematic screening of "homologous medicine and food" substances, and the safety and feasibility of long-term use have not been clarified; the research on the mechanism of action focuses on the regulation of monoamine neurotransmitters, and there is insufficient research on the intervention of hyperfunction of the HPA axis and activation of the JAK2 / STAT3 inflammatory pathway; the crude process leads to the loss of active ingredients. For example, the retention rate of thermosensitive flavonoid components by the traditional water decoction method is less than 60%.

[0005] Homologous medicine and food substances have become a new direction for the research and development of antidepressant drugs due to their safety and health care functions. However, there are obvious defects in the existing technology: mainly single-drug research, lacking the compound synergistic mechanism, and unable to explain the phenomenon of enhanced drug efficacy of "1 + 1 > 2"; the preparation process is backward, and the traditional water decoction process (boiling at 100°C) leads to the degradation of thermosensitive components and a low extract yield; the action target is single, and it is difficult to improve severe depression accompanied by hippocampal neuron damage.

[0006] In summary, the current field of antidepressants faces a triple technical barrier: drug side effects, unclear TCM mechanisms, and fragmented research on the homology of medicine and food. There is a need to provide a TCM composition with low side effects, excellent TCM efficacy, and that meets the requirements of the homology of medicine and food. Summary of the Invention

[0007] To address one of the aforementioned technical problems in the prior art, the present invention provides a food-medicine homology composition for the prevention and treatment of depression, and further provides a compound preparation made from this composition. Furthermore, the present invention also provides one method for preparing this compound preparation.

[0008] According to a first aspect of the present invention, a traditional Chinese medicine composition for treating depression is provided, comprising the following traditional Chinese medicinal materials in parts by weight: 3-10 parts of rose, 3-10 parts of peony, 3-10 parts of bitter orange, 0.5-2 parts of dendrobium, and 0.2-1 parts of notoginseng.

[0009] According to an embodiment of the present invention, the traditional Chinese medicine composition comprises the following traditional Chinese medicinal materials in parts by weight: 4-6 parts of rose, 4-6 parts of peony, 4-6 parts of bitter orange, 0.8-1.5 parts of dendrobium, and 0.4-0.6 parts of notoginseng.

[0010] According to one preferred embodiment of the present invention, the traditional Chinese medicine composition comprises the following traditional Chinese medicinal materials in parts by weight: 5 parts rose, 5 parts peony, 5 parts bitter orange, 1 part dendrobium, and 0.5 parts notoginseng.

[0011] According to another preferred embodiment of the present invention, the traditional Chinese medicine composition comprises the following traditional Chinese medicinal materials in parts by weight: 6 parts rose, 6 parts peony flower, 6 parts bitter orange flower, 1.5 parts dendrobium flower, and 0.6 parts notoginseng flower.

[0012] According to another preferred embodiment of the present invention, the traditional Chinese medicine composition comprises the following traditional Chinese medicinal materials in parts by weight: 4 parts rose, 4 parts peony flower, 4 parts bitter orange flower, 0.8 parts dendrobium flower, and 0.4 parts notoginseng flower.

[0013] Rose petals are the dried flower buds of *Rosaceae* (Rosaceae family). They are slightly warm in nature, sweet and slightly bitter in taste, and enter the liver, spleen, and stomach meridians. Their main functions are to soothe the liver and relieve depression, harmonize blood and regulate menstruation, open the orifices and remove blood stasis, and soothe the liver and invigorate the spleen. The *Materia Medica Usage Research* states that they "relieve qi stagnation, alleviate liver depression, promote blood circulation, and regulate the stomach." Rose petals mainly contain volatile oils, flavonoids, phenolic acids, and polysaccharides. Bitter orange blossom, also known as bitter orange flower or sour orange flower, is the dried flower stamen of *Citrus aurantium* L. var. *amara Engl.*, belonging to the Rutaceae family. First mentioned in the *Kaibao Materia Medica*, it is widely distributed in my country. It is slightly cold in nature, fragrant, and slightly bitter in taste. It has the functions of soothing the liver and regulating qi, relieving depression and calming the mind, regulating qi and resolving phlegm, and promoting digestion and harmonizing the stomach. Clinically, it is suitable for symptoms such as chest and abdominal distension and pain, indigestion, and phlegm retention. The main components of bitter orange blossom are rich in volatile oils, as well as flavonoids and alkaloids such as naringin and hesperidin, which have various pharmacological effects, including antioxidant, anti-inflammatory, and anti-tumor properties. Panax notoginseng flower is the dried product of the unopened inflorescences of Panax notoginseng (Burk.) FHChen, a plant of the Araliaceae family that has grown for more than two years. Panax notoginseng is a perennial herb, sweet and cool in nature, primarily used for clearing heat and detoxifying, calming the liver and improving eyesight, promoting body fluid production and quenching thirst, and lowering blood pressure. Clinically, it is mainly used to treat dizziness, tinnitus, insomnia, and hypertension, and has sedative and anti-inflammatory effects. It mainly contains saponins, flavonoids, and polysaccharides as active ingredients. Peony flower is the flower of Paeonia suffruticosa Andr., a plant of the Ranunculaceae family. It has a bitter and bland taste and is neutral in nature, and can be used for women's irregular menstruation and menstrual cramps. Peony flowers contain a rich variety of active ingredients, including proteins and amino acids, vitamins, flavonoids, volatile oils, and various trace elements. Studies have shown that the flavonoids in peony flowers have anti-inflammatory, anti-cancer, cardiovascular protective, and neurodegenerative disease prevention effects. Dendrobium flowers are the dried flowers of the orchid *Dendrobium officinale* (*Dendrobium officinale* Kimura et Migo). Recorded in books such as the *Shennong Bencao Jing*, they can promote gastric juice secretion, aid digestion, enhance metabolism, nourish yin and clear heat, and benefit the stomach and promote the production of body fluids. They are slightly cold in nature and sweet in taste, primarily possessing the effects of nourishing yin and clearing heat, and benefiting the stomach and promoting the production of body fluids. Dendrobium flowers mainly contain polysaccharides, flavonoids, terpenes, esters, and other effective components, and the total phenols and total flavonoids in *Dendrobium officinale* flowers are higher than those in the *Dendrobium officinale* stem. The alkaloids contained in Dendrobium flowers have anti-inflammatory effects, inhibiting the release of inflammatory factors and reducing inflammatory responses.

[0014] According to traditional Chinese medicine theory, depression is classified as "depressive disorder". Historically, doctors have associated emotional distress and qi stagnation with depression. Traditional Chinese medicine believes that the occurrence and development of depression are related to the disorder of zang-fu organ functions and the imbalance of qi and blood. Therefore, the main treatment principle is to regulate the functions of zang-fu organs and soothe the liver to relieve depressive symptoms. By summarizing the clinical application experience and theoretical basis of traditional Chinese medicine in treating depression, the inventor screened out five herbal medicines, namely rose flower, bitter orange flower, pseudo-ginseng flower, dendrobium flower and peony flower, from food and medicine substances to form a traditional Chinese medicine composition.

[0015] According to the second aspect of the present invention, there is provided a traditional Chinese medicine composition preparation, which comprises the above-mentioned traditional Chinese medicine composition and pharmaceutically acceptable excipients. <​​​​​​​​​​​​​​​​​​​​​A fourth aspect of this invention provides the use of these traditional Chinese medicine compositions and related preparations in the preparation of health foods that assist in improving memory. Memory loss is a common symptom in patients with depression or anxiety. Given that the compositions provided by this invention have a clear antidepressant effect, they can also be used for memory decline caused by mood problems.

[0023] Beneficial effects This application is the first to use a traditional Chinese medicine composition determined based on the theory of food and medicine sharing the same origin, containing anti-inflammatory and antioxidant substances such as flavonoids, alkaloids, fatty acids, and other secondary metabolites. These bioactive substances work synergistically to improve depressive symptoms. The traditional Chinese medicine composition provided in this application can significantly improve depressive symptoms: it can effectively alleviate weight loss, reduce anhedonia, alleviate despair, and lower anxiety levels, and has a significant protective effect on damaged neurons. Comprehensive evidence demonstrates that the intervention effect of the aforementioned traditional Chinese medicine composition can effectively improve depressive symptoms. Attached Figure Description

[0024] Figure 1 The TIC peak diagram is shown, displaying the characteristic ion peaks of the sample under different modes, where... Figure 1 A is the cation mode base peak diagram. Figure 1 B is the base peak diagram in anion mode.

[0025] Figure 2 The effect of the aforementioned food-medicine homology composition for preventing and treating depression on food intake in rats was shown: compared with the normal group, P<0.05, P<0.01, n≥6.

[0026] Figure 3 The effect of the aforementioned food-medicine homology composition for the prevention and treatment of depression on rat body weight is shown. Figure 3 In the diagram, A represents changes in body weight, and B represents the trend of those changes. Compared to the normal group, P<0.05, P<0.001, compared with the model group, #P<0.05, ###P<0.001, n=10.

[0027] Figure 4 The effect of the aforementioned food-medicine homology composition for preventing and treating depression on sucrose preference rate in rats was shown: compared with the normal group, P<0.05, P<0.001, compared with the model group, #P<0.05,###P<0.001, n=10.

[0028] Figure 5 The effect of the aforementioned food-medicine homology composition for preventing and treating depression on immobility time in the forced swimming test in rats was shown: compared with the normal group, P<0.05, P < 0.001, compared with the model group, # P < 0.05, n = 8.

[0029] Figure 6 The effects of the aforementioned food-medicine homology composition for the prevention and treatment of depression on the behavior of rats in an open field experiment are shown. Figure 6 A represents the number of times the pattern is worn. Figure 6 B represents the number of times the person stood up. Compared to the normal group, P<0.05, P < 0.001, compared with the model group, #P < 0.05, ###P < 0.001, n = 6.

[0030] Figure 7 The effects of the aforementioned food-medicine homology composition for the prevention and treatment of depression on the morphology of rat hippocampal cells are shown.

[0031] Figure 8 The effects of the aforementioned food-medicine homology composition for the prevention and treatment of depression on serum indicators in rats were shown. Figure 8 A represents serum 5-HT. Figure 8 B represents serum dopamine (DA) levels. Compared to the normal group, P<0.05, P<0.01, P < 0.001, compared with the model group, #P < 0.05, ##P < 0.01, ###P < 0.001, n = 5. Detailed Implementation

[0032] This application provides a traditional Chinese medicine composition comprising rose, peony, bitter orange, dendrobium, and notoginseng.

[0033] In some embodiments, the traditional Chinese medicine composition can be prepared into preparations, including but not limited to dried flower tea, decoction, paste, extract, pill, powder, granule, tablet and capsule.

[0034] Pharmacological analysis revealed that the traditional Chinese medicine composition described in this application contains anti-inflammatory and antioxidant substances such as flavonoids, alkaloids, fatty acids, and other secondary metabolites. These bioactive substances work synergistically to improve depressive symptoms.

[0035] This application used a CUMS rat model to demonstrate the antidepressant effect of a traditional Chinese medicine composition and evaluated its efficacy through intergroup comparisons. Compared to the control group, CUMS rats exhibited significant depressive characteristics, including weight loss, decreased interest, reduced curiosity about new things, and hopeless behavior. Rats treated with the traditional Chinese medicine composition showed significant improvement in these behavioral characteristics, specifically reduced weight loss, alleviated anhedonia, improved hopelessness, and reduced anxiety levels. These results indicate that the traditional Chinese medicine composition has a therapeutic effect on depressed rats. Histomorphological observations further revealed the potential mechanism of action of the traditional Chinese medicine composition provided in this application. In the hippocampus of CUMS rats, the traditional Chinese medicine composition showed a protective effect on nerve cells, demonstrating that it may improve hippocampal neural function by inhibiting nerve cell damage and promoting neuronal repair and regeneration. The traditional Chinese medicine composition may also promote neuronal remodeling, which is of great significance for restoring normal neural network function and improving depressive symptoms. Serum analysis showed that the levels of serotonin (5-HT) and dopamine (DA) in the model group rats were significantly reduced, and the levels of related factors in the serum of the traditional Chinese medicine composition group were also significantly improved. Serotonin, a monoamine neurotransmitter, plays a role in the central and peripheral nervous systems, participating in many physiological and behavioral processes, including mood, sleep, activity, suicide, and sexual behavior. Related studies have shown that serotonin levels are significantly reduced in the brains of patients with mood disorders and schizophrenia. Depression is associated with a reduction in central adrenergic receptor sites, particularly an absolute or relative decrease in norepinephrine (NE) and dopamine (DA).

[0036] Compared with the normal control group, the rats in the traditional Chinese medicine composition group experienced a decrease in body weight, but the degree of weight loss was significantly less than that in the CUMS model group, indicating that the traditional Chinese medicine composition provided in this application can effectively alleviate the weight loss phenomenon in the depressed rat model. Regarding food intake, the rats in the traditional Chinese medicine composition group showed a significant increase in food intake compared with the model group, further confirming the positive effect of the traditional Chinese medicine composition in improving depression-related behaviors and physiological indicators. Furthermore, based on the rats' growth and daily indicators, the dosage of the traditional Chinese medicine composition used did not induce significant toxic side effects, demonstrating good safety and tolerability.

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0038] definition Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0039] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0040] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0041] As used herein, the term "pharmaceuticalally acceptable excipient" refers to an excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, and is well known in the art. Examples include one or more of the following: carriers, excipients, adjuvants, fillers, diluents, disintegrants, lubricants, flow aids, binders, solubilizers, surfactants, emulsifiers, preservatives, antioxidants, flavoring agents, colorants, osmotic pressure regulators, and support agents.

[0042] The following examples are provided to aid in understanding the invention. However, it should be understood that these examples are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the invention. The reagents and / or kits used in the following examples are commercially available or can be synthesized by known methods.

[0043] It should be noted that, unless specific conditions are specified in the examples, experimental conditions should be performed according to standard conditions, manufacturer recommendations, or publicly reported experimental conditions. Reagents or instruments whose manufacturers are not specified are all commercially available, standard products. For reagents whose manufacturers are specified, similar products from other manufacturers are substitutes.

[0044] Preparation Example 1 Weigh 5g of rose petals, 5g of peony petals, 5g of bitter orange blossom, 1g of dendrobium flower, and 0.5g of Panax notoginseng flower. Add 12 times the amount of water and soak twice at 80℃ for 0.5 hours each time. Then filter and combine all the filtrates, keeping the total filtrate for later use. Concentrate the filtrate under reduced pressure at -0.06 to -0.10 MPa and 50 to 70℃ to obtain a fluid extract with a relative density of 1.00 to 1.04 (measured at 60±5℃). After high-speed centrifugation, collect the centrifuged liquid. The yield of the extract is 19.95%. Dry to obtain a compound dried powder.

[0045] Without preservatives, it should be stored in a refrigerator at 4°C.

[0046] Preparation Example 2 Take 10 kg of rose petals, 8 kg of peony petals, and 7 kg of bitter orange blossoms. Add 10 times the amount of water and distill for 3 hours, collecting 0.2 kg of volatile oil. Prepare an inclusion complex with 0.3 kg of β-cyclodextrin, and retain the residue. Mix the residue with 1.5 kg of dendrobium flowers and 0.8 kg of notoginseng flowers, add 8 times the amount of water, and decoct twice (2 hours each time). Combine the decoctions, filter, and concentrate to a clear extract with a relative density of 1.15 (60℃). Dry the residue and pulverize it into an 80-mesh fine powder. Mix the clear extract with the pulverized residue powder, 12 kg of pregelatinized starch, and 2 kg of crospovidone. Add 5% hydroxypropyl methylcellulose solution to prepare a soft mass, granulate through a 16-mesh sieve, dry at 60℃ until the moisture content is ≤5%, and granulate through a 14-mesh sieve. After granulation, add 0.6 kg of magnesium stearate, 0.5 kg of the remaining crospovidone, and 0.5 kg of the volatile oil inclusion complex, mix well, and compress into 0.4 g / tablets (approximately 110,000 tablets).

[0047] Preparation Example 3 5 kg of rose petals, 4 kg of peony petals, 10 kg of bitter orange blossoms, 2 kg of dendrobium flowers, and 0.5 kg of Panax notoginseng flowers (total 21.5 kg) were decocted three times with 10 times the amount of water, each time for 1.5 hours. The decoctions were combined, filtered, and concentrated under reduced pressure to a clear extract (approximately 18 kg) with a relative density of 1.30 (50°C). The clear extract was mixed with 15 kg of maltodextrin and 0.1 kg of aspartame, and 50% ethanol was added to adjust the moisture content of the soft material. Granulation was performed using an 18-mesh sieve. The mixture was dried at 45°C until the moisture content was ≤6%, and then granulated using a 16-mesh sieve to obtain approximately 55 kg of granules.

[0048] Preparation Example 4 3 kg of rose petals, 5 kg of peony petals, 6 kg of bitter orange blossoms, 2 kg of dendrobium flowers, and 1 kg of Panax notoginseng flowers (total 17 kg) are soaked in 12 times the amount of water for 2 hours, then decocted three times (2.5 hours the first time, and 2 hours each for the second and third times). The decoctions are combined, filtered, and allowed to stand for 48 hours (to allow impurities to settle). The supernatant is then concentrated to a thick paste with a relative density of 1.45 (60℃) (approximately 16 kg). The paste is heated to 90℃, and 30 kg of refined honey (refined to a relative density of 1.38) is added. The mixture is stirred until it reaches a "flag-hanging" consistency, then 0.08 kg of potassium sorbate is added, stirred well, and then packaged (150 g per bottle).

[0049] Preparation Example 5 4 kg of rose petals, 3 kg of peony petals, 5 kg of bitter orange blossoms, 0.5 kg of dendrobium flowers, and 0.3 kg of notoginseng flowers (total 12.8 kg). After cleaning, grind the rose petals, bitter orange blossoms, dendrobium flowers, and notoginseng flowers separately into a 16-mesh coarse powder. Grind the peony flowers separately and sift them. First, mix 0.3 kg of notoginseng flowers with 0.5 kg of dendrobium flowers, then gradually add the other flowers. Use an automatic packaging machine to package the mixture into 3g tea bags (approximately 4260 bags in total). Suitable for frequent daily brewing (1-2 bags per day).

[0050] Preparation Example 6 6 kg of rose petals, 3 kg of peony petals, 7 kg of bitter orange blossoms, 1 kg of dendrobium flowers, and 0.7 kg of Panax notoginseng flowers (total 17.7 kg) were decocted twice (1.2 hours each time) with 7 times the amount of water. The decoctions were combined, filtered, and concentrated to 40 L. Ethanol was added to bring the alcohol content to 70%. The mixture was allowed to stand for 36 hours, and the supernatant was collected to recover the ethanol until no alcohol odor remained, yielding approximately 25 L of concentrated liquid. 0.05 kg of steviol glycosides and 0.15 kg of sodium benzoate were added to the concentrated liquid, stirred to dissolve, and purified water was added to 150 L. The mixture was then filtered through a 0.22 μm filter membrane, dispensed into 10 ml bottles, and sterilized at 100 °C for 30 minutes.

[0051] Experimental Example 1: Analysis of Active Ingredients S1. Drug treatment: The components of the food-medicine homology composition for the prevention and treatment of depression obtained in Example 1 were analyzed by LC-MS / MS. The specific method is as follows: (1) Accurately weigh 50 mg of the compound powder from Preparation Example 1 and place it in a 2 mL EP tube. Add 2 homogenizing beads to each EP tube. Then add 500 μL of isotope-labeled extraction solution to each EP tube in a ratio of methanol:acetonitrile:water = 2:2:1. After adding all reagents, vortex the EP tubes for 30 seconds to ensure that the drug and extraction solution are fully mixed.

[0052] (2) Place the above EP tube into a homogenizer and homogenize it at a frequency of 35 Hz for 240 seconds. After homogenization, transfer the EP tube to ice water for ultrasonic treatment for 5 minutes each time, repeating 3 times. After ultrasonic treatment, place the EP tube in a -40℃ refrigerator for 30 minutes to promote precipitation.

[0053] (3) Take out the EP tube after it has been standing, place it in a centrifuge, and centrifuge at 12000 rpm for 15 minutes at 4°C to separate the supernatant and precipitate. Carefully aspirate the supernatant and place it in a -40°C freezer for 10 minutes. Then, place the EP tube back in the centrifuge and centrifuge again at 12000 rpm for 15 minutes at 4°C to further separate impurities and ensure the purity of the supernatant.

[0054] (4) Take the supernatant from the centrifuged EP tube and filter it with a 0.22μm microporous membrane to remove any possible small particles. Carefully transfer the filtered liquid to a vial and then perform instrumental analysis to determine the content and characteristics of the drug components.

[0055] S2, On-machine testing The target compound was detected using an ultra-high performance liquid chromatograph (Vanquish, Thermo Fisher Scientific) with a Phenomenex Kinetex C18 liquid chromatography column (2.1 mm × 100 mm, 2.6 μm).

[0056] The chromatographic settings are as follows: Phase A of the liquid chromatography is an aqueous solution containing 0.01% acetic acid, and Phase B is a mixed solvent of isopropanol and acetonitrile (volume ratio 1:1); during chromatographic analysis, the sample pan temperature is set to 4℃, and the injection volume is 2μL.

[0057] The OrbitrapExploris 120 mass spectrometer, controlled by Xcalibur software (Thermo, version 4.4), completed primary and secondary mass spectrometry data acquisition. Detailed mass spectrometry analysis parameters were set as follows: sheath gas flow rate was set to 50 Arb, auxiliary gas flow rate to 15 Arb, capillary temperature to 320℃, full scan mass spectrometry and secondary mass spectrometry resolutions to 60,000 and 15,000 respectively, collision energy to SNCE20 / 30 / 40, and spray voltage to 3.8 kV in positive ion mode or -3.4 kV in negative ion mode.

[0058] S3. Results Analysis The raw data was converted to mzXML format using ProteoWizard software to ensure data format compatibility and standardization. Metabolites were identified using collaboratively developed R packages, with the databases TCM (V1.0) and BT-HERB (V1.0) used in the identification process. After metabolite identification, the results were visualized using a self-developed R package to intuitively display the distribution and characteristics of the metabolites, providing a basis for subsequent qualitative analysis.

[0059] Experimental results (1) TIC (Total Ion Chromatography) peak chromatogram To comprehensively analyze the mass spectrometry characteristics of the samples, a detailed analysis of the mass spectra at each time point was performed in both positive and negative ion modes. The ion intensities of the strongest signals in the mass spectra at each time point were continuously labeled to ensure the capture of the most representative signals. These signals were then ranked according to their scores, and the top 10 substances were selected. These peak charts illustrate the characteristic ion peaks of the samples in different modes. The relevant results are as follows: Figure 1 as shown

[0060] (2)Qualitative analysis By using the above software to analyze the experimental results and comparing them in detail with the relevant data in the database, the main component composition of Wuhua Yin was determined. The specific results are shown in Table 1

[0061] Table 1 Ion mode TIC peak list

[0062] Based on the results shown in Table 1, the main bioactive substances contained in the traditional Chinese medicine composition of food and medicine homology for preventing and treating depression are flavonoids, alkaloids, fatty acids and other secondary metabolites.国内外研究已证实这些物质的抗炎和抗氧化特性。作为一种多酚类化学物质,黄酮类化合物具有多种生物活性,它们可以通过调节神经递质水平、激活脑源性神经营养因子信号通路,促进神经可塑性以及发挥抗炎和抗氧化作用,从而实现抗抑郁效果。例如,所述中药组合物中含有的橙皮苷能够通过调节5-HT水平来发挥其抗抑郁特性。此外,生物碱类物质可以通过调节单胺类神经递质水平(如5-HT和NE)来改善情绪低落的情况。脂肪酸类物质则可通过促进神经细胞的生长和修复、减少神经炎症以及调节炎症反应来发挥其抗抑郁作用。这些生物活性物质的协同作用,使得组合物在改善抑郁症状方面展现出显著的潜力。The anti-inflammatory and antioxidant properties of these substances have been confirmed by domestic and foreign research. As a polyphenolic chemical substance, flavonoids have a variety of biological activities. They can achieve antidepressant effects by regulating neurotransmitter levels, activating the brain-derived neurotrophic factor signaling pathway, promoting neural plasticity, and exerting anti-inflammatory and antioxidant effects. For example, hesperidin contained in the traditional Chinese medicine composition can exert its antidepressant properties by regulating the 5-HT level. In addition, alkaloid substances can improve the situation of low mood by regulating the levels of monoamine neurotransmitters (such as 5-HT and NE). Fatty acid substances can exert their antidepressant effects by promoting the growth and repair of nerve cells, reducing neuroinflammation, and regulating the inflammatory response. The synergistic effect of these bioactive substances makes the composition show significant potential in improving depressive symptoms

[0063] Experimental Example 2. Pharmacodynamic test of the traditional Chinese medicine composition of food and medicine homology for preventing and treating depression on CUMS depressive rats The WKY rats and the CUMS modeling method were selected to evaluate the pharmacodynamics of the traditional Chinese medicine composition from multiple aspects

[0064] 1. Experimental materials 1.1. Animals All animals were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., including 40 clean-grade, healthy male 6-week-old WKY rats with the same genetic background (license number: SCXK[Beijing]2021-0006, Beijing, China). The rats were housed in separate cages, and the whole experiment was carried out at the SPF-level animal center of Beijing University of Chinese Medicine (ethical batch number: BUCM-2024022602-1080, Beijing, China) under the environment of 12-hour day-night cycle (8:00-20:00), with the temperature of 22±2°C and the relative humidity of 50%-60%. They had free access to drinking water (purified water) and food

[0065] 1.2. Drugs The compound powder prepared in Preparation Example 1 was used to conduct an intervention efficacy test on CUMS depressed rats; the positive control drug was fluoxetine hydrochloride capsules (manufacturer: Pathoon France; repackaging plant: Eli Lilly Suzhou Pharmaceutical Co., Ltd.; batch number: HJ20181215, Suzhou, China).

[0066] 2. Experimental Methods 2.1 Preparation of Traditional Chinese Medicine Compositions The compound powder and fluoxetine hydrochloride powder prepared in Example 1 were dissolved separately in physiological saline and adjusted to the predetermined dosage concentration. After the drugs were completely dissolved, the solution was vortexed for 5 minutes. The mixed solution was then placed in an ultrasonic instrument and sonicated at 50 Hz at 40°C for 30 minutes to further promote drug dissolution. After treatment, the solution was transferred to a refrigerator at 4°C for refrigeration to maintain the stability and activity of the drugs. Before each daily gavage procedure, the refrigerated drug solution was taken out and preheated in a water bath at 37°C for half an hour to ensure it reached the appropriate temperature.

[0067] 2.2 Animal modeling and grouping Before the experiment, all rats underwent a seven-day acclimatization period to adapt to the experimental environment. After the initial weight measurement and sucrose preference test, all rats were randomly assigned to four groups using a random number table: control group, model group, fluoxetine group, and traditional Chinese medicine composition (compound powder) group, with 10 rats in each group. Except for the control group, the rats in the other groups received a chronic unpredictable mild stress (CUMS) daily, including restraint, solitary confinement, one-minute tail clamping, swimming in 4°C water for 5 minutes, 24-hour continuous light exposure, 24-hour fasting, or 24-hour water deprivation. To ensure the unpredictability of the stress, these stressors were randomly rearranged each week during the five-week experiment.

[0068] 2.3 Administration method and dosage Gavage administration began in the first week. The control group (1 mL / 100 g / day of normal saline), the model group (1 mL / 100 g / day of normal saline), the fluoxetine group (10 mg / kg / day of fluoxetine), and the traditional Chinese medicine composition group (0.38 mg / g / day of the traditional Chinese medicine composition prepared in step 2.1 above) were all administered with normal saline 1 hour before modeling each day.

[0069] 2.4 Routine Indicator Testing During the experiment, the daily condition of the rats was observed, with a focus on indicators such as their mental state, level of excitement, and coat luster to assess their overall health. The rats' weight (once a week) and food intake (three times a week) were recorded regularly to monitor their growth and development.

[0070] 2.5 Behavioral Testing The behavioral tests were scheduled as follows: the sucrose preference test was conducted on day 0 (before the start of the experiment) and day 28 (at the end of the experiment) to assess the rats' preference for sucrose solution; the forced swimming test was conducted on day 31 to assess the rats' despair by measuring the time they remained still in the water; and the open field test was conducted on day 34 to observe the rats' activities in the open field, including horizontal movement distance and vertical movement frequency, to assess their anxiety and depression-like behaviors.

[0071] 2.5.1 Sucrose Preference Experiment Before conducting the sucrose preference test (SPT), all rats underwent two days of acclimatization training: On the first day, two bottles of 1% sucrose solution were prepared, which the rats could freely consume for 24 hours. On the second day, one bottle was replaced with ordinary drinking water for the rats' daily consumption. After a 12-hour period of fasting and water restriction, the formal sucrose preference test began. In each cage, one bottle of ordinary drinking water and one bottle of 1% sucrose solution were placed simultaneously, with both liquids having the same initial volume, allowing the rats to choose freely. To prevent the rats from influencing the results due to positional preferences, the positions of the two bottles were switched every 2 hours. After 8 hours, both bottles were removed, and their consumption was recorded.

[0072] The sucrose preference rate is calculated by determining the proportion of sucrose-containing water consumed to total fluid consumption. This indicator reflects the rats' preference for sucrose-containing water and serves as an assessment of the rats' interest level.

[0073] Sucrose preference rate (%) = Sucrose water consumption + Drinking water consumption × 100% 2.5.2 Forced Swimming Experiment The day before the formal Forced Swimming Test (FST), all rats were placed sequentially in a cylindrical container filled with water for 15 minutes of acclimatization training. After training, the rats were removed from the water and dried with a towel. On the day of the formal experiment, the rats were placed in the same experimental setup for a 5-minute test. After the initial 2-minute acclimatization, the behavior of each rat was recorded for the following 5 minutes, with particular attention paid to immobility time to assess the degree of behavioral despair. Immobility was defined as minimal movement to maintain head above water, remain afloat, or make slight turning movements when the animal ceased struggling and attempting to escape. This metric measures the negative coping behaviors exhibited by rats in the face of an inescapable situation and is used to assess the severity of depressive-like behavior.

[0074] 2.5.3 Open Field Experiment In the Open Field Test (OFT), a self-made black open field box (80×80×80cm) was used. The day before the actual experiment, each rat was placed in the open field box for 3 minutes of acclimatization training. On the day of the actual experiment, the number of times the rat stood up within 5 minutes (including jumping or climbing the side walls of the open field box) and the frequency of crossing the grid at the bottom of the open field box were recorded (a crossing was defined as when three of the rat's paws entered another empty grid). After each rat's experiment, the urine and feces in the open field box were cleaned and disinfected with 75% medical alcohol. The experiment continued after the alcohol had evaporated, and the entire process was conducted in a quiet environment to ensure consistency.

[0075] 2.6 Sample Preparation Thirty-five days after drug administration, all rats were fasted for 12 hours but allowed free access to water. They were then anesthetized by intraperitoneal injection of 10% sodium pentobarbital (0.4 mL / 100 g). After confirming anesthesia, blood was drawn from the abdominal aorta of five rats in each group. Immediately after death, brain tissue was removed, and the prefrontal cortex, hippocampus, and liver were separated. All procedures were performed on an ice tray. The extracted tissues were placed in cryovials. The brains of the remaining five rats were fixed by perfusion with 4% paraformaldehyde and placed in fixative. The collected blood was allowed to stand at room temperature for 3 hours, followed by further centrifugation at 4°C (3000 rpm, 10 minutes). The resulting serum was aliquoted and stored at -20°C for later use.

[0076] 2.7. Use enzyme-linked immunosorbent assay (ELISA) to detect the levels of serotonin (5-HT) and dopamine (DA) in rat serum according to the instructions in each kit.

[0077] 2.8. Tissue sectioning and staining: Paraffin sections of the whole rat brain were prepared, stained with H&E (hematoxylin and eosin), and Nissl staining was performed.

[0078] 2.9 Statistical Analysis In this study, data analysis was performed using IBM SPSS Statistics 23.0. For the description of continuous data, the statistical expression mean ± standard deviation (mean ± SD) was used to visually represent the central tendency and dispersion of the data. Hypothesis testing results provided statistics and their corresponding p-values, with p < 0.05 used as the criterion for statistical significance (compared to the normal group). P<0.05, P<0.01, P < 0.001, compared with the model group, #P < 0.05, ##P < 0.01, ###P < 0.001. Nonparametric tests were used for non-normally distributed data, and one-way ANOVA was used for normally distributed data. Within-group comparisons were performed using repeated measures ANOVA with paired t-tests. Comparisons between groups were performed using one-way ANOVA: when data met homogeneity of variance, Fisher's Least Significant Difference Test (LSD test) was used for pairwise comparisons; when data did not meet homogeneity of variance, Dunnett's T3 post-hoc test (Dunnett's T3 test) was used for pairwise comparisons.

[0079] 3. Experimental Results 3.1 Effects of the compound powder on the daily condition and food intake of rats Before modeling and drug administration, there were no significant differences among the groups of rats. With subsequent continuous, chronic, unpredictable, and mild stimulation, the rats in the model group gradually became lethargic, their fur became dry, their daily activity decreased, their sleep time increased, and their food intake also decreased. Similar conditions were observed in the fluoxetine group and the traditional Chinese medicine combination group, but these were improvements compared to the model group.

[0080] One week later, the rats' food intake was measured, and the results were as follows: Figure 2 As shown in the figure, starting from the third week, the food intake of rats treated with CUMS was significantly reduced compared to the control group. By the fourth week, there was a statistically significant difference in food intake between the model group and the normal control group (P<0.01), indicating that CUMS treatment had a significant inhibitory effect on the feeding behavior of rats. Meanwhile, compared to the model group, the feeding behavior of rats in both the fluoxetine group and the traditional Chinese medicine combination group was improved (P<0.05).

[0081] 3.2 Effect of compound powder on rat body weight Before the experiment, the body weight of rats in each group was statistically analyzed, and the results showed no significant difference between the groups. During the experiment, the body weight of rats in the control group showed a stable increasing trend, and no abnormal conditions occurred throughout the experiment. After 5 weeks of CUMS modeling and corresponding drug intervention, the body weight of rats in the model group was significantly lower than that in the control group, and the difference was statistically significant (P<0.001). This result indicates that CUMS treatment successfully induced weight loss in rats, simulating one of the typical physiological characteristics of a depression model. At the same time, compared with the model group, the body weight of rats in the traditional Chinese medicine composition group and the fluoxetine group was improved, showing the alleviating effect of drug intervention on weight loss. The body weight of rats in the traditional Chinese medicine composition group was significantly different from that in the model group (P<0.05), suggesting that the compound powder has a positive effect on improving CUMS-induced weight loss.

[0082] During the 5-week modeling period, the body weight of rats in all four groups increased normally in the first 3 weeks. After 3 weeks, except for the control group, the body weight of all rats decreased to varying degrees. The model group continued to lose weight during subsequent feeding, and the difference between the model group and the control group was statistically significant at week 4 (P=0.026). At week 5, the body weight decreased significantly (P<0.001). Compared with the model group, the fluoxetine group showed the most significant improvement in body weight loss at week 5, and the traditional Chinese medicine combination group also showed some improvement. The results indicate that the compound powder improved the body weight loss in CUMS rats. The changes in body weight before and after treatment and the trend of body weight change within 5 weeks are shown below. Figure 3 As shown in Table 2, detailed weekly weight changes are presented.

[0083] Table 2. Effects of compound powder on body weight in rats over 5 weeks ( ±SD, n=10)

[0084] Note: Compared with the normal group P<0.05, P<0.01, P<0.001, compared with the model group, #P<0.05,###P<0.001.

[0085] 3.3 Effects of Traditional Chinese Medicine Combinations on Behavioral Outcomes in Rats 3.3.1 Sucrose Preference Test Before the experiment officially began, all rats underwent a sucrose preference test to assess their preference for sucrose solution. The results showed no significant difference in sucrose preference rates among the groups initially. After five weeks of CUMS model construction and corresponding drug intervention, the sucrose preference rate in the model group was significantly lower than that in the control group (P<0.001). This result indicates that CUMS treatment successfully induced anxieties in rats, consistent with typical characteristics of a depression model. Compared with the model group, the sucrose preference rate in each drug-treated group was significantly increased (P<0.001), further confirming the significant ameliorative effect of the compound powder on CUMS-induced anxieties and demonstrating its role in alleviating depressive-like behavior. The changes in sucrose preference rates before and after the experiment are shown below. Figure 4 As shown.

[0086] 3.3.2 Forced Swimming Experiment In the forced swimming experiment, after 5 weeks of CUMS treatment, the immobility time of rats in the model group was significantly increased, showing a highly significant difference compared with the control group (P<0.001), indicating that CUMS treatment successfully induced a state of despair in the rats, leading to a significant decrease in the survival drive of the rats in the model group. Compared with the model group, the immobility time of rats in the fluoxetine group was significantly reduced (P=0.034), and the immobility time of rats in the traditional Chinese medicine composition group was also significantly reduced compared with the model group (P=0.029), indicating that the compound powder can significantly improve the despair behavior of CUMS rats and enhance their survival drive. In conclusion, the compound powder can alleviate the despair state of depressed rats to some extent. The specific results of the forced swimming experiment are as follows: Figure 5 As shown.

[0087] 3.3.3 Open Field Experiment The open field test reflects an animal's exploratory drive and curiosity about new things. In this experiment, compared with the control group, the model group rats showed a significant decrease in the number of square crossings and standing time (P<0.001), indicating a lack of interest in novelty, consistent with a depressive state. The fluoxetine group showed a significant improvement in the number of standing and square crossings compared to the model group (P<0.001); the traditional Chinese medicine composition group showed a significant increase in the number of square crossings (P<0.001), and the fluoxetine group also showed a significant increase in standing time (P<0.001). The traditional Chinese medicine composition group also showed some improvement compared to the model group, but the differences were not statistically significant. The results of the open field test are as follows: Figure 6 As shown in Table 3, the results of all the above behavioral experiments are presented.

[0088] Table 3. Results of the behavioral experiment ( ±SD)

[0089] Note: Compared with the normal group P<0.05, P < 0.001, compared with the model group, #P < 0.05, ###P < 0.001.

[0090] 3.4 Effects of Traditional Chinese Medicine Composition on the Morphology of Rats' Hippocampus In this study, rat thalamic tissue was fixed, then embedded in paraffin and sectioned. After sectioning, H&E staining and Nissl staining were performed, and the staining results are shown below. Figure 7 As shown.

[0091] H&E and Nissl staining results showed that in the control group rats, the hippocampal cells were neatly arranged with normal nuclei, intact neuronal cell membranes, abundant Nissl bodies, and rich cellular layers. Compared to normal rats, the model group rats showed a reduced number of hippocampal cell layers, unclear structure, neuronal atrophy, cell membrane rupture, and many neurons exhibiting vacuolization and shrinkage, with some cells detaching to form vacuoles, and a reduction in Nissl bodies. Compared to the model group rats, the fluoxetine group and the traditional Chinese medicine composition group rats still showed some cell atrophy, smaller nuclei, and vacuolization and shrinkage of neurons in the hippocampus, but the cells were more neatly arranged, with richer cellular layers and relatively intact neuronal cell membranes, demonstrating that the traditional Chinese medicine composition can protect and repair damaged nerve cells.

[0092] 3.5 Effects of Traditional Chinese Medicine Combinations on Serum 5-HT and DA Levels in Rats Serum ELISA analysis revealed changes in the levels of neurotransmitters 5-HT and DA in the serum of rats in each group: the serum 5-HT and DA levels in the model group were significantly lower than those in the control group (P<0.001). Further analysis showed that the serum 5-HT level in the fluoxetine group was significantly higher than that in the model group (P=0.003), indicating that fluoxetine can effectively regulate 5-HT levels. Meanwhile, the serum 5-HT level in the traditional Chinese medicine composition group also showed a significant improvement, with a statistically significant difference compared to the model group (P=0.015), suggesting that the traditional Chinese medicine composition has a certain effect on regulating 5-HT levels. Regarding serum DA levels, the serum DA content in the model group was significantly lower than that in the control group (P=0.001), further confirming the inhibitory effect of CUMS on neurotransmitters. The DA level in the traditional Chinese medicine composition group was significantly higher than that in the model group (P=0.002), indicating that the traditional Chinese medicine composition can improve the reduction of DA in CUMS rats, thereby alleviating depressive-like behavior to some extent.

[0093] The traditional Chinese medicine composition effectively regulated the serum levels of 5-HT and DA in CUMS rats, demonstrating its effect in improving depression-related neurotransmitter disorders. Specific data are as follows: Figure 8 As shown in Table 4.

[0094] Table 4. Data on serum 5-HT and DA levels in rats ( ±SD, n=5)

[0095] Note: Compared with the normal group P<0.01, P < 0.001, compared with the model group, #P < 0.05, ##P < 0.01.

[0096] In summary, this study successfully established a rat depression model using the CUMS method and systematically evaluated the antidepressant effects of the traditional Chinese medicine composition from three aspects: behavioral manifestations, histological characteristics, and biochemical indicators. Behavioral tests assessed the depressive-like behavioral changes in rats, histological observations analyzed the pathological changes in nerve tissue, and biochemical indicator detection explored the effects of the traditional Chinese medicine composition on neurotransmitters and related biomarkers at the molecular level. These comprehensive evaluation results provide a scientific basis for the application of traditional Chinese medicine compositions in the treatment of depression.

[0097] The results showed that no significant toxic side effects were observed in rats treated with the traditional Chinese medicine composition for 5 weeks, proving that this dosage was safe in application. During the first three weeks of modeling, there were no significant differences in baseline conditions among the groups; body weight, food intake, and behavioral indicators showed no statistically significant differences. From the fourth week onwards, CUMS rats clearly exhibited a depressive state, demonstrating the effectiveness of the chronic, unpredictable, and mild stimulation method for modeling. Further analysis after five weeks of modeling and drug administration revealed that the traditional Chinese medicine composition significantly improved weight loss, anhedonia, despair, and anxiety levels in depressed rats. H&E staining and Nissl body staining results also demonstrated the protective effect of the traditional Chinese medicine composition against neuronal damage. ELISA analysis showed that the traditional Chinese medicine composition significantly increased serum 5-HT and DA levels. These results indicate that the traditional Chinese medicine composition, as a novel traditional Chinese medicine compound, has significant antidepressant potential, and its mechanism of action may be related to regulating neurotransmitter levels and protecting neurons.

[0098] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A traditional Chinese medicine composition for treating depression, comprising the following traditional Chinese medicine materials in parts by weight: rose flowers 3-10 parts, peony flowers 3-10 parts, Daidai flowers 3-10 parts, dendrobium flowers 0.5-2 parts, and Sanqi flowers 0.2-1 part.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, The traditional Chinese medicine composition comprises the following traditional Chinese medicine materials in parts by weight: rose flowers 4-6 parts, peony flowers 4-6 parts, Daidai flowers 4-6 parts, dendrobium flowers 0.8-1.5 parts, and Sanqi flowers 0.4-0.6 part.

3. The traditional Chinese medicine composition according to claim 1, characterized in that, The traditional Chinese medicine composition comprises the following traditional Chinese medicine materials in parts by weight: rose flowers 5 parts, peony flowers 5 parts, Daidai flowers 5 parts, dendrobium flowers 1 part, and Sanqi flowers 0.5 part; or rose flowers 6 parts, peony flowers 6 parts, Daidai flowers 6 parts, dendrobium flowers 1.5 parts, and Sanqi flowers 0.6 part; or rose flowers 4 parts, peony flowers 4 parts, Daidai flowers 4 parts, dendrobium flowers 0.8 parts, and Sanqi flowers 0.4 part. 4.A traditional Chinese medicine composition preparation comprising the traditional Chinese medicine composition of any one of claims 1-3 and pharmaceutically acceptable excipients. 5.The traditional Chinese medicine composition preparation of claim 4, which comprises, but is not limited to, dry flower tea, decoction, paste, extract, pill, powder, granule, tablet, and capsule. 6.A method for preparing the traditional Chinese medicine preparation of claim 4 or 5, comprising the following steps: weighing rose flowers 3-10 parts, peony flowers 3-10 parts, Daidai flowers 3-10 parts, dendrobium flowers 0.5-2 parts, and Sanqi flowers 0.2-1 part, adding 8-15 times the amount of water, soaking at 60-90℃ for 2-3 times, each time for 0.5-2 hours, then filtering, combining all the filtrates, and reserving the total filtrate; reducing pressure to concentrate the filtrate to obtain a flow extract.

7. The production method according to claim 6, characterized by, The reducing pressure concentration is performed at-0.06 to-0.10 Mpa and 50-70℃.

8. The preparation method according to claim 6, characterized in that, The relative density of the concentrated product is 1.00-1.04 g / mL (60±5℃).

9. The preparation method according to claim 6, characterized in that, The method further comprises centrifuging the flow extract at high speed, taking the centrifugal liquid, and drying to obtain a powder. 10.The use of the traditional Chinese medicine composition of claims 1-3, the traditional Chinese medicine preparation of claims 4-5, or the product prepared by the method of claims 6-9 in preparing health food for assisting in improving memory.

Images