A traditional Chinese medicine composition for treating insomnia and / or anxiety, an acupoint application and a preparation method thereof

Abstract

The application discloses a traditional Chinese medicine composition for treating insomnia and / or anxiety, acupoint application and a preparation method, and relates to the technical field of traditional Chinese medicines. According to weight parts, the traditional Chinese medicine composition comprises the following components: 1-15 parts of fried jujube seed, 1-10 parts of raw gardenia, 1-5 parts of poria, 1-5 parts of anemarrhena, 1-5 parts of chuanxiong and 1-5 parts of sophora flavescens. The traditional Chinese medicine composition is scientific and reasonable in compatibility, has the effects of soothing liver and purging heat and nourishing blood and tranquilizing, is safe in medication, good in curative effect, can be effectively used for treating insomnia and / or anxiety of the blood deficiency and liver heat type, is a great innovation for the treatment method of insomnia and / or anxiety of the blood deficiency and liver heat type, and will bring remarkable economic and social benefits.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology, and in particular to a traditional Chinese medicine composition, acupoint application, and preparation method for treating insomnia and / or anxiety. Background Technology

[0002] Insomnia is a subjective experience characterized by dissatisfaction with sleep duration and / or quality despite adequate sleep opportunities and environment, impacting daytime social functioning. It is primarily characterized by nighttime insomnia and daytime functional impairment. Symptoms often include difficulty falling asleep, sleep maintenance difficulties, early awakening, and difficulty falling back asleep. Typical daytime symptoms include fatigue, mood swings, general malaise, and cognitive decline, severely affecting the patient's physical and mental health and social functioning. Globally, approximately 27% of the population experiences sleep problems, meaning at least one in four people suffers from insomnia. About 30% of adults experience insomnia each year; if acute insomnia is included, the incidence of primary insomnia reaches as high as 50%. Insomnia is prone to recurrence and chronic persistence; among patients meeting the diagnostic criteria for insomnia, 31%–75% have chronic insomnia, with more than two-thirds of these patients having a disease duration of more than one year. However, the insomnia rate continues to increase year by year. Data from the "2021 White Paper on Exercise and Sleep," released in Beijing on March 18, 2021, by the Chinese Sleep Research Society and other institutions, shows that over 300 million people in China currently suffer from sleep disorders. Therefore, effectively preventing and treating insomnia is urgently needed.

[0003] Anxiety disorder, also known as anxiety neurosis, is the most common type of neurosis. It is characterized primarily by anxious emotional experiences and is divided into two forms: panic attacks and generalized anxiety disorder. The main symptoms include: objectless tension and worry, restlessness, symptoms of autonomic nervous system dysfunction such as palpitations, tremors, sweating, frequent urination, and motor restlessness.

[0004] Anxiety is a risk factor for insomnia, and insomnia significantly increases the risk of developing anxiety. Insomnia, a common sleep disorder, is both a physiological and psychological illness; long-term insomnia can easily lead to mood disorders such as anxiety and depression. Simultaneously, anxiety can induce insomnia or exacerbate its severity, eventually developing into a comorbid state. Insomnia and anxiety are mutually causal, influencing and aggravating each other, creating a vicious cycle. Liver qi stagnation, leading to internal heat, depletes yin and blood, resulting in insufficient blood to nourish the heart and restlessness of the mind—this is the common pathogenesis of both conditions in Traditional Chinese Medicine (TCM). Currently, there is a lack of effective TCM compound formulas for treating blood deficiency and liver heat type insomnia and / or anxiety.

[0005] Currently, the treatment of insomnia and anxiety mainly relies on oral medications. While Western medicine can improve insomnia and alleviate anxiety symptoms to some extent, it has many adverse reactions such as drug dependence, symptom progression, and drug resistance, leading to poor patient adherence and thus affecting clinical efficacy. Although traditional Chinese medicine has fewer adverse reactions, some patients find its taste unpalatable, affecting further treatment. In addition, oral medications have many side effects, and significant drug loss occurs after gastrointestinal absorption, resulting in low efficacy; furthermore, adherence is poor for elderly and young patients. It is under these circumstances that the technique of treating anxiety and insomnia through acupoint application has emerged, attracting great attention from the industry.

[0006] Acupoint application therapy, based on the holistic concept and syndrome differentiation of Traditional Chinese Medicine (TCM), and guided by the theory of meridians, involves applying medicinal patches to specific points on the body surface. This therapy treats diseases through the pathway of the body surface-acupoint-meridian-organ system. TCM acupoint application therapy "cuts into the skin, penetrates deep into the flesh, is absorbed through the breath, and integrates with the exudate." On one hand, the stimulation of acupoints by the medicine adjusts organ function and balances Yin and Yang; on the other hand, the direct action of the medicine, when applied to corresponding acupoints, penetrates the skin and enters the bloodstream, reaching the site of organ dysfunction and exerting its meridian-specific and functional effects to improve and enhance the body's immunity, thereby reducing morbidity and alleviating symptoms. From a modern medical perspective, the mechanism of acupoint application is consistent with the transdermal absorption of drugs; the medicine diffuses through different layers of the skin, enters the bloodstream, and directly reaches the target point to exert its therapeutic effect. Small molecular weight drugs are absorbed through the epidermis into the stratum corneum, passively transported by the concentration gradient between the inside and outside of the skin, diffuse from the dermis to the capillaries, and finally enter the systemic circulation to be delivered to the target site. Large molecular weight drugs are absorbed through accessory organs such as sebaceous glands and sweat glands. Compared with non-acupoint areas, acupoints have a thinner stratum corneum and richer nerve endings, exhibiting external sensitivity and amplification effects, and possess unique physiological functions, producing effects that cannot be achieved through non-acupoint drug delivery. Transdermal drug delivery systems, using traditional Chinese medicine acupoint application, reduce the first-pass effect of liver metabolism, the influence of pH value and digestive enzymes on drug concentration, retain more active ingredients, improve bioavailability, reduce drug toxicity and adverse reactions, and maintain stable blood drug concentrations, preventing local accumulation of drug concentrations and non-target tissue specific delivery. Therefore, transdermal drug delivery systems are a new research hotspot internationally.

[0007] Therefore, developing an acupoint patch for treating insomnia and anxiety caused by blood deficiency and liver heat, which is effective, has no toxic side effects, is minimally irritating to the skin, is safe, efficient, and easy to use, and is suitable for large-scale industrial production, and its preparation method meets market demand and has broad market value and application prospects. Summary of the Invention

[0008] The purpose of this invention is to provide a traditional Chinese medicine composition, acupoint application, and preparation method for treating insomnia and / or anxiety, thereby solving the problems existing in the prior art. This traditional Chinese medicine composition is scientifically and rationally formulated, possessing the effects of soothing the liver and purging fire, nourishing blood and calming the mind. It is safe to use, has good efficacy, and can be effectively used to treat insomnia and / or anxiety caused by blood deficiency and liver heat. This represents a major innovation in the treatment of insomnia and / or anxiety caused by blood deficiency and liver heat, and will bring significant economic and social benefits.

[0009] To achieve the above objectives, the present invention provides the following solution:

[0010] This invention provides a traditional Chinese medicine composition for treating insomnia and / or anxiety caused by blood deficiency and liver heat. The traditional Chinese medicine composition comprises the following components by weight: 1-15 parts of stir-fried jujube seed, 1-10 parts of raw gardenia, 1-5 parts of poria cocos, 1-5 parts of anemarrhena asphodeloides, 1-5 parts of chuanxiong rhizome, and 1-5 parts of fermented soybean.

[0011] Preferably, the traditional Chinese medicine composition comprises the following components by weight: 15 parts of stir-fried jujube seed, 3 parts of raw gardenia, 2 parts of poria cocos, 2 parts of anemarrhena asphodeloides, 2 parts of chuanxiong rhizome, and 2 parts of fermented soybean.

[0012] The present invention also provides the use of the above-mentioned traditional Chinese medicine composition in the preparation of a medicine for treating insomnia and / or anxiety of blood deficiency and liver heat type.

[0013] The present invention also provides a medicine for treating insomnia and / or anxiety of blood deficiency and liver heat type, the raw materials of which include the above-mentioned traditional Chinese medicine composition.

[0014] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0015] Furthermore, the medication is an acupoint patch.

[0016] Furthermore, the acupoint patch comprises, from the inside out, a peeling layer, a base fabric layer, an ointment layer, and a backing layer;

[0017] The release layer and the base fabric layer, the base fabric layer and the ointment layer, and the ointment layer and the backing layer are each independently bonded to each other by adhesives;

[0018] The release layer is release paper; the base fabric layer is made of non-woven fabric; the backing layer is made of non-woven fabric.

[0019] The raw materials for preparing the ointment layer include the traditional Chinese medicine composition.

[0020] Furthermore, the ointment layer is formulated from a mixture of powders of the traditional Chinese medicine composition, glycerin, carbomer homopolymer, and capsule gelatin.

[0021] Furthermore, the mass ratio of the mixed powder of the traditional Chinese medicine composition, the glycerin, the carbomer homopolymer, and the capsule gelatin is 3:5:1:1.

[0022] The present invention also provides a method for preparing the above-mentioned drug, comprising the following steps:

[0023] After pulverizing and sieving each component of the traditional Chinese medicine composition, they are mixed evenly to obtain a mixed powder of traditional Chinese medicine.

[0024] After the glycerin, carbomer homopolymer and capsule are mixed evenly with gelatin, the traditional Chinese medicine powder is added and stirred evenly to make a drug coating.

[0025] The drug coating is evenly applied to non-woven fabric, cut into ointment pieces, and dried to obtain dried ointment pieces.

[0026] The dried ointment is cut into small pieces and applied to a pre-cut adhesive mesh cloth to obtain the medicine.

[0027] The therapeutic principle of the traditional Chinese medicine composition of this invention is: to soothe the liver and clear heat, nourish blood and calm the mind.

[0028] In the traditional Chinese medicine composition of the present invention:

[0029] Stir-fried jujube seeds are sweet and sour in taste and neutral in nature; they enter the heart, liver, and gallbladder meridians; they nourish the heart and liver, calm the mind and soothe the nerves, reduce sweating, and promote the production of body fluids.

[0030] Raw gardenia is bitter and cold in nature; it enters the heart, lung, and triple burner meridians; it clears heat and relieves irritability, clears heat and promotes diuresis, cools the blood and detoxifies.

[0031] Poria cocos has a sweet and bland taste and a neutral nature; it enters the heart, lung, spleen, and kidney meridians; it calms the mind, soothes the nerves, and promotes urination.

[0032] Anemarrhena asphodeloides has a bitter and sweet taste and is cold in nature; it enters the lung, stomach, and kidney meridians; it clears heat and drains fire, nourishes yin and moistens dryness.

[0033] Lightly fermented soybeans are pungent and bitter in taste, and cool in nature; they enter the lung and stomach meridians; they relieve exterior syndromes, eliminate irritability, and disperse stagnant heat.

[0034] Chuanxiong (Ligusticum striatum) is pungent and warm in nature; it enters the liver, gallbladder, and pericardium meridians; it invigorates blood circulation, promotes qi circulation, dispels wind, and relieves pain.

[0035] The above-mentioned components are scientifically and rationally combined according to the principles of traditional Chinese medicine (TCM) formulation, specifically the combination of principal, assistant, adjuvant, and guide herbs. Ziziphus jujuba seed is sour and sweet, nourishing the heart and liver, calming the mind and soothing the nerves; Gardenia jasminoides is bitter and cold, purging fire and relieving irritability, guiding heat downwards. These two herbs together serve as the principal herbs. The assistant herbs are Poria cocos, calming the mind and soothing the nerves; Anemarrhena asphodeloides, clearing heat and relieving irritability; and fermented soybean, pungent and bitter, light and cold in nature, which can both dispel external pathogens and disperse stagnant heat, harmonizing the heart and kidneys, guiding kidney water upwards to nourish heart yang and relieve irritability, thus assisting the principal herbs in calming the mind and relieving irritability. The adjuvant herb, Ligusticum chuanxiong, is a qi-regulating herb in the blood, pungent and aromatic, dispersing and warming, regulating liver blood and soothing liver qi. Combined with stir-fried Ziziphus jujuba seed, the combination of sour and astringent properties, and pungent and dispersing properties, nourishes and promotes blood circulation, effectively nourishing blood and regulating liver qi. The combined effects of these herbs soothe the liver, purge heat, nourish blood, and calm the mind, making it effective in treating insomnia and / or anxiety due to blood deficiency and liver heat.

[0036] The present invention discloses the following technical effects:

[0037] The traditional Chinese medicine composition provided by this invention is scientifically and rationally formulated, and has the effects of soothing the liver and purging heat, nourishing blood and calming the mind. It is safe to use and has good efficacy. It can be effectively used to treat insomnia and / or anxiety caused by blood deficiency and liver heat. It is a major innovation in the treatment of insomnia and / or anxiety caused by blood deficiency and liver heat, and will bring significant economic and social benefits.

[0038] The acupoint patch for treating insomnia and / or anxiety caused by blood deficiency and liver heat provided by this invention is made of fine powder of Chinese herbal medicine, glycerin, carbomer homopolymer and capsule gelatin in a mass ratio of 3:5:1:1. Through the mutual cooperation and interaction of the components, the patch can be well absorbed by the human body, the efficacy can be fully utilized, and it has little skin irritation, no toxic side effects, and is safer to use.

[0039] The method for preparing acupoint patches for treating insomnia and / or anxiety caused by blood deficiency and liver heat provided by this invention only requires stacking the layers, without the need for complicated procedures and special equipment. It has high preparation efficiency, a good preparation environment, low labor intensity, and is suitable for large-scale continuous production. Attached Figure Description

[0040] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0041] Figure 1 The ion flow diagram of blank serum under negative ion mode;

[0042] Figure 2 The ion flow diagram of drug-containing serum in negative ion mode;

[0043] Figure 3 The ion chromatogram of blank serum in positive ion mode;

[0044] Figure 4 This is an ion chromatogram of drug-containing serum in positive ion mode;

[0045] Figure 5 Venn diagram of drug targets and disease targets;

[0046] Figure 6 A network diagram of PPIs for drug and disease targets;

[0047] Figure 7 A schematic diagram of the core target;

[0048] Figure 8 This is a drug-ingredient-disease-target network diagram;

[0049] Figure 9 Bubble chart for KEGG enrichment analysis;

[0050] Figure 10 This is a diagram analyzing the GO functionality. Detailed Implementation

[0051] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0052] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0053] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0054] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0055] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0056] Example 1

[0057] An acupoint patch comprises, from the inside out, a release layer, a base fabric layer, an ointment layer, and a backing layer; the release layer and the base fabric layer, the base fabric layer and the ointment layer, and the ointment layer and the backing layer are each independently bonded by adhesives; the release layer is ordinary release paper; the base fabric layer is made of non-woven fabric; the backing layer is made of non-woven fabric; the ointment layer is prepared by mixing traditional Chinese medicine powder, glycerin, carbomer homopolymer (Xi'an Tianzheng Pharmaceutical Excipients Co., Ltd.; CP2020), and capsule gelatin (Shaanxi Zhengyi Pharmaceutical Excipients Co., Ltd.; 9000-70-8) in a mass ratio of 3:5:1:1; by weight, the raw materials of the traditional Chinese medicine powder are: 15 parts of stir-fried jujube seed, 3 parts of raw gardenia, 2 parts of poria cocos, 2 parts of anemarrhena asphodeloides, 2 parts of chuanxiong rhizome, and 2 parts of fermented soybean.

[0058] The specific preparation method for acupoint patches is as follows:

[0059] (1) Grind the stir-fried jujube seeds, raw gardenia, poria cocos, anemarrhena asphodeloides, chuanxiong rhizome and fermented soybean into fine powder, pass them through an 8-mesh sieve (150 mesh), and mix them evenly according to the above mass ratio to obtain a mixed powder of Chinese medicine for later use.

[0060] (2) Add glycerin to a tank mixer and keep it at a constant temperature of 50°C. Then add carbomer homopolymer and capsule gelatin in sequence. Stir continuously for 2 hours, then add the traditional Chinese medicine powder and stir for 1 hour at a stirring speed of 20 rpm. After stopping the stirring, the drug coating is prepared.

[0061] (3) Adjust the roller spacing of the coating machine to 12 mils, hang the non-woven fabric and silicone anti-stick paper according to the "Standard Operating Procedures for Coating Machine for Bapogel", add the drug coating into the material trough of the coating machine in batches, coat it evenly on the non-woven fabric, and cut it into 37cm×15cm single pieces of ointment.

[0062] (4) Place the coated ointment sheets one by one on a stainless steel tray, put them in a drying oven and dry them at 50°C for 1.5 hours. Then stop drying and place them at room temperature for later use.

[0063] (5) Cut the dried ointment into small round pieces with a diameter of 3cm.

[0064] (6) Apply the small circular ointment pieces obtained in the previous step to the cut adhesive mesh cloth with a diameter of 4.5cm to obtain the acupoint patch, named Zaozhi Anshen Patch.

[0065] Example 2

[0066] Same as Example 1, except that the raw materials of the traditional Chinese medicine mixed powder are: 1 part of fried wild jujube seeds, 10 parts of raw gardenia fruits, 1 part of poria, 5 parts of anemarrhena rhizome, 1 part of chuanxiong rhizome, and 5 parts of fermented soya beans.

[0067] Example 3

[0068] Same as Example 1, except that the raw materials of the traditional Chinese medicine mixed powder are: 5 parts of fried wild jujube seeds, 1 part of raw gardenia fruits, 5 parts of poria, 1 part of anemarrhena rhizome, 5 parts of chuanxiong rhizome, and 1 part of fermented soya beans.

[0069] Animal experiment of Example 4

[0070] 1. Experimental animals and feeding conditions

[0071] Specific pathogen-free 6-week-old Wistar rats, male, weighing 200±20 g, were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd., and the animal license number was: SCXK (Beijing) 2021-0006. The rats were housed at the Institute of Psychology, Chinese Academy of Sciences, with a temperature of 23±2°C and a relative humidity of 50-70%, and a 12h:12h light / dark cycle. All animals were housed individually in a quiet environment in the same animal room, fed with sterilized maintenance feed and sterilized water, and cleaned and disinfected regularly. This experiment passed the review of the Ethics Committee of the Institute of Psychology, Chinese Academy of Sciences (H23160).

[0072] 2. Animal grouping and administration method

[0073] Grouping: After 1 week of adaptive feeding, 36 male Wistar rats were divided into a blank group (Control), a model group (Model), and a Zaozhi Anshen Patch group (ZZAST) according to the random number table method, with 12 rats in each group, and each rat was housed individually in a cage.

[0074] Model Preparation: Except for the control group, two other groups established a generalized anxiety disorder (GAD) model using uncertain empty bottle drinking and chronic restraint stress. After one week of acclimatization feeding, rats were trained to drink water at set times for seven days: 10 minutes each time during the morning (8:00-8:10 AM) and evening (8:00-8:10 PM), with the water bottle removed at the remaining times. The stress experiment began at 8:00 AM on the eighth day. Random stimuli were provided during the morning and evening time slots: a full water bottle was given for 10 minutes during one of the morning or evening time slots, while an empty water bottle was given during the other time slot. After the morning drinking stimulation, the rats were restrained for 4 hours continuously using a transparent plastic rat restraint device for two weeks. The control group had free access to food. Behavioral assessments were performed on all rats on day 14 of the stress period, with crossover between groups. All animals entered the testing laboratory two hours in advance to acclimatize to the environment.

[0075] Administration method: Starting from day 8, the drug was administered after an uncertain empty bottle drinking stimulation and before restraint. The Control group received no intervention, the ZZAST group received the Jujube and Gardenia Tranquilizing Patch prepared in Example 1 for acupoint application, and the Model group received a blank acupoint patch. Both groups were intervened continuously for 2 weeks.

[0076] The preparation method of the blank acupoint patch is the same as that in Example 1, except that the ointment layer does not contain mixed Chinese medicine powder.

[0077] Acupoint location: The bilateral Taiyang, Neiguan and Qimen acupoints of rats were selected for the experiment. The location of acupoints was based on "Experimental Acupuncture" and related literature (the effect of penetrating acupoints on the expression of nerve growth factor in the brain tissue around the hematoma of rats with cerebral hemorrhage; the effect of applying acupoints to the Yu and Mu acupoints on serum uric acid and its mechanism in rats with hyperuricemia).

[0078] Acupoint application procedure: After the rats were not stimulated by drinking water from an empty bottle, one person restrained them while another person trimmed the hair near the bilateral temples, Neiguan (PC6), and Qimen (LR14) acupoints. The hair was then removed using a cotton swab dipped in a suitable amount of depilatory cream, followed by disinfection with 75% alcohol. The Model group and ZZAST group received blank acupoint patches and jujube and gardenia calming patches, respectively, applied to the corresponding acupoints and secured with adhesive tape. The patch diameter was 0.5 cm. The dosage was calculated based on the literature "Study on the effect of Xingpi Jieyu formula on hippocampal neuronal synaptic remodeling in depressed rats based on the AMPK / SIRT1 / PGC-1α pathway," with each acupoint patch containing approximately 0.1 g of drug. The treatment was administered once daily in the morning for 4 hours each time, for 14 consecutive days. After application and fixation, the rats were restrained in a restraint device.

[0079] 3. Methods for evaluating drug efficacy

[0080] 3.1 Behavioral Evaluation

[0081] Macroscopic representation:

[0082] Before each drug intervention and restraint, observe the mental state, fur color and activity of rats in each group to determine the degree of resistance of rats to application, gavage and restraint, and record their diet and feces.

[0083] Changes in body weight of rats in each group:

[0084] Rats were weighed before and weekly after the start of the experiment, and the drug dosage was adjusted according to the weight changes. The changes in body weight of rats in each group were calculated and compared before modeling and at weeks 2 and 4 after modeling.

[0085] 3.2 Elevated Cross Maze Experiment

[0086] After the last administration, the rats in each group were tested using an elevated cross maze experiment. One hour before the experiment, the rats were placed in the experimental environment to allow them to acclimatize to the maze and their surroundings. During the testing procedure, the lights were turned off to maintain a dim and quiet environment in the laboratory. The elevated cross maze apparatus consisted of two open arms and two closed arms. Each arm was 10 cm wide and 50 cm long, and the closed arm was 40 cm high. The height of all four arms from the ground was 100 cm. The rats were placed in the center of the maze, with their heads facing the closed arms, and the observer was at least 1 meter away from the center of the maze. Record the number of times the mouse entered the open arm (OE), the time spent in the open arm (OT), the number of times it entered the closed arm (CE), and the time spent in the closed arm (CT) within 5 minutes. Calculate: the total number of entries into the open and closed arms (OE+CE), representing the mouse's motor ability; the percentage of entries into the open arm (OE%), calculated as OE / (OE+CE)×100%; and the percentage of time spent in the open arm (OT%), calculated as OT / (OT+CT)×100%. Clean the equipment promptly before and after each rat experiment, wiping the sides and bottom of the equipment with 75% alcohol to remove any information left by the previous animal (such as feces, odor, etc.) and avoid affecting the results of subsequent experiments.

[0087] 3.3 Open Field Experiment

[0088] During the test, a single rat was placed in a circular, clean, open box with a diameter of 100cm × 100cm. The video tracking and analysis system automatically recorded the rat's spontaneous activities over 5 minutes, and the animal movement trajectory tracking system calculated the total distance traveled and the time spent in the central area. The laboratory was kept dimly lit and quiet during the test, and each rat was cleaned and disinfected promptly before and after the experiment.

[0089] 3.4 Sample Collection and Processing

[0090] Rats in each group underwent behavioral testing, and samples were collected. Rats were anesthetized via intraperitoneal injection of 1% sodium pentobarbital (40 mg / kg). Blood was rapidly collected from the abdominal aorta, allowed to stand at room temperature for 30 minutes, and then centrifuged (4℃, 3000 rpm, 10 minutes). Serum was separated, aspirated, and aliquoted into EP tubes. After blood collection from the abdominal aorta, the brain was harvested from the head on ice, and hippocampal tissue was collected. Blood vessels were removed, and the tissue was washed in physiological saline. Serum and hippocampal tissue were stored at -80℃ for later analysis.

[0091] 3.5 Blood Component Detection of Jujube and Gardenia Sedative Patches

[0092] The components of traditional Chinese medicine in rat serum were detected by UHPLC-OE-MS. UHPLC was performed on a SCIEX Triple TOF 6600+.

[0093] 3.6 Network Pharmacology

[0094] 3.6.1 Screening of blood-borne target components of Jujube and Gardenia Sedative Patch

[0095] By searching the TSCMP database, relevant targets of the blood-entering components of Zaozhi Anshen Patch were retrieved, and the SMILLES structural information of the blood-entering components was obtained from the PubChem database. The SMILLES were then imported into SwissTargetPrediction to predict the possible targets of the blood-entering components of Zaozhi Anshen Patch. The screening criteria were set to "Possibility>0", and known targets of unpredicted active compounds were supplemented by targets confirmed by published literature.

[0096] 3.6.2 Screening of GAD-related targets

[0097] Using "generalized anxiety disorder" as the keyword, disease targets related to GAD were collected from the GeneCards database (https: / / www.genecards.org), OMIM database (http: / / www.omim.org), TTD database (http: / / bidd.nus.edu.sg / group / cjttd), and DRUGBANK database (https: / / www.drugbank.ca). After merging and deduplication, the intersection of these targets with the component targets was used to obtain the potential targets for treating GAD with Zaozhi Anshen Patch.

[0098] 3.6.3 Construction of the blood-entry component of Jujube and Gardenia Tranquilizing Patch - PPI network construction targeting generalized anxiety disorder

[0099] The chemical component targets and GAD-related targets of Zaozhi Anshen Patch were analyzed using an online Venn diagram (version 2.1.0) to screen for overlapping targets. The gene names of the obtained blood-entry component targets and potential GAD targets were standardized using the Uniprot database (https: / / www.uniprot.org). The overlapping targets were then imported into the STRING platform (http: / / string.db.org), with a confidence score ≥ 0.400. TSV data files were downloaded and imported into Cytoscape 3.7.2 software to construct a "component-target-disease" visualization network.

[0100] 3.6.4 GO and KEGG enrichment analysis

[0101] We used the Metascape database (https: / / metascape.org / gp / index.html# / main / step1) to perform gene ontology (GO) functional annotation and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis on potential disease targets, with P < 0.01 as a constraint. The results were then visualized on the Bioinformatics website (http: / / www.bioinformatics.com.cn).

[0102] Enzyme-linked immunosorbent assay (ELISA) was used to detect serum interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) in model rats.

[0103] Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect PI3K / Akt pathway mRNA.

[0104] Western blot (WB) was used to detect the neurotransmitters 5-hydroxytryptamine (5-HT), dopamine (DA), and γ-aminobutyric acid (GABA), as well as key proteins brain-derived neurotrophic factor (BDNF), cyclic adenosine monophosphate responsive element binding protein 1 (CREB1), and PI3K / Akt pathway-related proteins.

[0105] 4. Results

[0106] 4.1 Blood Component Analysis of Jujube and Gardenia Sedative Patches

[0107] This experiment used UHPLC-QE-MS technology to detect and compare the blank serum of the model group rats and the drug-containing serum of rats after intervention with Zaozhi Anshen Patch. Based on the secondary spectrum score, mass deviation value, and relevant literature review, 18 components were identified in the blood. The chemical components identified in the drug-containing serum of rats under positive and negative ion modes are shown in Table 1 and [Table data missing]. Figure 1-4 .

[0108] Table 1. Information on Blood-Entering Components of Jujube and Gardenia Tranquilizing Patches

[0109]

[0110]

[0111] 4.2 Network pharmacology results

[0112] 4.2.1 Obtaining the target points of blood components for the Jujube and Gardenia Sedative Patch

[0113] By searching the TSCMP database, 132 therapeutic targets of 18 blood-entering components of Zaozhi Anshen Patch were retrieved. Based on the SMILLES structural information of the blood-entering components obtained from the PubChem database, the SMILLES were imported into SwissTargetPrediction to predict potential targets for the possible effects of Zaozhi Anshen Patch. The screening criteria were set to "Possibility>0" and targets confirmed by published literature. After merging, a total of 258 targets were obtained. After removing duplicate values, a total of 191 targets were finally obtained.

[0114] 4.2.2 Acquisition of GAD-related targets

[0115] 6159 targets related to GAD were obtained from the Genecards database. Targets with a relevance score > 16.83 were designated as potential targets for GAD, resulting in 770 targets. Further related targets were added by combining data from the OMIM (642 targets), TTD (4 targets), and DRUGBANK (40 targets) databases. After merging and removing duplicates, 1358 potential targets related to GAD were finally obtained.

[0116] 4.2.3 Construction of the blood-entry component-GAD target PPI network of Jujube and Gardenia Tranquilizing Patch

[0117] The relevant targets of the jujube and gardenia calming patch in the blood and the relevant targets of GAD were input into the Venny 2.1 online graphing platform to draw a Venn diagram. The intersection of the two diagrams yielded 62 common drug-disease targets. Figure 5 Sixty-two potential targets for the treatment of GAD with jujube and gardenia calming patches were imported into the String database. Data with a confidence score ≥ 0.400 was selected. TSV data files were downloaded and imported into Cytoscape 3.7.2 software to construct a PPI network diagram. (See attached image.) Figure 6 The Degree algorithm of the CytoHubba plugin was used to identify the core targets of the PPI network. The top ten core targets include BDNF, TNF, IL6, PTGS2, IL1β, FOS, and CREB1, etc. (See [link]). Figure 7 In the diagram, the darker the color of a node, the higher its Degree score, meaning that the node is more important in the PPI network.

[0118] 4.2.4 Construction of the Drug-Blood Component-Disease-Target Network Diagram

[0119] like Figure 8 As shown, in the "Drug Ingredient-Target-Pathway" network diagram constructed using Cytoscape 3.7.2 software, pink represents the disease name (GAD), orange represents roasted jujube seed (SZR), poria cocos (FS), anemarrhena asphodeloides (ZM), raw gardenia (ZZ), chuanxiong rhizome (CX), and fermented soybean (DC), yellow represents the blood-entering components of traditional Chinese medicine, and purple represents potential targets. Since betulinic acid, mangiferin, and neomangiferin have no overlapping targets with GAD, only 15 blood-entering components are presented in the "Drug Ingredient-Target-Pathway" network diagram. The Degree value of the active ingredient was calculated using the NetworkAnalyzer tool in Cytoscape 3.7.2 software. The larger the Degree value, the more biological processes involved, and the greater the importance of that node.

[0120] 4.2.5 Enrichment analysis of target function and pathway

[0121] KEGG pathway enrichment and GO analysis were performed on the core targets using the Metascape database. Key KEGG pathways involved include neuroactive ligand-receptor interactions, PI3K-Akt, dopaminergic synapses, 5-HTergic synapses, and calcium signaling channels. (See [link to Metascape database]). Figure 9 The top ten pathways and their related genes are shown in Table 2. GO functional analysis includes three aspects: biological process (BP), cellular component (CC), and molecular function (MF). Biological process BP mainly involves chemical synaptic transmission, anterosynaptic signal transmission, transsynaptic signal transduction, synaptic signaling, and regulation of postsynaptic membrane potential; CC mainly involves the synaptic membrane, postsynaptic membrane, dendrites, dendritic trees, and presynaptic membrane; MF mainly involves neurotransmitter receptor activity, postsynaptic neurotransmitter receptor activity, benzodiazepine receptor activity, extracellular ligand-gated single-atom ion channel activity, and GABA-gated chloride ion channel activity, etc. (See Table 2 for details). Figure 10 .

[0122] Table 2. Top 10 KEGG pathways and associated genes

[0123]

[0124]

[0125] 4.3 Animal Experiment Validation Results

[0126] 4.3.1 Macroscopic Characterization

[0127] Before the modeling process began, all groups of rats were in good mental condition, active, with steady breathing, clear eyes without discharge, rosy ears and noses, thick and shiny fur, and spherical feces. At the onset of stress in week 2, rats in the Model and ZZAST groups exhibited irritability and erect hair. In week 3, rats in the Control group showed no abnormalities in mental state or behavior except for physical growth. Rats in the Model group were more alert when handled, jumping around in their cages. When restrained or bound, they cried out, resisted violently, breathed rapidly, and urinated and defecated more frequently, with disheveled fur. Rats in the ZZAST group also showed similar reactions when restrained, but to a lesser degree than those in the Model group. In week 4, rats in the Control group increased in size, with no other significant abnormalities. Rats in the Model group showed a more pronounced stress response, exhibiting strong rejection, tense bodies, erect ears, a defensive posture, irritability, disheveled and dry fur, and loose or unformed feces. The ZZAST group showed improvement in these symptoms.

[0128] 4.3.2 Changes in body weight of rats in each group

[0129] There was no statistically significant difference in body weight among the groups at baseline (P>0.05). During weeks 2 and 4 of the experiment, the body weight of rats in all groups increased, but the Control group and ZZAST group had higher body weights than the Model group (P<0.01), as shown in Table 3.

[0130] Table 3 Comparison of rat body weight among groups

[0131]

[0132] Note: Compared to Control, * P<0.05, ** P<0.01; compared with the Model, ▲ P<0.05, ▲▲ P<0.01.

[0133] 4.3.3 Behavioral comparison of rats in different groups

[0134] (1) Elevated Cross Maze Experiment

[0135] Compared with the Control group, the OE% and OT% of the Model group were significantly lower, with statistically significant differences (P<0.01). Compared with the Model group, the ZZAST group significantly increased the percentage of anxious rats entering the open arm and the percentage of rats remaining in the open arm, with statistically significant differences (P<0.01), as shown in Table 4.

[0136] Table 4 Comparison of results of elevated cross maze test in rats of different groups

[0137]

[0138] Note: Compared to Control, * P<0.05, ** P<0.01; compared with the Model, ▲ P<0.05, ▲▲ P<0.01.

[0139] (2) Open field experiment

[0140] Compared with the Control group, the Model group showed a significant decrease in both the number of times rats entered the central zone and the duration of stay in the central zone (P<0.01). Compared with the Model group, the ZZAST group showed a significant increase in the number of times anxious rats entered the central zone (P<0.05) and the duration of stay in the central zone (P<0.01), as shown in Table 5.

[0141] Table 5 Comparison of open field test results among different groups of rats

[0142]

[0143] Note: Compared to Control, * P<0.05, ** P<0.01; compared with the Model, ▲ P<0.05, ▲▲ P<0.01.

[0144] 4.3.4 Comparison of serum inflammatory factors among different groups of rats

[0145] Compared with the control group, the serum levels of IL-1β, TNF-α, and IL-6 in the model group were significantly increased (P<0.01). Compared with the model group, the serum levels of IL-1β, TNF-α, and IL-6 in the ZZAST group were all decreased (P<0.05, P<0.01), as shown in Table 6.

[0146] Table 6. Serum IL-1β, IL-6, and TNF-α levels in rats of each group (pg / ml)

[0147]

[0148] Note: Compared to Control, * P<0.05, ** P<0.01; compared with the Model, ▲ P<0.05, ▲▲ P<0.01.

[0149] 4.3.5 Comparison of hippocampal neurotransmitters among different groups of rats

[0150] The levels of 5-HT, DA, and GABA in the hippocampus of rats in each group were detected. The results showed that compared with the Control group, the levels of all three neurotransmitters, 5-HT, DA, and GABA, were significantly lower in the Model group (P<0.01). Compared with the Model group, the levels of all three neurotransmitters were significantly higher in the ZZAST group (P<0.05, P<0.01), as shown in Table 7.

[0151] Table 7. Relative expression levels of hippocampal neurotransmitters in each group of rats (n=3)

[0152]

[0153] Note: Compared to Control, * P<0.05, ** P<0.01; compared with the Model, ▲ P<0.05, ▲▲ P<0.01.

[0154] 4.3.6 Comparison of key proteins BDNF and CREB1 among different groups of rats

[0155] The levels of BDNF and CREB1 in the hippocampus of rats in each group were measured. The results showed that compared with the control group, the levels of BDNF and CREB1 in the model group were significantly decreased, and the differences were statistically significant (P<0.05, P<0.01). Compared with the model group, the levels of the two key proteins in the ZZAST group were increased, and the differences were statistically significant (P<0.05), as shown in Table 8.

[0156] Table 8. Relative expression levels of BDNF and CREB1 in the hippocampus of rats in each group (n=3)

[0157]

[0158] Note: Compared to Control, * P<0.05, ** P<0.01; compared with the Model, ▲ P<0.05, ▲▲ P<0.01.

[0159] 4.3.7 Comparison of PI3K / Akt pathway proteins among different groups of rats

[0160] The PI3K / Akt pathway proteins in the hippocampus of rats in each group were detected. The results showed that there was no statistically significant difference in the relative expression levels of PI3K and Akt proteins among the groups (P>0.05). Compared with the Control group, the levels of p-PI3K and p-Akt proteins in the Model group were significantly decreased (P<0.01). Compared with the Model group, the levels of p-PI3K and p-Akt pathway proteins in the ZZAST group were increased (P<0.05), as shown in Table 9.

[0161] Table 9. Relative expression levels of PI3K / Akt pathway proteins in the hippocampus of rats in each group (n=3)

[0162]

[0163] Note: Compared to Control, * P<0.05, ** P<0.01; compared with the Model, ▲ P<0.05, ▲▲ P<0.01.

[0164] 4.3.8 Comparison of PI3K mRNA and Akt mRNA among different groups of rats

[0165] The expression levels of PI3K and Akt mRNA in the hippocampus of rats in each group were detected. Compared with the control group, the expression levels of PI3K and Akt mRNA in the model group were significantly decreased (P<0.01); compared with the model group, the expression levels of PI3K and Akt mRNA in the ZZAST group were significantly increased (P<0.05), as shown in Table 10.

[0166] Table 10. Relative expression levels of PI3K / Akt pathway mRNA in the hippocampus of rats in each group (n=3)

[0167]

[0168] Note: Compared to Control, * P<0.05, ** P<0.01; compared with the Model, ▲ P<0.05, ▲▲ P<0.01.

[0169] 5. Summary

[0170] This invention demonstrates that the Jujube and Gardenia Sedative Patch can improve anxiety-like behavior in GAD model rats, reduce serum levels of pro-inflammatory factors IL-1β, IL-6, and TNF-α, increase hippocampal neurotransmitters 5-HT, DA, and GABA, and promote the expression of PI3K / Akt pathway-related proteins and key proteins BDNF and CREB1. Therefore, the mechanism by which the Jujube and Gardenia Sedative Patch treats GAD may be related to activating the PI3K / Akt pathway, regulating neurotransmitters, inhibiting inflammatory responses, and exerting neuroprotective effects.

[0171] Example 5 Clinical Trial

[0172] Objective: To evaluate the clinical efficacy and safety of Zaozhi Anshen Patch in treating GAD and its improvement of sleep disorder symptoms through clinical trials.

[0173] I. Methods

[0174] 1. Source of cases

[0175] The cases in this clinical trial were patients diagnosed with GAD at Dongfang Hospital of Beijing University of Chinese Medicine, whose TCM diagnosis was blood deficiency and liver heat syndrome.

[0176] 2. Diagnostic criteria

[0177] The Western medical diagnostic criteria refer to the diagnostic criteria for GAD in CCMD-III formulated by the Chinese Medical Association's Psychiatry Branch: it refers to an anxiety disorder characterized by a lack of clear object and specific content, with a predominantly fearful and tense feeling, accompanied by significant autonomic nervous system symptoms, muscle tension, and motor restlessness. Patients experience suffering because they find it unbearable and unable to escape it.

[0178] (1) Symptom criteria: Meets the diagnostic criteria for neurosis; mainly characterized by persistent primary anxiety symptoms, and meets the following two criteria: ① Frequent or persistent fear or apprehension without a clear object or fixed content; ② Accompanied by autonomic symptoms or motor restlessness.

[0179] (2) Disease duration criteria: The symptoms must have been met for at least 6 months.

[0180] The diagnostic criteria for anxiety disorders in Traditional Chinese Medicine (TCM) are based on the diagnostic and treatment guidelines for common internal diseases in TCM (2008, China Association of Traditional Chinese Medicine). The specific details are as follows:

[0181] Symptom criteria: The main symptom is persistent primary anxiety symptoms, and meets the following two criteria: (1) Frequent or persistent fear or apprehension without a clear object or fixed content that is difficult to control; (2) Autonomic nervous system dysfunction or motor restlessness.

[0182] Severity criteria: The patient's social functioning is impaired, and he or she suffers from unbearable and inescapable pain.

[0183] Disease duration criteria: Symptoms must have been present for at least 6 months.

[0184] The diagnostic criteria for blood deficiency and liver heat syndrome are as follows: emotional instability, inability to concentrate, restlessness, palpitations, forgetfulness, dizziness, irritability, insomnia and dreaminess, pale complexion, fatigue, dry mouth and bitter taste, pale tongue with thin coating, and wiry, thready and rapid pulse.

[0185] 3. Inclusion criteria

[0186] It meets the diagnostic criteria for both Western medicine GAD and traditional Chinese medicine blood deficiency and liver heat syndrome;

[0187] 14 points≤HAMA<29 points;

[0188] Hamilton Depression Rating Scale (HAMD-17) score <7;

[0189] 18 years old ≤ age ≤ 65 years old;

[0190] Sign the informed consent form.

[0191] 4. Exclusion Criteria

[0192] Patients with severe anxiety whose HAMA score is ≥29;

[0193] Anxiety attacks secondary to other mental or physical illnesses and accompanied by severe psychotic symptoms;

[0194] Individuals with substance use disorders (including addiction to or abuse of substances such as alcohol, tobacco, caffeine, sedatives, and psychoactive drugs);

[0195] Accompanied by severe diseases of the heart, brain, liver, kidneys, and other systems;

[0196] I have used anti-anxiety and antidepressant medications in the past month;

[0197] Pregnant or breastfeeding women;

[0198] Those allergic to the test drug or its components;

[0199] Any participant who meets any of the above criteria will be excluded.

[0200] 5. Case withdrawal, dropout / loss to follow-up and their management

[0201] (1) Researcher's decision to withdraw

[0202] If a subject's disease progresses or worsens, requiring a change in treatment plan, and although the treatment plan was not completed as required, researchers may decide to terminate the trial and allow the subject to receive other treatments for the purpose of protecting the subject. After the completion of treatment, the relevant tests in this case will be counted as "invalid" eligible cases.

[0203] If a subject's HAMA score drops to below 14 during the trial, the treatment is considered effective and the subject is considered cured. At this point, the trial can be terminated and the subject can be counted as a "effective" qualified case.

[0204] If a subject develops other comorbidities, complications, or other special physiological or pathological changes during the trial, the trial may be terminated after research and assessment.

[0205] Subjects who experience serious adverse events, significant adverse events, or adverse events of grade 3 or above in the common adverse reaction evaluation criteria that are related to the investigational drug should not continue the trial.

[0206] Poor subject compliance (e.g., failure to take medication or undergo examinations as prescribed, use of concomitant medications prohibited by the protocol, and other behaviors that affect the evaluation of the safety and effectiveness of the trial).

[0207] Researchers discovered subjects who did not meet the inclusion criteria but were mistakenly included after enrollment.

[0208] (2) The subject decided to withdraw.

[0209] If a participant no longer wishes to participate in a clinical trial, they may request to withdraw from the trial or retract their informed consent. There are various reasons for a participant's voluntary withdrawal, such as feeling that the treatment is not effective enough, finding certain adverse reactions intolerable, financial reasons, or not providing a reason. The reasons for withdrawal should be investigated and recorded as much as possible.

[0210] Participants who did not explicitly indicate their withdrawal from the trial but were lost to follow-up due to failure to take medication as prescribed or failure to complete the tests.

[0211] Management of withdrawal or dropout cases: After a subject decides to withdraw from the trial, the researcher should contact the subject as much as possible to inquire about the reasons for refusing to continue participating in the trial, such as poor efficacy (worsening of the condition or occurrence of complications), loss to follow-up without explanation, poor compliance, disclosure of blinding, adverse events (inability to tolerate certain adverse reactions, including adverse drug reactions and allergic reactions), etc. The researcher should also record the time of the subject's last medication, complete the assessment items, and properly preserve the trial data related to dropout cases.

[0212] 6. Sample size estimation

[0213] Following the sample size calculation method for comparing rates in a two-independent-sample randomized controlled trial, PASS 15.0 software was used to calculate the sample size. Based on the references, the efficacy rate in the treatment group was taken as 50%, and the efficacy rate in the placebo group as 20%, with a 2:1 allocation scheme and R = 2. With α = 0.05 and β = 0.20, the calculated sample size was 75 cases. Considering an approximately 10% dropout rate, the design required the collection of approximately 82.5 cases, rounded to 90 cases, with 60 cases in the treatment group and 30 cases in the placebo group.

[0214] 7. Design Type

[0215] This invention employs a prospective, randomized, double-blind, placebo-controlled trial.

[0216] 8. Intervention measures

[0217] Treatment group: The Jujube and Gardenia Tranquilizing Patch prepared in Example 1 was used for treatment.

[0218] Acupoints for application: bilateral temples, Neiguan (PC6), and Qimen (LR14).

[0219] Instructions for use: Before application, disinfect the application area with 75% alcohol and then wipe it with ginger slices. Apply the Jujube and Gardenia Tranquilizing Patch to the corresponding acupoints once every night for a seven-day course of treatment. Begin use 30-60 minutes before bedtime and end the next morning.

[0220] Control group: treated with placebo acupoint patches (the preparation method of the placebo acupoint patches is the same as in Example 1, the only difference being that the ointment layer does not contain mixed Chinese medicine powder).

[0221] The acupoints for application and the method of use were the same as those for the treatment group.

[0222] All of the above-mentioned drugs were prepared by Beijing Xiucheng Pharmaceutical Co., Ltd.

[0223] 9. Test cycle

[0224] Each group of subjects will receive acupoint patch treatment for 4 consecutive weeks after enrollment, and will undergo planned visits at the end of the screening period (baseline) and the end of the 4th week.

[0225] 10. Medication and treatment are prohibited.

[0226] The following medications are prohibited during the trial: anti-anxiety medications (including but not limited to benzodiazepines). Benzene dinitrates and non-benzodiazepines Antidepressants, anti-anxiety Chinese medicines and prepared Chinese medicines, etc.

[0227] 11. Permitted medication and treatment

[0228] Patients with underlying diseases diagnosed and in stable condition before enrollment can continue to use their original medications, but the type and dosage of medication should remain stable.

[0229] Record in detail the medications and other therapies that were continued or added during the trial, including the name of the medication, dosage, indication, frequency of use, and duration.

[0230] 12. Observation Indicators

[0231] (1) General information: including gender, age, education level, course of disease, comorbidities, medication use, etc.

[0232] (2) Efficacy indicators:

[0233] Key therapeutic indicators:

[0234] Hamilton Anxiety Scale (HAMA).

[0235] Secondary efficacy endpoints:

[0236] Traditional Chinese Medicine Symptom Scale;

[0237] Self-rating anxiety scale (SAS);

[0238] Pittsburgh Sleep Quality Index (PSQI);

[0239] The MOS item short from health survey (SF-36).

[0240] (3) Security assessment:

[0241] Observe the patient's vital signs (respiration, heart rate, blood pressure, body temperature) during treatment and whether there are adverse reactions such as redness, itching, blisters, or ulceration at the application site.

[0242] 13. Criteria for Evaluating Therapeutic Effect

[0243] (1) Clinical efficacy evaluation criteria

[0244] The efficacy was evaluated based on the reduction rate calculated from the HAMA scores before and after treatment, using a grading system: HAMA reduction rate = (HAMA score before treatment - HAMA score after treatment) / (HAMA score before treatment) × 100%. The specific grading based on the reduction rate is as follows:

[0245] Cure: HAMA score reduction rate ≥75%;

[0246] Effective: 50% ≤ HAMA score reduction rate ≤ 75%;

[0247] Effective: 30% ≤ HAMA deduction rate < 50%

[0248] Invalid: HAMA deduction rate <30%.

[0249] Overall effective rate (%) = (Number of cured cases + Number of cases with significant improvement + Number of cases with improvement) / Total number of cases × 100%.

[0250] (2) Evaluation criteria for TCM syndrome differentiation and efficacy

[0251] According to the "Guiding Principles for Clinical Research of New Traditional Chinese Medicine Drugs," the reduction rate is calculated based on the difference in TCM syndrome scores before and after treatment: TCM syndrome score reduction rate = (TCM syndrome score before treatment - TCM syndrome score after treatment) / TCM syndrome score before treatment × 100%. The specific classifications based on the reduction rate are as follows:

[0252] Cure: After treatment, the clinical symptoms and signs in traditional Chinese medicine disappear or basically disappear, and the score reduction rate is ≥95%;

[0253] Significant effect: After treatment, the clinical symptoms and signs in traditional Chinese medicine were significantly improved, with a reduction rate of ≥70% in 95% of the patients.

[0254] Effective: After treatment, the clinical symptoms and signs in traditional Chinese medicine all improved, with a reduction rate of ≥30% in 70% of cases;

[0255] Ineffective: No significant improvement in TCM clinical symptoms and signs after treatment, with a score reduction rate of <30%;

[0256] Overall effective rate = (number of cured cases + number of cases with significant improvement + number of cases with improvement) / total number of cases × 100%.

[0257] 14. Ethics: This clinical study will be conducted in accordance with the Declaration of Helsinki and the requirements of clinical medical ethics, and has been approved by the Ethics Committee of Dongfang Hospital of Beijing University of Chinese Medicine (No. JDF-IRB-2023050802).

[0258] 15. Statistical Analysis: SPSS 25.0 statistical software package was used for statistical analysis, and GraphpadPrism 9.0 was used for plotting. Quantitative data were expressed as mean ± standard deviation. For those following a normal distribution, paired-samples t-tests were used for comparisons within a group before and after treatment, and independent-samples t-tests were used for comparisons between groups; for those not following a normal distribution, nonparametric tests were used. Categorical data were expressed as percentages. Unordered data were analyzed using the Pearson chi-square test or Fisher's exact test, and ordered data were analyzed using the rank-sum test. All statistical analyses were two-tailed tests; P < 0.05 indicated statistical significance, and P < 0.01 indicated significant difference.

[0259] II. Results

[0260] 1. Research Completion Status

[0261] This study included 90 patients with GAD of blood deficiency and liver heat type, with 60 in the treatment group and 30 in the control group. During the treatment, 3 patients dropped out of the treatment group (1 dropped out due to personal reasons for refusing follow-up, 1 dropped out due to being unable to cooperate with follow-up due to work relocation, and 1 dropped out due to personal reasons), and 2 patients dropped out of the control group (1 dropped out due to taking a prohibited drug during the trial, and 1 dropped out due to personal reasons). The final number of participants who completed the experiment was: 57 in the treatment group and 28 in the control group.

[0262] 2. HAMA scale scoring results

[0263] Compared with baseline, after 4 weeks of treatment, a paired-samples t-test revealed that the treatment group showed a significant decrease in HAMA total score and scores of each sub-item (P<0.05, P<0.01); while the control group showed a significant decrease in HAMA total score, anxiety mood, tension, fear, muscular symptoms, sensory symptoms, cardiovascular symptoms, respiratory symptoms, gastrointestinal symptoms, and behavioral performance during interviews (P<0.05, P<0.01). Compared with the control group, the treatment group showed a significant decrease in HAMA total score, anxiety mood, fear, gastrointestinal symptoms, and autonomic nervous system symptoms after treatment (P<0.05, P<0.01), as shown in Table 11.

[0264] Table 11 Comparison of HAMA scores between the two groups of patients

[0265]

[0266]

[0267] Note: Compared with the same group before treatment, *P<0.05, **P<0.01; compared with the control group after treatment, ▲ P<0.05,▲▲ P<0.01.

[0268] 3. Comparison of HAMA clinical efficacy

[0269] As shown in Table 12, according to the clinical efficacy evaluation criteria, in the treatment group, after treatment with Zaozhi Anshen Patch, 4 cases were cured, 19 cases showed significant improvement, 23 cases showed improvement, and 11 cases were ineffective, with a total effective rate of 80.70%. In the control group, after treatment with placebo patches, 0 cases were cured, 6 cases showed significant improvement, 5 cases showed improvement, and 17 cases were ineffective, with a total effective rate of 39.29%.

[0270] Table 12 Comparison of clinical efficacy between the two groups of patients

[0271]

[0272] 4. Results of Traditional Chinese Medicine Symptom Scale Scoring

[0273] Compared with baseline, after 4 weeks of treatment, a paired-samples t-test revealed that the total score and scores of all 13 sub-items of the Traditional Chinese Medicine (TCM) Symptom Scale in the treatment group significantly decreased (P<0.01). In contrast, the total score, scores for emotional instability, palpitations, dizziness, irritability, insomnia, dry mouth and bitter taste, tongue appearance, and pulse in the control group all decreased significantly compared to before treatment (P<0.05, P<0.01). Compared with the control group, the total score, scores for emotional instability, mental instability, forgetfulness, pale complexion, dry mouth and bitter taste, and pulse in the treatment group all significantly decreased (P<0.05, P<0.01), as detailed in Table 13.

[0274] Table 13 Comparison of Traditional Chinese Medicine Symptom Scale Scores Between the Two Groups of Patients

[0275]

[0276]

[0277] Note: Compared with the same group before treatment, *P<0.05, **P<0.01; compared with the control group after treatment, ▲ P<0.05, ▲▲ P<0.01.

[0278] 5. Comparison of TCM syndrome differentiation and treatment efficacy

[0279] As shown in Table 14, according to the TCM syndrome efficacy evaluation criteria, in the treatment group, after treatment with Zaozhi Anshen Patch, there were 0 cases of complete cure, 4 cases of significant improvement, 39 cases of improvement, and 14 cases of no effect, with a total effective rate of 75.44%. In the control group, after treatment with placebo patches, there were 0 cases of complete cure, 0 cases of significant improvement, 7 cases of improvement, and 21 cases of no effect, with a total effective rate of 25.00%.

[0280] Table 14 Comparison of TCM syndrome efficacy between the two groups of patients

[0281]

[0282] 6. SAS score

[0283] Compared with baseline, after 4 weeks of treatment, nonparametric tests showed that the SAS scores of both the treatment and control groups decreased significantly (P<0.01). A two-sample t-test compared with the control group revealed that the SAS score of the treatment group was significantly lower than that of the control group (P<0.01), as detailed in Table 15.

[0284] Table 15 Comparison of SAS scores between the two groups of patients

[0285]

[0286] Note: Compared with the same group before treatment, *P<0.05, **P<0.01; compared with the control group after treatment, ▲ P<0.05, ▲▲ P<0.01.

[0287] 7. PSQI

[0288] Compared with baseline, after 4 weeks of treatment, a paired-samples t-test showed that the PSQI total score and scores of each sub-item in the treatment group decreased significantly (P<0.01); while there was no statistically significant difference in the PSQI total score and scores of each sub-item in the control group before and after treatment (P>0.05). Compared with the control group, the PSQI total score, sleep duration, sleep efficiency, sleep disorder, and daytime functioning scores in the treatment group decreased significantly after treatment (P<0.05), as detailed in Table 16.

[0289] Table 16 Comparison of PSQI scores between the two groups of patients

[0290]

[0291]

[0292] Note: Compared with the same group before treatment, *P<0.05, **P<0.01; compared with the control group after treatment, ▲ P<0.05, ▲▲ P<0.01.

[0293] 8. SF-36 rating

[0294] Compared with baseline, after 4 weeks of treatment, a paired-samples t-test revealed that all SF-36 scores in the treatment group decreased significantly (P<0.01); while the scores for physical functioning and energy in the control group decreased significantly compared with before treatment (P<0.05, P<0.01). Compared with the control group, the treatment group showed a more significant decrease in scores for bodily pain, general health status, and emotional functioning after treatment (P<0.05, P<0.01), as shown in Table 17.

[0295] Table 17 Comparison of SF-36 scores between the two groups of patients

[0296]

[0297]

[0298] Note: Compared with the same group before treatment, *P<0.05, **P<0.01; compared with the control group after treatment, ▲ P<0.05, ▲▲ P<0.01.

[0299] 9. Security Analysis

[0300] Two patients in both the treatment and control groups experienced allergic reactions, both manifesting only as redness at the skin contact area of ​​the patch (one at the temple, the other at the Qimen acupoint), while the ointment itself did not cause redness or itching. After the patch was replaced with adhesive tape, the redness did not recur. It is considered that the patients were allergic to the patch rather than to the herbal medicine itself. Therefore, the Zaozhi Anshen Patch has good clinical safety in treating GAD.

[0301] 10. Summary

[0302] This invention, based on clinical trials, aims to evaluate the clinical efficacy and safety of Zaozhi Anshen Patch in treating patients with GAD (Gastrointestinal Disorder) of blood deficiency and liver heat syndrome. The results show that Zaozhi Anshen Patch can improve anxiety and somatic symptoms in patients with GAD of blood deficiency and liver heat type, reduce scores on the Traditional Chinese Medicine Symptom Scale, alleviate clinical symptoms, improve quality of life, promote the recovery of social function, and has good safety profile.

[0303] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An acupoint patch for treating insomnia and / or anxiety due to blood deficiency and liver heat, characterized in that, Raw materials include traditional Chinese medicine compositions and pharmaceutically acceptable excipients; The traditional Chinese medicine composition is made from the following components: 15 parts of stir-fried jujube seed, 3 parts of raw gardenia, 2 parts of poria cocos, 2 parts of anemarrhena asphodeloides, 2 parts of chuanxiong rhizome, and 2 parts of fermented soybean.

2. The acupoint patch according to claim 1, characterized in that, The acupoint patch comprises, from the inside out, a peeling layer, a base fabric layer, an ointment layer, and a backing layer; The release layer and the base fabric layer, the base fabric layer and the ointment layer, and the ointment layer and the backing layer are each independently bonded to each other by adhesives; The release layer is release paper; the base fabric layer is made of non-woven fabric; the backing layer is made of non-woven fabric. The raw materials for preparing the ointment layer include the traditional Chinese medicine composition.

3. The acupoint patch according to claim 2, characterized in that, The ointment layer is formulated from the mixed powder of the traditional Chinese medicine composition, glycerin, carbomer homopolymer and capsule gelatin.

4. The acupoint patch according to claim 3, characterized in that, The mass ratio of the mixed powder of the traditional Chinese medicine composition, the glycerin, the carbomer homopolymer, and the gelatin for capsules is 3:5:1:1.

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