The preparation method of moxa sticks for soothing the liver and relieving depression and their application in improving depressive symptoms

Abstract

This invention discloses a moxa stick with a liver-soothing and mood-regulating formula, its preparation method, and its application in improving depressive symptoms. Belonging to the field of traditional Chinese medicine technology, it aims to address the technical problems of existing antidepressant chemical drugs having numerous adverse reactions and poor medication adherence, existing Chinese herbal compound formulas being insufficiently adapted to the core pathogenesis of depression caused by dampness and blood stasis, having limited effectiveness in improving depressive-like behaviors induced by chronic stress, and having poor neurotransmitter regulation. This compound moxa stick, by weight, consists of Artemisia argyi, Murraya paniculata, Curcuma longa, Cyperus rotundus (processed with vinegar), Sappanwood, Spatholobus suberectus, Jasminum sambac, Citronella foenum-graecum, Citrus reticulata peel, Ficus hirta, Smilax glabra, Saussurea costus, Zingiber officinale, Osmanthus fragrans (from Guangxi Zhuang Autonomous Region), and Albizia julibrissin. It is mainly used to prepare antidepressant preparations, which can improve depressive-like behaviors induced by chronic unpredictable stress, meeting the needs of antidepressant treatment.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to a moxa stick for soothing the liver and relieving depression, its preparation method, and its application in improving depressive symptoms. Background Technology

[0002] Depression is a common affective disorder in clinical practice. Chronic, unpredictable stress is a significant risk factor for the development and progression of depression. In recent years, the global incidence of this disease has continued to rise, becoming a public health issue affecting the physical and mental health and quality of life of the public, and there is a significant clinical need for treatment. Currently, the mainstream antidepressant treatments are mainly chemical drugs. Although these drugs can alleviate depressive symptoms to some extent, they generally have problems such as long onset of action, high incidence of adverse reactions, poor long-term patient adherence, and easy relapse of symptoms after discontinuation, which limits their clinical application. Traditional Chinese medicine has accumulated rich clinical experience in the intervention of depression. Existing Chinese herbal medicine formulas for treating depression are mostly formulated around the core pathogenesis of liver qi stagnation, deficiency of both heart and spleen, and insufficiency of liver and kidney. When the body has internal obstruction of dampness and turbidity, and qi stagnation and blood stasis, it is easy to induce emotional depression. Existing formulas are difficult to accurately match the core pathogenesis of depression in this type of person with dampness and blood stasis. They have limited effect on improving depressive-like behaviors induced by chronic stress. At the same time, they are not good at regulating the disorder of key neurotransmitter levels such as serotonin and dopamine in the body under the depressive state. They are difficult to meet the clinical needs for precise antidepressant treatment for regional populations. Summary of the Invention

[0003] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0004] Another objective of this invention is to provide a compound moxa stick containing a liver-soothing and mood-regulating formula, which can effectively improve depressive-like behavior induced by chronic unpredictable stress, upregulate the levels of serotonin and dopamine in the body's serum, correct neurotransmitter imbalances in a depressive state, compensate for the shortcomings of existing antidepressants with many adverse reactions, and provide a safe and effective TCM formula option for clinical intervention of depression.

[0005] To achieve these objectives and other advantages of the present invention, a compound moxa stick for soothing the liver and relieving depression is provided. The raw materials are weighed according to the following parts by weight: 14-16 parts by weight of Artemisia argyi, 0.5-0.7 parts by weight of Murraya paniculata, 0.8-1.0 parts by weight of Curcuma longa, 0.5-0.7 parts by weight of Cyperus rotundus (processed with vinegar), 0.2-0.4 parts by weight of Sappanwood, 0.5-0.7 parts by weight of Spatholobus suberectus, 0.5-0.7 parts by weight of Jasminum sambac, 0.5-0.7 parts by weight of Citronella foenum-graecum, 0.2-0.4 parts by weight of Citrus reticulata peel, 0.4-0.6 parts by weight of Ficus hirta, 0.4-0.6 parts by weight of Smilax glabra, 0.4-0.6 parts by weight of Ligusticum sinense, 0.2-0.4 parts by weight of fresh ginger, 0.5-0.7 parts by weight of Osmanthus fragrans from Guangxi Zhuang Autonomous Region, and 0.5-0.7 parts by weight of Albizia julibrissin.

[0006] A method for preparing a compound moxa stick based on a liver-soothing and mood-regulating formula includes the following steps: Weigh all the raw materials in the above-mentioned weight proportions; All raw materials except mugwort leaves are placed in a pulverizer and pulverized. After pulverization, the powder is passed through a 140-160 mesh sieve to obtain fine powder of traditional Chinese medicine. Artemisia leaves are processed into Artemisia floss with a floss ratio of 5-10:1; All the prepared moxa wool and all the fine powder of traditional Chinese medicine were placed in a mixing container and stirred continuously to make the fine powder of traditional Chinese medicine evenly dispersed in the moxa wool, thus obtaining the mixed moxa wool and medicinal herbs. The mixture of medicinal and moxa wool was weighed quantitatively according to the fixed filling amount of a single moxa stick; Mulberry bark paper is used as the paper for rolling. The quantitatively measured mixture of medicinal moxa wool is placed evenly in the moxa stick rolling machine, and the rolling machine is started to complete the rolling, resulting in a moxa stick blank with a diameter of 1.8-2.2 cm and a length of 19-21 cm. The joints and end faces of the moxa stick blank are sealed with special sealing adhesive for moxa sticks; The sealed moxa stick blank is placed in a cool and ventilated place to air dry naturally, resulting in the finished compound moxa stick.

[0007] Preferably, all the prepared moxa wool and all the fine powder of traditional Chinese medicine are placed in a mixing container and continuously stirred and turned. The specific steps are as follows: First, divide all the fine powder of Chinese medicine into two portions, A and B, by weight. At the same time, divide all the moxa wool into the first moxa wool and the second moxa wool by weight ratio of 15:85. A portion of fine Chinese medicine powder and the first portion of Artemisia flakes were placed together in a sealed pressure-resistant container. The ambient temperature inside the container was stabilized at 0-4 ℃ and the internal air pressure was stabilized at 0.22-0.28 MPa. Clean and dry compressed air was introduced for airflow mixing for 10 minutes. After completion, the mixture was discharged at room temperature and pressure to obtain drug-loaded locked fiber powder. The prepared drug-loaded locked fiber powder, B part of traditional Chinese medicine fine powder and the second moxa wool are then placed in a three-dimensional motion mixer and mixed for 25 minutes at a speed of 16-19 r / min and an ambient relative humidity of 38%-42% to obtain a uniformly dispersed drug-moxa wool mixture.

[0008] Preferably, the specific steps for introducing clean and dry compressed air for air mixing are as follows: First, reserve 3%-5% of the total mass of the fine powder of Chinese medicine A as a pilot fine powder. Put the remaining fine powder of Chinese medicine A and the first batch of moxa wool into a sealed pressure-resistant container. After sealing, adjust the temperature inside the container to stabilize at 0-4 ℃ and pre-pressurize the air pressure to 0.12-0.15 MPa. Let it stand for 8 min. Take the reserved pilot fine powder and place it in an external ultrasonic atomizing can, add an equal mass of anhydrous ethanol, and atomize it for 12 min at 0-4 ℃ and 180W ultrasonic power to obtain the saturated atomized gas of compound volatile oil. Mix the atomizing gas with clean, dry compressed air at a volume ratio of 1:9, stabilize the temperature of the mixed carrier gas at 0-4℃, and introduce it into the container at a constant rate of 0.02 MPa / min until the gas pressure stabilizes at 0.22-0.28 MPa. Maintain this condition for airflow circulation and mixing for 10 min. After mixing, the pressure was uniformly released to atmospheric pressure at a rate of 0.01 MPa / min, and the drug-loaded locked fiber powder was obtained by discharging the material.

[0009] The application of the compound moxa stick of the liver-soothing and mood-relieving formula in the preparation of antidepressant preparations.

[0010] Preferably, the antidepressant is used to improve depressive-like behavior induced by chronic unpredictable stress.

[0011] The present invention has at least the following beneficial effects: First, the compound moxa sticks of the Liver-Soothing and Depression-Relieving Formula use mugwort leaves as the core, combined with a variety of authentic medicinal materials such as Murraya paniculata, Curcuma longa, Cyperus rotundus (processed with vinegar), Sappanwood, Spatholobus suberectus, Jasminum sambac, Citronella foenum-graecum, Citrus reticulata peel, Ficus hirta, Smilax glabra, Saussurea costus, ginger, Osmanthus fragrans from Guangxi Zhuang Autonomous Region, and Albizia julibrissin. The formula closely addresses the core pathogenesis of depression caused by internal dampness and turbidity, qi stagnation and blood stasis. It takes into account the multi-dimensional effects of resolving dampness and turbidity, promoting qi circulation, clearing blood stasis, and relieving liver depression. It can intervene from the root cause of depression, rather than just regulating a single symptom.

[0012] Secondly, in the preparation process of this compound moxa stick, the medicinal materials other than the mugwort leaves are first pulverized and passed through a 140-160 mesh sieve to obtain fine powder of traditional Chinese medicine. The mugwort leaves are processed into moxa floss with a floss ratio of 5-10:1. Then, the moxa floss and the fine powder of traditional Chinese medicine are thoroughly mixed, weighed quantitatively, rolled in mulberry paper, sealed, and naturally dried in a cool and ventilated place to obtain the finished product. The entire preparation process can retain the volatile active ingredients in the medicinal materials to the greatest extent, avoid damage to the medicinal substances caused by high-temperature processing, and at the same time, it can make the fine powder of traditional Chinese medicine evenly dispersed in the moxa floss fiber system, ensuring that the finished moxa stick burns stably and the medicinal components can be released stably and continuously with the moxa smoke.

[0013] Third, the mixing of moxa wool and fine Chinese medicine powder can be completed in a graded and step-by-step manner. First, divide all the fine Chinese medicine powder into two equal parts by mass, and at the same time, divide all the moxa wool into two parts by fixed mass. First, place the first part of fine Chinese medicine powder and the first part of moxa wool together in a sealed pressure-resistant container, control a stable low temperature and high pressure environment, and introduce clean and dry compressed air for airflow mixing. After restoring to normal temperature and pressure, discharge the material to obtain drug-loaded locked fiber powder. Then, place the obtained powder, the second part of fine Chinese medicine powder and the second part of moxa wool together in a three-dimensional motion mixer, control the appropriate speed and ambient humidity to complete the mixing, and obtain a uniformly dispersed mixture of medicine and moxa wool. Low temperatures allow for reversible expansion of the cell wall pores of the moxa wool plant fibers. Combined with the power of airflow, this allows the fine powder of traditional Chinese medicine to enter the fiber pores. Once the temperature and pressure return to normal, the fiber pores naturally shrink, allowing the fine powder of traditional Chinese medicine to be stably bound inside the moxa wool fibers. Subsequent uniform mixing ensures that the powder is evenly dispersed throughout the moxa wool system, preventing powder from falling off, settling, or agglomerating during subsequent processing, transportation, and combustion. This ensures a stable and consistent drug load in a single moxa stick and between different batches, and prevents excessive inhalable particulate matter from falling off during combustion. The smoke release is also more gentle and controllable.

[0014] Fourth, during the airflow mixing process, a fixed proportion of pilot fine powder can be reserved from the first batch of Chinese herbal powder. The remaining first batch of Chinese herbal powder and the first part of moxa wool are put into a sealed pressure-resistant container. After sealing, a stable low-temperature environment is regulated and the pressure is pre-pressurized to the set pressure and then left to stand. The reserved pilot fine powder is placed in an external ultrasonic atomizing tank, and an equal mass of anhydrous ethanol is added. The compound volatile oil saturated atomized gas is obtained by ultrasonic atomization in a low-temperature environment. The atomized gas is mixed with clean and dry compressed air at a fixed volume ratio. The temperature of the mixed carrier gas is regulated to be stable in the low-temperature range. The gas is introduced into the container at a fixed rate to the set pressure. The airflow circulation mixing is maintained under these conditions. After the mixing is completed, the pressure is released at a fixed rate to the normal pressure and then discharged. The carrier gas prepared in this way can achieve a balance with the volatile active ingredients in the raw medicinal materials, avoiding the extraction and dissipation of the core volatile medicinal components in the medicinal materials during the purging process of the dry airflow, and retaining the antidepressant active substances in the formula to the greatest extent. The trace amount of ethanol in the atomized gas can gently promote the swelling of the cell walls of the moxa fibers and improve the synchronicity of fiber pore expansion. Combined with the uniform pressurization and depressurization process, it can avoid the moxa fibers from becoming brittle and electrostatically agglomerated, further improving the binding stability of the medicinal powder and moxa fibers, and ensuring the uniformity of the burning performance and medicinal release of the moxa stick.

[0015] Fifth, this compound moxa stick can be used for antidepressant treatment, especially for intervention in depressive-like behaviors induced by chronic unpredictable stress. Treatment with this compound moxa stick can effectively improve symptoms such as anhedonia, anxiety-like behavior, and food suppression in new environments during a depressive state. Simultaneously, it can upregulate the levels of key neurotransmitters such as serotonin and dopamine in the body's serum, correcting monoamine neurotransmitter imbalances during a depressive state. The compound moxa stick of this invention can be administered via inhalation of moxa smoke, allowing the active ingredients to take effect rapidly, avoiding the first-pass effect of oral administration, reducing common adverse reactions of traditional chemical antidepressants, and providing a gentler administration method with better safety and compliance with long-term use.

[0016] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Detailed Implementation

[0017] The present invention will now be described in further detail so that those skilled in the art can implement it based on the description.

[0018] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0019] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0020] Example 1 This embodiment provides a compound moxa stick for a liver-soothing and mood-lifting formula, which contains the following ingredients by weight: 15 parts mugwort leaves, 0.6 parts Murraya paniculata, 0.9 parts Curcuma longa, 0.6 parts Cyperus rotundus (processed with vinegar), 0.3 parts Sappanwood, 0.6 parts Spatholobus suberectus, 0.6 parts Jasminum sambac, 0.6 parts Citronella foenum-graecum, 0.3 parts dried tangerine peel, 0.5 parts Ficus hirta, 0.5 parts Smilax glabra, 0.5 parts Clematis chinensis, 0.3 parts fresh ginger, 0.6 parts Osmanthus fragrans (from Guangxi Zhuang Autonomous Region), and 0.6 parts Albizia julibrissin.

[0021] The specific preparation method of the compound moxa stick of the Liver-Soothing and Depression-Relieving Formula in this embodiment is as follows: Weigh all the above-mentioned raw materials by weight, and put all the raw materials except for the mugwort leaves into a pulverizer for pulverization. After pulverization, pass the powder through a 150-mesh sieve to obtain fine powder of traditional Chinese medicine for later use. Process the weighed mugwort leaves into mugwort floss with a floss ratio of 8:1 for later use. All the prepared moxa wool and all the fine powder of traditional Chinese medicine were placed in a mixing container and stirred continuously for 30 minutes to make the fine powder of traditional Chinese medicine evenly dispersed in the moxa wool, thus obtaining the mixed moxa wool of medicine and moxa wool. The mixture of medicinal moxa wool was weighed quantitatively according to a fixed filling amount of 20 g per moxa stick. Mulberry bark paper was used as the paper for rolling. The quantitatively measured mixture of medicinal moxa wool was placed evenly in the moxa stick rolling machine, and the rolling machine was started to complete the rolling, resulting in a moxa stick blank with a diameter of 2.0 cm and a length of 20 cm. The joints and end faces of the moxa stick blank are sealed with special sealing adhesive for moxa sticks; The sealed moxa stick blanks are placed in a cool, ventilated place to air dry naturally for 72 hours to obtain the finished compound moxa sticks.

[0022] I. Effect Test 1. Materials and Methods 1.1 Laboratory Animals Thirty SPF-grade male ICR mice, 6-8 weeks old and weighing 18-22g, were purchased from Hunan Silek Jingda Experimental Animal Co., Ltd. The mice were housed in an SPF-grade barrier animal facility with strictly controlled environmental parameters: temperature (25±2)℃, relative humidity 40%-60%, and a 12h / 12h alternating light and dark regime. They were allowed free access to irradiated sterilized SPF-grade mouse maintenance feed and autoclaved purified water. Bedding was autoclaved and changed weekly to ensure a clean and stable environment. After one week of acclimatization, and once no abnormalities were observed, the mice were randomly divided into five groups using a random number table: a blank control group, a model group, a single moxa stick group (moxa stick group), a compound moxa stick group (compound moxa group), and a positive control group (fluoxetine group), with six mice in each group.

[0023] 1.2 Main Reagents and Instruments The formula for soothing the liver and relieving depression was prepared in Example 1.

[0024] Fluoxetine hydrochloride (positive control, Eli Lilly and Company, USA).

[0025] Cyclophosphamide for injection, for future use.

[0026] Mouse 5-HT and DA ELISA kit (Wuhan Beilaiyin Company).

[0027] Single moxa stick (Guai Ai product, 27.5 g / stick).

[0028] 1% sodium pentobarbital injection, analytical grade anhydrous ethanol, and sterile physiological saline.

[0029] Open field test chamber (60 cm×60 cm×60 cm, with matching animal behavior video analysis system), sucrose preference test cage, new environment feeding inhibition test cage, self-made moxa smoke fumigation device (60 cm×60 cm×180 cm), high-speed refrigerated centrifuge, full-wavelength microplate reader, electronic analytical balance, high-speed grinder, moxa stick rolling machine, constant temperature water bath.

[0030] 1.3 Experimental Methods 1.3.1 Preparation and grouping of depression model Following previously reported classical methods and optimized for our experimental conditions, a mouse model of depression was established using chronic unpredictable stress (CUS) combined with isolation. The modeling period lasted four weeks. Except for the control group, which was housed in groups of five, all other groups were housed individually. Each day, one or two different stressors were randomly selected using a random number table to provide stimulation. The same stimulus was not administered consecutively to avoid adaptation in the mice. The stressors used included: 24-hour fasting, 24-hour water deprivation, tail clamping for 1 minute, horizontal restraint for 2 hours, moist bedding for 24 hours, cage tilting at 45° for 12 hours, reversed day-night cycle for 24 hours, swimming in 4°C cold water for 5 minutes, and continuous noise stimulation for 2 hours.

[0031] After one week of acclimatization feeding, mice were randomly divided into 5 groups of 6 mice each using a random number table. The treatment protocols for each group are as follows: Control group: Normal group rearing, no stress stimulation, daily gavage with an equal volume of sterile saline, no other intervention.

[0032] Model group: Individually housed in single cages, with continuous CUS stimulation and daily gavage administration of an equal volume of sterile saline, without drug intervention.

[0033] Single moxa stick group: Individually housed in single cages, receiving CUS stimulation throughout the process, and being given an equal volume of sterile saline by gavage daily, while simultaneously receiving single moxa stick smoke inhalation intervention.

[0034] Compound moxa stick group: Individually housed in single cages, receiving CUS stimulation throughout the process, and being given an equal volume of sterile saline by gavage daily, while simultaneously receiving compound moxa stick smoke inhalation intervention.

[0035] Fluoxetine positive control group (Western medicine group): Individually housed and subjected to CUS modeling stimulation throughout the process, while simultaneously administered fluoxetine hydrochloride 10 mg / kg / d by gavage at a fixed time each day, with an administration volume of 10 mL / kg.

[0036] 1.3.2 Methods of Intervention with Artemisia Smoke The moxibustion smoke intervention and model establishment were initiated simultaneously. Starting from the first day of model establishment, the intervention was conducted at a fixed time every evening, once a day, for four consecutive weeks. During the intervention, mice in the single moxa stick group and the compound moxa stick group were placed in a self-made closed moxibustion smoke fumigation device measuring 60 cm × 60 cm × 180 cm. The device was equipped with smoke distribution channels and exhaust gas adsorption and filtration modules. The temperature and humidity inside the device were kept consistent with the breeding environment during the intervention. The corresponding moxa sticks were lit, and 2 / 3 of a single moxa stick (27.5 g per stick, approximately 18.3 g burned per session) was burned each time. The fumigation time for each session was controlled to be 30 minutes.

[0037] Mice in the control group and the model group were placed in a smokeless ventilation device of the same specification for 30 minutes at the same time to ensure that the environmental exposure and operation procedures of mice in each group were completely consistent except for the intervention factor of smoke, and to eliminate the interference of confounding factors.

[0038] 1.3.3 Behavioral Testing All behavioral tests were completed on days 1-3 after modeling and intervention. The test order was sucrose preference test, open field test, and new environment feeding inhibition test, with each test spaced 24 hours apart to avoid interference from stress effects between experiments. All tests were conducted in a quiet behavioral testing room with uniform and constant lighting. Mice were allowed to acclimatize in the testing room environment for 30 minutes before the test.

[0039] Sucrose Preference Experiment (SPT): Sucrose acclimatization training was conducted on days 26-27 after model establishment, and the formal test was performed on day 28. During the acclimatization training phase, each cage of mice was simultaneously given two bottles of 1% sucrose solution. After 24 hours, these were replaced with one bottle of 1% sucrose solution and one bottle of pure water, allowing the mice to adapt to the two-bottle selection pattern of sucrose solution and pure water. Before the formal test, mice were deprived of water for 24 hours. During the test, they were placed in individual cages and simultaneously given one pre-weighed bottle of 1% sucrose solution and one bottle of pure water. The consumption of the two bottles was recorded continuously for 6 hours. During the test, the positions of the two bottles were swapped every 3 hours to eliminate the influence of positional preference on the results. The sucrose preference rate was calculated using the formula: Sugar water preference rate (%) = Sugar water consumption / (Sugar water consumption + Pure water consumption) × 100%.

[0040] Open field test (OFT): An open field test chamber measuring 60 cm × 60 cm × 60 cm was used. The bottom of the chamber was divided into a peripheral area and a central activity area (20 cm × 20 cm in the center). During the formal test, mice were gently placed in a fixed corner of the open field chamber facing the chamber wall and allowed to explore freely for 5 minutes. The SuperMaze animal behavior video analysis system was used to automatically record the total distance traveled by the mice, the number of times they entered the central area, and the time spent in the central area. After each mouse was tested, the inner walls and bottom of the open field chamber were immediately wiped thoroughly with 75% ethanol. The next mouse was tested only after the ethanol had completely evaporated to eliminate any odor residue from the previous mouse.

[0041] New Environment Feeding Inhibition Test (NSFT): Mice were fasted and deprived of water for 24 hours before the test. The new cages used in the test were identical in size to the rearing cages, with a 2 cm thick layer of clean bedding at the bottom and one pellet of standard mouse maintenance food placed in the center. During the test, the mouse was gently placed in a fixed corner of the cage, facing the wall. The time interval from when the mouse was placed in the cage until it picked up the food with its forelimbs and began to eat was recorded as the feeding latency. If the mouse did not exhibit feeding behavior within 300 seconds (5 minutes) after being placed in the cage, the experiment was terminated, and the feeding latency was recorded as 300 seconds. After each mouse was tested, the cage was thoroughly cleaned and disinfected with 75% ethanol to eliminate any residual odor.

[0042] 1.4 Statistical Methods Data analysis was performed using SPSS 22.0 statistical software, and statistical graphs were generated using GraphPad Prism 9.0 software. All continuous data were first tested for normality using the Shapiro-Wilk method. Data conforming to a normal distribution were expressed as mean ± standard deviation (x ± s). One-way ANOVA was used to compare means among multiple groups. When variances were homogeneous, pairwise comparisons between groups were performed using the LSD-t test; when variances were unequal, pairwise comparisons between groups were performed using Dunnett's T3 test. A p-value < 0.05 was considered statistically significant.

[0043] II. Test Results 2.1 Sucrose Preference Experiment The sucrose preference test was used to assess anhedonia-like depressive behavior in mice. The results are shown in Table 1. Compared with the control group, the sucrose preference rate in the CUS model group was significantly lower (P<0.05), indicating that the model mice exhibited typical anhedonia behavior, and the depression model was successfully established in this experiment. Compared with the model group, the sucrose preference rate in the single moxa stick group, compound moxa stick group, and fluoxetine positive control group was significantly higher (P<0.05). The compound moxa stick group showed a similar improvement in sucrose preference rate as the fluoxetine positive control group, suggesting that moxa smoke inhalation intervention can effectively reverse CUS-induced anhedonia-like depressive behavior in mice.

[0044] Table 1. Effect of moxa sticks on the sugar water preference rate in depressed mice (x±s, n=6) Note: *Compared with the blank group P <0.05; # Comparison with the model group P <0.05.

[0045] 2.2 Open Field Experiment The open field test was used to assess anxiety-like behaviors, autonomous exploration ability, and spontaneous activity levels associated with depression in mice. The results are shown in Table 2. Compared with the control group, the CUS model group showed a significant decrease in the number of central area entries and a significant shortening of the central area dwell time (P<0.05), indicating that the model mice exhibited significant anxiety-like behaviors, a significantly reduced willingness to autonomously explore unfamiliar environments, and a decreased spontaneous activity ability, thus successfully establishing a depression model. Compared with the model group, the compound moxa stick group showed a significant increase in the central area dwell time (P<0.05), while the fluoxetine positive control group showed a significant increase in the number of central area entries and a significant increase in the central area dwell time (P<0.05). The single moxa stick group showed a certain upward trend in the number of central area entries and dwell time compared with the model group, but the difference was not statistically significant (P>0.05). The results indicate that compound moxa stick inhalation intervention can effectively improve CUS-induced anxiety-like depressive behavior in mice and enhance their autonomous exploration ability.

[0046] Table 2. Effects of moxa sticks on the number of times moxa sticks crossed the central region and the time spent in the central region in depressed mice (x±s) Note: *Compared with the blank group P <0.05; # Comparison with the model group P <0.05.

[0047] 2.3 New Environment Feeding Inhibition Experiment The novel environment feeding inhibition test is a classic behavioral indicator for evaluating the effect of antidepressant interventions. It is mainly used to assess anxiety-like behaviors accompanying depression in mice and the feeding inhibition effect induced by novel environment stress. The experimental results are shown in Table 3. Compared with the control group, the feeding latency of mice in the CUS model group was significantly prolonged (P<0.05), indicating that the model mice exhibited significant anxiety-like behaviors and feeding inhibition in novel environments, consistent with the core behavioral characteristics of depression models. The depression model was successfully established in this experiment. Compared with the model group, the feeding latency of mice in the compound moxa stick group and the fluoxetine positive control group was significantly shortened (P<0.05); the feeding latency of mice in the single moxa stick group showed a decreasing trend compared with the model group, but the difference was not statistically significant (P>0.05). The results indicate that compound moxa stick inhalation intervention can effectively reverse CUS-induced novel environment anxiety-like behaviors in mice, alleviate feeding inhibition under stress, and exert a clear antidepressant and anti-anxiety effect.

[0048] Table 3 Effect of moxa sticks on the feeding latency of depressed mice (x±s, n=6) Note: *Compared with the blank group P <0.05; # Comparison with the model group P <0.05.

[0049] 2.4 Detection of serum neurotransmitter 5-HT and DA levels After the behavioral experiment, mouse serum was collected and the 5-HT and DA ELISA kit was strictly followed according to the instructions. The steps of sample addition, incubation, washing, binding of enzyme-labeled secondary antibody, color development and termination of reaction were completed in sequence. The absorbance (OD value) of each well was measured using a full-wavelength microplate reader, and the content of 5-HT and DA in serum was calculated according to the standard curve.

[0050] The results are shown in Table 4. Compared with the control group, the serum 5-HT and DA levels in the CUS model group mice were significantly decreased (P<0.05), suggesting that chronic unpredictable stress can significantly reduce the levels of monoamine neurotransmitters in mice. Compared with the model group, the serum 5-HT and DA levels in the single-acid moxa group, compound moxa group, and fluoxetine group were significantly increased (P<0.05), and all recovered to levels close to those of the control group, with no significant differences among the three groups. This indicates that moxa smoke inhalation can correct neurotransmitter imbalances in depressed model mice by upregulating serum 5-HT and DA levels, thereby exerting an antidepressant effect.

[0051] Table 4. Effects of moxa sticks on serum 5-HT and DA levels in depressed mice (x±s) Note: *Compared with the blank group P <0.05; # Comparison with the model group P <0.05.

[0052] In summary, behavioral tests showed that the intervention significantly increased the sucrose preference rate in the model mice, significantly increased the number of times they entered and stayed in the central area of ​​the open field experiment, and significantly shortened the latency period for feeding in the new environment. Serum test results showed that the levels of serotonin and dopamine in the mouse serum significantly rebounded, which can effectively improve the depressive-like behavior of mice and correct the monoamine neurotransmitter disorder in the depressive state.

[0053] Example 2 This embodiment provides a compound moxa stick based on a formula for soothing the liver and relieving depression. The difference between this embodiment and Example 1 is that the specific preparation method for this compound moxa stick is as follows: Weigh all the above-mentioned raw materials by weight, and put all the raw materials except for Artemisia argyi into a grinder for grinding. After grinding, pass the powder through a 150-mesh sieve to obtain fine powder of Chinese medicine for later use. The weighed mugwort leaves are processed into mugwort floss with a floss-making ratio of 8:1, and set aside for later use. The obtained fine powder of Chinese medicine is divided into two parts, A and B, by equal mass. At the same time, all the moxa wool is divided into the first moxa wool and the second moxa wool by mass ratio of 15:85, for later use. A portion of fine Chinese medicine powder and the first portion of mugwort floss were placed together in a sealed pressure-resistant container. The ambient temperature inside the container was stabilized at 2 ℃ and the internal air pressure was stabilized at 0.25 MPa. Clean and dry compressed air was introduced for airflow mixing for 10 min. After completion, the mixture was slowly restored to room temperature and pressure before being discharged to obtain drug-loaded locked fiber powder. The prepared drug-loaded locked fiber powder, B part of traditional Chinese medicine fine powder and the second moxa wool are then placed together in a three-dimensional motion mixer and mixed for 25 minutes at a speed of 18 r / min and an ambient relative humidity of 40% to obtain a uniformly dispersed drug-moxa wool mixture. The mixture of medicinal moxa wool was weighed quantitatively according to a fixed filling amount of 20 g per moxa stick. Mulberry bark paper was used as the paper for rolling. The quantitatively measured mixture of medicinal moxa wool was placed evenly in the moxa stick rolling machine, and the rolling machine was started to complete the rolling, resulting in a moxa stick blank with a diameter of 2.0 cm and a length of 20 cm. The joints and two ends of the moxa stick blank are sealed with special sealing adhesive for moxa sticks; the sealed moxa stick blank is placed in a cool and ventilated place to air dry naturally for 72 hours to obtain the finished compound moxa stick.

[0054] Example 3 This embodiment provides a compound moxa stick based on a formula for soothing the liver and relieving depression. The difference between this embodiment and Embodiment 2 is that the specific preparation method for this compound moxa stick is as follows: Weigh all the above-mentioned raw materials by weight, and put all the raw materials except for Artemisia argyi into a grinder for grinding. After grinding, pass the powder through a 150-mesh sieve to obtain fine powder of Chinese medicine for later use. The weighed mugwort leaves are processed into mugwort floss with a floss-making ratio of 8:1, and set aside for later use. The obtained fine powder of Chinese medicine is divided into two parts, A and B, by equal mass. At the same time, all the moxa wool is divided into the first moxa wool and the second moxa wool by mass ratio of 15:85, for later use. First, reserve 4% of the total mass of the fine powder of Chinese medicine A as a pilot fine powder. Then, put the remaining fine powder of Chinese medicine A and the first moxa wool into a sealed pressure-resistant container. After sealing, adjust the temperature inside the container to stabilize at 2 ℃ and pre-pressurize the air pressure to 0.14 MPa, and let it stand for 8 min. Take the reserved pilot fine powder and place it in an external ultrasonic atomizing can, add an equal mass of anhydrous ethanol, and atomize it for 12 min at 2 ℃ and 180 W ultrasonic power to obtain the saturated atomized gas of compound volatile oil. The atomizing gas and clean, dry compressed air were mixed at a volume ratio of 1:9. The temperature of the mixed carrier gas was stabilized at 2 ℃ and introduced into the container at a constant rate of 0.02 MPa / min until the gas pressure stabilized at 0.25 MPa. The gas flow was maintained under these conditions for 10 min. After mixing, the pressure was uniformly released to atmospheric pressure at a rate of 0.01 MPa / min, and the drug-loaded locked fiber powder was obtained by discharging the material. The prepared drug-loaded locked fiber powder, B part of traditional Chinese medicine fine powder and the second moxa wool are then placed together in a three-dimensional motion mixer and mixed for 25 minutes at a speed of 18 r / min and an ambient relative humidity of 40% to obtain a uniformly dispersed drug-moxa wool mixture. The mixture of medicinal moxa wool was weighed quantitatively according to a fixed filling amount of 20 g per moxa stick. Mulberry bark paper is used as the rolling paper. The quantitatively measured mixture of medicinal moxa wool is placed evenly in the moxa stick rolling machine, and the rolling machine is started to complete the rolling, resulting in a moxa stick blank with a diameter of 2.0 cm and a length of 20 cm. The joints and two ends of the moxa stick blank are sealed with special sealing adhesive for moxa sticks. The sealed moxa stick blanks are placed in a cool, ventilated place to air dry naturally for 72 hours to obtain the finished compound moxa sticks.

[0055] I. Effect Test 1. Verification of core quality indicators of finished moxa sticks 1.1 Detection Method Ten moxa sticks each from Examples 3, 2, and 1 were prepared, and the following indicators were tested: Drug loading uniformity and powder shedding rate: Using eucalyptol and turmeric as indicators, the RSD of single-branch axial drug loading, the RSD of batch drug loading, and the powder shedding rate during the rolling process were calculated. 1.2 Relative Standard Deviation (RSD): This reflects the dispersion of a set of test data. The smaller the value, the better the uniformity of drug loading. The calculation formula is: ; The standard deviation (SD) is calculated using the Bessel formula: ; In the formula: Xi is the detection value of the i-th time. is the arithmetic mean of all detected values, and n is the number of tests.

[0056] Drug loading: The content of the index component is indicated by eucalyptol in Artemisia argyi and turmeric dione in Curcuma longa. The commonly used unit is mg / g (the number of milligrams of the index component per gram of sample).

[0057] 1.3 Calculation method for single-branch axial drug loading RSD Testing procedure: Take one compound moxa stick to be tested, cut it into three equal parts (upper, middle, and lower) along the axial direction, weigh each part, and use HPLC to determine the drug loading of the index components in the three parts of the sample to obtain three drug loading test values.

[0058] Step-by-step calculation: Calculate the arithmetic mean of the drug load in the three sections of this moxa stick. : = (X on + X 中 +X 下 ) / 3 Calculate the standard deviation (SD) of the three test values ​​using the Bessel formula; The axial drug load (RSD) of a single moxa stick is calculated using the general RSD formula, and the result is expressed as a percentage.

[0059] 1.4 Method for calculating the inter-batch drug loading RSD Testing method: Take 10 compound moxa sticks from the same batch and measure the overall average drug loading of each moxa stick (by crushing the whole moxa stick), and obtain 10 individual drug loading test values.

[0060] Step-by-step calculation Calculate the arithmetic mean of the drug loading values ​​of 10 moxa sticks. ; Calculate the standard deviation (SD) of the 10 test values ​​using the Bessel formula; The inter-batch drug loading RSD of this batch of moxa sticks was calculated using the general RSD formula, and the result was expressed as a percentage.

[0061] The inter-batch drug loading RSD is essentially the intra-batch uniformity of drug loading among different moxa sticks within the same batch. This indicator reflects the quality uniformity of the same batch of products. The smaller the RSD value, the smaller the difference in drug loading between different moxa sticks in the same batch, and the better the inter-batch quality stability of the product.

[0062] 1.5 Calculation method for powder shedding rate during the rolling process Testing procedure: During the moxa stick rolling process, keep the rolling equipment and process parameters constant, record the total mass of the mixed moxa wool and medicinal herbs put into the rolling process, and after the quantitative moxa stick rolling is completed, collect all the free medicinal powder that has fallen off in the rolling machine, the spreading platform, and the gaps in the equipment (pass through a 150-mesh sieve to remove moxa wool fiber debris), and weigh the dry weight of the fallen medicinal powder.

[0063] Calculation formula: The powder loss rate during the rolling process (%) = (total mass of fine Chinese medicine powder lost during the rolling process / total mass of fine Chinese medicine powder contained in the mixed moxa wool added to the rolling process) × 100%.

[0064] 1.6 Drug Powder Lock-in Rate: The drug-loaded locking fiber powder prepared in each example (for Example 1, the mixed drug-artemisia floss after stirring) was placed in a 200-mesh standard test sieve and horizontally purged with clean compressed air at 0.1 MPa for 5 min. The mass of drug powder floating under the sieve was collected, and the drug powder lock-in rate was calculated according to the formula: Lock-in rate (%) = (Total mass of added powder - Mass of free powder) / Total mass of added powder × 100%; 1.7 Retention rate of volatile active ingredients: The content of the core antidepressant volatile active ingredients eucalyptol, cyperone, osmanthus aldehyde and turmeric dione in the formulation were determined by HPLC. The relative retention rate of each indicator component was calculated based on the theoretical content corresponding to the theoretical amount of raw materials.

[0065] 1.8 Serum Neurotransmitter Detection: After satisfactory anesthesia of mice via intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg), whole blood samples were collected by enucleation and placed in enzyme-free coagulation tubes. After standing at room temperature for 60 min, the blood samples were centrifuged at 4 ℃ and 3500 r / min (centrifugation radius 8 cm) for 15 min. The supernatant serum was carefully separated, aliquoted into enzyme-free EP tubes, and stored at -80 ℃ for later use. Repeated freeze-thaw cycles should be avoided before testing.

[0066] Samples were processed and tested strictly in accordance with the instructions for the mouse 5-hydroxytryptamine (5-HT) and dopamine (DA) ELISA kit. The absorbance (OD) values ​​at the corresponding wavelengths were measured using a full-wavelength microplate reader. The actual content of 5-HT and DA in each sample was calculated based on the standard curve plotted with the kit's accompanying standards.

[0067] 1.2 Test Results Table 5 Comparison of core quality indicators of compound moxa sticks prepared using three different processes The results show that, compared with the conventional stirring and mixing process in Example 1, the stepwise mixing process used in Example 2 can significantly reduce the difference in drug loading in the axial direction of a single moxa stick and between batches, and greatly reduce the shedding of medicinal powder during processing. This fundamentally solves the technical problems of easy shedding and sedimentation of fine Chinese herbal powder and uneven drug loading, ensuring the stability of the release of medicinal components in the finished moxa sticks. Compared with Example 2, the compound moxa stick prepared in Example 3 shows a significantly improved drug powder locking rate, further optimized drug loading uniformity, and a lower drug powder shedding rate during processing. Simultaneously, it can retain the core volatile antidepressant active ingredients in the formula to the greatest extent, fundamentally solving the technical problems of volatile medicinal component loss during airflow mixing, electrostatic aggregation and brittleness of moxa fibers, low drug powder locking rate, and large batch-to-batch differences, providing a quality foundation for the stable efficacy of the finished moxa sticks.

[0068] 2. Pharmacodynamic validation test 2.1 The compound moxa sticks prepared in this embodiment were used to intervene in the depression model mice induced by chronic unpredictable stress (CUS). The experimental animals, modeling methods, grouping treatment, intervention plan, and serum neurotransmitter indicators were completely consistent with those in Example 1. The intervention was carried out once a day for 30 minutes each time, starting from the first day of modeling, for 4 consecutive weeks.

[0069] 2.2 Serum neurotransmitter detection results Table 6. Effects of Compound Moxa Sticks from Example 3 on Serum 5-HT and DA Levels in Depressed Mice (x±s, n=6) Note: *Compared with the blank group P<0.05; # Comparison with the model group P <0.05.

[0070] Serum serotonin (5-HT) and dopamine (DA) are core monoamine neurotransmitters that regulate emotional responses and depressive behavior. Abnormal downregulation of their levels is one of the core pathological features of depression induced by chronic unpredictable stress (CUS). This experiment used ELISA to detect the levels of 5-HT and DA in the serum of mice in each group, aiming to verify the corrective effect of compound moxa sticks prepared using different processes on neurotransmitter imbalances in a depressive model mouse. The results are shown in Table 6.

[0071] Compared with the control group, the serum levels of 5-HT and DA in mice in the CUS model group were significantly reduced (P<0.05), indicating that chronic unpredictable stress has successfully induced typical monoamine neurotransmitter disorders in mice, and the depression model of this experiment has been successfully established.

[0072] Compared with the model group, the single moxa stick intervention significantly upregulated the serum 5-HT and DA levels in depressed model mice (P<0.05), confirming that simple moxa smoke inhalation has a certain neurotransmitter regulation and depressive state improvement effect. On this basis, the compound moxa stick of the present invention, prepared by conventional stirring and mixing process in Example 1, had a similar upregulation effect on serum 5-HT and DA in mice as the single moxa stick group, with no significant statistical difference between the two. Both could significantly restore the disordered neurotransmitter levels in the model mice, confirming that the compound moxa stick of the present invention can synergistically play a role in correcting neurotransmitter disorders on the basis of pure moxa sticks, and has a clear antidepressant pharmacological basis.

[0073] Building upon this, Example 2, using a graded and stepwise mixing process, produced a compound moxa stick that significantly upregulated serum 5-HT and DA levels in mice. The 5-HT level increased from 201.56±4.98 ng / mL in Example 1 to 210.75±4.62 ng / mL, and the DA level increased from 808.51±33.67 pg / mL to 812.47±32.85 pg / mL, both levels being closer to the normal physiological levels of the control group. These results indicate that the graded and stepwise mixing process, through low-temperature, high-pressure airflow mixing, physically locks the fine powder of traditional Chinese medicine within the moxa fibers. The three-dimensional motion mixing then constructs a uniform and stable drug-carrying system, fundamentally preventing powder loss and medicinal component loss during processing, transportation, and combustion. This ensures the stable and continuous release of antidepressant active ingredients in the moxa smoke, thereby more effectively correcting monoamine neurotransmitter disorders in depressed mice and further enhancing the antidepressant intervention effect of the compound moxa stick.

[0074] Compared with Example 2, the compound moxa stick prepared by Example 3 using an optimized airflow mixing process showed a more prominent effect in correcting the neurotransmitter disorder in the serum of depressed model mice. The serum 5-HT level in the mice was further increased to 214.82±4.31 ng / mL, and the DA level was increased to 815.94±32.16 pg / mL. Both indicators have basically returned to the normal physiological levels of the blank group, and the upregulation effect on neurotransmitters is better than the positive control of fluoxetine, a first-line antidepressant in clinical practice. The results indicate that the design of the compound volatile oil saturated atomization carrier gas can avoid the loss of the core volatile antidepressant active ingredients in the formula during the airflow mixing process based on the principle of phase equilibrium. At the same time, through process optimization such as the mild swelling of fiber cell walls mediated by trace amounts of ethanol, pre-pressurization and static setting, and uniform pressure increase and decrease, the locking rate and dispersion uniformity of the powder in the moxa wool fiber are further improved. This ensures the stable and continuous release of the active ingredients during the burning of the moxa stick, and retains the volatile antidepressant active substances in the formula to the greatest extent. It can completely reverse the CUS-induced monoamine neurotransmitter disorder in mice, and ultimately achieve a significant improvement in the antidepressant efficacy of the compound moxa stick.

[0075] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. A compound moxa stick with a formula for soothing the liver and relieving depression, characterized in that, The raw materials are weighed according to the following parts by weight: 14-16 parts by weight of Artemisia argyi, 0.5-0.7 parts by weight of Murraya paniculata, 0.8-1.0 parts by weight of Curcuma longa, 0.5-0.7 parts by weight of Cyperus rotundus (processed with vinegar), 0.2-0.4 parts by weight of Sappanwood, 0.5-0.7 parts by weight of Spatholobus suberectus, 0.5-0.7 parts by weight of Jasminum sambac, 0.5-0.7 parts by weight of Citronella foenum-graecum, 0.2-0.4 parts by weight of Citrus reticulata peel, 0.4-0.6 parts by weight of Ficus hirta, 0.4-0.6 parts by weight of Smilax glabra, 0.4-0.6 parts by weight of Ligusticum sinense, 0.2-0.4 parts by weight of fresh ginger, 0.5-0.7 parts by weight of Osmanthus fragrans from Guangxi Zhuang Autonomous Region, and 0.5-0.7 parts by weight of Albizia julibrissin.

2. The method for preparing the compound moxa stick of the liver-soothing and mood-regulating formula according to claim 1, characterized in that, Includes the following steps: Weigh all the raw materials in the above-mentioned weight proportions; All raw materials except mugwort leaves are placed in a pulverizer and pulverized. After pulverization, the powder is passed through a 140-160 mesh sieve to obtain fine powder of traditional Chinese medicine. Artemisia leaves are processed into Artemisia floss with a floss ratio of 5-10:1; All the prepared moxa wool and all the fine powder of traditional Chinese medicine were placed in a mixing container and stirred continuously to make the fine powder of traditional Chinese medicine evenly dispersed in the moxa wool, thus obtaining the mixed moxa wool and medicinal herbs. The mixture of medicinal and moxa wool was weighed quantitatively according to the fixed filling amount of a single moxa stick; Mulberry bark paper is used as the paper for rolling. The quantitatively measured mixture of medicinal moxa wool is placed evenly in the moxa stick rolling machine, and the rolling machine is started to complete the rolling, resulting in a moxa stick blank with a diameter of 1.8-2.2 cm and a length of 19-21 cm. The joints and end faces of the moxa stick blank are sealed with special sealing adhesive for moxa sticks; The sealed moxa stick blank is placed in a cool and ventilated place to air dry naturally, resulting in the finished compound moxa stick.

3. The method for preparing the compound moxa stick of the liver-soothing and mood-regulating formula according to claim 1, characterized in that, All the prepared moxa wool and all the fine powdered Chinese medicine were placed in a mixing container and stirred continuously. The specific steps are as follows: First, divide all the fine powder of Chinese medicine into two portions, A and B, by weight. At the same time, divide all the moxa wool into the first moxa wool and the second moxa wool by weight ratio of 15:

85. A portion of fine Chinese medicine powder and the first portion of Artemisia flakes were placed together in a sealed pressure-resistant container. The ambient temperature inside the container was stabilized at 0-4℃ and the internal air pressure was stabilized at 0.22-0.28 MPa. Clean and dry compressed air was introduced for airflow mixing for 10 minutes. After completion, the mixture was discharged at room temperature and pressure to obtain drug-loaded locked fiber powder. The prepared drug-loaded locked fiber powder, B part of traditional Chinese medicine fine powder and the second moxa wool are then placed in a three-dimensional motion mixer and mixed for 25 minutes at a speed of 16-19 r / min and an ambient relative humidity of 38%-42% to obtain a uniformly dispersed drug-moxa wool mixture.

4. The method for preparing the compound moxa stick of the liver-soothing and mood-regulating formula according to claim 1, characterized in that, The specific steps for introducing clean and dry compressed air for air mixing are as follows: First, reserve 3%-5% of the total mass of the fine powder of Chinese medicine A as a pilot fine powder. Put the remaining fine powder of Chinese medicine A and the first batch of moxa wool into a sealed pressure-resistant container. After sealing, adjust the temperature inside the container to be stable at 0-4 ℃ and pre-pressurize the air pressure to 0.12-0.15MPa. Let it stand for 8 minutes. Take the reserved pilot fine powder and place it in an external ultrasonic atomizing can, add an equal mass of anhydrous ethanol, and atomize it for 12 min at 0-4 ℃ and 180 W ultrasonic power to obtain the saturated atomized gas of compound volatile oil. Mix the atomizing gas with clean, dry compressed air at a volume ratio of 1:9, stabilize the temperature of the mixed carrier gas at 0-4 ℃, and introduce it into the container at a constant rate of 0.02 MPa / min until the gas pressure stabilizes at 0.22-0.28 MPa. Maintain this condition for airflow circulation and mixing for 10 min. After mixing, the pressure was uniformly released to atmospheric pressure at a rate of 0.01 MPa / min, and the drug-loaded locked fiber powder was obtained by discharging the material.

5. The application of the compound moxa stick of the liver-soothing and mood-relieving formula according to claim 1 in the preparation of antidepressant preparations.

6. The application according to claim 5, characterized in that, The antidepressant is used to improve depressive-like behavior induced by chronic unpredictable stress.

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