A method for forming an animal model of spleen yang deficiency syndrome based on ice coptis chinensis decoction

Abstract

The present invention discloses a method for forming an animal model of spleen yang deficiency syndrome based on a decoction of coptis chinensis, comprising taking male SD rats as model animals and adaptively raising them for several days; gavage-feeding the model animals with the decoction of coptis chinensis; then placing the model animals in a cold environment for 60 to 90 minutes for cold stimulation; after the cold stimulation is completed, removing the model animals and placing them in a room temperature environment for normal feeding; and continuously treating them for multiple days to obtain an animal model of spleen yang deficiency syndrome. The present invention uses a decoction of coptis chinensis combined with cold stimulation in a cold environment to induce spleen yang deficiency syndrome in rats. The high-dose decoction of coptis chinensis is a bitter and cold medicine, which can have a similar effect on spleen yang damage caused by the bitter and cold purgative effects of rhubarb and senna. At the same time, the coptis chinensis is treated at low temperatures before gavage, which further aggravates its cold effect. Therefore, although the coptis chinensis has no purgative effect, its enhanced cold property can still contribute to the formation of spleen yang deficiency syndrome.

Description

Technical Field

[0001] The invention relates to the technical field of animal modeling, and in particular to a method for forming an animal model of spleen yang deficiency syndrome based on a water decoction of coptis chinensis. Background Art

[0002] Spleen Yang Deficiency Syndrome refers to a deficiency of spleen Yang, resulting in a failure to warm and transport the spleen, leading to the internal generation of Yin and cold, manifested by symptoms of deficiency and cold, including loss of appetite, abdominal distension, pain, and loose stools. This syndrome is often caused by further development of spleen Qi deficiency; or by excessive consumption of raw and cold foods, direct external cold, or excessive consumption of bitter and cold foods, which damage spleen Yang; or by kidney Yang deficiency, which weakens the Mingmen Fire, preventing fire from nourishing the earth. This leads to spleen Yang deficiency, impaired warming and transport, internal generation of cold, and failure to transport food and transform moisture. Spleen Yang Deficiency is generally caused by spleen Yang deficiency and impaired transport and transformation, resulting in abdominal distension and loss of appetite. Yang deficiency leads to internal generation of cold, which stagnates Qi, resulting in abdominal pain that responds to warmth and pressure. Yang deficiency prevents the transformation of moisture, leading to loose, thin, and watery stools. Spleen Yang deficiency fails to warm the extremities, resulting in coldness. Weakened middle Yang leads to internal retention of moisture and dysfunctional bladder Qi transformation, resulting in difficulty urinating. Flowing through the skin can cause heaviness and discomfort in the limbs, even swelling. Leukorrhea in women can result in excessive and watery vaginal discharge. A pale, puffy tongue with a slippery white coating, and a deep, slow, and weak pulse are all symptoms of Yang Qi deficiency and internal cold and dampness. Clinical manifestations include loss of appetite, abdominal distension, persistent abdominal pain, a preference for warmth and pressure, a fear of cold, cold extremities, a pale, pale, or puffy complexion, a bland mouth without thirst, loose stools or even undigested food, edema of the limbs, scanty urination, or clear, thin, and profuse vaginal discharge. A pale, puffy tongue with teeth marks, a slippery white coating, and a deep, slow, and weak pulse.

[0003] Traditional Chinese Medicine (TCM) syndrome models utilize certain biological characteristics of animals to simulate human syndromes. Spleen deficiency syndrome is a common syndrome in TCM clinical practice. To explore the nature of spleen deficiency, modern researchers have established a series of distinctive animal models, each of which simulates the clinical manifestations and biological characteristics of spleen deficiency to varying degrees.

[0004] The methods for establishing animal models of spleen yang deficiency have been published in many documents. From the perspective of modeling mechanism, they can generally be divided into the following methods: bitter and cold purgative method, cold and dampness obstructing the spleen method, irregular diet method, Western medicine method, surgery method and complex factor method.

[0005] Reference 1: Zeng Meiyan et al. published "A Review of Research on the Construction and Evaluation of Animal Models for Spleen Yang Deficiency Syndrome," which outlines methods for modeling spleen yang deficiency syndrome. These methods include single-factor and composite-factor approaches, with single-factor approaches further divided into Traditional Chinese Medicine (TCM) and Western Medicine (WM) approaches.

[0006] In early research on animal models of spleen yang deficiency syndrome, single-factor modeling was widely used. Based on analysis of the pathogenesis and clinical manifestations of spleen yang deficiency syndrome, methods such as bitter and cold purgation, cold-dampness obstructing the spleen, dietary irregularities, Western medication, and surgery were developed. Single-factor modeling methods in Traditional Chinese Medicine (TCM) include bitter and cold purgation, cold-dampness obstructing the spleen, and dietary irregularities; while single-factor modeling methods in Western medicine include the use of reserpine and interscapular brown fat resection. The specific modeling procedures are described below.

[0007] Bitter and cold purgative method: The bitter and cold purgative method involves gavage of purgatives to rats, inducing spleen yang deficiency through the cooling and purgative effects of the drugs. Commonly used purgatives include rhubarb and senna leaves.

[0008] Cold-Dampness Spleen Stagnation Method: The spleen favors dryness and dislikes dampness. Invasion of cold and dampness can easily lead to dampness in the spleen and dysfunction of its transport and transformation. This method replicates the syndrome of spleen yang deficiency by simulating a cold and damp environment. Rat cages were lined with thick sawdust and placed in an artificial climate chamber to simulate a cold and damp environment. The temperature was maintained at (6±2)°C and the humidity at (90±4)%. After 30 days of modeling, Wistar rats developed typical symptoms of spleen yang deficiency.

[0009] Dietary Disorders: The Suwen Bilun Pian states, "Excessive consumption of food and drink can damage the stomach and intestines." Based on this theory, researchers used dietary disorder to construct an animal model of spleen yang deficiency. By feeding mice cabbage and then adding lard once every two days (in any amount), the mice developed symptoms of spleen yang deficiency after nine days.

[0010] Western medicine single factor modeling method

[0011] Western medicine modeling: Animal models of spleen yang deficiency can be established using modern Western medicine, with reserpine being a common treatment. Reserpine affects the central nervous system, depleting catecholamines in the body, leading to sympathetic dysfunction and symptoms similar to spleen yang deficiency. Guo Jian et al. injected Wistar rats intraperitoneally with reserpine at a dose of 0.2 mg / kg once daily for three days. The rats developed symptoms such as weight loss, lethargy, diarrhea, and shrivelled fur.

[0012] Surgical Method: Interscapular brown fat is the primary heat-producing substance in adult animals. Resection of interscapular brown fat can reduce heat production and attenuate Yang Qi. A spleen Yang deficiency model was established by resection of interscapular brown fat tissue. The mice were fed a normal diet for the first 41 days. On the 42nd day, scapular brown fat resection was performed. The mice were fed a normal diet for 28 days after surgery.

[0013] Composite Factor Modeling: Modern people are prone to spleen yang deficiency due to factors such as a preference for raw and cold foods, a preference for cold foods, and irregular sleep and rest schedules. This leads to more complex clinical symptoms. Therefore, single-factor modeling is no longer sufficient for constructing animal models of spleen yang deficiency. Given the complexity of pathogenic factors and clinical manifestations, a growing number of studies are employing composite factor modeling. The following summarizes and discusses the use of composite factor modeling to establish animal models of spleen yang deficiency.

[0014] Traditional Chinese Medicine composite factor modeling method:

[0015] Most Traditional Chinese Medicine (TCM) composite factor modeling methods first establish a spleen-qi deficiency model, then construct a yang-qi deficiency model through bitter and cold purgatives. This approach has the advantage of being closer to the clinical pathogenesis of spleen-yang deficiency, and the resulting model more closely reflects the comprehensive clinical manifestations of spleen-yang deficiency. In the first phase, Xiong Rui et al. treated SD rats with dietary instability and excessive fatigue to injure their spleen. They were fed 10g of cabbage per rat on odd days and gavaged with 2mL of lard per rat on even days. They were then forced to swim to exhaustion in 4°C water each afternoon. In the second phase, they used bitter and cold purgatives to injure spleen-yang, administering 1.5mL of a 1g / mL senna leaf extract daily. Successful modeling was indicated when the rats exhibited symptoms such as curling up, diarrhea, and inability to swim.

[0016] Hu Yuanxiang et al. used the bitter and cold purgative method plus hunger and satiety disorder to replicate the spleen yang deficiency syndrome model. They gave rats 10 mL / kg of rhubarb decoction once a day, fasting on odd days and not fasting on even days to establish the spleen yang deficiency syndrome model. Ji Aidong et al.

[14] used the bitter and cold purgative method combined with the cold and dampness obstructing the spleen method to replicate the spleen yang deficiency syndrome model. They gavaged SD rats with 9 g / kg of rhubarb powder solution and cultured the rats in a climate modeling box simulating a cold and damp environment for 9 days to obtain the spleen yang deficiency syndrome model.

[0017] Document 2: "Experimental Study on Changes in MDA, SOD, GSH-Px, T-AOC, 8-OHdG, and Telomere Length in Rats with Spleen Qi Deficiency, Spleen Yin Deficiency, and Spleen Yang Deficiency Syndrome" published by Shang Bing; the article also discloses the evaluation criteria for the symptoms of the spleen yang deficiency syndrome model in rats, which mainly include decreased swimming endurance, fatigue, squinting, arched back, decreased rectal temperature, curling up, weight loss, decreased food intake, loose stools, and dry fur.

[0018] The modeling effect evaluation criteria or contents disclosed in Document 2 are basically the same as most recognized modeling evaluation methods. It can be seen that the evaluation methods for spleen yang deficiency syndrome in the prior art are relatively similar. The present invention can use the above-mentioned evaluation method to evaluate modeling animals and judge the modeling effect.

[0019] Literature 3: Li Qin et al. summarized the evaluation methods of animal models obtained by the animal modeling method of spleen yang deficiency in "Research Progress on Modeling Methods and Model Evaluation of Animal Models of Spleen Yang Deficiency Syndrome".

[0020] Common symptoms of spleen yang deficiency in animal models induced by the above-mentioned modeling methods include decreased food intake, fatigue, loose stools, withered fur, decreased rectal temperature, and a tendency to gather in groups. However, each modeling method also has its own unique individual symptoms. Models induced by bitter and cold purgatives are often accompanied by persistent diarrhea and rectal prolapse; models induced by cold and dampness obstructing the spleen are accompanied by decreased water intake and joint swelling; models induced by irregular diet are often accompanied by dry stools followed by loose stools; and the Traditional Chinese Medicine (TCM) complex etiology method, which involves multiple factors, is more clinically relevant and lacks distinct individual characteristics. Neither Western medicine nor surgical methods conform to TCM etiology. Western medicine models are often accompanied by depression, while surgical methods have relatively few repeated trials and their individual characteristics are not yet clear.

[0021] The microscopic indicators of the spleen yang deficiency model are generally detected and compared with the biochemical indicators of animals. The specific microscopic indicators are as follows:

[0022]

[0023]

[0024] The article also discloses the use of bitter and cold purgation method to establish a spleen yang deficiency model, which is a modeling method that simulates the cause of disease in traditional Chinese medicine. It is simple to operate, the modeling conditions are easy to control, and the repeatability is good. However, most literature does not report the natural recovery time of the model, and the stability of the model cannot be known.

[0025] Reference 4: Yang Yanqun's "Study on a Composite Factor Modeling Method for Spleen Yang Deficiency in Rats" discloses that the composite factor modeling method replicates the symptoms of a spleen yang deficiency rat model, with most symptoms generally consistent with the syndromic manifestations of spleen yang deficiency. In particular, the reduced urinary D-xylose concentration and increased urine volume in the model group are new findings. However, the model's natural recovery time is short, and the experimental methodology still needs to be improved to make the resulting model more suitable for clinical practice and research needs.

[0026] Reference 5: "Establishment of a Rat Model of Spleen Yang Deficiency and Observation of the Efficacy of Lizhong Decoction" published by Yang Yanqun et al.; This document discloses that rats modeled with reserpine injection combined with a multifactorial composite method developed clinical symptoms consistent with spleen yang deficiency. These symptoms did not recover even three weeks after the modeling process was discontinued, indicating that the model established using this method is relatively stable and that the natural recovery period can exceed three weeks. After treatment with Lizhong Decoction, the urinary D-xylose levels of the model rats returned to normal levels by the third week of treatment, a relatively shorter recovery time compared to the natural recovery group. Three weeks after the modeling factors were removed, gastric acidity increased in both the natural recovery group and the Lizhong Decoction group, with the latter group showing a more pronounced increase, but no significant difference compared to the normal group.

[0027] Published modeling methods demonstrate that Traditional Chinese Medicine (TCM) models disease by simulating etiology and pathogenesis, while Western medicine often models disease by simulating certain symptoms of spleen yang deficiency through simple pathological damage. For example, reserpine does not directly affect the spleen, but rather affects the central nervous system, depleting catecholamines in the body, leading to sympathetic dysfunction and symptoms similar to spleen yang deficiency. Surgical methods remove the main heat-producing substances, causing the body to become cold. This represents an organic injury and does not conform to the TCM theory of spleen yang deficiency, resulting in a loss of warmth and an internal accumulation of yin and cold.

[0028] Although the modeling method of single-factor or compound-factor Chinese medicine is more in line with the process and mechanism of the TCM etiology of spleen yang deficiency syndrome, it is simple to operate, the modeling conditions are easy to control, and the repeatability is good, but it has been found through experiments that the natural recovery time of most models is short and the stability of the models is poor. For example, Document 3 points out that the natural recovery time of most TCM single-factor or traditional TCM compound-factor modeling methods is not public, and its stability cannot be known. Document 4 mentions that the natural recovery time of the compound-factor modeling method to replicate the spleen yang rat model is short. Document 5 uses the Western medicine method of reserpine for modeling, and the natural recovery time exceeds 3 weeks, with good stability. It can be seen from this that although the modeling method under the theory of traditional Chinese medicine is more operational, its stability is short compared to the natural recovery time of Western medicine, and its stability is poor. In the process of pathological research on spleen yang deficiency syndrome, it is often necessary to verify the effects of various counter-evidence drugs. For example, Zhang Wentong et al. published "Experimental Study on the Effect of Fuzi Lizhong Pills on Relieving Low Heat Production in Rats with Spleen Yang Deficiency Syndrome". In this experimental study, Fuzi Lizhong Pills were used to regulate spleen yang deficiency syndrome, and the effect of Fuzi Lizhong Pills on the adaptive heat production of spleen yang deficiency model rats was verified. It was found that Fuzi Lizhong Pills upregulated the content of in situ heat-producing molecular proteins in skeletal muscle of spleen yang deficiency, reduced the energy charge of skeletal muscle and enhanced the heat production potential of rats with spleen yang deficiency, reduced the energy charge of liver and enhanced the heat production potential of rats with spleen yang deficiency, and reduced the ATPase activity of skeletal muscle of rats with spleen yang deficiency.

[0029] For example, Ning Wanling et al. published "Study on the Expression and Intervention Mechanism of Aconite Lizhong Decoction on APQ4 and ANP in Rats with Spleen Yang Deficiency Syndrome". This experimental study observed the effects of Aconite Lizhong Decoction on the levels of aquaporin 4 and atrial natriuretic peptide ANP in rats with spleen yang deficiency syndrome, and explored its mechanism of involvement in the regulation of water metabolism in rats with spleen yang deficiency syndrome.

[0030] In these experiments, the animals are usually first modeled, and then gavage-administered with the corresponding medication, such as Fuzi Lizhong Decoction or other traditional Chinese medicines that promote yang and strengthen the spleen, to determine their effects. After gavage with Fuzi Lizhong Decoction, the experimental animals are not gavaged with the modeling medication to avoid any interaction. In this case, if the modeling animals recover quickly, there will not be sufficient time to verify the effect of the counter-drug, or the differences in the comparison process will not be easy to distinguish.

[0031] Trigonosciadium infects the liver and spleen meridians; it promotes qi circulation and relieves pain, dissipates blood stasis, and is neutral in nature, with a tendency to descend and drain, particularly in the liver and spleen blood, guiding blood stasis downward. Coptis chinensis is bitter and cold in nature. It enters the heart, spleen, stomach, liver, gallbladder, and large intestine meridians. Coptis chinensis, bitter in flavor and cold in nature, clears heat and dampness, and purges fire and detoxifies. It primarily contains protoberberine-type alkaloids, inhibitors of topoisomerases I and II, larch resinol, and trans-(4-hydroxyphenyl)ethylferulate. Currently, no studies have identified its use as a modeling agent for animal models of spleen yang deficiency syndrome.

[0032] To sum up, although the existing modeling methods of traditional Chinese medicine or Western medicine have a long natural recovery time and stability, their modeling methods do not conform to the theory of traditional Chinese medicine and the mechanism of etiology. The modeling methods based on the theory of traditional Chinese medicine and Chinese medicine have a short natural recovery time and poor stability. Summary of the Invention

[0033] The purpose of the present invention is to provide a method for establishing an animal model of spleen yang deficiency syndrome based on ice coptis chinensis decoction, which can comply with the etiology theory of spleen yang deficiency syndrome in traditional Chinese medicine and has good stability.

[0034] To achieve the above object, one embodiment of the present invention provides a method for forming an animal model of spleen yang deficiency syndrome based on a decoction of Coptis chinensis, comprising the following steps:

[0035] Step (1) male SD rats weighing between 180 g and 220 g were used as model animals and were adaptively raised for several days;

[0036] Step (2) gavage-feeding the model animals with a decoction of Rhizoma Coptidis once a day at a dose of 10 ml / kg to 15 ml / kg each time, with the temperature of the decoction of Rhizoma Coptidis being 3° C. to 5° C.; then placing the model animals in a cold environment for 60 min to 90 min for cold stimulation, and after the cold stimulation is completed, removing the model animals and placing them in a room temperature environment for normal feeding with free access to water;

[0037] Repeat step (2) and continue treating according to step (2) for 14 to 18 days to obtain an animal model of spleen yang deficiency syndrome.

[0038] In a preferred embodiment of the present invention, the modeling animals in step (1) are male SD rats weighing 200 g to 210 g. After obtaining the modeling animals, they are adaptively raised for 3 to 7 days.

[0039] In a preferred embodiment of the present invention, in step (2), the patient is gavage-fed at a dose of 12 ml / kg each time every day, and the temperature of the icy coptis chinensis decoction is 4°C during gavage.

[0040] In a preferred embodiment of the present invention, the cold environment in step (2) is a breeding environment with a temperature of -2°C to 2°C. The model animals are prohibited from drinking water and eating during the cold stimulation process, and each model animal is raised individually; the duration of each cold stimulation is 60 minutes.

[0041] In a preferred embodiment of the present invention, the preparation method of the ice coptis chinensis decoction is as follows:

[0042] A1. Take 25g-28g of Coptis chinensis and 5g-8g of Trigonosciadium japonicum, add water and soak them at a material-liquid ratio of 1:20-22 for the first decoction. After decoction, filter the medicinal liquid. Add water to the medicinal residue at a material-liquid ratio of 1:15-16 for the second decoction. After the second decoction is completed, filter the medicinal liquid and combine the two medicinal liquids to obtain a total decoction.

[0043] A2. Use a rotary evaporator to concentrate the total decoction to 50% to 55% of the original volume to obtain a concentrated solution. After packaging the concentrated solution, refrigerate it at 3°C ​​to 5°C until use.

[0044] In a preferred embodiment of the present invention, the preparation method of the ice coptis chinensis decoction is as follows:

[0045] A1. Take 27g of Coptis chinensis and 6g of Trigonosciadium japonicum, add water and soak them at a solid-liquid ratio of 1:20 for the first decoction. After decoction, filter the medicinal liquid. Add water to the medicinal residue at a solid-liquid ratio of 1:15 for the second decoction. After the second decoction is completed, filter the medicinal liquid and combine the two medicinal liquids to obtain a total decoction.

[0046] A2. Concentrate the total decoction to 55% of the original volume using a rotary evaporator to obtain a concentrate. Aliquot the concentrate and store in a refrigerator at 4°C for later use.

[0047] In summary, the present invention has the following advantages:

[0048] The present invention uses a water decoction based on ice coptis root combined with cold stimulation in a cold environment to achieve spleen yang deficiency syndrome in rats. The high-dose water decoction of coptis root is a bitter and cold medicine, which can have a similar spleen yang-damaging effect as the bitter and cold purgative effect of rhubarb and senna leaves. At the same time, the coptis root is treated at low temperature before being fed, which further aggravates the cold effect of coptis root. Although coptis root has no purgative effect, its enhanced coldness can still contribute to the formation of spleen yang deficiency syndrome. At the same time, trifoliate vine is a qi-moving and qi-relieving medicine with a strong qi-moving effect. The water decoction forms an interaction between the bitter and cold effects of ice coptis root and trifoliate vine to move qi and relieve qi. The spleen yang deficiency syndrome formed after long-term treatment has significant symptoms. It also has good stability, which is conducive to subsequent related experimental research.

[0049] Trigonosciadium japonicum has the effect of breaking up blood, so it is generally contraindicated in patients with qi deficiency and physical weakness. It should also be used with caution in patients with both qi and blood deficiency, or spleen and stomach weakness without stagnation. The reason for this is that Trigonosciadium japonicum has a strong effect of promoting qi and relieving qi, which can aggravate qi deficiency in patients with qi deficiency or weakness, leading to a worsening of their condition. Trigonosciadium japonicum also breaks up qi in the blood, making recovery from qi rupture slow and difficult, resulting in a long recovery time. The "Compendium of Materia Medica" records the theory that "spleen deficiency should not be treated with purgation, descending, or promoting qi." This suggests that spleen deficiency, which is inherently low in qi, will exacerbate or prolong symptoms of spleen deficiency by taking Trigonosciadium japonicum. DETAILED DESCRIPTION

[0050] The present invention provides a method for forming an animal model of spleen yang deficiency syndrome based on a decoction of coptis chinensis, comprising the following steps:

[0051] Step (1) male SD rats weighing between 180 g and 220 g were used as model animals and were adaptively raised for several days;

[0052] Step (2) gavage-feeding the model animals with a decoction of Rhizoma Coptidis once a day at a dose of 10 ml / kg to 15 ml / kg each time, with the temperature of the decoction of Rhizoma Coptidis being 3° C. to 5° C.; then placing the model animals in a cold environment for 60 min to 90 min for cold stimulation, and after the cold stimulation is completed, removing the model animals and placing them in a room temperature environment for normal feeding with free access to water;

[0053] Repeat step (2) and continue treating according to step (2) for 14 to 18 days to obtain an animal model of spleen yang deficiency syndrome.

[0054] Example 1:

[0055] A method for forming an animal model of spleen yang deficiency syndrome based on a decoction of coptis chinensis, comprising the following steps:

[0056] Step (1) Male SD rats weighing 200 g ± 3 g were used as model animals and were adaptively raised for 5 days;

[0057] Step (2) gavage-feeding the model animals with a decoction of Rhizoma Coptidis (Coptidis Rhizoma) once a day at a dose of 12 ml / kg each time, with the temperature of the decoction of Rhizoma Coptidis (Coptidis Rhizoma) at 4°C; then placing the model animals in a cold environment at 0°C for 60 minutes for cold stimulation. During the cold stimulation, the model animals were prohibited from drinking water or eating, and each model animal was housed individually. After the cold stimulation was completed, the model animals were removed and placed in an environment at room temperature of 20°C for normal feeding, with free access to water;

[0058] Repeat step (2) and continue to treat according to step (2) for 15 days to obtain an animal model of spleen yang deficiency syndrome.

[0059] Example 2:

[0060] A method for forming an animal model of spleen yang deficiency syndrome based on a decoction of coptis chinensis, comprising the following steps:

[0061] Step (1) Male SD rats weighing about 210 g were used as model animals and adaptively raised for 4 days;

[0062] Step (2) gavage-feeding the model animals with a decoction of Rhizoma Coptidis (Coptidis chinensis) once a day at a dose of 13 ml / kg each time, with the temperature of the decoction at 3°C; then placing the model animals in a cold environment at -1°C for 70 minutes for cold stimulation. During the cold stimulation, the model animals were prohibited from drinking water or eating, and each model animal was housed individually. After the cold stimulation was completed, the model animals were removed and placed in an environment at room temperature of 22°C for normal feeding, with free access to water;

[0063] Repeat step (2) and continue treating according to step (2) for 14 days to obtain an animal model of spleen yang deficiency syndrome.

[0064] Example 3:

[0065] A method for forming an animal model of spleen yang deficiency syndrome based on a decoction of coptis chinensis, comprising the following steps:

[0066] Step (1) Male SD rats weighing about 200 g were used as model animals and adaptively raised for 6 days;

[0067] Step (2) gavage-feeding the model animals with a decoction of Rhizoma Coptidis (Coptidis chinensis) once a day at a dose of 14 ml / kg each time, with the temperature of the decoction of Rhizoma Coptidis (Coptidis chinensis) at 3°C; then placing the model animals in a cold environment at 1°C for 75 minutes for cold stimulation. During the cold stimulation, the model animals were prohibited from drinking water or eating, and each model animal was housed individually. After the cold stimulation was completed, the model animals were removed and placed in an environment at room temperature of 22°C for normal feeding, with free access to water;

[0068] Repeat step (2) and continue treating according to step (2) for 16 days to obtain an animal model of spleen yang deficiency syndrome.

[0069] Example 4

[0070] The preparation method of the ice coptis chinensis decoction is as follows:

[0071] A1, take 26g of Coptis chinensis and 6g of Sparganium, add 640g of water to soak at a solid-liquid ratio of 1:20 and decoct for the first time, filter the medicinal liquid after decoction, the quality of the medicinal residue is slightly larger than the original medicine, and the medicinal residue adds 575g of water to carry out the second decoction at a solid-liquid ratio of 1:15. After the second decoction is completed, filter the medicinal liquid, and merge the two medicinal liquids to obtain a total decoction;

[0072] A2. Use a rotary evaporator to concentrate the total decoction to 50% of its original volume to obtain a concentrated solution, i.e., concentrate the total decoction to half. After the concentrated solution is divided into aliquots, it is placed in a refrigerator at 4°C for later use.

[0073] Example 5

[0074] The preparation method of the ice coptis chinensis decoction is as follows:

[0075] A1. Take 27g of Coptis chinensis and 6g of Trigonosciadium japonicum, add water and soak them at a solid-liquid ratio of 1:20 for the first decoction. After decoction, filter the medicinal liquid. Add water to the medicinal residue at a solid-liquid ratio of 1:15 for the second decoction. After the second decoction is completed, filter the medicinal liquid and combine the two medicinal liquids to obtain a total decoction.

[0076] A2. Concentrate the total decoction to 55% of the original volume using a rotary evaporator to obtain a concentrate. Aliquot the concentrate and store in a refrigerator at 4°C for later use.

[0077] In Example 1, the icy coptis chinensis water decoction of Example 4 was used, and in Examples 2 and 3, the icy coptis chinensis water decoction of Example 5 was used for research and testing.

[0078] Experimental Example 1: Evaluation of Modeling Results

[0079] The model animals in Example 1 were observed and physically and chemically evaluated. There are many evaluation indicators for the animal model of spleen yang deficiency syndrome. In terms of its macroscopic manifestations, many experimenters use the symptoms of weight loss, poor appetite, fatigue, weakness, cold limbs, chills, loose stools, and withered fur to determine whether the model is successful. Generally, the following six indicators can be used to evaluate the spleen yang deficiency syndrome rat model:

[0080] (1) Chills: The rectal temperature of rats was measured and showed a significant decrease;

[0081] (2) Emaciation: The rats' body weight decreased significantly;

[0082] (3) Weakness: The gripping strength of both forelimbs is significantly reduced;

[0083] (4) Poor food intake: The daily food and water intake of individual rats decreased significantly;

[0084] (5) Withered fur;

[0085] (6) If the stool is loose, the water content is tested.

[0086] Take 20 rats for the modeling test in Example 1, and use SD rats of the same weight as the control group for comparison. The control group is raised normally and fed with the same weight of water instead of the ice coptis chinensis decoction. The forelimb gripping force is tested by using the YLS-13A rat and mouse gripping force tester. First, press the rat on the gripping plate with your right hand, push the gripping plate forward with your left hand, then slide your right hand backward to the tail of the rat, and gently release the gripping plate with your left hand. When the rat grabs the gripping plate with force, apply force and pull it back in time to obtain the maximum gripping force. A dedicated person measures continuously for 3 times and takes the average value. There are 20 rats in both Example 1 and the control group of this experiment. The average value of the comparison data is taken as the experimental data. The experimental results are as follows:

[0087]

[0088]

[0089] From the above experimental results, it can be seen that the macroscopic characteristics of the experimental animals obtained by modeling in Example 1 of the present invention are consistent with the situation of spleen yang deficiency in traditional Chinese medicine theory, with characteristics such as emaciation, hypothermia, fatigue, and loose stools, indicating that the modeling was successful.

[0090] Experimental Example 2: Animal Model Stability Test

[0091] (1) After the modeling is completed, the animals are evaluated and analyzed. If the modeling is successful, a stability analysis can be performed to determine the natural recovery time of the modeled animals under the condition of spleen yang deficiency. The longer the natural recovery time, the better the stability, and the more conducive it is to subsequent counter-evidence drugs or other animal drug experimental studies. Natural recovery can be determined based on the macroscopic characteristics of the modeled rats, that is, when the weight of the modeled animals returns to the pre-modeling value, the rectal temperature returns to normal, the food intake returns to normal, the water content of the stool returns to normal, and the forelimb grip strength returns to normal, the modeled animals are considered to have fully recovered; because the judgment of hair condition is prone to large errors, when judging natural recovery, hair condition is used as an auxiliary analysis and is not used as one of the detection items for whether natural recovery has occurred.

[0092] (2) The modeling animals, SD rats, were placed in an environment of 20°C and raised normally. During the feeding process, they were provided with sufficient feed and water. The rats' housing was disinfected and replaced regularly to keep it dry. Body weight, rectal temperature, food intake, stool moisture content, and forelimb grip strength were tested at a fixed time every day. When the above five indicators all reached normal values, it was determined that the day was the natural recovery time of the SD rats. This time period was the natural recovery period of the modeling animals. The natural recovery period of each experimental rat was recorded, and the average natural recovery period of the experimental rats in this group was taken as the stability evaluation data of Experimental Example 2.

[0093] (3) Processing of discrete data: During the recovery process of SD rats under normal feeding, if the natural recovery time of a rat is significantly longer than that of other SD rats, or if the recovery time of any of the five indicators of the model animal is significantly longer than that of other SD rats or the average value, it is considered that the recovery of the five indicators of the SD rat may be affected by other reasons, such as the influence of other diseases. When the above situation occurs, the experimental data of the SD rat are discarded.

[0094] Experimental Example 3: Stability of different modeling methods

[0095] Experimental Example 3-1: Using the same method as in Example 1

[0096] Experimental Example 3-2: Reserpine Modeling Method

[0097] The same SD rats as in Example 1 were taken and reserpine was intraperitoneally injected into the SD rats at a dose of 0.2 mg·kg-1·d-1 for each rat for 13 consecutive days.

[0098] Experimental Example 3-3: Bitter and Cold Purgative Method

[0099] The same SD rats as in Example 1 were used for modeling. The rats were gavaged with 10 g / kg of 4°C senna solution once a day and swam for 10 minutes in 30°C water for 3 consecutive weeks.

[0100] Experimental Example 3-4:

[0101] SD rats were used as in Example 1. The model group rats were subjected to the following cycles over a 15-day period: (1) a full meal for 1 day, followed by a 2-day fast, with no water withdrawal, for a total of 5 cycles; (2) daily swimming in water at 35-37°C until exhaustion. After completion of the above two steps, proceed to step (3). (3) 20% senna leaf aqueous extract (2 mL / 100 g) was administered orally daily, once in the morning and evening, for 7 consecutive days.

[0102] The rats in the above-mentioned multiple experimental examples are all SD male rats from the same batch. Each group of examples preferably has 10 rats as test samples. The weight difference of each rat is small. Relevant experiments are carried out at the same time. After the model is obtained and the experimental animals are confirmed to have become spleen yang deficiency animal models through evaluation, they are immediately continued to be raised in a natural recovery state, and various macroscopic characteristics are detected. The time of complete natural recovery is recorded, as shown below:

[0103] category Experimental Example 3-1 Experimental Example 3-2 Experimental Example 3-3 Experimental Example 3-4 Mean time to recovery 20.5±1.4 days 16.4±0.8 days 12.2±0.9 days 14.8±1.3 days

[0104] As can be seen from Experimental Example 3, the natural recovery time of the model animals obtained by the method of the present invention is the longest and the most stable. The recovery time of the model animals obtained by the Western medicine modeling method of reserpine is second, and the stability is slightly better than that of the bitter and cold purgation method. This shows that the present invention uses the bitter and cold effects of Bing Huanglian to form spleen yang deficiency syndrome, and at the same time adds Sanleng to break up the qi in the blood, which aggravates the deficiency syndrome while also having a longer recovery time than the purgation method.

[0105] Experimental Example 4: Stability of different bitter and cold medicines

[0106] Experimental Example 4-1: Using the same method as in Example 1

[0107] Experimental Example 4-2: Using Dachengqi Decoction to Replace Binghuanglian

[0108] Step (1) Male SD rats weighing 200 g ± 3 g were used as model animals and were adaptively raised for 5 days;

[0109] Step (2) gavage-feed the modeling animals with 4°C iced Dachengqi decoction once a day at a dose of 12 ml / kg each time; then place the modeling animals in a cold environment at 0°C for 60 minutes for cold stimulation. After the cold stimulation is completed, the modeling animals are removed and placed in a room temperature environment at 20°C for normal feeding with free access to water; the treatment according to step (2) is continued for 15 days to obtain an animal model of spleen yang deficiency syndrome. The ratio of Dachengqi decoction is: 12 grams of rhubarb, 24 grams of magnolia bark, 12 grams of immature bitter orange, and 9 grams of sodium sulfate.

[0110] Experimental Example 4-3: Using Curcuma zedoaria to replace Trigonella ternata

[0111] Step (1) Male SD rats weighing 200 g ± 3 g were used as model animals and were adaptively raised for 5 days;

[0112] Step (2) gavage-feeding the model animals with a decoction of Rhizoma Coptidis (Coptidis Rhizoma) once a day at a dose of 12 ml / kg each time, with the temperature of the decoction of Rhizoma Coptidis (Coptidis Rhizoma) at 4°C; then placing the model animals in a cold environment at 0°C for 60 minutes for cold stimulation. During the cold stimulation, the model animals were prohibited from drinking water or eating, and each model animal was housed individually. After the cold stimulation was completed, the model animals were removed and placed in an environment at room temperature of 20°C for normal feeding, with free access to water;

[0113] Repeat step (2) and continue to treat according to step (2) for 15 days to obtain an animal model of spleen yang deficiency syndrome.

[0114] The preparation method of the ice coptis chinensis decoction is as follows:

[0115] A1, take 26g of Coptis chinensis and 6g of Curcuma zedoaria, add 640g of water to soak at a solid-liquid ratio of 1:20 and decoct for the first time, filter the medicinal liquid after decoction, the quality of the medicinal residue is slightly larger than that of the original medicine, and the medicinal residue adds 575g of water to carry out the second decoction at a solid-liquid ratio of 1:15. After the second decoction is completed, filter the medicinal liquid, and merge the two medicinal liquids to obtain a total decoction;

[0116] A2. Use a rotary evaporator to concentrate the total decoction to 50% of its original volume to obtain a concentrated solution, i.e., concentrate the total decoction to half. After the concentrated solution is divided into aliquots, it is placed in a refrigerator at 4°C for later use.

[0117] Example 4-4: Using Citrus aurantium to replace Trigonella ternata

[0118] Step (1) Male SD rats weighing 200 g ± 3 g were used as model animals and were adaptively raised for 5 days;

[0119] Step (2) gavage-feeding the model animals with a decoction of Rhizoma Coptidis (Coptidis Rhizoma) once a day at a dose of 12 ml / kg each time, with the temperature of the decoction of Rhizoma Coptidis (Coptidis Rhizoma) at 4°C; then placing the model animals in a cold environment at 0°C for 60 minutes for cold stimulation. During the cold stimulation, the model animals were prohibited from drinking water or eating, and each model animal was housed individually. After the cold stimulation was completed, the model animals were removed and placed in an environment at room temperature of 20°C for normal feeding, with free access to water;

[0120] Repeat step (2) and continue to treat according to step (2) for 15 days to obtain an animal model of spleen yang deficiency syndrome.

[0121] The preparation method of the ice coptis chinensis decoction is as follows:

[0122] A1, take 26g of Coptis chinensis and 6g of Immature Citrus aurantium, add 640g of water at a solid-liquid ratio of 1:20 to soak and carry out the first decoction, filter the medicinal liquid after decoction, the quality of the medicinal residue is slightly larger than that of the original medicine, and the medicinal residue adds 575g of water at a solid-liquid ratio of 1:15 to carry out the second decoction, filter the medicinal liquid after the second decoction is complete, and merge the two medicinal liquids to obtain a total decoction;

[0123] A2. Use a rotary evaporator to concentrate the total decoction to 50% of its original volume to obtain a concentrated solution, i.e., concentrate the total decoction to half. After the concentrated solution is divided into aliquots, it is placed in a refrigerator at 4°C for later use.

[0124] The rats in multiple experimental examples in Experimental Example 4 were all SD male rats from the same batch, with little difference in weight. The relevant experiments were conducted at the same time. After the model was established and the experimental animals were confirmed to have become spleen yang deficiency animal models through evaluation, they were immediately continued to be raised in a natural recovery state, and various macroscopic characteristics were tested. The time for complete natural recovery was recorded, as shown below:

[0125] category Experimental Example 4-1 Experimental Example 4-2 Experimental Example 4-3 Experimental Example 4-4 Mean time to recovery 20.5±1.3 days 13.6±1.3 days 15.8±0.8 days 14.5±0.6 days

[0126] As can be seen from Experimental Example 4, the model animals obtained by the method used in the present invention have the longest natural recovery time and the best stability. Among them, Experimental Example 4-2 used Dachengqi Decoction instead of iced coptis chinensis. Dachengqi Decoction also used ice-cold drugs for gavage and simultaneously applied cold stimulation. The natural recovery time of the animal model produced was also longer. However, since no drugs that break up blood and qi were added, its natural recovery time was also shorter than that of Experimental Example 4-1. Similarly, the mechanism of spleen yang deficiency formed in Experimental Example 4-4 is somewhat similar to that of Experimental Example 4-2, but coptis chinensis is different from rhubarb. Rhubarb is a bitter and cold purgative, while coptis chinensis is a bitter and cold heat-clearing drug and does not have a purgative effect. Rhubarb is a bitter and cold purgative, which also allows some coldness to be expelled during the experiment, mainly due to increased urine. Coptis chinensis does not have a purgative effect. It mainly accumulates coldness in the rats and aggravates it when combined with an ice-cold decoction. The immature bitter orange in Example 4-4 has the effects of regulating qi, eliminating accumulation, and resolving phlegm and dispersing abdominal distension. It is used to treat internal stagnation, fullness and pain in the abdomen, heaviness after diarrhea, constipation, phlegm stagnation and qi obstruction in the chest, chest congestion, gastroptosis, rectal prolapse, and uterine prolapse. It is often used in combination with liquorice, tangerine peel, huangling, betel nut, cyperus rotundus, qingpi, bupleurum, Shenqu, and platycodon.

[0127] Experimental Example 4-3 uses the replacement of Curcuma and Trigonella lobata. Both Curcuma and Trigonella lobata have the effect of breaking blood and qi, but Curcuma is mild in nature and Trigonella lobata is flat and bitter. Curcuma belongs to a warming medicine and can reduce the coldness of Coptis chinensis, so that the coldness pathogenic process of Coptis chinensis is adversely affected during the deficiency syndrome modeling process, resulting in prolonged modeling time, and the stability after modeling is worse than that of the control group. However, since Curcuma is still a blood and qi-breaking drug, it can also have an adverse effect on the qi in the blood of rats, so that the deficiency syndrome recovery time is also longer. It can be seen that the present invention accumulates coldness in rats by ice-cold Coptis chinensis decoction, gradually breaks the qi in the blood, deprives its essence, blood and qi, makes its qi and blood weak, and qi and blood recover more slowly, thereby making the animal model of spleen yang deficiency syndrome in the case of qi and blood weakness, the natural recovery time is very long, and the obtained rat spleen yang deficiency animal model has better stability.

Claims

1. A method for forming an animal model of spleen yang deficiency syndrome based on a decoction of Coptis chinensis, characterized in that: The following steps are involved: Step (1) Male SD rats weighing between 180 g and 220 g were used as model animals and were adaptively raised for several days; Step (2) gavage-feeding the model animals with a decoction of Rhizoma Coptidis once a day at a dose of 10 ml / kg to 15 ml / kg each time, with the temperature of the decoction of Rhizoma Coptidis being 3°C to 5°C; then placing the model animals in a cold environment for 60 min to 90 min for cold stimulation, wherein the cold environment is a breeding environment with a temperature of -2°C to 2°C. After the cold stimulation is completed, the model animals are taken out and placed in a room temperature environment for normal breeding with free access to water; Repeat step (2) for 14 to 18 days to obtain an animal model of spleen yang deficiency syndrome; The preparation method of the ice coptis chinensis decoction is as follows: A1. Take 25g-28g of Coptis chinensis and 5g-8g of Trigonosciadium japonicum, add water and soak at a material-liquid ratio of 1:20-22 for the first decoction. After decoction, filter the medicinal liquid. Add water to the medicinal residue at a material-liquid ratio of 1:15-16 for the second decoction. After the second decoction is completed, filter the medicinal liquid and combine the two medicinal liquids to obtain a total decoction. A2. Use a rotary evaporator to concentrate the total decoction to 50%-55% of the original volume to obtain a concentrated solution. Aliquot the concentrated solution and refrigerate at 3°C-5°C until use.

2. The method according to claim 1, wherein: The modeling animals in step (1) are male SD rats weighing 200 g to 210 g. After obtaining the modeling animals, they are adaptively raised for 3 to 7 days.

3. The method according to claim 1, wherein: In step (2), the patient is gavaged at a dose of 12 ml / kg each time each day, and the temperature of the icy coptis chinensis decoction is 4°C during gavage.

4. The method according to claim 1, wherein: In the step (2), the model animals were prohibited from drinking water and eating during the cold stimulation process, and each model animal was housed individually; the duration of each cold stimulation was 60 minutes.

5. The method according to claim 1, wherein: The preparation method of the ice coptis chinensis decoction is as follows: A1. Take 27g of Coptis chinensis and 6g of Trigonosciadium japonicum, add water and soak them at a solid-liquid ratio of 1:20 for the first decoction. After decoction, filter the medicinal liquid. Add water to the medicinal residue at a solid-liquid ratio of 1:15 for the second decoction. After the second decoction is completed, filter the medicinal liquid and combine the two medicinal liquids to obtain a total decoction. A2. Use a rotary evaporator to concentrate the total decoction to 55% of its original volume to obtain a concentrate. Aliquot the concentrate and store in a refrigerator at 4°C until use.

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