Application of antrodia camphorata triterpenes in treatment of depression

Abstract

The invention discloses an application of antrodia camphorata total triterpenes in treating depression. The antrodia camphorata total triterpenes are obtained by extracting antrodia camphorata sporocarp or mycelium. The antrodia camphorata total triterpenes are the main active substance in the antrodia camphorata sporocarp, and the content of the triterpenes in the antrodia camphorata is far higher than that of the ganoderma lucidum. The antrodia camphorata triterpenes in the invention can significantly improve depressive symptoms and restore neurotransmitter and inflammatory factor levels. In addition, the antrodia camphorata triterpene also has the effects of dispelling the effects of alcohol, protecting the liver, resisting hepatic fibrosis, tumor, fatigue, cancer, inflammation and virus, reducing blood pressure, regulating immunity, regulating intestinal flora and the like. The antrodia camphorata triterpene is wide in source, remarkable in effect, free of side effects and diversified in function, and has great development value and good application prospects in treating or relieving depression.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to the application of total triterpenes of Antrodia camphorata in treating depression. Background Art

[0002] Depression, also known as depressive disorder, is mainly manifested by low mood, reduced exercise, dislike of socializing, loss of appetite, weight loss, etc. It is a complex mental health disease that can cause sleep disorders, endocrine disorders, autonomic dysfunction, low immunity and cardiovascular and cerebrovascular diseases, etc. It has the characteristics of high morbidity, high disability rate, high suicide risk and prolonged course of disease. Depression has become the second largest disease in the world after heart disease. Due to the rapid development of society, people are now generally facing greater pressure. In recent years, the risk of depression has increased year by year. However, since the pathogenesis of depression has not been fully clarified, it is very difficult to treat. At present, drug therapy is still a common means of treating depression in clinical practice. Based on the monoamine neurotransmitter deficiency hypothesis, many antidepressants have been developed, including fluoxetine, paroxetine, etc., but these drugs generally have a slow effect and have side effects such as gastrointestinal and cardiovascular diseases. Therefore, people are in urgent need of developing some new antidepressant drugs with stronger specificity and fewer side effects. In recent years, medicine and food have attracted more and more attention and favor from researchers due to their advantages such as abundant sources, few side effects, rich targets and diverse functions.

[0003] Antrodia cinnamomea, also known as Antrodia camphorata, Antrodia camphorata mushroom, etc., is a rare medicinal and edible fungus belonging to the phylum Basidiomycetes, family Polyporaceae, genus Microporus. It has multiple biological activities such as sobering up and protecting the liver, anti-cancer, anti-tumor, antioxidant, anti-inflammatory, antibacterial, lowering blood sugar, lowering blood lipids, lowering blood pressure, regulating immunity and neuroprotection. At present, more than 220 compounds have been isolated from the fruiting bodies or mycelium of Antrodia camphorata, including triterpenes, polysaccharides, nucleotides, maleic acid and succinic acid derivatives, ubiquinone derivatives, etc. Among them, triterpenes are the most important active substances in the fruiting bodies of Antrodia camphorata. The content of triterpenes in the fruiting bodies of Antrodia camphorata is more than 10 times that of Ganoderma lucidum. In the wild Antrodia camphorata fruiting bodies that have grown for more than 10 years, the content of bitter substances such as terpenes can reach about 45%. Antrodia camphorata triterpenes have been proven to have the effects of sobering up and protecting the liver, anti-liver fibrosis, anti-tumor, anti-fatigue, anti-cancer, anti-inflammatory, lowering blood sugar, lowering blood pressure, regulating immunity and regulating intestinal flora. Based on the rich content and excellent biological activity of Antrodia cinnamomea triterpenes, more and more scholars have begun to explore and develop the application of Antrodia cinnamomea triterpenes in the fields of medicine and functional food. Summary of the invention

[0004] Purpose of the invention: In view of the deficiencies in the prior art, the present invention provides an application of total triterpenes of Antrodia camphorata in the treatment of depression. The present invention proposes for the first time that triterpenes of Antrodia camphorata have a very good antidepressant effect, and that triterpenes of Antrodia camphorata are rich in sources, safe, have no side effects, have significant effects, and have diverse functions, and can be used to prepare antidepressant drugs or antidepressant health products.

[0005] Technical solution: In order to achieve the above purpose, the present invention uses the triterpenoids of Antrodia cinnamomea in the preparation of antidepressant drugs or health products.

[0006] The antrodia cinnamomea triterpenes are extracted and purified from antrodia cinnamomea fruiting bodies or mycelium.

[0007] The camphorata fruiting bodies include wild fruiting bodies, artificial basswood cultivated fruiting bodies, solid-state cultured fruiting bodies and / or dish-cultured fruiting bodies.

[0008] Wherein, the Antrodia camphorata mycelium includes liquid fermentation mycelium and / or solid fermentation mycelium.

[0009] Wherein, the total mass fraction of triterpenoid compounds in the total triterpenes of Antrodia camphorata is above 60%.

[0010] Specifically, the triterpenoid compounds in the purified extract account for more than 60% of the total triterpenoid mass fraction of Antrodia camphorata.

[0011] The extraction process of total triterpenes of Antrodia camphorata comprises the following steps: crushing Antrodia camphorata fruiting bodies or mycelium, extracting by refluxing with high-concentration ethanol, concentrating and drying the extract to obtain a crude extract of total triterpenes of Antrodia camphorata; subsequently, dispersing the crude extract of total triterpenes of Antrodia camphorata in pure water, defatting with petroleum ether, and then adsorbing with a macroporous resin chromatography column, eluting with pure water and low-concentration ethanol to remove impurities, eluting with high-concentration ethanol and collecting the eluate; finally, concentrating and drying the eluate to obtain total triterpenes powder of Antrodia camphorata.

[0012] The solid-liquid ratio of the crushed Antrodia camphorata fruiting bodies or mycelium to ethanol is 1:20-60 mg / mL, the low-concentration ethanol is ethanol with a volume fraction of 5-30%, and the high-concentration ethanol is ethanol with a volume fraction of 80-100%.

[0013] Wherein, the reflux extraction time is 2-6 hours, and the volume of the eluent is 2-8 times of the column volume.

[0014] Preferably, the Antrodia camphorata fruiting body is crushed, anhydrous ethanol is added at a solid-liquid ratio of 1:50 mg / mL, and the mixture is refluxed and extracted for 4 hours. The extract is concentrated to about 1 / 5 of the original volume, and placed in a 75°C constant temperature drying oven for drying to obtain a crude extract of total triterpenes of Antrodia camphorata; subsequently, the crude extract of total triterpenes of Antrodia camphorata is ultrasonically dispersed in 10 times the mass of pure water, an equal volume of petroleum ether is added, and the mixture is oscillated and defatted at 65°C for 10 minutes, and the defatting is repeated twice; the pH of the defatted aqueous phase is adjusted to 2.5, and then adsorbed on an AB-8 macroporous resin chromatography column, with the sample loading volume being 0.5 times the column volume each time; then, 3 times the column volume of pure water and 20% ethanol are used in sequence to elute and remove impurities, and then 6 times the column volume of anhydrous ethanol is used to elute and collect the eluate, with a flow rate of 1 mL / min; finally, the eluate is concentrated to about 1 / 5 of the original volume, and placed in a 75°C constant temperature drying oven for drying to obtain a powder of total triterpenes of Antrodia camphorata.

[0015] Furthermore, the extract and purified product of the Antrodia camphorata fruiting body or mycelium is used in the preparation of antidepressant drugs or health products, and the extraction method is as shown above.

[0016] The oral dosage of the total triterpenes of Antrodia cinnamomea of ​​the present invention is 10-40 mg / kg·bw. Preferably, the oral dosage of the total triterpenes of Antrodia cinnamomea is 20 mg / kg·bw.

[0017] The present invention uses antrodia cinnamomea triterpenoids to achieve significant antidepressant effects, especially at the optimal dose. Among them, taking 10-40mg / kg of antrodia cinnamomea triterpenoids shows different degrees of improvement effects for different depressive symptoms, but the antidepressant effect is best at a medium dose (20mg / kg). At the optimal dose of antrodia cinnamomea triterpenoids, the effect of improving depressive symptoms such as anhedonia, autonomous exploratory activity and behavioral despair is significantly better than that of the positive drug fluoxetine, and the effect of controlling and restoring inflammatory factors and neurotransmitter levels is not less than that of the positive drug fluoxetine. But overall, antrodia cinnamomea triterpenoids have more development value and application prospects than fluoxetine. Because fluoxetine is a pure compound, and the optimal dose is 15mg / kg, although the optimal dose of antrodia cinnamomea triterpenoids in the present invention is 20mg / kg, which is slightly higher than the dose of fluoxetine, the antrodia cinnamomea triterpenoids in the present invention are total triterpenoids, not pure compounds. But even so, at the optimal dose, the improvement effect of antrodia cinnamomea total triterpenoids on many depressive symptoms is still significantly better than fluoxetine. The real antidepressant effect of the total triterpenoids of Antrodia camphorata is probably only one or more triterpenoid compounds. If the one or more triterpenoid compounds of Antrodia camphorata can be further separated and used alone or in combination, the dosage will be much lower than that of fluoxetine. Therefore, the triterpenoids of Antrodia camphorata in the present invention have great development potential in the development of antidepressant drugs or health products.

[0018] In addition, the content of the camphor triterpenoids of the present invention in the camphor fruiting bodies is extremely high, which is not only conducive to improving its extraction yield, but also reduces the extraction cost. The camphor triterpenoids in the present invention can significantly improve depressive symptoms and restore the levels of neurotransmitters and inflammatory factors. In addition, the camphor triterpenoids also have the effects of sobering up and protecting the liver, anti-liver fibrosis, anti-tumor, anti-fatigue, anti-cancer, anti-inflammatory, anti-viral, lowering blood pressure, immune regulation and intestinal flora regulation. Therefore, the anti-depressant camphor triterpenoids provided by the present invention have a wide range of sources, significant effects, no side effects, and diverse functions, and have great development value and good application prospects.

[0019] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0020] 1. The Antrodia camphorata in the present invention is a medicinal and edible mushroom with a long history of consumption, and its safety has been verified by a large number of animal experiments and clinical trials, proving that Antrodia camphorata is safe and non-toxic. Therefore, Antrodia camphorata triterpenes, as the main natural active substance in Antrodia camphorata, also have the advantages of high safety and no side effects.

[0021] 2. The triterpenoids of Antrodia camphorata in the present invention are the active substances with the highest content in the fruiting bodies of Antrodia camphorata, and their content is usually above 10%. At the same time, the fruiting bodies of Antrodia camphorata have been artificially cultivated on a large scale, and the cultivation methods are diverse, including log cultivation, solid-state fermentation and dish cultivation, etc., which provide a rich source of raw materials for the extraction of triterpenoids of Antrodia camphorata. In addition, the extraction yield of triterpenoids of Antrodia camphorata is high and the cost is low. The above characteristics greatly enhance the economy and practicality of the present invention.

[0022] 3. The triterpenoids of Antrodia camphorata provided by the present invention have significant antidepressant effects. At the optimal dosage (20 mg / kg·bw), the effect of relieving certain depressive symptoms is even better than that of the positive drug fluoxetine, and has great development value and good application prospects.

[0023] 4. The triterpenoids of Antrodia camphorata provided by the present invention not only have antidepressant effects, but also have the effects of sobering up and protecting the liver, anti-liver fibrosis, anti-tumor, anti-fatigue, anti-cancer, anti-inflammatory, anti-viral, lowering blood pressure, immune regulation and intestinal flora regulation. While relieving depression, it can also play a health-care role in preventing diseases and promoting health, which further greatly increases the research and development value and application prospects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 To study the effects of reserpine on the horizontal movement, tail suspension immobility time and 5-hydroxytryptamine (5-HT) concentration in mice;

[0025] Figure 2 The effect of total triterpenes of Antrodia camphorata on horizontal movement and tail suspension immobility time of mice;

[0026] Figure 3The effect of total triterpenes of Antrodia cinnamomea on the concentration of inflammatory factors in mouse serum;

[0027] Figure 4 The effect of total triterpenes of Antrodia cinnamomea on the concentration of monoamine neurotransmitters in the hippocampus of mice.

[0028] The figures that do not contain the same lowercase letters indicate significant differences, p < 0.05. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0030] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. Experimental methods without specific conditions specified in the examples are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0031] The Antrodia camphorata strain was purchased from the American Type Culture Collection (ATCC) with the number ATCC 200183. The Antrodia camphorata fruiting bodies were obtained by the applicant using the ATCC 200183 strain to culture conventional Cinnamomum camphora basswood, with a culture period of 1 year, specifically: without adding any materials to the wood, Antrodia camphorata was directly inoculated on the sterilized Cinnamomum camphora wood, and the fruiting bodies were collected after culturing for 1 year under natural conditions.

[0032] Example 1

[0033] Extraction and detection of triterpenes from Antrodia cinnamomea

[0034] (1) Extraction of Crude Triterpenes from Antrodia Camphorata

[0035] 5 g of Antrodia camphorata fruiting body powder was accurately weighed, and 250 mL (solid-liquid ratio 1:50 mg / mL) of anhydrous ethyl was added for reflux extraction for 4 h, and the extract was concentrated to about 1 / 5 of the original volume, and placed in a 75°C constant temperature drying oven for drying to obtain a crude extract of total triterpenes of Antrodia camphorata; subsequently, the crude extract of total triterpenes of Antrodia camphorata was ultrasonically dispersed in 10 times the mass of pure water, and an equal volume of petroleum ether was added for oscillation degreasing at 65°C for 10 min, and the degreasing was repeated twice; the pH of the defatted aqueous phase was adjusted to 2.5, and then adsorbed with an AB-8 macroporous resin chromatography column, and the loading volume was 0.5 times the column volume each time; then, 3 times the column volume of pure water and 20% ethanol were used to elute and remove impurities respectively, and then 6 times the column volume of anhydrous ethanol was used for elution and the eluate was collected, and the flow rate was 1 mL / min; finally, the eluate was concentrated to about 1 / 5 of the original volume, and placed in a 75°C constant temperature drying oven for drying to obtain total triterpenes powder of Antrodia camphorata.

[0036] (2) Detection of triterpenoid compounds in Antrodia cinnamomea

[0037] ① Accurately weigh 5 mg of the above-mentioned total triterpenoid powder of Antrodia cinnamomea and dissolve it in 500 μL of anhydrous ethanol;

[0038] ②Pipette 20 μL of crude triterpene ethanol solution into a 10 mL stoppered test tube (set up 5 replicate test tubes) and evaporate to dryness in a 60°C water bath;

[0039] ③Add 0.3 mL of freshly prepared 5% vanillin-glacial acetic acid solution and 1 mL of perchloric acid respectively, react at 60°C for 20 min, and then cool with ice water immediately;

[0040] ④Add 10mL of glacial acetic acid and shake well;

[0041] ⑤ Use an empty stoppered test tube, add 0.3 mL of freshly prepared 5% vanillin-glacial acetic acid solution, 1 mL of perchloric acid and 10 mL of glacial acetic acid mixed solution as a blank control, and measure the absorbance at a wavelength of 550 nm.

[0042] ⑥ Draw a standard curve using oleanolic acid as the standard and calculate the triterpene content based on the standard curve.

[0043] According to the test, the total content of triterpenoid compounds in the extracted and purified total triterpenoid powder of Antrodia camphorata is about 84.7%, which is almost all triterpenoid compounds.

[0044] Example 2

[0045] Construction of a mouse model of depression

[0046] (1) Animal grouping and drug administration

[0047] Thirty-six 6-week-old C57BL / 6 male mice were randomly divided into 6 groups (n=6): normal group (denoted as "CK"), solvent control group (denoted as "Nor"), 0.2 mg / kg·bw (body weight) (hereinafter referred to as "mg / kg") reserpine injection group (denoted as "Res0.2"), 0.4 mg / kg reserpine injection group (denoted as "Res0.4"), 0.6 mg / kg reserpine injection group (denoted as "Res0.6") and 0.8 mg / kg reserpine injection group (denoted as "Res0.8"). The mice were injected with reserpine at 2 pm every day for 10 consecutive days. The mice were weighed and recorded before each injection. On the 7th and 8th days after the injection of mice, the mice were trained to adapt to the preference for sugar water. Food and water were withdrawn at 12 pm on the 8th day until 8 am on the 9th day, and the sugar water preference experiment was started. Ten days after the injection, the mice were subjected to open field test and tail suspension test 1h, 4h and 24h after intraperitoneal injection of reserpine.

[0048] (2) Open field test

[0049] One of the typical symptoms of depression in mice is a significant decrease in free exploration activities, which is manifested as a significant decrease in the number of horizontal movement grids in the open field test. During the test, the environment was kept quiet throughout the whole process. An open box with a length*width*height of 50 cm was used, and the bottom of the box was divided into several squares of equal area. The mice were placed in the central grid, and a mobile phone was used to record and time the video. The number of horizontal movement grids of the mice within 5 minutes was observed and recorded (only the grids that the four limbs completely passed through were counted). The results are as follows: Figure 1 As shown in A.

[0050] (3) Tail suspension test

[0051] Another typical symptom of depression in mice is behavioral despair, which is manifested by a significant increase in the immobility time of the tail suspension test. The tail of the mouse (2 cm from the tip of the tail) was attached to the suspension rod of the tail suspension equipment analyzer test box (30 cm from the ground) with medical tape, and the mouse activity was monitored for 6 minutes using a video tracking system. The proportion of immobility time was calculated using SuperTst software. The results are shown in the figure. Figure 2 B. Immobility is defined as the absence of voluntary or escape-oriented movement.

[0052] (4) Detection of 5-hydroxytryptamine (5-HT) levels in mouse hippocampus

[0053] After measuring the behavioral data, the mice were killed by dislocating the neck and dissected. The hippocampal tissue was removed from the mouse brain and a certain PBS solution (pH 7.2-7.4) was added to fully homogenize the hippocampal tissue. The supernatant was collected by centrifugation (2500r / min, 20min). Subsequent operations were strictly carried out in accordance with the instructions of the "Mouse 5-HT ELISA" kit (Nanjing Senbeijia Biotechnology Co., Ltd.). The results are shown in the figure. Figure 1 As shown in C.

[0054] Depend on Figure 1 A and 1B show that the injection of 0.2-0.8mg / kg of reserpine significantly reduced the horizontal exercise volume of mice compared with the control group and significantly increased the tail suspension immobility time compared with the control group, which is a typical symptom of depression, indicating that reserpine can indeed effectively induce depression in mice. When the injection dose of reserpine is higher than (including) 0.6mg / kg, the dose is obviously too high, and the mice are already severely depressed. At this time, it is difficult for drug treatment to show efficacy; when the injection dose of reserpine is lower than (including) 0.2mg / kg, the dose seems too low, and the various indicators of the mice are relatively close to normal, indicating that the depression symptoms of the mice are relatively mild; Figure 1C shows that the concentration of 5-HT in the mouse brain tissue is seriously dose-dependent with the injection dose of reserpine, and decreases with the increase of the dose of reserpine. Combining the results of the mine field experiment and the tail suspension experiment, and taking into account the severity of depression in mice and the difficulty of drug treatment, the injection dose of reserpine for inducing depression in mice was finally determined to be 0.4 mg / kg.

[0055] Example 3

[0056] Example 1: Improvement of the Antrodia camphorata triterpenoids on reserpine-induced depressive-like behavior in mice

[0057] (1) Experimental animal grouping and drug administration

[0058] 36 experimental mice were randomly divided into 6 groups (n=6): normal group (denoted as "Nor"), model group (denoted as "Mod"), group treated with 10 mg / kg of Antrodia cinnamomea triterpenoids by gavage (denoted as "Tri-10"); group treated with 20 mg / kg of Antrodia cinnamomea triterpenoids by gavage (denoted as "Tri-20"); group treated with 40 mg / kg of Antrodia cinnamomea triterpenoids by gavage (denoted as "Tri-40"); and positive control group treated with 15 mg / kg of fluoxetine by gavage (denoted as "Flu"). Subsequently, during the entire experimental period, mice in the Nor group and Mod group were gavaged with the same volume of 0.9% saline as the triterpenoid treatment group; the Antrodia cinnamomea triterpenoid treatment group was gavaged with Antrodia cinnamomea triterpenoids according to the designed dose; and the positive control group was gavaged with fluoxetine at a dose of 15 mg / kg. The gavage operation was performed at 2 pm every day. One hour after intragastric administration, mice in the normal group were injected with 0.1% DMSO solvent, and the other treatment groups were injected with 0.4 mg / kg reserpine (dissolved in 0.1% DMSO). The mice were weighed and recorded before each injection. After 10 consecutive days of injection, the open field test, tail suspension test and sugar water preference test were performed on the mice 4 hours after intraperitoneal injection of reserpine.

[0059] (2) Sugar water preference test

[0060] One of the typical symptoms of depression in mice is anhedonia, loss of desire for pleasure such as sweet stimulation, which is manifested as a significant decrease in the sugar water preference value in the sugar water preference test. The experiment is divided into two stages. Before the formal experiment begins, the mice are first given adaptive training. First, each group of mice is provided with two bottles of 2% sucrose water, which are fed without fasting for 24 hours. Then one of the bottles of sucrose water is replaced with pure water, which is fed without fasting for 24 hours. Finally, the mice are fasted and deprived of water for 8 hours before the formal test. One bottle of 2% sucrose water and one bottle of pure water are placed on the mouse cage respectively. After the mice drink freely for 24 hours, the consumption of sucrose water and pure water is recorded (the position of the kettle is changed every 12 hours during this period), and the preference value of sugar water (Sucrose Preference, SP) is calculated. The calculation formula is: SP = sucrose water consumption / (sucrose water consumption + pure water consumption)*100%, and the results are shown in Table 1.

[0061] Table 1

[0062]

[0063] As shown in Table 1, the model group mice (depressed mice) had a significantly lower sugar preference value than the normal group (from 77% to 55%) due to the influence of reserpine. After intragastric administration of Antrodia cinnamomea triterpenoids, the sugar preference values ​​of mice (61%-75%) were significantly increased compared with the model group, indicating that the appropriate dose of Antrodia cinnamomea triterpenoids has a significant improvement effect on the depression symptoms of anhedonia in mice. Among them, the effect of intragastric administration of medium dose (20 mg / kg) of Antrodia cinnamomea triterpenoids on the improvement of anhedonia in mice was significantly better than that of intragastric administration of medium (10 mg / kg) and high dose (40 mg / kg) of Antrodia cinnamomea triterpenoids, which made the sugar preference value of mice (75%) basically restored to the level of normal mice, and the effect was even better than the positive control fluoxetine (71%).

[0064] (3) Open field test and tail suspension test

[0065] The open field test and tail suspension test were performed in the same manner as in Example 2. Figure 2 shown.

[0066] Figure 2 The results showed that the horizontal exercise volume (autonomous exploratory activity) of the mice in the model group was significantly lower than that of the normal group, and the tail suspension time (behavioral despair) was significantly higher than that of the normal group, showing typical symptoms of depression. Figure 2A shows that oral administration of 10 mg / kg (low dose) of Antrodia cinnamomea triterpenoids has a certain promoting effect on the autonomous exploratory activities of depressed mice; oral administration of 40 mg / kg (high dose) of Antrodia cinnamomea triterpenoids has a significant promoting effect on the autonomous exploratory activities of depressed mice, and the promoting effect is equivalent to that of the positive drug fluoxetine, which increases the horizontal exercise volume of mice by about 163% compared with the model group; oral administration of 20 mg / kg (medium dose) of Antrodia cinnamomea triterpenoids has the best promoting effect on the autonomous exploratory activities of depressed mice, which increases the horizontal exercise volume of mice by about 375% compared with the model group, which is significantly better than the promoting effect of high doses of triterpenoids and fluoxetine. At the same time, Figure 2 B shows that oral administration of 40 mg / kg of Antrodia cinnamomea triterpenoids has a certain improvement effect on the behavioral despair of depressed mice; oral administration of 10 mg / kg of Antrodia cinnamomea triterpenoids has a significant improvement effect on the behavioral despair of depressed mice, and the improvement effect is equivalent to that of the positive drug fluoxetine; oral administration of 20 mg / kg of Antrodia cinnamomea triterpenoids has the best improvement effect on the behavioral despair of depressed mice, which reduces the tail suspension immobility time of mice from 170s in the model group to 115s (the tail suspension immobility time of normal mice is 92s), and even significantly improves the improvement effect of fluoxetine (the tail suspension immobility time is 138s). The above results show that the appropriate dose of Antrodia cinnamomea triterpenoids has a significant improvement effect on the autonomous exploratory activities and behavioral despair of reserpine-induced depressed mice, and the improvement effect is significantly better than the positive drug fluoxetine.

[0067] Example 4

[0068] Effects of Antrodia camphorata triterpenoids on the concentration of inflammatory factors in mouse serum

[0069] After the mouse behavior experiment in Example 3 was completed, the mouse eyeballs were removed to collect blood. The blood was centrifuged at 3000 r / min, 4°C, for 15 min, and the supernatant was collected for the detection of inflammatory factors (TNF-α and IL-6). The relevant operations were strictly carried out in accordance with the instructions of the kit (Nanjing Senbeijia Biotechnology Co., Ltd.). The results are as follows: Figure 3 shown.

[0070] Depend on Figure 3 It can be seen that compared with the normal group of mice, the concentrations of inflammatory factors IL-6 and TNF-α in the serum of the model group mice were significantly increased, among which the concentration of TNF-α increased significantly to more than 4 times that of the normal group of mice. Neuroinflammation theory is one of the pathogenesis of depression. Therefore, controlling the level of inflammatory factors in serum is crucial for the treatment or relief of depression. Figure 3The results showed that high dose (40 mg / kg) of Antrodia cinnamomea triterpenoids by intragastric administration had no significant effect on reducing the concentration of IL-6 and TNF-α in the serum of depressed mice; however, low dose (10 mg / kg) and medium dose (20 mg / kg) of Antrodia cinnamomea triterpenoids by intragastric administration could significantly reduce the concentration of IL-6 and TNF-α in the serum of depressed mice. Among them, medium dose of Antrodia cinnamomea triterpenoids had the best effect, which was equivalent to the effect of positive drug fluoxetine, and could reduce the concentration of inflammatory factors IL-6 and TNF-α in the serum of depressed mice from 73 ng / mL and 418 μg / mL to 52 ng / mL and 120 μg / mL, respectively, basically returning to the level of normal mice. In summary, medium dose (20 mg / kg) of Antrodia cinnamomea triterpenoids by intragastric administration could significantly reduce the concentration of inflammatory factors TNF-α and IL-6 in the serum of depressed mice, and the reduction effect was not lower than or even slightly better than that of positive drug fluoxetine.

[0071] Example 5

[0072] Effects of Antrodia camphorata triterpenoids on the concentration of monoamine neurotransmitters in the hippocampus of mice

[0073] After the mouse behavior test in Example 3 was completed, the mouse hippocampus tissue was removed on ice, immediately frozen in liquid nitrogen and stored in a -80°C refrigerator. After all mice were treated, the levels of neurotransmitters 5-hydroxytryptamine (5-HT) and dopamine (DA) were uniformly tested. All operations were strictly carried out in accordance with the instructions of the kit (Nanjing Senbeijia Biotechnology Co., Ltd.). The results are shown in the figure. Figure 4 shown.

[0074] Depend on Figure 4 It can be seen that the monoamine neurotransmitters 5-hydroxytryptamine (5-HT) and dopamine (DA) in the model group mice were significantly reduced compared with the normal group mice, and the concentration of 5-hydroxytryptamine was significantly reduced by about 65% compared with the normal group mice, which is consistent with the literature report that reserpine induces depression by depleting monoamine neurotransmitters. Figure 4 A shows that oral administration of high dose (40 mg / kg) of Antrodia cinnamomea triterpenoids has no significant effect on restoring dopamine concentration in depressed mice; oral administration of low dose (10 mg / kg) and medium dose (20 mg / kg) of Antrodia cinnamomea triterpenoids can significantly increase dopamine levels in depressed mice, among which medium dose of Antrodia cinnamomea triterpenoids can increase dopamine concentration from 50 ng / mL in depressed mice to 79 ng / mL, basically restoring it to the level of normal mice (80 ng / mL), and the effect is equivalent to that of fluoxetine; Figure 4B shows that oral administration of 10-40 mg / kg of Antrodia cinnamomea triterpenes can significantly increase the 5-hydroxytryptamine level of depressed mice; among them, oral administration of low and high concentrations of Antrodia cinnamomea triterpenes can increase the 5-hydroxytryptamine concentration of mice by 65% ​​and 132% respectively compared with the model group mice; but oral administration of medium doses of Antrodia cinnamomea triterpenes has the best effect on increasing the 5-hydroxytryptamine concentration of depressed mice, which can increase the 5-hydroxytryptamine concentration of mice by about 240% compared with the model group mice, basically restoring to the level of normal mice, and the effect is equivalent to fluoxetine. It can be seen that oral administration of appropriate concentrations of Antrodia cinnamomea triterpenes can alleviate the depletion of neurotransmitters by reserpine by significantly increasing the levels of neurotransmitters 5-hydroxytryptamine and dopamine in the hippocampus of depressed mice, thereby improving or treating the depressive symptoms of mice, and the increase effect is not lower than or even slightly better than the positive drug fluoxetine.

[0075] In summary, according to Examples 3-5, the triterpenoids of Antrodia camphorata in the present invention have a very good effect on improving depression. Among them, the triterpenoids of Antrodia camphorata with an intragastric administration of 10-40 mg / kg showed different degrees of improvement effects on different depressive symptoms, but the antidepressant effect was best at a medium dose (20 mg / kg). At the optimal intragastric administration dose of triterpenoids of Antrodia camphorata, the improvement effect on depressive symptoms such as anhedonia, autonomous exploratory activity and behavioral despair was significantly better than that of the positive drug fluoxetine, and the effect of controlling and restoring the levels of inflammatory factors and neurotransmitters was no less than that of the positive drug fluoxetine.

[0076] Example 6

[0077] tablet

[0078] The total triterpenes of Antrodia cinnamomea prepared in Example 1 (10 g), hydroxypropyl methylcellulose E (30 g), starch (40 g), appropriate amount of povidone K30 and magnesium stearate (0.2 g) were mixed, granulated and tableted.

[0079] In addition, according to the conventional preparation method of the Pharmacopoeia 2015 edition, the total triterpenes of Antrodia camphorata obtained in the example can be added with different pharmaceutical excipients to prepare capsules, powders, tablets, granules, pills, injections, syrups, oral liquids, inhalants, ointments, solutions, creams, gels, powders, lotions, tinctures, suppositories or patches.

Claims

1. Application of total triterpenes of Antrodia cinnamomea in the preparation of antidepressant drugs or health products.

2. The use according to claim 1, characterized in that: The total triterpenes of Antrodia camphorata are obtained by extraction and purification from Antrodia camphorata fruiting bodies or mycelium.

3. The use according to claim 1, characterized in that: The camphorata fruiting bodies include wild fruiting bodies, artificial basswood cultivated fruiting bodies, solid-state cultured fruiting bodies and / or dish-cultured fruiting bodies.

4. The use according to claim 1, characterized in that: The camphorata mycelium includes liquid fermentation mycelium and / or solid fermentation mycelium.

5. The use according to claim 1, characterized in that: The total mass fraction of triterpenoid compounds in the total triterpenes of Antrodia camphorata is above 60%.

6. The use according to claim 2, characterized in that: The extraction and purification process of total triterpenes of Antrodia camphorata comprises the following steps: crushing Antrodia camphorata fruiting bodies or mycelium, extracting by refluxing with high-concentration ethanol, concentrating and drying the extract to obtain a crude extract of total triterpenes of Antrodia camphorata; subsequently, dispersing the crude extract of total triterpenes of Antrodia camphorata in pure water, defatting with petroleum ether, adsorbing with a macroporous resin chromatography column, eluting with pure water and low-concentration ethanol to remove impurities, eluting with high-concentration ethanol and collecting the eluate; finally, concentrating and drying the eluate to obtain total triterpenes powder of Antrodia camphorata.

7. The use according to claim 6, characterized in that: The solid-liquid ratio of the crushed Antrodia camphorata fruiting bodies or mycelium to ethanol is 1:20-60 mg / mL, the low-concentration ethanol is ethanol with a volume fraction of 5-30%, and the high-concentration ethanol is ethanol with a volume fraction of 80-100%.

8. The use according to claim 6, characterized in that: The reflux extraction time is 2-6 hours, and the volume of the eluent is 2-8 times the column volume.

9. An antidepressant drug composition, characterized in that: The composition uses the antrodia cinnamomea triterpenoids described in claim 1 as an effective raw material, and also includes pharmaceutically acceptable carriers and excipients.

10. The pharmaceutical composition according to claim 9, characterized in that The dosage form of the composition is preferably tablets, capsules, granules, lyophilized powders, granules, injections, intravenous drip solutions, suspensions, emulsions, solutions, syrups, sprays or aerosols.

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