WO2006072213A1 - Anti-depression formulations - Google Patents

Abstract

A formulations for the treatment of depression and related symptoms prepared from Psoralea corylifolia extract and Poria cocos extract.

Description

 Drug for treating depression

Technical field

 The present invention relates to the field of pharmaceutical compositions, nutritional supplements, health foods, and other related components. In particular, the present invention relates to a formulation comprising an herbal extract for alleviating depression and related conditions. BACKGROUND The references cited in this disclosure are not necessarily prior art. Therefore, references to these references do not imply that such references are prior art in any jurisdiction. Depression is a common and frequently-occurring disease that seriously endangers the physical and mental health of human beings. With the increasing social competition, the physical and psychological pressures on people are increasing, and the incidence of depression is on the rise. The latest statistics from the World Health Organization show that the global prevalence rate is about

11%, and predict that 2020 will become the second medical disease in the world. Therefore, it is of great strategic significance to strengthen the research on the pathogenesis and prevention of depression.

The pathogenesis of depression is complicated, and its development is related to systemic disorders such as central nervous system, neuroendocrine system and immune system. When clinically relied on drug treatment or control of depression, the existing Western medicine has a narrow anti-depression spectrum, a single site of action, and a large toxic side effect, which often limits the patient's choice. Therefore, there is an increasing demand worldwide for highly effective and safe drugs for the treatment of complex causes of depression. Therefore, it is necessary to propose new and more effective drugs for treating depression in view of the above disadvantages. Summary of the invention

 According to an aspect of the invention, a pharmaceutical composition for treating depression, characterized in that the pharmaceutical composition comprises an extract of a leguminous psoralen and an extract of a polyporaceae fungus. In a preferred embodiment, the pharmaceutical composition consists essentially of an extract of the leguminous psoralen and an extract of the Polyporaceae fungus. In a more preferred embodiment, the pharmaceutical composition consists of an extract of the leguminous psoralen and an extract of the Polyporaceae fungus. In another preferred embodiment, the pharmaceutical composition is composed of an extract of psoralen (dried mature fruit of leguminous psoralen) and an extract of sputum (dried sclerotia of polyporaceae fungus). In a more preferred embodiment, the weight ratio of the psoralen extract to the alfalfa extract is about 1:1 to 1:7. In a more preferred embodiment, the weight ratio of the psoralen extract to the alfalfa extract is from about 1 : 1 to about 1:3. In a more preferred embodiment, the weight ratio of the psoralen extract to the anthraquinone extract is about 1:1 to 1:2. In a most preferred embodiment, the weight ratio of the psoralen extract to the alfalfa extract is about 1:1.5.

 In another preferred embodiment, the psoralen extract comprises 80-99% furanocoumarin, wherein the furocoumarin comprises 4-12% psoralen and about 3-11 % of the psoralen. In a more preferred embodiment, the psoralen extract comprises 95% furocoumarin, wherein the furocoumarin contains about 7-9% psoralen and 6-8% different Psoralen.

 In another preferred embodiment, the alfalfa extract contains about 57-62% of the Lycium barbarum polysaccharide. In another preferred embodiment, the pharmaceutical composition does not comprise a Chinese herbal medicine selected from the group consisting of ginseng, astragalus, scorpion, salvia miltiorrhiza, Polygonum multiflorum, Ophiopogon japonicus, Codonopsis, Atractylodes, Chonglou, Xiangfu, Atractylodes, Windproof, medlar, medlar, comfrey, licorice.

According to another aspect of the present invention, a pharmaceutical composition comprising furocoumarin and an anthraquinone polysaccharide, wherein the furocoumarin is provided Includes psoralen and isopsoralen. In a preferred embodiment, the content ratio of the psoralen, isopsoralen and scorpion polysaccharide is from 7 to 9:6 to 8:86 to 93.

 According to another aspect of the present invention, there is provided a method of preparing the above pharmaceutical composition, comprising:

 a. extracting psoralen to form a psoralen extract;

 b. extracting cockroaches to form cockroach extract;

 c combining the psoralen extract and the sputum extract.

 In a preferred embodiment, psoralen and strontium are prepared by the following weight ratios: psoralen 1 to 2 parts, 茯苓 1 to 2 parts. In a more preferred embodiment, the psoralen and bismuth are prepared by the following weight ratios: 1-2 parts of psoralen, 1 part of 茯苓. In a more preferred embodiment, the psoralen and strontium are prepared by the following weight ratios: 2 parts of psoralen, 1 part.

 In another preferred embodiment, the step a. extracting the psoralen further comprises:

 i) extracting the psoralen with a first extract to form a first extraction solution; and extracting the first extraction solution with a second extract to form a second extraction solution comprising a residue;

 Iii) separating and drying the residue from the second extraction solution;

 It is characterized in that one of the extracts is extracted with a highly polar solvent and the other extract is extracted with a low polar solvent.

In a more preferred embodiment, the first extract is ethanol, methanol or acetone and the second extract is ethyl acetate, diethyl ether, chloroform or dichloromethane. In a more preferred embodiment, the first extract is 55-75% (v/v) ethanol and the second extract is 80-100% ethyl acetate. In a most preferred embodiment, the first extract is 65% (v/v) ethanol and the second extract is 100% acetic acid B Ester.

 In another preferred embodiment, the extracting of the step b.茯苓 further comprises:

 i) extracting hydrazine with water to obtain a water-soluble extract and a non-water-soluble hydrazine residue;

 Ii) extracting the water-insoluble hydrazine residue with an alkaline solvent to obtain an alkali-soluble extract;

 Iii) combining the water-soluble extract and the alkali-soluble extract to form the alfalfa extract.

 In a more preferred embodiment, the method according to claim 22, step i) further comprising:

 a. boiling the crucible in water to form an aqueous solution of hydrazine;

 b. filtering and concentrating the aqueous hydrazine solution to form a concentrated aqueous hydrazine solution; c. precipitating the concentrated aqueous hydrazine solution to form a precipitate;

 d. The precipitate is separated and dried to form a water soluble extract of hydrazine.

 In a more preferred embodiment, the precipitate in step C. is formed by the addition of an alcohol. In a more preferred embodiment, the alcohol is 80-100% (v/v) ethanol. In a most preferred embodiment, the alcohol is 95% (v/v) ethanol.

 In another more preferred embodiment step (ii) further comprises:

 a. extracting the mash residue with an alkaline solvent to form an extract solution, wherein the pH of the extract solution is = 11-13;

 b. neutralizing the extract solution with acid to a pH of 6-7 to form a neutralized extract solution;

 c precipitating the neutralized extract solution to form a precipitate;

d. The precipitate is separated and dried to obtain the lysine-soluble extract. In a more preferred embodiment, the alkaline solvent is 1 M NaOH. In another more preferred embodiment, the precipitation is obtained by the addition of an alcohol. In a more preferred embodiment, the alcohol is 80-100% (v/v) ethanol. In a most preferred embodiment, the alcohol is 95% (v/v) ethanol.

 According to another aspect of the present invention, there is provided a nutritional supplement comprising a psoralen extract and a sputum extract. In a preferred embodiment, the weight ratio of the psoralen extract to the anthraquinone extract is about 1:1 to 1:7. In a more preferred embodiment, the weight ratio of the psoralen extract to the alfalfa extract is about 1:1 to 1:3. In a more preferred embodiment, the weight ratio of the psoralen extract to the alfalfa extract is about 1:1-1:2. In a most preferred embodiment, the weight ratio of the psoralen extract to the extract of the mash is about 1:1.5.

 According to another aspect of the present invention, a health food comprising psoralen extract and sputum extract is provided. In a preferred embodiment, the weight ratio of the psoralen extract to the sputum extract is about 1:1 to 1:7. In a more preferred embodiment, the weight ratio of the psoralen extract to the alfalfa extract is about 1:1 to 1:3. In a more preferred embodiment, the weight ratio of the psoralen extract to the alfalfa extract is about 1:1 to 1:2. In a most preferred embodiment, the weight ratio of the psoralen extract to the extract of the mash is about 1:1.5.

According to another aspect of the present invention, there is provided an application of a psoralen extract and a sputum extract as a preparation for treating depression. In a preferred embodiment, the weight ratio of the psoralen extract to the alfalfa extract is from 1:1 to 1:7. In a more preferred embodiment, the weight ratio of the psoralen extract to the alfalfa extract is from 1:1 to 1:3. In a more preferred embodiment, the weight ratio of the psoralen extract to the alfalfa extract is from about 1:1 to 1:2. In a most preferred embodiment, the weight ratio of the psoralen extract to the alfalfa extract is about 1:1.5. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a process for preparing a mash extract provided in accordance with one aspect of the present invention. Figure 2 shows the HPLC chromatogram of standard psoralen and isostoral fat. Figure 3 shows an HPLC chromatogram of a psoralen extract prepared in accordance with another aspect of the invention. Figure 4 shows HPLC and UV spectra of a sample of psoralen mixture

 Figure 5 shows the HPLC and UV spectra of psoralen (BGZ-1).

 The psoralen and sputum described herein are defined as follows.

 Psoralen is a dry and mature fruit of Psoral corylifolia L., which is a commonly used traditional Chinese medicine for warming kidney and aphrodisiac. Its sexual taste is Xin Wen, into the kidney, the main five labors and seven injuries, warm kidney yang, cure kidney effusion, through the door, warm dantian, convergence spirit. Wenshen Zhuangyang Chinese medicine can achieve the effect of "tonifying kidney" and "centering" by regulating HPA axis function and immune function. Modern research has shown that psoralen contains coumarins, flavonoids, volatile oils, etc., which regulates the central nervous system, neuroendocrine system, enhances immunity, fights cancer, and inhibits nitric oxide synthase. In the clinical practice of traditional Chinese medicine, psoralen is often used to treat menopausal syndrome. Foreign clinical studies have confirmed that coumarin in the psoralen can treat seasonal affective disorder by inhibiting melatonin metabolism.

As used herein, "extract""herbextract" or "herbal extract" refers to an active ingredient that is extracted from a plant by the general methods or equivalent methods described herein. "water The soluble extract "refers to a water-soluble extract. "Alkali-soluble extract" refers to an extract soluble in an alkaline solvent.

 The "psoralen extract" as used herein refers to a composition which is isolated and extracted from the dried fruit of the psoralen plant or the psoralen by a specific extraction method in a general case. Preferably, the composition is isolated from the dried fruit of the psoralen plant by a specific extraction method. This extract contains furocoumarin, psoralen, and psoralen.

 茯苓 is a polyporaceae fungus Ρ Ροπ' cocos (Schw.) Wolf. Dry sclerotia. In the Han Dynasty, Zhongjing Fangzhong reused it to achieve the goal of "Ningxin and God".茯苓 mainly contains 茯苓 polysaccharides, triterpenoid organic acids and other ingredients. The hydrophobic extract has a calming effect on the central nervous system. Hydrophobic extract can correct the intracellular calcium level to provide a scientific basis for the treatment of central nervous system diseases. Lycium barbarum polysaccharides have immunomodulatory functions such as enhancing killer cell activity of natural killer cells and lymphokine activation, regulating cytokine levels, and inhibiting tumor necrosis factor.

 As used herein, "sputum extract" refers to a specific extraction process, which is isolated from the dried sclerotium, mycelium, or other parts of the fungus of the genus Polyporus. Active composition. Preferred are active compositions which are isolated from the dried sclerotia of the fungus sputum by a specific extraction process. This extract includes lycium polysaccharide. The lycium polysaccharide contains gluten, dextran, sucrose, sucrose and the like. As used herein, "茯苓 polysaccharide" refers to a polysaccharide extracted from alfalfa.

 As used herein, "highly polar solvent" refers to an organic solvent having a Snyder polarity index greater than 5. "Low polar solvent" refers to an organic solvent having a Snyder polarity index of less than 5. Antidepressant effect

Tail Suspension Test (TST), Forced Swimming Test (FST) and chronic mild stress model (Chronic Mild) Stress, CMS) was used to study the antidepressant effect of the pharmaceutical composition. The characteristics of these models are as follows - mouse tail-tail model

 One of the main symptoms of depression is a reduction in motivational behavior. In the experimental model of the tail suspension, the tail-suspended mice struggled to overcome the abnormal position. This is the motive behavior of the mice, but after a certain period of time, there is intermittent motion, showing a "disappointment" state. The immobility referred to here reflects a state called "behavioral despair and variant" or "failure to adapt to stress", which is a negative Unmotivated desperate state. The effect of the drug on antidepressant in mice can be evaluated by measuring the immobility time of the mice after a period of suspension. (The shorter the immobility time, the more significant the antidepressant effect of the drug is shown). The desperate model of mouse tail suspension behavior is sensitive to most antidepressants, and its operation is simple and fast, so it is widely used in the screening of antidepressants. Mouse forced swimming model

One of the main symptoms of depression is a reduction in motivational behavior. In the forced swimming model, mice were forced to swim in a confined space. They first showed the motivational behavior of struggling and trying to escape, and then in a state of immobility called "desperation." The immobility referred to here reflects a state called "behaviour despair and variant" or "failure to adapt to stress", which is a negative Unmotivated desperate state. By measuring the immobility time of mice after forced swimming for a period of time, the anti-depressant effect of the drug on mice can be measured. (The shorter the immobility time, the more significant the antidepressant effect of the drug is shown). The desperate model of forced swimming behavior in mice is sensitive to most antidepressants, and its operation is simple and fast, so it is widely used in the screening of such drugs. Chronic Mild Stress (CMS) model

 In a chronic mild stimulation model, rats were stimulated with a chronic mild procedure to observe recovery of sucrose intake, inhibition of brain monoamine oxidase-A (MAO-A) and monoamine oxidase-B (MAO-B) activity, and cortisol. The degree of reduction in content is used to determine the effectiveness of antidepressants.

 In the chronic mild stimulation model, rats received multiple mild stimuli in sequence within a few weeks. The effect of chronic mild stimulation on the behavior of rats was evaluated by using a rat to reduce the intake of sweet solution. Antidepressants antagonize this effect, which increases the sucrose intake of rats in chronic mild stimulation models. Further studies have shown that chronic mild stimuli can cause damage to the biochemical and physiological processes in rats. Administration of antidepressants such as tricyclic antidepressants, selective serotonin reuptake inhibitors, monoamine oxidase inhibitors, etc., can improve the damage of chronic mild irritations to animals, while non-depressant drugs are ineffective in this model. This model has good predictive validity (because of chronic stimuli, behavioral changes can be recovered by multiple antidepressants), appearance effectiveness (almost all manifestable depressive symptoms have been shown), and intuitive effectiveness (chronic mild stimuli) This leads to a general reduction in reward response, similar to the core symptom of anxedonia (Willner R 1997).

 Chronic mild stimulation reduces sucrose intake in animals and increases the activity of brain monoamine oxidase-A (MAO-A) and monoamine oxidase-B (MAO-B) as well as serum cortisol levels. Therefore, an agent that restores sucrose intake to animals, inhibits the activity of brain MAO-A and MAO-B, and lowers serum cortisol levels is considered to be an effective antidepressant. Example 1

 Preparation of psoralen extract

Medicines and instruments

• Buguzhi: It was purchased from Jiangsu Province, China in May 2001 and was certified by the Department of Biology, School of Life Sciences, Nanjing University, China. Sample (No.NJ-34060) stored in Nanjing College of Life Sciences, Herbarium, Nanjing, China, 210043. 95% (Wv) Ethanol: purchased from Nanjing Chemical Reagent No. 1, Product Code: 1370400201.

 • 65% (Wv) Ethanol: Prepare 684ml of 95% (v/v) ethanol in 316ml of water.

 • 100% ethyl acetate: purchased from Nanjing Chemical Reagent No. 1 Factory, product number: 1370401001. • Infiltration tube (1000ml): Purchased from Tianjin Youfeng Technical Glass Co., Ltd. Product number:

119-02-10.

 • Separating funnel (1000ml): Purchased from Nanjing Sanai Glass Instrument Co., Ltd. Product number: 8543 ο

 • Rotary vacuum concentrator: Purchased from Buchi LabortechnikAQ Switzerland. Grind 100 g of dried psoralen into powder, add 65% ethanol and 75 mL and mix well. After suffocating for 2 h, the powder should be evenly filled into the percolating tube. Soak in 65 % ethanol 200 mL for 24 h, then add 1300 ml of 65 % ethanol to the percolating tube. 1500.0 mL of percolate was collected at a rate of 1.5 mL/min. Concentrate under reduced pressure at 60 ° C until the percolate has no ethanol flavor, and obtain 2'50.0 mL of the drug solution. The solution was extracted 8 times with 250 ml of 100% ethyl acetate. The ethyl acetate layer was collected, concentrated under reduced pressure at 60 ° C, and ethyl acetate was evaporated. The remaining residue was dried under reduced pressure at 60 ° C to obtain 12.10 g of psoralen extract. (Yield 12.10%) Example 2.

 Preparation of alfalfa extract

Medicines and instruments

 • 茯苓: Purchased from Jiangsu Province, China, in May 2001, and identified by the Department of Biology, School of Life Sciences, Nanjing University, China. The sample (No.NU-62004) was stored in the Herbarium of the School of Life Sciences, Nanjing University, Nanjing, China, 210093.

• 95% (v/v) ethanol: purchased from Nanjing Chemical Reagent First Factory, product number: 1370400201. • Sodium hydroxide: purchased from Nanjing Chemical Reagent No. 1 Factory. Product Description: 1370500701. A 1 mol/L sodium hydroxide solution was prepared by dissolving 40 g of the sodium hydroxide in 1000 ml of water.

 • 99% v/v acetic acid: purchased from Nanjing Chemical Reagent No.1, product number: 1370401201.

 100 ml of the acetic acid was added to 900 ml of water to prepare 10% acetic acid.

 • Beaker (2000ml): Purchased from Shanghai Shenbo Instrument Co., Ltd., China. Product number: SF-1101-15 o

 • Beaker (5000ml): Purchased from Shanghai Shenbo Instrument Co., Ltd., China. Product Code: SF-1101-17.

 • Separating funnel (1000ml): Purchased from Nanjing Sanai Glass Instrument Co., Ltd. Product number: 8543 ο

 • Flask (2000ml): purchased from Nanjing Sanai Glass Instrument Co., Ltd. Product Code: 1117ο • Spherical Condensing Tube: Purchased from Beijing Glass Instrument Factory. Product Code: yq230503. • Infiltration tube (1000ml): Purchased from Tianjin Youfeng Technical Glass Co., Ltd. Product number: 119-02-10.

 • RE52-1 Rotary Evaporator: Purchased from Shanghai Huxi Analytical Instrument Factory Co., Ltd. ·

• HY30-01: Electronic vacuum drying oven: purchased from Taijiang Medical Instrument Factory, Fuzhou, China.

• HH-S electric thermostatic water bath: purchased from Dongtai Electrical Appliance Factory, Jiangsu Province, Jiangsu Province, China. As shown in Fig. 1, 100 g of sputum is ground into a coarse powder, and 1000 mL of distilled water is added to the mashed powder in step A, soaked for 0.5 h, and then decocted at 95-100 ° C for 2 hours, and filtered to obtain an aqueous solution of hydrazine (I). And 茯苓 residue (1). In the step Β, the hydrazine residue (1) is extracted in the same manner as in the step ,, to obtain an aqueous solution of hydrazine (Π), (III) and hydrazine residue (11). In the step C, the aqueous hydrazine solutions (1), (?), and (III) are combined to form an aqueous hydrazine solution (IV). The hydrazine aqueous solution (IV) was concentrated in a step D using a rotary evaporator to obtain 300 ml of a concentrated aqueous hydrazine solution. In step E, 840 ml of 95% ethanol is stirred and added to the concentrated hydrazine solution. The final ethanol concentration was 70% (v/v), and it was allowed to stand at 4 °C for 24 h to form a precipitate. It was filtered, and the obtained precipitate was dried under reduced pressure at 60 ° C to obtain 1.23 g of a dry powdery bismuth extract (ΠΙ) and a hydrazine-ethanol solution. In step F, the residue (II) is soaked in 1000 ml of 1 M sodium hydroxide solution for 10 hours (room temperature), and then filtered to obtain a sodium hydroxide extraction solution (I) (filtrate) and an antimony residue (IV) in the step. In G, the hydrazine residue (IV) is further extracted twice by the method of the step F to obtain a hydrazine residue (V) and a sodium hydroxide extraction solution (11), (111) of hydrazine. In the step H, the combined sodium hydroxide extraction solutions (1), (Π), and (III) were combined to form 2900 ml of a sodium hydroxide extraction solution (IV). In the step I, the solution (IV) was extracted with sodium hydroxide of 10% acetic acid to pH-6, and then 8120 ml of 95% ethanol was added thereto with stirring, and allowed to stand at 4 ° C for 24 hours to form a precipitate. Filtration, and the resulting precipitate was dried under reduced pressure at 60 ° C to obtain 35.69 g of a dry powdery bismuth extract VI.

 The alfalfa extracts (III) and (VI) were combined to obtain 36.92 茯苓 extract (yield 36.92%). Example 3

 Method for analyzing psoralen composition

 The components of the psoralen extract (psoralen and isopsoralen) prepared in Example 1 were analyzed by high performance liquid chromatography (HPLC) in combination with ultraviolet detection. This method has a lower detection limit and good accuracy, accuracy, and linearity. Medicine

Spectral grade acetonitrile and methanol were purchased from the International Laboratory (USA). Purified water was prepared by the Mili-Q system (Millipore). The standard psoralen and isopsoralen were purchased from the China National Institute for the Control of Pharmaceutical and Biological Products. The standard psoralen and isopsoralen were dissolved in methanol solution to form a standard solution, which was used as an external standard solution. Instrument and chromatographic conditions

 Chromatography was performed using an Agilent 1100 liquid chromatograph and a Diode Array Detector (DAD). HPLC separation of psoralen and isopsoralen using Zorbax XDB RP-C 18 analytical column (column length: 4.6 mm id x 25 cm, particle size: 5 m) and selection of RP-C18 as a guard column (column length: 4.6 mm id x 1.25 cm). The solvent was filtered through a 0.45 m HV filter (Milipore) and degassed in an ultrasonic bath for 20 minutes before use. The mobile phase was eluted with acetonitrile-water (40:60, v/v) at a flow rate of 1.0 ml/min. The injection volume was 1.0 1, and the detection wavelength was 246 nm. All chromatographic analyses were carried out at 22 °C. Sample Preparation

 Weigh 15 mg of psoralen extract accurately, dissolve in methanol, and dilute to a 25 ml volumetric flask. Prior to HPLC analysis, it was filtered through a microporous membrane (0.45 μM). Result

 HPLC analysis showed that the compounds in the sample were able to achieve baseline separation and analysis was completed within 20 minutes (psoralen retention time was 9.9 minutes and iso-psoralen retention time was 10.6 minutes). Figure 2 shows the HPLC chromatogram of the standard psoralen and isopsoralen. Figure 3 is an HPLC chromatogram of psoralen extract. There were no other impurity peak interferences at the control retention time. Comparing Fig. 2 with Fig. 3, according to the retention time of the standard and the corresponding position of the sample peak, it can be seen that the psoralen extract contains psoralen and isopsoralen.

数据The least squares method was used for data regression. The results showed that psoralen had a good linear relationship in the range of 0.00219-0.351 μg. Iso-psoralen has a good linear relationship in the range of 0.00206-0.3301 μ _§ . Psoralen and psoralen The detection limit of the prime is 0.0003 μ _§ . The minimum detectable amount of psoralen was 0.0011 μ _§ , while the lowest detectable amount of isopsoralen was 0.0010 μ _§ .

Linear regression equation for psoralen: y = 7289.9 -14.593 x, r = 0.9998 Linear regression equation for isopsoralen: y = 7214.4 -12.456 x, r = 0.9997 where = peak area ratio, X = injection Amount (μ _§ ), r = correlation coefficient analysis of psoralen Five batches of psoralen extract were prepared according to the procedure of Example 1. The content of total furocoumarin in the psoralen extract sample was determined by TLC comparison and colorimetric method, about 95%. The contents of psoralen and isopsoralen in five batches of psoralen extract were determined by HPLC analysis. The results are shown in Table 1. Table 1 Content of psoralen and isopsoralen in five batches of psoralen extract (me _an ±SD)

SD, standard deviation; standard deviation of five determinations The psoralen extract prepared according to Example 1 contained about 8.01% psoralen and 6.86% isopsoralen. Example 3.1

 Method for analyzing bismuth components

The hydrazine component was analyzed by the phenol-suli ric acid method. The phenol sulfuric acid method (Dubosis et al., 1956) is a colorimetric reaction. Due to its simplicity, it is commonly used to determine sugars, oligosaccharides, polysaccharides and their derivatives in samples. Medicine

 • Sulfuric acid, 95.5 %, reagent grade, purchased from Sigma-Aldrich (St. Louis, MO, USA).

 Product number: 32,050-1.

 • Reagent grade phenol, available from Sigma-Aldrich (St. Louis, MO, USA). Product Number: P4161 o

 • D-Glucose: purchased from Sigma-Aldrich (St Louis, MO, USA). Product Code: G5250.

 • 5 g/dl phenol-water solution: 5 g of re-distilled phenol is dissolved in 100 ml of distilled water.

• 10 g/dl glucose-water solution: 10 g of glucose is dissolved in 100 ml of distilled water. Instrument

 U-3000 Spectrophotometer: Hitachi, Ltd., Tokyo, Japan. Sample Preparation

Weigh accurately 5 mg of sputum extract, dissolve in water, and dilute to a 100 ml volumetric flask. Phenol sulfuric acid

Glucose is selected as the standard. The measurement was carried out according to the basic operating procedure of Dubosis et al. (1956). Precisely measure 0.2 g, dl, 0.6, 0.8, 1 and 1.2 ml of 10 g/dl glucose solution. Add the tubes separately and add water to make up the final volume of 2ml (containing 20-120 mg of glucose). An additional 1 ml of a 5% (w/v) phenol solution was added. Then, 5 ml of concentrated sulfuric acid was quickly added along the surface of the liquid to achieve a good mixing effect. The tube was allowed to stand for 10 minutes, shaken, and placed in a 25-30 ° C water bath for 15 minutes, and the absorbance was measured at 490 nm. Take a blank with distilled water. Taking the glucose reference quantity as the abscissa and the absorbance as the ordinate, the least squares method was used for data regression. The linear regression equation - y = 0.0068 +6.6929x, r = 0.9994

Among them, _y = absorbance value, ^:= glucose amount (111 _§ ) ^ = correlation coefficient 含量 extract polysaccharide content analysis

 The polysaccharide of the alfalfa extract was determined by the phenol sulfuric acid method. Standard curves were prepared using different concentrations of D-glucose (20-120 mg) as standard solutions. Accurately measure 2 ml of the sputum extract solution and add them to the test tube. Same as above, add 1 ml of 5% (w/v) phenol solution. Then, 5 ml of concentrated sulfuric acid was quickly added. After standing for 10 minutes, the mixture was shaken, placed in a 25-30 ° C water bath for 15 minutes, and the absorbance was measured at 490 nm. The blank sample is replaced by distilled water. Distilled water was used as a blank control. Calculate the amount of polysaccharide in the sample based on the standard curve. The sample results of the five batches of alfalfa extract are shown in Table 2. Table 2 Determination of polysaccharide content in five batches of cockroach extract 茯苓 extract 茯苓 polysaccharide (%)

1 60.31 ± 1.39

2 58.36 ± 1.11 茯苓 extract 茯苓 polysaccharide (%)

3 59.19 ±1.48

4 58.56 ± 1.06

5 59.95 ± 1.19 Example 4

 Preparation of medicament

The following compositions were prepared in this example:

Composition I: psoralen extract: 茯苓 extract =1: 1.5, equivalent to psoralen: 茯 苓 = 2: 1. Composition Π: psoralen extract: 茯苓 extract =1: 3, equivalent to psoralen: 茯苓 =1: 1.

Composition III: psoralen extract: 茯苓 extract =1: 6, equivalent to psoralen: 茯 苓 =1: 2. These compositions will be subjected to the following animal biological activity evaluation experiments. Experiment 1

Material

 Animal

 • ICR mice, male, 26±2g, provided by the Jiangsu Experimental Animal Center, housed in a 25±2 oC room, fed with free water for one week. All animal testing procedures are in accordance with the regulations of the China Animal Management Committee.

2. Reagent

• Bakuchi extract prepared according to the procedure of Example 1 • Alfalfa extract prepared according to the procedure of Example 2

 • Composition I: psoralen extract: 茯苓 extract =1: 1.5

 • Composition II: psoralen extract: 茯苓 extract =1:3

 • Composition III: Psoralea extract: 茯苓 extract =1:6

 • Fluoxetine hydrochloride (positive control group): purchased from Sigma-Aldrich, (St. Louis MO, USA), product number: F132

 • Saline (negative vs. control): purchased from Sigma-Aldrich, (St. Louis, MO USA), Product Code: 150-3

3. Mode of administration and dosage

The mode and dose of each group of mice to be administered are as follows:

• Saline (negative control group): 5 ml/kg/day

 • Bakuchi extract group: 80mg/kg/day, 50 mg/kg/day, 28.5 mg/kg/day

• 茯苓 extract group: 171.5 mg/kg/day, 150 mg/kg/day, 120 mg/kg/day

• Composition Group I: 200 mg/kg/day

 • Composition Group II: 200 mg/kg/day

 • Composition Group III: 200 mg/kg/day

• 'Fluoxetine hydrochloride group: 26 mg/kg/day The mice were fasted for 1.5 hours before dosing. The above drug is dissolved or suspended in physiological saline to prepare the concentration. According to the mouse weight drug. Oral, once daily, at 13:00-14:00 daily. As shown in Tables 3 and 4, the mice were administered for 3 days and 7 days, respectively. No abnormalities in the mice were observed during the administration. Behavioral experiments were performed 1 hour after the last administration. method

 1. Mouse tail suspension experiment

 Refer to the Stem (1985) method. Clamp the mouse 1 cm from the end of the tail, hang it upside down in the box (30 cmx30 cmx25 cm), the head is about 5 cm from the bottom of the box, observe for 6 min, and the immobile time of the mouse tail within 4 min after calculation .

 2. Mouse forced swimming experiment

 Refer to the Porsolt (1977) method. A single mouse was placed in a water depth 10 cm beaker (18 cm l4 cm) at a water temperature of 25 ± 1. C. After 6 minutes of observation, the immobility time of the mice swimming within 4 min was calculated. After the experiment, the animals were tested, placed in a hot air cage for 15 minutes, and then returned to the feeding conditions.

3. Statistical processing

 All data are expressed in Mean ± S.E.M.进行 ANOVA analysis method for statistical testing. PO.05 indicates a significant difference. At the same time, the Siw/erra ' /;?/^ correction method is used to prevent false positive results. Result

The results of the effect on the time of suspension of mice were shown in Table 3. The results of the effect on the swimming time of the mice are shown in Table 4.

Table 3 affects the time of mouse tail suspension

Compared with the control group, ΡΟ.05, PO.01, PO.001.

in conclusion

 1. The composition prepared from the psoralen extract and the alfalfa extract in an appropriate ratio can effectively reduce the immobility time of the tailed mice and the forced swimming mice at a suitable dose.

2. The effect of the composition from the strongest to the weakest is: Composition 1 > Composition 11 > Composition III. Experiment 2

 Study on Depressive Behavior and Biochemical Indexes in Rats Caused by Chronic Mild Stimulation

Material

 Animal

 • Male Wistar rats (200 ± 20) g, provided by the Jiangsu Experimental Animal Center. The experiment was carried out in the laboratory for 1 week before the experiment, and the temperature was kept at 25±2 °C for 12 h/12 h. Except for special stimuli and requirements, animals are kept in a single cage and fed freely. All animal testing procedures are in accordance with the regulations of the China Animal Management Committee.

2. Reagent

 • Composition I: Same as used in Experiment 1. Gp, psoralen extract: 茯苓 extract =1:1.5

 • 5-Hydroxytrptamine (5-HT): for monoamine oxidase-A (MAO-A) specific substrate, purchased from Sigma-Aldrich (St. Louis, MO, USA), product number: H 9523

 • P-phenylethylamine (β-ΡΕΑ): for monoamine oxidase-B (MAO-B) specific substrate, purchased from Sigma-Aldrich (St Louis, MO, USA), product number: P 2641

 • Fluoxetine hydrochloride: purchased from Sigma-Aldrich (St. Louis, MO, USA), product number: F132

 • Saline: for negative control, purchased from Sigma-Aldrich (St. Louis, MO, USA), part number 150-3

 • Sucrose: purchased from Sigma-Aldrich (St. Louis, MO, USA), product number: S7903.

 The sucrose aqueous solution l% (w/v) is made of 10 g of sucrose dissolved in 1000 ml of water.

 • Bovine serum albumin: purchased from Sigma-Aldrich (St.

Louis, MO, USA), Product Code: P 0914 • Butyl acetate: purchased from Nanjing Chemical Reagent Co., Ltd., product number: 1370401001

• Cyclohexane: purchased from Nanjing Chemical Reagent Co., Ltd., product number: 1370400701 • Sodium hydroxide: purchased from Nanjing Chemical Reagent Co., Ltd., product number: 1370500701

• Hydrochloric acid: purchased from Nanjing Chemical Reagent Co., Ltd., Product No.: 1370500101

• Sodium dihydrogen phosphate: purchased from Nanjing Chemical Reagent Co., Ltd., product number: 1370503001

• Disodium hydrogen phosphate: purchased from Nanjing Chemical Reagent Co., Ltd., product number: 1370503101

• WH-3 Micro Oscillator: It is a product of Shanghai Huxi Analytical Instrument Factory.

 • XHF-1 high speed disperser: It is a product of Shanghai Jinda Biochemical Instrument Co., Ltd.

 • U-3000 Spectrophotometer: Hitachi, Ltd., Tokyo, Japan.

 • Eppendorf centrifuge: Germany, 5415 R

 • HH-S electric thermostatic water bath: It is a product of Jiangsu Dongtai Electric Appliance Factory.

 • GC-1200r radioimmunoassay counter: for the science and technology industry company (USTCTEK)

3. Mode of administration and dosage

 • Blank saline group (not stimulated by CMS): 10 ml/kg/day

* Model saline group (stimulated by CMS): 10 ml/kg/day

 • Composition Group I (200): 200 mg/kg/day

 • Composition Group I (150): 150 mg/kg/day

• Fluoxetine hydrochloride group: 10 mg/kg/day The above substances were dissolved or suspended in physiological saline to make the concentration. According to the body weight of the mice. Oral, once a day for six weeks. The respective measurement methods of the CMS model are as follows. Sucrose intake test

 After one week of rat adaptation, all animals were trained in sucrose-based intake twice a week. Each time after being deprived of food and water for 14 h, 1% aqueous sucrose solution was supplied for 1 h and weighed. The sucrose intake of the rats was basically stable after 3 weeks. (How to calculate sucrose intake: The weight of the container before ingestion (g) - The weight of the container after ingestion (g)) In the following experiment, the sucrose intake was measured once a week (9: 00a. Rn.-10: 00 am), the method is the same as above.

2. CMS method

 After three weeks of training in sucrose-based intake, the rats were randomized into two groups, the empty group (not stimulated by CMS) and the model group (stimulated by CMS). The model group underwent a 4-week low-intensity mild stimulus. The stimulation included: deprivation of food or water, oblique cage 45 °C, interval illumination (alternating light/dark/2 h), contaminated squirrel cage (200 mL water added to the bottom), pairing Feeding, low intensity strobe lighting (150 times / mim). Stimulate twice a week in the above order, 12 to 14 hours each time.

 The blank group was housed in another room, and there was no contact with the animals in the stimulation group, except for the determination of sucrose intake after 14 hours of deprivation of food and water, and free drinking water.

3. Administration

 After 4 weeks of stimulation, the sucrose intake of the animals was significantly reduced. Then randomized and given the following drugs:

 (1) Composition (I) group: dose 200 mg/kg/day composition (I)

 (2) Composition (I) group: dose 150 mg/kg/day composition (I)

 (3) Fluoxetine hydrochloride group: dose of 10 mg/kg/day fluoxetine hydrochloride (positive control group)

(4) Model saline group: Dose 10 ml/kg/day saline (negative control group) All routes of administration were oral. All animals were dosed once daily at 10 am for 6 weeks. Except for the blank group animals, each group was stimulated during the administration period, and each was performed. 4. Blood and brain tissue sampling

 After the last determination of sucrose intake for 24 h, the whole blood was immediately decapitated during 10 a.m.-l la.m., centrifuged at 3000 °C for 10 min at 4 °C, and serum was taken at -20 °. C storage for cortisol determination. The whole brain was quickly taken on an ice table, and the blood was washed with frozen physiological saline and stored at -80 °C for the activity of monoamine oxidase A and B (MAO-A and MAO-B).

5. Determination of MAO-A and MAO-B activity

 The mitochondrial part of the rat brain was prepared as described by Schurr and Livne (1976). The whole brain MAO-A and MAO-B activity assays were performed by reference to the method of spectrophotometry by Yu et al. (2002).

The mitochondria fraction was suspended in 9 volumes of cold sodium dihydrogen phosphate buffer (10 mM, pH 7.4, containing 320 mM sucrose) at 4. Mix and stir for 20 minutes under C. The resulting mixture was centrifuged at 4000 rpm for 10 minutes at 4 ° C, and the resulting supernatant was centrifuged again at 15000 rpm for 30 minutes at 4 ° C to obtain a protein precipitate. The precipitate was resuspended in the same buffer. The protein concentration was adjusted to 1 mg/ml. The protein content was determined by the modified Lowry (1951) method. Bovine serum albumin is used as a standard. In the reaction system, 4 mM 5-HT or 2 mM β-oxime was used as a specific substrate for ΜΑΟ-Α and ΜΑΟ-Β. ΙΟηιΜ sodium dihydrogen phosphate buffer (pH 7.4) was added to the above mixture to a final volume of 1 ml. The reaction was carried out at 37 ° C for 20 minutes, and the reaction was quenched by the addition of 1 M hydrochloric acid. This was followed by extraction with 4 ml of n-butyl acetate (for MAO-A) or 4 ml of cyclohexane (for MAO-B). The activity of MAO-A and MAO-B in the organic layer was measured at 280 nm and 242 nm, respectively. The blank control shell lj was added with 200 ΠΜ hydrochloric acid before the reaction, and the other steps were the same as above. W

6. Determination of cortisol content

 Cortisol content was determined by radioimmunoassay. Follow the instructions of the manufacturer (manufacturer: Beijing Furui Biotechnology Co., Ltd., product number: FR-FJ-055).

7. Statistical processing

 All data are represented by Mean ± S.E.M. Statistical analysis was performed using the ANOVA analysis method. PO.05 indicates a significant difference. Also use the iw/erra ' correction method to prevent false positive results. - Results

 1. Effect on sucrose intake

 The results are shown in Table 5. There was no significant change in the intake of 1% sucrose water in the blank group during the whole experiment, but the sucrose intake in the model group was significantly lower than that in the blank group, and the negative control group in the model group was stimulated in the later stage. This has been consistently reduced in this way.

 In the model group (CMS) animals, oral administration of psoralen extract and sputum extract composition composition I (1:1.5) and fluoxetine reversed rat sucrose intake to varying degrees compared with week 0. The amount is reduced. After 2 weeks of administration, the composition 200 mg/kg significantly increased the sucrose intake of the model group (CMS) and returned to normal after 4 weeks of administration. The composition 150 mg/kg significantly increased the sucrose intake after 3 weeks of administration, and returned to normal after 5 weeks of administration. It takes 6 weeks for fluoxetine to restore sucrose intake to animals. It was shown that composition I (1:1.5) was more effective than fluoxetine in model group (CMS) animals. Table 5. Effect of composition I on sucrose intake

 Group dosing cycle (weeks)

 0 1 2 3 4 5 6 Blank group (not subject to 13.4 13.5 ± 13.8 14.1 13.8 13.4 ± 14.6 ±

±1.3 1.6 ± 1.6 ± 1.9 ± 1.1 1.9 1.7

 There was a statistically significant difference between the drug-treated animals in the model group at week 0: #Ρ <0.01,##Ρ<0.001

2. Effect on MAO-A and MAO-B activity

The results are shown in Table 6. Compared with the normal animal blank group, CMS can significantly increase the activity of MAO-A and MAO-B in the whole brain of rats. Oral administration of psoralen extract and alfalfa extract composition 1 (1: 1.5) 200 mg/kg and 150113⁄4/13⁄4 significantly inhibited the activity of ^^ 0 and 1^^0-:8, and inhibited the activity of MAO-A. The inhibition rates of MAO-B activity were 44.09% and 42.88%, respectively, which were 45.49% and 12.88%, respectively. Fluoxetine also had an inhibitory effect, and the inhibition rates of MAO-A and MAO-B activities were 16.43% and 54.05%, respectively. Table 6. Effect of Composition I on MAO-A and MAO-B Activity

There was a statistically significant difference compared with normal rats: ^# PO.05, ^m P<0.01, statistically significant difference compared with CMS-excited rats *P<0.05, **P<0.01

P<0.001

3. Effects on cortisol levels

The results are shown in Table 7. Compared with the normal animal blank group, CMS can significantly increase serum cortisol levels in rats. Oral administration of psoralen extract and alfalfa extract composition I (1:1.5) can significantly reduce serum cortisol levels in CMS animals, wherein the composition 200 mg/kg can be Cortisol levels in CMS rats returned to normal. Under the experimental conditions, fluoxetine failed to significantly change serum cortisol levels in CMS rats.

Table 7. Effect of composition I on cortisol levels

 Statistically significant differences compared with normal rats: # P<0.01

 Statistically significant difference compared with CMS-stimulated rats * *P<0.05, **P<0.01

1. The psoralen extract and alfalfa extract composition can effectively restore the sucrose intake of CMS-stimulated rats at a suitable ratio and dose, and there is a statistically significant difference compared with the saline group.

 2. The psoralen extract and alfalfa extract composition can effectively inhibit the increase of MAO-A and MAO-B activity in rats induced by CMS at a suitable ratio and dosage, and have statistically significant differences compared with the saline group. .

3. The psoralen extract and alfalfa extract composition can effectively reduce the increase of cortisol level in rats induced by CMS at a suitable ratio and dosage, and have statistically significant differences compared with the saline group. 4. Oral Composition I can reverse the abnormalities in behavior and biochemical indicators produced by CMS-stimulated rats. Significantly increased sucrose intake, resulting in significant antidepressant effects. The composition may achieve antidepressant effects by inhibiting the activity of MAO-A and MAO-B and down-regulating the function of the hypothalamic-pituitary-adrenal axis (HPA axis). Composition I with antidepressant effect is more effective for treating senile depression. Although Example 1 demonstrates a method of preparing a psoralen extract, there are many other methods for obtaining a psoralen extract. For example, 100 g of dried psoralen is ground into powder, placed in a 2500 ml round bottom bottle, and added with 800 mL of 65% ethanol, and extracted three times in a water bath at 85-90 ° C for 1.5 h each time to obtain a psoralen extraction solution. . The resulting extract was concentrated under reduced pressure at 60 ° C (rotary pressure concentrator: Buchi Labortechnik AG, Switzerland) to obtain 250 ml of a drug solution. The above-mentioned liquid was extracted 8 times with 250 ml of 100% ethyl acetate, and the ethyl acetate layer was combined and concentrated under reduced pressure at 60 ° C to remove ethyl acetate. The remaining residue was dried under reduced pressure at 60 ° C to obtain 12.21 g of psoralen extract (yield 12.21%). Supplementary experiment Part 1: The effectiveness of the extract and compound (psoralen: 茯苓 = 2:1)

First, the sample

 The compound is prepared by combining 2:1 compatibility with psoralen and medicinal materials, and then extracting with water and ethanol according to the conventional method. (In contrast to the above-mentioned composition I, the above-mentioned composition I is a psoralen medicine and a medicinal material, respectively, and is combined together).

(1) Extract sample name and abbreviation

The water extract and the ethanol extract of the three substances were formed by extracting the compound, psoralen and sputum with ethanol and water, respectively. For the psoralen ethanol extract and then petroleum ether, ' Extraction with chloroform, ethyl acetate and n-butanol. The sample names of various extracts are abbreviated as follows: Compound ethanol extract: Compound alcohol;

 Compound water extract: rehydration;

 Psoralen ethanol extract: supplemental alcohol;

 Buguzhi water extract: hydration;

 Petroleum ether extraction site of psoralen ethanol extract: supplemental phenol;

 Chloroform extraction site of psoralen ethanol extract: supplemental alcohol chlorine;

 Ethyl acetate extraction site of psoralen ethanol extract: supplemental alcohol ester;

 The n-butanol extraction site of the psoralen ethanol extract: supplemental alcohol;

 Extraction residue of psoralen ethanol extract: supplemental alcohol residue;

 Water extract: water;

Ethanol extract: sterol. The above extraction and extraction methods are carried out in a conventional manner. (2) The yield of the extract sample, and the determination results of its psoralen and isopsoralen are shown in Table, Table 8, Table C and Table D.

Table A extract sample yield

* The sample is not dry Table B Contents of psoralen and isopsoralen in medicinal materials, psoralen, psoralen, psoralen, psoralen sample

 Quantity (g) Content (%) Element content (%) Prime amount (g) Prime amount (g) (g) Polyol 667 0.405 0.356 2.701 , 2.375 5.076 Rehydration 667 0.405 0.356 2.701 2.375 5.076 Supplementary alcohol 1000 0.405 0.356 4.050 3.560 7.610 hydration 1000 0.405 0.356 4.050 3.560 7.610 supplemental alcohol

 1000 0.405 0.356 4.050 3.560 7.610 Stone

 Supplemental alcohol

 1000 0.405 0.356 4.050 3.560 7.610 supplemental alcohol

 1000 0.405 0.356 4.050 3.560 7.610 Ester

 Supplemental alcohol

 1000 0.405 0.356 4.050 3.560 7.610 normal supplemental alcohol

1000 0.405 0.356 4.050 3.560 7.610

Table c Extracts of psoralen and isopsoralen in different extracts Bone extract Extract psoralen Isopsoralen Bone fat B匕

 Sample, day, tea

 Amount (g) Content (%) Content (%) Amount (g) (g)

 (g)

 Polyol 188.73 1.367 1.196 2.580 2.257 4.837 Rehydration 195.1 0.110 0.084 0.215 0.164 0.378 supplemental alcohol 260.07 1.524 1.35 3.963 3.511 7.474 hydration 242.43 0.129 0.099 0.313 0.240 0.553 supplemental alcohol

 77.11 0.897 0.995 0.692 0.767 1.459 stone

 Supplemental alcohol

 133.66 2.148 1.758 2.871 2.350 5.221 Chlorine

 Supplemental alcohol

 18.48 0.749 0.612 0.138 0.113 0.252 ester

 Supplemental alcohol

 19.78 0.033 0.038 0.007 0.008 0.014 正补醇

52.63 0.012 0.01 0.006 0.005 0.012

Transfer rate of psoralen and isopsoralen in extract

 Psoralen transfer rate

Sample isopsoralen transfer rate (%) total transfer rate (%)

 (%) Surface 95.52 95.03 95.29 Rehydration 7.96 6.91 7.45 Supplemental alcohol 97.85 98.62 98.21 Hydration 7.73 6.74 7.27 Supplementary alcohol

 17.09 21.54 19.17 stone

Supplemental alcohol

 70.89 66.01 68.61 supplemental alcohol

 3.41 3.17 3.31 ester

Supplemental alcohol

 0.17 0.22 0.18 positive

Supplemental alcohol

0.15 0.14 0.16

(3) Dose design and its basis

 According to the Chinese Pharmacopoeia, the amount of Chinese herbal medicines is calculated in the same way as the previous method.

 H (high dose) 9 g/kg

 L (low dose) 6 g/kg

 H (high dose) 15 g/kg

 L (low dose) 9 g/kg

 Compound H (high dose) (9+4.5) g/kg

 (Psoralen: 茯苓 = 2:1) L (low dose) (6+3 ) g/kg

Table E Extract dose

Sample dose high dose low dose dose extract dose

 (g/kg) (mg/kg) psoralen water extraction H 1.17 283.643 substances L 0.78 189.095 water extract H 1.95 56.121

 L 1.17 33.673 compound (psoralen: H 1.755 342.401 茯苓 = 2: 1) water extraction L 1.17 228.267

Psoralen extraction H 1.17 304.282 L 0.78 202.855 sterol extract H 1.95 18.135

 L 1.17 10.881

H 1.755 331.221 Compound (psoralen: H 1.755 331.221 茯苓 = 2:1) Alcohol L 1.17 220.814

Take

After drowning water H 1.95 362.700

Residual alkali extract L 1.17 217.620 Bakuchiol extraction H 1.17 90.219

L 0.78 60.146

Petroleum ether extraction department

Minute

Bakuchiol extraction H 1.17 156.382

L 0.78 104.255 chloroform extraction section

Bakuchiol extraction H 1.17 21.622

Ethyl acetate extraction L 0.78 14.414

Take part

Bakuchiol extraction H 1.17 23.143

N-butanol extraction L 0.78 15.428

section

Bakuchiol extraction H 1.17 61.577

Extract residue L 0.78 41.0151 II. Behavioral despair model

 Animal

Male ICR mice (clean grade, body weight 20-25g). The animal room temperature is controlled at 22 ± 2 V. Maintain a 12 hour light and dark interval. Dosage at 14: 00 daily. Free water food. Adapted to support for 4 days, Remove abnormal animals. The administration time was one, three, seven, and fourteen days, respectively. One hour after the last administration, a mouse forced swimming test (FST) and a tail suspension experiment (TST) were performed. The behavior observation time is from 9: 00 to 15: 30 of the day.

 2. Mouse forced swimming test (FST)

 The mice were placed in a beaker having a depth of 10 cm (20 cm in height and 14 cm in diameter) at a water temperature of 25 °C. After 6 minutes of observation, the immobility time of the mice in the administration group and the control group was compared within 4 minutes.

 3. Mouse tail suspension experiment (TST)

 2 cm of the tail of the mouse was adhered to a horizontal wooden stick, and the head was placed upside down. The head was about 5 cm away from the table, and the sides of the line of sight of the mouse were blocked by a plate. After 6 min of suspension, the immobility time of the mice in the administration group and the control group was compared within 4 min.

 All experimental data above are expressed as ±. Statistical analysis was performed using the Student-t analysis method. PO.05 indicates a significant difference. Third, the experimental results

 (1) Comparison of antidepressant activity of ethanol and water extracts of scented psoralen, sputum, and psoralen: 茯苓(2:1) The experimental results are shown in Table F and Table G, respectively. It can be seen from the table:

 1. Single psoralen ethanol and water extract

 The experimental results in the animal TST and FST experiments were basically the same. After 3, 7 and 14 days of administration, the psoralen ethanol extract was stronger than the water extract, and the low-dose group of skeletal ethanol extract was slightly stronger than the high dose group.

 Comparing the content of ingredients, the content of psoralen and isopsoralen in psoralen ethanol extract was much higher than that of psoralen water extract.

2. Single miso alcohol and water extract The experimental results on animal TST and FST experiments were also basically the same. After 3, 7 and 14 days of administration, the hydrophobic extract was stronger than the alcohol extract, and the hydrophobic extract was slightly stronger than the high dose group.

 3. Compound psoralen: 茯苓 (2: 1) ethanol and water extract

 In the animal TST experiment, the animal suspension time was shortened to some extent after 3 and 7 days of administration. After 14 days of administration, the compound ethanol extract was stronger than the water extract, and the low dose group was slightly stronger than the high dose group.

 In the animal FST experiment, the compound ethanol extract was stronger than the water extract after 3 days of administration, and the dose relationship was not obvious. After 7 days of administration, the high-dose group of the compound water extract was slightly stronger than the low-dose group of the ethanol extract. After 14 days of administration, the low-dose group of the compound ethanol extract was stronger than the water extract group, and the high-dose group was basically equivalent. In general, the compound ethanol extract acts stronger than its water extract.

 Comparing the content of the ingredients, the content of psoralen and isopsoralen in the compound ethanol extract was much higher than that of the compound water extract.

 4. Single psoralen, ethanol extract of medlar, and compound psoralen: ethanol extract of 茯苓 (2: 1)

 In the animal TST experiment, there was no significant difference between the 3 and 7 days of administration. After 14 days of administration, the single-flavored psoralen ethanol extract had a stronger effect.

 In the animal FST experiment, the effect of the single extract of psoralen ethanol extract and the compound ethanol extract was basically the same, with dose effect.

 The active ingredient in the compound alcohol extract may be derived from psoralen.

 5. Single psoralen, water extract of medlar, and compound psoralen: water extract of 茯苓 (2: 1)

In the animal TST experiment, there was no significant difference after 3 days of administration. After 7 days of administration, the compound water extract was slightly more potent. After 14 days of administration, the water extract of the single miso Stronger.

 In the animal FST experiment, the water extract of the single miso was stronger after 3 days of administration. After 14 days of administration, the high and low dose effects of the aqueous extract of the single miso were comparable to those of the combined aqueous extract.

 The active ingredient in the compound water extract may be derived from strontium.

6. Residue alkali extract after hydrophobic extraction

The alkaline extract of the residue after hydrophobic extraction was slightly stronger on the TST in the animal FST experiment. It was further confirmed that the residue alkali extract after the hydrophobic extraction had antidepressant activity.

Table F The effects of single-flavored psoralen, ethanol and water extracts of alfalfa, and the antidepressant activity of ethanol and water extracts of compound psoralen: 茯苓 (2:1) on the time of suspension in mice

Table G The effects of single-flavor psoralen, ethanol and water extracts of medullary blush, and compound psoralen: 茯苓(2:1) on the anti-depressant activity of ethanol and water extracts on the swimming time of mice

(B) the anti-depressant activity of the skeletal fat extract organic solvent in turn, the anti-depressant activity comparison activity results are shown in Table H and Table I. In the animal TST experiment, the n-butanol extraction site of the psoralen ethanol extract had no effect. The extraction residue of psoralen ethanol extract was administered for 7 days. And after 14 days there is a certain effect. The petroleum sputum extraction site, the chloroform extraction site and the ethyl acetate extraction site of the psoralen ethanol extract were effective after 3 days, 7 days and 14 days of administration, and the relationship between the three strengths and the dose was not obvious. There was also no significant difference in the effect with the psoralen extract.

 In the animal FST experiment, the n-butanol extraction site of the psoralen ethanol extract and the psoralen residue had no effect. The petroleum ether extraction site, the chloroform extraction fraction and the ethyl acetate extraction site of the psoralen ethanol extract were effective after 3 days, 7 days and 14 days of administration, and the dose relationship was obvious. The high dose effect of the low-dose, chloroform extraction site and ethyl acetate extraction site of the petroleum ether extract of psoralen ethanol extract was particularly significant. After 14 days of administration, the high-dose effect of the chloroform extraction site and the ethyl acetate extraction site of the psoralen ethanol extract was stronger than that of the psoralen extract.

 Comparing the components, the psoralen ethanol extract was extracted in stages by organic solvent, and the chloroform extraction site was mainly concentrated in the content of psoralen and isopsoralen.

Fourth, the conclusion

1. Samples derived from psoralen rich in the above coumarin components have strong antidepressant effects.

2. The water extract and alkali extract samples derived from the high total sugar content of the cockroach have a strong antidepressant effect.

Table H Effect of sequential extraction of organic solvent from psoralen extract on the time of suspension of mice. Group dose High or low Suspension time (s) (mean ± standard error)

 3 days 7 days 14 days Blank control 109.70±8.38 124.89±8.35 112.40 soil 11.17 Fluoxetine 57.70±8.94 82.78 ± 6.69 69.60 ± 6.56 Perm Fe H 98.33 ± 12.35 117.70±7.91 79.74±9.88*

L 80.44士11.37* 114.64±5.12* 81.05±11.23* Supplemental alcohol chlorine H 83.75±8.94* 98.71±9.30 ^¥ 82.57±11.4f

 L 79.15±6.59" 121.19±9.26 71.25±8.74" supplemental alcohol ester H 80.93±8.37* 98.50±8.24* 82.29±7.68 *

L 89.45±6.87 ^¥ 81.20±7.69* 97.60±9.14 Supplemental alcohol positive H 90.42±8.36 114.89±10.81 86.73±11.49

 L 97.00±9.24 101.13±6.13* 108.85±10.86 Supplementary alcohol residue H 93.35±8.75 98.50±5.76* 106.60±5.64

 L 96.39士 7.62 95.95±6.78" 88.58±10.94 * supplemental alcohol · H 64.16士 7.24 87.84±7.80" 87.99±11.92

L 79.17±7·9 ( 102.62±4.67* 68.23±11.78 **

Influence

The second part of the mixture of psoralen and isopsoralen and psoralen on animal behavior and

 Influence of the ΗΡΑ axis

(The mixture of psoralen and isopsoralen and psoralen are both coumarins) 1. Preparation and analysis of psoralen monomer 1. Preparation of a mixture of psoralen and isopsoralen 1 kg of psoralen crude powder, 95% ethanol reflux 3 times, 3 hours each time. The extracts were combined, and the extract was concentrated to a non-alcoholic taste under reduced pressure, diluted with water to 1000 mL, and extracted with an equal volume of chloroform for 3 times. The chloroform extract was concentrated under reduced pressure to give 165 g of extract. The aqueous layer after chloroform extraction was extracted with an equal amount of ethyl acetate for three times. The ethyl acetate extract was concentrated under reduced pressure to give a 15 g g.

 Take 60 g of chloroform extract, dissolve it in 60 mL of methanol, load it on Sephadex LH-20 (column 500 g, equilibrated with methanol), elute with methanol, and collect the fractions when the sample is eluted. 20 mL, until the sample is completely eluted, a total of about 40 parts. Each fraction was tested by TLC, and psoralen and isopsoralen (purchased from China National Institute for the Control of Pharmaceutical and Biological Products) were used as reference materials, and the fractions rich in psoralen and isopsoralen were combined. Concentrate to an appropriate volume and load on a Sephadex LH-20 column, elute with methanol, collect fractions, and measure by TLC. This was repeated 5 times, and a sample of 2.20 g of a mixture of psoralen and isopsoralen was obtained, and the yield was about 0.61%.

 The TLC test conditions were as follows: Silica gel GF 254 prefabricated plate, observed with cyclohexane-ethyl acetate (3:1) as a developing solvent under a 365 nm UV lamp.

The HPLC conditions were as follows: Column: Zorbax XDB RP-C ₁₈ (4.6 mm 250 mm, 5 m); Mobile phase: Methanol-water (40: 60); Flow rate: lO mL . min'¹; Detection wavelength: 246 Wake up .

1. Take 1.5g of psoralen and isopsoralen mixed sample, colorless needle crystal (acetone), which is identified as psoralen and isopsoralen mixture by HPLC compared with control TLC. The chromatographic conditions are the same as before. The UV spectrum is shown in Figure 4. 2. Preparation of psoralen (BGZ-1) in psoralen

 15 g of the above ethyl acetate extract was taken, dissolved in 40 mL of methanol, and applied to a column of Sephadex LH-20 (column 500 g, equilibrated with methanol), eluted with methanol, and collected while the sample was eluted. Divided into 20 mL each, until the sample was completely eluted, a total of about 30 parts. Each fraction was tested by TLC, and the psoralen was used as a control. The fractions rich in psoralen were combined, concentrated to an appropriate volume, and loaded on a Sephadex LH-20 column, eluted with methanol, and collected. Flow fraction, TLC detection. This was repeated 4 times, and 0.42 g of psoralen was obtained, and the yield was about 0.04%.

 The TLC test conditions were as follows: Silica gel GF 254 prefabricated plate, observed with cyclohexane-ethyl acetate (3:1) as a developing solvent under a 365 nm UV lamp.

HPLC conditions were as follows: Column: Zorbax XDB RP-C ₁₈ (4.6 mm 250 mm, 5 m) _; mobile phase: methanol-water (65: 35); flow rate: lO mL . min'¹; detection wavelength: 340 nm . Take psoralen to 0.2g. Light yellow powder (acetone), identified as 1 phloem by ¹ H NMR and MS spectroscopy, HPLC (chromatographic conditions are the same as Chapter 1), UV spectrum is shown in Figure 5.

Second, the experimental method

1. The behavioral experiment method is the same as before.

 2. Biological samples

 Serum and brain sections were quickly taken in the usual way. Store at -80 ° C for HPA

3. Determination of corticotropin releasing factor (CRF) and adrenocorticotropic Hormone (ACTH) Samples were processed as usual and determined by radioimmunoassay

4. Determination of serum cortisol (Cortisol)

 Samples were processed as usual and determined by magnetic separation enzyme-linked immunosorbent assay. Third, the experimental results

 (1) a mixture of psoralen and isopsoralen

1. Impact on the behavior of depressed animals

 The results are shown in Table A and Table B. The experimental results of psoralen and isopsoralen mixed samples on animals TST and FST were basically the same. Compared with the model blank control group, after 1 day, 3 days, 7 days and 14 days of administration, the mixed samples of psoralen and isopsoralen showed a significant inverse U-shaped dose-effect relationship. The optimal dose was 20 Mg/kg, followed by 40 mg/kg. Under the experimental conditions, the effect was similar to that of fluoxetine with a prolonged administration time, and slightly weaker than amitriptyline. This result further confirmed that the mixed sample of psoralen and isopsoralen has an antidepressant effect.

 2. Effect of mixed samples of psoralen and isopsoralen on CRF levels

 Cortex: FST significantly increases cortical CRF levels. After 1 day of administration, each dose group significantly decreased the increase in cortical CRF levels caused by FST. After 3, 7 and 14 days of dosing, there was a trend to reduce cortical CRF levels in each dose group, but there was no significant effect.

 Hippocampus: FST increases hippocampal CRF levels. One day after administration, the dose group of 20-60 mg/kg significantly reversed the increase in CRF levels in the hippocampus caused by FST, and there was substantially no significant effect in each dose group after 3, 7 and 14 days of administration.

Corpus striatum: FST does not substantially affect striatum CRF levels. There was also no significant effect in each dose group after administration. Medulla oblongata: FST can significantly increase the level of CRF in the medulla oblongata. Both the 1 and 3 day low dose groups significantly reduced the elevation of CMR caused by FST. After 7 and 14 days of dosing, each dose group substantially reduced the medullary CRF level.

 Hypothalamus: FST can significantly increase hypothalamic CRP levels. After 14 days of administration, each dose group substantially reduced hypothalamic CRF levels.

 Serum: FST increases serum CRF levels. After 3 days of administration, each dose group had a certain decrease in serum CRF levels caused by FST.

 3. Effect of mixed samples of psoralen and isopsoralen on serum cortisol and ACTH levels

 FST increases serum cortisol levels. After 1, 7, and 14 days of dosing, each dose group significantly reduced the increase in serum cortisol levels caused by FST.

FST has the effect of lowering serum ACTH levels. There was essentially no significant effect after administration.

Effect of mixed samples of psoralen and isopsoralen on the time of mouse tail suspension (n=15)

(mg/kg) 1 3 7 14 (days) blank control 97.12±6.19 97.70±6.48 114.23±8.03 109.61±7.48

Mixed sample of psoralen and isopsoralen

 10 82.26±5.98 74.30±7.56 82.98±7.28 104.07±9.24

 20 75.17士 6.98 65.83±5.24 87.05±5.21 68.31±9.52

 40 74.59±5.95 75.10±8.39 89.68士 12.17 89.39±8.22

 60 83.88±5.75 80.98士 4.30 92.63±7.72 106.80±4.64

Amitriptyline 10 55.69±5.78 40.38±5.74 73.66±5.60 52.15±7.37 Fluoxetine 26 59.12±5.70 70.37±9.01: 74.65 soil 10.52 64.55±7.69'

Group 2 Effect of mixed samples of psoralen and isopsoralen on forced swimming time in mice (n=15)

 Different agent

(mg/kg) 1 3 7 14 (days) blank control - 105.37±7.27 95.76±8.41 104.66±7.81 105.419±8.08

Mixed sample of psoralen and isopsoralen

 10 53.22士 6.42*** 82.88±11.30 82.75±7.94 74.94±10.90

 20 67.95±6.38*** 64.94±10.04* ' 67.56±9.19** 54.23±9.46

 40 61.18士 6.95*** 74.16±7.30* 71.86士 10.85 63.48±8.41

60 82.54±8.22* 70.53±10.36* 77.20±10.54: 78.88±7.73 ^H

Amitriptyline 10 53.21±8.02*** 50.08±8.82' 60.33±11.36 40.68±7.75

Fluoxetine 26 60.94±7.02*** 39.14±8.31: 74.35±12.42* 58.78±11.36*

Effect of psoralen mixture sample on CRF level in mouse brain under FST model

Table 3-2

Table 3-3

Group Qi [J amount (mg/kg) CRF (ng/g wet tissue weight)

Day 7 Cortex Hippocampus Striatum Medullary Hypothalam Normal control - 74.91 ±5.18 300.84 ± 16.82 422.01 ± 19.29 89.80 ±5.12 437.07 ±22.03

FST control - 100.14 ± 7.75 + 332.58 ± 20.36 434.91 ± 32.17 109.15 Ϊ 5.07 457.18 ± 19.37 psoralen mixture 10 108.98 ± 11.07 + 320.81 ± 26.60 471.58 ± 42.60 89.44 ± 4.68 * 465.99 ± 28.71 psoralen mixture 20 98.23 ± 9.76 + 292.87 ±10.17 457.52 ±51.98 84.19 ±7.27' 477.27 ±32.41 psoralen mixture 40 88.45 ±6.89 285.67 ± 17.08 378.76 ±17.62 81.74 ±8.18" 452.27 ±18.87 psoralen mixture 60 81.82 ±5.18 278.93 ± 26.94 359.52 ±39.36 85.40 ± 8.27' 527.05 ±27.77+ amitriptyline 10 112.82 士^ 321.59 ±22.54 430.58 ±73.81 96.58 ±4.89 364.61 ±30.35, fluoxetine 26 95.30 ±6.38+ 349.59 ±33.31 471.40 ±47.26 95.46 ±7.53 479.76 ±37.17

Table 3-4

Psoralen, fluoxetine and amitriptyline were administered orally for 1, 3, 7, and 14 days, respectively. The data was taken in a mean SEM with an animal number _n = 10. *P<0.05, **P<0.01, ***P<0.001 compared with the FST stimulation control group. Compared with the normal control group, +Ρ<0.05, ^Ρ^.ΟΙ, ^PO.OOL

2005/002432 Table 4-1 Serum CRF levels, cortex of ICR mice in the FST model

 Table 4-2

 Group dose serum

 (mg/kg) CRP (ng/ml) ACTH (pg/ml) Cortisol (ng/ml)

Day 3 Normal blank - 3.97 ±0.53 76.20 ± 6.27 4.44 ± 0.22

FST

Blank - 5.06 ±0.31 73.85 ± 6.46 6.48 ± 0.38 ⁺⁺⁺ psoralen mixture 10 3.32 ± O 104.28 ± 19.02 5.41 ±0.38+

 20 3.15 ±0.39" 93.34 ± 10.27 5.32 ±0.29+

40 2.71 ± 0.38TM 88.33 ± 10.75 6.81 ± 0.37 ⁺⁺⁺

60 3.45 ±0.48 70.97 ±8.75 6.56 ±0.36+ amitriptyline 10 5.14±0.52 63.67 ± 13.14 5.17 ±0.26 Fluoxetine 26 3.94 ±0.33' 57.21 ±6.01 5.44±0.25 ⁺ Table 4-3

Table 4-4

Psoralen, fluoxetine and amitriptyline were orally administered 1, 3, 7, and 14, respectively. The data is in the mean SEM, the number of animals is n=10. *P<0.05, **P<0.01, ***P<0.001 compared with the FST-stimulated control group. Compared with the normal control group, + P<0.05, ⁺⁺ P<0.01, PO.OOl o

(2) Buguzhiding

 1. Impact on the behavior of depressed animals

 The results are shown in Table E and Table F. The results of BGZ-1 in animal TST and FST were basically the same. Compared with the model blank control group, BGZ-1 significantly shortened the animal's tail suspension and swimming time after 3 days of administration. The low dose group was more effective than the high dose group. The optimal dose is 20 mg/kg.

On TST, BGZ-1 (20 mg/kg) is slightly stronger than fluoxetine, but Weak than amitriptyline. On FST, BGZ-1 (20 mg/kg) was weaker than fluoxetine and amitriptyline.

 This indicates that BGZ-1 has an antidepressant effect. Table E Effect of BGZ-1 on the time of mouse tail suspension (n=15) Time of suspension tail (s) (mean ± standard error)

(mg/kg) (days) blank control - 97.70±6.48

Buguzhiding 20 57.58±6.41

 60 80.70±6.16 *

Amitriptyline 10 40.38±5.74

Fluoxetine 26 68.20±7.04 **

Effect of Buguzhi (BGZ-1) on swimming time in mice (n=15)

Swimming time (s) (mean ± standard error)

(mg/kg) (days)

96.74±7.34

 56.29±6.54

 74.39±9.16

 46.36±5.54

39.14±8.31

2. Effect of psoralen on HPA axis function

 Under the above conditions, BGZ-1 administration significantly reduced serum CRF, serum cortisol and ACTH levels after 3 days of administration, and had a dose-effect relationship. See Table G.

Table G. Effect of BGZ-1 on serum CRF levels, cortisol levels, and ACTH levels in ICR mice that were not stimulated and stimulated by FST ^a

Fourth, the conclusion

1. The coumarin ingredients in psoralen such as psoralen, isopsoralen, and psoralen have antidepressant effects. It is further demonstrated that samples derived from psoralen rich in the above coumarin components have a strong antidepressant effect.

 2. The mixture of psoralen and isopsoralen has different effects on the function of the sacral axis in different brain regions, and there is a quantitative and time-dependent relationship.

3. The mixture of psoralen and isopsoralen can reverse the serum CRF caused by FST There is a quantitative and time-dependent relationship with elevated serum cortisol levels.

 4. Mixed samples of psoralen and isopsoralen may achieve antidepressant effects by improving HPA axis dysfunction.

 5. Buguzhiding can reverse serum-related HPA axis function indicators.

Part III: Proportion of different extract ratios 1. Compatible samples Converted according to human dose

Group Number Psoralen (g) Medicinal Herbs (g)

5-1 5 10

 5-2 5 12

 5-3 5 15

 6-1 7 10

 6-2 7 12

 6-3 7 15

 7-1 9 10

 7-2 9 12

7-3 9 15

Extract dose: mouse

Single extract

 According to the dosage of human medicine (70kg person)

 Group Number Psoralen (g) Medicinal Herbs (g)

8-1 5 0

 8-2 7 0

 8-3 9 0

 9-1 0 10

 9-2 0 12

9-3 0 15 Group number psoralen extract (mgkg) 茯苓 extract (mgkg)

8-1 48.3 0

 8-2 67.6 0

 8-3 86.9 0

 9-1 0 20.0

 9-2 0 24.0

9-3 0 30.0

Effect of Table A on the time of suspension of TST mice (n=10, Mean±S.E.M.)

Group agent hanging time (s)

(mg/kg) Day 7 Day 14 Blank 115.8 ± 14.5 117.6 ± 11.8

Flu 26 56.5 ± 7.7" 61.0 ± 7.4***

5-1 - 89.9 ±7.0 108.0 ±8.5

5-2 - 72.2 ‧ 8.8* 88.9 ± 9.5

5-3 - 90.4 ± 10.7 88.9 ±8.0

6-1 - 62.8 ± 10.5 79.4 ±8.8*

6-2 - 70.1 ±8.9* 97.2 ± 11.0

6-3 - 91.7 ±9.8 107.8 ± 14.0

7-1 - 87.6 ± 11.9 104.0 ± 12.6

7-2 - 81.8 ± 8.2 84.4 ± 12.5

7-3 - 72.4 ± 10.5* 84.1 ±13.1

8-1 - 81.0 ±8.6 90.2 ±8.0

8-2 - 85.8 ±8.6 81.5 ±9.9*

8-3 - 72.3 ± 8.4* 67.6 ± 12.1

9-1 - 78.0 ± 12.3 66.0 ±10.8**

9-2 - 63.0 ±7.3 81.2 ± 12.0*

9-3 - 80.2 ±5.1* 116.5 ±8.5

Compared with the blank control group, *P<0.05;P<0.01;P< 0.001 Effect of Table B on swimming time in FST mice (n=10, Mean±SEM)

(mg/kg) Day 7 Day 14 Blank 120.7 ± 13.3 93.3 ±9.2

Flu 26 79.4 ± 7.69* 70.8 ± 12.3

5-1 - 82.2 ±6.5* 66.4 ± 11.9

5-2 - 80·0± 11. 59.6士 11,0*

5-3 - 103.6 ± 13.8 92.4 ± 10.7

6-1 - 99.0 ±9.9 85.8 ± 10.5

6-2 - 96.9 ±7.1 90.3 ±9.1

6-3 - 68.1 ±6. Wide 62.3 ± 10.4*

7-1 - 88.9 ±7.6 71.9 ± 10.6

7-2 - 91.1 ±6.0 62.8 士 8.

7-3 - 84.1 ±6 63.3 ±6.5*

8-1 - 74.1 ±7.4** 86.8 ±8.9

8-2 - 78.8 ± 11.5* 88.6 ± 16.0

8-3 - 93.6 ±5.7 84.0 ±8.4

9-1 - 74.9 8.6 ** 66.0 ± 4.4*

9-2 - 82.4 ±5.9* 54.2 ± 7.4"

9-3 85.2 ±6.2* 70.8 ± 10.8

Compared with the blank control group, P<0.05: P<0.01: P< 0.001 The preparation of the compositions of the present invention is exemplified in the above description. Although the examples exemplify specific ways of practicing the invention, those skilled in the art will be able to prepare compositions encompassed by the present invention using similar methods.

 Unless otherwise indicated, all scientific and technical terms used in this specification have the same meaning as commonly understood by those skilled in the art. The present invention is only illustrative of the preferred methods and materials, and other similar methods and materials are also suitable for practicing the present invention. Those skilled in the art of preparing Chinese medicines can easily practice the present invention in the above manner.

 Although traditional Chinese medicines are generally prepared from dry materials, it should be understood that the purpose of drying plant materials is simply for storage, transportation, and subsequent processing. Drying is not for the benefit of herbal medicine. Therefore, it should be understood that the present invention can be practiced in the same manner as fresh herbal plants. It is within the scope of the invention to prepare a traditional Chinese medicine using fresh plant material in a suitable ratio and amount.

 It is also noted that although certain parts of the plant contain higher amounts of active ingredients, the plant and fungal materials disclosed herein are also specific plant and fungal parts according to the Pharmacopoeia of the People's Republic of China. However, the active ingredient may also be present in other parts of the same plants and fungi. Therefore, it is within the scope of the invention to extract relevant active ingredients from other parts of the same plants and fungi. It should also be understood that in the Pharmacopoeia, many plant species within a genus belong to the same plant, and plants of different species within the same genus can sometimes be substituted for each other.

Those skilled in the art will know how to grow herbaceous cells and tissues in vitro using plant cell and tissue culture techniques, and extract the desired activity from these cells and tissues. Minute. Thus, while the present invention discloses how to extract active ingredients from dried plant and fungal parts, it is within the scope of the invention to extract relevant active ingredients in plant cells and tissues.

 The general extraction process involves reducing the volume of the herb material and then extracting it with a suitable extract, for example, by reflux. The herb extract can be obtained by immersing the whole plant of psoralen or sputum, the leaves, stems, roots, sclerotia, mycelium and/or fruit of the plant in an extract, or refluxing the extract . The type of extract is not limited. Usable extracts such as organic solvents such as methanol, ethanol, propanol, butanol, propanol, ... 1,3 butanediol, glycerol, acetone, butanone, ethyl acetate, diethyl ether, chloroform, dichloromethane And water. The solvent may be used singly or in combination. A preferred embodiment of the invention is the use of methanol, ethanol, ethyl acetate or a mixture of these solvents with water. A more preferred embodiment is the use of ethanol or a mixture of water and ethanol, which is considered to be safe for organisms (low toxicity).

 The reduction in the volume of the herb can be, but is not limited to, achieved by: cutting, damage, shearing, twisting, twisting, mashing, grinding, pulverizing, and the like. As long as it is reduced, it can increase the surface area of the herb. All materials and methods for achieving such objectives are within the scope of the invention.

 While the invention has been described as being illustrative of the embodiments of the present invention, it is to be understood by those skilled in the art that

When administered to different species of mammals, fish or birds, adjustments to the drug, dosage form, dosage, administration time and interval are inevitable. In some cases, a dose lower than the lowest dose described herein is far enough. In other cases, even if the dose used is higher than the highest dose, it will not cause harmful side effects, as long as it will be higher before administration. The dose is divided into multiple smaller doses in one day and administered in multiple doses.

 The active ingredient may be administered alone or in admixture with other pharmaceutically acceptable carriers or diluents. Administration can be carried out one or more times. The active ingredient can be administered in a number of different dosage forms, such as pills, capsules, troches, tablets, hard candies, powders, sprays, creams, ointments, suppositories, gels, gels, pastes, lotions, Ointments, suspensions, injectable solutions, medicinal liquors, canes and the like.

 Carriers include solid diluents or fillers, sterile aqueous vehicles, various non-toxic organic solvents, and the like. The oral pharmaceutical composition may also be suitably sweetened or flavored. The concentration of active compound in these dosage forms is from about 5.0% to about 70%.

 When administered orally, the pill may contain various excipients such as microcrystalline cellulose, sodium citrate, calcium carbonate, dicalcium phosphate, glycine, or may be used together with various disintegrating agents, such as starch (preferably Corn, potato, tapioca starch, alginic acid, and specific silicides may also be mixed with granulation binders such as polyvinylpyrrolidone, sucrose, bone glue and gum arabic. In addition, a lubricant such as magnesium stearate, sodium lauryl sulfate, talc or the like may be added, which is a good adjuvant for preparing the pellet. A similar solid composition can also be added to the capsule as a filler. Preferred materials include lactose, milk sugar, and polymeric polyethylene glycol.

 In the preparation of aqueous suspensions and/or medicinal liquors for oral administration, the active compounds may be mixed with a plurality of sweetening, flavoring, colouring or colouring agents. And, if necessary, it can be mixed with an emulsifier and/or a suspending agent. It may also be mixed with a diluent such as water, ethanol, propanol, glycerin or the like.

In the preparation of parenteral injection forms, the active ingredient can be dissolved in sesame oil and peanut oil. An oily solution, or dissolved in propylene glycol to make an aqueous solution. The aqueous solution must be suitably buffered (preferably at a pH greater than 8) and the liquid used for dilution must be isostatic. The aqueous solution is suitable for intravenous injection. The oil solution is suitable for intra-articular, intramuscular or subcutaneous injection. All of the above solutions are prepared under sterile conditions and follow standard pharmaceutical techniques well known to those of ordinary skill in the art.

 Alternatively, the active compound can be administered topically. Useful means include: creams, gels, gels, pastes, ointments, ointments and the like, prepared by standard pharmaceutical techniques well known to those of ordinary skill in the art.

 In the case of administration to animals other than humans, such as cattle or poultry livestock, the active compound can be administered orally, for example, by means of a drug.

 The active compound can also be administered via a liposome delivery system, such as a single layer of small vesicles, a single layer of large vesicles, multiple layers of vesicles, and the like. The vesicles can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholine.

 When prepared as a nutritional supplement, it can be mixed with other foods that do not affect the activity of the drug. For example, starch, glucose, oil, cellulose, and the like.

The references cited in this manual, including the materials listed below, are references to the entire document.

Archer, J., 1973. Tests for emotionality in rats and mice: A review. Animal Behavior 21, 205-235.

 Porsolt, R.D., Bertin, A., Jalfre, M., 1977. Behavioural despair in mice: a primary screening test for antidepressant. Archives Internationales de Pharmacodynamie et de Therapie 229, 327-336.

 Stem L, Chermat R, Thierry B, Simon P. 1985. The tail suspension test: a new method for screening antidepressants in mice. Psychopharmacology (Berl) 85: 367-370.

 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. 1951. Protein measurement with folin phenol reagent. J. Biol. Chem. 193, 265-275. Schurr, A., Livne, A., 1976. Different inhibition of mitochondrial monoamine Oxidase from brain by hashish components. Biochemical Pharmacology 25, 1201-1203.

 Yu, Z.F., Kong, L.D., Chen, Y" 2002. Antidepressant activity of 161-165.

 Willner P. 1997. Validity, reliability and utility of the chronic mild stress model of depression: a 10-year review and evaluation. Psychopharmacology (Berl) 134, 319-29.

Claims

Claim

A pharmaceutical composition for treating depression, characterized in that the composition comprises an extract of a leguminous plant psoralen and an extract of a polyporaceae fungus.

 The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition consists essentially of an extract of a leguminous psoralen and an extract of a polyporaceae fungus.

 The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is composed of an extract of a leguminous psoralen and an extract of a polyporaceae fungus.

The pharmaceutical composition according to Claim 1, 2, or 3, wherein the pharmaceutical composition is an extract of psoralen (dried mature fruit of leguminous psoralen) and mash

(Dry sclerotia of Polyporaceae fungus )) Extract composition.

 The pharmaceutical composition according to claim 4, wherein the weight ratio of the psoralen extract to the alfalfa extract is from 1:1 to 1:7.

 The pharmaceutical composition according to claim 4, wherein the weight ratio of the psoralen extract to the alfalfa extract is 1: 1 to 1:3.

 The pharmaceutical composition according to claim 4, wherein the weight ratio of the psoralen extract to the alfalfa extract is 1:1.5.

 The pharmaceutical composition according to claim 4, wherein the psoralen extract comprises 80-99% of furocoumarin, wherein the furocoumarin comprises 4-12% of the supplement Osteolipin and 3-11% isostorphage.

The pharmaceutical composition according to claim 4, wherein the psoralen is extracted The extract contains 95% furocoumarin, wherein the furocoumarin comprises 7-9% psoralen and 6-8% isopsoralen.

 The pharmaceutical composition according to claim 4, wherein the alfalfa extract contains 50-70% of a Lycium barbarum polysaccharide.

 A pharmaceutical composition for treating depression, characterized in that the pharmaceutical composition comprises furocoumarin and anthraquinone polysaccharide, wherein the furocoumarin comprises psoralen and isopsoralen.

 The pharmaceutical composition according to claim 11, wherein the content ratio of the psoralen, isopsoralen and Lycium polysaccharide is 7-9:6-8:86-93.

 13. A method of preparing a pharmaceutical composition according to claim 4, comprising:

 a. extracting psoralen to form a psoralen extract;

 b. extracting cockroaches to form cockroach extract;

 c combining the psoralen extract and the sputum extract.

 14. A method according to claim 13 wherein the psoralen and bismuth are made by the following weight ratios:

 Psoralen 1~2 parts 茯苓 1~2 parts.

15. A method according to claim 13 wherein the psoralen and strontium are made by the following weight ratios:

 Psoralen 1~2 servings 茯苓 1 serving

16. The method according to claim 13, wherein the psoralen and the sputum are made by the following weight ratio -

茯苓 1 serving.

17. The method according to claim 13, wherein said step a. extracting psoralen further comprises:

 i) extracting the psoralen with a first extract to form a first extraction solution; ii) extracting the first extraction solution with a second extract to form a second extraction solution comprising a residue;

 Iii) separating and drying the residue from the second extraction solution;

It is characterized in that one of the extracts is a highly polar solvent and the other extract is a low polarity solvent.

 18. The method of claim 17, wherein the first extract is ethanol, methanol or acetone.

 The method according to claim 17, wherein the first extract is 55-75% (v/v) ethanol.

 The method according to claim 17, wherein the second extract is ethyl acetate, diethyl ether, chloroform or dichloromethane.

 21. The method of claim 17, wherein the second extract is 80-100% ethyl acetate.

 The method according to claim 17, wherein the extracting of the step b. 进 further comprises:

 i) extracting hydrazine with water to obtain a water-soluble extract and a non-water-soluble hydrazine residue;

ϋ) extracting the water-insoluble hydrazine residue with an alkaline solvent to obtain an alkali-soluble extract; Iii) combining the water-soluble extract and the alkali-soluble extract to form the alfalfa extract.

 23. The method of claim 22, wherein step i) further comprises:

 a. boiling the crucible in water to form an aqueous solution of hydrazine;

 b. filtering and concentrating the aqueous hydrazine solution to form a concentrated aqueous hydrazine solution; c. precipitating the concentrated aqueous hydrazine solution to form a precipitate;

 d. The precipitate is separated and dried to form a water soluble extract of hydrazine.

 24. The method of claim 23, wherein the precipitate in step c. is formed by the addition of an alcohol.

 25. The method of claim 24 wherein the alcohol is 80-100% (v/v) ethanol.

26. The method of claim 22, wherein step (ii) further comprises:

 a. extracting the water-insoluble hydrazine residue with an alkaline solvent to form a extract solution, wherein the pH of the extract solution is 11-13;

 b. neutralizing the extract solution with acid to a pH of 6-7 to form a neutralized extract solution;

 c precipitating the neutralized extract solution to form a precipitate;

 d. The precipitate is separated and dried to obtain the alkali-soluble extract.

 27. The method of claim 26, wherein the alkaline solvent is 1 M NaOH.

28. The method of claim 26, wherein the precipitation is obtained by adding an alcohol.

29. The method of claim 28, wherein the alcohol is 95% (v/v) ethanol.

30. A nutritional supplement comprising psoralen extract and alfalfa extract.

The nutritional supplement according to claim 30, wherein the psoralen extract The weight ratio with the alfalfa extract is about 1:1 to 1:7.

 The nutritional supplement according to claim 30, wherein the psoralen extract and the medlar extract have a weight ratio of about 1:1 to 1:3.

 A nutritional supplement according to claim 30, wherein the weight ratio of the psoralen extract to the alfalfa extract is about 1:1.5.

 34. The application of psoralen extract and alfalfa extract as a preparation for treating depression.

 The use according to claim 34, wherein the weight ratio of the psoralen extract to the sputum extract is 1:1 to 1:7.

 36. The use according to claim 34, wherein the weight ratio of the psoralen extract to the sputum extract is 1:1 to 1:3

 37. The use according to claim 34, wherein the weight ratio of the psoralen extract to the sputum extract is 1:1.5.