USE OF MAITAKE AND MAITAKE GRIFOLAN D COMPONENT IN PREPARATION OF ANTIDEPRESSANT DRUG

Abstract

The present invention “use of Maitake and Maitake polysaccharide D Fraction in manufacture of medicaments for anti-depression” belongs to the field of pharmaceuticals, which characterized in that the Maitake whole herb, Maitake extract or Maitake polysaccharide D fraction is used in the preparation of antidepressants. The medicament can be used to treat or prevent depression, which has a significant antidepressant effect, not only strong effect, but also no conventional antidepressant medicaments toxicity, side effects, such as induced epilepsy, ataxia and other shortcomings. Therefore, they can be used as an excellent antidepressant candidate medicament.

Claims

1. An application of Maitake in the preparation of antidepressant medicaments is characterized as that the medicinal component of the antidepressant is selected from group of consisting of Maitake whole herb, Maitake extract, Maitake Polysaccharides D fraction, or Maitake Polysaccharides D fraction salts, esters or the glycosides synthesized by Maitake Polysaccharides D fraction and polysaccharides, wherein said Maitake extract refers to product from following process: marinating the powder of the Maitake fruit body in pure hot water of 98° C. for 3 hours, the quality ratio of material to water is 1:30, pH 6.5˜7.0, and then concentrating the solution.

2. The application according to claim 1, the mentioned antidepressant medicament refers to a medicament that used for preventing or treating depression.

3. The use according to claim 1, the mentioned medicament is in dosage form of powder, table, capsule, solution, suspension, injecta or drip.

4. A kind of antidepressant medicament, it is characterized in: the medicinal component is selected from group of consisting of Maitake whole herb, Maitake extract, Maitake Polysaccharides D fraction, Maitake Polysaccharides D fraction salts, esters or the glycosides synthesized by Maitake Polysaccharides D fraction and polysaccharides, wherein said Maitake extract refers to product from following process: marinating the powder of the Maitake fruit body in pure hot water of 98° C. for 3 hours, the quality ratio of material to water is 1:30, pH 6.5˜7.0, and then concentrating the solution.

5. The medicament according to claim 4, the mentioned antidepressant refers to preventing or treating depression.

6. The medicament according to claim 5, the medicament is in the dosage form of powder, table, capsule, solution, suspension, injecta or drip.

7. The medicament according to claim 4, the mentioned medicament further contains pharmaceutically acceptable adjuvant.

8. The medicament according to claim 4, the mentioned medicament further contains pharmaceutical ingredient that plays a positive role in treating depression together with Maitake whole herb, Maitake extract, or Maitake Polysaccharides D fraction, or application a component that enhances the stability of Maitake whole herb, Maitake extract, or Maitake Polysaccharides D fraction.

9. An antidepressant method which characterized in that the treatment comprises providing an effective doses of medicament to subject on time, wherein medicinal component of mentioned medicament is selected from group of consisting of the Maitake whole herb, Maitake extract, or Maitake Polysaccharides D fraction, wherein said Maitake extract refers to product from following process: marinating the powder of the Maitake fruit body in pure hot water of 98° C. for 3 hours, quality ratio of material to water is 1: 30, pH 6.5˜7.0, and then concentrating the solution.

10. The method according to claim 9, said providing an effective doses of medicament to subject on time refers to providing Maitake Polysaccharides D fraction or Maitake extract with a dose of from 0.5 mg˜1000 mg/kg of body weight per day, or providing the Maitake whole herb together with food by a quality ratio of 1:1-4 between the Maitake whole herb and food.

11. The method according to claim 9, said providing effective doses of medicament to subject on time refers to providing Maitake Polysaccharides D fraction or Maitake extract by 5-15 mg/kg body weight per day.

12. The method of claim 9, the method of administration is oral, infusion or injection; the subject is a mammal, and the mentioned mammal comprising human.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0043] FIG. 1. The chemical structural formula of Maitake Polysaccharides D fraction.

[0044] FIG. 2. Treatment with Maitake Polysaccharides D fraction to mice after 60 minutes—data in tail suspension test (TST) :

[0045] Vertical coordinate: tail suspension immobility (%control);

[0046] Horizontal coordinate: from left to right: Maitake Polysaccharides D fraction control (0.9% saline), Maitake Polysaccharides D fraction low dose (5 mg/kg), Maitake Polysaccharides D fraction medium dose (8 mg/kg), Maitake Polysaccharides D fraction high dose (12.5 mg/kg) , Imipramine control (15 mg/kg).

[0047] FIG. 3. Treatment with Maitake Polysaccharides D fraction to mice after 60 minutes—data in forced swimming test (FST):

[0048] Vertical coordinate: forced swimming immobility (%control);

[0049] Horizontal coordinate: from left to right: Maitake Polysaccharides D fraction control (0.9% saline), Maitake Polysaccharides D fraction low dose (5 mg/kg), Maitake Polysaccharides D fraction medium dose (8 mg/kg), Maitake Polysaccharides D fraction high dose (12.5 mg/kg) , Imipramine control (15 mg/kg).

[0050] FIG. 4. Treatment with Maitake Polysaccharides D fraction to mice after 5 days—data in tail suspension test (TST):

[0051] Vertical coordinate: tail suspension immobility (%control);

[0052] Horizontal coordinate:from left to right: Maitake Polysaccharides D fraction control (0.9% saline), Maitake Polysaccharides D fraction low dose (5 mg/kg), Maitake Polysaccharides D fraction medium dose (8 mg/kg), Maitake Polysaccharides D fraction high dose (12.5 mg/kg) , Imipramine control (15 mg/kg).

[0053] FIG. 5. Treatment with Maitake Polysaccharides D fraction to mice after 5 days—data in forced swimming test (FST):

[0054] Vertical coordinate: forced swimming immobility (%control);

[0055] Horizontal coordinate: from left to right: Maitake Polysaccharides D fraction control (0.9% saline), Maitake Polysaccharides D fraction low dose (5 mg/kg), Maitake Polysaccharides D fraction medium dose (8 mg/kg), Maitake Polysaccharides D fraction high dose (12.5 mg/kg) , Imipramine control (15 mg/kg).

[0056] FIG. 6. Treatment with Maitake Polysaccharides D fraction to mice after 5 days—data in Open field test (FST):

[0057] Vertical coordinate: the center area distance (% meter);

[0058] Horizontal coordinate: from left to right: Maitake Polysaccharides D fraction control (0.9% saline), Maitake Polysaccharides D fraction low dose (5 mg/kg), Maitake Polysaccharides D fraction medium dose (8 mg/kg), Maitake Polysaccharides D fraction high dose (12.5 mg/kg) , Imipramine control (15 mg/kg).

[0059] FIG. 7. Treatment with Maitake Polysaccharides D fraction to mice after 5 days—data in Open field test (FST):

[0060] Vertical coordinate: the total area distance (%meter);

[0061] Horizontal coordinate: from left to right: Maitake Polysaccharides D fraction control (0.9% saline), Maitake Polysaccharides D fraction low dose (5 mg/kg), Maitake Polysaccharides D fraction medium dose (8 mg/kg), Maitake Polysaccharides D fraction high dose (12.5 mg/kg) , Imipramine control (15 mg/kg).

[0062] FIG. 8. Body weight of the mice after 5 days of treatment with Maitake Polysaccharides D fraction.

[0063] FIG. 9. The photo of Grifola frondosa.

[0064] FIG. 10. After one day Grifola frondosa treatment to mice—data in tail suspension test:

[0065] Vertical coordinate: tail suspension immobility (% of control);

[0066] Horizontal coordinate: from left to right: Negative control (0.9% saline), Imipramine positive control (15 mg/kg), Grifola frondosa (1:4), Grifola frondosa (1:2), Grifola frondosa (1:1).

[0067] FIG. 11. After one day Grifola frondosa treatment to mice—data in forced swimming test:

[0068] Vertical coordinate: forced swimming immobility (% of control);

[0069] Horizontal coordinate: from left to right: Negative control (0.9% saline), Imipramine positive control (15 mg/kg), Grifola frondosa (1:4), Grifola frondosa (1:2), Grifola frondosa (1:1).

[0070] FIG. 12. The treatment of Grifola frondosa to mice after three days—data in Open field test (FST):

[0071] Vertical coordinate: the distance traveled in the total area (% meter);

[0072] Horizontal coordinate: from left to right: Negative control (0.9% saline), Grifola frondosa (1:4), Grifola frondosa (1:2), Grifola frondosa (1:1).

[0073] FIG. 13. The treatment with Grifola frondosa to mice for three days—data in Open field test (FST):

[0074] Vertical coordinate: the distance traveled in the center area (% meter);

[0075] Horizontal coordinate: from left to right: Negative control (0.9% saline), Grifola frondosa (1:4), Grifola frondosa (1:2), Grifola frondosa (1:1).

[0076] FIG. 14. Treatment with Maitake extract after 3 days to mice—data in tail suspension test (TST):

[0077] From left to right: Negative control (0.9% saline), Maitake extract low dose (5 mg/kg), Maitake extract medium dose (8 mg/kg), Maitake extract high dose (12.5 mg/kg) , Imipramine control (15 mg/kg).

[0078] FIG. 15. Treatment with Maitake extract after 3 days to mice—data in forced swimming test (FST):

[0079] From left to right: Negative control (0.9% saline), Maitake extract low dose (5 mg/kg), Maitake extract medium dose (8 mg/kg), Maitake extract high dose (12.5 mg/kg) , Imipramine control (15 mg/kg).

[0080] FIG. 16. Treatment with Maitake extract to mice after 4 days—data in Open field test (OFT) to determine the distance traveled:

[0081] From left to right: Negative control (0.9% saline), Maitake extract low dose (5 mg/kg), Maitake extract medium dose (8 mg/kg), Maitake extract high dose (12.5 mg/kg) , Imipramine control (15 mg/kg).

[0082] FIG. 17. Treatment with Maitake extract to mice after 4 days in Open field test (OFT) to determine the distance traveled in the center area:

[0083] From left to right: Negative control (0.9% saline), Maitake extract low dose (5 mg/kg), Maitake extract medium dose (8 mg/kg), Maitake extract high dose (12.5 mg/kg), Imipramine control (15 mg/kg).

DETAILED DESCRIPTION

[0084] The following implementation plan are only intended to illustrate and demonstrate the discovery of the new use of the Maitake extract of the invention that found in resisting depression, without limiting this invention.

EXAMPLE 1

Antidepressant Experiments of Maitake Polysaccharides D Fraction

[0085] Experimental materials and methods:

I. Experimental Details:

[0086] 1. Animal species: mouse, CD-1 (ICR)

[0087] 2. Level: SPF level

[0088] 3. Animal specifications: purchase of initial age and weight (5 weeks old: 20-22 g)

[0089] Formal laboratory weeks and body weight (7 weeks old: 30-39 g)

[0090] 4. Gender: Male

[0091] 5. Quality inspection unit: Beijing Vital River Laboratories

[0092] 6. Use: scientific research

[0093] 7. Sale of the unit: Beijing Vital River Laboratories

[0094] 8. License number: SCXK (jing) 2012-0001

II. Experimental Conditions

[0095] 1. Experimental animals were received on Jul. 15, 2014 from the Beijing Capital Airport on early flight (8:10 take off), at 13:30 pm animals arrived Kunming Changshui Airport cargo center. We chartered a car from Yunnan University to Kunming Changshui Airport cargo center to take experimental animals back to the Yunnan University biological pavilion of East Building, Room 301, animal housing room. Then, let the mice stay in the original box to rest for 2 hours after sub-cage feeding.

[0096] 2. Each cage housed 4 mice, drinking water and feeding free.

[0097] 3. Drinking distilled water (laboratory equipment homemade), and mouse food was purchased from Animal Experimental Department of the Kunming Medical University.

[0098] 4. Twelve hours Light / dark cycle: at 9:00 a.m. lights were on, and at 21:00 p.m. lights were off .

[0099] 5. Room temperature was at 23° C.

[0100] 6. The bedding of the cage was replaced every two days. Let the mice have a comfortable living environment.

[0101] 7. Mice adapted the environment for one week in the new cage before the formal experiment.

III. Maitake Polysaccharides D Fraction

[0102] Maitake D-fraction was prepared from alcohol precipitation from the Maitake drops (Milk Healthy Milk Company), and this method removes any impurities such as glycerin and water, and proceeds as follows:

[0103] 1) Prepared and marked ten 1.5-ml centrifuge tube, and then weighed empty tube for product calculation;

[0104] 2) For each 1.5-ml centrifuge tube, we took 200 ul of Maitake drops, added 600 ul 95% ethanol, and then set at room temperature for 2 hours. The precipitation at the bottom of 1.5 ml centrifuge tube was clearly observed.

[0105] 3) The tubes were centrifuged with high-speed refrigerated centrifuge at 12000 rpm, at 4° C. for 1 hour. At the bottom of the centrifuge tube, we could clearly observe the precipitation. The supernatant was removed into another tube.

[0106] 4) The 1.5 ml centrifuge tubes with the precipitation were placed in a 37° C. incubator for one hour.

[0107] 5) We took the 1.5 ml centrifuge tube from the incubator, air dried at room temperature, then determine the weight of the Maitake D-fraction by weight loss method.

[0108] 6) Approximately 10 mg of Maitake D-fraction was obtained from each 1.5 ml centrifuge tube.

[0109] Reference method: 3 times the volume of 95% ethanol alcohol precipitation, wash with acetone, ether, respectively, freeze-dried with a vacuum freeze dryer to get Maitake D-fraction.

IV. Preparation of the Different Concentrations of Maitake D-Fraction Required Concentration of the Allocation Method for the Mouse Injection:

[0110] 1. A total of 108.4 mg Maitake D-fraction was obtained from 10 centrifuge tubes by weight loss method, and then 200 ul of 0.9% saline (purchased from the hospital) was added to each 1.5-ml centrifuge tube to dissolve the precipitation at the bottom of the centrifuge tube, and then transfer the thoroughly dissolved solution from the ten 1.5-ml centrifuge tubes to a sterile 7 ml centrifuge tube with a 200 ul pipetting, then add 2 ml 0.9% saline diluted (with a total of 4 ml 0.9% saline).

[0111] The final concentration of the Maitake D-fraction solution was 108.4mg/4m1=27.1 mg/ml.

[0112] 2. The different dose dilutions of Maitake D-fraction were as follows:

TABLE-US-00001 Concentration of Dilution Diluted Medicament The final dose stock solution factor concentration volume required 27.1 mg/ml 1:54.2  0.5 mg/ml 300 ul/30 g   5 mg/kg 27.1 mg/ml 1:33.9  0.8 mg/ml 300 ul/30 g   8 mg/kg 27.1 mg/ml 1:21.7 1.25 mg/ml 300 ul/30 g 12.5 mg/kg

[0113] Aliquots of Maitake D-fraction was prepared into 1.5 ml centrifuge tubes according to the dilution factor above, wrapped in the entire 1.5 ml of the centrifuge tube with aluminum foil, sealed, and placed in -80° C. freezer. The aliquots were taken out before use, and then diluted in 0.9% saline according to the calculation of dilution ratio, mixed well before use.

[0114] 3. The dose dilution factor required for imipramine is as follows:

TABLE-US-00002 Concentration of Dilution Diluted Medicament The final dose stock solution factor concentration volume required 15 mg/ml 1:10 1.5 mg/ml 300 ul/30 g 15 mg/kg

[0115] Thirty miligram imipramine was weighed, placed in sterile 7m1 centrifuge tube, and then 2ml 0.9% saline dissolution was added (the final concentration of imipramine solution was 30 mg/2 ml=15 mg/ml) to the tube to make the solution. The imipramine solution was aliqoted, sealed, and freezed down in −80° C. freezer. Just before use, the imipramine solution was taken out, and diluted in 0.9% saline according to the calculation of dilution ratio. The solution was mixed well before use.

V. Behavioral Experiment:

[0116] 1. The mice had adapted in our new breeding environment for a week before the experiment (the mice we purchased were 5 weeks old, so the mice in our new breeding environment adapt to two weeks before the formal experiment);

[0117] 2. To start the experiment on Jul. 30, 2014, we prepared the medicine and the equipment needed for the experiment before 10:00 am. Since then the mice were weighed and injected every day at 10:00 am;

[0118] 3. Experimental arrangements:

TABLE-US-00003 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 After 60 Mice Mice After 60 At the same At the same minutes of rest rest minutes time every time every injection, the at the at the of day After day After suspension same same injection, 60 minutes 60 minutes test or forced time time the open of injection, of swimming after the after the field test the injection, test was day of day of was suspension anatomical performed injection injection performed test or forced experiments swimming were test was performed performed in mice

[0119] 4. Forced swimming conditions: room temperature: 23° C., water temperature: 24° C., humidity: 62-67%, replaced the water after two groups of experiments.

[0120] 5. Experimental results: [0121] Behavioural experiment results of CD-1 mice were treated with Maitake D-fraction of different doses: [0122] 1) The results of the tail suspension test from mice treated with Maitake D-fraction (10-12 repeat), after 60 minutes or 5 days of treatment, were shown in FIG. 2 and FIG. 4, respectively. [0123] 2) The results of the forced swimming test from mice treated with Maitake D-fraction (10-12 repeat), after 60 minutes or 5 days of treatment, were shown in FIG. 3 and FIG. 5, respectively.

Analysis of Experimental Results:

[0124] The tail suspension test results of histogram from Maitake D-fraction (10-12 repeat) treated mice after 60 minutes or 5 days were presented in FIG. 2 and FIG. 4. It could be seen that Maitake D-fraction of low dose (5 mg/kg) showed significant antidepressant effect (after 60 minutes: P=0.0099, P<0.01; 5 days: P=0.0151, P<0.05), the medium dose of Maitake D-fraction (8 mg/kg) showed significant antidepressant effect (after 60 minutes: P=0.0068, P<0.001; 5 days: P=0.0078, P<0.01), and the high dose of Maitake D-fraction (12.5 mg/kg) showed significant antidepressant effect (after 60 minutes: P=0.0018, P<0.001; 5 days: P=0.0014, P<0.01). (As shown in FIG. 2 and FIG. 4)

[0125] The forced swimming test results of histogram from Maitake D-fraction (10-12 repeat) treatment after 60 minutes or 5 days were presented in FIG. 3 and FIG. 5. It could be seen that Maitake D-fraction of low dose (5 mg/kg) showed significant antidepressant effect (after 60 minutes: P=0.0095, P<0.01; 5 days: P=0.0431, P<0.05), the medium dose of Maitake D-fraction (8 mg/kg) showed significant antidepressant effect (after 60 minutes: P=0.0080, P<0.01; 5 days: P=0.0046, P<0.01), and the high dose of Maitake D-fraction (12.5 mg/kg) showed significant antidepressant effect (after 60 minutes: P=0.0062, P<0.001; 5 days: P=0.0012, P<0.01). (As shown in FIG. 3 and FIG. 5)

[0126] 3) The results of distance traveled in the center area in open field test after 3 days of Maitake D-fraction (8 repeat) treatment were shown in FIG. 6.

[0127] 4) The results of distance traveled in the total area in open field test after 3 days of Maitake D-fraction (8 repeat) treatment were shown in FIG. 7.

Analysis of Experimental Results:

[0128] The results of histogram of distance traveled in the center area in open field test after 3 days of Maitake D-fraction (8 repeat) treatment was presented in FIG. 6. It can be seen that Maitake D-fraction of low dose (5 mg/kg), medium dose (8 mg/kg), high dose (12.5 mg/kg) showed no significant effect (P>0.05), and the results further implied that the shortening of the immobilized time of the mice is not caused by the central excitatory effect of the medicament (Maitake D-fraction). (As shown in FIG. 6)

[0129] The results of histogram of distance traveled in the total area in open field test after 3 days of Maitake D-fraction (8 repeat) treatment were demonstrated in FIG. 6. It can be seen that Maitake D-fraction of low dose (5 mg/kg), medium dose (8 mg/kg), high dose (12.5 mg/kg) showed no significant effect (P >0.05), and the results further implied that the shortening of the immobilized time of the mice is not caused by the central excitatory effect of the medicament (Maitake D-fraction). (As shown in FIG. 7)

[0130] Body weights of the mice after 5 days of treatment were shown in FIG. 8.

[0131] In other examples of the present invention, a directly extracted Maitake extract is used. That means the fine powder of the Maitake fruiting body was extracted with pure hot water (material to water ratio of 1:30). The optimal technological conditions were as follows: pH 6.5-7.0, extracted at 98° C. for 3 hours after the concentration derived. In the mouse experiment, almost exactly the same results as those shown in FIG. 4-7 were obtained, please see FIG. 14-17.

EXAMPLE 2

Antidepresaant Experiment of Grifola frondosa Whole Herb (Maitake Whole Herb)

[0132] A. Experimental Materials and Methods:

[0133] (A) Experimental animal details

[0134] 1. Animal species: mouse, CD-1 (ICR)

[0135] 2. Level: SPF level

[0136] 3. Animal specifications: the initial age and weight purchased (6 weeks old: 22-25 g); Formal laboratory weeks and body weight (7 weeks old: 30-37 g)

[0137] 4. Gender: Male

[0138] 5. Quality inspection unit: Beijing Vital River Laboratories

[0139] 6. Use: scientific research

[0140] 7. Sale of the unit: Beijing Vital River Laboratories

[0141] 8. License number: SCXK (jing) 2012-0001

[0142] (B) . Experimental Animal Feeding Conditions:

[0143] 1. Experimental animals arrived on Apr. 21, 2015 from the Beijing Capital Airport early flight (7:40 take off). At 13:30 pm, animals arrived Kunming Changshui Airport cargo center. We chartered a car from Yunnan University to Kunming Changshui Airport cargo center to take experimental animals back to the Yunnan University biological pavilion of East Building, Room 301, animal housing room. Then, let the mice stay in the original box to rest for 2 hours after sub-cage feeding.

[0144] 2. Each cage housed 4 mice, drinking water and feeding free.

[0145] 3. Drinking distilled water (laboratory equipment homemade), and mouse food was purchased from Animal Experimental Department of the Kunming Medical University.

[0146] 4. Twelve hours Light/dark cycle : at 9:00 a.m. lights were on, and at 21:00 p.m. lights were off .

[0147] 5. Room temperature was at 23° C.

[0148] 6. The bedding of the cage was replaced every two days.

[0149] 7. Mice adapted the environment for one week in the new cage before the formal experiment.

[0150] (C) Grifola frondosa Details:

[0151] 1. Grifola frondosa: Purchased from Yunnan wild mushroom Industry Co., Ltd. Vegetatively: Fujian Province

[0152] (D) Grifola frondosa and Regular Food with Different Ratio of the Production of Grifola Frondosa-Containing Food.

[0153] 1. The dry Grifola frondosa were placed in a sterilization pot. Then we set constant temperature at 100° C. and steamed the dry Grifola frondosa for 30 minutes.

[0154] 2. Then we removed the steamed Grifola frondosa, placed on a clean gauze, dried in the natural sunlight, until dry.

[0155] 3. The dried Grifola frondosa was then crushed into the powder with a universal crusher and packed in a clean plastic bag.

[0156] 4. In order to prepare the Grifola frondosa-containing food, we went to mice food production room, in the Experimental Animal Department, Kunming Medical University. The production of different ratio of Grifola frondosa-containing food was performed, according to the weight ratio. Details are as follows:

TABLE-US-00004 Grifola frondosa dry powder:normal mice food base material 1:4 Grifola frondosa dry powder:normal mice food base material 1:2 Grifola frondosa dry powder:normal mice food base material 1:1

[0157] (E) The Different Experimental Treatment Groups Were Set Up As Follows, Each Group Had 15 Mice:

[0158] Negative control group: eating normal mice food, daily intraperitoneal injection of 0.9% saline according to weight;

[0159] Positive control group: eating normal mice food, daily intraperitoneal injection of imipramine (15mg/kg) according to weight;

[0160] Grifola frondosa mice food: eating 1:4 Grifola frondosa-containing food, daily intraperitoneal injection of 0.9% saline according to weight;

[0161] Grifola frondosa mice food: eating 1:2 Grifola frondosa-containing food,daily intraperitoneal injection of 0.9% saline according to weight;

[0162] Grifola frondosa mice food: eating 1:1 Grifola frondosa-containing food, daily intraperitoneal injection of 0.9% saline according to weight;

[0163] In order to ensure the consistency of the experiment, due to the establishment of a positive control for the intraperitoneal injection of imipramine, so the other groups were corresponding daily intraperitoneal injection of 0.9% saline according to weight.

[0164] (F) The Positive Control Medicament Imipramine for Mice Injection was Prepared as Follows:

TABLE-US-00005 Concentration of Dilution Diluted Medicament The final dose stock solution factor concentration volume required 15 mg/ml 1:10 1.5 mg/ml 300 ul/30 g 15 mg/kg

[0165] Sixty miligram imipramine was weighed, placed in sterile 7 ml centrifuge tube, and then 4 ml 0.9% saline dissolution was added (the final concentration of imipramine solution was 60 mg/4 ml=15 mg/ml) to the tube to make the solution. The imipramine solution was aliqoted, sealed, and freezed down in −80° C. freezer. Just before use, the imipramine solution was taken out, and diluted in 0.9% saline according to the calculation of dilution ratio. The solution was mixed well before use.

[0166] (G) Behavioral Experiment:

[0167] 1. The mice had adapted in our new breeding environment for a week before the experiment;

[0168] 2. To start the experiment on Apr. 28 2015, we prepared the medicine and the equipment needed for the experiment before 10:00 am. Since then the mice were weighed and injected every day at 10:00 am;

[0169] 3. Experimental arrangements:

TABLE-US-00006 Day 1 Day 3 Day 5 At 10:00 am of injection At 10:00 am of At 10:00 am of injection after 60 minutes, the tail injection after 60 after 60 minutes, the suspension test test was minutes, the open field forced swimming test performed test was performed was performed

[0170] 4. Experimental environmental conditions [0171] (1) Tail suspension test conditions: room temperature: 24° C., humidity: 62-67%. [0172] (2) Forced swimming test conditions: room temperature:24° C., water temperature: 24° C., humidity: 62-67%, each two groups of experiments replace the water once.

[0173] B. The EXPERIMENTAL RESULTS AND ANALYSIS:

[0174] (1) Experimental results:

[0175] Behavioral results after different doses of Grifola frondosa treatment to CD-1 mice: [0176] 1) The results of tail suspension test after treatment with Grifola frondosa (15 repeat) for one day were shown in FIG. 10. [0177] 2) The results of forced swimming test after treatment with Grifola frondosa (15 repeat) for five days were shown in FIG. 11. [0178] 3) The results of the open field test after treatment with Grifola frondosa (15 repeat) for three days (total area moving distance and middle area moving distance) were shown in FIG. 12 and FIG. 13.

[0179] (2) Analysis of experimental results:

[0180] Analysis of the experimental results from the tail suspension test: the tail suspension test results of histogram after 1 day of Grifola frondosa (15 repeat) treatment were show in FIG. 10. It can be seen that Grifola frondosa (1:4) showed significant antidepressant effect (P=0.0449, P<0.01), Grifola frondosa (1:2) showed significant antidepressant effect (P=0.0075, P<0.001), and Grifola frondosa (1:1) showed significant antidepressant effect (P=0.0128, P<0.05). (As shown in FIG. 10)

[0181] Analysis of experimental results in the forced swimming test: the results of histogram of the forced swimming test after 5 days of Grifola frondosa (15 repeat) were presented in FIG. 11. It can be seen that Grifola frondosa (1:4) showed significant antidepressant effect (P=0.0079, P<0.01), Grifola frondosa (1:2) showed significant antidepressant effect (P=0.0030, P<0.001), and Grifola frondosa (1:1) showed significant antidepressant effect (P=0.0186, P<0.05). (As shown in FIG. 11)

[0182] Analysis of the Results in Open Field Test:

[0183] (1) The results of the distance traveled in the total area from the open field test after 3 days of Grifola frondosa (15 repeat) treatment were presented in FIG. 12. It can be seen that Grifola frondosa (1:4), (1:2), (1:1) showed no significant effect (P>0.05), and the results implied that the shortening of the immobilized time of the mice is not caused by the central excitatory effect of the medicament (Grifola frondosa). (As shown in FIG. 12)

[0184] (2) The results of the distance traveled in the center area of open field test after 3 days of the Grifola frondosa (15 repeat) treatment was presented in FIG. 13. It can be seen that Grifola frondosa (1:4), (1:2), (1:1) showed no significant effect (P>0.05), and the results further implied that the shortening of the immobilized time of the mice is not caused by the central excitatory effect of the medicament (Grifola frondosa). (As shown in FIG. 13)