Patent Publication Number: US-2021188865-A1

Title: Compound, preparation method and use thereof

Description:
CROSS REFERENCE TO RELATED APPLICATION 
     This application is a continuation-in-part application of PCT application No. PCT/CN2019/111538, filed on Oct. 16, 2019, which claims priority of Chinese patent application No. 201811482596.8 filed on Dec. 5, 2018. The contents of the above-identified applications are all hereby incorporated by reference. 
    
    
     BACKGROUND 
     Field of the Invention 
     The present application relates to the field of biomedical chemistry, and specifically, to a compound, a preparation method and use thereof. 
     Related Arts 
     Drugs refer to medicines that are used repeatedly and continuously for non-medical purposes and can cause dependence (that is, addiction). There is an increasing number of drug addicts, and their ages are getting younger. Only in China, there are more than 14 million drug addicts, while the number is more than 200 to 400 million in the whole world. Drugs are becoming more and more harmful to the world, and the types of drugs are becoming more and more diverse, from opium and marijuana to heroin, ketamine, methamphetamine, and the like. The proliferation of drugs can directly endanger physical and mental health of people, and brings a huge threat to economic development and social progress. 
     Detoxication refers to the withdrawal of drug addicts from taking and injecting drugs and the drug addiction. Detoxification treatment for drug addicts may generally include 3 stages: detoxification, rehabilitation, and counseling for re-entering society. Currently, there are 3 commonly used methods for detoxication: natural withdrawal, drug withdrawal, and non-drug withdrawal. The natural withdrawal method is a mandatory detoxication method and lacks humanity. The drug withdrawal method, also known as drug detoxification treatment, refers to a detoxication method by providing and lacks humanity. The drug withdrawal method, also known as drug detoxification treatment, refers to a detoxication method by providing drug addicts with withdrawal drugs to relieve and reduce the withdrawal symptoms in a manner of replacement and decreasing dosage and gradually achieve detoxification, which is the most important detoxication method. Common detoxication drugs may also be classified into 3 categories: opioid chemicals, non-opioid chemicals, and traditional Chinese medicines. However, neither of those detoxication methods can help drug addicts to get rid of drugs physically and psychologically, and they are prone to lapse and relapse. To date, there is no good solutions to this. Therefore, drug detoxication and prohibition have become a worldwide problem. For this reason, people have been trying to find a safe detoxication drug with no dependence. 
     The traditional Chinese medicines have a long history in detoxication. Compared with the most commonly used replacement treatments such as methadone and naltrexone (both are opioid chemicals), it has the advantages of good compliance, no addiction, definite efficacy on protracted symptoms, and high ethical rate (the proportion of drug addicts who do not relapse after detoxication). Currently, the traditional Chinese medicines used in detoxication are mostly compound preparations evolved on the basis of traditional detoxication recipes. They have many complex effective active ingredients, and their detoxication mechanism cannot be clearly determined. In addition, there are few researches on pharmacology and toxicology, thereby limiting the scientization, standardization, and internationalization of the traditional Chinese medicine compound preparations for detoxication. Therefore, the research on the active ingredients and detoxication mechanism of a single Chinese medicine is beneficial to the use and development of the Chinese medicine detoxication drugs. 
     Limax is a pulmonate mollusk, belonging to the gastropod Gastropoda. Limacidae. Because its body surface is covered with mucus, it is commonly known as “sticky worm, slug, shellless snail, snail, and attached snail”. From the perspective of Chinese Medicine. Limax is “salty and cold; acting in the lung meridian, the liver meridian, and the large intestine meridian”. It has effects of expelling wind for relieving convulsion, and clearing away heat and removing toxic substances. It is mainly used for treating crooked eye and mouth due to apoplexy, tendon and vessel contracture, infantile convulsion, wheeze, pharyngeal swelling, sore throat, carbuncle, subcutaneous nodule, and swelling and pain due to hemorrhoid. The traditional Chinese medicine uses Limax to treat disorders such as tumors, asthma, and bronchitis. Existing studies have indicated that Limax contains various ingredients such as protein and polysaccharide. Furthermore, the pharmacological studies have found that Limax plays a role in anti-tumor, anti-asthma and the like. In recent years, studies on the efficacy of the Limax extract have mostly focused on anti-tumor and its effects on respiratory diseases. Among others, the researches on its anti-tumor effects have become a hot spot in domestic and foreign researches. In the researches on the folk prescriptions and secret recipes of Chinese herbal medicine, the inventor found that the Limax extract has the efficacy of detoxication treatment, and has been committed to this research. The related active ingredients have been extracted and separated from the Limax extract. 
     SUMMARY 
     An object of the present application is to provide a compound and a preparation method thereof, which provides a new idea for the research and development of Chinese medicine detoxication products. 
     The present application uses the following technical solutions: 
     A compound is provided, which has a structure of: 
     
       
         
         
             
             
         
       
     
     Further, the compound has the chiral C configurations of: C3, R: C6, S: C7, S; C10, S; C11, R; C12, R; C13, R; C14, R; C15, S; C18, S: C19, S; and C22, R. 
     Further, the compound has a molecular formula: C 36 H 58 O 10 ; molecular weight: 650; melting point: 228-229° C.; and solubility: white needle-like crystal, insoluble in water, hardly soluble in acid and alkali, easily soluble in ethyl acetate and acetic acid, and soluble in methanol, ethanol, acetone, and chloroform. 
     Further, the compound is prepared by extracting and separating from Limax, wherein the Limax comprises one or more of  Vaginulus alte  (Ferussac),  Limax maximus  L.,  L. flavus  L.,  Agriolimax agrestis  L., and  Phiolomycus bilineatus.    
     A method for preparing the compound as described above is provided, including a supercritical CO 2  extraction method and a solvent extraction method. 
     Further, in the method for preparing the compound as described above, the supercritical CO 2  extraction method includes the following steps: 
     R1. sorting Limax, removing impurities, and pulverizing, to obtain Limax powder; 
     R2. putting the Limax powder into a supercritical CO 2  extractor for extraction to obtain an extract: 
     R3. adding vegetable oil to the extract, heating and mixing evenly, cooling, standing, and filtering to obtain a precipitate, washing the precipitate with a washing solvent, and drying to obtain a dried precipitate; and 
     R4. adding a crystallization solvent to the dried precipitate, heating to dissolve, cooling, standing to precipitate white needle-like crystals, and filtering to obtain the crystals, recrystallizing, and drying, to obtain the compound. 
     Furthermore, in the method for preparing the compound as described above, the pulverizing in R1 includes pulverizing into 10-30 meshes. 
     Furthermore, in the method for preparing the compound as described above, the conditions for the supercritical CO 2  extraction in R2 include: pressure: 20-30 kPa, temperature: 60-70° C., flow: 400-500 PV, and extraction time: 3-5 h. 
     Furthermore, in the method for preparing the compound as described above, the vegetable oil in R3 is tea seed oil,  camellia  seed oil, soybean oil, or olive oil; 
     the vegetable oil is added in a weight ratio of “extract: vegetable oil=2-6:1-3” 
     the heating is carried out at a temperature of 60-80° C.; 
     the time for the standing is 7-10 days: and 
     the washing includes washing with n-hexane, petroleum ether, or 120 # gasoline for 3-5 times, with an amount of 200-500 ml each time. 
     Furthermore, in the method for preparing the compound as described above, the crystallization solvent in R4 is methanol, acetone, ethyl acetate, or chloroform added at an amount of 3-10 times the weight of the dried precipitate; and the time for the standing is 7-10 days. 
     Further, in the method for preparing the compound as described above, the solvent extraction method includes the following steps: 
     S1. sorting Limax, removing impurities, and pulverizing, to obtain Limax powder: 
     S2. heating reflux extraction the Limax powder with a solvent, filtering, leaving a filtrate and recovering the solvent from the filtrate under reduced pressure until the recovery is complete, to obtain a thick paste; 
     S3. adding the thick paste to a silica gel chromatography column, eluting with an elution solvent, collecting an eluate, and recovering the elution solvent from the eluate under reduced pressure, to obtain a thick substance: and 
     S4. adding a dissolution solvent to the thick substance, heating to dissolve completely, cooling, and freezing to precipitate white crystals, filtering, leaving a filtrate, recovering the dissolution solvent from the filtrate under reduced pressure, and re-standing to precipitate white needle-like crystals, filtering, and drying, to obtain the crystals, recrystallizing, and drying, to obtain the compound. 
     Furthermore, in the method for preparing the compound as described above, the pulverizing in S1 includes pulverizing into 10-30 meshes. 
     Furthermore, in the method for preparing the compound as described above, the solvent in S2 is one or more of ethanol, methanol, acetone, chloroform, 120 # gasoline, n-hexane, petroleum ether, diethyl ether, and ethyl acetate; and the heating reflux extraction includes heating reflux extraction 1-3 times with the solvent, with an amount of 5-15 times the weight of the Limax powder each time, for 1-3 h. 
     Furthermore, in the method for preparing the compound as described above, the adding to the silica gel chromatography column in S3 specifically includes adding silica gel to the thick paste at an amount of 4-6 times the weight of the thick paste, mixing evenly, and adding to the chromatography column pre-filled with silica gel at an amount of 2-4 times the weight of the thick paste; and 
     the elution solvent is methanol, acetone, or ethyl acetate, and the amount of the elution solvent used is: the volume of the elution solvent: the total weight of the silica gel in the chromatography column=1.5-3:1-2. 
     Furthermore, in the method for preparing the compound as described above, the dissolution solvent in S4 is methanol or ethanol added in an amount of 4-8 times the volume of the thick substance: the freezing is carried out at a temperature of 2-10° C. for 24 hours; the recovering the solvent from the filtrate under reduced pressure comprises concentrating to 50-60% of the original volume; and the time for the re-standing is 7-10 days. 
     Furthermore, in the method for preparing the compound as described above, before the eluting with an elution solvent in S3, a step of pre-elution with a pre-elution solvent is performed, wherein the pre-elution solvent is petroleum ether, n-hexane, or 120 # gasoline, and the amount of the pre-elution solvent used is: the volume of the pre-elution solvent: the total weight of the silica gel in the chromatography column=−3:0.5-1.5. 
     Use of the above-mentioned compound in the preparation of medicines, health foods, and food for preventing or treating withdrawal or withdrawal-like symptoms is provided. 
     Use of the above-mentioned compound in the preparation of medicines, health food, and food for inhibiting withdrawal symptoms in morphine-dependent animals is provided. 
     The compound of the present application is prepared by separating from the extract of Chinese medicine Limax (scientific name:  Agriolima agrestis ) through a simple method, and named 3,12,13-triacetyl limaxol A after identification, with a chemical name of (3R,5aS,7aS,8R,9R,10R,10aR,10bS)-dodecahydro-8-((S)-2-hydroxy-4-((1S,2S,5R)-1,4,4-trimethyl-3,8-dioxa-bicyclo[3.2.1]octan-2-yl)butan-2-yl)-4,4,7a,10b-tetramethyl-1H-indeno[5,4-b]oxepine-3,9,10-triol triacetate. Pharmacological tests have proved that the compound has a significant inhibitory effect on withdrawal jumping symptoms in morphine-dependent animals 1 hour after intragastric administration, and still shows an inhibitory trend after 3 hours, indicating that the compound has significant effects on physiological or psychological dependent detoxification or detoxication, and has potential application value in the preparation of medicines, health food, and food for detoxification, detoxication, or similar drug-dependent treatment. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows an X-ray crystal structure of the compound according to the present application. 
         FIG. 2  to  FIG. 5  show carbon (C) NMR spectra data and hydrogen (H) NMR spectra data of the compound according to the present application. 
         FIG. 6  shows mass spectrometry (MS) data of the compound according to the present application. 
         FIG. 7  shows infrared (IR) spectroscopy data of the compound according to the present application. 
         FIG. 8  shows inhibition rates of methadone, Limax extract, and different doses of 3,12,13-triacetyl limaxol A on morphine-withdrawal jumping symptoms, 
       where: 1: methadone 20 mg/kg (1 h);
         2: Limax crude extract 10 g/kg (1 h);   3: 3,12,13-triacetyl limaxol A 0.5 g/kg (1 h);   4: 3,12,13-triacetyl limaxol A 0.25 g/kg (1 h);   5: 3,12,13-triacetyl limaxol A 0.125 g/kg (1 h); and   6: 3,12,13-triacetyl limaxol A 0.5 g/kg (3 h).       

         FIG. 9 a    shows the retention time on the rotarod of the treatment groups 30 minutes after administration in a mouse rotarod model according to Example 11. 
         FIG. 9 b    shows the retention time on the rotarod of the treatment groups 60 minutes after administration in a mouse rotarod model according to Example 11. 
         FIG. 9 c    shows the retention time on the rotarod of the treatment groups 120 minutes after administration in a mouse rotarod model according to Example 11. 
         FIG. 10  shows the effect of a single oral administration of triacetyl limaxol A on morphine-induced conditioned place preference behavior in rats according to Example 12. 
         FIG. 11  shows the effect of triacetyl limaxol A on the formation of conditioned place preference induced by morphine in rats according to Example 13. 
         FIG. 12 a    shows the experimental data of morphine hydrochloride induced conditioned place preference in rats according to Example 14. 
         FIG. 12 b    shows the experimental data of triacetyl limaxol A induced conditioned place preference in rats according to Example 14. 
         FIG. 13  shows the results of displacement binding assay by the displacement of tritiated U69593, DAMGO and DPDPE with AK and reference ligands at (A) CHO κ, (B) CHO μ and (C) CHO δ cell membranes. Data are means±SEM of n&gt;3 experiments for all cell lines. Reference ligands: U50488, DAMGO and SNC80. 
     
    
    
     DETAILED DESCRIPTION 
     I. A Compound 
     Example 1 
     The present application provides a compound having a specific structure of: 
     
       
         
         
             
             
         
       
     
     The specific information of the compound is as follows: 
     (1) The compound is named “3,12,13-triacetyl limaxol A”: 
     (2) Chiral C configurations of the compound include: C3, R: C6, S; C7, S; C10, S; C11, R; C12, R; C13, R; C14, R: C15, S; C18, S: C19, S; and C22, R with the crystal structure shown in  FIG. 1 ; 
     (3) Physical and chemical properties and spectra data include: 
     {circle around (1)} molecular formula: C 36 H 58 O 10 ; molecular weight 650: 
     {circle around (2)} state: white needle-like crystals; 
     {circle around (3)} melting point: 228-229° C.: 
     {circle around (4)} solubility: insoluble in water, hardly soluble in acid and alkali, easily soluble in ethyl acetate and acetic acid, and soluble in methanol, ethanol, acetone, and chloroform: 
     {circle around (5)} carbon (C) and hydrogen (H) NMR spectra data, as shown in Table 1 and  FIG. 2  to  FIG. 5 ; 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 NMR data MR2 
               
               
                   13 C and  1 H-NMR data (500 MHz, in CD 3 OD) 
               
            
           
           
               
               
               
               
            
               
                   
                 NO 
                 δ C   
                 δ H  (J in Hz) 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 1 
                   
                   
               
               
                   
                 2 
                 76.5 
               
               
                   
                 3 
                 81.0 
                 4.82 (d, J = 9.8 Hz, 1H) 
               
               
                   
                 4 
                 27.5 
                 2.01, 2.05 (m, 2H) 
               
               
                   
                 5 
                 42.1 
                 1.36 (m, 2H) 
               
               
                   
                 6 
                 41.8 
               
               
                   
                 7 
                 77.5 
                 3.37 (dd, J = 11.6, 4.5 Hz, 1H) 
               
               
                   
                 8 
                 28.7 
                 1.51, 1.72 (m, 2H) 
               
               
                   
                 9 
                 39.9 
                 1.51, 1.98 (m, 2H) 
               
               
                   
                 10 
                 46.3 
               
               
                   
                 11 
                 60.6 
                 1.50 (d, J = 11.5 Hz, 1H) 
               
               
                   
                 12 
                 80.0 
                 5.31 (dd, J = 11.5, 2.5 Hz, 1H) 
               
               
                   
                 13 
                 83.4 
                 5.42 (dd, J = 7.6, 2.5 Hz, 1H) 
               
               
                   
                 14 
                 59.9 
                 1.84 (d, J = 7.6 Hz, 1H) 
               
               
                   
                 15 
                 76.5 
               
               
                   
                 16 
                 41.8 
                 1.76-1.79 (m, 2H) 
               
               
                   
                 17 
                 27.2 
                 1.57, 1.91 (m, 2H) 
               
               
                   
                 18 
                 82.8 
                 3.22 (dd, J = 10.8, 4.0 Hz, 1H) 
               
               
                   
                 19 
                 81.8 
               
               
                   
                 20 
                 37.7 
                 1.66, 2.06 (m, 2H) 
               
               
                   
                 21 
                 25.9 
                 1.83, 2.14 (m, 2H) 
               
               
                   
                 22 
                 84.0 
                 3.85 (d, J = 7.0 Hz, 1H) 
               
               
                   
                 23 
                 74.2 
               
               
                   
                 24 
               
               
                   
                 25 
                 23.4 
                 1.12 (s, 3H) 
               
               
                   
                 26 
                 24.8 
                 1.17 (s, 3H) 
               
               
                   
                 27 
                 15.1 
                 0.97 (s, 3H) 
               
               
                   
                 28 
                 18.2 
                 1.36 (s, 3H) 
               
               
                   
                 29 
                 26.7 
                 1.28 (s, 3H) 
               
               
                   
                 30 
                 22.6 
                 1.26 (s, 3H) 
               
               
                   
                 31 
                 27.0 
                 1.04 (s, 3H) 
               
               
                   
                 32 
                 27.2 
                 1.38 (s, 3H) 
               
               
                   
                 33 
                 171.6 
               
               
                   
                 34 
                 21.6 
                 2.10 (s, 3H) 
               
               
                   
                 35 
                 172.3 
               
               
                   
                 36 
                 21.4 
                 2.02 (s, 3H) 
               
               
                   
                 37 
                 172.3 
               
               
                   
                 38 
                 21.2 
                 2.00 (s, 3H) 
               
               
                   
                   
               
            
           
         
       
     
     {circle around (6)} mass spectrometry (MS) data, as shown in  FIG. 6  and 
     {circle around (7)} infrared (IR) spectroscopy data, as shown in  FIG. 7 . 
     II. A Method for Preparing a Compound 
     Example 2 
     A method for preparing a compound (a supercritical CO 2  extraction method) includes the following steps: 
     R1. Limax, after sorting and removing impurities, was pulverized into 30 mesh powder for later use; 
     R2. 50 kg of Limax powder was put into a supercritical CO 2  extractor for extraction under the extraction conditions: pressure 20 kPa, temperature 60° C., flow 400-500 PV, and extraction time 3 h, obtaining 2.9 kg of ointment-like extract for later use; 
     R3. tea seed oil was added to the extract in a weight ratio of “extract: tea seed oil=2:1”, that is, 2.9 kg of the extract and 1.45 kg of tea seed oil were put into a mixing tank; the mixture was heated at 60° C., mixed evenly, cooled and stood for 7 days to precipitate, and then filtered to obtain 198 g of precipitate; the precipitate was washed with petroleum ether (200 ml×3) under heating, and then dried, to obtain 123 g of precipitate; and 
     R4. the precipitate was added to 800 ml of methanol, and dissolved by heating, cooled, and stood for 7 days to precipitate white needle-like crystals, filtered to obtain the crystals; an appropriate amount of ethyl acetate was added to dissolve the crystals, filtered, and the filtrate was stood for 7 days to precipitate white needle-like crystals, filtered and dried to obtain 95.4 g of the compound, 3,12,13-triacetyl limaxol A. 
     Example 3 
     A method for preparing a compound (a supercritical CO 2  extraction method) includes the following steps: 
     R1. Limax, after sorting and removing impurities, was pulverized into 20 mesh powder for later use: 
     R2. 60 kg of Limax powder was put into a supercritical CO 2  extractor for extraction under the extraction conditions: pressure 25 kPa, temperature 65° C., flow 400-500 PV, and extraction time 4 h, obtaining 3.5 kg of ointment-like extract for later use; 
     R3. soybean oil was added to the extract in a weight ratio of “extract: soybean oil=4:2”, that is, 3.5 kg of the extract and 1.75 kg of soybean oil were put into a mixing tank; the mixture was heated at 65° C., mixed evenly, cooled and stood for 8 days, and then filtered to obtain 235 g of precipitate; the precipitate was washed with n-hexane (300 ml×3), and then dried, to obtain 151 g of precipitate; and 
     R4. the precipitate was added to 500 ml of ethyl acetate, and heated to dissolve, cooled, and stood for 8 days to precipitate white needle-like crystals, and filtered to obtain the crystals; an appropriate amount of ethyl acetate was added to dissolve the crystals, filtered, and the filtrate was stood for 8 days to precipitate white needle-like crystals, filtered and dried to obtain 116.0 g of the compound, 3,12,13-triacetyl limaxol A. 
     Example 4 
     A method for preparing a compound (a supercritical CO 2  extraction method) includes the following steps: 
     R1. Limax, after sorting and removing impurities, was pulverized into 20 mesh powder for later use; 
     R2. 100 kg of Limax powder was put into a supercritical CO 2  extractor for extraction under the extraction conditions: pressure 28 kPa, temperature 68° C., flow 400-500 PV, and extraction time 4.5 h, obtaining 5.9 kg of ointment-like extract for later use; 
     R3. olive oil was added to the extract in a weight ratio of “extract: olive oil=5:2.5”, that is, 5.9 kg of the extract and 2.95 kg of olive oil were put into a mixing tank; the mixture was heated at 70° C., mixed evenly, cooled and stood for 9 days, and then filtered to obtain 393 g of precipitate; the precipitate was washed with 120 # gasoline (400 ml×4), and then dried, to obtain 255 g of precipitate; and 
     R4. the precipitate was added to 2200 ml of acetone, and heated to dissolve, cooled, and stood for 9 days to precipitate white needle-like crystals, and filtered to obtain the crystals; an appropriate amount of ethyl acetate was added to dissolve the crystals, filtered, and the filtrate was stood for 9 days to precipitate white needle-like crystals, filtered and dried to obtain 196.1 g of the compound, 3,12,13-triacetyl limaxol A. 
     Example 5 
     A method for preparing a compound (a supercritical CO 2  extraction method) includes the following steps: 
     R1. Limax, after sorting and removing impurities, was pulverized into 10 mesh powder for later use: 
     R2. 150 kg of Limax powder was put into a supercritical CO 2  extractor for extraction under the extraction conditions: pressure 30 kPa, temperature 70° C., flow 400-500 PV, and extraction time 5 h, obtaining 8.7 kg of ointment-like extract for later use; 
     R3.  camellia  seed oil was added to the extract in a weight ratio of “extract:  camellia  seed oil=6:3”, that is, 8.7 kg of the extract and 4.36 kg of  camellia  seed oil were put into a mixing tank; the mixture was heated at 80° C., mixed evenly, cooled and stood for 10 days, and then filtered to obtain 581 g of precipitate; the precipitate was washed with n-hexane (500 ml×5), and then dried, to obtain 372 g of precipitate; and 
     R4. the precipitate was added to 5000 ml of chloroform, and heated to dissolve, cooled, and stood for 10 days to precipitate white needle-like crystals, and filtered to obtain the crystals; an appropriate amount of ethyl acetate was added to dissolve the crystals, filtered, and the filtrate was stood for 10 days to precipitate white needle-like crystals, filtered and dried to obtain 287.3 g of the compound, 3,12,13-triacetyl limaxol A. 
     Example 6 
     A method for preparing a compound (a solvent extraction method) includes the following steps: 
     S1. Limax, after sorting and removing impurities, was pulverized into 20 mesh powder for later use; 
     S2. 100 kg of Limax powder was put into a multi-functional extraction tank, and extracted at reflux with n-hexane for 2 times, firstly with an amount of 10 times the weight of the Limax powder for 1.5 h, and then with an amount of 8 times the weight of the Limax powder for 1.0 h; each of the extracts was filtered, and the filtrates were combined, and the n-hexane was recovered under reduced pressure until the recovery was complete, to obtain 8.10 kg of an oily thick paste: 
     S3. 40.5 kg of silica gel was added to the thick paste and mixed evenly; the mixture was added to a chromatography column pre-filled with 24.3 kg of silica gel, and pre-eluted with petroleum ether in the ratio of “the volume of petroleum ether: the total weight of the silica gel in the chromatography column=2:1” (that is, 129.6 L of petroleum ether), and then eluted with ethyl acetate in the ratio of “the volume of ethyl acetate: the total weight of the silica gel in the chromatography column=2.25:1.5” (that is, 97.2 L of ethyl acetate), the ethyl acetate eluate was collected, and concentrated under reduced pressure to recover ethyl acetate, thereby obtaining 4.3 kg of a thick substance; and 
     S4. 27.0 L of methanol was added to the thick substance, heated to dissolve completely, and frozen at 6° C. for 24 hours, to precipitate white crystals, filtered, and the filtrate was concentrated under reduced pressure to recover about 14.9 L of methanol, and then stood for 9 days to precipitate white needle-like crystals, filtered and dried to obtain 359 g of crystals, 1.0 L of ethyl acetate was added to the crystals, heated to dissolve, cooled, and stood for 9 days, to precipitate white needle-like crystals, filtered and dried to obtain 210.4 g of the compound, 3,12,13-triacetyl limaxol A. 
     Example 7 
     A method for preparing a compound (a solvent extraction method) includes the following steps: 
     S1. Limax, after sorting and removing impurities, was pulverized into 30 mesh powder for later use; 
     S2. 50 kg of Limax powder was put into a multi-functional extraction tank, and extracted at reflux with a mixed solvent (methanol:diethyl ether:ethyl acetate=1:1:1) at an amount of 15 times the weight of the Limax powder for 3 h, filtered, and the mixed solvent was recovered from the filtrate under reduced pressure until the recovery was complete, to obtain 3.98 kg of an oily thick paste; 
     S3. 23.8 kg of silica gel was added to the thick paste and mixed evenly: the mixture was added to a chromatography column pre-filled with 15.9 kg of silica gel, and pre-eluted with 120 # gasoline in the ratio of “the volume of 120 # gasoline: the total weight of the silica gel in the chromatography column=1:0.5” (that is, 79.4 L of 120 # gasoline), and then eluted with methanol in the ratio of “the volume of methanol: the total weight of the silica gel in the chromatography column=1.5:1” (that is, 59.5 L of methanol), the methanol eluate was collected, and concentrated under reduced pressure to recover methanol, thereby obtaining 2.07 kg of a thick substance; and 
     S4. 13.0 L of ethanol was added to the thick substance, heated to dissolve completely, and frozen at 10° C. for 24 hours, to precipitate white crystals, filtered, and the filtrate was concentrated under reduced pressure to recover about 6.5 L of ethanol, and then stood for 7 days to precipitate white needle-like crystals, filtered and dried to obtain 178.1 g of crystals, 500 ml of ethyl acetate was added to the crystals, heated to dissolve, cooled, and stood for 7 days, to precipitate white needle-like crystals, filtered and dried to obtain 103.8 g of the compound, 3,12,13-triacetyl limaxol A. 
     Example 8 
     A method for preparing a compound (a solvent extraction method) includes the following steps: 
     S1. Limax, after sorting and removing impurities, was pulverized into 10 mesh powder for later use: 
     S2. 150 kg of Limax powder was put into a multi-functional extraction tank, and extracted at reflux with a mixed solvent (ethanol:acetone=1:1) for 3 times, firstly with an amount of 12 times the weight of the Limax powder for 2.5 h, secondly with an amount of 8 times the weight of the Limax powder for 1.5 h, thirdly with an amount of 5 times the weight of the Limax powder for 1 h; each of the extracts was filtered, and the filtrates were combined, and the mixed solvent was recovered under reduced pressure until the recovery was complete, to obtain 11.98 kg of an oily thick paste; 
     S3. 48.4 kg of silica gel was added to the thick paste and mixed evenly; the mixture was added to a chromatography column pre-filled with 24.3 kg of silica gel, and pre-eluted with n-hexane in the ratio of “the volume of n-hexane: the total weight of the silica gel in the chromatography column=3:1.5” (that is, 146.3 L of n-hexane), and then eluted with acetone in the ratio of “the volume of acetone: the total weight of the silica gel in the chromatography column=3:2” (that is, 110.8 L of acetone), the acetone eluate was collected, and concentrated under reduced pressure to recover acetone, thereby obtaining 6.3 kg of a thick substance; and 
     S4. 40.0 L of methanol was added to the thick substance, heated to dissolve completely, and frozen at 2° C. for 24 hours, to precipitate white crystals, filtered, and the filtrate was concentrated under reduced pressure to recover about 24 L methanol, and then stood for 10 days to precipitate white needle-like crystals, filtered and dried to obtain 534 g of crystals, 1.5 L of ethyl acetate was added to the crystals, heated to dissolve, cooled, and stood for 10 days, to precipitate white needle-like crystals, filtered and dried to obtain 311.2 g of the compound, 3,12,13-triacetyl limaxol A. 
     Example 9 
     A method for preparing a compound (a solvent extraction method) includes the following steps: 
     S1. Limax, after sorting and removing impurities, was pulverized into 20 mesh powder for later use; 
     S2. 70 kg of Limax powder was put into a multi-functional extraction tank, and extracted at reflux with a mixed solvent (chloroform: petroleum ether: 120 # gasoline: n-hexane=1:2:1:6) for 2 times, firstly with an amount of 13 times the weight of the Limax powder for 2 h, and then with an amount of 6 times the weight of the Limax powder for 1.0 h: each of the extracts was filtered, and the filtrates were combined, and the mixed solvent was recovered under reduced pressure until the recovery was complete, to obtain 5.52 kg of an oily thick paste; 
     S3. 24.8 kg of silica gel was added to the thick paste and mixed evenly: the mixture was added to a chromatography column pre-filled with 13.8 kg of silica gel, and pre-eluted with petroleum ether in the ratio of “the volume of petroleum ether: the total weight of the silica gel in the chromatography column=2.4:1.2” (that is, 77.2 L of petroleum ether), and then eluted with ethyl acetate in the ratio of “the volume of ethyl acetate: the total weight of the silica gel in the chromatography column=1.8:1.2” (that is, 57.9 L of ethyl acetate), the ethyl acetate eluate was collected, and concentrated under reduced pressure to recover ethyl acetate, to obtain 2.90 kg of a thick substance; and 
     S4. 18.3 L of ethanol was added to the thick substance, heated to dissolve completely, and frozen at 8° C. for 24 hours, to precipitate white crystals, filtered, and the filtrate was concentrated under reduced pressure to recover about 9.6 L of ethanol, and then stood for 8 days to precipitate white needle-like crystals, filtered and dried to obtain 252.0 g of crystals, 0.7 L of ethyl acetate was added to the crystals, heated to dissolve, cooled, and stood for 8 days, to precipitate white needle-like crystals, filtered and dried to obtain 148.2 g of the compound, 3,12,13-triacetyl limaxol A. 
     III. Pharmacological Tests 
     Example 10 
     Experiment on the effect of the Limax extract on morphine-induced physical dependence 
     1. Object of Experiment 
     This experiment aims to study the effect of the Limax extract on morphine-induced physical dependence symptoms. 
     2. Materials and Method 
     2.1 Materials 
     2.1.1 Test samples 
     The Limax extract was provided by Guangxi Jiufu Biotechnology Co., Ltd., the Limax crude extract was an ointment-like extract obtained by the supercritical CO 2  extraction in R2 in Example 3 of the present application, and the 3,12,13-triacetyl limaxol A was a sample prepared in Example 3 of the present application; the morphine hydrochloride injection was commercially available from Shenyang No. 1 Pharmaceutical Factory of NORTHEAST PHARM; and the methadone was commercially available from Tianjin Central Pharmaceutical Industry Co., Ltd. 
     2.1.2 Experiment Animals 
     Kunming mice, males, weighing 18-22 g, were commercially available from Shanghai SiJie Laboratory Animal Co., Ltd. 
     2.2 Method 
     2.2.1 Effect on Morphine-Induced Physical Symptoms 
     The animals were randomized into a normal saline group, a model group, a positive control group (methadone), and test medicine groups at different doses. Each group has at least 8 mice. The normal saline group was given normal saline every day. The animals in the morphine model group, the positive control group, and the test medicine groups were given morphine at increasing doses of 10, 20, 40, 80, 100 mg/kg by subcutaneous administration twice a day, with a 6-hour interval between each administration. From day 3, 100 mg/kg of morphine was administrated for 7 consecutive days. On day 8, 4 mg/kg of naloxone was administered intraperitoneally 4 hours after the administration of morphine, and the number of jumpings within 30 minutes was recorded. At different time points (1 hour or 3 hours) before naloxone administration, the vehicle control, the positive control, and the test medicine were dosed intragastrically. 
     2.2.2 Statistical Analysis 
     The experiment results were represented by Mean±SEM, and differences between groups are tested by t-test. P&lt;0.05 showed a significant difference. 
     3. Results 
     Effect of the Limax Extract on Morphine-Induced Physical Dependence Symptoms 
     The experiment results were shown in Table 2 to Table 4. After chronic morphine treatment, naloxone was administrated to induce withdrawal responses, and the animals showed significant withdrawal jumping symptoms. The withdrawal jumping symptoms of the animals were significantly reduced 1 hour after the methadone (20 mg/kg) administration, 3 hours after the Limax crude extract (10 g/kg) administration, and 1 hour after the 3,12,13-triacetyl limaxol A (0.5 g/kg) administration. Table 5 showed that the withdrawal jumping symptoms of the animals tended to be reduced 3 hours after the 3,12,13-triacetyl limaxol A (0.5 g/kg) administration, but there was no significant difference from the model group.  FIG. 8  showed inhibition rates of three drugs on morphine-withdrawal jumping symptoms, in an order of Limax crude extract (10 g/kg, −3 h)&gt;3,12,13-triacetyl limaxol A (0.5 g/kg, −1 h)≈methadone (20 mg/kg, −1 h). 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Effect of methadone on morphine-withdrawal jumping symptoms 
               
            
           
           
               
               
               
            
               
                   
                   
                 % Inhibition of 
               
               
                   
                 Jumping times 
                 morphine-withdrawal 
               
               
                 Group 
                 (Mean ± SEM) 
                 jumping 
               
               
                   
               
               
                 Normal saline 
                 0.3 ± 0.3 
                 — 
               
               
                 Morphine 
                   62.4 ± 12.7*** 
                 — 
               
               
                 Morphine + methadone 
                 33.8 ± 3.3* 
                 46.1 ± 5.2 
               
               
                 20 mg/kg 
               
               
                   
               
            
           
         
       
     
     The results in Table 2 showed that there was a significant difference between the morphine group and the control group (**P&lt;0.001), indicating that naloxone administration after chronic morphine treatment can induce significant withdrawal jumping symptoms. There was a significant difference between the morphine+methadone group and the morphine group (*P&lt;0.05), indicating that the withdrawal jumping symptoms induced by naloxone administration can be significantly reduced 1 hour after the methadone (20 mg/kg) administration. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Effect of the Limax crude extract on 
               
               
                 morphine-withdrawal jumping symptoms 
               
            
           
           
               
               
               
            
               
                   
                   
                 % Inhibition of 
               
               
                   
                 Jumping times 
                 morphine-withdrawal 
               
               
                 Group 
                 (Mean ± SEM) 
                 jumping 
               
               
                   
               
               
                 Normal saline 
                 0.0 ± 0.0 
                 — 
               
               
                 Morphine 
                  54.8 ± 14.1** 
                 — 
               
               
                 Morphine + Limax crude 
                 14.3 ± 4.7* 
                 73.9 ± 8.6 
               
               
                 extract 10 g/kg 
               
               
                   
               
            
           
         
       
     
     The results in Table 3 showed that there was a significant difference between the morphine group and the control group (**P&lt;0.01), indicating that naloxone administration after chronic morphine treatment can induce significant withdrawal jumping symptoms. There was a significant difference between the morphine+Limax crude extract group and the morphine group (*P&lt;0.05), indicating that the withdrawal jumping symptoms induced by naloxone administration can be significantly reduced 3 hours after the Limax crude extract (10 g/kg) administration. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Effect of 3,12,13-triacetyl limaxol A on morphine-withdrawal 
               
               
                 jumping symptoms 1 hour after administration 
               
            
           
           
               
               
               
            
               
                   
                   
                 % Inhibition of 
               
               
                   
                 Jumping times 
                 morphine-withdrawal 
               
               
                 Group 
                 (Mean ± SEM) 
                 jumping 
               
               
                   
               
               
                 Normal saline 
                 1.3 ± 0.8 
                 — 
               
               
                 Morphine 
                  34.5 ± 5.2*** 
                 — 
               
               
                 Morphine + 
                 18.5 ± 2.4* 
                 48.2 ± 7.5  
               
               
                 3,12,13-triacetyl limaxol A 
               
               
                 0.5 g/kg 
               
               
                 Morphine + 
                 27.5 ± 9.4  
                 21.1 ± 28.2 
               
               
                 3,12,13-triacetyl limaxol A 
               
               
                 0.25 g/kg 
               
               
                 Morphine + 
                 33.5 ± 4.1  
                  3.0 ± 12.2 
               
               
                 3,12,13-triacetyl limaxol A 
               
               
                 0.125 g/kg 
               
               
                   
               
            
           
         
       
     
     The results in Table 4 showed that there was a significant difference between the morphine group and the control group (***P&lt;0.001), indicating that naloxone administration after chronic morphine treatment can induce significant withdrawal jumping symptoms. There was a significant difference between the morphine+high-dose 3,12,13-triacetyl limaxol A group and the morphine group (*P&lt;0.05), indicating that the withdrawal jumping symptoms induced by naloxone administration can be significantly reduced 1 hour after the morphine+3,12,13-triacetyl limaxol A (0.5 g/kg) administration. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Effect of 3,12,13-triacetyl limaxol A on morphine-withdrawal 
               
               
                 jumping symptoms 3 hours after administration 
               
            
           
           
               
               
               
            
               
                   
                   
                 % Inhibition of 
               
               
                   
                 Jumping times 
                 morphine-withdrawal 
               
               
                 Group 
                 (Mean ± SEM) 
                 jumping 
               
               
                   
               
               
                 Normal saline 
                 0.6 ± 0.5 
                 — 
               
               
                 Morphine 
                  29.8 ± 9.5** 
                 — 
               
               
                 Morphine + 
                 16.7 ± 4.9  
                 45.0 ± 17.0 
               
               
                 3,12,13-triacetyl limaxol A 
               
               
                 0.5 g/kg 
               
               
                   
               
            
           
         
       
     
     The results in Table 5 showed that there was a significant difference between the morphine group and the control group (***P&lt;0.01), indicating that naloxone administration after chronic morphine treatment can induce significant withdrawal jumping symptoms. There was no significant difference between the morphine+high-dose 3,12,13-triacetyl limaxol A group and the morphine group (P&gt;0.05), indicating that the withdrawal jumping symptoms induced by naloxone administration tended to be reduced 3 hours after the morphine+3,12,13-triacetyl limaxol A (0.5 g/kg) administration, but there was no significant difference. 
     4. Conclusion 
     The Limax crude extract (10 g/kg) has a significant inhibitory effect on withdrawal jumping symptoms in morphine-dependent animals 3 hours after intragastric administration, while the 3,12,13-triacetyl limaxol A (0.5 g/kg) has a significant inhibitory effect on withdrawal jumping symptoms in morphine-dependent animals 1 hour after intragastric administration, and still shows an inhibitory trend after 3 hours. 
     Example 11 
     Evaluation experiment on the sedative effect of the 3,12,13-triacetyl limaxol A (referred to as triacetyl limaxol A below) 
     1. Object of Experiment 
     This experiment aims to evaluate whether the triacetyl limaxol A has a sedative effect on mice. 
     2. Materials 
     2.1 Test Samples 
     (1) Triacetyl limaxol A: a sample prepared in Example 3 of the present invention. To an appropriate amount of the triacetyl limaxol A was added 0.5% of Carboxymethyl cellulose sodium salt (0.5% of CMC-Na) to prepare a test sample. 
     (2) Limax crude extract: an ointment-like extract obtained by the supercritical CO 2  extraction in R2 in Example 3 of the present invention. To an appropriate amount of the Limax crude extract was added an appropriate amount of the soybean oil to prepare a test sample. 
     (3) U50488: commercially available from Sigma Chemical Company. To an appropriate amount of U50488 was added an appropriate amount of the normal saline to prepare 0.5 mg/ml of a control sample. 
     2.2 Experiment Animals 
     ICR mice, males, weighing 18-22 g, were commercially available from Shanghai Lingchang Biotechnology Co., Ltd. 
     Animal grouping: The experiment comprised a normal saline control group, a U50488 group (5 mg/kg), a triacetyl limaxol A group (500 mg/kg), and a Limax crude extract group (10 g/kg). Each group had 10 mice. The sedative effects of the medicines were tested 30 minutes, 60 minutes, and 120 minutes after administration. 
     2.3 Main Instrument: 
     Rotarod instrument, model JL Behv-RRTG-5, commercially available from Shanghai Jiliang Software Technology Co., Ltd. 
     3. Method 
     3.1 Dosage Design 
     The dose of the triacetyl limaxol A was set to 500 mg/kg, the dose of the Limax crude extract was set to 10 g/kg, and the dose of the control sample U50488 was set to 5 mg/kg; and the normal saline (10 ml/kg) was administered to the normal saline control group. 
     3.2 Dosing Regimen 
     Preparation of animals before administration: The animals were allowed to eat and drink freely. 
     Dosage determination: The dosages were calculated based on the actual weights of the animals weighed before administration. 
     Route of administration: The test sample was administrated orally, which was consistent with the proposed clinical route; and the control sample U50488 was administrated by intraperitoneal injection. 
     Frequency of administration: single dose 
     Volume of administration: 20 ml/kg of the test sample, and 10 ml/kg of the control sample 
     Site of administration: The test sample was dosed by intragastric administration, and the control sample was dosed by intraperitoneal injection. 
     3.3 Measurement of Observation Index 
     Observation index: the retention time on the rotarod (s) 
     Measurement method: The male mice were put on the rotarod instrument with a rotarod speed set to increase evenly from 4 r/min to 40 r/min in 240 s. The retention time of the mice was used as an observation index. Two baseline measurements were determined before administration. The animals with a retention time of less than 120 s were excluded, and the animals meeting the requirements were selected for testing, with 10 in each group. The retention times of each animal 30 minutes, 60 minutes, and 120 minutes after administration were recorded. The experiment was performed for 240 s. 
     3.4 Statistical Analysis 
     The retention times of the mice in each group were represented by Mean±SEM. 
     A two-tailed t test was performed on the retention times in the test sample group and the normal saline control group. 
     4. Results 
     As shown in  FIG. 9 a    to  FIG. 9 c    (in  FIG. 9 a    to  FIG. 9 c   , * indicated that the p value was less than 0.05 compared with the normal saline control group, and ** indicated that the p value was less than 0.01 compared with the normal saline control group), the retention times of the animals 30 minutes after the administration of 5 mg/kg U50488 were significantly lower than that of the normal saline control group, indicating that 5 mg/kg of U50488 had a significant sedative effect, which was consistent with literature reports. The retention times of the animals 30, 60, and 120 minutes after oral administration of 500 mg/kg triacetyl limaxol A were respectively 196.7±24.3 s, 209.7±20.4 s, and 236.9±2.0 s. Compared with the normal saline control group, the retention times were not significantly reduced, indicating that 500 mg/kg of the triacetyl limaxol A did not have a significant sedative effect. In contrast, the retention times of the animals 120 minutes after oral administration of 10 g/kg Limax crude extract were significantly lower than that of the normal saline control group, indicating that the Limax crude extract had a sedative effect. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Experimental data in a mouse rotarod model 
               
               
                 (Mean ± SEM) (retention time on the rotarod, s) 
               
            
           
           
               
               
               
               
            
               
                   
                 30 min after 
                 60 min after 
                 120 min after 
               
               
                 Group 
                 administration 
                 administration 
                 administration 
               
               
                   
               
               
                 Normal saline control group 
                 204.0 ± 22.8 
                 202.1 ± 24.1 
                 201.9 ± 24.1 
               
               
                 U50488 group (5 mg/kg) 
                  100.7 ± 23.9* 
                  181.2 ± 1.966 
                 223.7 ± 11.7 
               
               
                 Triacetyl limaxol A group (500 mg/kg) 
                 196.7 ± 24.3 
                 209.7 ± 20.4 
                 236.9 ± 2.0  
               
               
                 Limax crude extract group (10 g/kg) 
                 167.5 ± 25.5 
                 129.6 ± 24.2 
                  118.5 ± 21.9** 
               
               
                   
               
               
                 *indicates that the p value is less than 0.05 compared with the normal saline control group, and 
               
               
                 **indicates that the p value is less than 0.01 compared with the normal saline control group. 
               
            
           
         
       
     
     5. Conclusion 
     500 mg/kg of the triacetyl limaxol A does not show a significant sedative effect in the mouse rotarod model 30 minutes, 60 minutes, and 120 minutes after oral administration, while 10 g/kg of the Limax crude extract shows a significant sedative effect 120 minutes after administration. 
     Example 12 
     Effect of the triacetyl limaxol A on morphine-induced conditioned place preference behavior in rats 
     1. Object of Experiment 
     This experiment aims to evaluate whether a single oral administration of the triacetyl limaxol A can inhibit the morphine-induced conditioned place preference behavior in rats. 
     2. Materials 
     2.1 Test Samples 
     (1) Triacetyl limaxol A: a sample prepared in Example 3 of the present invention. 
     (2) Morphine hydrochloride injection: commercially available from Shenyang No. 1 Pharmaceutical Factory of NORTHEAST PHARM. 
     (3) Solvent in Self-Micro emulsifying Drug Delivery System (SMEDDS) (referred to as SMEDDS solvent below): provided by Guangxi Jiufu Biotechnology Co., Ltd. and used as a stock solution, which was composed of: medium-chain triglyceride (MCT): polyoxyethylene (35) castor oil (EL): polyethylene glycol 400 (PEG-400): absolute ethanol=30:72:24:24 (by mass). 
     2.2 Experiment Animals 
     SD rats, males, weighing 180-200 g, were commercially available from SIPPR-BK Laboratory Animal Co., Ltd. 
     Animal grouping: The experiment comprised a normal saline control group, a normal saline+morphine group, a SMEDDS solvent+morphine group, and a triacetyl limaxol A (70 mg/kg)+morphine group. Each group had 5-7 male rats. In the experiment, the animals were orally administered, and the residence times of each animal in the left and right compartments of a box were recorded 60 minutes after administration. 
     2.3 Main Instrument: 
     Conditioned place preference (CPP) video analysis system: model JL Behv-CPPG-4, commercially available from Shanghai Jiliang Software Technology Co., Ltd. 
     3. Method 
     3.1 Dosage Design 
     Based on the physical dependence experiment in mice, the dose of the triacetyl limaxol A was set to 70 mg/kg, the volume of the SMEDDS solvent for intragastric administration was set to 7 ml/kg, and 7 ml/kg of the normal saline was administered intragastrically to the normal saline control group and the morphine groups. 
     3.2 Dosing Regimen 
     Preparation of animals before administration: The animals were allowed to eat and drink freely. 
     Dosage determination: The dosages were calculated based on the actual weights of the animals weighed before administration. 
     Route of administration: The test sample was administrated orally, which was consistent with the proposed clinical route. 
     Frequency of administration: once a day, for four consecutive days 
     Volume of administration: 7 ml/kg of the test sample 
     Site of administration: The test sample was dosed by intragastric administration. 
     3.3 Measurement of Observation Index 
     Observation index: CPP SCORE (s)=the time in the drug compartment after administration−the time in the drug compartment before administration 
     Measurement method: After acclimation to the box, the male rats were allowed to explore the two compartments of the box for 15 minutes. According to the residence times of each animal in the left and right compartments during the exploration, the animals with a residence time of greater than 720 s or less than 180 s in any compartment were excluded. The compartment where each of the animals stayed for a short time was defined as its drug compartment. Based on the residence time in the drug compartment, the qualified animals were equally assigned to each dose group. Except for the animals in the normal saline control group, which were injected with the normal saline (1 ml/kg) subcutaneously for four consecutive days, the animals in the other three groups were subcutaneously injected with morphine (10 mg/kg) daily for four consecutive days. Immediately after the injection, the animals were put into their drug compartments to pair for 50 minutes, 2 days after the last pairing, each animal was put into the box to test for 15 minutes, 60 minutes before the testing, the normal saline was administrated intragastrically to the animals in the normal saline control group and the normal saline+morphine group, the SMEDDS solvent was administrated intragastrically to the animals in the SMEDDS solvent+morphine group, and a mixed solution of the triacetyl limaxol A and the SMEDDS solvent was administrated intragastrically to the animals in the triacetyl limaxol A+morphine group. The residence times of each animal in the left and right compartments were recorded, and the CPP SCOREs (conditioned place preference scores) were calculated. In the mixed solution of the triacetyl limaxol A and the SMEDDS solvent, the concentration of the triacetyl limaxol A was 10 mg/g. In an example, the mixed solution may be prepared by using the following method: 
     (1) 30.0 g of MCT, 72.0 g of EL, and 24.0 g of PEG-400 were evenly mixed; 
     (2) 1.5 g of the triacetyl limaxol A and 24.0 g of absolute ethanol were heated at 80° C. until the triacetyl limaxol A was completely dissolved; and 
     (3) the solution of the triacetyl limaxol A in the absolute ethanol from step (2) was added to the mixed solution from step (1), and evenly mixed to obtained a clear solution (that is, the mixed solution of the triacetyl limaxol A and the SMEDDS solvent). 
     3.4 Statistical Analysis 
     The CPP SCOREs of the rats in each group were represented by Mean±SEM. 
     A two-tailed t test was performed on the CPP SCOREs of the test sample group and the normal saline control group. 
     4. Results 
     As shown in  FIG. 10  (in  FIG. 10 , * indicated that the p value was less than 0.05 compared with the normal saline control group), the CPP SCOREs of the normal saline+morphine group, the SMEDDS solvent+morphine group, and the triacetyl limaxol A+morphine group 60 minutes after oral administration were significantly increased compared with that of the normal saline control group, which showed a conditioned place preference behavior. Moreover, the CPP SCORE of the triacetyl limaxol A (70 mg/kg)+morphine group was 318.5±90.9 s, which was not significantly different from the normal saline+morphine group and the SMEDDS solvent+morphine group, indicating that a single oral administration of 70 mg/kg of the triacetyl limaxol A cannot affect the morphine-induced conditioned place preference behavior in rats. 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Effect of the triacetyl limaxol A on morphine-induced 
               
               
                 conditioned place preference behavior in rats 
               
               
                 Experimental data CPP SCORE (Mean ± SEM) (s) 
               
            
           
           
               
               
               
            
               
                   
                 Group 
                 CPP SCORE (s) 
               
               
                   
                   
               
               
                   
                 Normal saline control group 
                  −43.9 ± 81.9, n = 7 
               
               
                   
                 Normal saline + morphine group 
                 269.8 ± 78.8*, n = 6 
               
               
                   
                 SMEDDS solvent + morphine group 
                 359.6 ± 73.6*, n = 5 
               
               
                   
                 Triacetyl limaxol A (70 mg/kg) + 
                 318.5 ± 90.9*, n = 7 
               
               
                   
                 morphine group 
               
               
                   
                   
               
               
                   
                 *indicates that the p value is less than 0.05 compared with the normal saline control group. 
               
            
           
         
       
     
     5. Conclusion 
     A single oral administration of 70 mg/kg of the triacetyl limaxol A cannot affect the morphine-induced conditioned place preference behavior in rats. 
     Example 13 
     Effect of the concomitant administration of triacetyl limaxol A on the formation of conditioned place preference induced by morphine in rats 
     1. Object of Experiment 
     This experiment aims to evaluate whether the concomitant administration of the triacetyl limaxol A can inhibit the formation of conditioned place preference induced by morphine in rats. 
     2. Materials 
     2.1 Test Samples 
     (1) Triacetyl limaxol A: a sample prepared in Example 3 of the present invention. 
     (2) Morphine hydrochloride injection: commercially available from Shenyang No. 1 Pharmaceutical Factory of NORTHEAST PHARM. 
     (3) SMEDDS solvent: the same as that in Example 12, commercially available from Guangxi Jiufu Biotechnology Co., Ltd. and used as a stock solution. 
     2.2 Experiment Animals 
     SD rats, males, weighing 180-200 g, were commercially available from SIPPR-BK Laboratory Animal Co., Ltd. 
     Animal grouping: The experiment comprised a normal saline control group, a normal saline+morphine group, a SMEDDS solvent+morphine group, and a triacetyl limaxol A (70 mg/kg)+morphine group. Each group had 6-8 male rats. In the experiment, the animals were orally administered. 30 minutes after the administration, they were injected subcutaneously with morphine (10 mg/kg), and then were put into the drug compartments to pair for 50 minutes. 
     2.3 Main Instrument: 
     Conditioned place preference video analysis system: model JL Behv-CPPG-4, commercially available from Shanghai Jiliang Software Technology Co., Ltd. 
     3. Method 
     3.1 Dosage Design 
     Based on the physical dependence experiment in the mice, the dose of the triacetyl limaxol A was set to 70 mg/kg, the volume of the SMEDDS solvent for intragastric administration was set to 7 ml/kg, and the volume of the normal saline for intragastric administration was set to 7 ml/kg. 
     3.2 Dosing Regimen 
     Preparation of animals before administration: The animals were allowed to eat and drink freely. 
     Dosage determination: The dosages were calculated based on the actual weights of the animals weighed before administration. 
     Route of administration: The test sample was administrated orally, which was consistent with the proposed clinical route. 
     Frequency of administration: once a day, for four consecutive days 
     Volume of administration: 7 ml/kg of the test sample 
     Site of administration: The test sample was dosed by intragastric administration. 
     3.3 Measurement of Observation Index 
     Observation index: CPP SCORE (s)=the time in the drug compartment after administration−the time in the drug compartment before administration 
     Measurement method: After acclimation to the box, the male rats were allowed to explore the two compartments of the box for 15 minutes. According to the residence times of each animal in the left and right compartments during the exploration, the animals with a residence time of greater than 720 s or less than 180 s in any compartment were excluded. The compartment where each of the animals stayed for a short time was defined as its drug compartment. Based on the residence time in the drug compartment, the qualified animals were equally assigned to each dose group. Except for the animals in the normal saline control group, which were injected with the normal saline (1 ml/kg) subcutaneously for four consecutive days, the animals in the other three groups were subcutaneously injected with morphine (10 mg/kg) daily for four consecutive days. Immediately after the injection, the animals were put into their drug compartments to pair for 50 minutes. 30 minutes before the injection of morphine, the normal saline was administrated intragastrically to the animals in the normal saline+morphine group, the SMEDDS solvent was administrated intragastrically to the animals in the SMEDDS solvent+morphine group, and a mixed solution of the triacetyl limaxol A and the SMEDDS solvent (see Example 12) was administrated intragastrically to the animals in the triacetyl limaxol A (70 mg/kg)+morphine group. 2 days after the last pairing, each animal was put into the box to test for 15 minutes. The residence times of each animal in the left and right compartments were recorded, and the CPP SCOREs were calculated. 
     3.4 Statistical Analysis 
     The CPP SCOREs of the rats in each group was represented by Mean±SEM. 
     A two-tailed t test was performed on the CPP SCOREs of the test sample group and the normal saline control group. 
     4. Results 
     As shown in  FIG. 11  (in  FIG. 11 , * indicated that the p value was less than 0.05 compared with the normal saline control group, ** indicated that the p value was less than 0.01 compared with the normal saline control group, and *** indicated that the p value was less than 0.001 compared with the normal saline control group), the CPP SCOREs of the normal saline+morphine group, the SMEDDS solvent+morphine group, and the triacetyl limaxol A+morphine group 60 minutes after oral administration were significantly increased compared with the normal saline control group, which showed a conditioned place preference behavior. Moreover, the CPP SCORE of the triacetyl limaxol A (70 mg/kg)+morphine group was 318.5±90.9 s, which was not significantly different from the normal saline+morphine group and the SMEDDS solvent+morphine group, indicating that a single oral administration of 70 mg/kg of the triacetyl limaxol A cannot affect the morphine-induced conditioned place preference behavior in rats. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Effect of the triacetyl limaxol A on the formation of 
               
               
                 conditioned place preference induced by morphine in rats 
               
               
                 Experimental data CPP SCORE (Mean ± SEM) (s) 
               
            
           
           
               
               
               
            
               
                   
                 Group 
                 CPP SCORE (s) 
               
               
                   
                   
               
               
                   
                 Normal saline control group 
                      −62.0 ± 25.4, n = 7 
               
               
                   
                 Normal saline + morphine group 
                 152.2 ± 32.2***, n = 7 
               
               
                   
                 SMEDDS solvent + morphine group 
                  121.0 ± 36.6**, n = 8 
               
               
                   
                 Triacetyl limaxol A (70 mg/kg) + 
                 223.8 ± 34.0***, n = 6 
               
               
                   
                 morphine group 
               
               
                   
                   
               
               
                   
                 *indicates that the p value is less than 0.05 compared with the normal saline control group, 
               
               
                   
                 **indicates that the p value is less than 0.01 compared with the normal saline control group, and 
               
               
                   
                 ***indicates that the p value is less than 0.001 compared with the normal saline control group. 
               
            
           
         
       
     
     5. Conclusion 
     70 mg/kg of the triacetyl limaxol A orally administrated concomitantly with the morphine cannot affect the formation of conditioned place preference induced by morphine in rats. 
     Example 14 
     Evaluation experiment on the addictive potential of the triacetyl limaxol A 
     1. Object of Experiment 
     This experiment aims to evaluate whether the triacetyl limaxol A can induce rats to form conditioned place preference. 
     2. Materials 
     2.1 Test Samples 
     (1) Triacetyl limaxol A (test sample): a sample prepared in Example 3 of the present invention, used as a stock solution. 
     (2) Morphine hydrochloride injection: commercially available from Shenyang No. 1 Pharmaceutical Factory of NORTHEAST PHARM. 
     (3) SMEDDS solvent: the same as that in Example 12, commercially available from Guangxi Jiufu Biotechnology Co., Ltd. and used as a stock solution. 
     2.2 Experiment Animals 
     SD rats, males, weighing 180-200 g, were commercially available from SIPPR-BK Laboratory Animal Co., Ltd. 
     Animal grouping: The experiment comprised a normal saline s.c group (s.c represents subcutaneous administration), a morphine group, a normal saline p.o group (p.o represents oral administration), a SMEDDS solvent control group, a 17.5 mg/kg triacetyl limaxol A group, a 35 mg/kg triacetyl limaxol A group, and a 70 mg/kg triacetyl limaxol A group. Each group had 9-12 male rats. The animals in the normal saline p.o group, the SMEDDS solvent control group, and all of the triacetyl limaxol A groups were administrated intragastrically, and 30 minutes later, they were put into the drug compartments to pair for 50 minutes, while the animals in the normal saline s.c group and the morphine group were injected subcutaneously with the normal saline and the morphine (10 mg/kg) respectively, and then immediately put into the drug compartments to pair for 50 minutes. 
     2.3 Main Instrument: 
     Conditioned place preference video analysis system: model JL Behv-CPPG-4, commercially available from Shanghai Jiliang Software Technology Co., Ltd. 
     3. Method 
     3.1 Dosage Design 
     Based on the physical dependence experiment in the mice, the doses of the test sample groups were set to 17.5 mg/kg, 35 mg/kg, and 70 mg/kg, and the volumes for intragastric administration were set to 7 ml/kg, and the volumes for subcutaneous injection were set to 1 ml/kg. 
     3.2 Dosing Regimen 
     Preparation of animals before administration: The animals were allowed to eat and drink freely. 
     Dosage determination: The dosages were calculated based on the actual weights of the animals weighed before administration. 
     Route of administration: The test sample was administrated orally, which was consistent with the proposed clinical route. 
     Frequency of administration: once a day, for four consecutive days 
     Volume of administration: 7 ml/kg of the test sample 
     Site of administration: The test sample was dosed by intragastric administration. 
     3.3 Measurement of observation index 
     Observation index: CPP SCORE (s)=the time in the drug compartment after administration−the time in the drug compartment before administration 
     Measurement method: After acclimation to the box, the male rats were allowed to explore the two compartments of the box for 15 minutes. According to the residence times of each animal in the left and right compartments during the exploration, the animals with a residence time of greater than 720 s or less than 180 s in any compartment were excluded. The compartment where each of the animals stayed for a short time was defined as its drug compartment. Based on the time in the drug compartment, the qualified animals were equally assigned to each dose group. The animals in the normal saline s.c group were subcutaneously injected with the normal saline (1 ml/kg) daily for four consecutive days, and the animals in the morphine group were subcutaneously injected with morphine (10 mg/kg) daily for four consecutive days, and then the animals of the two groups were immediately put into their drug compartments to pair for 50 minutes. The animals in the normal saline p.o group were intragastrically administered with the normal saline for four consecutive days, the animals in the SMEDDS solvent control group were intragastrically administered with the SMEDDS solvent for four consecutive days, and the animals in the triacetyl limaxol A groups were intragastrically administered with the mixed solutions of the triacetyl limaxol A and the SMEDDS solvent with different doses (see Example 12) for four consecutive days. 30 minutes later, the animals of the foregoing groups were put into their drug compartments to pair for 50 minutes. 2 days after the last pairing, each animal was put into the box to test for 15 minutes. The residence times of each animal in the left and right compartments were recorded, and the CPP SCOREs were calculated. 
     3.4 Statistical Analysis 
     The CPP SCOREs of the rats in each group were represented by Mean±SEM. 
     A two-tailed t test was performed on the CPP SCOREs of the test sample groups and the normal saline control group. 
     4. Results 
     As shown in  FIG. 12 a    and  FIG. 12 b    (in the figures, * represented p&lt;0.05 compared with the normal saline s.c group), the CPP SCORE of the morphine group after administration for four consecutive days was 208.3±16.2 s, which was significantly increased (p&lt;0.05) compared with the normal saline s.c group ( FIG. 12 a   ), suggesting that the morphine induced conditioned place preference behavior in rats. The CPP SCOREs of the 17.5 mg/kg, 35 mg/kg, and 70 mg/kg triacetyl limaxol A groups were respectively 96.8±42.4 s, 36.4±60.5 s, and 13.4±60.6 s (Table 9), which had no significant changes compared with the SMEDDS solvent control group, indicating that different doses of the triacetyl limaxol A cannot induce the rats to form the conditioned place preference, and thus had no significant psychological dependence potential. 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Experimental data of CPP SCORE (Mean ± SEM) (s) for 
               
               
                 inducing the rats to form the conditioned place preference 
               
            
           
           
               
               
            
               
                 Group 
                 CPP SCORE (s) 
               
               
                   
               
               
                 Normal saline s.c group 
                 73.0 ± 41.7, n = 10 
               
               
                 Morphine group 
                 208.3 ± 16.2*, n = 10  
               
               
                 Normal saline p.o group 
                 115.7 ± 46.8, n = 10  
               
               
                 SMEDDS solvent control group 
                 −46.0 ± 79.2, n = 8  
               
               
                 Triacetyl limaxol A group (17.5 mg/kg) 
                 96.8 ± 42.4, n = 12 
               
               
                 Triacetyl limaxol A group (35 mg/kg) 
                 36.4 ± 60.5, n = 9  
               
               
                 Triacetyl limaxol A group (70 mg/kg) 
                 13.4 ± 60.6, n = 10 
               
               
                   
               
               
                 *indicates that the p value is less than 0.05 compared with the normal saline s.c group. 
               
            
           
         
       
     
     5. Conclusion 
     17.5 mg/kg, 35 mg/kg, and 70 mg/kg of the triacetyl limaxol A cannot induce the rats to form the conditioned place preference, and thus have no psychological dependence potential. 
     Example 15 
     Evaluation of the effect of the Limax extracts (crude extract, limaxol A, and triacetyl limaxol A) on morphine-induced physical dependence 
     1. Test Samples 
     (1) Triacetyl limaxol A: a sample prepared in Example 3 of the present invention. 
     (2) Morphine hydrochloride injection: commercially available from Shenyang No. 1 Pharmaceutical Factory of NORTHEAST PHARM. 
     (3) Limaxol A: provided by Guangxi Jiufu Biotechnology Co., Ltd., having a structure of: 
     
       
         
         
             
             
         
       
     
     2. Experiment Animals 
     Kunming mice, males, weighing 18-22 g, were commercially available from the experiment animal center of Shanghai Institute of Materia Medica, Chinese Academy of Sciences. 
     3. Method 
     The animals were randomized to a normal saline control group, a morphine group, a morphine+1 g/kg triacetyl limaxol A group, and a morphine+1 g/kg limaxol A group. A mouse model of physical dependence was established by administrating morphine subcutaneously twice a day (60 mg/kg on the first day, and 80 mg/kg on the second day), with a 6-hour interval between each administration, and from day 3, administrating 100 mg/kg of morphine 7 consecutive days. On day 8, 4 mg/kg of naloxone was injected intraperitoneally 4 hours after the administration of morphine to induce withdrawal responses. 3 hours before the injection of the naloxone, the animals in each group were administrated different drugs, that is, 1 g/kg of the triacetyl limaxol A was administrated to the animals in the morphine+1 g/kg triacetyl limaxol A group, and 1 g/kg of the limaxol A was administrated to the animals in the morphine+1 g/kg limaxol A group, to detect the effect on the morphine-induced physical dependence symptoms. 
     4. Results: as shown in Table 10 
     
       
         
           
               
               
               
             
               
                 TABLE 10 
               
               
                   
               
               
                   
                   
                 Inhibition of 
               
               
                   
                 Jumping times 
                 morphine-withdrawal 
               
               
                 Group 
                 (Mean ± SEM) 
                 jumping 
               
               
                   
               
             
            
               
                 Morphine group 
                 38.1 ± 8.8 
                 — 
               
               
                 Morphine + 1 g/kg triacetyl 
                 21.7 ± 6.9 
                 43.13% 
               
               
                 limaxol A group 
               
               
                 Morphine + 1 g/kg limaxol 
                 30.0 ± 9.7 
                 21.26% 
               
               
                 A group 
               
               
                   
               
            
           
         
       
     
     The experimental data shows that under the same dosage conditions, the inhibition rate of the triacetyl limaxol A on the morphine-dependent mice is more than twice that of the limaxol A, and thus has a stronger biological activity and a better effect. 
     Example 16 
     Evaluation of the affinity and selectivity of triacetyllimaxol A for three subtypes of opioid receptors κ, μ, and δ 
     1. Object of Experiment 
     This experiment aims to evaluate the affinity and selectivity of triacetyllimaxol A for three subtypes of opioid receptors κ, μ, and δ. 
     2. Materials 
     2.1 Test sample: a mixed solution of triacetyllimaxol A (code name AK) and SMEDDS solvent. Preparation method: AK was mixed with SMEDDS solvent (the same as Example 12) to prepare a stock solution with a concentration of 1×10 −1.81  M, which was aliquoted into 10 μl/EP tube and preserved at −20° C. for use, and diluted to a desired concentration on the day of use. 
     2.2 Main reagents 
     2.2.1 Membrane receptor protein: prepared by cell membranes extracted from CHO cells stably expressing receptors κ, μ, and δ which were constructed by Shanghai Institute of Materia Medica, Chinese Academy of Sciences (referring to 3.1 below); 
     2.2.2 Radioligands 
       3 H-DAMGO (50.1 Ci/mmol, μ opioid receptor agonist), lot number 2479555; 
       3 H-DPDPE (48.6 Ci/mmol, δ opioid receptor agonist), lot number 2764670: and 
       3 H-U69593(39.1 Ci/mmol, κ opioid receptor agonist), lot number 2230633: 
     all of the radioligands were commercially available from PE Company. 
     2.2.3 Selective ligands (Reference ligands) 
     High-selective μ opioid receptor agonist DAMGO, commercially available from TOCRIS Bioscience Company, lot number 32A: 
     High-selective κ opioid receptor agonist (±)-trans-U50488, commercially available from TOCRIS Bioscience Company, lot number 4B/242199; and 
     High-selective δ opioid receptor agonist SNC80, commercially available from Abcam Corporation, lot number APN11310-7-8. 
     2.2.4 Other reagents 
     PPO (2,5-diphenyloxazole): commercially available from Sinopharm Chemical Reagent Co., Ltd, lot number 20180305; 
     POPOP (1,4-bis(5-phenyloxazol-2-yl)-benzene): commercially available from SigmaCorporation; 
     Toluene: commercially available from Sinopharm Chemical Reagent Co., Ltd. lot number 20191101; 
     Fetal bovine serum: commercially available from Shanghai Sunub Bio-Tech Development Inc.: 
     Ham&#39;s F-12: commercially available from Shanghai BasalMedia Technologies Co., LTD., lot number K120803: 
     G418: commercially available from Sigma Corporation: 
     Tris (tris(hydroxymethyl)aminomethane): commercially available from Sigma Corporation; 
     HEPES (N-2-Hydroxyethylpiperazine-N′-2-ethanesulfonic acid): commercially available from Amresco Inc.; 
     EDTA (ethylenediaminetetraacetic acid): commercially available from Invitrogen Corporation, lot number 2730C504; 
     EGTA (ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid): commercially available from Amresco Inc.; 
     NaCl (sodium chloride): commercially available from Sinopharm Chemical Reagent Co., Ltd, lot number 20191031; 
     MgCl 2  (magnesium chloride): commercially available from Sinopharm Chemical Reagent Co., Ltd, lot number 20181214; 
     NaH 2 PO 4  (sodium dihydrogen phosphate): commercially available from Sinopharm Chemical Reagent Co., Ltd, lot number 20140115: and 
     Grade GF/C glass fiber filter paper: commercially available from Whatman Inc. 
     2.3 Main Instrument 
     Perkin Elmer liquid scintillation counter: model PRI-CARB 2910, commercially available from PE Company. 
     3. Method 
     3.1 Preparation of Membrane Receptor Proteins 
     Cells were cultured in a 10 cm 2  of culture dish (Ham&#39;s F-12 culture medium+10% fetal bovine serum) for several days. After the cells were grown to confluence on the culture dish, the medium was aspirated, and 3 ml of PBS/EDTA solution (0.1 M NaCl, 0.01 M NaH 2 PO 4 , and 0.04% EDTA) was added to digest for 3-5 min. The cells were completely detached by pipetting several times. The cells were collected in a 40 ml of centrifuge tube, centrifuged at 5000 rpm for 5 min, and the supernatant was discarded. Ice-cold homogenization solution (50 mM HEPES, 3 mM MgCl 2 , and 1 mM EGTA, pH 7.4) was added to the centrifuge tube, and the solution and the precipitate obtained above were transferred to a homogenizer for homogenization. Then, the homogenate was transferred to the centrifuge tube, and centrifuged twice at 15000 rpm for 20 min to obtain a precipitate. The precipitate was homogenized by adding an appropriate amount of 50 mM Tris-HCl buffer solution, pH 7.4, to measure the protein concentration. The resulting homogenate was then aliquoted into EP tubes, and preserved in a refrigerator at −80° C. for use. 
     3.2 Radioligand-Receptor Binding Assay 
     Corresponding reagents were respectively added to flow tubes according to Table 11 to Table 13 below: 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Affinity profiles of κ opioid receptors 
               
            
           
           
               
               
               
               
               
            
               
                   
                 κ opioid 
                   
                 Final 
                 Final 
               
               
                   
                 membrane 
                   3 H-U69593 
                 concentration 
                 concentration 
               
               
                   
                 receptor (μg) 
                 (μl) 
                 of U50488 (mol/L) 
                 of AK(mol/L) 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                 Total binding tube 
                 50 
                 10 
                 — 
                 — 
               
               
                 Nonspecific binding tube 
                 50 
                 10 
                 1*10 −5   
                 — 
               
               
                 Sample tube 1 
                 50 
                 10 
                 1*10 −4   
                 — 
               
               
                 Sample tube 2 
                 50 
                 10 
                 1*10 −5   
                 — 
               
               
                 Sample tube 3 
                 50 
                 10 
                 5*10 −6   
                 — 
               
               
                 Sample tube 4 
                 50 
                 10 
                 1*10 −6   
                 — 
               
               
                 Sample tube 5 
                 50 
                 10 
                 1*10 −7   
                 — 
               
               
                 Sample tube 6 
                 50 
                 10 
                 1*10 −8   
                 — 
               
               
                 Sample tube 7 
                 50 
                 10 
                 1*10 −9   
                 — 
               
               
                 Sample tube 8 
                 50 
                 10 
                     1*10 −10   
                 — 
               
               
                 Sample tube 9 
                 50 
                 10 
                 — 
                 1.537*10 −3   
               
               
                 Sample tube 10 
                 50 
                 10 
                 — 
                 1.537*10 −4   
               
               
                 Sample tube 11 
                 50 
                 10 
                 — 
                 1.537*10 −5   
               
               
                 Sample tube 12 
                 50 
                 10 
                 — 
                 1.537*10 −6   
               
               
                 Sample tube 13 
                 50 
                 10 
                 — 
                 1.537*10 −7   
               
               
                 Sample tube 14 
                 50 
                 10 
                 — 
                 1.537*10 −8   
               
               
                 Sample tube 15 
                 50 
                 10 
                 — 
                 1.537*10 −9   
               
               
                   
               
               
                 Note: 
               
               
                 “—” represents no adding. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Affinity profiles of δ opioid receptors 
               
            
           
           
               
               
               
               
               
            
               
                   
                 δ opioid 
                   
                 Final 
                 Final 
               
               
                   
                 membrane 
                   3 H-DPDPE 
                 concentration 
                 concentration 
               
               
                   
                 receptor (μg) 
                 (μl) 
                 of SNC80 (mol/L) 
                 of AK (mol/L) 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                 Total binding tube 
                 50 
                 10 
                 — 
                 — 
               
               
                 Nonspecific binding tube 
                 50 
                 10 
                 1*10 −6   
                 — 
               
               
                 Sample tube 1 
                 50 
                 10 
                 1*10 −5   
                 — 
               
               
                 Sample tube 2 
                 50 
                 10 
                 1*10 −6   
                 — 
               
               
                 Sample tube 3 
                 50 
                 10 
                 1*10 −7   
                 — 
               
               
                 Sample tube 4 
                 50 
                 10 
                 1*10 −8   
                 — 
               
               
                 Sample tube 5 
                 50 
                 10 
                 1*10 −9   
                 — 
               
               
                 Sample tube 6 
                 50 
                 10 
                     1*10 −10   
                 — 
               
               
                 Sample tube 7 
                 50 
                 10 
                     1*10 −11   
                 — 
               
               
                 Sample tube 8 
                 50 
                 10 
                 — 
                 1.537*10 −3   
               
               
                 Sample tube 9 
                 50 
                 10 
                 — 
                 1.537*10 −4   
               
               
                 Sample tube 10 
                 50 
                 10 
                 — 
                 0.7685*10 −5   
               
               
                 Sample tube 11 
                 50 
                 10 
                 — 
                 1.537*10 −5   
               
               
                 Sample tube 12 
                 50 
                 10 
                 — 
                 1.537*10 −6   
               
               
                 Sample tube 13 
                 50 
                 10 
                 — 
                 1.537*10 −7   
               
               
                 Sample tube 14 
                 50 
                 10 
                 — 
                 1.537*10 −8   
               
               
                   
               
               
                 Note: 
               
               
                 “—” represents no adding. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 Affinity profiles of μ opioid receptors 
               
            
           
           
               
               
               
               
               
            
               
                   
                 μ opioid 
                   
                 Final 
                 Final 
               
               
                   
                 membrane 
                   3 H-DAMGO 
                 concentration 
                 concentration 
               
               
                   
                 receptor (μg) 
                 (μl) 
                 of DAMGO (mol/L) 
                 of AK(mol/L) 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                 Total binding tube 
                 50 
                 10 
                 — 
                 — 
               
               
                 Nonspecific binding tube 
                 50 
                 10 
                 1*10 −5   
                 — 
               
               
                 Sample tube 1 
                 50 
                 10 
                 1*10 −4   
                 — 
               
               
                 Sample tube 2 
                 50 
                 10 
                 1*10 −5   
                 — 
               
               
                 Sample tube 3 
                 50 
                 10 
                 1*10 −6   
                 — 
               
               
                 Sample tube 4 
                 50 
                 10 
                 1*10 −7   
                 — 
               
               
                 Sample tube 5 
                 50 
                 10 
                 1*10 −8   
                 — 
               
               
                 Sample tube 6 
                 50 
                 10 
                 1*10 −9   
                 — 
               
               
                 Sample tube 7 
                 50 
                 10 
                     1*10 −10   
                 — 
               
               
                 Sample tube 8 
                 50 
                 10 
                 — 
                 1.537*10 −3   
               
               
                 Sample tube 9 
                 50 
                 10 
                 — 
                 1.537*10 −4   
               
               
                 Sample tube 10 
                 50 
                 10 
                 — 
                 1.537*10 −5   
               
               
                 Sample tube 11 
                 50 
                 10 
                 — 
                 1.537*10 −6   
               
               
                 Sample tube 12 
                 50 
                 10 
                 — 
                 1.537*10 −7   
               
               
                 Sample tube 13 
                 50 
                 10 
                 — 
                 1.537*10 −8   
               
               
                 Sample tube 14 
                 50 
                 10 
                 — 
                 1.537*10 −9   
               
               
                   
               
               
                 Note: 
               
               
                 “—” represents no adding. 
               
            
           
         
       
     
     The final volume of each of the foregoing tubes was 100 μl, and the tubes were incubated at 37° C. for 30 min, and finally quenched in ice water. The content of each tube was filtered under negative pressure by using GF/C glass fiber filter paper on a Millipore sample collector. The filter paper was washed with 4 ml of 50 mM Tris-HCl (pH 7.4) for three times, and dried. It was then placed in a 0.5 ml of Eppendorf tube, and 0.5 ml of lipophilic scintillation cocktail was added. Radioactive intensity was measured by using Perkin Elmer PRI-CARB 2910 liquid scintillation counter, and the inhibition rate was calculated. The experiment was repeated for more than three times in triplicate. 
       Inhibition rate=(total binding tube dpm−sample tube dpm)/(total binding tube dpm−nonspecific binding tube dpm)×100%.
 
     3.3 Measurement of Protein Concentration by Using a BCA Protein Assay Kit 
     A 10 μl of protein standard (BSA standard protein) was diluted to a final concentration of 0.5 mg/ml. 0, 1, 2, 4, 8, 12, 16, and 20 μl of BSA standard protein and test samples (i.e., the foregoing total binding tube, the nonspecific binding tubes, and the sample tubes) were respectively added to a 96-well plate, and were added up to 20 μl with the diluted BSA standard protein. 200 μl of BCA working solution was added to each well, and incubated at 37° C. for 30 min. The absorbance at a wavelength of 562 nm was measured with a plate reader, and the protein concentration of each of the tubes was calculated according to the standard curves. 
     3.4 Statistical Analysis 
     The affinity dissociation constant K i  was calculated with Prism 8.0 software, where the concentration of the labeled ligand added was 2.0 nM for  3 H-DAMGO, 1.1 nM for  3 H-DPDPE, and 1.5 nM for  3 H-U69593. The dissociation constant K d  was 0.93 nM for the μ receptor-ligand complex, 0.77 nM for the δ receptor-ligand complex, and 1.1 nM for the κ receptor-ligand complex. 
     4. Results 
     As shown in  FIG. 13  and Table 14, AK had a high affinity for δ opioid receptor (Ki: 47.71±21.25 μM), and had low affinities for μ and κ opioid receptors. 
     Table 14. Affinity values (Ki) for the binding to opioid receptors in CHO cells stably expressing opioid receptors. Membranes were incubated with varying concentrations of ligands in the presence of 1.9-2.0 nM  3 H-DAMGO, 1.0-1.1 nM  3 H-DPDPE and 1.4-1.5 nM  3 H-U69593. Data are expressed as the means±SEM for at least three independent experiments performed in triplicate. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 14 
               
               
                   
               
               
                   
                 μ binding affinity 
                 δ binding affinity 
                 κ binding affinity 
               
               
                 Compounds 
                 K i  (nM) 
                 K i  (nM) 
                 K i  (nM) 
               
               
                   
               
             
            
               
                 AK 
                 — 
                 4771.2 ± 2125.2 
                 — 
               
               
                 DAMGO 
                 5.01 ± 0.59 
                 — 
                 — 
               
               
                 SNC80 
                 — 
                 10.08 ± 0.16  
                 — 
               
               
                 U50488 
                 — 
                 — 
                 2.19 ± 1.2 
               
               
                   
               
            
           
         
       
     
     5. Conclusion 
     In vitro radioligand-receptor binding assays show that AK has a certain affinity for δ opioid receptor (K i : 47.71±21.25 μM), and has no detectable affinities for μ and κ opioid receptors.