Abstract:
The invention relates to a compound, 1,3-diolein-2-linolein, having a structure of formula (I), which is prepared by a process comprising the preparation of  Coix  seed oil from the  Coix  seed powder; the preliminary separation of 1,3-diolein-2-linolein; and the double-separation of 1,3-diolein-2-linolein. The invention also relates to pharmaceutical preparations of 1,3-diolein-2-linolein, and the use of this compound and pharmaceutical preparations thereof in the treatment of tumors.

Description:
CROSS REFERENCE TO RELATED PATENT APPLICATION 
       [0001]    The present application claims a priority of the Chinese patent application CN201410342454.7 with filing date Jul. 18, 2014, which application is incorporated herein by reference. 
       FIELD OF THE INVENTION 
       [0002]    The present invention relates to fields of organic chemistry and pharmaceuticals, specifically, the present invention relates to a compound, 1,3-diolein-2-linolein, pharmaceutical preparations thereof, the process for the preparation of same and the use thereof in the treatment of tumors. 
       BACKGROUND OF THE INVENTION 
       [0003]      Coix  seeds are dried ripe seeds of  Coix lacryma - jobi  L. var  ma - yuen  (Roman.), Stapf, a genus of plant in the Poaceae family. It is a dampness-eliminating drug and has been used as a medicinal and edible plant for a long time. Modern researches have found that  Coix  seeds have many pharmacological effects, such as analgesic anti-inflammatory, immunomodulatory, anti-ulcer, hypolipidemic and anti-obesity effects. In recent years, researchers, domestic and abroad, have studied the chemical composition of the  Coix  seed by using TLC, HPLC-MS, GC, etc., and found that the primary active ingredients in it includes esters, unsaturated fatty acids, saccharides and lactams. Among them, esters are the first discovered components having anti-tumor activities and the most reported chemical components attracting the most attention. The active ingredients of Kanglaite injection, which has been widely used in Chinese clinical applications, are esters extracted from  Coix  seed. It is shown in Yang Ling&#39;s research (Food Science, 2001, 22(5): 59-62) that the esters extracted from  Coix  seed contain triglycerides from 87.64% to 97.66% in mole percentage, and also monoglycerides, diglycerides and fatty acid esters. Further researches have indicated that triglycerides in  Coix  seed contain up to 8 ingredients in a relative content 3%, such as trilinolein (1), dilinolein-olein (2), palmitin-dilinolein (3), linolein-diolein (4) palmitin-linolein-olein (5), dipalmitin-linolein (6), triolein (7) and palmitin-diolein (8). The total contents of the 8 ingredients are over 90% of triglycerides. The complexity of the ingredients in  Coix  seed oil will inevitably be a great challenge which needs to be faced to in the quality control of the practical production process and in the safety of clinical applications. 
         [0004]    Therefore, the development of a safe, effect and controllable medicine for the treatment of tumors became a focused issue of the invention. In this invention, one of the ingredients in triglycerides in  Coix  seed oil has been separated. A compound, 1,3-diolein-2-linolein, having a structure of formula (I) has been prepared for the first time. There is no report on the use of this compound and pharmaceutical preparations thereof in the treatment of tumors till now. 
       SUMMARY OF THE INVENTION 
       [0005]    The first aspect of the invention is to provide a compound, 1,3-diolein-2-linolein, having a structure of formula (I): 
         [0000]    
       
                 
         
             
             
         
       
     
         [0006]    The compound of formula (I) is prepared by a process comprising steps of:
       (1) preparation of  Coix  seed oil;   (2) preliminary separation of 1,3-diolein-2-linolein; and   (3) double-separation of 1,3-diolein-2-linolein.       
 
         [0010]    The preparation of  Coix  seed oil in step (1) comprises steps of: 
         [0011]    crushing  Coix  seeds having a moisture content ≦10% into 10-300 mesh powder and extracting said powder using a supercritical fluid extraction to afford a crude  Coix  seed oil; adding petroleum ether in an amount of 40-58% by weight of crude  Coix  seed oil and caustically refining this crude  Coix  seed oil with aqueous alkali, q.s.; after layering, adding neutral alumina and/or kaolin to the organic phase; filtering and sterilizing the filtrated oil via dry heat sterilization under vacuum at 150-180° C.; then cooling and filtering the sterilized oil to give  Coix  seed oil. 
         [0012]    Preferably, the supercritical fluid extraction is the supercritical CO 2  extraction with an extraction temperature of 30-60° C., an extraction pressure of 19-24 Mpa, a separation temperature of 30-60° C., a separation pressure of 6-15 Mpa, a carbon dioxide flow rate of 10-5000 L/h, and an extraction time of 1-4 h; 
         [0013]    the aqueous alkali can be a 0.5-3% KOH or NaOH aqueous solution; and 
         [0014]    the step of adding neutral alumina and/or kaolin includes adding 3-8% activated neutral alumina by weight of crude  Coix  seed oil; filtering the mixture and heating the filtrate to 40-50° C.; adding 2-6% activated Kaolin by weight of crude  Coix  seed oil and filtering the mixture; removing solvent from the filtrate under vacuum; and adding 8-12% activated neutral alumina by weight of crude  Coix  seed oil, then filtering the mixture. 
         [0015]    More preferably, the preparation of  Coix  seed oil in step (1) especially comprises steps of: 
         [0016]    crushing  Coix  seeds having a moisture content ≦10% into 10-300 mesh powder and extracting the powder in a supercritical CO 2  extraction system wherein the CO 2  preheater, extractor and separation column are heated to reach an extraction temperature of 30-60° C. and a separation temperature of 30-60° C., then pressurized to reach an extraction pressure of 19-24 Mpa and a separation pressure of 6-15 Mpa; the CO 2 , post the extraction of  coix  seed oil, enters into one or more resolution column(s), as needed, having a resolution temperature of 30-60 and a resolution pressure of 2-6 Mpa. The flow rate of CO 2  in the whole cycle is kept at 10-5000 L/h. The continuous extraction for 1-4 h affords a crude  Coix  seed oil from the lower end of the separation column; and 
         [0017]    adding petroleum ether in an amount of 40-58% by weight of crude  Coix  seed oil to the crude  Coix  seed oil, and caustically refining the mixture with 2% NaOH solution, q.s.; after layering, collecting the organic phase and washing it with purified water; demulsifying the washed organic phase with acetone (q.s.); discarding the lower layer, adding activated neutral alumina in an amount of 3-8% by weight of crude  Coix  seed oil, and filtering the mixture; heating the filtrate up to 40-50 and adding activated kaolin in an amount of 2-6% by weight of crude  Coix  seed oil, and filtering the mixture; removing solvent from the filtrate under vacuum; and adding activated neutral alumina in an amount of 8-12% by weight of crude  Coix  seed oil, then filtering the mixture to give an oil; sterilizing the filtrated oil for 1-3 h via dry heat sterilization under vacuum at 150-180° C.; then cooling and filtering the sterilized oil through a ≦0.2 μm microporus membrane to give  Coix  seed oil. 
         [0018]    The preliminary separation of 1,3-diolein-2-linolein in step (2) is especially as follows: 
         [0019]    Instrument and method: using a preparative high performance liquid chromatograph in combination with an evaporative light-scattering detector, wherein the preparative chromatographic column is Agilent Zorbax SB-C18 (250 mm×21.2 mm, 7 μm); mobile phase A is acetonitrile and mobile phase B is tetrahydrofuran/acetonitrile (1:1); gradient conditions are mobile phase B: 0-27 min: 50%-60%, 27-35 min: 60-90%, 35-45 min: 90-100%; the flow rate is 18 mL/min; the injection volume is 200-3000; and the collection response is set at the peak level min=30 mV; 
         [0020]    Preparation of sample: 0.1 g/ml  Coix  seed oil solution, prepared with mobile phase B; and 
         [0021]    Collection of fraction: collecting the fraction of the chromatographic peak at the retention time of about 16 min and removing the solvent at a low temperature and a reducing pressure to obtain a preliminarily prepared sample. 
         [0022]    The double-separation of 1,3-diolein-2-linolein in step (3) is especially as follows: 
         [0023]    First separation: dissolving the sample obtained in the preliminary separation in n-hexane, q.s., with an ultrasonic wave to obtain a solution; injecting a sample (60 μl) of this solution in an analytical high performance liquid chromatograph (Column: Venusil XBP C18 (L) (10*250 mm, 5 μm, 150 Å)); eluting the sample with a mobile phase: acetonitrile/tetrahydrofuran (75:25) at a flow rate of 4 mL/min; detecting fractions with a DAD detector at 205 nm; collecting the fraction of chromatographic peak at the retention time of about 19 min and concentrating this fraction with nitrogen flow blowing; and 
         [0024]    second separation: dissolving the sample obtained in the first separation in n-hexane, q.s., with an ultrasonic wave, to obtain a solution; injecting a sample (60 μl) of this solution in a high performance liquid chromatograph (column: Venusil CA (10*250 mm, 5 μm, 1000 Å)); eluting the sample with a mobile phase: n-hexane/ethanol (95:5) at a flow rate of 4 mL/min; detecting fractions with an ultraviolet detector at 205 nm; collecting the fraction of the chromatographic peak at retention time of about 2.5-5 min and concentrating the fraction with a nitrogen flow; and cryopreserving the final product at a low temperature. 
         [0025]    The structure of the compound of the invention has been identified by its physiochemical properties, mass spectra, NMR, UV spectroscopy and IR spectroscopy. 
         [0026]    1. Experimental Apparatus 
         [0027]    HPLC: Agilent 1100 (DAD, ELSD), Agilent, USA; 
         [0028]    NMR spectrometer: BRUKER AVANCE 500 MHz, Bruker, USA; 
         [0029]    Mass spectrometer: Macromass GCT time of flight mass spectrometer (TOF), Waters, USA; 
         [0030]    Finnigan TRACE DSQ-MS, Thermo Finnigan, USA; 
         [0031]    HPLC-MS: Finnigan LCQ advantage MAX, Thermo Finnigan, USA; 
         [0032]    IR spectrometer: Nicolet 6700, Thermo Finnigan, USA; 
         [0033]    Polarimeter: Jasco P-1030, JASCO, Japan. 
         [0034]    2. Experimental Methods 
         [0035]    HPLC: 10 μl Solution of 20 mg pure product in 1 ml dichloromethane was determined in Agilent 1100 HPLC (Column: Agilent Zorbax SB-C18 (250 mm×4.6 mm, i.d. 5 μm); Mobile phase: dichloromethane/acetonitrile (35:65, v/v)). 
         [0036]    LC-MS: The sample was determined in APCI positive ion mode in Finnigan LCQ advantage MAX in accordance of LC conditions. 
         [0037]    IR: Pure product coated on the crystal surface of potassium bromide was determined in IR spectrometer (Nicolet 6700). 
         [0038]    HR-EI and Qu.-EI: 5 μg pure product coated in the cell was determined in Macromass GCT time of flight mass spectrometer (TOF) in conditions of Source Temp 200° C., Sample Temp 400° C. and DIRECT 70 eV, and in Finnigan TRACE DSQ-MS in conditions of Source Temp 250° C., Sample Temp 250° C. and DIRECT 70 eV. 
         [0039]    NMR: To 30-40 mg Pure product in a NMR tube was added 0.5 ml deuterated chloroform (CDCl 3 ) and injected highly pure argon. The NMR tube was closed meltingly in Argon shield with an alcohol blast burner, then  1 H-NMR,  13 C-NMR, HSQC and HMBC were determined in a BRUKER 500M NMR spectrometer. 
         [0040]    Specific rotation: Pure product, q.s., was dissolved in 1.50 ml chloroform, shaken up, and determined in JASCO P-1030. 
         [0041]    3. Analysis of Spectrograms 
         [0042]    The compound of formula (I) is a faint yellow oily liquid at room temperature. 
         [0043]    HR-EI-MS: m/z=882.7678 (Calcd.=882.7676, C 57 H 102 O 6 ), Degree of unsaturation=7 ( FIG. 1 ). 
         [0044]    APCI-MS: 883.4, [M+H] +  signal, and fragment signals [M+H-linoleic acid] + =603.3 and [M+H-oleic acid] + =601.2 simultaneously appeared. It is deduced that there are two acyl-chains, oleic acid and linoleic acid ( FIG. 2 ). 
         [0045]    IR (KBr flim): 1747, 1163, 1097; 2925, 2855, 723; 3007, 1655 cm −1  ( FIG. 3 ). 
         [0046]      1 H-NMR: δ5.27 (m, 1H), 4.30 (dd, J=4.29 Hz, J=11.9 Hz, 2H), 4.15 (dd, J=5.9 Hz, J=11.9 Hz, 2H), 2.33 (m, 6H), 2.77 (t, 2H), 5.36 (m, 8H), 0.88 (m, 9H) ( FIG. 4 ). 
         [0047]      13 C-NMR: δ 14.10, 14.14, 22.60, 22.71, 24.86, 24.90, 25.65, 27.2, 27.22, 27.24, 29.07, 29.79, 31.55, 31.93, 34.05, 34.21, 62.12, 68.89, 127.90, 128.10, 129.73, 130.00, 130.03, 130.24, 172.87, 173.29 ( FIG. 5 ). 
         [0048]    The second aspect of the invention is to provide a pharmaceutical composition, comprising a therapeutically effective amount of at least one active ingredient including the compound of formula (I) and one or more pharmaceutically acceptable carriers. 
         [0049]    Preferably, the pharmaceutical composition is a pharmaceutical preparation in a form of unit dose, comprising 0.1 mg-1000 mg of compound of formula (I) and 0.01%-99.99% pharmaceutically acceptable carriers by weight of the pharmaceutical preparation. 
         [0050]    The pharmaceutical composition of the invention can be any form of medicine, such as common tables, sugar coating tables, film coating tables, enteric coating tables, capsules including hard capsules and soft capsules, oral liquids, buccal preparations, granules, electuaries, pills, powders, mastics, sublimed preparations, suspensions, pulvis, solutions, injections, suppositories, ointments, plasters, creams, sprays, drops and patches. 
         [0051]    The pharmaceutically acceptable carriers are selected from pharmaceutically conventional dilutions, excipients, fillers, emulsifiers, binders, lubricants, absorption accelerators, surfactants, disintegrants, lubricants and antioxidants, if necessary, flavoring agents, sweeteners, preservative and/or coloring agents. 
         [0052]    Especially, the pharmaceutically acceptable carriers are selected from one or more in the group consisting of: mannitol, sorbitol, sodium metabisulfite, sodium bisulfite, sodium thiosulfate, cysteine hydrochloride, thioglycolic acid, methionine, soybean lecithin, vitamin C, vitamin E, EDTA disodium, EDTA calcium sodium, a monovalent alkali metal carbonate, acetate, phosphate or its aqueous solution, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acids, sodium chloride, potassium chloride, sodium lacetate, ethylparaben solution, benzoic acid, potassium sorbate, chlorhexidine acetate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, and silicic derivatives, cellulose and its derivatives, alginates, gelatin, polyvinyl pyrrolidone, glycerin, Tween 80, agar-agar, calcium carbonate, calcium bicarbonate, surfactant, polyethylene glycol, cyclodextrin, β-cyclodextrin, phospholipid material, kaolin, talc, and calcium stearate or magnesium stearate. 
         [0053]    Preferably, the pharmaceutical preparation of the invention can be an oral solid preparation, an oral liquid preparation, or an injection. 
         [0054]    Preferably, the oral solid preparation is selected from any one of capsules, tablets, dripping pills, granules and concentrated pills. 
         [0055]    Preferably, the oral liquid preparation is selected from any one of aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, and a dry product that can be reconstructed by water or other suitable carrier prior to use. 
         [0056]    Preferably, the injection is selected from any one of injection, lyophilized powder and aqueous injection. 
         [0057]    Preferably, the pharmaceutical preparation of the invention is an injection comprising the following components: 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 1,3-diolein-2-linolein 
                 100-300 
                 g 
               
               
                   
                 Soybean lecithin for Injection or 
                 10-40 
                 g 
               
               
                   
                 soybean lecithin acceptable for injection 
               
               
                   
                 Glycerin for Injection or 
                 15-50 
                 g 
               
               
                   
                 glycerin acceptable for injection 
               
               
                   
                 Water for injection adds to 
                 1000 
                 mL. 
               
               
                   
                   
               
             
          
         
       
     
         [0058]    This injection is prepared by a method comprising steps of: 
         [0059]    adding appropriate amount of water for injection to a formulated amount of soybean lecithin for injection or soybean lecithin acceptable for injection; dispersing the mixture with a high shear dispersing emulsifier to give a dispersion without bulk or granular solid; adding a formulated amount of glycerin for injection or glycerin acceptable for injection; then adding water for injection to a specified amount, and stirring the mixture to give a water phase; 
         [0060]    weighing a formulated amount of 1,3-diolein-2-linolein; heating the weighed glyceride and the water phase separately to 60-70° C., then mixing them and emulsifying the mixture in a high pressure homogenizer, wherein the low pressure is 5-12 MPa and the high pressure is 25-50 MPa; repeating the cycle of homogenization for 3-6 times until the amount of particles below 2 μm is no less than 95% and particles above 5 μm are undetectable; if necessary, using NaOH or HCl to adjust pH to 4.8 to 8.5, preferably 6.8 to 7.0, most preferably 6.8; and 
         [0061]    filtering the resulting homogeneous emulsion by nitrogen pressure through a ≦3 μm microporus filter; filling the emulsion with nitrogen, sterilizing and cooling the emulsion to afford the injection of the invention. 
         [0062]    Preferably, the pharmaceutical preparation of the invention is a capsule comprising the following components: 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 1,3-diolein-2-linolein 
                 200-800 
                 g 
               
               
                   
                 Antioxidant(s) and/or emulsifier(s) 
                 0.20-0.60 
                 g 
               
               
                   
                 To give 
                 1000 
                 capsules. 
               
               
                   
                   
               
             
          
         
       
     
         [0063]    The above capsule is prepared by a method comprising steps of: 
         [0064]    preparing a glue solution: weighing gelatin, purified water, glycerin and a preservative at a weight ratio of 1:0.6-1.2:0.3-0.8:0.0001-0.01; adding glycerin, purified water and preservative sequentially into a glue melting tank; heating to 70-90; then adding gelatin and constantly stirring the mixture under vacuum until the gelatin is completely dissolved; filtering the glue solution and storing the filtered glue solution at 56-62° C. for use; 
         [0065]    preparing a drug liquid: adding formulated amount of 1,3-diolein-2-linolein, antioxidant(s) and/or emulsifier(s) into an dosing tank, and stirring the mixture constantly until being homogeneously mixed; and 
         [0066]    pressing capsules: choosing proper pellet dies according to the capsule size; pressing capsules at a temperature of 15-30 and a relative humidity of less than 35%; drying the pressed and shaped capsules; after removing capsules of abnormal size, washing the normal capsules with 95% medicinal ethanol, and drying them continuously to a moisture content of less than 12%; visually inspecting and removing unqualified capsules; finally printing and packaging to afford capsules. 
         [0067]    In the above capsule, the preservative is selected from any one of 10% ethylparaben solution, benzoic acid, potassium sorbate and chlorhexidine acetate; the antioxidant is vitamin E; and the emulsifier is Tween 80. 
         [0068]    It is shown in pharmacological experiments that the compound, 1,3-diolein-2-linolein, and pharmaceutical preparations thereof inhibit 7 human tumor cell lines in varying degrees, thus can be used as therapeutical drugs of neoplastic diseases. 
         [0069]    Therefore, another aspect of the invention is to provide a use of the compound of formula (I) and pharmaceutical preparations thereof in the treatment of a tumor selected from a group consisting of pancreatic cancer, breast cancer, ovarian cancer, liver cancer, lung cancer, gastric cancer and colon cancer. 
         [0070]    The following experimental data are used to illustrate beneficial anti-tumor effects of the compound of formula (I) and the pharmaceutical preparations thereof. 
       I. Inhibition of the Compound of Formula (I) and Preparations Thereof on 7 Human  Tumor Cell Lines in MTT Method In Vitro 
       [0071]    A. Experimental Materials and the Preparation Thereof:
       (1) Cell lines: PANC-1 (human pancreatic cancer cells), SKOV3 (human ovarian cancer cells), MCF-7 (human breast cancer cells), SMMC-7721 (human liver cancer cells), A549 (human lung cancer cells), BGC-823 (human gastric cancer cells), COLO205 (human colonic cancer cells), storaged and passaged maintainably in Research and Evaluation Center for Pharmacology, Shanghai Institute of Pharmaceutical Industry;   (2) DMEM complete medium supplied with 10% newborn calf serum (GIBCO BRL), 1% of penicillin (100 U/mL)+streptomycin (100 μg/mL);   (3) 0.25% trypsin solution, purchased from Invitrogen Corp. and storaged at −20;   (4) Phosphate buffer (PBS): NaCl 8 g, KCl 0.2 g, Na 2 HPO 4  1.15 g and KH 2 PO 4  0.2 g, dissolved in 1 L double-distilled water and autoclaved at 121 for 20 min, then storaged at 4;   (5) MTT (AMRESCO) solution: 5 mg/ml in PBS;   (6) Formazan crystal dissolving solution: SDS 10 g, isobutanol 5 ml and concentrated hydrochloric acid 0.1 ml, dissolved in 100 ml of deionized double distilled water.       
 
         [0078]    B. Experimental Method 
         [0079]    The inhibition effects of the compound of formula (I) on the above-mentioned cell lines were detected by using MTT method. The specific procedures were as follows: 
         [0080]    (1) Cell culture: (a) Storaged cells were taken out from the liquid nitrogen, thawed quickly in a 37 water bath, then aseptically transferred into 6 ml of cellular medium in a 10 ml centrifugal tube, centrifuged at 1000 rpm for 5 min. The supernatant was discarded, then the precipitated cells were re-suspended in 5-6 ml cellular media by pipetting and transferred into a flask in a 37 incubator for cell culture; (b) Next day, the flask was taken out from the incubator and the used medium was discarded, then the cells were incubated in 5-6 ml fresh medium in the 37 incubator; (c) On the third day, the flask was taken out from the incubator and the used medium was discarded, then 2-3 ml of PBS (pH7.4) was added into the flask with rocking for cleaning it and the used PBS was discarded. Such a cellular cleaning step was repeated once again. 3-5 drops of 0.25% trypsin solution were added into the flask with sloshing, thus well-distributed in it. The flask was capped and placed in a 37 incubator for about 3 min, and the separation of cells from the flask wall was observed under the microscope. 2 ml of cellular medium was added and cells were separated completely from the flask wall by pipetting, then the cell suspension was transferred into 2 separate clean flasks, each containing 5-6 ml medium. The cell suspension was well-distributed by pipetting, then the flask was placed in a 37 incubator. (d) Step(c) was repeated every other day. In the whole cultivation process, adherent cells were not allowed to grow too dense and suspension cells were always maintained at a logarithmic growth stage. 
         [0081]    (2) Preparation of the sample and the control: A proper amount of sample of the compound of formula (I) was dissolved in DMSO to obtain a solution in a concentration of 10 mg/ml. This solution was diluted in a gradient dilution with PBS to obtain a set of sample solutions in the concentration of 10 mg/ml, 5000 μg/ml, 2500 μg/ml, 1250 μg/ml, 625 μg/ml and 312.5 μg/ml, respectively. 
         [0082]    (3) Each diluted sample solution was added into duplicated wells of a 96 well flat-bottom microplate (10 μl/well). The correspondingly diluted DMSO solutions, as controls, were added into the wells of the microplate. 
         [0083]    (4) Cells in a logarithmic growth stage were trypsinized and washed, then re-suspended in the medium containing 10% calf serum. The number of living cells was counted in Trypan blue dye exclusion method and cell suspensions were adjusted into a density of 2×10 5  cell/ml. 
         [0084]    (5) The cell-contained 96 well flat-bottom microplate was placed in a 37 incubator and cells were incubated under 5% CO 2  for 48 h. 
         [0085]    (6) 20 it of 5 mg/ml MTT solution was added into each well and cells were incubated continuously in the incubator for 3-4 h. 
         [0086]    (7) 100 it of crystal dissolving solution was added into each well and cells were incubated continuously in the incubator overnight, so as to dissolve the resulted formazan crystals sufficiently. Then, the absorbance value was measured at 570 nm for each well. 
         [0087]    (8) Based on absorbance values, inhibition rates on the cell growth were calculated for sample groups of various concentrations. The calculation formula was as follows: 
         [0000]      (1−mean absorbance of experimental wells/mean absorbance of control wells)×100%
 
         [0088]    C. Experimental Results 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Inhibition rates of the compound of formula (I) in various 
               
               
                 concentrations on the cell growth in 7 cell lines (%) 
               
             
          
           
               
                   
                 Concentration of the compound 
               
             
          
           
               
                   
                 1000 
                 500 
                 250 
                 125 
                 62.5 
                 31.25 
               
               
                 Cell line 
                 μg/ml 
                 μg/ml 
                 μg/ml 
                 μg/ml 
                 μg/ml 
                 μg/ml 
               
               
                   
               
             
          
           
               
                 PANC-1 
                 75.00 
                 48.15 
                 25.89 
                 2.01 
                 0.80 
                 / 
               
               
                 SKOV3 
                 99.35 
                 93.83 
                 79.37 
                 76.76 
                 33.97 
                 9.97 
               
               
                 MCF-7 
                 99.73 
                 68.80 
                 67.09 
                 62.26 
                 31.48 
                 13.30 
               
               
                 SMMC-7721 
                 98.82 
                 80.05 
                 62.30 
                 38.52 
                 13.36 
                 8.81 
               
               
                 A549 
                 98.23 
                 67.03 
                 50.48 
                 46.39 
                 25.57 
                 5.16 
               
               
                 BGC-823 
                 98.52 
                 84.96 
                 76.15 
                 78.96 
                 63.83 
                 8.31 
               
               
                 COLO205 
                 93.77 
                 82.30 
                 43.93 
                 12.62 
                 10.66 
                 8.95 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 IC 50  values of the compound of formula (I) in 7 cell lines in vitro (μg/ml) 
               
             
          
           
               
                   
                 Cell line 
               
             
          
           
               
                 Sample 
                 PANC-1 
                 SKOV3 
                 MCF-7 
                 SMMC-7721 
                 A549 
                 BGC-823 
                 COLO205 
               
               
                   
               
             
          
           
               
                 Positive control (Taxol) 
                 0.44 
                 0.38 
                 0.34 
                 0.12 
                 0.49 
                 0.24 
                 0.75 
               
               
                 compound of formula (I) 
                 508.9 
                 92.61 
                 105.7 
                 150.2 
                 155.6 
                 84.98 
                 209.3 
               
               
                   
               
             
          
         
       
     
         [0089]    D. Conclusion 
         [0090]    It is shown in the results that the compound of formula (I) and preparations thereof in various concentrations have obvious inhibition on 7 human tumor cell lines. They can be used as antitumor drugs. Since the active ingredient in the pharmaceutical preparations has a simple construct and definite content, the clinical medication is safer. The less dose and controllable process quality of the compound and preparations thereof have avoided defects of more side effects resulting from the complex ingredients when  Coix  seed oil extracted from  Coix  seeds is administered directly. 
     
    
     
       DESCRIPTION OF FIGURES 
         [0091]      FIG. 1  is an EI mass spectrum of 1,3-diolein-2-linolein. 
           [0092]      FIG. 2  is an APCI mass spectrum of 1,3-diolein-2-linolein. 
           [0093]      FIG. 3  is an IR spectrum of 1,3-diolein-2-linolein. 
           [0094]      FIG. 4  is a  1 H-NMR spectrum of 1,3-diolein-2-linolein. 
           [0095]      FIG. 5  is a  13 C-NMR spectrum of 1,3-diolein-2-linolein. 
           [0096]      FIG. 6  is a HSQC spectrum of 1,3-diolein-2-linolein. 
           [0097]      FIG. 7  is a HMBC spectrum of 1,3-diolein-2-linolein. 
       
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
       [0098]    The following examples further illustrate the invention, but are not construed as a limitation of the invention. 
       Example 1 
     Preparation of  Coix  Seed Oil 
       [0099]    1000 g  Coix  seeds having a moisture ≦10% were crushed into 10 mesh powder and extracted in a supercritical CO 2  extraction system, wherein the extraction temperature was 40° C., the extraction pressure was 20 Mpa, the separation temperature was 30, the separation pressure was 6 Mpa, and the flow rate of liquid CO 2  was 50 L/h. The  Coix  seed powder was continuously extracted for 2 h to give a crude  Coix  seed oil. 
         [0100]    To the crude  Coix  seed oil obtained in the supercritical CO 2  extraction were added 40% petroleum ether by weight of the crude  Coix  seed oil and 0.5% NaOH aqueous solution to caustically refine the crude oil. After layering, to the organic phase was added 3% activated neutral alumina by weight of the crude oil, and the mixture was filtered. The filtrate was heated to 40, and 2% activated Kaolin by weight of the crude oil was added. The mixture was filtered and the filtrate was concentrated under a reduced pressure to remove the solvent. 8% activated neutral alumina by weight of the crude oil was added. The mixture was filtered, and the filtrated oil underwent dry heat sterilization by vacuum at 180. After cooling, the oil was filtered to give the  Coix  seed oil. 
       Example 2 
     Preparation of  Coix  Seed Oil 
       [0101]    1000 g  Coix  seeds having a moisture ≦10% were crushed into 100 mesh powder and extracted in a supercritical CO 2  extraction system, wherein the extraction temperature was 50° C., the extraction pressure was 22 Mpa, the separation temperature was 40 and the separation pressure was 8 Mpa, and the flow rate of liquid CO 2  was 500 L/h. The  Coix  seed powder was continuously extracted for 3 h to give a crude  Coix  seed oil. 
         [0102]    To the crude  Coix  seed oil obtained in the supercritical CO 2  extraction were added 46% petroleum ether by weight of the crude oil and 1% NaOH aqueous solution to caustically refine the crude oil. After layering, to the organic phase was added 5% activated neutral alumina by weight of the crude oil, and the mixture was filtered. The filtrate was heated to 50, then added 4% activated Kaolin by weight of the crude oil and the mixture was filtered. The filtrate was concentrated under a reduced pressure to remove the solvent. Then 10% activated neutral alumina by weight of the crude oil was added. The mixture was filtered, and the filtrated oil underwent dry heat sterilization by vacuum at 150. After cooling, the oil was filtered to give the  Coix  seed oil. 
       Example 3 
     Preparation of  Coix  Seed Oil 
       [0103]    1000 g  Coix  seeds having a moisture ≦10% were crushed into 200 mesh powder and extracted in a supercritical CO 2  extraction system, wherein the extraction temperature was 60° C., the extraction pressure was 24 Mpa, the separation temperature was 50, the separation pressure was 10 Mpa, and the flow rate of liquid CO 2  was 1500 L/h. The  Coix  seed powder was continuously extracted for 1.5 h to give a crude  Coix  seed oil. 
         [0104]    To the crude  Coix  seed oil obtained in the supercritical CO 2  extraction were added 50% petroleum ether by weight of the crude oil and 2% NaOH aqueous solution to caustically refine the crude oil. After layering, to the organic phase was added 6% activated neutral alumina by weight of the crude oil, and the mixture was filtered. The filtrate was heated to 45 and 5% activated Kaolin by weight of the crude oil was added. Then the mixture was filtered and the filtrate was concentrated under a reduced pressure to remove the solvent. 10% Activated neutral alumina by weight of the crude oil was added. The mixture was filtered and the filtrated oil underwent dry heat sterilization by vacuum at 160. After cooling, the oil was filtered to give the  Coix  seed oil. 
       Example 4 
     Preparation of  Coix  Seed Oil 
       [0105]    1000 g  Coix  seeds having a moisture ≦10% were crushed into 100 mesh powder and extracted in a supercritical CO 2  extraction system.  Coix  seed powder was put in an extractor. The CO 2  preheater, extractor and separation column were heated, so that the extraction temperature was 60, the extraction pressure was 24 Mpa, the separation temperature was 60 and the separation pressure was 15 Mpa. The supercritical CO 2  gas with the extracted oil was introduced, as needed, into 5 resolution columns wherein the resolution temperature was 50 and the resolution pressure was 6 Mpa. The flow rate of CO 2  in the whole cycle was kept at 3000 L/h. The continuous extraction for 4 h afforded a crude  Coix  seed oil from the lower end of the separation column. 
         [0106]    To the crude  Coix  seed oil obtained in the supercritical CO 2  extraction were added 58% petroleum ether by weight of the crude oil and 2% NaOH aqueous solution, q.s., to caustically refine the crude oil. After layering, the organic phase was washed with purified water and the washed organic phase was demulsified with acetone, q.s. To the upper oil layer was added 8% activated neutral alumina by weight of the crude oil, and the mixture was filtered. The filtrate was heated to 46 and 6% activated Kaolin by weight of the crude oil was added. Then the mixture was filtered, and the filtrate was concentrated under a reduced pressure to remove the solvent. 12% Activated neutral alumina by weight of the crude oil was added. The mixture was filtered and the filtrated oil underwent dry heat sterilization by vacuum at 170 for 2 h. After cooling, the oil was filtered through a 0.2 μm millipore filter to give the  Coix  seed oil. 
       Example 5 
     Preparation of  Coix  Seed Oil 
       [0107]    1000 g  Coix  seeds having a moisture 10% were crushed into 300 mesh powder and extracted in a supercritical CO 2  extraction system.  Coix  seed powder was put in an extractor. The CO 2  preheater, extractor and separation column were heated, so that the extraction temperature was 50, the extraction pressure was 20 Mpa, the separation temperature was 45 and the separation pressure was 12 Mpa. The supercritical CO 2 , post extraction of  coix  seed oil, was introduced, as needed, into 3 resolution columns wherein the resolution temperature was 45 and the resolution pressure was 5 Mpa. The flow rate of CO 2  in the whole cycle was kept at 2000 L/h. The continuous extraction for 3 h affords a crude  Coix  seed oil from the lower end of the separation column. 
         [0108]    To the crude  Coix  seed oil obtained in the supercritical CO 2  extraction were added 48% petroleum ether by weight of the crude oil and 2% NaOH aqueous solution, q.s., to caustically refine the crude oil. After layering, the organic phase was washed with purified water and the washed organic phase was demulsified with acetone, q.s. To the upper oil layer was added 6% activated neutral alumina by weight of the crude oil, and the mixture was filtered. The filtrate was heated to 42 and 4% activated Kaolin by weight of the crude oil was added. Then the mixture was filtered, and the filtrate was concentrated under a reduced pressure to remove the solvent. 9% Activated neutral alumina by weight of the crude oil was added. The mixture was filtered and the filtrated oil underwent dry heat sterilization by vacuum at 165 for 3 h. After cooling, the oil was filtered through a ≦0.2 μm millipore filter to give the  Coix  seed oil. 
       Example 6 
     Preliminary Separation of 1,3-Diolein-2-Linolein 
       [0109]    Instrument and method: A preparative Gilson high performance liquid chromatograph was used in combination with an evaporative light-scattering detector, wherein the preparative chromatographic column was Agilent Zorbax SB-C18 (250 mm×21.2 mm, 7 μm); mobile phase A was acetonitrile and mobile phase B was tetrahydrofuran/acetonitrile (1:1); gradient conditions were mobile phase B: 0-27 min: 50%-60%, 27-35 min: 60-90%, 35-45 min: 90-100%; the flow rate was 18 mL/min; the injection volume was 200-300 μl; and the collection response was set at the peak level min=30 mV. 
         [0110]    Preparation of samples: 0.1 g/ml  Coix  seed oil solution was prepared with mobile phase B. 
         [0111]    Collection of fractions: The fraction of the chromatographic peak at retention time of about 16.0 min was collected and the solvent was removed at a low temperature and a reducing pressure to obtain a crude 1,3-diolein-2-linolein. 
       Example 7 
     Double-Separation of 1,3-diolein-2-linolein 
       [0112]    First separation: The crude 1,3-diolein-2-linolein obtained in Example 6 was dissolved in n-hexane, q.s., with an ultrasonic wave, to obtain a solution; a sample (60 μl) of this solution was analyzed in an analytical Agilent 1100 high performance liquid chromatograph (column: Venusil XBP C18 (L) (10*250 mm, 5 μm, 150 Å)) with a mobile phase, acetonitrile/tetrahydrofuran (75:25), at a flow rate of 4 mL/min. Fractions were detected with a DAD detector at 205 nm. The fraction of the chromatographic peak at the retention time of about 19 min was collected and concentrated with nitrogen flow blowing. 
         [0113]    Second separation: The product obtained in the first separation was dissolved in n-hexane, q.s., with an ultrasonic wave, to obtain a solution. A sample (60 μl) of this solution was injected in a high performance liquid chromatograph (column: Venusil CA (10*250 mm, 5 μm, 1000 Å)) and enluted with a mobile phase, n-hexane/ethanol (95:5), at a flow rate of 4 mL/min. Fractions were detected with an ultraviolet detector at 205 nm and the chromatographic peak at retention time of about 2.5-5 min was collected. The fraction was concentrated with nitrogen flow blowing, and the final product, 1,3-diolein-2-linolein, was cryopreserved at a low temperature. 
       Example 8 
     Preparation of the Injection of 1,3-diolein-2-linolein 
       [0114]    Formulation: 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 1,3-diolein-2-linolein 
                 200 
                 g 
               
               
                   
                 Soybean lecithin for injection 
                 25 
                 g 
               
               
                   
                 Glycerin acceptable for injection 
                 30 
                 g 
               
               
                   
                 Water for injection adds to 
                 1000 
                 mL 
               
               
                   
                   
               
             
          
         
       
     
         [0115]    Process: 
         [0116]    To a formulated amount of soybean lecithin for injection was added an appropriate amount of water for injection. The mixture was dispersed with a high shear dispersing emulsifier into a dispersion without bulk or granular solid. Formulated amount of glycerin acceptable for injection was added. Then water for injection was added to a specified amount, and the mixture was stirred to give a water phase. 
         [0117]    A formulated amount of 1,3-diolein-2-linolein was weighed. The weighed oil and the water phase prepared above were heated separately to 65, then mixed and emulsified in a high pressure homogenizer, wherein the low pressure was 8 MPa and the high pressure was 30 MPa. The homogenization was repeated for 4 times until the amount of particles below 2 μm was no less than 95% and particles above 5 μm were undetectable. If necessary, NaOH or HCl was used to adjust the pH to 6.8. 
         [0118]    The resulting homogeneous emulsion was filtered by nitrogen pressure through a ≦3 μm microporus filter, then filled under nitrogen, and finally sterilized and cooled to afford the injection. 
       Example 9 
     Preparation of the Capsule of 1,3-diolein-2-linolein 
       [0119]    Formulation: 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 1,3-diolein-2-linolein 
                 500 
                 g 
               
               
                   
                 Vitamin E 
                 0.40 
                 g 
               
               
                   
                 to give 
                 1000 
                 capsules 
               
               
                   
                   
               
             
          
         
       
     
         [0120]    Process: 
         [0121]    Glue formulation: Gelatin, purified water, glycerin and potassium sorbate solution were weighed at a weight ratio of 1:0.9:0.6:0.005. Glycerin, purified water and potassium sorbate solution were sequentially added into a glue melting tank and the mixture was heated to 80. Then gelatin was added and the mixture was constantly stirred under vacuum until the gelatin was completely dissolved. The glue was filtered and stored at 56-62 for use. 
         [0122]    Drug liquid formulation: Formulated amount of 1,3-diolein-2-linolein and Vitamin E were added into an ingredient tank and the mixture was stirred constantly until being thoroughly mixed. 
         [0123]    Capsule pressing: Proper pellet dies were chosen according to the capsule size. Capsules were pressed at a temperature of 15 and a relative humidity of less than 35%, then shaped and dried. After excluding capsules of abnormal size, normal capsules were washed with 95% medicinal ethanol and dried continuously till the moisture content was less than 12%. Unqualified capsules were removed by visual inspection, and final products were printed and packaged. 
       Example 10 
     Preparation of the Injection of 1,3-diolein-2-linolein 
       [0124]    Formulation: 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 1,3-diolein-2-linolein 
                 100 
                 g 
               
               
                   
                 Soybean lecithin for injection 
                 10 
                 g 
               
               
                   
                 Glycerin for injection 
                 15 
                 g 
               
               
                   
                 Water for injection adds to 
                 1000 
                 mL 
               
               
                   
                   
               
             
          
         
       
     
         [0125]    Process: 
         [0126]    To a formulated amount of soybean lecithin for injection was added an appropriate amount of water for injection. The mixture was dispersed with a high shear dispersing emulsifier into a dispersion without bulk or granular solid. Formulated amount of glycerin for injection was added. Then water for injection was added to a specified amount, and the mixture was stirred to give a water phase. 
         [0127]    A formulated amount of 1,3-diolein-2-linolein was weighed. The weighed oil and the water phase prepared above were heated separately to 60, then mixed and emulsified in a high pressure homogenizer, wherein the low pressure was 5 MPa and the high pressure was 35 MPa. The homogenization was repeated for 6 times until the amount of particles below 2 μm was no less than 95% and particles above 5 μm were undetectable. If necessary, NaOH or HCl was used to adjust the pH to 4.8. 
         [0128]    The resulting homogeneous emulsion was filtered by nitrogen pressure through a ≦3 μm microporus filter, then filled under nitrogen, and finally sterilized and cooled to afford the injection. 
       Example 11 
     Preparation of the Injection of 1,3-diolein-2-linolein 
       [0129]    Formulation: 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 1,3-diolein-2-linolein 
                 300 
                 g 
               
               
                   
                 Soybean lecithin acceptable for injection 
                 40 
                 g 
               
               
                   
                 Glycerin acceptable for injection 
                 50 
                 g 
               
               
                   
                 Water for injection adds to 
                 1000 
                 mL 
               
               
                   
                   
               
             
          
         
       
     
         [0130]    Process: 
         [0131]    To a formulated amount of soybean lecithin acceptable for injection was added an appropriate amount of water for injection. The mixture was dispersed with a high shear dispersing emulsifier into a dispersion without bulk or granular solid. Formulated amount of glycerin acceptable for injection was added. Then water for injection was added to a specified amount, and the mixture was stirred to give a water phase. 
         [0132]    A formulated amount of 1,3-diolein-2-linolein was weighed. The weighed oil and the water phase prepared above were heated separately to 70, then mixed and emulsified in a high pressure homogenizer, wherein the low pressure was 11 MPa and the high pressure was 45 MPa. The homogenization was repeated for 3 times until the amount of particles below 2 μm was no less than 95% and particles above 5 μm were undetectable. If necessary, NaOH or HCl was used to adjust the pH to 8.5. 
         [0133]    The resulting homogeneous emulsion was filtered by nitrogen pressure through a ≦3 μm microporus filter, then filled under nitrogen, and finally sterilized and cooled to afford the injection. 
       Example 12 
     Preparation of the Injection of 1,3-diolein-2-linolein 
       [0134]    Formulation: 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 1,3-diolein-2-linolein 
                 150 
                 g 
               
               
                   
                 Soybean lecithin acceptable for injection 
                 35 
                 g 
               
               
                   
                 Glycerin for injection 
                 30 
                 g 
               
               
                   
                 Water for injection adds to 
                 1000 
                 mL 
               
               
                   
                   
               
             
          
         
       
     
         [0135]    Process: 
         [0136]    To a formulated amount of soybean lecithin acceptable for injection was added an appropriate amount of water for injection. The mixture was dispersed with a high shear dispersing emulsifier into a dispersion without bulk or granular solid. Formulated amount of glycerin for injection was added. Then water for injection was added to a specified amount, and the mixture was stirred to give a water phase. 
         [0137]    A formulated amount of 1,3-diolein-2-linolein was weighed. The weighed oil and the water phase prepared above were heated separately to 68, then mixed and emulsified in a high pressure homogenizer, wherein the low pressure was 9 MPa and the high pressure was 40 MPa. The homogenization was repeated for 5 times until the amount of particles below 2 μm was no less than 95% and particles above 5 μm were undetectable. If necessary, NaOH or HCl was used to adjust the pH to 7.2. 
         [0138]    The resulting homogeneous emulsion was filtered by nitrogen pressure through a ≦3 μm microporus filter, then filled under nitrogen, and finally sterilized and cooled to afford the injection. 
       Example 13 
     Preparation of the Capsule of 1,3-diolein-2-linolein 
       [0139]    Formulation: 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 1,3-diolein-2-linolein 
                 200 
                 g 
               
               
                   
                 Vitamin E 
                 0.20 
                 g 
               
               
                   
                 to give 
                 1000 
                 capsules 
               
               
                   
                   
               
             
          
         
       
     
         [0140]    Process: 
         [0141]    Glue formulation: Gelatin, purified water, glycerin and 10% ethylparaben solution were weighed at a weight ratio of 1:1.2:0.8:0.01. Glycerin, purified water and 10% ethylparaben solution were sequentially added into a glue melting tank and heated to 70. Then gelatin was added and the mixture was constantly stirred under vacuum until the gelatin was completely dissolved. The glue was filtered and stored at 60 for use. 
         [0142]    Drug liquid formulation: Formulated amount of 1,3-diolein-2-linolein and Vitamin E were added into an ingredient tank and the mixture was stirred constantly until being thoroughly mixed. 
         [0143]    Capsule pressing: Proper pellet dies were chosen according to the capsule size. Capsules were pressed at a temperature of 30 and a relative humidity of less than 35%, then shaped and dried. After excluding capsules of abnormal size, normal capsules were washed with 95% medicinal ethanol and dried continuously till the moisture content was less than 12%. Unqualified capsules were removed by visual inspection, and final products were printed and packaged. 
       Example 14 
     Preparation of the Capsule of 1,3-diolein-2-linolein 
       [0144]    Formulation: 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 1,3-diolein-2-linolein 
                 800 
                 g 
               
               
                   
                 Tween 80 
                 0.60 
                 g 
               
               
                   
                 to give 
                 1000 
                 capsules 
               
               
                   
                   
               
             
          
         
       
     
         [0145]    Process: 
         [0146]    Glue formulation: Gelatin, purified water, glycerin and benzoic acid were weighed at a weight ratio of 1:1.2:0.8:0.01. Glycerin, purified water and benzoic acid were sequentially added into a glue melting tank and heated to 90. Then gelatin was added and constantly stirred under vacuum until the gelatin was completely dissolved. The glue was filtered and stored at 56 for use. 
         [0147]    Drug liquid formulation: Formulated amount of 1,3-diolein-2-linolein and Tween 80 were added into an ingredient tank and the mixture was stirred constantly until being thoroughly mixed. 
         [0148]    Capsule pressing: Proper pellet dies were chosen according to the capsule size. Capsules were pressed at a temperature of 25 and a relative humidity of less than 35%, then shaped and dried. After excluding capsules of abnormal size, normal capsules were washed with 95% medicinal ethanol and dried continuously till the moisture content was less than 12%. Unqualified capsules were removed by visual inspection, and final products were printed and packaged. 
       Example 15 
     Preparation of the Capsule of 1,3-diolein-2-linolein 
       [0149]    Formulation: 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 1,3-diolein-2-linolein 
                 600 
                 g 
               
               
                   
                 Tween 80 
                 0.30 
                 g 
               
               
                   
                 to give 
                 1000 
                 capsules 
               
               
                   
                   
               
             
          
         
       
     
         [0150]    Process: 
         [0151]    Glue formulation: Gelatin, purified water, glycerin and chlorhexidine acetate were weighed at a weight ratio of 1:1.0:0.5:0.008. Glycerin, purified water and chlorhexidine acetate were sequentially added into a glue melting tank and heated to 85. Then gelatin was added and the mixture was constantly stirred under vacuum until the gelatin was completely dissolved. The glue was filtered and stored at 62 for use. 
         [0152]    Drug liquid formulation: Formulated amount of 1,3-diolein-2-linolein and Tween 80 were added into an ingredient tank and the mixture was stirred constantly until being thoroughly mixed. 
         [0153]    Capsule pressing: Proper pellet dies were chosen according to the capsule size. Capsules were pressed at a temperature of 28 and a relative humidity of less than 35%, then shaped and dried. After excluding capsules of abnormal size, normal capsules were washed with 95% medicinal ethanol and dried continuously till the moisture content was less than 12%. Unqualified capsules were removed by visual inspection, and final products were printed and packaged.