Abstract:
Two crystalline forms of pinocembrin of formula (I): α and β, their preparation and their use for manufacture of pharmaceutical compositions. There exists difference between them in bioavailability. They are used for treating and preventing cerebral ischemic diseases by protective action of neurovascular unit, and enhancing blood drug level in vivo.

Description:
FIELD OF INVENTION 
     The present invention relates to two crystalline forms of the compound pinocembrin, and active pharmaceutical ingredients, pharmaceutical compositions and dosage forms containing the two crystalline forms of pinocembrin, and the use thereof for manufacture of pharmaceutical compositions and treating diseases, and the method for preparing the two crystalline forms of pinocembrin. 
     BACKGROUND OF THE INVENTION 
     Pinocembrin (chemical name: 5,7-dihydroxy-2-phenyl-4-chromanone) is a flavone compound, widely found in the nature. Its chemical structure is as below: 
                                
exiting in 1-isomer, d-isomer, 1-isomer- or d-isomer-enriched mixture, and racemate.
 
     Previous pharmacological experiments showed that pinocembrin had strong bacteriostasis, antivirus, and antifungal activities. For example, honey, a Chinese traditional health care food, is rich in pinocembrin. So eating honey sugar frequently is not only harmless to the teeth, but also can sterilize the oral cavity, for example, relieving oral ulcer and accelerating wound healing. Chinese Patent CN1695608A, titled “Use of pinocembrin for manufacture of pharmaceutical compositions for preventing and treating diseases related to nerve cell injury” [1] , disclosed the use of pinocembrin for manufacture of pharmaceutical compositions for preventing or treating diseases related to cerebral ischemia, sequelae of cerebral ischemia, nerve cell injury and function alteration. 
     DESCRIPTION OF THE INVENTION 
     It was found surprisingly by the inventors that pinocembrin had two different crystalline forms α and β, and the inventors developed the preparations thereof. The inventors also found that there was a significant difference between the two crystalline forms in the uptake by organism, wherein the uptake rate of β crystalline form was greater than that of α crystalline form, for example, the uptake rate of β crystalline form can be 2 times or more greater than that of α crystalline from. The biological activities thereof in medicament therapy are different due to differences in drug blood concentrations. 
     One embodiment of the present invention provides α crystalline form, β crystalline form, or the mixture of α crystalline form and β crystalline form in different proportions, with respect to pinocembrin. Preferably, these crystalline forms or the mixture thereof contains no crystal water or other organic solvents. 
     One embodiment of the present invention provides the method for preparing α crystalline form, β crystalline form, or the mixture of α crystalline form and β crystalline form of pinocembrin in different proportions. 
     One embodiment of the present invention provides a pharmaceutical composition comprising pure α crystalline form, pure β crystalline form, or the mixture of α crystalline form and β crystalline form of pinocembrin in different proportions. The pharmaceutical composition can also comprise one or more pharmaceutically acceptable carriers. There is no limitation on the pharmaceutically acceptable carriers, provided that they are suitable for formulation and would not substantially affect the effect of pinocembrin crystalline form of the present invention. 
     One embodiment of the present invention relates to dosage forms comprising the pinocembrin crystalline forms solid. There are no limitations on specific dosage forms. For example, they can be tablets, capsules, pills, injections, sustained-release preparations, controlled-release preparations and the like. 
     One embodiment of the present invention provides use of the crystalline forms solid of pinocembrin, including α crystalline form, β crystalline form or the mixture of α crystalline form and β crystalline from in different proporations, for bringing about difference of drug uptake in treatment. 
     One embodiment of the present invention relates to the use of α crystalline form, β crystalline form, or the mixture of α crystalline form and β crystalline form of pinocembrin in different proportions in the manufacture of a medicament for treating diseases related to cerebral ischemia or a medicament for preventing diseases related to cerebral ischemia by protecting neurovascular unit function. 
     One embodiment of the present invention provides the use of pinocembrin for protecting neurovascular unit function in the treatment of diseases related to cerebral ischemia, and for improving the blood concentration in an organism due to the crystal form effect. 
     Morphologic Features of a Sample of α Crystalline Form of Pinocembrin According to an Embodiment: 
     For a sample of α crystalline form of pinocembrin obtained according to an embodiment of the present invention, when analyzed by X-ray single crystal diffraction, it showed monoclinic crystal system symmetry, the space group was P2 1 /c, and the crystal cell parameter values were as follows: a=5.189 Å, b=24.149 Å, c=10.472 Å, α=90°, β=102.31° and γ=90°. 
       FIG. 1  is an illustration showing the relative configuration of the molecule,  FIG. 2  is an illustration showing the stereo-structure projection of the molecule,  FIG. 3  is an illustration showing the unit cell stacking of the molecule along the a axis. Table 1 shows atomic coordinate parameters and equivalent temperature factors. Table 2 shows bond length values of bonding atoms. Table 3 shows bond angle values of bonding atoms. As some carbon atoms of B ring adopt a disordered orientation state, the four atoms of C 2′ , C 3′ , C 5′  and C 6′  occupy two positions, with an occupancy rate of 0.5 respectively. 
     
       
                 
         
             
             
         
      
     
     Formula 1 relative molecule configuration of α crystalline form of pinocembrin 
     
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Atomic coordinate parameters (relative coordinate) of a  
               
               
                 sample of α crystalline form of pinocembrin  
               
             
          
           
               
                   
                   
                   
                   
                 tem-  
                   
               
               
                   
                   
                   
                   
                 per- 
                   
               
               
                   
                   
                   
                   
                 ature 
                 occu- 
               
               
                   
                   
                   
                   
                 fac- 
                 pan- 
               
               
                   
                 x 
                 y 
                 z 
                 tors 
                 cy 
               
               
                   
               
             
          
           
               
                 O 1 
                 −.2023(8) 
                 −.6574(2) 
                 −.7376(4) 
                 3.4(2) 
                 1.0 
               
               
                 O 2 
                 −.4261(9) 
                 −.8310(2) 
                 −.5601(4) 
                 4.3(2) 
                 1.0 
               
               
                 O 3 
                 −.8660(9) 
                 −.6732(2) 
                 −.3557(4) 
                 4.2(2) 
                 1.0 
               
               
                 O 4 
                 −.6128(8) 
                 −.7370(2) 
                 −.4969(4) 
                 3.6(2) 
                 1.0 
               
               
                 C 2 
                 −.4094(24) 
                 −.6318(3) 
                 −.6954(12) 
                 3.5(8) 
                 1.0 
               
               
                 C 3 
                 −.5019(13) 
                 −.6513(3) 
                 −.5885(6) 
                 3.6(3) 
                 1.0 
               
               
                 C 4 
                 −.4859(12) 
                 −.7131(3) 
                 −.5720(6) 
                 3.0(3) 
                 1.0 
               
               
                 C10 
                 −.3269(12) 
                 −.7427(2) 
                 −.6435(6) 
                 2.8(3) 
                 1.0 
               
               
                 C 5 
                 −.2968(12) 
                 −.8009(2) 
                 −.6366(6) 
                 3.1(3) 
                 1.0 
               
               
                 C 6 
                 −.1407(13) 
                 −.8289(2) 
                 −.7054(6) 
                 3.3(3) 
                 1.0 
               
               
                 C 7 
                 −.0117(12) 
                 −.7979(2) 
                 −.7862(6) 
                 3.1(3) 
                 1.0 
               
               
                 C 8 
                 −.0336(12) 
                 −.7405(2) 
                 −.7966(6) 
                 2.8(3) 
                 1.0 
               
               
                 C 9 
                 −.1894(12) 
                 −.7138(2) 
                 −.7262(6) 
                 2.9(3) 
                 1.0 
               
               
                 C 1′ 
                 −.4049(18) 
                 −.5701(3) 
                 −.7176(9) 
                 6.7(5) 
                 1.0 
               
               
                 C 2′ 
                 −.316(5) 
                 −.5292(7) 
                 −.6099(19) 
                 7.4(2) 
                 0.5 
               
               
                 C 3′ 
                 −.326(5) 
                 −.4730(7) 
                 −.6436(21) 
                 7.4(1) 
                 0.5 
               
               
                 C 4′ 
                 −.3988(18) 
                 −.4566(3) 
                 −.7567(10) 
                 7.5(1) 
                 1.0 
               
               
                 C 5′ 
                 −.456(5) 
                 −.4929(8) 
                 −.8580(22) 
                 7.9(2) 
                 0.5 
               
               
                 C 6′ 
                 −.435(6) 
                 −.5510(7) 
                 −.8288(23) 
                 7.8(2) 
                 0.5 
               
               
                 C 2″ 
                 −.194(4) 
                 −.5453(7) 
                 −.6870(25) 
                 7.1(1) 
                 0.5 
               
               
                 C 3″ 
                 −.178(4) 
                 −.4859(7) 
                 −.700(3) 
                 7.3(2) 
                 0.5 
               
               
                 C 5″ 
                 −.630(4) 
                 −.4859(7) 
                 −.7856(22) 
                 7.4(1) 
                 0.5 
               
               
                 C 6″ 
                 −.640(4) 
                 −.5432(6) 
                 −.7752(22) 
                 8.8(1) 
                 0.5 
               
               
                 H 2 
                 −.591 
                 −.644 
                 −.779 
                 7.6 
                 1.0 
               
               
                 H 3A 
                 −.701 
                 −.637 
                 −.587 
                 5.0 
                 1.0 
               
               
                 H 3B 
                 −.369 
                 −.632 
                 −.502 
                 5.0 
                 1.0 
               
               
                 H 6 
                 −.116 
                 −.874 
                 −.697 
                 4.3 
                 1.0 
               
               
                 H 8 
                 .070 
                 −.717 
                 −.861 
                 3.6 
                 1.0 
               
               
                 HO 2 
                 −.525 
                 −.802 
                 −.507 
                 3.2 
                 1.0 
               
               
                 HO 3 
                 −.775 
                 −.699 
                 −.420 
                 3.2 
                 1.0 
               
               
                 H 2′ 
                 −.177 
                 −.548 
                 −.528 
                 3.2 
                 0.5 
               
               
                 H 3′ 
                 −.160 
                 −.447 
                 −.578 
                 3.2 
                 0.5 
               
               
                 H 4′ 
                 −.430 
                 −.413 
                 −.771 
                 3.2 
                 1.0 
               
               
                 H 5′ 
                 −.591 
                 −.477 
                 −.952 
                 3.2 
                 0.5 
               
               
                 H 6′ 
                 −.533 
                 −.577 
                 −.908 
                 3.2 
                 0.5 
               
               
                 H 2″ 
                 −.095 
                 −.557 
                 −.569 
                 3.2 
                 0.5 
               
               
                 H 3″ 
                 −.072 
                 −.456 
                 −.601 
                 3.2 
                 0.5 
               
               
                 H 5″ 
                 −.723 
                 −.469 
                 −.896 
                 3.2 
                 0.5 
               
               
                 H 6″ 
                 −.742 
                 −.570 
                 −.857 
                 3.2 
                 0.5 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Bond length values of bonding atoms of a sample of α crystalline form 
               
               
                 of pinocembrin (Å) 
               
             
          
           
               
                 Bonding Atoms 
                 Bond Length 
                 Bonding Atoms 
                 Bond Length 
               
               
                   
               
             
          
           
               
                 O(1)—C(2) 
                 1.391(9) 
                 C(6)—C(7) 
                 1.402(8) 
               
               
                 O(1)—C(9) 
                 1.369(7) 
                 C(6)—H(6) 
                 1.098(16) 
               
               
                 O(2)—C(5) 
                 1.360(7) 
                 C(7)—0(3) 
                 1.351(7) 
               
               
                 O(2)—Ho(2) 
                 1.090(20) 
                 C(7)—C(8) 
                 1.393(8) 
               
               
                 O(3)—C(7) 
                 1.351(7) 
                 C(8)—C(9) 
                 1.366(8) 
               
               
                 O(3)—Ho(3) 
                 1.100(21) 
                 C(8)—H(8) 
                 1.099(23) 
               
               
                 O(4)—C(4) 
                 1.268(7) 
                 C(1′)—C(2′) 
                 1.497(22) 
               
               
                 C(2)—C(3) 
                 1.391(11) 
                 C(1′)—C(6′) 
                 1.230(30) 
               
               
                 C(2)—C(1′) 
                 1.509(10) 
                 C(2′)—C(3′) 
                 1.399(25) 
               
               
                 C(2)—H(2) 
                 1.183(24) 
                 C(2′)—H(2′) 
                 1.090(30) 
               
               
                 C(3)—C(4) 
                 1.503(8) 
                 C(3′)—C(4′) 
                 1.229(24) 
               
               
                 C(3)—H(3A) 
                 1.087(21) 
                 C(3′)—H(3′) 
                 1.160(30) 
               
               
                 C(3)—H(3B) 
                 1.109(21) 
                 C(4′)—C(5′) 
                 1.360(30) 
               
               
                 C(4)—C(10) 
                 1.420(8) 
                 C(4′)—H(4′) 
                 1.072(17) 
               
               
                 C(10)—C(5) 
                 1.416(8) 
                 C(5′)—C(6′) 
                 1.430(3) 
               
               
                 C(10)—C(9) 
                 1.417(8) 
                 C(5′)—H(5′) 
                 1.150(3) 
               
               
                 C(5)—C(6) 
                 1.371(8) 
                 C(6′)—H(6′) 
                 1.080(3) 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Bond angle values of bonding atoms of a sample of α crystalline form 
               
               
                 of pinocembrin (°) 
               
             
          
           
               
                   
                 Bond angle  
                   
                 Bond angle  
               
               
                 Bonding atoms 
                 value 
                 Bonding atoms 
                 value 
               
               
                   
               
             
          
           
               
                 C(2)—O(1)—C(9)  
                 116.2(5) 
                 C(5)—C(6)—H(6) 
                 121.2(13) 
               
               
                 C(5)—O(2)—Ho(2) 
                 107.8(11) 
                 C(7)—C(6)—H(6) 
                 121.1(13) 
               
               
                 C(7)—O(3)—Ho(3) 
                 113.3(11) 
                 O(3)—C(7)—C(6) 
                 116.2(5) 
               
               
                 C(4)—O(4)—Ho(2) 
                 102.1(9) 
                 O(3)—C(7)—C(8) 
                 121.3(5) 
               
               
                 C(4)—O(4)—Ho(3) 
                 117.2(8) 
                 C(6)—C(7)—C(8) 
                 122.5(5) 
               
               
                 O(1)—C(2)—C(3) 
                 121.9(7) 
                 C(7)—C(8)—C(9) 
                 118.3(5) 
               
               
                 O(1)—C(2)—C(1′) 
                 110.5(6) 
                 C(7)—C(8)—H(8) 
                 120.8(10) 
               
               
                 O(1)—C(2)—H(2) 
                 101.5(12) 
                 C(9)—C(8)—H(8) 
                 120.9(10) 
               
               
                 C(3)—C(2)—C(1′) 
                 118.5(7) 
                 O(1)—C(9)—C(10) 
                 121.4(5) 
               
               
                 C(3)—C(2)—H(2) 
                 99.1(13) 
                 O(1)—C(9)—C(8) 
                 116.5(5) 
               
               
                 C(1′)—C(2)—H(2) 
                 99.8(11) 
                 C(10)—C(9)—C(8) 
                 122.1(5) 
               
               
                 C(2)—C(3)—C(4) 
                 114.0(6) 
                 C(2)—C(1′)—C(2′) 
                 123.3(10) 
               
               
                 C(2)—C(3)—H(3A) 
                 113.3(13) 
                 C(2)—C(1′)—C(6′) 
                 120.9(11) 
               
               
                 C(2)—C(3)—H(3B) 
                 104.8(14) 
                 C(1′)—C(2′)—C(3′) 
                 117.4(16) 
               
               
                 C(4)—C(3)—H(3A) 
                 109.6(10) 
                 C(1′)—C(2′)—H(2′) 
                 111.2(16) 
               
               
                 C(4)—C(3)—H(3B) 
                 107.7(10) 
                 C(2′)—C(3′)—C(4′) 
                 122.9(16) 
               
               
                 H(3A)—C(3)—H(3B) 
                 106.9(15) 
                 C(2′)—C(3′)—H(3′) 
                 113.0(20) 
               
               
                 O(4)—C(4)—C(3) 
                 120.1(5) 
                 C(4′)—C(3′)—H(3′) 
                 115.5(20) 
               
               
                 O(4)—C(4)—C(10) 
                 122.2(5) 
                 C(3′)—C(4′)—C(5′) 
                 121.0(13) 
               
               
                 C(3)—C(4)—C(10) 
                 117.6(5) 
                 C(3′)—C(4′)—H(4′) 
                 117.3(16) 
               
               
                 C(4)—C(10)—C(5) 
                 123.0(5) 
                 C(5′)—C(4′)—H(4′) 
                 121.5(16) 
               
               
                 C(4)—C(10)—C(9) 
                 120.0(5) 
                 C(4′)—C(5′)—C(6′) 
                 118.0(18) 
               
               
                 C(5)—C(10)—C(9) 
                 117.0(5) 
                 C(4′)—C(5′)—H(5′) 
                 116.5(18) 
               
               
                 O(2)—C(5)—C(10) 
                 119.8(5) 
                 C(6′)—C(5′)—H(5′) 
                 120.9(24) 
               
               
                 O(2)—C(5)—C(6) 
                 117.9(5) 
                 C(1′)—C(6′)—C(5′) 
                 123.9(17) 
               
               
                 C(10)—C(5)—C(6) 
                 122.3(5) 
                 C(1′)—C(6′)—H(6′) 
                 117.0(19) 
               
               
                 C(5)—C(6)—C(7) 
                 117.8(5) 
                 C(5′)—C(6′)—H(6′) 
                 113.9(25) 
               
               
                   
               
             
          
         
       
     
     Note: in table 2 and 3, for the atoms C 2′ , C 3′ , C 5′  and C 6′  of B ring, only the bond length and bond angle values of one position are given in Table 2 and 3. 
     Powder (polycrystal) X-ray diffraction (CuK α  radiation) was performed on the α crystalline form solid of pinocembrin, and the diffraction peak locations: 2-Theta value (°) or d value (Å), and the relative strength of diffraction peak: peak height value (Height %) or peak area value (Area %) show the following characteristics (see Table 4,  FIG. 4 ). 
     
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 characteristic peak values of powder X-ray diffraction  
               
               
                 for a sample of α crystalline form of pinocembrin 
               
             
          
           
               
                   
                 Peak 
                 2-Theta 
                 d(Å) 
                 Height % 
                 Area % 
               
               
                   
                   
               
               
                   
                  1 
                  7.32 
                 12.07 
                 100.0 
                 100.0 
               
               
                   
                  2 
                  9.37 
                  9.44 
                   2.3 
                   2.5 
               
               
                   
                  3 
                 11.30 
                  7.82 
                   2.1 
                   2.2 
               
               
                   
                  4 
                 13.93 
                  6.35 
                   1.5 
                   2.5 
               
               
                   
                  5 
                 14.65 
                  6.04 
                  27.3 
                  27.1 
               
               
                   
                  6 
                 17.04 
                  5.20 
                   5.5 
                   8.7 
               
               
                   
                  7 
                 17.31 
                  5.12 
                  11.8 
                  18.2 
               
               
                   
                  8 
                 17.80 
                  4.98 
                   5.6 
                  26.6 
               
               
                   
                  9 
                 18.16 
                  4.88 
                   2.2 
                   1.8 
               
               
                   
                 10 
                 18.83 
                  4.71 
                   1.6 
                   2.1 
               
               
                   
                 11 
                 21.47 
                  4.13 
                   0.9 
                   0.5 
               
               
                   
                 12 
                 22.06 
                  4.03 
                  12.5 
                  13.8 
               
               
                   
                 13 
                 22.40 
                  3.97 
                   2.2 
                   2.4 
               
               
                   
                 14 
                 23.09 
                  3.85 
                   2.6 
                   2.0 
               
               
                   
                 15 
                 23.74 
                  3.74 
                   1.8 
                   1.6 
               
               
                   
                 16 
                 25.83 
                  3.45 
                   1.5 
                   1.8 
               
               
                   
                 17 
                 27.30 
                  3.26 
                   7.2 
                   9.6 
               
               
                   
                 18 
                 27.51 
                  3.24 
                   7.1 
                  14.1 
               
               
                   
                 19 
                 28.29 
                  3.15 
                   5.7 
                   6.4 
               
               
                   
                 20 
                 29.53 
                  3.02 
                   1.7 
                   1.9 
               
               
                   
                 21 
                 30.88 
                  2.89 
                   0.8 
                   1.0 
               
               
                   
                 22 
                 34.51 
                  2.60 
                   1.2 
                   1.2 
               
               
                   
                 23 
                 35.37 
                  2.54 
                   0.5 
                   0.6 
               
               
                   
                 24 
                 37.21 
                  2.41 
                   2.4 
                   2.4 
               
               
                   
                 25 
                 38.27 
                  2.35 
                   0.8 
                   1.4 
               
               
                   
                 26 
                 40.53 
                  2.22 
                   0.5 
                   0.8 
               
               
                   
                 27 
                 41.40 
                  2.18 
                   0.3 
                   0.3 
               
               
                   
                 28 
                 42.09 
                  2.15 
                   1.1 
                   1.1 
               
               
                   
                 29 
                 45.96 
                  1.97 
                   2.8 
                   3.2 
               
               
                   
                 30 
                 46.67 
                  1.94 
                   0.5 
                   0.7 
               
               
                   
                 31 
                 56.49 
                  1.63 
                   0.3 
                   0.5 
               
               
                   
                   
               
             
          
         
       
     
     In an embodiment of the present invention, when analyzed by DSC, α crystalline form solid of pinocembrin shows a decalescence transition temperature of about 206° C. (see  FIG. 5 ). 
     IR analysis with KBr pellet was performed on α crystalline form solid of pinocembrin (see  FIG. 6).The  characteristic peaks were as follows: 3090.6, 3011.6, 2889.1, 2747.4, 2636.2, 1631.5, 1602.5, 1584.3, 1487.7, 1466.2, 1454.5, 1435.6, 1354.9, 1302.4, 1257.0, 1217.0, 1168.2, 1088.6, 1064.9, 1028.0, 1014.6, 1001.3, 975.8, 918.0, 887.7, 861.8, 825.9, 789.9, 766.4, 715.2, 698.1, 663.7, 646.7, 620.3, 587.3, 574.9, 560.5, 526.9 and 487.9 cm −1 , wherein the peaks of 2891.1, 2747.4, 2636.2, 1631.5 and 1354.9 cm −1  were the main characteristic peaks of α crystalline form solid of pinocembrin. 
     Morphologic Features of a Sample of β Crystalline Form of Pinocembrin According to an Embodiment: 
     For β crystalline form of pinocembrin obtained according to an embodiment of the present invention, when analyzed by powder (polycrystal) X-ray diffraction (CuK α  radiation), it showed the diffraction peak location:2-Theta value (°) or d value (Å) and the relative strength of diffraction peak: peak height value (Height %) or peak area value (Area %), shown as the following characteristic peak values (see Table 5,  FIG. 7 ) 
     
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 characteristic peak values of powder X-ray diffraction  
               
               
                 for a sample of β crystalline form of pinocembrin 
               
             
          
           
               
                   
                 Peak 
                 2-Theta 
                 d(Å) 
                 Height % 
                 Area % 
               
               
                   
                   
               
               
                   
                  1 
                  7.33 
                 12.06 
                 100.0 
                  66.5 
               
               
                   
                  2 
                  9.41 
                  9.40 
                   6.2 
                   4.9 
               
               
                   
                  3 
                 11.33 
                  7.81 
                   6.1 
                   4.5 
               
               
                   
                  4 
                 14.07 
                  6.29 
                   2.9 
                   2.2 
               
               
                   
                  5 
                 14.69 
                  6.03 
                  27.2 
                  17.6 
               
               
                   
                  6 
                 17.49 
                  5.07 
                  63.9 
                 100.0 
               
               
                   
                  7 
                 19.01 
                  4.67 
                   2.8 
                   1.5 
               
               
                   
                  8 
                 21.49 
                  4.13 
                   0.1 
                   0   
               
               
                   
                  9 
                 22.11 
                  4.02 
                  12.9 
                  11.2 
               
               
                   
                 10 
                 23.17 
                  3.84 
                  19.4 
                   9.5 
               
               
                   
                 11 
                 25.91 
                  3.44 
                   3.3 
                   2.5 
               
               
                   
                 12 
                 27.41 
                  3.25 
                  31.7 
                  27.4 
               
               
                   
                 13 
                 28.31 
                  3.15 
                  19.0 
                  15.1 
               
               
                   
                 14 
                 29.51  
                  3.02 
                   1.6 
                   0.3 
               
               
                   
                 15 
                 31.03 
                  2.88 
                   0.7 
                   0.4 
               
               
                   
                 16 
                 34.47 
                  2.60 
                   1.0 
                   1.5 
               
               
                   
                 17 
                 35.45 
                  2.53 
                   0.4 
                   0.2 
               
               
                   
                 18 
                 37.24 
                  2.41 
                   1.5 
                   0.7 
               
               
                   
                 19 
                 38.15 
                  2.36 
                   1.2 
                   0.9 
               
               
                   
                 20 
                 40.61 
                  2.22 
                   3.1 
                   2.8 
               
               
                   
                 21 
                 42.83 
                  2.11 
                   1.5 
                   1.4 
               
               
                   
                 22 
                 44.89 
                  2.02 
                   1.6 
                   1.3 
               
               
                   
                 23 
                 46.01 
                  1.97 
                   2.4 
                   1.8 
               
               
                   
                 24 
                 50.36 
                  1.81 
                   0.7 
                   0.8 
               
               
                   
                 25 
                 56.63 
                  1.62 
                   1.0 
                   0.9 
               
               
                   
                   
               
             
          
         
       
     
     In an embodiment of the present invention, when analyzed by DSC, β crystalline form solid of pinocembrin shows a decalescence transition temperature of about 204° C. (see  FIG. 8 ). 
     IR analysis with KBr pellet was performed on β crystalline form solid of pinocembrin according to an embodiment of the present invention (see  FIG. 9 ). The characteristic peaks were as follows: 3090.8, 2890.0, 2748.9, 2638.3, 1633.5, 1602.9, 1585.0, 1487.9, 1466.1, 1454.3, 1344.4, 1302.7, 1216.7, 1168.2, 1088.4, 1065.5, 1028.8, 1014.3, 1001.5, 975.8, 917.8, 888.2, 861.8, 826.6, 789.1, 766.6, 741.1, 715.4, 698.0, 663.7, 646.0, 620.5, 587.9, 574.8, 560.9, 527.2 and 488.4 cm −1 , wherein the peaks of 2890.0. 2748.9. 2638.3, 1633.5 and 1344.4 cm −1  were the main characteristic peaks of β crystalline form solid of pinocembrin. 
     Method for Preparing a Sample of α Crystalline Form of Pinocembrin According to an Embodiment of the Present Invention 
     (1) dissolving a sample completely in a solvent selected from the group consisting of methanol, ethanol, chloroform, acetone, ethyl acetate, n-butanol, isopropanol, acetonitrile, THF, dioxane, 95% ethanol, glacial acetic acid, formic acid, ether, dichloromethane, toluene, benzene, n-hexane, cyclohexane, dioxane, DMF, petroleum ether, ammonia, n-propanol, or a mixture thereof, then, 
     (a) placing the mixture in a condition of temperature 4-50° C. and relative humidity 10%-75% to allow recrystallization for 1 to 60 days, or 
     (b) adding water to allow precipitate, then obtaining α crystalline form of pinocembrin through filtration under reduced pressure, freeze-drying or cold spray. 
     Method for Preparing a Sample of β Crystalline Form of Pinocembrin According to an Embodiment of the Present Invention 
     using a sample of α crystal form solid as the material, then 
     obtaining a sample of β crystalline form of pinocembrin through 
     (a) crystal transition by grinding, or 
     (b) dissolving the material completely in a solvent of pyridine or DMSO, adding water to allow precipitate, and performing filtration under reduced pressure, freeze-drying or cold spray. 
     Pharmacodynamics Characteristics of Pinocembrin Sample: 
     Pure α crystalline form, pure β crystalline form or the mixture of α and β crystalline forms of pinocembrin in any proportion according to the present invention have an effect on treating diseases related to cerebral ischemia or preventing diseases related to cerebral ischemia by protecting neurovascular unit function. 
     There is a difference in bioavailability between pure α crystalline form and pure β crystalline form of present invention. For oral administration, the bioavailability of β crystalline form is more than 2 times higher than that of α crystalline form. For a mixture of the two crystalline forms in any proportion, the bioavailability thereof can vary, dependent on different contents of β crystalline form. 
     Dosage and Preparation Characteristics: 
     For a pharmaceutical composition or a preparation comprising pure α crystalline form, pure β crystalline form or a mixture thereof in any proportion of pinocembrin according to an embodiment of present invention, daily dosage is 5˜250 mg, based on pinocembrin crystalline forms solid. Preparations include tablets, capsules, pills, injections, sustained-release preparations, controlled-release preparations and the like. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is an illustration showing the relative configuration of the molecule. 
         FIG. 2  is an illustration showing the stereo-structure projection of the molecule. 
         FIG. 3  is an illustration showing the unit cell stacking of the molecule (along the a axis). 
         FIG. 4  is a powder X-ray diffraction pattern for a sample of α crystalline form of pinocembrin. 
         FIG. 5  is a DSC trace for a sample of α crystalline form of pinocembrin. 
         FIG. 6  is an infrared absorption spectra for a sample of α crystalline form of pinocembrin. 
         FIG. 7  is a powder X-ray diffraction pattern for a sample of β crystalline form of pinocembrin. 
         FIG. 8  is a DSC trace for a sample of β crystalline form of pinocembrin. 
         FIG. 9  is an infrared absorption spectra for a sample of β crystalline form of pinocembrin. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The following exemplary examples are provided for the purpose of better description of the present invention, however, it should be appreciated that the present invention is not limited to the given examples. 
     Instruments and Test Conditions for the Following Examples: 
     
         
         1. Monocrystal X-ray analysis
       Instrument: MAC DIP-2030K area detector   Test condition: tube voltage: 50 KV, tube flow: 80 mA, ω scanning, MoK α , 2θ≦50.0°, scan range: 0-180°, pivot angle: 6°, step: 6°, scan rate: 1.8°/min.   
     
         2. Powder X-ray analysis
       Instrument: Rigaku D/max 2550 powder X-ray diffractometer   Test conditions: voltage: 40 KV, current: 150 mA, scan rate: 8°/min.   
     
         3. DSC analysis
       Instrument type: Seiko Instruments Inc. Differential Scanning Calorimeter   Test conditions: purge gas: N 2 , heating rate: 10° C. /min.   Temperature range: 25-250° C.   
     
         4. IR absorbance spectra
       Instrument Nicolet FT-IR spectrometer: IMPACT 400   Test conditions: KBr pellet   
     
         5. HPCL analysis
       Instrument: SHIMADZU LC-10Avp high performance liquid chromatography, SPD-M10Avp diode array detector, CLASS-VP chromatography data system; column: Alltch C 18 (5μ, 150×4.6 mm); test conditions: column temperature: room temperature; wavelength: 290 nm; mobile phase: methanol/phosphate saline pH 3.0 (64/36); flow rate : 1.0 mL/min; injection volume: 20 μL; injection concentration: 500.0 μg/mL
 
Preparation 1: Synthesis of Pinocembrin Sample
   
     
       
    
     To 1000 ml hydrogenation reaction kettle, was added 5 g (19.7 mmol) of 5,7-dihydroxyflavone, 650 ml of anhydrous ethanol, and 1.5 g of 10% palladium on carbon. Under a hydrogen pressure of 0.13 Mpa, the reaction was performed for 4 h at 40° C. When the reaction was finished, the palladium on carbon was filtered. The filtrate was concentrated, then separated and purified by column chromatography (eluted by methanol:acetic ether:petroleum ether=2:10:100 (V:V:V)) under vacuum. The solvent was evaporated to dryness, and 3.9 g of white amorphous solid powder was obtained (purity: 98.6%, detected by HPLC) in a yield of 52% [2] . 
     Preparation of a Sample of α Crystalline Form of Pinocembrin 
     EXAMPLE 1 
     Method 1 for Preparing a Sample of α Crystalline Form of Pinocembrin 
     5 g pinocembrin sample was added to 20 ml of 95% ethanol, and was heated to be dissolved completely, then cooled to room temperature and allowed to stand for 24 h. White solid was precipitated, and was filtered and dried. 4.5 g of white crystalline (purity: 98.8%, detected by HPLC) was obtained in a recovery of 90%. 
     The obtained crystalline was analyzed by X-ray single crystal diffraction. It showed monoclinic symmetry, space group was P2  1 /c, and the crystal cell parameter values were a=5.189 Å, b=24.149 Å, c=10.472 Å, α=90°, β=102.31° and γ=90°. 
     Powder (polycrystal) X-ray diffraction (CuK α  radiation) was performed on the obtained crystalline. The characteristic peak values of the diffraction peak location: 2-Theta value (°) or d value (Å) and the relative strength of diffraction peak: peak height value (Height %) or peak area value (Area %) were shown in Table 4, and the obtained trace was shown in  FIG. 4 . 
     DSC analysis was performed on the obtained crystalline, and decalescence transition temperature was 206° C. 
     IR analysis with KBr pellet was performed on the crystalline obtained, and the characteristic peaks were as follows: 3090.6, 3011.6, 2889.1, 2747.4, 2636.2, 1631.5, 1602.5, 1584.3, 1487.7, 1466.2, 1454.5, 1435.6, 1354.9, 1302.4, 1257.0, 1217.0, 1168.2, 1088.6, 1064.9, 1028.0, 1014.6, 1001.3, 975.8, 918.0, 887.7, 861.8, 825.9, 789.9, 766.4, 715.2, 698.1, 663.7, 646.7, 620.3, 587.3, 574.9, 560.5, 526.9 and 487.9 cm −1 . 
     The above spectra data showed that the crystalline form obtained in the present example was α crystalline form. 
     EXAMPLES 2 TO 10 
     Methods 2 to 10 for Preparing a Sample of α Crystalline Form of Pinocembrin 
     Referring to the preparation method of Example 1, using ethyl acetate, chloroform, acetone, acetonitrile, THF, ether, benzene, cyclohexane or DMF as the solvent, white crystalline of pinocembrin was obtained. The results of the experiments were shown in table 6. Powder X-ray diffraction, DSC and IR analysis were performed on the obtained crystalline, and the results showed that the crystalline form was α crystalline form of pinocembrin. 
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Results of the preparation of samples of α crystalline form of  
               
               
                 pinocembrin 
               
             
          
           
               
                   
                 reaction 
                 product weight  
                 HPLC purity  
                 recovery  
               
               
                 example 
                 solvent 
                 (g) 
                 (%) 
                 (%) 
               
               
                   
               
             
          
           
               
                 2 
                 ethyl acetate 
                 4.40 
                 99.0 
                 88 
               
               
                 3 
                 chloroform 
                 4.30 
                 98.7 
                 86 
               
               
                 4 
                 acetone 
                 4.30 
                 99.1 
                 86 
               
               
                 5 
                 acetonitrile 
                 4.20 
                 98.9 
                 84 
               
               
                 6 
                 THF 
                 4.25 
                 98.8 
                 85 
               
               
                 7 
                 ether 
                 4.35 
                 98.6 
                 87 
               
               
                 8 
                 benzene 
                 4.20 
                 98.9 
                 84 
               
               
                 9 
                 cyclohexane 
                 4.20 
                 98.6 
                 84 
               
               
                 10 
                 DMF 
                 4.20 
                 98.7 
                 84 
               
               
                   
               
             
          
         
       
     
     EXAMPLE 11 
     Method 11 for Preparing the Sample of α Crystalline Form of Pinocembrin 
     5 g pinocembrin sample was dissovled in 100 ml mixture of 95% ethanol and acetone (95% ethanol:acetone=1:1) completely at room temperature, then 100 ml water was added under stirring, and white precipitate appeared. The precipitate was filtered under reduced pressure and dried to obtain 4.00 g white crystalline (purity: 98.7%) in a recovery of 80.0%. Powder X-ray diffraction, DSC and IR analysis were performed on the obtained crystalline form, and the results showed that the crystalline form obtained was α crystalline form of pinocembrin. 
     EXAMPLES 12 TO 16 
     Methods 12 to 16 for Preparing a Sample of α Crystalline Form of Pinocembrin 
     Referring to the preparation method of Example 1, using the mixture of isopropanol and THF (isopropanol:THF=2:1), the mixture of acetonitrile and DMF (acetonitrile:DMF=4:1), the mixture of methanol and acetone (methanol:acetone=3:2), the mixture of ethanol and acetonitrile (ethanol:acetonitrile=1:1), and the mixture of ethanol, acetone and glacial acetic acid (ethanol:acetone:glacial acetic acid=2:1:0.1) as the solvents, white crystalline of pinocembrin was obtained. The results of the experiments were shown in Table 7. Powder X-ray diffraction, DSC and IR analysis were performed on the obtained crystalline, and the results showed that the crystalline form obtained was α crystalline form of pinocembrin. 
                                                                 TABLE 7                   Results of samples of α crystalline form of pinocembrin                reaction    product weight    HPLC purity    recovery        example   solvent   (g)   (%)   (%)                    12   2:1 isopropanol-THF   4.00   98.8   80       13   4:1 acetonitrile-DMF   3.80   98.7   76       14   3:2 methanol-acetone   3.90   98.9   78       15   1:1 ethanol-acetonitrile   4.05   99.0   81       16   2:1:0.1                       ethanol-acetone-glacial   4.10   98.9   82           acetic acid                                
Preparation of a Sample of β Crystalline Form of Pinocembrin
 
     EXAMPLE 17 
     Method 1 for Preparing a Sample of β Crystalline Form of Pinocembrin: 
     10 g sample of α crystalline form of pinocembrin was placed in a mortar, grinded evenly in the same direction for 1 hour at room temperature, and white crystalline was obtained, which is different from α crystalline form. 
     Powder (polycrystal) X-ray diffraction (CuK α  radiation) was performed on the obtained crystalline. The characteristic peak values of the diffraction peak location:2-Theta value (°) or d value (Å) and the relative strength of diffraction peak: peak height value (Height %) or peak area value (Area %), were shown in Table 5, and the obtained trace was shown in  FIG. 7 . 
     DSC analysis was performed on the obtained crystalline, and decalescence transition temperature was 204° C., as showed in the DSC trace. 
     IR analysis with KBr pellet was performed on the crystalline obtained, and the characteristic peaks were as follows: 3090.8, 2890.0, 2748.9, 2638.3, 1633.5, 1602.9, 1585.0, 1487.9, 1466.1, 1454.3, 1344.4, 1302.7, 1216.7, 1168.2, 1088.4, 1065.5, 1028.8, 1014.3, 1001.5, 975.8, 917.8, 888.2, 861.8, 826.6, 789.1, 766.6, 741.1, 715.4, 698.0, 663.7, 646.0, 620.5, 587.9, 574.8, 560.9, 527.2 and 488.4 cm −1 . 
     The above spectra date showed that the crystalline form obtained in the present example was β crystalline form. 
     EXAMPLE 18 
     Method 2 for Preparing β Crystalline Form of Pinocembrin 
     At room temperature, 5 g pinocembrin sample was dissolved in 75 ml DMSO, then to which 150 ml of water was added under stirring, and white precipitate appeared. The precipitate was filtered and dried to obtain 4.2 g white crystalline (purity: 98.8%, detected by HPLC) in a recovery of 84.0%. Powder X-ray diffraction, DSC and IR analysis were performed on the obtained crystalline, and the results showed that the crystalline form obtained was β crystalline form of pinocembrin. 
     EXAMPLE 19 
     Method 3 for Preparing a Sample of β Crystalline Form of Pinocembrin 
     Except that pyridine was used as the solvent, the same preparation method as that of Example 18 was used, and 8.8 g white crystalline was obtained (purity: 98.6%, detected by HPLC) in a recovery of 88.0%. Powder X-ray diffraction, DSC and IR analysis were performed on the obtained crystalline, and the results showed that the crystalline form obtained was β crystalline form of pinocembrin. 
     EXAMPLE 20 
     Method for Preparing a Sample of Pinocembrin Mixture of α Crystalline Form and β crystalline form in a ratio 1:1 
     10 g of pinocembrin sample α and β crystalline form were respectively weighed and placed into a sealable vessel. The vessel was sealed and shook to mix the solid evenly. The mixture sample of α and β crystalline form in a ratio 1:1 was obtained. 
     Formulation 
     EXAMPLE 21 
     Method 1 for Preparing a Combined Pharmaceutical Preparation (Tablet) 
     The samples of pure α crystalline form, pure β crystalline form, or solid mixture of (α+β) crystalline form (α:β=1:1) of pinocembrin were mixed with excipients in different proportions to obtain a solid of the combined pharmaceutical active ingredients. Tablets containing 5˜60 mg of active ingredients were prepared. The tablet formula were given in Table 8. 
     
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 Preparation formula for combined pinocembrin tablets 
               
             
          
           
               
                   
                 amount (g/1000 tablets) 
               
             
          
           
               
                   
                 Formula 
                 Formula 
                 Formula 
                 Formula 
                 Formula 
                 Formula 
                 Formula 
               
               
                 components 
                 1 
                 2 
                 3 
                 4 
                 5 
                 6 
                 7 
               
               
                   
               
             
          
           
               
                 Pinocembrin (g)  
                 5.0 
                 10.0 
                 20.0 
                 30.0 
                 40.0 
                 50.0 
                 60.0 
               
               
                 Lactose (g) 
                 100.0 
                 100.0 
                 100.0 
                 100.0 
                 100.0 
                 100.0 
                 100.0 
               
               
                 Starch (g) 
                 35 
                 30 
                 20 
                 10 
                 — 
                 — 
                 — 
               
               
                 Low-substituted 
                 3.0 
                 3.0 
                 3.0 
                 3.0 
                 3.0 
                 3.0 
                 3.0 
               
               
                 hydroxypropyl 
                   
                   
                   
                   
                   
                   
                   
               
               
                 cellulose (g) 
                   
                   
                   
                   
                   
                   
                   
               
               
                 MCC (g) 
                 — 
                 — 
                 — 
                 — 
                 3.0 
                 3.0 
                 3.0 
               
               
                 Talc powder (g) 
                 6.0 
                 6.0 
                 6.0 
                 6.0 
                 6.0 
                 6.0 
                 6.0 
               
               
                 Magnesium 
                 1.0 
                 1.0 
                 1.0 
                 1.0 
                 1.0 
                 1.0 
                 1.0 
               
               
                 stearate (g) 
                   
                   
                   
                   
                   
                   
                   
               
               
                 1% Sodium 
                 q.s. 
                 q.s. 
                 q.s. 
                 q.s. 
                 q.s. 
                 q.s. 
                 q.s. 
               
               
                 hydroxymethyl 
                   
                   
                   
                   
                   
                   
                   
               
               
                 cellulose 
               
               
                   
               
             
          
         
       
     
     The detailed preparation method was as follows: mixing the excipients with pinocembrin evenly, then adding appropriate amount of 1% sodium hydroxymethyl cellulose solution to make a dough. The dough was screened to obtain granules. The wet granules were dried and sieved. Then magnesium stearate and talc powder were added and mixed evenly, and the product was obtained by tabletting. 
     EXAMPLE 22 
     Method 2 for Preparing a Combined Pharmaceutical Formulation (Capsule) 
     The samples of pure α crystalline form, pure β crystalline form, or solid mixture of (α+β) crystalline form (α:β=1:1 or 1:3) of pinocembrin were mixed with excipients in different proportions to obtain a solid of the combined pharmaceutical active ingredients. The capsules containing 5˜60 mg of active ingredients were prepared. The capsule formulas were given in Table 9. 
     
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                 Preparation formulas for combined pinocembrin capsules 
               
             
          
           
               
                   
                 Amount (g/1000 capsules) 
               
             
          
           
               
                   
                 Formula 
                 Formula 
                 Formula 
                 Formula 
                 Formula 
                 Formula 
                 Formula 
               
               
                 components 
                 1 
                 2 
                 3 
                 4 
                 5 
                 6 
                 7 
               
               
                   
               
               
                 Pinocembrin 
                   5.0 
                  10.0 
                  20.0 
                  30.0 
                 40.0 
                 50.0 
                 60.0 
               
               
                 (g) 
                   
                   
                   
                   
                   
                   
                   
               
               
                 Lactose(g) 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
               
               
                 Starch(g) 
                 100.0 
                 100.0 
                 100.0 
                 100.0 
                 — 
                 — 
                 — 
               
               
                 MCC(g) 
                 — 
                 — 
                 — 
                 — 
                 70.0 
                 60.0 
                 50.0 
               
               
                 Magnesium 
                   1.0 
                   1.0 
                   1.0 
                   1.0 
                  1.0 
                  1.0 
                  1.0 
               
               
                 stearate(g) 
                   
                   
                   
                   
                   
                   
                   
               
               
                 1% Sodium 
                 q.s. 
                 q.s. 
                 q.s. 
                 q.s. 
                 q.s. 
                 q.s. 
                 q.s. 
               
               
                 hydroxymethyl 
                   
                   
                   
                   
                   
                   
                   
               
               
                 cellulose 
               
               
                   
               
             
          
         
       
     
     The detailed preparation method was as follows: mixing the excipients with pinocembrin evenly, then appropriate amount of 1% sodium hydroxymethyl cellulose solution was added to make wet granules, the wet granules were dried and sieved. Then magnesium stearate was added and mixed evenly, and the product was obtained by filling the mixture above into empty capsules. Alternatively, without granulation, the product was obtained by filling the excipients and pinocembrin into empty capsules directly, after they were mixed evenly and sieved. 
     EXAMPLE 23 
     Method 3 for Preparing Combined Pharmaceutical Formulation (Injection Solution and Freeze-dried Powder for Injection) 
     Pure α crystalline form, pure β crystalline form, or solid mixture of (α+β) crystalline form (α:β=1:1) of pinocembrin were mixed with excipients in different proportions to obtain a solid of the combined pharmaceutical active ingredients. Then the injections containing 5˜60 mg of active ingredients per ampoule were obtained. The injection formulas were given in Table 10. 
                                                                 TABLE 10                   Preparation formula for combined pinocembrin injections                Formula    Formula    Formula    Formula        components   1   2   3   4                    Pinocembrin (g)   1   1   1   1       Hydroxypropy1-β-cyclodextrin (g)   40   40   20   20       Distilled water (ml)   400   100   10000   10000       Ethanol (ml)   20   20   20   20       Sodium chloride (g)           90           Dextrose (g)               500                    
Formula 1: Preparation of Pinocembrin Injection Solution
     (1) To 400 ml of distilled water, 40 g of hydroxypropyl-β-cyclodextrin was added and dissolved with stirring;   (2) To 20 ml ethanol, 1 g of pinocembrin was added and dissolved, then the obtained solution was added into the hydroxypropyl-β-cyclodextrin solution mentioned above;   (3) The mixed solution was stirred magnetically for 20 min at 40˜50° C. When the solution became clear and transparent, 0.5 g of actived carbon for injection was added. Then the mixture was heated to 80° C. with stirring and kept at this temperature for 15 min, then the carbon was filtered. The filtrate was subpackaged into ampoule at 4 ml each. Pinocembrin injection solution was obtained after sterilizing at 121° C. for 15 min.
 
Formula 2: Preparation of Pinocembrin Freeze-dried Powder for Injection
   (1) In the sterile room, 40 g of hydroxypropyl-β-cyclodextrin was weighed and dissolved in water to make 150 ml solution. 0.1 g actived carbon was added, and the mixture was heated to mild boiling for 15 min, and then the carbon was filtered out;   (2) 1 g of pinocembrin was dissolved in 20 ml anhydrous ethanol, then the obtained solution was added into the hydroxypropyl-β-cyclodextrin solution mentioned above;   (3) The mixture was stirred magnetically for 20 min at 40˜50° C.- When the solution became clear and transparent, the inclusion complex solution of pinocembrin in hydroxypropyl-β-cyclodextrin was obtained;   (4) Water was added to the inclusion complex solution to 200 ml. The mixture was filtered through 0.22 μm filter membrane. The filtrate was subpackaged into vial of 10 ml (2 ml/vial), and placed into freeze dryer to freeze-dry. The sterile powder for injection was obtained after the vials&#39; stoppers were sealed.
 
Formula 3: Preparation of Pinocembrin Sodium Chloride Infusion
   (1) To 200 ml of distilled water, 20 g of hydroxypropyl-β-cyclodextrin was added and dissolved with stirring. 0.5 g actived carbon for infusion was added. The mixture was heated to 80° C. with stirring and kept at this temperature for 15 min, then the carbon was filtered;   (2) 1 g of pinocembrin was weighed and dissolved in 20 ml anhydrous ethanol, then the obtained solution was poured into the hydroxypropyl-β-cyclodextrin solution mentioned above;   (3) The mixture was stirred magnetically for 20 min at 40˜50° C. When the solution became clear and transparent, the inclusion complex solution of pinocembrin in hydroxypropyl-β-cyclodextrin was obtained;   (4) Water was added to the inclusion complex solution to 800 ml. After adding 90 g sodium chloride for injection, the solution was adjusted to a pH of 8˜9 and diluted to 10000 ml with water. Then 10 g actived carbon for injection was added, and stirred for 20 min;   (5) After the carbon was removed, the solution was subpackaged in 100 ml per bottle. The product was obtained after sterilizing at 121° C. for 30 min.
 
Formula 4: Preparation of Pinocembrin Dextrose Infusion
   (1) 20 g of hydroxypropyl-β-cyclodextrin was added to 200 ml of distilled water and dissolved with stirring. 0.5 g actived carbon for infusion was added. The mixture was heated to 80° C. by stirring and kept at this temperature for 15 min, then the carbon was filtered;   (2) 1 g of pinocembrin was dissolved in 20 ml anhydrous ethanol, then the obtained solution was poured into the hydroxypropyl-β-cyclodextrin solution mentioned above;   (3) The mixture was stirred magnetically for 20 min at 40˜50° C. When the solution became clear and transparent, the inclusion complex solution of pinocembrin in hydroxypropyl-β-cyclodextrin was obtained;   (4) Water was added to the inclusion complex solution to 800 ml. After adding 500 g glucose for injection, the solution was adjusted to a pH of 8˜9 and diluted to 10000 ml with water. Then 10 g actived carbon for injection was added, and stirred for 20 min;   (5) After the carbon was removed, the solution was subpackaged in 100 ml per bottle. The product was obtained after sterilizing at 121° C. for 30 min.   

     EXAMPLE 24 
     In vivo Absorption and Blood Concentration Characteristics for Pinocembrin Solid Active Ingredient of α and β Crystalline Forms: 
     18 of SD rats, female and male each half, with body weight of 230˜250 g, were randomized into 3 groups, with 6 rats each group and female half. After 10 hours of fasting but free water intake, the rats were administrated with pinocembrin solid active ingredient powder of α, β or 1:1 (α+β) mixed crystalline forms, at a dose of 50 mg/kg to stomach. Then the arterial blood samples at different times were taken and the contents of pinocembrin were determined. The result showed that for pinocembrin of different crystalline forms, at the same dosage by oral administration, the blood concentrations and the time to reach the peak concentration were different, wherein the blood concentration of α crystalline form was obviously lower than that of β crystalline form. 
     
       
         
               
             
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 11 
               
             
             
               
                   
               
               
                 Blood concentrations at different time for rats having been administered 
               
               
                 orally with pinocembrin samples of different crystalline forms (detected  
               
               
                 by HPLC, peak area value) 
               
             
          
           
               
                   
                 time 
               
             
          
           
               
                 crystalline forms 
                 10 min 
                 20 min 
                 30 min 
                 40 min 
               
               
                   
               
             
          
           
               
                 β crystalline from 
                 13.2 
                 48.6 
                 14.6 
                 14.2 
               
               
                 1:1 (α + β) crystalline 
                 8.5 
                 15.6 
                 7.9 
                 7.1 
               
               
                 forms 
                   
                   
                   
                   
               
               
                 α crystalline form 
                 3.8 
                 9.7 
                 4.4 
                 3.5 
               
               
                   
               
               
                 Note: 
               
               
                 the blood concentrations listed in the table were the average for the rats in each group. 
               
             
          
         
       
     
     REFERENCES 
     1. Chinese patent: publication number CN1695608A 
     2. Cheng Yonghao, etc. synthesis of 5,7-dihydricflavanone, chemical reagents, 2006, 28(7): 437