Abstract:
The present invention relates to a selectively purified tanshinone compounds containing extract from the root of a  Salvia  spp comprising Cryptotanshinone, Dihydrotanshinone, Tanshinone I, and Tanshinone IIA. It comprises at least 15%, by weight of the said identified tanshinone compounds and at least 4% by weight, of cryptotanshinone. The extract and formulations thereof have been found to exhibit excellent anti-microbial properties against, in particular MRSA.

Description:
TECHNICAL FIELD OF THE INVENTION 
       [0001]    The present invention relates to an antibacterial botanical product and more particularly to a selectively purified tanshinone compounds containing extract which exhibits activity against, in particular: 
         [0002]    Methicillin resistant  Staphylococcus aureus  (MRSA), 
         [0003]    Methicillin susceptible  Staphylococcus aureus  (MSSA), 
         [0004]    Coagulase negative Staphylococci (CNS), and 
         [0005]      Streptococcus , inparticular,  Steptococcus pneumoniae.    
         [0006]    The extract also demonstrates activity against  Propionibacterium acnes  and thus may be used therapeutically or cosmetically for the treatment of acne. 
         [0007]    The invention also relates to a scalable method of extraction including purification, formulations of said extracts and methods of treatment. 
       BACKGROUND OF THE INVENTION 
       [0008]    The extract exhibiting these beneficial properties is derived from the root and rhizome of  Salvia miltiorrhiza  Bunge, a perennial herb from the Labiatae family. In Traditional Chinese Medicine (TCM) it is also referred to as Danshen. 
         [0009]    Danshen was recorded as a top-grade herbal medicine in Shennong&#39;s Classic of Materia Medica, as well as in Compendium of Materia Medica and Annotations to the Divine Husbandman&#39;s Classic of Materia Medica. 
         [0010]    It has broad clinical applications. 
         [0011]    In the medical monographs of Coverage of the Materia Medica, Compendium of Materia Medica and Renewal of Herbal, Danshen is said to evacuate puss with detoxication, a reference to its anti-bacterial and anti-inflammatory effects. 
         [0012]    It should be noted that in TCM whole extracts, usually obtained as decoctions, are typically used in combination with a number of other herbs. 
         [0013]    Modern scientific research on Danshen started in 1930&#39;s. 
         [0014]    The chemical constituents of Danshen can be divided into two main categories of chemicals: 
         [0015]    lipid-soluble, and 
         [0016]    water-soluble. 
         [0017]    Earlier studies on “active” compounds of Danshen have mainly been concentrated on the lipid-soluble compounds, where around 40 compounds have been found so far. These can be further divided into two groups: 
         [0018]    Tanshinones (o-quinone structure) and 
         [0019]    Rosiglitazones (o-hydroxy rosiglitazone, paraquinoid structure). 
         [0020]    Most of the tanshinone compounds are diterpenes, of which they are mainly diterpene quinones. 
       Studies on Lipid-Soluble Chemicals 
       [0021]    Over 40 different compounds have been identified, including, for example: tanshinone, cryptotanshinone, tanshinone IIA, tanshinone IIB, methyltanshinone, hydroyltanshinone IIA, isotanshinone I, isotanshinone II, isocryptotanshinone, miltirone, L-dihydrotanshinone I, neotanshinone A, B, C, and salviol. 
         [0022]    The structures of a few of these compounds are illustrated below: 
         [0000]    
       
                 
         
             
             
         
       
     
       Antimicrobial and Anti-Inflammation Effects 
       [0023]    The early research on the lipid soluble compounds of Danshen focused on its antimicrobial effects and a series of screens on anti-bacterial, anti-fungi and anti-tubercle bacillus were carried out by the Institute of Materia Medica, Chinese Academy of Medical Sciences. The screen results showed that the total tanshinones significantly inhibited  Staphylococcus aureus  and an inhibition zone still appeared on sensitive strain 209P at the low concentration (6.25 μg/per tablet) on filter paper disc. The test on 50 erythromycin-resistant  Staphylococcus aureus  isolates from the clinic also showed activity. 
         [0024]    A test comparing the activity of tanshinone and 10 antibiotics has been carried out and the results showed that  Staphylococcus aureus  which was resistant to antibiotics was susceptible to tanshinone. 
         [0025]    With filter paper disc, 5 out of 10 chemicals isolated from Danshen demonstrated anti- Staphylococcus aureus  activity. These chemicals were:
       Cryptotanshinone,   Dihydrotanshinone,   Hydroyltanshinone,   Tanshinone IIB, and   Methyltanshinone.       
 
         [0031]    Tanshinone IIA, tanshinone I, and neotanshinone A, B, and C did not show activity. 
         [0032]    A study on the anti-microbial activity of tanshinone HA, and its correlation with the solvent selection, was carried out by ZHU Jiarong et al. When tanshinones were dissolved in chloroform tanshinone IIA did not demonstrate any anti-microbial activity but when it was dissolved in dimethylformamide (DMF) tanshinone IIA and IIB showed activity against:
         Escherichia coli  at the minimum inhibitory concentration (MIC) 50 or 25 μg/ml,     Staphylococcus aureus  ATCC225923 with MIC 100 or 50 μg/ml,     Bacillus  aeruginosus ATCC227853 with MIC 50 or 25 μg/ml, and   Haemolytic  streptococcus  with MIC 121.5 or 25 μg/ml.       
 
         [0037]    LUO Houwei et al reported tanshinone and 42 related compounds were tested against Tubercle  bacillus  in a structure-activity correlation study. It demonstrated that the quinone group was the principle structure responsible for the activity. 19 compounds with quinone group isolated from Danshen showed potent anti-bacterial activity and the MIC ranged between 0.31-5 mg/l. The bacteriostasis activity of o-quinone compounds was stronger than that of p-quinone compounds. A-ring hydroxylation or dehydrogenation of the inter ring resulted in less bacteriostasis activity. Different substitutions at a-H furan ring of tanshinone clearly affected bacteriostasis activity. 
         [0038]    LI Jiangqin et al. reported the study on tanshinone IIA, cryptotanshinone and their zinc iron complex against  Escherichia coli  and  Staphylococcus aureus  activity. Compared with each other, cryptotanshinone was more potent than tanshinone IIA. Cryptotanshinone showed better inhibition effect against  Staphylococcus aureus  than  Escherichia coli . The bacteriostasis activity was enhanced when cryptotanshinone complex was formed with metal ions, especially with zinc. 
         [0039]    LUO Yongjian et al reported that a bacteriostasis experiment was carried out on a product called “Xiao Yan Kun” which was extracted from Gansu Danshen. The main compounds of the product were cryptotanshinone and tanshinone IIA. In the test, acetone was used as solvent and berberine and oxytetracycline were used as the positive controls. The bacterial stains included  Staphylococcus aureus, Bacillus subtilis, Streptococcus agalactiae, Pseudomonas aeruginosa, Escherichia coli , and  Streptococcus dysgalactiae , respectively. The test samples showed better activity against  Staphylococcus aureus, Bacillus subtilis  and  Streptococcus agalactiae  than that of berberine. Cryptotanshinone was more active than tanshinone IIA but both samples were less active than oxytetracycline. Both samples showed no effect on  Pseudomonas aeruginosa, Escherichia coli  and  Streptococcus dysgalactiae.    
         [0040]    The patent literature makes reference to a number of Salviae extracts. 
         [0041]    CN101073599 discloses an extract of total ketone of comprising cryptotanshinone, tanshinone I, tanshinone IIA, methyl tanshinon, dihyderotanshinon I and ramification for use as a medicine or food. 
         [0042]    CN1927265 discloses a process for increasing cryptotanshinone content in Salviae miltoiorrhizae extract. 
         [0043]    CN1670019 discloses a method for extracting tanshinone in which a first extraction gives cryptotanshinone and dihydrotanshinone and a second extraction gives tanshinone IIA and tanshinone I. 
         [0044]    CN1944455 discloses a method of increasing the content of cryptotanshinone, dihydrotanshinone, tanshinone IIA and tanshinone I using column chromatography with a given simultaneous solvent extraction. 
         [0045]    None of the above specifically disclose an extract as characterized by the claims of the present invention. 
         [0046]    Additional prior art includes: 
         [0047]    US2003/0031690 which discloses a cosmetic composition comprising cryptotanshinone which is said to inhibit 5 alpha reductase activation. KR20020028041 which discloses the use of a composition containing Salviae miltoiorrhizae extract to treat pimples based on it&#39;s inhibition of 5 alpha reductase. 
         [0048]    CN1317308 which discloses a cryptotanoshine containing cream to treat acne. 
         [0049]    Biosci Biotechnol. Biochem 63 (12) 2236-2239 suggests that superoxidase radical formation might be the cause of the antibacterial activity of cryptotanshinone, dihydrotanshinone I. 
         [0050]    More recently, in the Journal of Microbiology, August 2007 p350-357 it has been reported that Salvia miltiorrhiza shows anti-microbial activity against MRSA. Different extracts were studied, including methanol, hexane, chloroform, ethyl acetate, butanol and water. The best activity was found in hexane and chloroform fractions. MIC for the hexane fraction against various MRSA specimens was 64&lt;MIC&#39;s&gt;128 μg/ml. 
         [0051]    With drug resistance proving such a major problem today, any active medicines or bactericidal compositions would be highly desirable. 
         [0052]    It is an aim of the present invention to provide a medicine or bactericidal compositions which is/are effective against MRSA in low doses and which can be produced effectively on a commercial, as opposed to laboratory scale. 
       SUMMARY OF THE INVENTION 
       [0053]    According to a first aspect of the present invention there is provided a selectively purified tanshinone compounds containing extract from the root of a  Salvia  spp comprising:
       Cryptotanshinone,   Dihydrotanshinone,   Tanshinone I, and   Tanshinone IIA, characterized in that the above identified tanshinone compounds comprise at least 15%, by weight, of the selectively purified extract and the cryptotanshinone comprises at least 4%, by weight, of the selectively purified extract.       
 
         [0058]    Preferably, the  Salvia  spp is Salvia miltiorrhiza Bunge although other Salvia Spp such as
         Salvia apiana        Salvia argentea        Salvia arizonica        Salvia azurea        Salvia carnosa        Salvia clevelandii        Salvia coccinea        Salvia divinorum        Salvia dorrii        Salvia farinacea        Salvia forreri        Salvia fulgens        Salvia funerea        Salvia glutinosa        Salvia greggii        Salvia guaranitica        Salvia hispanica        Salvia leucantha        Salvia leucophylla        Salvia libanotica        Salvia longistyla        Salvia lyrata        Salvia mexicana        Salvia officinalis.        Salvia patens        Salvia polystachya        Salvia potus.        Salvia pratensis        Salvia roemeriana        Salvia sclarea        Salvia spathacea        Salvia splendens        Salvia verticillata        Salvia viridis    
may be used.
       
 
         [0093]    More preferably still the identified tanshinone compounds comprises at least 35%, by weight, of the selectively purified extract and the cryptotanshinone comprises at least 15%, by weight, of the selectively purified extract. 
         [0094]    Yet more preferably still the identified tanshinone compounds comprise at least 45%, by weight, of the selectively purified extract and the cryptotanshinone comprises at least 25% by weight, of the selectively purified extract. 
         [0095]    In one embodiment the cryptotanshinone comprises at least 20%, more preferably at least 25%, more preferably still at least 40% and maybe as much as 60% of the four identified tanshinone compounds. 
         [0096]    Similarly, the tanshinone IIA preferably comprises less than 55% of the four identified tanshinone compounds, more preferably still less than 50%, yet more preferably still less than 40% and may comprise as little as 20% or less of the four identified tanshinone compounds. 
         [0097]    In a preferred embodiment, the selectively purified tanshinone compound containing extract is characterized in that it comprises the four identified tanshinone compounds in an amount of 42.89% (plus or minus 40%, through 30% to 20%):
       a cryptotanshinone content of 18.95% (plus or minus 40%, through 30% to 20%),   a dihydrotanshinone content of 3.65% (plus or minus 40%, through 30% to 20%),   a tanshinone I content of 3.82% (plus or minus 40%, through 30% to 20%), and   a tanshinone IIA content of 16.47% (plus or minus 40%, through 30% to 20%).       
 
         [0102]    The selectively purified tanshinone compound containing extract may be characterized in that it has an HPLC fingerprint substantially as illustrated in  FIG. 13  with characteristic peaks as indicated. 
         [0103]    The extract may be used in the manufacture of an antibacterial medicament, more particularly one for use in the treatment of a drug resistant bacterium. It is particularly useful to treat:
       Methicillin resistant  Staphylococcus aureus  (MRSA),   Methicillin susceptible  Staphylococcus aureus  (MSSA),   Coagulase negative  Staphylococci  (CNS), and     Streptococcus , in particular,  Steptococcus pneumoniae.          
 
         [0108]    It may also be used in the medical or cosmetic treatment of acne, a skin condition caused by infection with  Propionibacterium acnes.    
         [0109]    The extract may be included as the active ingredient of a pharmaceutical or cosmetic formulation with one or more excipients. It may also be added to a carrier to form compositions, e.g. hand cleaning or surface cleaning compositions which may take the forms of solutions, gels, sprays and impregnated wipes. 
         [0110]    Preferred pharmaceutical or cosmetic formulations are for topical or oral delivery. 
         [0111]    An effective concentration can be less than 64 μg/ml, more particularly less than 32 μg/ml and as low as 16 μg/ml or lower. Levels of activity at such low concentrations show a significant improvement over, for example, the teaching in The Journal of Microbiology, August 2007 p350-357. 
         [0112]    According to a further aspect of the present invention there is provided a scalable method of manufacturing a selectively purified tanshinone compounds containing extract of the root of a  Salvia  spp comprising the steps of:
       soaking raw material in strong ethanol for a time sufficient to solublize the tanshinone compounds,   extracting the tanshinone compounds containing fraction using a percolation method, and   concentrating the desired fraction under vacuum and recovering the ethanol.       
 
         [0116]    Preferably the method further comprises one or more purification steps to concentrate the tanshinone compounds containing fraction and/or cryptotanshinone. 
         [0117]    In one embodiment a first purification step comprises:
       a. dissolving the extract in sufficient water,   b. allowing the desired fraction to precipitate out,   c. discarding the aqueous solution, and   d. collecting the precipitate.       
 
         [0122]    In the preferred embodiment, a second purification step is conducted, comprising a separation on a macroporous resin column. This second purification step comprises:
       a) dissolving the precipitate in a mid strength ethanol; loading it onto an AB 8 macroporous resin column, manufactured by Lioayuan New Materials Ltd, (or other suitable column),   b) adding further mid strength ethanol to wash the column, discarding the eluent, and   c) eluting the desired extract with a higher than mid strength ethanol.       
 
         [0126]    Preferably the mid strength ethanol is 60% strength and the eluting ethanol is 70% ethanol. 
     
    
     
         [0127]    The invention will be further described, by way of example only, with reference to the following examples in which: 
           [0128]      FIG. 1  is a flow diagram illustrating an ethyl acetate extraction process; 
           [0129]      FIG. 2   a  is an HPLC chromatogram of reference samples under conditions as set out in Table 3 (gradient 1); 
           [0130]      FIG. 2   b  is an HPLC chromatogram of reference samples under conditions as set out in Table 4 (gradient 2); 
           [0131]      FIG. 3   a  is an HPLC chromatogram of a CO 2  extract under conditions as set out in Table 3 (gradient 1); 
           [0132]      FIG. 3   b  is an HPLC chromatogram of a CO 2  extract under conditions as set out in Table 4 (gradient 2); 
           [0133]      FIG. 4   a  is an HPLC chromatogram of ethyl acetate extract under conditions as set out in Table 3 (gradient 1); 
           [0134]      FIG. 4   b  is an HPLC chromatogram of ethyl acetate extract under conditions as set out in Table 4 (gradient 2); 
           [0135]      FIGS. 5   a - c  are TLC fingerprints in which, lane 1 is a CO 2  extract, lane 2 is an ethyl acetate extract and lane 3 is a referenced sample containing (bottom to top) dihydrotanshinone, cryptotanshinone, tanshinone I, and tanshinone HA; 
           [0136]      FIG. 6  is a flow diagram illustrating an improved extraction process; 
           [0137]      FIG. 7  is a TLC fingerprint showing (from left to right and top to bottom) 37 consecutive silica gel purified fractions from the process illustrated in  FIG. 6 ; 
           [0138]      FIG. 8  is a TLC fingerprint showing (from left to right) 19 variously combined silica gel purified fractions from the process illustrated in  FIG. 6 ; 
           [0139]      FIG. 9  is a TLC fingerprint showing (from left to right) the 7 th  to 13 th  merged silica gel purified fractions from  FIG. 8  (JZ061); 
           [0140]      FIG. 10   a  is an HPLC chromatogram of a percolation extract under conditions as set out in Table 3 (gradient 1) (SL0601); 
           [0141]      FIG. 10   b  is an HPLC chromatogram of an ethyl acetate purified extract under conditions as set out in Table 3 (gradient 1) (YY0601); 
           [0142]      FIG. 10   c  is an HPLC chromatogram of a silica gel purified extract under conditions as set out in Table 3 (gradient 1) (JZ0601); 
           [0143]      FIG. 10   d  is an HPLC chromatogram of reference compounds under conditions as set out in Table 3 (gradient 1); 
           [0144]      FIG. 11  is a flow diagram illustrating a scalable extraction process giving a characterized product rich in cryptotanishone as per section 6.0; 
           [0145]      FIG. 12  is a TLC fingerprint in which lane 1 is an extract from the process described with reference to  FIG. 11  and lane 2 is a mixed reference samples from bottom to top dihydrotanshinone, cryptotanshinone, tanshinone I, and tanshinone IIA; 
           [0146]      FIG. 13  is an HPLC chromatogram of an extract from the process described with reference to  FIG. 11  under conditions as set out in Table 4 (gradient 2); 
           [0147]      FIG. 14  is an HPLC chromatogram of reference compounds under conditions as set out in Table 4 (gradient 2); and 
           [0148]      FIG. 15  is a table showing the activity of a number of gel formulations. 
       
    
    
       [0149]    In the HPLC figs the identified compound peaks read left to right are: dihydrotanshinone, tanshinone I, cryptotanshinone, and tanshinone HA. 
       DETAILED DESCRIPTION 
     1.0 Extraction Methodology 
       [0150]    In order to identify selectively purified tanshinone compound containing extracts from the root of a  Salvia  spp, a number of alternative methodologies were examined. 
         [0151]    Initially, a super critical fluid extraction (SCFE) and an ethyl acetate extraction (EAE) were conducted: 
       1.1 SCFE 
       [0152]    Put the pulverized dry raw material of Salvia miltiorrhiza into the SCE-CO2 extractor. Set up the pressure at 20 MPa and temperature at 45 degree C. Add 30% (relative to the raw material) ethanol (95%) as the entrainer to the system. Set the flow rate at 1 ml/min and continuously extract for 60 min. The dark red crystal obtained was code numbered SME-1. 
       1.2 EAE 
       [0153]    The EAE methodology used is that set out with reference to  FIG. 1 . 
         [0154]    The Danshen raw material (50 g) is crushed and subjected to an ethanol extraction with 95% ethanol (added to four times volume) and left for about 1 hour. The process was repeated 3 times and the solvent extracts combined. 
         [0155]    Any residue was subjected to a water extraction (added to four times volume) and left for about an hour. Repeat twice. 
         [0156]    The ethanol extract underwent a percolation extraction in which the extract was dissolved in 15 ml of water and extracted with petroleum ether. The process was repeated three times adding 15 ml of water each time. 
         [0157]    The water fraction was then extracted with ethyl acetate (15 ml), and the process repeated three times. The resulting ethyl acetate fraction is referred to generally as SME-2. 
       2.0 Testing 
       [0158]    The resulting extracts were tested for their antimicrobial activity by the National Institute for the Control of Pharmaceutical and Biological Products (NICPBP), National Center for Drug Resistance of Bacteria Beijing, PR China. 
       Results: 
       [0159]    The extracts were tested for antimicrobial activity against 401 strains, over 95% of which were clinical isolates with drug resistance including 41 strains of MRSA and 17 strains of MRCNS. 
         [0160]    The pre-experiment showed that BC-SME-2 had:
       a high activity against Gram-positive bacteria, and   a low activity against Gram-negative bacteria with a strong selective antibacterial action.       
 
         [0163]    The samples showed a strong antibacterial action against BOTH
       MRSA, and   MRCNS.       
 
         [0166]    The samples had a strong action against  Staphylococcus aureus  and  Staphylococcus epidermidis.    
         [0167]    The information and numbers of the tested strains are listed in Table 1 and the MIC50 value and range for the important strains are listed in Table 2. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Test strains and results 
               
             
          
           
               
                   
                   
                   
                 Average 
               
               
                   
                   
                   
                 MIC50 
               
               
                   
                 Name of Bacteria 
                 Nos. 
                 (μg/ml) 
               
               
                   
                   
               
             
          
           
               
                   
                 
                   Enterococcus faecalis 
                 
                 72 
                 &gt;512 
               
               
                   
                   Enterococcus  sp. 
                 5 
                 &gt;512 
               
               
                   
                 
                   Escherichia coli 
                 
                 4 
                 &gt;512 
               
               
                   
                 
                   Staphylococcus aureus 
                 
                 120 
                 24 
               
               
                   
                 
                   Staphylococcus epidermidis 
                 
                 120 
                 22 
               
               
                   
                 
                   Streptococcus pneumoniae 
                 
                 43 
                 47 
               
               
                   
                   Klebsiella pneumoniae  ss. 
                 1 
                 16 &gt; 512 
               
               
                   
                 
                   Pseudomonas aeruginosa 
                 
                 1 
                 &gt;512 
               
               
                   
                 
                   Streptococcus equi 
                 
                 1 
                 &gt;512 
               
               
                   
                 
                   Streptococcus equinus 
                 
                 1 
                 128 
               
               
                   
                 
                   Streptococcus equisimilis 
                 
                 1 
                 &gt;512 
               
               
                   
                   Streptococcus iridans , alpha-hem. 
                 5 
                 64 
               
               
                   
                   Streptococcus , beta-haem. Group A 
                 1 
                 64 
               
               
                   
                   Streptococcus , beta-haem. Group B 
                 2 
                 256, 64 
               
               
                   
                   Streptococcus , beta-haem. Group C 
                 2 
                 64 
               
               
                   
                   Streptococcus , beta-haemolytic 
                 15 
                 32 &gt; 512 
               
               
                   
                   Streptococcus  sp. 
                 6 
                 256 
               
               
                   
                   Streptococcus  sp. 
                 1 
                 256 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Value and range of MIC50 for the important bacteria strains 
               
             
          
           
               
                   
                   
                 IC50 
                 MIC50 range 
               
               
                 Bacteria Name 
                 Strains 
                 (μg/ml) 
                 (μg/ml) 
               
               
                   
               
             
          
           
               
                 
                   Staphylococcus aureus 
                 
                 120 
                 16 
                  2-1024 
               
               
                 
                   Staphylococcus epidermidis 
                 
                 120 
                 16 
                  1-1024 
               
               
                   Enterococcus  sp. 
                 77 
                 1024 
                 16-1024 
               
               
                 
                   Streptococcus pneumoniae 
                 
                 43 
                 32 
                 16-1024 
               
               
                   Streptococcus  sp. 
                 35 
                 256 
                 32-1024 
               
               
                   
               
             
          
         
       
     
       3.0 Extract Analysis 
       [0168]    HPLC and TLC chromatographic fingerprints of the active extracts were obtained as set out below: 
       3.1 HPLC Analysis 
     3.1.1 Samples 
       [0000]    
       
         
           
             BC-SME 1 Batch No. CL0501 (CO 2  extract) 
             BC-SME 2 Batch No. YY0501 (Ethyl acetate extract) 
           
         
       
     
         [0171]    3.1.2 Apparatus Shimadzu LC-10A HPLC 
         [0000]    3.1.3 Chromatographic conditions 
         [0172]    Column: Atlantis® dC18 (5 μm, 250×4.6 mm) 
         [0173]    Detection wavelength: 255 nm 
         [0174]    Temperature: 25° C. 
         [0175]    Flow Speed: 1 ml/min 
         [0176]    Mobile phase: Methanol (A)-Water (B) gradient elution 
         [0177]    Two differing gradients were used as set out in Tables 3 and 4. 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 (Gradient 1) 
               
             
          
           
               
                   
                   
                 Mobile 
                 Mobile 
               
               
                   
                 Time 
                 Phase 
                 Phase 
               
               
                   
                 (min) 
                 A (%) 
                 B (%) 
               
               
                   
                   
               
               
                   
                  0~55 
                 63 
                 37 
               
               
                   
                 55~75 
                 63→70 
                 37→30 
               
               
                   
                 75~95 
                 70→90 
                 30→10 
               
               
                   
                   
               
             
          
         
       
     
         [0000]                                      TABLE 4                   (Gradient 2)                    Mobile   Mobile           Time   Phase   Phase           (min)   A (%)   B (%)                        0~35    70   30           35~45   70→100   30→0           45~50   100    0                        
Gradient  1  was the conditions used pre-experiment. Because of a long analytical period, Gradient  2  was found to be preferred.
 
       3.1.4 Reagents 
       [0178]    Methanol: chromatography pure, and Water: steam-distilled, the rest of reagents: analytical pure. 
         [0000]    3.1.5 Reference compounds
       Dihydrotanshinone,   Cryptotanshinone,   Tanshinone I, and   Tanshinone IIA (for content assay only)       
 
         [0183]    All supplied by National Institute for the Control of Pharmaceutical and Biological Products. 
       3.1.6 Preparation of Reference Solution 
       [0184]    Measure precisely:
       1 mg of Dihydrotanshinone,   1 mg of Cryptotanshinone,   1 mg of Tanshinone, and   2 mg of Tanshinone IIA, put them all into a 10 ml flask, add 8 ml of the mixed solution of methanol-methylene dichloride (9:1), ultrasound for 5 minutes, add the methanol-methylene dichloride (9:1) solution to volume, shake thoroughly and allow to stand.       
 
       3.1.7 Preparation of Sample Solution 
       [0189]    Measure appropriate amount of BC-SME 1 (CO 2  extract) and BC-SME 2 (ethyl acetate extract) respectively, put them into two 10 ml flasks, add 8 ml of the methanol-methylene dichloride (9:1) solution, dissolve with ultrasound, add the methanol-methylene dichloride (9:1) solution to volume, shaking and filter. The subsequent filtrate was taken as a sample solution. 
       3.1.8 Sample Loading 
       [0190]    Measure precisely 5 μl of each of the reference solution and the sample solution. Carry out the HPLC as described above. 
       3.1.9 The Experimental Results 
       [0191]    According to the two gradient conditions mentioned above, the peaks of the different tanshinone indicators achieved baseline separation. Gradient  2  was used due to the advantage of a shorter detection time, and the saving of solution. 
         [0192]    The HPLC fingerprints of the referent samples are shown in  FIGS. 2   a  and  FIG. 2   b  (under the different gradient conditions) and those of the two extracts are shown with reference to  FIGS. 3   a  and  3   b  (BC SME I) and  FIGS. 4   a  and  4   b  (BC SME II). From Left to Right the peaks are:
       Dihydrotanshinone,   Tanshinone I,   Cryptotanshinone, and   Tanshinone IIA.       
 
         [0197]    The content assay for the samples is given in Table 5 below. (Gradient  1  was used for the content assay.) 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Content Assay 
               
             
          
           
               
                 Name 
                 BC-SME-1 Content (%) 
                 BC-SME-2 Content (%) 
               
               
                   
               
             
          
           
               
                 Dihydrotanshinone 
                 1.04 
                 1.44 
               
               
                 Tanshinone I 
                 5.26 
                 4.64 
               
               
                 Cryptotanshinone 
                 2.75 
                 3.74 
               
               
                 Tanshinone IIA 
                 39.40 
                 8.56 
               
               
                 Total: 
                 48.45 
                 18.38 
               
               
                   
               
             
          
         
       
     
       3.1.10 Discussion 
       [0198]    From the HPLC content assay, it was found that the total tanshinone content was 48.5% in BC-SME 1, much higher than that in BC-SME 2. This result can explain the dissolvability difference of the two samples in polar solvents. 
         [0199]    The content of tanshinone IIA was as high as 39.40% in BC-SME 1, but the antibacterial activity level was low. 
         [0200]    The contents of dihydrotanshinone and cryptotanshinone in BC-SME 2 were higher than those in BC-SME 1, so it is presumed that the better antibacterial activity level of BC-SME 2 is due to the presence of these compounds. 
       3.2 TLC Analysis 
     3.2.1 Samples 
       [0000]    
       
         
           
             BC-SME 1 Batch No: CL0501 
             BC-SME2 Batch No: YY0501 
           
         
       
     
         [0203]    3.2.2 Standards and reagents
       Dihydrotanshinone,   Cryptotanshinone,   Tanshinon I, and   Tanshinone IIA.       
 
         [0208]    All supplied by National Institute for the Control of Pharmaceutical and Biological Products. 
         [0209]    Methanol: chromatographic pure (US Fisher), Water: re-distilled, and the rest was analytical pure. 
       3.2.3 Methods 
       [0210]    Take 15 mg each of BC-SME 1 and BC-SME 2, add 10 ml of the methanol-methylene dichloride (9:1) mixture, dissolve with ultrasound and filter. Take 1 mg each of dihydrotanshinone, cryptotanshinone, tanshinone I and tanshinone IIA, add 2 ml of the methanol—methylene dichloride (9:1) mixture respectively for the mixed solution standards. Following the TLC method (Chinese Pharmacopoia 2005 Version Vol. I Appendix VI B) take 50 of each the test solutions, together with 3 μl each of the above-mentioned mixed solution standards, place them respectively on the same silica gel G plate, using petroleum ether—tetrahydrofuran—methanol (10:2:1) as the developing system, examine under the sunlight. 
       3.2.4 Results 
       [0211]    Put the sample drops on the same silica gel G plate with the reference drops. The spots corresponding to the reference compounds showed the same color at the same positions. The TLC experiment was repeated three times. 
         [0212]    The Results are shown in  FIGS. 5   a ,  5   b  and  5   c    
         [0213]    In each:
       Lane 1: SME-1,   Lane 2: SME-2 and   Lane: reference compounds. Reading bottom to top these are:
           Dihydrotanshinone,   Cryptotanshinone,   Tanshinone I, and   Tanshinone IIA.   
               
 
       3.2.5 Discussion 
       [0221]    From the experimental results under these chromatographic conditions, the separation of the mixed reference compounds and samples of the tanshinone compounds were very good, and the clear spots of the reference compounds could be seen in the samples of BC-SME 1 and BC-SME 2. 
       4.0 Improved extraction-Comparison between thermal reflux and percolation 
       [0222]    Tanshinone compounds are lipid-soluble, so a high concentration ethanol (95% ethanol) was used as an extraction medium. A comparison between reflux and percolation extraction was made with a view to determining if a commercially scalable process giving a higher yield rate of cryptotanshinone and reduced impurity could be attained. 
       4.1 Percolation Extraction 
       [0223]    Soak 70 g of Danshen raw material in 95% ethanol for 12 hours and extract with 12 times its volume of 95% ethanol. The colature was collected and the ethanol recovered. The resulting extract was then dried with a vacuum concentrator and weighed. 40 mg of the dry solid extract was weighed and put it into a 50 ml volumeteric flask, dissolved with the mobile phase solution, diluted to volume, filtered and analysed with HPLC. 
       4.2 Reflux Methods 
       [0224]    Reflux 70 g of Danshen raw material with 6 times its own volume of 95% ethanol twice, for 1.5 hours on each occasion. The ethanol was recovered and the extract dried with a vacuum concentrator and weighed. 40 mg of the dry solid extract was placed into a 50 ml volumeteric flask; dissolved with the mobile phase solution, filtered and analysed with HPLC. 
       4.3 Results 
       [0225]    The Results showed that the extraction by using the percolation methods can raise the content and the conversion rate of cryptotanshinone but its yield rate was lower than that with the reflux methods. 
         [0226]    The different percolation methods selected, the yield rates, and the content and conversion rates of the extracts produced by the two different methods are set out in Table 6 below. 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Selection of the cryptotanshinone extraction methods 
               
             
          
           
               
                   
                   
                 Content 
                   
               
               
                 Methods 
                 Yield Rate (%) 
                 (mg/g extract) 
                 Conversion Rate (%) 
               
               
                   
               
               
                 Percolation 1 
                 4.96 
                 84.53 
                 95.29 
               
               
                 Percolation 2 
                 4.77 
                 87.41 
                 94.76 
               
               
                 Reflux 1 
                 9.28 
                 39.25 
                 82.78 
               
               
                 Reflux 2 
                 9.44 
                 40.00 
                 85.82 
               
               
                   
               
             
          
         
       
     
         [0227]    From the results it can be seen that percolation results in a significantly greater content (on a mg/g of extract basis) than reflux. Furthermore it is advantageous in that it uses simple equipment, is safe to operate, and is energy efficient. The extraction at room temperature also reduces damage to the active components, which are heat and light sensitive, and easily degraded. 
       4.4 Optimization of Percolation Method 
       [0228]    In order to optimize the process parameters of percolation extraction an orthogonal test was carried out and the conversion rate of cryptotanshinone used as an investigation indicator. 
       4.4.1. Design of Orthogonal Test 
       [0229]    An orthogonal test was designed to determine what factors might influence the conversion rate of cryptotanshinone. 
         [0000]    Three factors were selected for the orthogonal test:
       (A) Solvent consumption,   (B) Soaking time, and   (C) Outflow velocity.       
 
         [0233]    Three levels were selected for each factor. 
         [0234]    The conversion rate of cryptotanshinone was selected as the investigation indicator; analysis was carried out by using direct-vision methods and analysis of variance (ANOVA). 
         [0235]    The test was carried out as set out in Table 7. 
         [0000]    
       
         
               
               
               
               
             
           
               
                 TABLE 7 
               
               
                   
               
               
                   
                 A 
                 B 
                 C 
               
               
                   
                 Ethanol Consumption 
                 Soaking Time 
                 Outflow Velocity 
               
               
                 Level 
                 (folds) 
                 (h) 
                 (ml/min−1) 
               
               
                   
               
             
             
               
                 1 
                 10 
                 6 
                 10 
               
               
                 2 
                 12 
                 12 
                 15 
               
               
                 3 
                 14 
                 24 
                 20 
               
               
                   
               
             
          
         
       
     
       4.4.2 Design Methodology 
       [0236]    Measure 9 portions, 70 g each of the crude powder of Danshen raw material. Each portion was soaked respectively in an appropriate quantity of 95% ethanol for half an hour. Carry out the percolation under the conditions set out in Table 7. The respective solutions were collected and dried with vacuum concentration. The ethanol was recovered from each resulting solution and the extracts were dried under vacuum at 60° C., and weighed. 40 mg of dried extract was weighed into a 50 ml volumetric flask, dissolved into the mobile phase solution to volume, and filtered. The solutions were used for HPLC analysis. 
       4.4.3 Results of Orthogonal Test 
       [0237]    From the analysis of variance (ANOVA), three factors affected the conversion rate of Cryptotanshinone; the 3 influence degrees were A&gt;C&gt;B. The optimization grouping was A2B3C2, that is, in the conditions of 95% ethanol of 12 folds its volume, soaking for 24 hours, outflow velocity: 15 ml/min 
         [0238]    The results are shown in Table 8. 
         [0000]                                                              TABLE 8                                       Conversion Rate of       Nos   A   B   C   D (Blank)   Cryptotanshinone (%)               1   1   1   1   1   84.20       2   1   2   2   2   91.77       3   1   3   3   3   97.39       4   2   1   2   3   97.84       5   2   2   3   1   90.07       6   2   3   1   2   92.14       7   3   1   3   2   87.07       8   3   2   1   3   79.77       9   3   3   2   1   89.45       K1   273.36   269.11   256.11   263.72   G = ΣYi = 809.70       K2   287.00   261.61   279.06   270.98   CT = G2/9 = 72846.01       K3   256.29   278.98   274.53   275.00   Q = 1/3ΣK2i       Q   74259.75   72896.61   72944.51   72867.80   SS = Q-CT       SS   1413.74   50.60   98.50   21.79                    Source for                       ANOVA   SS   f   S   F               A   1413.74   2   706.87   64.88       B   50.60   2   25.30   0.51       C   98.50   2   49.25   4.52       Difference   21.79   2   10.89                    
4.4.4 Optimised percolation process
 
         [0239]    Based on the results of the orthogonal test, three validation tests were carried out for the optimized process. The results are illustrated in Table 9 
         [0000]    
       
         
               
               
               
               
             
           
               
                 TABLE 9 
               
               
                   
               
               
                   
                   
                 Content of 
                   
               
               
                 Proof 
                   
                 Cryptotanshinone (mg/g 
                 Conversion Rate of 
               
               
                 Tests 
                 Yield Rate (%) 
                 extract) 
                 Cryptotanshinone (%) 
               
               
                   
               
             
             
               
                 1 
                 4.82 
                 86.98 
                 95.28 
               
               
                 2 
                 4.85 
                 86.06 
                 94.86 
               
               
                 3 
                 4.84 
                 86.30 
                 94.93 
               
               
                   
               
             
          
         
       
     
       5.0 Activity Enhancement Step 
       [0240]    The applicant sought a methodology to selectively enhance the content of tanoshinone compounds in the extract and the methodology in this example demonstrates a process which, in the first instance increases the content of tanoshines approximately two fold but significantly increases the relative content of cryptotanoshinone content by an even greater factor. This is particular advantageous from a pharmaceutical activity perspective. 
       5.1 Design of the Experiment 
     Improved Extraction/Purification Process 
       [0241]    The process is illustrated in  FIG. 6  and comprises the following steps: 
         [0000]    1. Extract Danshen raw material with 95% ethanol with percolation and concentrate using vacuum drying;
 
2. Dissolve the extract with water;
 
3. Extract the solution with ethyl acetate; and
 
4. Purify with silica gel column;
 
       5.1.1 Purification of Danshen Fractions 
       [0242]    Danshen raw material: Batch No. DS0601. 
         [0243]    Select 106.5 g of clean raw material and crush to a powder. Soak in an appropriate quantity of 95% ethanol for 24 hours, distribute it well into the percolator, and extract with percolation with a flow rate of 15 ml/min. Collect the colature of 12 times the raw material, i.e. 1280 ml. Recover the ethanol under vacuum. Vacuum dry at 70° C. to get the final extract, 10.2 g. 
         [0244]    The final percolation extract of Danshen was given a batch number—SL0601. 
       5.1.2 Preparation of Ethyl Acetate Extract 
       [0245]    Take the final extract of Danshen mentioned in 5.1.1 above, add 100 ml water and extract it with 150 ml ethyl acetate twice; combine the two ethyl acetate solutions and dehydrate. Wash it with 100 ml and 50 ml water respectively and dehydrate. Concentrate the ethyl acetate extract under vacuum to obtain 5.765 g of a solid extract which was given a batch number—YY0601. 
       5.1.3 Silica Gel Purification 
       [0246]    Measure 2.0022 g of Danshen ethyl acetate extract (as 5.1.2), dissolve it with acetone, mix it fully with silica gel and remove the solvent. Apply the sample to a silica gel column, elute with petroleum ether—acetone in different proportions. Collect 50 ml eluate in each fraction 
         [0247]    The fractions collected are shown in Table 10 below: 
         [0000]    
       
         
               
               
               
               
             
               
               
               
               
             
           
               
                   
                 TABLE 10 
               
               
                   
                   
               
               
                   
                 Types of Eluate 
                 Volume (ml) 
                 Portions collected 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 petroleum ether - acetone 
                 900 
                 18 
               
               
                   
                 (97:3) 
               
               
                   
                 petroleum ether - acetone 
                 300 
                 6 
               
               
                   
                 (95:5) 
               
               
                   
                 petroleum ether - acetone 
                 300 
                 6 
               
               
                   
                 (90:10) 
               
               
                   
                 petroleum ether - acetone 
                 350 
                 7 
               
               
                   
                 (80:20) 
               
               
                   
                 petroleum ether - acetone 
                 500 
                 1 
               
               
                   
                 (50:50) 
               
               
                   
                 methanol 
                 500 
                 1 
               
               
                   
                   
               
             
          
         
       
     
         [0248]    Each fraction was applied on silica gel TLC plates, and petroleum ether—acetone was used as the developing system. Examination was under natural sunlight. The TLC fingerprints are shown in  FIG. 7   
         [0249]    Based on the TLC result a number of consecutive fractions were merged as follows:
       2nd and 3rd;   5th to 8th;   10th to 13th;   14th and 5th;   16th to 18th;   19th to 21st;   23rd and 24th;   25th and 26th;   28th to 30th;   31st to 34th;   35th and 36th.       
 
         [0261]    The resulting 20 new fractions were analyzed with Silical gel TLC and the results are shown in  FIG. 8 . 
         [0262]    The fractions showing the highest contents of cryptotanshinone and dihydrotanshinone, i.e. the 7th to the 13th samples, were combineed as “purified tanshinones” (0.420 g). This purified tanshinone containing compound extract was given a batch number: JZ0601. 
         [0263]    The ethyl acetate extract of Danshen (Lane 1), the purified tanshinones (Lane 2) and the mixed tanshinone standards were examined with silica gel TLC. The TLC fingerprint is shown in  FIG. 9 . The compounds from bottom to top are respectively:
       Dihydrotanshinone,   Cryptotanshinone,   Tanshinone I, and   Tanshinone IIA       
 
         [0268]    The chromatographic conditions were as described in 3.1.3 above (Table 3 gradient 1). 
       5.1.4 Samples 
       [0269]    The samples obtained at the three stages, namely:
       Danshen percolation extract, SL0601,   Danshen ethyl acetate extract, YY0601, and   Purified tanshinones, JZ0601, were additionally subjected to HPLC chromatographic analysis as described in 3.1.       
 
       5.1.5 Method and Analysis 
       [0273]    Weigh accurately:
       Danshen percolation extract 50.8 mg,   Danshen ethyl acetate extract 19.3 mg and   Purified tanshinones 11.9 mg,       
 
         [0277]    Put them into a 10 ml volumetric flask respectively; add 8 ml of the methanol-methylene dichloride (9:1) solution, dissolve with ultrasonication, add the methanol—methylene dichloride (9:1) solution to volume, shake thoroughly and filter. Carry out HPLC analysis. The resulting HPLC fingerprints are shown in  FIGS. 10   a  to  10   d.:    
         [0278]      FIG. 10   a  is an HPLC chromatogram of a percolation extract under conditions as set out in Table 3 (gradient 1) (SL0601); 
         [0279]      FIG. 10   b  is an HPLC chromatogram of a ethyl acetate purified extract under conditions as set out in Table 3 (gradient 1) (YY0601); 
         [0280]      FIG. 10   c  is an HPLC chromatogram of a silica gel purified extract under conditions as set out in Table 3 (gradient 1) (JZ0601); and the comparator 
         [0281]      FIG. 10   d  is an HPLC chromatogram of reference compounds under conditions as set out in Table 3 (gradient 1). 
         [0282]    The content of each compound from HPLC analysis is shown in Table 11. 
         [0000]    
       
         
               
               
               
             
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
                 TABLE 11 
               
             
             
               
                   
                   
               
               
                   
                 Danshen 
                   
               
             
          
           
               
                   
                 Percolation 
                 Ethyl Acetate Extract (%) 
                 Purified Tanshinones (%) 
               
             
          
           
               
                 Samples 
                 Extract (%) 
                   
                 Conversion 
                   
                 Conversion 
               
               
                 Peak Names 
                 Content 
                 Content 
                 Rate 
                 Content 
                 Rate 
               
               
                   
               
             
          
           
               
                 Dihydrotanshinone 
                 1.59 
                 2.64 
                 93.65 
                 4.96 
                 39.45 
               
               
                 Tanshinone I 
                 2.04 
                 3.57 
                 98.97 
                 5.34 
                 31.35 
               
               
                 Cryptotanshinone 
                 4.23 
                 6.46 
                 86.41 
                 30.18 
                 97.96 
               
               
                 Tanshinone IIA 
                 7.49 
                 13.39 
                 100.97 
                 8.57 
                 13.44 
               
               
                 Total: 
                 15.35 
                 26.06 
                   
                 49.06 
               
               
                 Inventory (g) 
                 106.50 
                 10.20 
                   
                 2.00 
               
               
                 Yield (g) 
                 10.20 
                 5.77 
                   
                 0.42 
               
               
                 Yield Rate (%) 
                 9.58 
                 56.57 
                   
                 21.00 
               
               
                   
               
             
          
         
       
     
         [0283]    The content of the four identified tanshinones in the purified tanshinone extract was 49.06% and the content of cryptotanshinone was 30% as the dominant component. 
         [0284]    Silica Gel Column Chromatography was demonstrated to be an effective method for purifying cryptotanshinone. The content of cryptotanshinone rose significantly to eliminate the non-active compounds from the ethyl acetate extraction. This purified tanshinones fraction was further studied for its antibacterial activity. 
       5.1.6 Activity 
       [0285]    Samples: purified Tanshinones, Batch No. JZ0601, 
         [0286]    Test Lab: National Institute for the Control of Pharmaceutical and Biological Products (NICPBP), National Center for Drug Resistance of Bacteria Beijing, PR China. 
         [0287]    The testing solution was prepared as follows:
       1. Place 6.40 mg of the sample into a 50 ml volumetric flask and add 15 ml DMF solution (N,N-Dimethylformamide)   2. Ultrasonicate for 10 minutes.   3. Add 15 ml of water to dilute the solution and ultrasonicate immediately for 5 minutes.   4. Add water to the volume and shake well, then ultrasonicate for another 5 minutes to obtain the testing solution of 0.128 mg/ml (30% DMF concentration).       
 
       Bacterial Strains: 
       [0292]    107 strains collected and kept by National Monitoring Center for Antibiotic Resistant Bacterial (China) were used to test the activity. The strains were evaluated with the Phoenix-100 automated Microbiology System. The testing strains included:
       87 strains of  Staphylococcus aureus  (SA) including 52 strains of methicillin resistant SA (MRSA) and 35 strains of methicillin susceptible SA (MSSA);   23 strains of Coagulase-negative Staphylococci (CNS) (MRCNS 4 and MSCNS19); and   7 strains of  Streptococcus  (5 strains of  Streptococcus pneumoniae ).       
 
       Drug Susceptibility Test: 
       [0296]    A microtitre broth dilution method (MH Broth, Oxoid Ltd UK) was used in the testing. The minimum inhibitory concentrations (MICs) of flucloxacillin/ampicillin on the isolated strains were tested based on the methods described on America CLSI/NCCLS Antimicrobial Susceptibility Testing (AST) (2006) 
         [0297]    Bacterial strains used for quality control:
         Staphylococcus aureus , (ATCC 29213) and     Streptococcus pneumoniae  (ATCC49619).       
 
       Statistical Analysis: 
       [0300]    WHONET software (version 5.3) supplied by WHO. 
       Results: 
       [0301]    1. The MIC of penicillin against  Staphylococcus aureus , (ATCC 29213) and  Streptococcus Pneumoniae  (ATCC49619) were conformed to CLSUNCCLS (2006).
 
2. The MICs of JZ0601 against  Staphylococcus  and  Streptococcus  were as set out in Tables 12.
 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 12 
               
               
                   
               
               
                   
                 Testing 
                 MIC50 
                 MIC90 
                 MIC 
               
               
                 Strains 
                 Nos. 
                 (μg/ml) 
                 (μg/ml) 
                 (μg/ml) Range 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 SA 
                 87 
                 8 
                 16 
                 0.25-16 
               
               
                 MSSA 
                 35 
                 8 
                 16 
                 0.25-16 
               
               
                 MRSA 
                 52 
                 8 
                 16 
                   4-16 
               
               
                 CNS 
                 23 
                 8 
                 16 
                   2-16 
               
               
                 
                   Streptococcus 
                 
                 7 
                 16 
                 16 
                 16 
               
               
                   
               
             
          
         
       
     
       Summary 
       [0302]    JZ0601 showed good activity against  Staphylococcus  including methicillin susceptible and resistant strains, as well as  Streptococcus , particularly  Streptococcus pneumoniae . The MICs of JZ0601 on all strains are as set out in Tables 13-16: 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 13 
               
             
             
               
                   
               
               
                 MICs of JZ0601 on 35 strains of methicillin 
               
               
                 susceptible  Staphylococcus aureus   
               
             
          
           
               
                 serial 
                   
                 MIC 
               
               
                 number 
                 Strain code 
                 (μg/ml) 
               
               
                   
               
             
          
           
               
                 1 
                 sau865 
                 8 
               
               
                 2 
                 guangzongsau 
                 8 
               
               
                 3 
                 Guangzongsau-2 
                 8 
               
               
                 4 
                 tjsau62 
                 4 
               
               
                 5 
                 tjsau52 
                 2 
               
               
                 6 
                 sau511524 
                 8 
               
               
                 7 
                 sau511837 
                 8 
               
               
                 8 
                 saubeisan50 
                 8 
               
               
                 9 
                 a838 
                 &gt;8 
               
               
                 10 
                 sau839 
                 &gt;8 
               
               
                 11 
                 mssa17 
                 8 
               
               
                 12 
                 a10 
                 &gt;8 
               
               
                 13 
                 a104 
                 8 
               
               
                 14 
                 sau59 
                 8 
               
               
                 15 
                 a85 
                 4 
               
               
                 16 
                 abeisan29 
                 4 
               
               
                 17 
                 ayou130 
                 0.25 
               
               
                 18 
                 mssa10 
                 8 
               
               
                 19 
                 mssa20 
                 &gt;8 
               
               
                 20 
                 mssa16 
                 8 
               
               
                 21 
                 saubeisan44 
                 2 
               
               
                 22 
                 mssa2 
                 8 
               
               
                 23 
                 mssa201 
                 8 
               
               
                 24 
                 mssa25 
                 8 
               
               
                 25 
                 mssa2949 
                 8 
               
               
                 26 
                 mssa8 
                 8 
               
               
                 27 
                 mssa855 
                 4 
               
               
                 28 
                 tjsau53 
                 0.25 
               
               
                 29 
                 saubeisan44 
                 2 
               
               
                 30 
                 tjsau52 
                 2 
               
               
                 31 
                 saubeisan45 
                 8 
               
               
                 32 
                 tjsau59 
                 8 
               
               
                 33 
                 tjsau60 
                 &gt;8 
               
               
                 34 
                 tjsau69 
                 4 
               
               
                 35 
                 ayou196 
                 8 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 14 
               
             
             
               
                   
               
               
                 MICs of JZ0601on 52 strains of methicillin 
               
               
                 resistant  Staphylococcus Aureus   
               
             
          
           
               
                 serial 
                   
                 MIC 
               
               
                 number 
                 Strain code 
                 (μg/ml) 
               
               
                   
               
             
          
           
               
                 1 
                 gdmrsa9103 
                 4 
               
               
                 2 
                 mrsa18 
                 8 
               
               
                 3 
                 beisanmrsa16 
                 4 
               
               
                 4 
                 beisanmrsa21 
                 8 
               
               
                 5 
                 mrsa15 
                 8 
               
               
                 6 
                 mu3* 
                 8 
               
               
                 7 
                 mu50* 
                 4 
               
               
                 8 
                 mrsa420 
                 8 
               
               
                 9 
                 mrsa13 
                 8 
               
               
                 10 
                 mrsa19 
                 8 
               
               
                 11 
                 mrsa2 
                 4 
               
               
                 12 
                 511400 
                 4 
               
               
                 13 
                 mrsa3409 
                 8 
               
               
                 14 
                 mrsa3479 
                 8 
               
               
                 15 
                 zjmrsa619057 
                 &gt;8 
               
               
                 16 
                 mrsa5002 
                 8 
               
               
                 17 
                 gdmrsa69 
                 4 
               
               
                 18 
                 mrsa5120 
                 8 
               
               
                 19 
                 mrsa60578 
                 8 
               
               
                 20 
                 wuhao 
                 8 
               
               
                 21 
                 mrsa5153 
                 8 
               
               
                 22 
                 zjmrsa614036 
                 8 
               
               
                 23 
                 zjmrsa614036 
                 8 
               
               
                 24 
                 zjmrsa613066 
                 &gt;8 
               
               
                 25 
                 zjmrsa612011 
                 &gt;8 
               
               
                 26 
                 zjmrsa708015 
                 &gt;8 
               
               
                 27 
                 zjmrsa705013 
                 4 
               
               
                 28 
                 zjmrsa611049 
                 8 
               
               
                 29 
                 zjmrsa709037 
                 &gt;8 
               
               
                 30 
                 zjmrsa611045 
                 8 
               
               
                 31 
                 mrsa40452 
                 4 
               
               
                 32 
                 gxmrsa7497 
                 8 
               
               
                 33 
                 zjmrsa160578 
                 4 
               
               
                 34 
                 zjmrsa607022 
                 4 
               
               
                 35 
                 zjmrsa608007 
                 &gt;8 
               
               
                 36 
                 gxmrsa7402 
                 8 
               
               
                 37 
                 mrsa516484 
                 4 
               
               
                 38 
                 mrsa516467 
                 8 
               
               
                 39 
                 mrsa516390 
                 4 
               
               
                 40 
                 gxmrsa3536 
                 8 
               
               
                 41 
                 gxmrsa4221 
                 8 
               
               
                 42 
                 gxmrsa5450 
                 8 
               
               
                 43 
                 zjmrsa809078 
                 &gt;8 
               
               
                 44 
                 gxmrsa7345 
                 &gt;8 
               
               
                 45 
                 mrsa127007 
                 8 
               
               
                 46 
                 gdmrsa12 
                 8 
               
               
                 47 
                 zjmrsa731066 
                 8 
               
               
                 48 
                 zjmrsa723053 
                 8 
               
               
                 49 
                 zjmrsa723017 
                 &gt;8 
               
               
                 50 
                 zjmrsa711067 
                 8 
               
               
                 51 
                 zjmrsa710008 
                 8 
               
               
                 52 
                 gxmrsa6372 
                 8 
               
               
                   
               
               
                 Note: 
               
               
                 *vancocin intermediary  Staphylococcus Aureus   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 15 
               
             
             
               
                   
               
               
                 MICs of JZ0601on 23 strains of Coagulase-negative  Staphylococci   
               
             
          
           
               
                 serial 
                   
                 MIC 
               
               
                 number 
                 Strain code 
                 (μg/ml) 
               
               
                   
               
             
          
           
               
                 1 
                 sep207481 
                 8 
               
               
                 2 
                 sep786 
                 8 
               
               
                 3 
                 sep207357 
                 8 
               
               
                 4 
                 who6 
                 8 
               
               
                 5 
                 sep999 
                 8 
               
               
                 6 
                 sep339 
                 4 
               
               
                 7 
                 209166 
                 &gt;8 
               
               
                 8 
                 sep207518 
                 8 
               
               
                 9 
                 sep474 
                 8 
               
               
                 10 
                 sep207742 
                 &gt;8 
               
               
                 11 
                 sep212419 
                 2 
               
               
                 12 
                 104023 
                 4 
               
               
                 13 
                 105510 
                 &gt;8 
               
               
                 14 
                 sep154 
                 8 
               
               
                 15 
                 537244 
                 8 
               
               
                 16 
                 212229 
                 2 
               
               
                 17 
                 212158 
                 2 
               
               
                 18 
                 SEP154-2 
                 8 
               
               
                 19 
                 1559 
                 &gt;8 
               
               
                 20 
                 Zhongshanyi# 
                 &gt;8 
               
               
                 21 
                 mrse511453# 
                 8 
               
               
                 22 
                 mrse36915# 
                 8 
               
               
                 23 
                 msse79647# 
                 8 
               
               
                   
               
               
                 Note: 
               
               
                 #methicillin resistant CNS 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 16 
               
             
             
               
                   
               
               
                 MICs of JZ0601 on 7 strains of  Streptococcus   
               
             
          
           
               
                 serial 
                   
                 MIC 
               
               
                 number 
                 Strain code 
                 (μg/ml) 
               
               
                   
               
               
                 1 
                 spy19615 
                 16 
               
               
                 2 
                 *spnA534 
                 16 
               
               
                 3 
                 *spn724 
                 16 
               
               
                 4 
                 *spn6305 
                 16 
               
               
                 5 
                 *spn16732 
                 16 
               
               
                 6 
                 *hb733-15 
                 16 
               
               
                 7 
                 sag13813 
                 16 
               
               
                   
               
               
                 Note: 
               
               
                 * Streptococcus pneumoniae . 
               
             
          
         
       
     
         [0303]    The above data demonstrates the benefits of using an extract which is not only characterized by its high tanoshinone compounds content but more particularly one with enhanced levels of particularly cryptotanoshinone. 
         [0304]    However, the methodology described to obtain this highly purified extract was not suitable for scale up and accordingly an alternative scalable methodology had to be developed. This is described below: 
       6.0 Scalable Methodology 
       [0305]    The preferred commercial scale production process for obtaining a selectively purified tanshinone compounds containing extract from the root of  Salvia  Spp, and more particularly one specifically enriched in cryptotanshinone, is set out with reference to  FIG. 11   
       6.1 General Methodology 
       [0000]    
       
         
           
             1. Take Danshen raw material; 
             2. Soak it with a sufficient volume of high concentration, typically 95%, ethanol for a time sufficient to solubilise the desired compounds, typically 24 hours; 
             3. Place the material into a percolator and extract using a percolation method; 
             4. Collect the ethanol solution with, typically, 12 times volume of its raw material at the desired percolation speed, preferably, 15 ml·min-1; 
             5. Concentrate the liquid extract under vacuum and recover the ethanol to obtain the ethanol extract. 
           
         
       
     
         [0311]    The yield rate is about 5-9%. 
         [0312]    The content of the total tanshinones is about 8%. 
       6.2 Purification 
       [0000]    
       
         
           
             1. Dissolve the ethanol extract with about 10 times of water, 
             2. Dispose of the aqueous solution and collect the precipitate. 
           
         
       
     
         [0315]    The yield rate is about 40% and the content of the total tanshinones is about 20%.
       3. Dissolve the precipitates with 60% ethanol and place the material onto an AB-8 macroporous resin column.   4. Elute with 60% ethanol and dispose of the fraction   5. Elute with 70% ethanol to obtain the selectively purified fraction containing tanshinones.       
 
         [0319]    The yield rate is 16-22% 
         [0320]    The content of the total tanshinones is up to 40%. 
       6.3 Specification 
       [0321]    The resulting purified extract has a specification as set out in Table 17 
         [0000]    
       
         
               
               
               
             
               
               
               
               
             
               
               
               
             
           
               
                   
                 TABLE 17 
               
               
                   
                   
               
               
                   
                 ITEMS 
                 SPECIFICATION 
               
               
                   
                   
               
             
             
               
                   
                 Appearance 
                 Redish-brown colour 
               
               
                   
                 Source 
                 Purified extract made from the dry root and 
               
               
                   
                   
                 rhizome of Salvia miltiorrhiza Bge. (Labiatae 
               
               
                   
                   
                 family) 
               
               
                   
                 Chemical 
                 Total Tanshinones ≧40% (including 
               
               
                   
                 Constituents 
                 Dihydrotanshinone, Cryptotanshinone, 
               
               
                   
                   
                 Tanshinone I and Tanshinone IIA) 
               
               
                   
                 Identification 
                 (1) TLC Identification corresponds to the 
               
               
                   
                   
                 standard chromatography 
               
               
                   
                   
                 (2) HPLC Identification corresponds to the 
               
               
                   
                   
                 standard chromatography 
               
             
          
           
               
                   
                 Inspection 
                 (1) Moisture 
                 &lt;5.0% 
               
               
                   
                   
                 (2) Total Ash 
                 &lt;5.0% 
               
               
                   
                   
                 (3) Acid Insoluble Ash 
                 &lt;2.0% 
               
               
                   
                   
                 (4) Heavy Metals 
                  &lt;10 ppm 
               
               
                   
                   
                 (5) Arsenic 
                   &lt;2 ppm 
               
               
                   
                 Microbial 
                 (1) Total Plate Count 
                 &lt;1000 cfu/g 
               
               
                   
                 Detection 
                 (2) Fungal&amp;Yeast 
                  &lt;100 cfu/g 
               
               
                   
                   
                 (3)  E. coli   
                 Neg. 
               
               
                   
                   
                 (4)  Salmonelia   
                 Neg. 
               
             
          
           
               
                   
                 Storage 
                 Store in a cool, dry place and avoid sunlight 
               
               
                   
                   
               
             
          
         
       
     
         [0322]    The extract can be identified chromatographically be either TLC or HPLC as set out below: 
       6.4 TLC 
     Preparation of the Standard Reference Solution 
       [0323]    1. Take 1 mg each of:
       Dihydrotanshinone,   Cryptotanshinone,   Tanshinone I, and   Tanshinone IIA standard reference chemicals
 
2. Add 2 ml of a mixed solution of methanol-methylene dichloride (9:1) to dissolve the substances to obtain the mixed standard reference solution.
       
 
       Preparation of the Test Solution 
       [0328]    1. Weigh 15 mg of JZ0702 (extract as  FIG. 11 ),
 
2. Add 10 ml of the mixed solution of methanol—methylene dichloride (9:1) and dissolve the sample by ultrasonication to obtain the test solution.
 
       Detection Method 
       [0329]    According to the TLC method described in the Chinese Pharmacopoeia 2005 Version, Vol. 1, Appendix VI B, 
         [0000]    1. Place 5 1 11 of above-mentioned test solution and 3 1 11 of the mixed standard solution onto a silica G plate,
 
2. Develop the plate with petroleum a mixed solvent: ether—tetrahydrofuran—methanol (10:2:1).
 
3. Dry the plate after development and observed the plate under daylight.
 
         [0330]    The test sample showed colour spots in the position corresponding to that of the reference chemicals in the chromatogram ( FIG. 12 ). 
         [0331]    The TLC methodology is qualitative rather than quantitative. 
       6.5 HPLC 
     Chromatographic Conditions 
       [0332]    As section 3.1.3. Table 4 (gradient 2) 
       Preparation of Reference Solution 
       [0333]    Weigh accurately 1 mg each of:
       Dihydrotanshinone,   Cryptotanshine, and   Tanshinone 1 and 2 mg of:   Tanshinone IIA       
 
         [0338]    Place them into a 10 ml volumetric flask, add 8 ml of the mixed solvent of methanol -methylene dichloride (9:1) and ultrasonicate for 5 min. 
         [0339]    Add the mixed solvent to the volume and shake thoroughly to obtain the reference solution. 
       Preparation of Sample Solutions 
       [0340]    1. Weigh accurately 10 mg of JZ0702 and put it into a 10 ml volumetric flask.
 
2. Add 8 ml of mixed solvent methanol—methylene dichloride (9:1) and ultrasonicate for 5 min.
 
3. Add the mixed solvent to the volume and shake fully to obtain the test solution.
 
       Assay Method 
       [0341]    Inject 5 μl each of both the test solution and the reference solution respectively into an HPLC column and run. The profile for the extract is illustrated in  FIG. 13  and compared to the reference sample  FIG. 14   
         [0342]    From this the four tanshinones were calculated to be 42.89% of the extract, calculated as:
       Dihydrotanshinone (3.65%),   Cryptotanshinone (18.95%),   Tanshinone I (3.82%), and   Tanshinone IIA (16.47%)       
 
       7.0 Activity against  Propionibacterium acnes    
     Objective: 
       [0347]    To evaluate the in vitro activity of a selectively purified tanshinone compounds containing extract against the anaerobic bacteria  Propionibacterium acnes  ( P. acnes ). 
       Methods: 
       [0348]    A selectively purified tanshinone compounds containing extract was added to wells containing  P. acnes  (ATCC 6919; 1×10 4  to 5×10 5  CFU/mL) in culture, grown under controlled conditions (reinforced clostridial medium, 37° C.). Final inoculum concentration was determined by reference to a standard optical density curve and adjusted as necessary. Wells were incubated for 48 hours at 37° C. and examined for growth of culture. Wells were scored positive (+) for inhibition of growth, or negative (−) for no effect on growth. Eight different concentrations ranging from 0.03 μg/mL-100 μg/mL were screened. Ampicillin was run at a concentration of 0.1 μg/mL as a positive control. MIC and MBC were calculated. 
       Results: 
       [0349]    The extract was tested at half-log concentrations of 0.03 μg/mL to 100 μg/mL for potential bactericidal activity against  P. acnes . From this, a minimum inhibitory concentration (MIC) of 10 μg/mL was determined, and a minimum bactericidal concentration (MBC) of 30 μg/mL was calculated. The results are shown in Table 18. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 18 
               
             
             
               
                   
               
               
                 Microbial analysis scoring of PYN6&#39;s inhibitory affect on  P. acnes   
               
             
          
           
               
                   
                 Growth inhibition 
                   
               
               
                 Concentration 
                 (MIC) 
                 Growth inhibition (MBC) 
               
               
                   
               
               
                 0.03 μg/mL   
                 − 
                 − 
               
               
                 0.1 μg/mL  
                 − 
                 − 
               
               
                 0.3 μg/mL  
                 − 
                 − 
               
               
                  1 μg/mL 
                 − 
                 − 
               
               
                  3 μg/mL 
                 − 
                 − 
               
               
                 10 μg/mL 
                 + 
                 − 
               
               
                 30 μg/mL 
                 + 
                 + 
               
               
                 100 μg/mL  
                 + 
                 + 
               
               
                   
               
               
                 (− = no inhibition; + = inhibition). 
               
             
          
         
       
     
       Conclusion 
       [0350]    The extract showed antibiotic activity against  P. acnes  with a MIC of 10 μg/mL (MBC of 30 μg/mL). 
       8.0 Experiment to Determine Potential for Resistance Development 
       [0351]    This experiment was conducted to test for the rapid development of resistance in  Staphylococcus aureus  in the presence of sub-inhibitory doses of the active extract. 
       Materials and Methods 
     Bacterial Strains 
       [0352]    Oxford  Staphylococcus aureus  (NCTC 6571) and 3 clinical isolates of MRSA were tested, T3, 102 and MRSA 99. All clinical strains were from The Royal London, St Bartholomew&#39;s or Newham hospitals in London. Each strain had been identified as an MRSA. 
         [0000]    Active extract/Ciprofloxacin/Gentamicin 
         [0353]    Active extract powder (Phynova) 
         [0354]    Ciprofloxacin (Bayer Pharmaceutical) 
         [0355]    Gentamicin powder (Sigma chemicals) 
       Solubilisation of PYN6 
       [0356]    The standard method using DMF was used. Manufacturer&#39;s method using DMF where the solution was unfiltered prior to use. Solution—640 mg/1 was added to 3 ml DMF, sonicated in a sonic water bath then made up to 10 ml with water as per instructions. 
       Development of Resistance Testing 
       [0357]    High concentrations of organisms (10 7 cfu/m1 −1 ) were grown in sub—inhibitory concentrations 0, 2 and 8 mg l −1  of the active extract. MICs had been determined previously as 16 mg l −‘ . 
         [0358]    Organisms were sub-cultured into fresh active extract media at day 4 and again at day 7. MICs were checked weekly using standard methods. Subcultures were carried out in triplicate. 
       Results 
       [0359]    No change in MIC was observed over the 3 week test period. No growth indicates the MIC level. Three MRSA clinical isolates have been tested and control Strain Oxford  Staphylococcus aureus  Tables 19, 20, 21 and 22. Table 23 shows the comparative development of resistance with Ciprofloxicin and table 24 for gentamicin. 
         [0360]    There was no development of resistance for the active extract or gentamicin. The MICs to ciprofloxacin increased after 2 weeks treatment. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 19 
               
             
             
               
                   
               
               
                 Subculture results showing MIC after extended 
               
               
                 culture at sub-inhibitory doses for MRSA T3 
               
             
          
           
               
                   
                 Sub culture con- 
                   
                 Growth 
                 Growth 
                 Growth 
                 Growth 
                 Growth 
               
               
                 Day 
                 centration mg l −1   
                 Organism 
                 Control 
                 2 mg l −1   
                 8 mg l −1   
                 16 mg l −1   
                 32 mg l −1   
               
               
                   
               
             
          
           
               
                 1 
                 0 
                 T3 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 1 
                 0 
                 T3 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 1 
                 4 
                 T3 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 1 
                 4 
                 T3 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 1 
                 8 
                 T3 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 1 
                 8 
                 T3 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 7 
                 0 
                 T3 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 7 
                 0 
                 T3 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 7 
                 4 
                 T3 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 7 
                 4 
                 T3 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 7 
                 8 
                 T3 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 7 
                 8 
                 T3 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 14 
                 0 
                 T3 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 14 
                 0 
                 T3 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 14 
                 4 
                 T3 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 14 
                 4 
                 T3 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 14 
                 8 
                 T3 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 14 
                 8 
                 T3 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 21 
                 0 
                 T3 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 21 
                 0 
                 T3 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 21 
                 4 
                 T3 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 21 
                 4 
                 T3 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 21 
                 8 
                 T3 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 21 
                 8 
                 T3 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 20 
               
             
             
               
                   
               
               
                 Subculture results showing MIC after extended 
               
               
                 culture at sub-inhibitory doses for MRSA 99 
               
             
          
           
               
                   
                 Sub culture con- 
                   
                 Growth 
                 Growth 
                 Growth 
                 Growth 
                 Growth 
               
               
                 Day 
                 centration mg l −1   
                 Organism 
                 Control 
                 2 mg l −1   
                 8 mg l −1   
                 16 mg l −1   
                 32 mg l −1   
               
               
                   
               
             
          
           
               
                 1 
                 0 
                 99 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 1 
                 0 
                 99 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 1 
                 4 
                 99 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 1 
                 4 
                 99 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 1 
                 8 
                 99 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 1 
                 8 
                 99 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 7 
                 0 
                 99 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 7 
                 0 
                 99 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 7 
                 4 
                 99 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 7 
                 4 
                 99 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 7 
                 8 
                 99 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 7 
                 8 
                 99 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 14 
                 0 
                 99 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 14 
                 0 
                 99 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 14 
                 4 
                 99 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 14 
                 4 
                 99 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 14 
                 8 
                 99 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 14 
                 8 
                 99 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 21 
                 0 
                 99 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 21 
                 4 
                 99 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 21 
                 4 
                 99 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 21 
                 8 
                 99 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 21 
                 8 
                 99 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 21 
               
             
             
               
                   
               
               
                 Subculture results showing MIC after extended 
               
               
                 culture at sub-inhibitory doses for MRSA 99 
               
             
          
           
               
                   
                 Sub culture con- 
                   
                 Growth 
                 Growth 
                 Growth 
                 Growth 
                 Growth 
               
               
                 Day 
                 centration mg l −1   
                 Organism 
                 Control 
                 2 mg l −1   
                 8 mg l −1   
                 16 mg l −1   
                 32 mg l −1   
               
               
                   
               
             
          
           
               
                 1 
                 0 
                 102 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 1 
                 0 
                 102 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 1 
                 4 
                 102 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 1 
                 4 
                 102 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 1 
                 8 
                 102 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 1 
                 8 
                 102 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 7 
                 0 
                 102 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 7 
                 0 
                 102 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 7 
                 4 
                 102 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 7 
                 4 
                 102 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 7 
                 8 
                 102 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 7 
                 8 
                 102 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 14 
                 0 
                 102 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 14 
                 0 
                 102 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 14 
                 4 
                 102 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 14 
                 4 
                 102 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 14 
                 8 
                 102 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 14 
                 8 
                 102 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 21 
                 0 
                 102 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 21 
                 0 
                 102 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 21 
                 4 
                 102 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 21 
                 4 
                 102 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 21 
                 8 
                 102 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 21 
                 8 
                 102 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 22 
               
             
             
               
                   
               
               
                 Subculture results showing MIC after extended culture 
               
               
                 at sub-inhibitory doses for Oxford  Staph aureus.   
               
             
          
           
               
                   
                 Sub culture con- 
                   
                 Growth 
                 Growth 
                 Growth 
                 Growth 
                 Growth 
               
               
                 Day 
                 centration mg l −1   
                 Organism 
                 Control 
                 2 mg l −1   
                 8 mg l −1   
                 16 mg l −1   
                 32 mg l −1   
               
               
                   
               
             
          
           
               
                 1 
                 0 
                 OX 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 1 
                 0 
                 OX 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 1 
                 4 
                 OX 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 1 
                 4 
                 OX 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 1 
                 8 
                 OX 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 1 
                 8 
                 OX 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 7 
                 0 
                 OX 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 7 
                 0 
                 OX 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 7 
                 4 
                 OX 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 7 
                 4 
                 OX 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 7 
                 8 
                 OX 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 7 
                 8 
                 OX 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 14 
                 0 
                 OX 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 14 
                 0 
                 OX 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 14 
                 4 
                 OX 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 14 
                 4 
                 OX 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 14 
                 8 
                 OX 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 14 
                 8 
                 OX 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 21 
                 0 
                 OX 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 21 
                 0 
                 OX 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 21 
                 4 
                 OX 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 21 
                 4 
                 OX 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 21 
                 8 
                 OX 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 21 
                 8 
                 OX 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 23 
               
             
             
               
                   
               
               
                 Subculture results showing MIC after extended culture at 
               
               
                 sub-inhibitory doses of Ciprofloxacin for MRSA T3 (MIC 0.5 mg l −1)   
               
             
          
           
               
                   
                   
                 Sub culture 
                   
                   
                   
               
               
                   
                   
                 concentration 
                   
                 Growth 
                 Growth 
               
               
                   
                 Day 
                 mg l −1   
                 Organism 
                 Control 
                 8 mg l −1   
               
               
                   
                   
               
             
          
           
               
                   
                 1 
                 0 
                 T3 
                 + 
                 − 
               
               
                   
                 1 
                 0 
                 T3 
                 + 
                 − 
               
               
                   
                 1 
                 0.06 
                 T3 
                 + 
                 − 
               
               
                   
                 1 
                 0.06 
                 T3 
                 + 
                 − 
               
               
                   
                 7 
                 0 
                 T3 
                 + 
                 − 
               
               
                   
                 7 
                 0 
                 T3 
                 + 
                 − 
               
               
                   
                 7 
                 0.06 
                 T3 
                 + 
                 − 
               
               
                   
                 7 
                 0.06 
                 T3 
                 + 
                 − 
               
               
                   
                 14 
                 0 
                 T3 
                 + 
                 − 
               
               
                   
                 14 
                 0 
                 T3 
                 + 
                 − 
               
               
                   
                 14 
                 0.06 
                 T3 
                 + 
                 + 
               
               
                   
                 14 
                 0.06 
                 T3 
                 + 
                 + 
               
               
                   
                 21 
                 0 
                 T3 
                 + 
                 − 
               
               
                   
                 21 
                 0 
                 T3 
                 + 
                 − 
               
               
                   
                 21 
                 0.06 
                 T3 
                 + 
                 + 
               
               
                   
                 21 
                 0.06 
                 T3 
                 + 
                 + 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 24 
               
             
             
               
                   
               
               
                 Subculture results showing MIC after extended culture at 
               
               
                 sub-inhibitory doses of Gentamicin for MRSA T3 (MIC 4 mg l −1 ) 
               
             
          
           
               
                   
                   
                 Sub culture 
                   
                   
                   
               
               
                   
                   
                 concentration 
                   
                 Growth 
                 Growth 
               
               
                   
                 Day 
                 mg l −1   
                 Organism 
                 Control 
                 8 mg l −1   
               
               
                   
                   
               
             
          
           
               
                   
                 1 
                 0 
                 T3 
                 + 
                 − 
               
               
                   
                 1 
                 0 
                 T3 
                 + 
                 − 
               
               
                   
                 1 
                 1 
                 T3 
                 + 
                 − 
               
               
                   
                 1 
                 1 
                 T3 
                 + 
                 − 
               
               
                   
                 7 
                 0 
                 T3 
                 + 
                 − 
               
               
                   
                 7 
                 0 
                 T3 
                 + 
                 − 
               
               
                   
                 7 
                 1 
                 T3 
                 + 
                 − 
               
               
                   
                 7 
                 1 
                 T3 
                 + 
                 − 
               
               
                   
                 14 
                 0 
                 T3 
                 + 
                 − 
               
               
                   
                 14 
                 0 
                 T3 
                 + 
                 − 
               
               
                   
                 14 
                 1 
                 T3 
                 + 
                 − 
               
               
                   
                 14 
                 1 
                 T3 
                 + 
                 − 
               
               
                   
                 21 
                 0 
                 T3 
                 + 
                 − 
               
               
                   
                 21 
                 0 
                 T3 
                 + 
                 − 
               
               
                   
                 21 
                 1 
                 T3 
                 + 
                 − 
               
               
                   
                 21 
                 1 
                 T3 
                 + 
                 − 
               
               
                   
                   
               
             
          
         
       
     
       Conclusions 
       [0361]    The extract is active against MRSA at inhibitory levels of 16 mg l −1  and above. There was no change in the MIC level over the test period for the strains tested. No rapid development of resistance occurred and the test period was beyond that used by Boos M. et al. (In Vitro Development of Resistance to Six Quinolones in  Streptococcus pneumoniae, Streptococcus pyogenes , and  Staphylococcus aureus . Antimicrob. Agents Chemother. 45, 938-942) to demonstrate a seven fold increase in resistance to quinilones within 10 days. 
       9.0 Experiment to Determine Activity of PYN 6 in a Gel Formulation 
       [0362]    A number of different gel formulations containing extracts of the invention were prepared and tested on clinical MRSA isolates to determine the suitability of the active extract for topical delivery. 
         [0363]    10 clinical isolates of MRSA were tested. 
         [0364]    Agar diffusion tests (based on BSAC standard methods) were used to compare the relative activity of different gels against MRSA. Zones of inhibition around each 100 μl sample were compared. PYN6 was prepared by dissolving in DMF and then water, final concentration 500 mg/L. 
       Results 
       [0365]      FIG. 15  illustrates graphically of the effect of PYN6 in water and in gel formulations against 10 different strains of MRSA 
         [0366]    Gel 1 and 1(2)-Faith in Nature gels (glycerin based) 
         [0367]    Ge16QM-QM thin gel (Proprietary—colloidal detergent based) 
         [0368]    Ge1MQM-QM medium gel (Proprietary—colloidal detergent based) 
         [0369]    Ge1FQM-QM full gel (Proprietary—colloidal detergent based) 
       Conclusions 
       [0370]    All gels showed activity at a level of 500 mg/L. All were comparable to the activity of 500 mg/L PYN6 in water. PYN6 works in gel or water formulation and has potential as a topical antimicrobial against MRSA. 
       10.0 Experiment to Determine Activity of PYN 6 Against Individual Compounds 
     Methods 
       [0371]    Minimum Inhibitory and Minimum Bactericidal activities (MIC and MBC) of PYN6 were determined for 2 strains of MRSA using standard microtitre well methods. MICs were determined by measuring growth over 201us using spectroscopy at 490 nm. MBCs were determined by subculture from these microtitre plates onto solid media after 20 hrs and determining survival of bacteria grown in the presence of PYN6. 
       PYN6 Compounds 
       [0372]      
         [0000]    
       
         
               
               
               
             
           
               
                   
                   
               
               
                   
                 Code 
                 PYN6 compound 
               
               
                   
                   
               
             
             
               
                   
                 A 
                 Tashinone 1 
               
               
                   
                 B 
                 Tashinone 11A 
               
               
                   
                 C 
                 Dyhydrotashinone 
               
               
                   
                 D 
                 Cryptotashinone 
               
               
                   
                 PYN6 
                 PYN6 
               
               
                   
                   
               
             
          
         
       
     
       Results 
       [0373]    TEST 1 MIC and MBC comparing PYN6 to A, B, C and D against MRSA 98 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Code 
                 PYN6 compound 
                 MIC mg/L 
                 MBC mg/L 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 A 
                 Tashinone 1 
                 125 
                 &lt;500 
               
               
                   
                 B 
                 Tashinone 11A 
                 125 
                 &lt;500 
               
               
                   
                 C 
                 Dyhydrotashinone 
                 8 
                 125 
               
               
                   
                 D 
                 Cryptotashinone 
                 15 
                 250 
               
               
                   
                 PYN6 
                 PYN6 
                 31 
                 125 
               
               
                   
                   
               
             
          
         
       
     
         [0374]    TEST 2 MIC and MBC comparing PYN6 to A, B, C and D against MRSA 2 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Code 
                 PYN6 compound 
                 MIC mg/L 
                 MBC mg/L 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 A 
                 Tashinone 1 
                 125 
                 &gt;500 
               
               
                   
                 B 
                 Tashinone 11A 
                 125 
                 &gt;500 
               
               
                   
                 C 
                 Dyhydrotashinone 
                 1.8 
                 62 
               
               
                   
                 D 
                 Cryptotashinone 
                 7.7 
                 125 
               
               
                   
                 PYN6 
                 PYN6 
                 7.7 
                 125 
               
               
                   
                   
               
             
          
         
       
     
         [0375]    The results demonstrate that PYN 6 an extract enriched in Cryptotashinone and Dyhydrotashinone (compared to Tashinone I and IIA-Table 5) performed very effectively.