Abstract:
A novel compound 5,8a-di-1-propyl 1-octahydronapthalen-1-(2H) One, and a process for isolating the compound and provides the process for isolation of the compound 5,8a-di-1-propyl 1-octahydronapthalen-1-(2H) One from  Trachyspermum ammi  (Ajowan caraway) seeds involving washing the seeds, drying the same and crushing it to form a powder; subjecting the powder to solvent extraction; isolating the active compound by chromatography; cooling the fractions; and subjecting the fractions to the step of crystallization in pure methanol.

Description:
FIELD OF INVENTION 
       [0001]    This invention relates to novel compound “5,8a-di-1-propyl 1-octahydronapthalen-1-(2H) one, from  Trachyspermum  (Ajowan caraway) seeds used for the prevention and treatment of Dental caries” 
       BACKGROUND OF THE INVENTION 
       [0002]    This is first isolated compound from Ajowan caraway seeds. Dental caries is most prevalent disease, next only to the common cold. It causes an irreversible damage to the grinding machinery involved in the intake of the food and hence, causes great discomfort. The changes in homeostasis of oral cavity with an overgrowth of  Streptococcus mutans  is recognized as the primary cause of the disease. Hence, most of the targets are now aimed at either elimination of this bacteria or suppression of its virulence.  S Mutans  strongly adhere and release acids by the fermentation of carbohydrates that leads to the demineralization of the tooth. This attachment is mediated mostly by the interaction of the surface proteins and extra cellular polysaccharide of bacteria. The ambiguity is basic treatment for dental caries like the use of fluoride and antibiotics vitalizes the deployment of herbal medicines. We therefore isolated and characterized a compound from Ajowan seeds to treat Dental caries with no side effects. 
       OBJECTS OF THE INVENTION 
       [0003]    An object of this invention is to propose a process for isolation of a novel compound 5,ba-di-1propy1-octachydronapthalen-1-(2H)one from  Trachyspermum  (Ajowan caraway) seeds 
         [0004]    Another object of this invention is to propose a compound which is used for the prevention and treatment of dental caries. 
         [0005]    Further object of this invention is to propose a novel compound which possesses anti-biofilm &amp; anti adherence properties. 
         [0006]    Still further object of this invention is to propose a compound which is anticariogenic activity. 
       BRIEF DESCRIPTION OF THE INVENTION 
       [0007]    According to this invention there is provided a novel compound 5,8a-di-1-propyl 1-octahydronapthalen-1-(2H) One. 
         [0008]    In accordance with this invention there is provided a process for isolation of a novel compound 5,8a-di-1-propyl 1-octahydronapthalen-1-(2H) One, from  Trachyspermum  (Ajowan caraway) seeds for the prevention and treatment of Dental caries comprising 
         [0009]    Washing the seeds, drying the same and crushing it to form a powder, 
         [0010]    Subjecting the powder to the step of solvent extraction 
         [0011]    Isolating the active compound by chromatography 
         [0012]    Cooling the fractions 
         [0013]    And subjecting the fractions to the step of crystallization in pure methanol. 
     
    
     
       BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS 
         [0014]      FIG. 1 : A reaction Scheme. 
           [0015]      FIG. 2 : Inhibitory effect of the compound on the extent of sucrose dependent cell adherence of  Streptococcus mutans . The % cell adherence is the relative amount (%) of adherent cells at different concentrations of the compound as compared to the cells attached in the absence of any compound. Each value is an average of triplicate assays and each bar indicates ±SD (n=3). 
           [0016]      FIG. 3 : Inhibitory effect of the compound on the biofilm formed by  Streptococcus mutans . The % biofilm formed is the relative (%) of biofilm formed at different concentrations of the compound as compared to that in the absence of any compound. Each value is an average of triplicate assays and each bar indicates ±SD (n=3) 
           [0017]    FIG. 4: CLSM images of  Streptococccus mutans  biofilm formed in the presence or absence of the compound after 24 h incubation. The results are expressed as the means ±standard deviations of triplicate assays. 
           [0018]      FIG. 5 : Inhibitory effect of compound on the hyrdophobicity of  Streptococcus mutans  where all the concentrations were taken in triplicates. 
           [0019]      FIG. 6 : Inhibitory effects of the compound on insoluble glucan synthesis by crude GTF of  Streptococcus mutans . The % insoluble glucan synthesis is the relative amount (%) ±SD (n=3) 
       
    
    
     DETAILED DESCRIPTION 
       [0020]    This invention relates to a process for the isolation of 5,8a-di-1propy 1-octachydronapthalen-1-(2H)-One. 
         [0021]    The seeds were washed, dried under shade and crushed to make powder. The powdered seeds were exhaustively extracted with solvents of increasing polarity. 
         [0022]    The petroleum ether extract was concentrated under reduced pressure and chromatographed over silica, gel (60-80 mesh) column (110×4cm) for the isolation of the active compound. Fractions of 150 ml were collected and evaporated. Different ratios of petroleum either and diethyl ether were run. The active compound was found in fractions 67 and 68 (petroleum ether: diethyl ether: 8:2), which showed similar pattern on the TLC plates and gave similar FTIR spectra. On crystallization with excess methanol, the active compound, with the help of different spectroscopic techniques was identified as (4aS, 5R, 8aS) 5,8a-di-1-propyl-octahyrdronapthalen-1-(2H)-one. The reaction scheme is shown in  FIG. 1 . 
         [0023]    The purified compound was obtained as white amorphous solid. Its melting point was measured as 82° C. and was uncorrected. The. FTIR spectrum band v (cm −1 ) at 2920, 2840, 1700, 1440, 1400 1310, 880 and 600 (Figure S 1 ).  1 H NMR spectrum displayed signals at δ0.89(t,J=7.2 H 3 -3′ and H 3 -3″, δ1.26 (brs, methylene protons), 1.64 (m, J=7.6 H-5 and H-8b) and 2.36 (t, J=7.6 H 2 -2) (Figure S 2 (a), (b)). The  13  C NMR gave signals at δc 210.16 (C-1), 40.25 (C-2), 22.69 (C-3), 24.75 (C-4) 35.36(C-5) (C-5), 31.93(C-6), 21.70 (C-7), 33.65(C-8), 52.60(C-8a), 46.45(C-8b), 35.09(C-1′), 19.26 (C-2′ and C-2″), 14.12 (C-3′ and C-3″) and 35.43 (C-1″0)(Figure S 3 ). The positive ion FAB MS spectrum showed peaks at m/z 236, 193,181, 166, 152, 150, 138, 98, 84, 70, 55, 43 and 42 (Figure S 4 ). The base peak was observed at m/z 55. The compound was identified as 5,8a-di-1-propyl-octahydronaptialen-1-(2H)-one. The structure is shown in figure 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    5,8 a -di-1 propyl-octahydronapthalen-1-(2H)-one 
       Determination of MIC And MBC 
       [0024]    The MIC of the compound against  Streptococcus mutans  was found to be 156.25 μg/ml. 
         [0025]    The effect of the adherence of  Streptococcus mutans  by different concentrations of the compound are given in Figure. The reduction in the adherence was found to be in a dose dependent manner. The least concentration to give at least 50% inhibition was 39.06 μg/ml. Even though the MIC is not very effective, that is, 156.25 μg/ml, the adherence properties were effected at a concentration as low as 9.77 μg/ml with 30% reduction as shown in  FIG. 2 . 
       Effect On  Streptococcus Mutans  Biofilm Formation 
       [0026]    The biofilm formation was assessed using increasing concentration of the compound. At 78.13 μg/ml., the compound inhibited the biofilm formation of  Streptococcus mutans . As observed in the adherence, the inhibitory effect was also found to be in a dose dependent manner. As expected, the compound completely inhibited the biofilm formation at 156.25 μg/ml (MIC) due to its antimicrobial effect as shown in  FIG. 3 . 
       Confocal Microscopy 
       [0027]    The biofilm of  Streptococcus mutans  was analyzed under the confocal laser scanning microscope to observe the changes in its morphology as shown in  FIG. 4 . In the absence of the compound, the cells showed clumps and aggregate that was absent when treated with sub inhibitory concentration of the compound. The cells in the treated sample were more spread out and dispersed. Each panel of the image is a representative view of 141.145 μm by 141.145 μm along the xy axis. The xy analysis provides the biofilm surface coverage, while the z-section analyses establish the thickness of the biofilm. The control cells have biofilms that are clumped and less spread along the xy lane, show a thickness of 19.74±0.5 μm. In the presence of 78.13 μ/ml of the compound, the cells show a complete absence of clumped cells and thickness is 12.8±0.7 pm. The cells wee individually scattered over the surface rather than in any arrangement. 
       Inhibition of Acid Production 
       [0028]    The pH of the culture medium was recorded after 24 h of treatment to determine the effect of this compound on acid production. The reduction in pH drop at various concentrations of the compound is reported in table. At a concentration of 78.13 μg/ml, there is maximum reduction in acid production as is evident by an increase in pH from 4.7 (control) to 7.5. This suggests that 78.13 μg/ml compound effectively inhibits acids production in  Streptococcus mutans . 
         [0000]    
       
         
               
               
               
             
               
               
               
             
           
               
                   
               
               
                 CONCENTRATION 
                 pH ± SD 
                 pH ± SD 
               
               
                 (μg/ml) 
                 (onset) 
                 (after 24 h) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 0 
                 7.44 ± 1.09 
                 4.71 ± 3.41 
               
               
                 2.44 
                 7.28 ± 0.09 
                 4.77 ± 1.69 
               
               
                 4.88 
                 7.24 ± 1.33 
                 4.84 ± 1.20 
               
               
                 9.76 
                 7.33 ± 1.11 
                 5.49 ± 1.05 
               
               
                 19.53 
                 7.38 ± 1.91 
                 5.62 ± 1.96 
               
               
                 39.01 
                 7.43 ± 1.13 
                 5.68 ± 6.78 
               
               
                 78.13 
                 7.43 ± 0.15 
                 6.48 ± 5.97 
               
               
                 156.25 
                 7.38 ± 1.29 
                 7.55 ± 2.65 
               
               
                   
               
             
          
         
       
     
       Effect of the Compound On Hydrophobocity of  Streptococcus Mutans    
       [0029]    It was shown that the cell surface hydrophobocity of  S. mutans  is affected following treatment with the compound obtained from  Trachyspermum ammi  as shown in figure. Exposing the bacteria to 9.76 μg/ml of the compound drastically reduced the cell surface hydrophobicity to more than half when compared to untreated condition as shown in  FIG. 5 . 
       Inhibition of Water Insoluble Glucan Synthesis 
       [0030]    The influence of different concentrations was assessed for the synthesis of insoluble glucan. The synthesis was found to be suppressed by the compound in dose dependent manner. There was a 25% reduction in the synthesis of insoluble glucan at a concentration as low as 4.88 μl/ml as shown in  FIG. 6 .