Abstract:
This invention relates to diterpenes of general formula (1) belonging to labdane class, isolated from  Leucas stelligera , being useful for prevention, treatment, inhibition or controlling growth and proliferation of mycobacterial activity in mammals. The invention further discloses process for isolation of the fractions containing the same.

Description:
RELATED APPLICATIONS 
     This application is a §371 of PCT/IN2012/000543 filed Aug. 6, 2012, and claims priority from Indian Patent Application No. 2224/DEL/2011 filed Aug. 5, 2011, both incorporated by reference in their entirety. 
     TECHNICAL FIELD OF INVENTION 
     This invention relates to diterpenes of general formula 1 belonging to labdane class, isolated from  Leucas stelligera , being useful for prevention, treatment, inhibition or controlling growth and proliferation of mycobacterial activity in mammals. The invention further discloses process for isolation of the fractions containing the same. 
     BACKGROUND AND PRIOR ART 
       Tuberculosis  (TB) is a major and still neglected cause of death and disability with around 2 million deaths worldwide in 2009 and 9 million infections. There is more  tuberculosis  today than at any other time in history. The emergence of drug resistant strains and confluence with HIV epidemic has turned TB into a global public health crisis. Although, available drug regimens can cure most patients, emergence of MDR, SDR and XDR-TB coupled with insufficient global drug pipeline, justifies continued efforts towards development of new drugs with new mode of action and novel structures. It has therefore now become imperative to identify new lead molecules for the development of new tuberculostatic drugs. 
     Family Lamiaceae and diterpenes class of compounds, particularly from  Leucas  genus has been the subject matter of interest for the study of anti-tubercular drugs. Reference may be made to International Journal of Pharm Tech Research, ISSN: 0974-4304 Vol. 3, No. 2, pp 872-880, published in. April-June 2011 titled “A Review on Anti-Tubercular Plants” by Vikrant Arya et.al discloses various plant drugs along with their chemical constituents like alkaloids, flavonoids, tannins, xanthones, triterpenes, quinones etc. responsible for anti-tubercular activity. Table 2 in the said article gives a brief description of common anti-tubercular plants from Ayurveda. Table 3 lists anti-tubercular plants of foreign origin wherein, diterpenes are isolated from plant (i)  Salvia hypargeia  of family Lamiaceae and (ii)  Salvia sclarea  of Family Lamiaceae using alcohol as a solvent. 
     Article titled ‘Antimycobacterial natural products’ by Brent R. Copp in Nat. Prod. Rep., 2003, 20, 535-557, having DOI: 10.1039/b212154a discloses natural products having anti-TB activity. The article further discloses Terpenes with anti-TB activity. (i) Halogenated monoterpenes originally isolated from the marine red alga  Plocamium cartilagineum , exhibits potent anti-algal activity as well as antimycobacterial activity towards  M. tuberculosis  H37Rv with an MIC of 32 μg/ml and  M. avium  (MIC 64 μg/ml). (ii) Also identified as exhibiting antitubercular ( M. tuberculosis  H37Rv) activity in this study were juniperexcelsic acid (MIC 14.4 μg/ml) (205) and sclareol (MIC6.0 μg/ml) (206). 
     
       
                 
         
             
             
         
      
     
     Article titled “Phytochemical Investigation and Antimicrobial Activity of  Leucas cephalotes  (Roth.) Spreng. Whole Herb by Katara Antariksh et.al in Der Pharmacia Lettre, 2010, 2(4):284-296; discloses  Leucas cephalotes  Roth. Spreng (Lamiaceae) to cure various disorders. The plant has antipyretic action and also considered to be stimulant, expectorant, aperient, diaphoretic, insecticidal and emmenagogue.  Leucas cephalotes  (Roth.) Spreng. whole herb contains new labdane, norlabdane and abietane type diterpenes and protostane type triterpenes, together with common triterpene, five sterols and eight flavones. 
     Different extracts of the transverse section of stem, leaf and root were subjected to various phytochemical tests, to identify the active constituents, shown in Table 2 in the said article. Ethanolic and methanolic extracts were found to have more active constituents compared to other extracts. Phytochemicals such as carbohydrate, proteins and amino acids, glycosides, flavonoids, tannins and phenols and steroids were commonly found in most of the extracts. (However, the extracts do not mention diterpenes as a phytoconstituent). 
     Article titled “Diterpenes from  Leucas aspera  Inhibiting Prostaglandin-Induced Contractions” by Samir Kumar Sadhu et.al in J. Nat. Prod., 2006, 69 (7), pp 988-994 and having DOI: 10.1021/np058118m relates to four new diterpenes, leucasperones A (1) and B (2) and leucasperols A (3) and B (4), and three new isopimarane glycosides, leucasperosides A, B, and C (5-7), together with the known compounds asperphenamate, maslinic acid, (−)-isololiolide, and linifolioside. leucasperone A (1), leucasperosides A (5) and B (6), and linifolioside showed inhibition of prostaglandin-induced contractions. 
     
       
                 
         
             
             
         
      
     
     In the search for such new molecules, a wide range of plant extracts were screened for their inhibitory potential against the  tuberculosis . The extracts of  Leucas stelligera  unexpectedly showed most potent anti-tubercular property among the extracts tested.  Leucas stelligera  is an edible plant with leaves eaten as vegetable. No chemical and pharmacological work is reported on this species. 
     OBJECTIVE OF THE INVENTION 
     Main objective of the present invention is to provide compounds of general formula 1 isolated from  Leucas stelligera  useful for the treatment of  Mycobacterium tuberculosis.    
     Another object of the present invention is to provide a process for isolation and characterization of diterpene compounds from  Leucas stelligera.    
     SUMMARY OF THE INVENTION 
     Accordingly, the present invention provides diterpenes of general formula 1 belonging to labdane class, isolated from  Leucas stelligera    
     
       
                 
         
             
             
         
      
     
     In an embodiment of the present invention compounds of general formula 1 wherein said compound is represented by group of the following compounds 
     
       
                 
         
             
             
         
      
     
     In another embodiment of the present invention Compounds of general formula 1 are useful for prevention, treatment, inhibition or controlling growth and proliferation of tubercular activity in mammals. 
     In yet another embodiment of the present invention Compounds of general formula 1 as claimed in claim  1 , wherein IC50 value is in the range of 5.02 to 9.8 μg/ml against  Mycobacterium tuberculosis.    
     In still another embodiment of the present invention Compounds of general formula 1 as claimed in claim  1 , wherein IC90 value is in the range of 10.7 to 46.52 μg/ml against  Mycobacterium tuberculosis.    
     Another embodiment of the present invention a process for the extraction of compounds of general formula 1, wherein the said process comprising the step of:
         i. Powdering aerial parts of  L. stelligera  followed by extraction with acetone at a temperature in the range of 25 to 30° C., filtering acetone solubles and concentrating under reduced pressure in the range of 50-100 mm Hg to obtain a greenish acetone extract,   ii. separating the extract of step i) by using column chromatography (CC) with gradient increasing polarity of the 10-50% acetone in pet ether to obtain 11 fractions (LS-1 to LS-11),   iii. subjecting fraction LS7 of step ii) to CC with gradient of acetonitrile from 1% to 3% in chloroform to obtain 18 sub-fractions (LS7a-r),   iv. subjecting sub-fraction LS7i of step iii) to CC in 15% acetone in petroleum ether to obtain compound 1 and 4,   v. subjecting fraction LS4 of step ii) to CC in 6% acetonitrile in chloroform to obtain 10 (LS4a-j) sub-fractions,   vi. subjecting fraction LS5 of step ii) to CC in 6% acetonitrile in chloroform to obtain 13(L S5a-m) sub-fractions,   vii. combining fractions LS4f, LS4h, LS4i of step v) and LS5j, LS5k of step vi) and subjecting to CC in 15% acetone-petroleum ether to obtain compound 2,   viii. subjecting fraction LS11 of step ii) to CC in gradient of methanol from 5-20% in chloroform to obtain 8 fractions (LS11a-h),   ix. subjecting sub-fraction LS11b of step viii) to CC in gradient of methanol in chloroform from 1-3% to obtain 6 (LS11bi-bvi) sub-fractions,   x. subjecting sub-fraction LS11bvi of step ix) to CC in gradient of acetone from 5-50% in petroleum ether to obtain further sub-fractions including sub-fraction LS11bvia,   xi. subjecting sub-fraction LS11bvia of step x) to successive preparative thin layer chromatographies (PTLC) in 25% acetonitrile in chloroform and 35% ethyl acetate in chloroform to obtain compound 3.       

     In still another embodiment of the present invention a pharmaceutical composition comprising a compound selected from the group of general formula 1 and a pharmaceutically acceptable carrier. 
    
    
     
       DESCRIPTION OF FIGURES 
         FIG. 1 :  FIG. 1 a    gives  1 HNMR of compound 1 
         FIG. 1 b    gives  13 CNMR of compound 1 with DEPT in inset. 
         FIG. 2 :  FIG. 2 a    gives  1 HNMR of compound 2 
         FIG. 2 b    gives  13 CNMR of compound 2 with DEPT in inset. 
         FIG. 3 :  FIG. 3 a    gives  1 HNMR of compound 3 
         FIG. 3 b    gives  13 CNMR of compound 3 with DEPT in inset. 
         FIG. 4 :  FIG. 4 a    gives  1 HNMR of compound 4 
         FIG. 4 b    gives  13 CNMR of compound 4 with DEPT in inset. 
     
    
    
     DETAILED DESCRIPTION OF INVENTION 
     Entire mature plants of  Leucas stelligera , in flowering, are collected from agricultural fields from Mulshi area, District, Pune on 3 Jan. 2008. A herbarium is deposited in Botanical Survey of India, Western Circle, Pune (voucher no. SPJ-5). Plant material is cleaned off adhering dust and unwanted plant material, roots are removed, aerial parts dried in shade, cut and pulverized. Powdered plant material is extracted with aetone to prepare acetone extract. From the acetone extract, active compounds 1- 4 are isolated as shown in scheme 1. 
     
       
                 
         
             
             
         
      
     
     In one preferred embodiment, the pulverized aerial parts of  Leucas stelligera  are subject to acetone extraction; separation of fractions and isolation of diterpenes belonging to Labdane class from sub fractions are depicted in Scheme 1 as above. The acetone extract of the aerial parts of  L. stelligera  by repeated chromatography resulted in the isolated 4 compounds all of which are diterpenes belonging to Labdane class. The structures of the compounds as confirmed by NMR (Tables land 2) are enlisted here in Formula 1. 
     
       
                 
         
             
             
         
      
     
     The compounds are isolated from the fractions/extracts using at least one separation technique selected from partition(s), precipitation(s), crystallization, normal phase chromatography, reversed phase chromatography, size exclusion chromatography and ion exchange chromatography or combinations thereof. 
     All the compounds 1 to 4 isolated are characterized by NMR, the details of which is provided in Tables 1 and 2. 
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                   13 C NMR shifts for compounds 1, 2, 3 and 4. 
               
             
          
           
               
                 Position 
                 1 
                 2 
                 3 
                 4 
               
               
                   
               
             
          
           
               
                 1 
                 31.8, CH 2   
                 33.0, CH 2   
                 31.9, CH 2   
                 36.5, CH 2   
               
               
                 2 
                 18.6, CH 2   
                 18.7, CH 2   
                 18.6, CH 2   
                 18.6, CH 2   
               
               
                 3 
                 41.6, CH 2   
                 41.8, CH 2   
                 41.7, CH 2   
                 42.2, CH 2   
               
               
                 4 
                 33.3, C 
                 33.5, C 
                 33.3, C 
                 32.9, C 
               
               
                 5 
                 46.3, CH 
                 47.0, CH 
                 46.4, CH 
                 46.2, CH 
               
               
                 6 
                 21.5, CH 2   
                 21.7, CH 2   
                 21.6, CH 2   
                 20.7, CH 2   
               
               
                 7 
                 31.2, CH 2   
                 32.1, CH 2   
                 31.3, CH 2   
                 37.8, CH 2   
               
               
                 8 
                 36.6, CH 
                 35.8, CH 
                 36.9, CH 
                 74.2, C 
               
               
                 9 
                 76.8, C 
                 95.0, C 
                 77.3, C 
                 61.2, CH 
               
               
                 10 
                 43.2, C 
                 42.5, C 
                 43.2, C 
                 38.9, C 
               
               
                 11 
                 32.2, CH 2   
                 29.3, CH 2   
                 33.0, CH 2   
                 20.9, CH 2   
               
               
                 12 
                 22.2, CH 2   
                 33.4, CH 2   
                 31.9, CH 2   
                 45.0, CH 2   
               
               
                 13 
                 135.1, CH 
                 89.9, C 
                 144.7, C 
                 73.6, C 
               
               
                 14 
                 143.7, C 
                 45.5, CH 2   
                 126.0, C 
                 146.2, CH 
               
               
                 15 
                 70.1, CH 2   
                 99.2, CH 
                 60.6, CH 2   
                 111.1, CH 2   
               
               
                 16 
                 174.5, C 
                 77.3, CH 2   
                 58.5, CH 2   
                 27.3, CH 3   
               
               
                 17 
                 16.2, CH 3   
                 17.7, CH 3   
                 16.5, CH 3   
                 32.0, CH 3   
               
               
                 18 
                 33.7, CH 3   
                 22.0, CH 3   
                 33.7, CH 3   
                 33.1, CH 3   
               
               
                 19 
                 22.0, CH 3   
                 33.3, CH 3   
                 22.0, CH 3   
                 21.4, CH 3   
               
               
                 20 
                 16.3, CH 3   
                 18.2, CH 3   
                 16.2, CH 3   
                 24.8, CH 3   
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                   1 H NMR shifts for compounds 1, 2, 3 and 4. 
               
             
          
           
               
                 Position 
                 1 
                 2 
                 3 
                 4 
               
               
                   
               
             
          
           
               
                  1 
                 1.49 (m) 
                 1.36 m 
                 1.48 m 
                 1.10 m, 
               
               
                   
                   
                   
                   
                 1.57 m 
               
               
                  2 ax 
                 1.48 m 
                 1.55 m 
                 1.55 m, 1.48 m 
                 1.68 m 
               
               
                  2 eq 
                 1.48 m 
                 1.48 m 
                   
                 1.43m 
               
               
                  3 ax 
                 1.16 dt (3.5, 
                 1.17 dt (3.6, 
                 1.15 dt (3.5, 
                 1.15 dt 
               
               
                   
                 12.9) 
                 13.0) 
                 13.2) 
                 (3.92, 13.62) 
               
               
                  3 eq 
                 1.34 m 
                 1.33 m 
                 1.35 m 
                 1.37 m 
               
               
                  4 
                 — 
                 — 
                 — 
                 — 
               
               
                  5 
                 1.42 m 
                 1.37 m 
                 1.39 m 
                 1.06 dd 
               
               
                   
                   
                   
                   
                 (12.64, 2.76) 
               
               
                  6 ax 
                 1.54 m 
                 1.55 m 
                 1.53 m 
                 1.55 m 
               
               
                  6 eq 
                 1.29 m 
                 1.27 m 
                 1.28 m 
                 1.25 m 
               
               
                  7 α 
                 1.49 m 
                 1.48 m 
                 1.27 m 
                 1.55 m 
               
               
                  7 β 
                 1.29 m 
                 1.22 m 
                 1.27 m 
                 1.55 m 
               
               
                  8 
                 1.79 m 
                 1.76 m 
                 1.78 m 
                 — 
               
               
                  9 
                 — 
                 — 
                 — 
                 1.11 
               
               
                 10 
                 — 
                 — 
                 — 
                 — 
               
               
                 11 
                 1.66 m, 1.85 m 
                 1.76 m, 2.03 m 
                 1.78 m, 1.57 m 
                 1.78 m, 
               
               
                   
                   
                   
                   
                 1.50 m 
               
               
                 12 
                 2.39 dt (1.34, 
                 2.00 (m) 
                 2.2 m 
                 1.52-1.63 m 
               
               
                   
                 8.43) 
               
               
                 13 
                 — 
                 — 
                 — 
                 — 
               
               
                 14 
                 7.11 bt (1.6) 
                 2.32 d (13.10), 
                 5.63 t (6.91) 
                 5.95 dd 
               
               
                   
                   
                 1.98 m 
                   
                 (17.30, 
               
               
                   
                   
                   
                   
                 10.38) 
               
               
                 15 
                 4.77 bq (1.99) 
                 5.43 bs 
                 4.19 bs 
                 5.22 (d, 
               
               
                   
                   
                   
                   
                 17.30), 
               
               
                   
                   
                   
                   
                 5.03 d 
               
               
                   
                   
                   
                   
                 (10.38) 
               
               
                 16 
                 — 
                 4.37 d (8.5), 
                 4.20 bs 
                 1.29 s 
               
               
                   
                   
                 β3.62 d (8.5) 
               
               
                 17 
                 0.92 d (6.65) 
                 0.91 d (6.62) 
                 0.89 d (6.99) 
                 1.47 s 
               
               
                 18 
                 0.88 s 
                 0.81 s 
                 0.88 s 
                 0.87 s 
               
               
                 19 
                 0.84 s 
                 0.86 s 
                 0.84 s 
                 0.79 s 
               
               
                 20 
                 0.94 s 
                 0.91 s 
                 0.94 s 
                 1.07 s 
               
               
                   
               
             
          
         
       
     
     In a preferred embodiment the novel labdane class of the invention were evaluated for their anti-tubercular activity. Three compounds, 1, 2 and 3 are found to be specifically active against  Mycobacterium . The compounds are also evaluated for cytotoxicity as exemplified herein and found to be non toxic. Compound 4 is also found to have potent anti-tubercular activity. 
     The following examples, will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purpose of illustrative discussion of preferred embodiments of the invention. 
     EXAMPLE 1 
     Powdered aerial parts of  L. stelligera  (1.8 kg) were extracted with acetone (3×3 L, 14 h) at a temperature of 25 to 30° C. The mixture was filtered and concentrated under reduced pressure to provide a greenish acetone extract of 57.0 g, 55.0 g of which was further purified by column chromatography (CC) with increasing polarity of acetone from 10 to 50%, in petroleum ether to give 11 fractions (LS1-LS11). 
     Fraction LS7 (4.1 g) was further subjected to CC with acetonitrile from 1% to 3% in chloroform to give 18 sub-fractions (LS7a-r). Sub-fraction LS7i (287.2 mg) was further subjected to CC in 15% acetone in petroleum ether to give compound 1 (120 mg) and 4 (35 mg). 
     Fraction LS4 (5.5 g) and LS5 (2.3 g) were further subjected to CC in 6% acetonitrile in chloroform to give 10 (LS4a-j) and 13 (LS5a-m) sub-fractions respectively. Fractions LS4f, LS4h, LS4i and LS5j, LS5k were pooled and further subjected to CC in 15% acetone-petroleum ether to give compound 2 (150 mg). 
     Fraction LS11 (4.9 g) was further subjected to CC in gradient of methanol from 5-20% in chloroform to give 8 fractions (LS11a-h). Sub-fraction LS11b (3 g) was further subjected to CC in methanol-chloroform from 1-3% to give 6 (LS11bi-bvi) sub-fractions. 
     Sub sub-fraction LS11bvi (300 mg) was further subjected to CC in gradient of acetone from 5-50% in petroleum ether to obtain further sub fractions including LS11bvia. Sub fraction LS11bvia was purified by successive preparative thin layer chromatography (PTLC) in 25% acetonitrile in chloroform and 35% ethyl acetate in chloroform to give compound 3 (20 mg). 
     EXAMPLE 2 
     Anti-Mycobacterial Activities of Compounds 1, 2, 3 and 4 
     All the isolated compounds and whole acetone extract were tested for their in vitro effect against  Mycobacterium tuberculosis  H37Ra. Compounds 1, 2, 3 and 4 showed significant inhibition against  Mycobacterium tuberculosis  H37Ra. The IC 50  and IC 90  values for the same are shown in Table 3. 
     To check the specificity of these compounds, Compounds 1, 2 and 3 were also tested at these IC 90  values against  Escherichia coli  and  Mycobacterium smegmatis , using cultures maintained in-house. The percent inhibition values are given in Table 4. 
     Protocol for Activity Against  M. smegmatis:    
     On the 3rd day of incubation, the microplate was taken out to remove the seal and measure the viable cells. Optical density was measured before addition of XTT at 470 nm. 200 μM XTT was added and incubated for 20 min at 37° C. after shaking for 1 min. After 20 min incubation, 60 μM menadione was added and mixed for 1 min and then incubated at 37° C. for another 20 min. The optical density was measured at 470 nm by using a micro plate reader. For Hypoxia induced XTT reduction microplate assay (HXRMA) on the 7th day of incubation, the plates were taken out and the seal was removed. A similar protocol as mentioned above was repeated for aerobically grown  M. smegmatis.    
     Protocol for Activity Against  E. coli:    
     The effect on growth was calculated by measuring the absorbance of culture at 620 nm after an incubation time of 6 h. 
     Results: 
     These compounds did not show any significant effect on  E. coli  and  M. smegmatis , thus indicating the specificity of these compounds against pathogenic mycobacteria. 
     
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 In vitro anti-mycobacterial activity of 1-4 and 
               
               
                 acetone extract against  M. tuberculosis   
               
             
          
           
               
                 Samples 
                 IC 50  (μg/ml) 
                 IC 90  (μg/ml) 
               
               
                   
               
             
          
           
               
                 1 
                 5.02 
                 19.67 
               
               
                 2 
                 5.55 
                 14.88 
               
               
                 3 
                 9.8 
                 46.52 
               
               
                 4 
                 5.95 
                 10.85 
               
               
                 Extract 
                 8.94 
                 43.98 
               
               
                 Isoniazid 
                   
                 0.05 ± 0.003 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Percent inhibition values of 1-4 and acetone 
               
               
                 extract showing biological activities 
               
             
          
           
               
                 Samples* 
                 
                   M. smegmatis 
                 
                 
                   E. coli 
                 
               
               
                   
               
               
                 1 
                 31.56 ± 6.09 
                  0.29 ± 3.56 
               
               
                 2 
                 47.83 ± 3.74 
                 23.15 ± 2.45 
               
               
                 3 
                 30.57 ± 3.58 
                 11.47 ± 1.65 
               
               
                 Extract 
                 19.47 ± 0.16 
                  3.48 ± 2.561 
               
               
                 Rifampicin** 
                  0.2 ± 0.03 
                  0.5 ± 0.04 
               
               
                   
               
               
                 *Compounds tested at IC 90  found against  M. tuberculosis   
               
               
                 **IC 90 , μg/ml 
               
             
          
         
       
     
     The experiment was carried out three times and the results are mean±S. D. 
     EXAMPLE 3 
     Composition (i) 
     Compound of formula 1 (viz., Compound for Compound 2 or Compound 3): 10.0% w/w 
     FDA approved colour: 0.3% w/w 
     FDA approved flavor: 0.7% w/w 
     Magnesium stearate: 2.0% w/w 
     Mannitol: q.s. to 100.0%w/w 
     Procedure: Dissolve mannitol in water, add color and flavor to it. Evaporate water to adsorb color and flavor on mannitol. Mix active ingredient and Magnesium stearate and fill in pouch or bottle. 
     Mode of Administration 
     Disperse the powder in water/juice. 
     Composition (ii) 
     
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 Corosolic acid 
                 5.0% 
                 w/w 
               
               
                   
                 FDA approved colour 
                 0.3% 
                 w/w 
               
               
                   
                 FDA approved flavor 
                 0.7% 
                 w/w 
               
               
                   
                 Magnesium stearate 
                 2.0% 
                 w/w 
               
               
                   
                 Mannitol 
                 q.s. to 100.0% 
                 w/w 
               
               
                   
                   
               
             
          
         
       
     
     Procedure: Dissolve mannitol in water, add color and flavor to it. Evaporate water to adsorb color and flavor on mannitol. Mix active ingredient and Magnesium stearate and may be filled in a capsule of suitable size. 
     Mode of Administration: 
     The capsule may be had with water or juice. 
     Advantages of Present Invention 
     After assaying the compounds 1, 2, 3, and 4 of the present invention, these compounds are found to be effective against  Mycobacterium tuberculosis  due to their anti-tubercular property. 
     Compounds 1, 2 and 3 were fond to be selective inhibitors of  M. tuberculosis.    
     The plant of the present investigation is edible and with the source anti-TB activity identified, it holds a promising for being used in the treatment of  tuberculosis.    
     Compound 4 belongs to the novel class of compounds called cis-labdane.