Biologically active chloroform fraction of an extract obtained from a mangroone plant Salvadora persica L

Abstract

The invention discloses a process of extracting, fractionating and purifying bioactive molecules from an associated mangrove plant, methods of screening for pharmacological activities of crude extract, its fractions and purified compounds and use of the chloroform fraction of the crude extract as anti-spasmodic, anti-arrhythmic and anti-cholinergic agent.

What is claimed is:  
     
         1 . A process for the extraction and purification of a biologically active extract from the plant  Salvadora persica , comprising a steps of 
 i) collecting and processing the plant parts of  Salvadora persica,      ii) preparing a crude extract from the plant parts of  Salvadora persica,      iii) testing the crude extract using methods of pharmacology,    iv) fractionating the crude extract,    v) testing the fractions using methods of pharmacology,    vi) isolating the pure compounds by column chromatography,    vii) testing the pure compounds by using methods of pharmacology, and    viii) identifying the compounds by spectroscopy.    
     
     
         2 . A process as claimed in  claim 1  wherein the plant parts of  Salvadora persica  are selected from leaves, stems and flowers.  
     
     
         3 . A process as claimed in  claim 1  wherein the steps for preparation of a crude extract are: 
 (i) air-drying the plant parts,  
 (ii) immersing the plant parts in 90% aqueous methanol for one week, at room temperature (28±2° C.),  
 (iii) filtering the methanolic extract by conventional methods, and  
 (iv) evaporating the methanolic extract under reduced pressure at room temperature (28±2° C.) to a minimum quantity to obtain a crude extract.  
 
     
     
         4 . A process as claimed in  claim 1  wherein the crude extract is partitioned into four fractions using solvents selected from petroleum ether, chloroform, butanol and aqueous fraction.  
     
     
         5 . A process as claimed in  claim 1  wherein the chloroform fraction exhibits anti-cholinergic activity, anti-spasmodic and anti-arrhythmic activity.  
     
     
         6 . A process as claimed in  claim 1  wherein the chloroform fraction is passed through a silica gel column and then eluted using petroleum ether ethyl acetate-chloroform-methanol gradient system.  
     
     
         7 . A process as claimed in  claim 1  wherein the thin layer chromatography (TLC) was carried out on silica gel.  
     
     
         8 . A process as claimed in  claim 1  wherein the TLC was visualized either by exposing plates to iodine vapours or by spraying with methanolic sulphuric acid.  
     
     
         9 . A process as claimed in  claim 1  wherein the fractions which showed anti-cholinergic activity were pooled and passed through the silica gel column and eluted with same set of solvents.  
     
     
         10 . A process as claimed in  claim 1  wherein Methyl palmitate in the extract showed higher anti-cholinergic activity and Betulin showed mild anticholinergic activity.  
     
     
         11 . A process as claimed in  claim 1  wherein β-amyrin (non-steroidalpolycyclic triterpene) molecule is characterised by: 
 Molecular formula: C 30 H 50 O  
 Molecular weight: 426  
 Melting point: 160° C.  
 
     
     
         12 . A process as claimed in  claim 1  wherein Betulin molecule is characterised by: 
 Molecular formula: C 30 H 50 O 2    
 Molecular weight: 442  
 Melting point: 255° C.  
 
     
     
         13 . A process as claimed in  claim 1  wherein Ursolic acid (triterpenic acid) molecule is characterised by: 
 Molecular formula: C 30 H 50 O 3    
 Molecular weight: 456  
 Melting point: 292° C.  
 
     
     
         14 . A process as claimed in  claim 1  wherein Methyl palmitate (Aliphatic Ester) molecule is characterised by: 
 Molecular formula: C 30 H 50 O 2    
 Molecular weight: 256  
 Melting point: 30° C.  
 
     
     
         15 . A process as claimed in  claim 1  wherein Lupeol (non-steroidalpolycyclic triterpene) molecule is characterised by: 
 Molecular formula: C 30 H 50 O  
 Molecular weight: 426  
 Melting point: 215° C.  
 
     
     
         16 . A Pharmaceutical composition exhibiting anti-cholinergic, anti-spasmodic, and anti-arrhythmic activity, said composition comprising the chloroform fraction of the extract obtained from the plant  Salvadora persica  optionally, with conventional additives.  
     
     
         17 . A composition as claimed in  claim 16  wherein the additives are selected from therapeutically acceptable additives.  
     
     
         18 . A composition as claimed in  claim 16  wherein the extract contains compounds selected from β-amyrin, betulin, ursolic acid, methyl palmitate and lupeol.  
     
     
         19 . A composition as claimed in  claim 16  wherein the amount of extract in the composition is 10 μgms.  
     
     
         20 . A composition as claimed in  claim 16  wherein the dosage of the extract is 3 μg/ml to 10 μg/ml.  
     
     
         21 . A method for the treatment of arrhythmias, said method comprising the step of administering a therapeutically effective amount of extract obtained from the plant  Salvadora persica , optionally with conventional additives to a subject afflicted with arrhythmias.  
     
     
         22 . A method as claimed in  claim 21  wherein the extract obtained from the plant  Salvadora persica , is a chloroform fraction.  
     
     
         23 . A method as claimed in  claim 21  wherein 3 to 10 μg/ml. amount of the extract is administered to the subject for a period of 10 minutes.  
     
     
         24 . A method as claimed in  claim 21  wherein the subject is a human or animal.  
     
     
         25 . A method for the treatment of spasmodic conditions such as muscular spasms, said method comprising the step of administering a therapeutically effective amount of extract obtained from the plant  Salvadora persica , optionally with conventional additives to a subject in need thereof.  
     
     
         26 . A method as claimed in  claim 25  wherein the extract obtained from the plant  Salvadora persica , is a chloroform fraction of the crude extract of the plant  Salvadora persica.    
     
     
         27 . A method as claimed in  claim 25  wherein 3 to 10 μg/ml. amount of the extract is administered to the subject for a period of 10 minutes.  
     
     
         28 . A method as claimed in  claim 25  wherein the subject is a human or animal.  
     
     
         29 . A method for the treatment of cholinergic conditions such as bronchial asthma, said method comprising the step of administering a therapeutically effective amount of extract obtained from the plant  Salvadora persica , optionally with conventional additives to a subject in need thereof.  
     
     
         30 . A method as claimed in  claim 29  wherein the extract obtained from the plant  Salvadora persica , is a chloroform fraction of the crude extract.  
     
     
         31 . A method as claimed in  claim 29  wherein 3 to 10 μg/ml. amount of the extract is administered to the subject for a period of 10 minutes.  
     
     
         32 . A method as claimed in  claim 29  wherein the subject is a human or animal.