Abstract:
According to this invention there is provided a herbal formulation for the prevention and management of neurodegenerative disorders comprising, hydro-methanolic extract of at least one plant selected from  Curcuma longa, Bacopa monnieri  and  Valeriana jatamansi , at 70-80° C., maintaining the pH of the solution between 7-10, Separating the active compounds chromatographically and Subjecting the active compounds to the step of molecular characterization.

Description:
FIELD OF INVENTION 
       [0001]    This invention relates to role of an herbal formulation in the prevention and management of age related neurodegenerative disorders with special reference to Senile Dementia. 
       BACKGROUND OF INVENTION 
       [0002]    Due to increase in life expectancy the number of aged are increasing throughout the world neurodegenerative disorders are the diseases of nervous system including brain, spinal cord and peripheral nerves. 10 percent of population over the age of 70 years has a significant memory loss out of which 50 percent cause is Alzheimer&#39;s diseases. The most common neurodegenerative disorders are Alzheimer&#39;s diseases (AD), Parkinson&#39;s disease (PD) and Huntington disease (HD). Protein aggregation oxidative stress, mitochondrial dysfunction and glutamate excito-toxicity are the bio-markers playing role in the process of neurodegeneration and death of neurons. Oxidative injury play a vital role in the degeneration and death of neurons. A number of reactive oxygen species (ROS) is found in the body with a wide variations in their site of formation, function and biological half life. An impaired uptake and increased accumulation of glucose and glutamate are found among AD and PD patients. It is reported that oxidative stress is not only related to protein aggregation rather is closely interrelated with mitochondrial dysfunction and glutamate excito-toxicity. The process of cell death also involves the excess release of inflammatory cytokines IL-6 and TNF-α. Thus a number of genetic and environmental factors play a role in the pathogenesis of neurodegeneration and neurodegenerative disorders. Senile dementia is characterized as loss of intellectual ability associated with old age like progressive deterioration in thinking, memory, behavior, personality and motor functions. Senile dementia is divided into two groups—
       1. Dementia due to generalized atrophy (SDAT) and dementia due to vascular problems mainly due to strokes.   2. Depression, poor nutrition, drug poisoning, alcoholism, hyperglycemia, obesity, elevated homocystein etc., are the other causes of dementia.       
 
         [0005]    In senile dementia the patient&#39;s brain function gradually deteriorates showing progressive loss of memory and mental ability with noticeable personality changes. Initially short term memory is affected—like affected person forgets what happened hours or minutes ago, feels difficulty in working routine work. Patient lose interest in various activities, he suffers from a kind of physical instability and possess aggression in his/her behavior and also exhibit moral inhibition. Recent conventional pharmacological strategies for the management of neurodegenerative disorders have shown decrease in progression of disease process rather than cure of disease, but the results are not very satisfactory and thus their application is restricted, particularly in reference to aging population. 
         [0006]    Ayurveda has described several medicinal plants under the concept Medhya-rasayana which refers to the agents acting on higher brain function by interacting on ability or power of acquisition (intelligence), ability or power of retention and ability or power of recall of memory by amelioration of seven Medha of the individuals. In Ayurveda several medhya rasayana drugs are kept under category of medhya which are responsible for facilitating learning and memory. Taking lead from ancient literature the present study was conducted in various experimental and clinical trials. 
       OBJECTS OF INVENTION 
       [0007]    The major object of present invention is to propose an Ayurvedic plant based formulation beneficial in the prevention and management of neurodegenerative disorders like senile dementia, vascular dementia and senile dementia of Alzheimer&#39;s type among aged population. 
         [0008]    Another object of present invention is to propose a plant based Ayurvedic formulation effective in the prevention and management of memory and attention span among elderly people. 
         [0009]    Still another object of present study is to proposed an Ayurvedic formulation beneficial to check the further loss of memory and cognition among aged. 
         [0010]    Further, object of present invention is to propose a novel formulation beneficial in amelioration of neuro-chemical markers like acetylcholine, serotonin, nor-adrenaline, dopamine particularly glutamate involved in neurodegeneration. 
         [0011]    Yet another object of present invention is to propose a plant based formulation showing effectivity on various oxidative stress markers like TBARS, glutathione, superoxide dismutase (SOD), Catalase and GPx. 
         [0012]    One of the objects of present invention is to propose a novel Ayurvedic formulation beneficial in reducing the brain inflammation by acting on inflammatory cytokines IL-6, TNF-α including hs. C-reactive protein. 
         [0013]    Still another object of present invention is to propose a plant based formulation beneficial in reducing the elevated homocysteine level among senile dementia subjects and others suffering from neurodegenerative disorders. 
         [0014]    Further, another object of present invention is to propose a plant based Ayurvedic formulation effective in improving the orientation, working memory, language and communication skill among elderly people having neurodegeneration. 
         [0015]    Another object of present invention is to propose an Ayurvedic formulation having beneficial role in improving the over all mental performance and also effective in maintenance of general health status among elderly population. 
       STATEMENT OF INVENTION 
       [0016]    According to this invention there is provided a novel Ayurvedic formulation showing beneficial role in the prevention and management of neurodegenerative disorders like senile dementia, senile dementia of Alzheimer&#39;s type, vascular dementia caused due to stroke and neurodegeneration associated with various etiological factors like diabetes mellitus, obesity, hypertension, dyslipidemia etc. 
         [0017]    Further, according to this invention there is provided a process for the preparation of novel Ayurvedic formulation as claimed in claim  1  comprising of preparing hydro-methanolic extract of  Valeriana jatamansi  (Tagar, rhizome),  Bacopa monnieri  (Brahmi, whole plant) and  Curcuma longa  (Haridra, rhizome) by using water (aqueous) and methanol (30:70) at 70-80° C. and maintaining the pH of solution between 7-10, separating chromatographically the active compound by using TLC, HPLC, and HPTLC supporting the molecular characterization of plant extract by using IR and NMR. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0018]      FIG. 1  is a flow diagram of the process. 
           [0019]      FIG. 2  is showing the effect of test drug on mini mental state examination in SDAT cases. 
           [0020]      FIG. 3  is showing the effect of test drug on mini mental state examination and attention span in SDAT cases. 
           [0021]      FIG. 4  is showing the effect of test drug on short term memory in SDAT cases. 
           [0022]      FIG. 5  is showing the effect of test drug on long term memory in SDAT cases. 
           [0023]      FIG. 6  is showing the effect of test drug on Anxiety level in SDAT cases. 
           [0024]      FIG. 7  is showing the effect of test drug on depression level in SDAT cases. 
           [0025]      FIG. 8  is showing the effect of test drug on hs C-Reactive Protein level in SDAT cases. 
           [0026]      FIG. 9  is showing the effect of test drug on Interleukin-6 level in SDAT cases. 
           [0027]      FIG. 10  is showing the effect of test drug on TNF-α level in SDAT cases. 
           [0028]      FIG. 11  is showing the effect of test drug on Homocysteine level in SDAT cases. 
       
    
    
     DETAILED DESCRIPTION OF INVENTION 
       [0029]    The hydro-methanolic extract of three Ayurvedic plants  Valeriana jatamansi, Curcuma longa  and  Bacopa monnieri  by using 30:70 ratio of water and methanol respectively is utilized for present study. The water utilized for extraction was decontaminated for any type of bacterial or abnormal growth by using reverse osmosis plant. After extraction the presence of active molecules in various plant extracts were identified by HPLC, HPTLC and NMR procedures. 
         [0030]    The biological activity was studied on the basis of mode of action of single plant selected for combined formulation as well as combined formulation by assessing their role on various targets involved in neurodegeneration as well as neurodegenerative disorders. The bio-molecular reaction following the interaction between the chemical and biological markers like cholinergic, dopaminergic, glutamatergic system, oxidative stress markers, inflammatory cytokines and also the neuropyschological assessments were evaluated. 
         [0031]    The pre-clinical toxicological studies of single as well as combined formulation were carried out to determine safety profile of present novel test formulation. The efficacy profile of test formulation were also done in pre-clinical animal model of Alzheimer&#39;s disease and age consistent status of various targets like neurotransmitters, neuro-inflammatory markers and oxidative stress markers. The mode of action of single plant candidate and combined formulation was determined in animal studies. 
         [0032]    The beneficial role of test formulation on cholinergic, dopaminergic and glutamate excito-toxicity effect, anti-neuro-inflammatory activity and anti-oxidant activity was determined in various animal models before utilizing the test formulation for human consumption. 
       Extraction Procedure: 
       [0033]    The dried rhizome of  Valeriana jatamansi , dried rhizome of  Curcuma longa , and dried whole plant of  Bacopa monnieri , were utilized for extraction. The hydro-methanolic extract of the plants were utilized for the identification of active compound present in the plants. After extraction, the extracted parts were taken for chromatographic separation by using TLC, HPLC, and HPTLC. After identification and separation of active compound, the molecular characterization was carried out by using IR and NMR. 
         [0034]    The extraction was done at the temperature of 70-80° C. The pH of the solution was maintained between 7-10. The steps carried out to isolate the active compound to assess the activity of test formulation are shown in  FIG. 1 . 
         [0035]    According to this invention, there is provided an Ayurvedic formulation for the prevention and management of neurodegenerative disorders particularly for senile dementia and senile dementia of Alzheimer&#39;s type. The present test formulation comprising of the following ingredients: 
         [0000]                                                              Name of the Plants   Parts used                                        1.     Curcuma longa  (Haridra)   rhizome           2.     Valeriana jatamansi  (Tagar)   rhizome           3.     Bacopa monnieri  (Brahmi)   whole plant                        
Preferably, the aforesaid plants are present in the formulation in the following doses—
 
         [0000]    
       
         
               
               
               
             
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Name of the Plants 
                 Dose range 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 1. 
                 
                   Curcuma longa 
                 
                 150-400 mg/day 
               
               
                   
                 2. 
                 
                   Valeriana jatamansi 
                 
                 200-450 mg/day 
               
               
                   
                 3. 
                 
                   Bacopa monnieri 
                 
                 250-500 mg/day 
               
               
                   
                   
               
             
          
         
       
     
         [0036]    The formulation also comprise known additive such as minerals, vitamins, salts filler (for capsulation or to prepare syrup) and binders, if required to present in trace amount. 
         [0037]    Thus any known additive or supplement is added to prepare the final formulation as required and present in trace amount. Reference is made here in capsule form (500 mg each). However, it would be apparent that the preparation may also be in the form of syrup/tablet. 
         [0038]    Preferably but without implying any limitation the preparation comprises— 
         [0000]    
       
         
               
               
               
             
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Name of the plant 
                 Dose 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 1. 
                 
                   Curcuma langa 
                 
                 250 mg/day 
               
               
                   
                 2. 
                 
                   Valeriana jatamansi 
                 
                 325 mg/day 
               
               
                   
                 3. 
                 
                   Bacopa monnieri 
                 
                 375 mg/day 
               
               
                   
                   
               
             
          
         
       
     
       Hypothesis: 
       [0039]    The present test formulation is based on the combined effect of three Ayurvedic plant extract namely  Curcuma longa, Valeriana jatamansi  and  Bacopa monnieri . This formulation has been proven for its neuro-chemical, neuro-modulatory, anti-inflammatory and anti-oxidant activity and the most important potential role is for memory and attention span enhancing activity among aged people. These effects are mainly mediated through its glutamate inhibitory activity as well as amelioration of cholinergic and dopaminergic neurons. Further, the novel formulation prepared by using specific doses of three plant extracts have exerted anti-oxidant role by regulation of oxidative enzymes. 
         [0040]    A number of changes take place in the brain during neurodegeneration or aging at molecular, cellular, structural and functional level. Neurotransmitters such as acetylcholine glutamate, dopamine play an important role in the regulation of neuronal circuits and influence the neurodegenerative process. Glutamate and GABA, the major excitatory and inhibitory neurotransmitters are responsible for the protection of neuronal survival and synaptic plasticity. Decrease 5-HT receptors were found in dementia. Serotonergic neurons either inhibits or increase the release of dopamine concentration, thus influence dementia directly. GABA is the primary inhibitory neurotransmitter in the CNS and abnormality is significantly associated with behavioral changes in neurodegenerative disorders. Regulation of 5-HT results in the regulation of aggression, mood, feeding, sleep, temperature, motor activity etc. 
         [0041]    Decrease in cholinergic markers cholin-acetyl-transferase (AchE) in the cortex, loss of neurons in nucleus basalis of meynert and also reduction in muscarinic type-II pre-synaptic receptor density are estimated in various type of dementia. It is reported that  Valeriana jatamansi  is a potent nervine tonic and has sedative effects thus it produces mental relaxation and induces sleep. 
         [0042]    As vascular inflammation is significantly involved with neurodegenerative disorders, high level of Interleukins, Tumor Necrosis Factor are responsible for neuro-inflammation. Further, high sensitive C-reactive protein is an important neuro-inflammatory factor manifesting vascular inflammation in the brain, particularly in Alzheimer&#39;s disease and Parkinson&#39;s disease. Oxidative injury plays a major role in the death and degeneration of neurons and also contributes in platelet aggregation and mitochondrial dysfunction and glutamate excitotoxicity. Recently various studies have suggested that apoptosis rather than necrosis is primarily responsible for loss of neurons and that caspases are the major executioners of apoptosis in common neurodegenerative diseases. This mechanism is important when studied in relation to aging brain. A number of acetylcholine esterase inhibitors, MAO-B inhibitors chelaters etc are being used for their multi-targeted action that prevents loss of mitochondrial permeability, cytochrome C release and down regulated the expression of pro-apoptonic genes, but the application of such agents are restricted due to unsatisfactory results and side effects. 
         [0043]    Keeping the above facts in view and effective role of plants that acted on various targets involved with neurodegenerative process determined in pre-clinical models, it was thought to propose a better remedial measure for the prevention and management of neurodegenerative process as well as neurodegenerative disorders particularly Senile Dementia, SDAT and vascular dementia caused due to stroke. 
       About the Plant: 
       [0044]      Curcuma longa : It belongs to Zingiberaceae family and found all over India in abundance. The active constituents of  curcuma longa  are flavonoid, curcumin and volatile oils including timerone, atlantone and zingiberone. It has anti-oxidant, hepato-protective, anti-oxidant and potent anti-inflammatory activity. It improves cognitive deficit and prevents amyloidal accumulation
   Bacopa monnieri: Bacopa monnieri  is one of the major ingredients of present test formulation that belongs to family scrophulariaceae. It has shown potential role in improving mental ability, intelligence, concentration and memory performance. It is a potent nervine tonic. It is reported that  Bacopa monnieri  crosses the blood brain barrier, regulates the cholinergic neuron, prevents the further loss of acetylcholine and thus is effective in the prevention and management of neuro-degenerative disorders. It has also shown anti-oxidant role.
   Valeriana Jatamansi  belongs to family Valerianceae also known as jatamansi. Rhizome is used medicinally. This species is globally distributed at altitude range of 1500-3600 m. Valerian, active compound of V-Jatamansi has been shown to improve sleep quality and reduce blood pressure. The rhizome of  Valeriana Jatamansi  is antispasmodic, carminative, diuretic, hypnotic, powerfully nervine, sedative and stimulant. It is also used in the treatment of painful menstruation, cramps, hypertension and irritable bowel syndrome.
 
       Example-I 
       [0045]    When the hydro-methanolic extract of  Bacopa monnieri  (40 mg/kg) and  Curcuma longa  (20 mg/kg) mixed and orally administered to experimental animals it restored the level of acetylcholine, dopamine, 5-HT and nor-adrenaline in aged mice brain where as it reduced the elevated glutamate content in the same experimental model. The drug also revealed reduction in inflammatory markers as well as the anti-oxidant activity in aged brain. 
       Example-II 
       [0046]    When the hydro-methanolic extract of  Bacopa monnieri  (35 mg/kg),  Curcuma longa  (20 mg/kg) and  Valeriana jatamansi  (25 mg/kg) mixed and given to streptozotocin neurotoxin induced Alzheimer&#39;s disease model rats a significant elevation in acetylcholine, ChAt activity, serotonergic, nor-adrenaline concentration were estimated in drug treated group. The rats exerted better learning ability following test formulation treatment. 
       Example-III 
       [0047]    In clinical trial when the hydro-methanolic extract of  Valeriana jatamansi  (350 mg/day) and  Bacopa monnieri  (450 mg/day) mixed and prescribed for oral administration to diagnosed senile dementia cases improvement in short and long term memory, attention span and reduction in anxiety and depression scores were noticed. 
       Example-IV 
       [0048]    The organic extract of  Bacopa monnieri  (425 mg/day) and  Curcuma longa  (400 mg/day) mixed and given orally to senile dementia cases a significant anti-oxidant role was observed as glutathione level increased and lipid peroxidase levels decreased following test formulation treatment. 
       Example-V 
       [0049]    The organic extract of  Curcuma longa  (425 mg/day) and  Valeriana jatamansi  (450 mg/day) mixed and prescribed for oral administration in two divided doses exerted significant reduction in inflammatory cytokines IL-6, and TNF-α indicating improvement in neurodegenerative process. CRP also reduced in test drug treatment group suggesting improved vascular inflammation. 
       Example-VI 
       [0050]    When the organic extract of  Bacopa monnieri  (425 mg/day) and  Valeriana jatamansi  (425 mg/day) mixed and given to diagnosed cases of Senile Dementia of Alzheimer&#39;s Type improvement in clinical symptoms like aggression, sleep disturbance, communication difficulties, psychotic feature (hallucination and delusion) was observed. 
       Example-VII 
       [0051]    When the hydro-methanolic extract of  Curcuma longa  (375 mg/day),  Bacopa monnieri  (450 mg/day) mixed and given to SDAT cases elevation in 5-HT, nor-adrenaline and dopamine level was noticed. A decrease in glutamate level was also estimated. 
       Example-VIII 
       [0052]    When the organic extract of  Valeriana jatamansi  450 mg/day and  Bacopa monnieri  425 mg/day mixed and given to SDAT cases improvement in sleep pattern, behavioral abnormalities were recorded after 3-6 months treatment. 
       Example-IX 
       [0053]    The most beneficial and promising results were noticed when the hydro-methanolic extract of  Bacopa monnieri  in the dose of 375 mg/day,  Valeriana jatamansi  in the dose of 325 mg/day and  Curcuma longa  250 mg/day mixed and given to neurodegenerative disorder patients both senile dementia and SDAT cases. An improvement in Mini Mental Scores, memory span, attention span was recorded and no further decline is reported. Further, an elevation in 5-HT, nor-adrenaline levels were estimated. The test drug exerted reduction in inflammatory bio-markers IL-6 and TNF-α. The test drug has shown overall improvement in mental health status by improving memory and cognition and also general feeling of well being among senile dementia, SDAT and other neurodegenerative disorders. The test formulation has anti-oxidant, anti-inflammatory and neuromodulatory activity. The drug is safe and can be given for longer time. 
       Experimental Evidence: 
     Study-I 
       [0054]      
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Age consistent status of neurotransmitter and beneficial role of test formulation 
               
             
          
           
               
                   
                   
                   
                 Nor- 
                   
                 Glutamate 
               
               
                   
                 Acetylcholine 
                 Dopamine 
                 adrenaline 
                 5-HT 
                 (μmol/gm 
               
               
                 Groups 
                 (nm/gm) 
                 (μg/gm) 
                 (μg/gm) 
                 (μg/gm) 
                 wt. tissue) 
               
               
                   
               
               
                 Group-I * (4 
                 26.82 ± 3.88 
                 0.42 ± 0.08 
                 0.45 ± 0.16 
                 0.67 ± 0.09 
                 10.73 ± 2.15 
               
               
                 months old mice) 
               
               
                 Group-II ** (10 
                 16.82 ± 2.81 
                 0.31 ± 0.09 
                 0.19 ± 0.07 
                 0.44 ± 0.08 
                 16.83 ± 3.41 
               
               
                 months old mice) 
               
               
                 Group-III *** 
                 23.59 ± 2.93 
                 0.39 ± 0.25 
                 0.38 ± 0.25 
                 0.59 ± 0.05 
                 14.09 ± 2.45 
               
               
                 (10 months old 
               
               
                 mice + test drug) 
               
               
                   
               
               
                 Comp. 
               
               
                 * vs ** 
                 P &lt; 0.01 
                 P &lt; 0.05 
                 P &gt; 0.05 
                 P &lt; 0.05 
                 P &lt; 0.05 
               
               
                 ** vs *** 
                 P &lt; 0.01 
                 P &gt; 0.05 
                 P &gt; 0.05 
                 P &gt; 0.05 
                 P &gt; 0.05 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Age consistent oxidative injury and benefits of novel 
               
               
                 Ayurveda formulation on oxidative stress markers 
               
             
          
           
               
                   
                   
                   
                   
                 Catalase (μM 
                 GPx (μM 
               
               
                   
                   
                   
                   
                 H2O2 
                 glutathione 
               
               
                   
                 TBARS 
                 Glutathione 
                   
                 oxidized/ 
                 oxidized/ 
               
               
                   
                 (nm/mg 
                 (μg/mg 
                 SOD (IU/mg 
                 min/mg 
                 min/mg 
               
               
                 Groups 
                 protein) 
                 protein) 
                 protein) 
                 protein) 
                 protein) 
               
               
                   
               
               
                 Group-I * (4 
                 0.297 ± 0.042 
                 4.251 ± 1.085 
                 47.994 ± 9.310 
                 17.091 ± 2.954 
                 3.194 ± 0.982 
               
               
                 months old mice) 
               
               
                 Group-II ** (10 
                 0.578 ± 0.039 
                 2.649 ± 0.102 
                 28.773 ± 5.901 
                 11.801 ± 1.013 
                 2.045 ± 0.172 
               
               
                 months old mice) 
               
               
                 Group-III *** (10 
                 0.429 ± 0.033 
                 3.287 ± 0.350 
                 36.801 ± 4.612 
                 14.914 ± 2.876 
                 2.985 ± 0.103 
               
               
                 months old mice + 
               
               
                 test drug) 
               
               
                   
               
               
                 Comp: 
               
               
                 * vs ** 
                 P &lt; 0.001 
                 P &gt; 0.05 
                 P &gt; 0.05 
                 P &gt; 0.05 
                 P &gt; 0.05 
               
               
                 ** vs *** 
                 P &lt; 0.05  
                 P &gt; 0.05 
                 P &gt; 0.05 
                 P &gt; 0.05 
                 P &gt; 0.05 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Age consistent elevation in inflammatory cytokines, homocysteine 
               
               
                 and beneficial role of novel formulation. 
               
             
          
           
               
                   
                   
                   
                   
                 Homocysteine 
               
               
                 Groups 
                 IL-6 (pg/ml) 
                 hs CRP (mg/L) 
                 TNF-α (pg/ml) 
                 (mmol/L) 
               
               
                   
               
               
                 Group-I * (4 
                  111.0 ± 12.15 
                 4.87 ± 0.85 
                  342.8 ± 15.26 
                 7.93 ± 1.05 
               
               
                 months old mice) 
               
               
                 Group-II ** 
                 302.0 ± 8.53 
                 7.99 ± 0.93 
                 604.85 ± 12.96 
                 13.81 ± 1.63  
               
               
                 (10 months old 
               
               
                 mice) 
               
               
                 Group-III *** (10 
                 187.0 ± 9.86 
                 5.69 ± 0.95 
                 481.21 ± 18.78 
                 9.85 ± 0.98 
               
               
                 months old mice + 
               
               
                 test drug) 
               
               
                   
               
               
                 Comp 
               
               
                 * vs ** 
                 P &lt; 0.001 
                 P &gt; 0.05 
                 P &lt; 0.001 
                 P &lt; 0.001 
               
               
                 ** vs *** 
                 P &lt; 0.001 
                 P &gt; 0.05 
                 P &gt; 0.05  
                 P &lt; 0.01  
               
             
          
         
       
     
       Study-II 
       [0055]      
         [0000]    
       
         
               
             
               
               
             
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Changes in Ach concentration following test formulation 
               
             
          
           
               
                   
                 Acetylcholine concentration (n mol/gm.) 
               
             
          
           
               
                   
                 Frontal cortex 
                 Hippocampus 
               
             
          
           
               
                 Treated groups 
                 Day 14 
                 Day 21 
                 Day 14 
                 Day 21 
               
               
                   
               
               
                 Vehicle (ACSF) * 
                 22.84 ± 3.45 
                 26.92 ± 5.06 
                 30.44 ± 4.98 
                 28.94 ± 3.75 
               
               
                 Td. with STZ ** 
                 11.84 ± 2.11 
                  6.92 ± 1.82 
                 15.87 ± 2.65 
                 12.99 ± 2.87 
               
               
                 Td. with test drug *** 
                 24.94 ± 3.45 
                 26.22 ± 4.04 
                 29.84 ± 3.11 
                 31.75 ± 4.14 
               
               
                 Td. with STZ + test drug **** 
                 17.87 ± 2.85 
                 16.92 ± 3.01 
                 23.97 ± 3.75 
                 22.20 ± 2.97 
               
               
                   
               
               
                 Comp. 
               
               
                 * vs ** 
                 p &lt; 0.001 
                 p &lt; 0.001 
                 p &lt; 0.001 
                 p &lt; 0.001 
               
               
                 ** vs ** 
                 p &lt; 0.001 
                 p &lt; 0.001 
                 p &lt; 0.001 
                 p &lt; 0.001 
               
               
                 *** vs **** 
                 p &lt; 0.001 
                 p &lt; 0.01  
                 p &lt; 0.01  
                 p &lt; 0.01  
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Effect of test formulation on ChAT activity 
               
             
          
           
               
                   
                 ChAT activity (n mol/mg protein/hour) 
               
             
          
           
               
                   
                 Frontal cortex 
                 Hippocampus 
               
             
          
           
               
                 Treated groups 
                 Day 14 
                 Day 21 
                 Day 14 
                 Day 21 
               
               
                   
               
               
                 Vehicle (ACSF) * 
                 21.84 ± 3.01 
                 23.97 ± 4.11 
                 22.05 ± 2.92 
                 19.85 ± 2.82 
               
               
                 Td. with STZ ** 
                 13.82 ± 2.02 
                 10.75 ± 1.68 
                  8.92 ± 1.72 
                  6.08 ± 1.01 
               
               
                 Td. with test drug *** 
                 20.85 ± 3.12 
                 22.70 ± 2.91 
                 18.22 ± 2.75 
                 19.75 ± 2.10 
               
               
                 Td. with STZ + test drug **** 
                 15.58 ± 2.33 
                 16.93 ± 3.11 
                 14.88 ± 3.14 
                 13.87 ± 2.91 
               
               
                   
               
               
                 Comp 
               
               
                 * vs ** 
                 p &lt; 0.001 
                 p &lt; 0.001 
                 p &lt; 0.001 
                 p &lt; 0.001 
               
               
                 ** vs *** 
                 p &lt; 0.001 
                 p &lt; 0.001 
                 p &lt; 0.001 
                 p &lt; 0.001 
               
               
                 *** vs **** 
                 p &lt; 0.01  
                 p &lt; 0.05  
                 p &lt; 0.05  
                 p &lt; 0.01  
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Effect of test formulation on Nor-adrenaline level 
               
             
          
           
               
                   
                 Nor-adrenaline (μ gm/gm tissue) 
               
             
          
           
               
                   
                 Frontal cortex 
                 Hippocampus 
               
             
          
           
               
                 Treated groups 
                 Day 14 
                 Day 21 
                 Day 14 
                 Day 21 
               
               
                   
               
               
                 Vehicle (ACSF) * 
                 0.264 ± 0.032 
                 0.271 ± 0.041 
                 0.262 ± 0.050 
                 0.265 ± 0.036 
               
               
                 Td. with STZ ** 
                 0.123 ± 0.022 
                 0.106 ± 0.028 
                 0.148 ± 0.031 
                 0.102 ± 0.026 
               
               
                 Td. with test drug *** 
                 0.271 ± 0.048 
                 0.284 ± 0.038 
                 0.275 ± 0.037 
                 0.288 ± 0.041 
               
               
                 Td. with STZ + test drug **** 
                 0.152 ± 0.030 
                 0.164 ± 0.034 
                 0.150 ± 0.036 
                 0.162 ± 0.028 
               
               
                   
               
               
                 Comp 
               
               
                 * vs ** 
                 p &lt; 0.05 
                 p &lt; 0.01 
                 p &gt; 0.05 
                 p &lt; 0.05 
               
               
                 ** vs *** 
                  p &lt; 0.001 
                 p &lt; 0.05 
                 p &gt; 0.05 
                 p &gt; 0.05 
               
               
                 *** vs **** 
                 p &lt; 0.05 
                 p &lt; 0.01 
                 p &lt; 0.05 
                  p &lt; 0.001 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Effect of test formulation on Serotonin level 
               
             
          
           
               
                   
                 Serotonin (μ gm/gm tissue) 
               
             
          
           
               
                   
                 Frontal cortex 
                 Hippocampus 
               
             
          
           
               
                 Treated groups 
                 Day 14 
                 Day 21 
                 Day 14 
                 Day 21 
               
               
                   
               
               
                 Vehicle (ACSF) * 
                 0.432 ± 0.036 
                 0.442 ± 0.020 
                 0.315 ± 0.024 
                 0.327 ± 0.021 
               
               
                 Td. with STZ ** 
                 0.289 ± 0.011 
                 0.221 ± 0.014 
                 0.240 ± 0.017 
                 0.210 ± 0.015 
               
               
                 Td. with test drug *** 
                 0.489 ± 0.032 
                 0.479 ± 0.027 
                 0.392 ± 0.036 
                 0.385 ± 0.030 
               
               
                 Td. with STZ + test drug 
                 0.354 ± 0.021 
                 0.362 ± 0.024 
                 0.288 ± 0.022 
                 0.268 ± 0.019 
               
               
                 **** 
               
               
                   
               
               
                 Comp 
               
               
                 * vs ** 
                 p &lt; 0.05 
                 p &lt; 0.01 
                 p &gt; 0.05 
                 p &lt; 0.05 
               
               
                 ** vs *** 
                  p &lt; 0.001 
                 p &lt; 0.05 
                 p &gt; 0.05 
                 p &gt; 0.05 
               
               
                 *** vs **** 
                 p &lt; 0.05 
                 p &lt; 0.01 
                 p &lt; 0.05 
                  p &lt; 0.001 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Effect of test drug on Retention of 
               
               
                 active avoidance learning (RAAL) 
               
             
          
           
               
                   
                 RAAL (no. of trials) 
                   
               
             
          
           
               
                 Treated group 
                 Day 14 
                 Day 21 
               
               
                   
               
               
                 Vehicle (ACSF) * 
                  7.11 ± 1.02 
                 6.82 ± 1.06 
               
               
                 Td. with STZ ** 
                 13.03 ± 2.13 
                 15.16 ± 2.12  
               
               
                 Td. with test drug *** 
                  6.58 ± 1.04 
                 5.21 ± 0.92 
               
               
                 Td. with STZ + test drug **** 
                 10.31 ± 1.30 
                 8.94 ± 1.04 
               
               
                   
               
               
                 Comp 
               
               
                 * vs ** 
                 p &lt; 0.001 
                 p &lt; 0.001 
               
               
                 ** vs *** 
                 p &lt; 0.001 
                 p &lt; 0.001 
               
               
                 *** vs **** 
                 p &lt; 0.001 
                 p &lt; 0.001 
               
             
          
         
       
     
       Clinical Evidence 
       [0056]      
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Effect of test drug on clinical complaints in SDAT cases 
               
             
          
           
               
                   
                 Complaints (In percent) 
               
             
          
           
               
                   
                   
                   
                 Communi- 
                   
                   
               
               
                   
                   
                 Sleep 
                 cation 
                   
                 Psychotic 
               
               
                   
                 Aggression 
                 disturbance 
                 difficulties 
                 Depression 
                 features 
               
             
          
           
               
                 Treated groups 
                 BT 
                 AT 
                 BT 
                 AT 
                 BT 
                 AT 
                 BT 
                 AT 
                 BT 
                 AT 
               
               
                   
               
             
          
           
               
                 Normal 
                 P td 
                 24 
                 24 
                 9 
                 10 
                 — 
                 — 
                 16 
                 17 
                 6 
                  9 
               
               
                 aged 
                 (N = 62) 
                 38.70 
                 38.70x 
                 14.51 
                 16.12x 
                   
                   
                 25.80 
                 27.41x 
                 9.67 
                 14.51x 
               
               
                   
                 T td 
                 25 
                 59 
                 22 
                 64 
                 — 
                 — 
                 15 
                 58 
                 9 
                 52 
               
               
                   
                 (N = 68) 
                 36.76 
                 86.76            
                 32.35 
                 94.11            
                   
                   
                 22.05 
                 85.29Ψ 
                 13.23 
                 76.47            
               
               
                 SDAT 
                 Pl td 
                 68 
                 70 
                 65 
                 68 
                 69 
                 70 
                 67 
                 66 
                 61 
                 68 
               
               
                 cases 
                 (N = 71) 
                 95.77 
                 98.59* 
                 91.54 
                 95.77* 
                 97.18 
                 98.59* 
                 94.36 
                 92.95* 
                 85.91 
                 95.77* 
               
               
                   
                 T td 
                 65 
                 69 
                 67 
                 66 
                 72 
                 70 
                 70 
                 68 
                 69 
                 70 
               
               
                   
                 (N = 78) 
                 83.33 
                 88.46# 
                 85.89 
                 84.61# 
                 92.30 
                 89.74# 
                 89.74 
                 87.17# 
                 88.46 
                 89.74# 
               
               
                   
               
               
                 (BT = Before therapy; AT = After 6 months therapy) 
               
               
                 Normal aged (Placebo treated) ATx = No improvement in percent; 
               
               
                 Normal aged (Test drug treated) AT            = Improvement in percent 
               
               
                 SDAT (Placebo treated) AT* = No improvement in percent; 
               
               
                 SDAT (Test drug treated) AT# = Improvement in percent 
               
             
          
         
       
     
         [0000]                                                                                  TABLE 2                   Effect of test drug on mini mental state examination in SDAT cases                Mini-mental state examination (Score)   Comp. initial vs 6            Treated groups   Initial   After 3 months   After 6 months   months treatment                    Normal   P td   18.75 ± 2.90   18.62 ± 3.01   17.82 ± 2.71   P &gt; 0.05       aged   (N = 62)           TD td   18.89 ± 3.01   19.83 ± 2.77   20.42 ± 3.04   P &lt; 0.01           (N = 68)       SDAT   P td   10.45 ± 1.34    9.23 ± 2.02    8.25 ± 1.01   P &lt; 0.001       cases   (N = 71)           T td   11.02 ± 1.66   11.68 ± 2.05   12.29 ± 2.26   P &lt; 0.001           (N = 78)                    
Results of the effect of test drug on mini mental state examination in SDAT cases are shown in  FIG. 2 .
 
         [0000]                                                                                  TABLE 3                   Effect of test drug on mini mental state examination       and attention span in SDAT cases                Attention Span (Score)   Comp. initial vs 6            Treated groups   Initial   After 3 months   After 6 months   months treatment                    Normal   P td   10.65 ± 2.82   10.42 ± 3.11   9.87 ± 2.13   P &gt; 0.05       aged   (N = 62)           Ttd   11.26 ± 2.17   11.10 ± 2.03   10.36 ± 1.93    P &lt; 0.05           (N = 68)       SDAT   P td    4.98 ± 1.21    4.11 ± 0.97   3.72 ± 0.75   P &lt; 0.001       cases   (N = 71)           T td    5.12 ± 1.05    5.83 ± 0.92   6.08 ± 1.23   P &lt; 0.001           (N = 78)                    
Results of the effect of test drug on mini mental state examination and attention span in SDAT cases are shown in  FIG. 3 .
 
         [0000]                                                                                  TABLE 4                   Effect of test drug on short term memory in SDAT cases                Short term memory (Score)   Comp. initial vs 6            Treated groups   Initial   After 3 months   After 6 months   months treatment                    Normal   P td   7.82 ± 2.13   7.73 ± 1.90   7.65 ± 1.75   P &gt; 0.05       aged   (N = 62)           T td   8.04 ± 1.55   8.93 ± 1.81   9.63 ± 2.12   P &lt; 0.001           (N = 68)       SDAT   P td   3.81 ± 0.97   3.26 ± 1.02   2.84 ± 0.85   P &lt; 0.001       cases   (N = 71)           T td   3.24 ± 0.77   3.88 ± 0.93   4.23 ± 1.04   P &lt; 0.001           (N = 78)                    
Results of the effect of test drug on short term memory in SDAT cases are shown in  FIG. 4 .
 
         [0000]                                                                                  TABLE 5                   Effect of test drug on long term memory in SDAT cases                Long term memory (Score)   Comp. initial vs 6            Treated groups   Initial   After 3 months   After 6 months   months treatment                    Normal   P td   6.13 ± 2.12   6.05 ± 1.75   5.48 ± 0.98   P &lt; 0.05       aged   (N = 62)           T td   5.85 ± 1.35   6.75 ± 1.22   6.90 ± 2.01   P &lt; 0.01           (N = 68)       SDAT   P td   1.75 ± 0.61   1.66 ± 0.25   1.52 ± 0.55   P &lt; 0.05       cases   (N = 71)           T td   1.68 ± 0.69   1.90 ± 0.52   2.23 ± 0.85   P &lt; 0.001           (N = 78)                    
Results of the effect of test drug on long term memory in SDAT cases are shown in  FIG. 5 .
 
         [0000]                                                                                  TABLE 6                   Effect of test drug on Anxiety level in SDAT cases                Anxiety Level (Score)   Comp. initial vs 6            Treated groups   Initial   After 3 months   After 6 months   months treatment                    Normal   P td   53.90 ± 4.11   55.82 ± 3.92   54.77 ± 5.01   P &gt; 0.05       aged   (N = 62)           T td   54.09 ± 3.11   52.75 ± 4.92   51.23 ± 3.75   P &lt; 0.05           (N = 68)       SDAT   P td   65.14 ± 4.35   64.90 ± 6.39   66.82 ± 4.22   P &lt; 0.01       cases   (N = 71)           T td   66.20 ± 5.16   64.81 ± 3.75   61.90 ± 6.08   P &lt; 0.001           (N = 78)                    
Results of the effect of test drug on Anxiety level in SDAT cases are shown in  FIG. 6 .
 
         [0000]                                                                                  TABLE 7                   Effect of test drug on depression level in SDAT cases                Depression Level (Score)   Comp. initial vs 6            Treated groups   Initial   After 3 months   After 6 months   months treatment                    Normal   P td   10.27 ± 3.04   11.08 ± 2.45   11.62 ± 2.14   P &lt; 0.01       aged   (N = 62)           T td    9.72 ± 2.46    8.20 ± 1.71    7.98 ± 2.85   P &lt; 0.001           (N = 68)       SDAT   P td   15.27 ± 2.45   15.98 ± 3.17   17.60 ± 2.13   P &lt; 0.001       cases   (N = 71)           T td   16.23 ± 3.10   14.38 ± 2.99   13.62 ± 4.02   P &lt; 0.001           (N = 78)                    
Results of the effect of test drug on depression level in SDAT cases are shown in  FIG. 7 .
 
         [0000]                                                                                  TABLE 8                   Effect of test drug on hs C-Reactive Protein level in SDAT cases                CRP (mg/L)   Comp. initial vs 6            Treated groups   Initial   After 3 months   After 6 months   months treatment                    Normal   P td   2.10 ± 0.48   1.97 ± 0.45   2.28 ± 0.70   P &lt; 0.05       aged   (N = 62)           T td   2.38 ± 0.76   1.85 ± 0.36   1.54 ± 0.84   P &lt; 0.001           (N = 68)       SDAT   P td   6.23 ± 1.09   5.98 ± 0.88   6.04 ± 1.12   P &gt; 0.05       cases   (N = 71)           T td   7.11 ± 1.02   6.25 ± 0.97   5.62 ± 1.23   P &lt; 0.001           (N = 78)                    
Results of the effect of test drug on hs C-Reactive Protein level in SDAT cases are shown in  FIG. 8 .
 
         [0000]                                                                                  TABLE 9                   Effect of test drug on Interleukin-6 level in SDAT cases                Interleukin-6 (pg/ml)   Comp. initial vs 6            Treated groups   Initial   After 3 months   After 6 months   months treatment                    Normal   P td   2.28 ± 0.41   2.19 ± 0.28   2.55 ± 0.24   P &lt; 0.001       aged   (N = 62)           T td   2.23 ± 0.24   1.68 ± 0.33   1.35 ± 0.20   P &lt; 0.001           (N = 68)       SDAT   P td   3.48 ± 0.37   3.98 ± 0.35   4.68 ± 1.02   P &lt; 0.001       cases   (N = 71)           T td   4.12 ± 1.06   3.68 ± 0.91   3.15 ± 0.68   P &lt; 0.001           (N = 78)                    
Results of the Effect of test drug on Interleukin-6 level in SDAT cases are shown in  FIG. 9 .
 
         [0000]                                                                                  TABLE 10                   Effect of test drug on TNF-α level in SDAT cases                TNF-α (pg/ml)   Comp. initial vs 6            Treated groups   Initial   After 3 months   After 6 months   months treatment                    Normal   P td   364.97 ± 53.41   410.85 ± 64.17    459.42 ± 71.28    P &lt; 0.001       aged   (N = 62)           T td   483.80 ± 85.64   422.90 ± 91.23    378.91 ± 48.42    P &lt; 0.001           (N = 68)       SDAT   P td    994.75 ± 128.42   972.40 ± 128.75   983.73 ± 121.05   P &gt; 0.05       cases   (N = 71)           T td   1124.06 ± 105.87   835.71 ± 128.45   744.55 ± 101.73   P &lt; 0.001           (N = 78)                    
Results of the Effect of test drug on TNF-α level in SDAT cases are shown in  FIG. 10 .
 
         [0000]                                                                                  TABLE 11                   Effect of test drug on Homocysteine level in SDAT cases                Homocysteine (mmol/L)   Comp. initial vs 6            Treated groups   Initial   After 3 months   After 6 months   months treatment                    Normal   P td   21.99 ± 2.85   23.41 ± 3.52   20.90 ± 3.05   P &lt; 0.05       aged   (N = 62)           T td   23.41 ± 2.75   21.06 ± 3.12   18.42 ± 3.29   P &lt; 0.001           (N = 68)       SDAT   P td   38.42 ± 4.16   37.88 ± 3.96   39.42 ± 4.11   P &gt; 0.05       cases   (N = 71)           T td   42.90 ± 5.62   34.82 ± 3.87   29.08 ± 4.16   P &lt; 0.001           (N = 78)                    
Results of the Effect of test drug on Homocysteine level in SDAT cases are shown in  FIG. 11 .