Abstract:
The present invention relates to a synergistic herbal composition for lowering the intra ocular pressure in different types of glaucoma and process for the preparation of the same in pharmaceutically acceptable dosage forms.

Description:
FIELD OF THE INVENTION 
     The present invention relates to a synergistic herbal composition for lowering the intra ocular pressure in different types of glaucoma and process for the preparation of the same in pharmaceutically acceptable dosage forms. 
     BACKGROUND OF THE INVENTION 
     Glaucoma is a disease characterized by high intraocular pressure (IOP) sufficient to cause either temporary or permanent impairment of vision. The rise in IOP might be due to:
         Increased rate of aqueous formation   Decreased rate of out flow   Raised pressure in the draining episcleral veins.       

     An obstruction to the circulation of the aqueous at the pupil or to its drainage through the angle of the anterior chamber causes glaucoma. The normal IOP of an individual ranges up to 20 mm Hg and can rise up to 60 to 70 mm of Hg in glaucoma patients. Raised IOP of this magnitude can result in loss of vision by causing damage to retinal nerve fibers. Optic nerve axons of the eyeball become compressed at the optic disc due to elevated IOP. This compression probably blocks the axonal flow of cytoplasm from the neuronal cell bodies in the retina to the extended optic nerve fibers entering the brain. It results in lack of nutrition of fibers and ultimately causes death of the neurons. Compression of retinal artery may increase the neuronal damage due to reduction in retinal nutrition. 
     Glaucoma is Generally Classified as 
     
         
         
           
             Primary 
             Developmental 
             Secondary 
           
         
       
    
     The commonest form of glaucoma is primary glaucoma; it can be: 
     Open Angle Glaucoma: 
     Angle of the anterior chamber is always open, at all stages of disease, and aqueous has access to the outflow channels at all times. There is increased resistance to outflow in the corneoscleral meshwork. IOP may be raised or elevated. 
     Angle Closure Glaucoma: 
     No abnormal resistance to outflow in the corneoscleral meshwork is observed. The sole cause of elevated tension is closure of the angle. The iris obstructs the access of aqueous humor to the outflow channels. 
     Magnitude of the Disease 
     Glaucoma is the third major cause of avoidable blindness. The global estimate of blindness is over 44 million with glaucoma accounting for more than 15% of the blind patients worldwide (2). Worldwide approximately two-thirds of all blind or visually impaired people are women (3). India has a high burden of blind (23.5%) in the world and 13% of the global blindness due to glaucoma is in India. Many population-based surveys carried out in the west and in Asia have shown that glaucoma remains undetected in nearly 50% of the cases and hence glaucoma related blindness and disability is often underestimated (4-5). 
     Prompt and effective management of glaucoma is necessary to reduce the incidence of cases of bilateral blindness due to progressive glaucoma. Biological revolution in medicine has provided new avenues for therapeutic intervention. Newer and innovative treatment strategies are being considered for the control of raised intraocular pressure (TOP) by the use of synthetic and herbal drugs in glaucoma. 
     Presently Available Glaucoma Therapy 
     The primary goal in the management of glaucoma is to lower IOP below 20 mm Hg in the patients with mild changes in the optic disc and below 15 mm Hg in the patients with more severe changes. Surgical intervention aiming at increasing the aqueous humor outflow is undertaken when IOP remains uncontrolled even with multiple drug therapy. 
     The following groups/drugs are widely used in the treatment of glaucoma: 
                                                                 Group of Drugs   Formulations                                TOPICAL DRUGS            Cholinergic agonists   Pilocarpine, carbechol, physostigmine       Adrenergic agonists   Forskolin, isoproterenol, salbutamol,           epinephrine       Adrenergic antagonists   Timolol, betaxolol, atenolol       Prostaglandin analogues   Latanoprost       Carbonic anhydrase   Trifluoromethazolmide, aminozolamide       inhibitors            SYSTEMIC DRUGS            Carbonic anhydrase   Acetazolamide, methazolamide,       inhibitor   dichlorphenamide       Hyper- osmotic agents   Glycerol, mannitol       Miscellaneous   Cannabinoids, prostaglandins, ACE           inhibitors, melatonin, calcium           channel blockers, haloperidol.                    
Drawbacks of the Presently Available Treatment
 
     Synthetic drugs are currently available for the control of intraocular pressure (IOP) but have the drawbacks of being toxic, expensive and often have to be administered multiple times a day resulting in poor patient compliance. Thus, an inexpensive drug with potent ocular hypotensive effect and devoid of side effects with low frequency of application is desirable and its identification will be major achievement for human welfare. Medicinal plant based formulations are being used since long for a variety of diseases. Pilocarpine is one example of antiglaucoma drug of plant origin. A variety of substances of plant origin are known to have antichlolonergic and diuretic activity. Such drugs might be useful antiglaucoma drugs. 
     Following are the adverse reactions observed for synthetic drugs: 
     
       
         
               
               
             
               
               
               
             
           
               
                   
                   
               
               
                   
                 Adverse reactions 
               
             
          
           
               
                 Pharmaceutical agents 
                 Ocular 
                 Systemic 
               
               
                   
               
               
                 Cholinergic agonist 
                 Stinging, irritation 
                 Headache, pain 
               
               
                 Pilocarpine 
                 Ciliary spasms (myopia) 
                 Sweating 
               
               
                   
                 Miosis (vision) 
                 Vomiting/diarrhea 
               
               
                   
                 Pupillary block 
                 Salivation, Bradycardia 
               
               
                   
                 Retinal detachment 
                 Arrhythmia 
               
               
                   
                   
                 Dyspnea 
               
               
                 Adrenergic agonist 
                 Stinging, burning 
                 Increased blood 
               
               
                 Epinephrine 
                 Mydriasis 
                 Pressure 
               
               
                   
                 Allergic sensitivity 
                 Increased heart rate 
               
               
                   
                 Pigment deposits 
                 Severe headaches 
               
               
                   
                 Cystoid macular edema 
                 Anxiety 
               
               
                   
                 Increased intraocular pressure (IOP) 
               
               
                 Alpha-2 agonists 
                 Allergic sensitivity 
                 Gastrointestinal 
               
               
                 Clonidine 
                 Minimal mydriasis 
                 Discomfort 
               
               
                   
                 Lid retraction 
                 Taste abnormalities 
               
               
                   
                 Conjunctival vasoconstriction 
                 Headache 
               
               
                   
                 Stinging, burning 
                 Fatigue/drowsiness 
               
               
                   
                 Foreign body sensation 
                 Oral dryness 
               
               
                   
                 Hyperemia 
               
               
                   
                 Conjunctival follicles 
               
               
                 Topical beta-blockers 
                 Stinging, burning 
                 Dyspnea 
               
               
                 Timolol 
                 Superficial punctate keratitis 
                 Bronchiole constriction 
               
               
                 Betaxolol 
                 Allergic sensitivity 
                 Decreased heart rate 
               
               
                   
                 Decreased corneal sensitivity 
                 Arrhythmias 
               
               
                   
                 Uveitis 4 
                 Decreased blood 
               
               
                   
                   
                 Pressure 
               
               
                   
                   
                 Depression, confusion 
               
               
                   
                   
                 Gastrointestinal 
               
               
                   
                   
                 Discomfort 
               
               
                 Oral carbonic 
                 None 
                 Malaise 
               
               
                 anhydrase inhibitors 
                   
                 Depression, confusion 
               
               
                 Acetazolamide 
                   
                 Metallic taste 
               
               
                   
                   
                 Anorexia 
               
               
                   
                   
                 Diarrhea 
               
               
                 Topical carbonic 
                 Stinging/burning 
                 Altered taste 
               
               
                 anhydrase inhibitors 
                 Allergic sensitivity 
               
               
                 Dorzolamide 
                 Blurred vision 
               
               
                   
                 Superficial punctate 
               
               
                   
                 keratitis 
               
               
                   
                 Corneal edema 
               
               
                 Prostaglandin analogs 
                 Blurred vision, Stinging, burning, 
                 Headaches 
               
               
                 Latanoprost 
                 Hyperemia 
                 Upper respiratory tract 
               
               
                   
                 Foreign body sensation 
                 Symptoms 
               
               
                   
                 Itching. 
               
               
                   
               
             
          
         
       
     
     OBJECTIVES OF THE INVENTION 
     The object of the present invention is to develop herbal synergistic composition for its IOP lowering and anti glaucoma potential. 
     Another object of the present invention is to formulate the synergistic herbal composition in to pharmaceutically acceptable ophthalmic dosage form with optimum anti glaucoma activity. 
     Yet another object of the present invention is to overcome disadvantages associated with commonly used allopathic medicine for glaucoma. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Accordingly present invention provides a synergistic herbal composition for lowering IOP in glaucoma comprising synergistically effective amount of extract of herbs selected from  Ocimum  species,  Curcuma  species,  Solanum nigrum  and  Areca catechu  optionally along with a pharmaceutically acceptable excipient or carrier. 
     In synergistic herbal composition of the present invention,  Ocimum  species is selected from the group consisting of  Ocimum basilicum, Ocimum canum, Ocimum kilimandscaricum, Ocimum sanctum, Ocimum viride  and  Curcuma  species is selected from the group consisting of  Curcuma longa, Curcuma avaleton, Curcuma amada, Curcuma aromatica Salisb.    
     A synergistic herbal composition of the present invention comprises: 
     
       
         
               
               
               
             
           
               
                   
               
               
                 S. No. 
                 Herbs 
                 % age by weight 
               
               
                   
               
             
             
               
                 1 
                 Extract of  Ocimum  species 
                 0.10 to 1.0% 
               
               
                 2 
                 Extract of  Curcuma  species 
                 0.01 to 0.50% 
               
               
                 3 
                 Extract of  Solanum nigrum   
                 0.10 to 0.75% 
               
               
                 4 
                 Extract of  Areca catechu   
                 0.10 to 1.0% 
               
               
                   
               
             
          
         
       
     
     A synergistic herbal composition of the present invention preferably comprises: 
     
       
         
               
               
               
             
           
               
                   
               
               
                 S. No. 
                 Herbs 
                 % age by weight 
               
               
                   
               
             
             
               
                 1 
                   Ocimum basilicum  extract 
                 0.50% 
               
               
                 2 
                   Curcuma longa  extract 
                 0.10% 
               
               
                 3 
                   Solanum nigrum  extract 
                 0.25% 
               
               
                 4 
                   Areca catechu  extract 
                 0.25% 
               
               
                   
               
             
          
         
       
     
     In the preferred embodiment, the synergistic herbal composition of the present invention preferably comprises  Ocimum basilicum, Curcuma longa  and  Solanum nigrum.    
     In yet another preferred embodiment, the synergistic herbal composition of the present invention preferably comprises  Ocimum basilicum, Curcuma longa  and  Areca catechu.    
     The use of the terms, “synergistic” and “synergistically effective,” are used in the present invention to mean a biological effect created from the application of two or more agents to produce a biological effect that is greater than the sum of the biological effects produced by the application of individual agents. 
     The synergistic herbal composition of the present invention optionally comprising the pharmaceutically acceptable excipients which are selected from the group comprising solubility enhancing agents such as polysorbate, cyclodextrin and their derivative, Cremophore RH 40 and other derivative, Viscosity increasing agents such as Hydroxy propyl methyl cellulose and other suitable cellulose derivative, poly vinyl alcohol, povidone, carbopol caragenin, etc, anti oxidants such as citric acid, EDTA and salts thereof, sodium metabisulphite and other approved water soluble anti oxidants, buffering agents such as citrate, borate, phosphate, citro phosphate and osmolarity adjusting agents such as sodium chloride, mannitol, glycerol, preservatives such as benzalkonium chloride, sorbic acid, methyl paraben, propyl paraben, and salts there of etc. 
     The synergistic herbal composition of the present invention is preferably formulated in the form of eye drop, ointment, cream, gel. 
     The present invention further comprises a process for preparing the said herbal formulation. The process of preparing the said herbal ophthalmic formulation comprises following steps:
         i. Coarsely grinding the individual herb such as herein described and extracting it with solvents having different polarity selected from the group such as water, alcohol, acetone, ethyl acetate, Petroleum ether, chloroform or their mixture in different ratio;   ii. Optionally concentrating the extract obtained in step (a) under vacuum and converting the same either in the form of thick paste having around 20-30% solids or in the form of dry powder;   iii. Formulating pharmaceutically acceptable herbal composition by combining the extracts obtained in step (a) or step (b) in different ratios such as herein described to yield the synergistic herbal composition in the form of eye drop, ointment, cream or gel;   iv. Alternatively the extraction can also be performed using distillation method and distillate can be directly used for formulating synergistic herbal composition such as herein described.       

     EXPERIMENTAL PROCEDURE 
     The invention is illustrated by the following examples which are not meant to restrict the scope of the invention in any manner 
     Although the invention has been described with reference to specific embodiments, this description is not meant to be construed in a limiting sense. Various modifications of the disclosed embodiments, as well as alternate embodiments of the invention, will become apparent to persons skilled in the art upon reference to the description of the invention. It is therefore contemplated that such modifications can be made without departing from the spirit or scope of the present invention as defined. In the examples, “part” and “parts” mean “part by weight” and “parts by weight”, respectively, unless otherwise specified. 
     The process for preparing the herbal composition comprises the following steps:
         (i) Extraction of herb: The herbs are cleaned, coarsely ground and extracted with water using 8 times the amount of water by heating it to boil for 10 min. The extract is filtered and concentrated to a thick paste having around 30% of solid content. It is then dried in vacuum drier at a temperature of 60 C or it can also be spray dried or freeze dried. The dried powder is collected, packed after sieving in double poly bag. Alternatively herbs can be distilled and distillate can be used for further formulation development.   (ii) The extracts as per formula are weighed and dissolved in water containing 0.25% HPMC as viscosity increasing agent under stirring. Additionally Cremophore RH 40 or its suitable derivative is added to increase the solubility.   (iii) The preparation in centrifuged at about 4000 rpm for 30-60 min to remove the insoluble impurities. Alternatively it can be filtered using what man filter paper.   (iv) To the clear filtrate suitable preservative (in this case, BKC, methyl and propyl paraben sodium and sorbic acid, phenyl ethyl alcohol and mercury containing preservatives are found to be suitable) are dissolved.   (v) Disodium EDTA and sodium meta bisulphite as anti oxidants are dissolved in it by stirring   (vi) Finally pH is adjusted with boric acid/borax, citric acid, sodium citrate, potassium and sodium phosphate to 5-7 and volume is made up with water for injection.   (vii) The liquid is then filtered using Millipore filter of 0.22 micron and filled in plastic eye drops vial under laminar flow and labeled.   (viii) The final product is clear, transparent having yellow to yellowish red color having osmolarity of 250-350 mosmol/kg and pH of 5-7.       

     The anti glaucoma activity of herbal ophthalmic solutions was evaluated in rabbits using following models of glaucoma:
     1. Normotensive model: Twelve Albino rabbits of either sex weighing 1.5-2 kg were subjected for baseline IOP measurements using Non Contact Tonometer. The drug (50 ul) was then instilled into one of the eye while the other eye served as the control eye and was instilled with normal saline (50 ul). IOP was then recorded at 30-minute interval for a period of 6 hours.   2. Water loading model: Twelve Albino rabbits of either sex were fasted overnight. The baseline IOP was then recorded next morning and drug (50 ul) was instilled in the test eye and normal saline (50 ul) in control eye. 30 minutes later rabbits were anaesthetized by injecting thiopentone sodium (16 mg/kg BW) in the marginal ear vein. Infant feeding tube was then inserted into the stomach followed by injection of tap water (100 ml/kg) through the tube. IOP was now recorded every 15-min for a period of 120 minutes. The concentration of drug showing best effect in normotensive model was further tested in water loading model. This model mimics the acute glaucomatous attacks.   3. Steroid induced glaucoma model: 12 young rabbits were trained to accept tonometry and then IOP was measured daily for 15 days so as to make a record of baseline TOP. These rabbits were then instilled with prednisolone 1% eye drops (10 ul) in the test eye and normal saline (20 ul) in the control eye, twice a day for a period of 40 days. IOP was measured twice a week during steroid treatment period. At the end of 40 days rabbits were subjected to evaluation of antiglucoma activity. The concentration of drug showing best effect in normotensive model was further tested in steroid induced glaucoma model. TOP measurements were done every 30-minute for a period of 6 hours. This model mimics the chronic open angle glaucoma.   

     The different ophthalmic formulations were prepared using following formulas: 
     (i)  Ocimum Basilicum  Ophthalmic Formulation (F1) Comprises: 
     
       
         
               
               
               
             
           
               
                   
               
               
                   
                   
                 Quantity for 
               
               
                 S.N. 
                 Ingredients 
                 100 ml (mg) 
               
               
                   
               
             
             
               
                 1 
                   Ocimum basilicum  extract 
                 500 
               
               
                 2 
                 Hydroxy propyl methyl cellulose 
                 250 
               
               
                 3 
                 Methyl paraben sodium 
                 200 
               
               
                 4 
                 Propyl paraben sodium 
                  20 
               
               
                 5 
                 Disodium EDTA 
                 100 
               
               
                 6 
                 Sodium metabisulphite 
                 100 
               
               
                 7 
                 Boric acid/borax quantity sufficient to pH 6.5 
                 q.s. 
               
               
                 8 
                 Water for injection q.s. to volume 
                 q.s. 
               
               
                   
               
             
          
         
       
     
     (ii)  Curcuma longa  Ophthalmic Formulation (F2) Comprises: 
     
       
         
               
               
               
             
           
               
                   
               
               
                   
                   
                 Quantity for 
               
               
                 S.N. 
                 Ingredients 
                 100 ml (mg) 
               
               
                   
               
             
             
               
                 1 
                   Curcuma longa  extract 
                 100 
               
               
                 2 
                 Hydroxy propyl methyl cellulose 
                 250 
               
               
                 3 
                 Methyl paraben sodium 
                 200 
               
               
                 4 
                 Propyl paraben sodium 
                  20 
               
               
                 5 
                 Disodium EDTA 
                 100 
               
               
                 6 
                 Sodium metabisulphite 
                 100 
               
               
                 7 
                 Boric acid/borax quantity sufficient to pH 6.5 
                 q.s. 
               
               
                 8 
                 Water for injection q.s. to volume 
                 q.s. 
               
               
                   
               
             
          
         
       
     
     (iii)  Solanum nigrum  Ophthalmic Formulation (F3) Comprises: 
     
       
         
               
               
               
             
           
               
                   
               
               
                   
                   
                 Quantity for 
               
               
                 S.N. 
                 Ingredients 
                 100 ml (mg) 
               
               
                   
               
             
             
               
                 1 
                   Solanum nigrum  extract 
                 250 
               
               
                 2 
                 Hydroxy propyl methyl cellulose 
                 250 
               
               
                 3 
                 Methyl paraben sodium 
                 200 
               
               
                 4 
                 Propyl paraben sodium 
                  20 
               
               
                 5 
                 Disodium EDTA 
                 100 
               
               
                 6 
                 Sodium metabisulphite 
                 100 
               
               
                 7 
                 Boric acid.borax quantity sufficient to pH 6.5 
                 q.s. 
               
               
                 8 
                 Water for injection q.s. to volume 
                 q.s. 
               
               
                   
               
             
          
         
       
     
     (iv)  Areca catechu  Ophthalmic Formulation (F4) Comprises: 
     
       
         
               
               
               
             
           
               
                   
               
               
                   
                   
                 Quantity for 
               
               
                 S.N. 
                 Ingredients 
                 100 ml (mg) 
               
               
                   
               
             
             
               
                 1 
                   Areca catechu  extract 
                 500 
               
               
                 2 
                 Hydroxy propyl methyl cellulose 
                 250 
               
               
                 3 
                 Methyl paraben sodium 
                 200 
               
               
                 4 
                 Propyl paraben sodium 
                  20 
               
               
                 5 
                 Disodium EDTA 
                 100 
               
               
                 6 
                 Sodium metabisulphite 
                 100 
               
               
                 7 
                 Boric acid.borax quantity sufficient to pH 6.5 
                 q.s. 
               
               
                 8 
                 Water for injection q.s. to volume 
                 q.s. 
               
               
                   
               
             
          
         
       
     
     (v) Combined  Ocimum basilicum, Curcuma Longa, Solanum Nigrum , ophthalmic formulation (F5) of the present invention comprises: 
     
       
         
               
               
               
             
               
               
               
             
           
               
                   
               
               
                   
                   
                 Quantity for 
               
               
                 S. N. 
                 Ingredients 
                 100 ml (mg) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                   Ocimum basilicum  extract 
                 500 
               
               
                 2 
                   Curcuma longa  extract 
                 100 
               
               
                 3 
                   Solanum nigrum  extract 
                 250 
               
               
                 4 
                 Hydroxy propyl methyl cellulose 
                 250 
               
               
                 5 
                 BKC 50% 
                 20 
               
               
                 6 
                 Disodium EDTA 
                 100 
               
               
                 7 
                 Sodium metabisulphite 
                 100 
               
               
                 8 
                 Borax/boric acid quantity sufficient to pH 6.5 
                 q.s. 
               
               
                 9 
                 Water for injection q.s. to volume 
                 q.s. 
               
               
                   
               
             
          
         
       
     
     (vi) Combined  Ocimum basilicum, Curcuma Longa, Areca Catechu  Ophthalmic formulation (F6) of the present invention comprises: 
                                                           Quantity for       S. N.   Ingredients   100 ml (mg)                                1     Ocimum basilicum  extract   500       2     Curcuma longa  extract   100       3     Areca catechu  extract   500       4   Hydroxy propyl methyl cellulose   250       5   Methyl paraben sodium   200       6   Propyl paraben sodium   20       7   Disodium EDTA   100       8   Sodium metabisulphite   100       9   Boric acid/borax quantity sufficient to pH 6.5   q.s.       10   Water for injection q.s. to volume   q.s.                    
Manufacturing Procedure for Formulation (F1, F2, F3 and F4):
         1. Hydroxy propyl methyl cellulose (HPMC) is dissolved in about 50 ml of water under stirring. Once it is dissolved completely, then main active ingredient as per above formula of Formulation (F1, F2, F3 and F4) are added to this and dissolved under stirring.   2. The solution is then centrifuged at 4000 rpm for 30 minutes and clear liquid is collected.   3. Methyl and propyl paraben sodium is dissolved in around 10 ml of water and added to clear filtrate of step 2 under stirring.   4. Item number 5 and 6 are then added and dissolved in main bulk under stirring and pH is checked   5. pH is then adjusted with dilute borax/boric acid solution and volume is made up.   6. The solution is filtered using 0.22 micron filter aseptically and product is filled in 10 ml three piece plastic vial.
 
Manufacturing Procedure for Formulation (F5 and F6):
   1. Hydroxy propyl methyl cellulose (HPMC) is dissolved in about 50 ml of water under stirring. Once it is dissolved completely, then main active ingredient as per above formula of Formulation (F1, F2, F3 and F4) are added to this and dissolved under stirring.   2. The solution is then centrifuged at 5000 rpm for 30 minutes and clear liquid is collected.   3. methyl and propyl paraben sodium is dissolved in around 10 ml of water and added to clear filtrate of step 2 under stirring.   4. Item number 7 and 8 are then added and dissolved in main bulk under stirring and pH is checked.   5. pH is then adjusted with dilute borax/boric acid solution and volume is made up.   6. The solution is filtered using 0.22 micron filter aseptically and product is filled in 10 ml three piece plastic vial.       

     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Effect of  Ocimum basilicum  (F1) on normotensive rabbit eye 
               
             
          
           
               
                   
                 Test Eye 
                   
                   
               
               
                 Time 
                 (% Change in 
                 Control eye (% 
                 % Difference between test and 
               
               
                 (Hours) 
                 IOP) 
                 Change in IOP) 
                 control eye 
               
               
                   
               
             
          
           
               
                 0 
                 0 
                 0 
                 0 
               
               
                 0.5 
                 −6.08 
                 5.92 
                 −12.00 
               
               
                 1 
                 −10.12 
                 5.79 
                 −15.92 
               
               
                 1.5 
                 −12.34 
                 3.67 
                 −16.01 
               
               
                 2 
                 −17.95 
                 4.56 
                 −22.51 
               
               
                 2.5 
                 −18.56 
                 3.86 
                 −22.43 
               
               
                 3 
                 −22.66 
                 −0.16 
                 −22.49 
               
               
                 3.5 
                 −20.64 
                 −0.43 
                 −20.22 
               
               
                 4 
                 −18.47 
                 1.56 
                 −20.03 
               
               
                 4.5 
                 −13.94 
                 1.58 
                 −15.52 
               
               
                 5 
                 −12.62 
                 3.56 
                 −16.18 
               
               
                 5.5 
                 −8.66 
                 2.43 
                 −11.09 
               
               
                 6 
                 −5.07 
                 −1.07 
                 −4.00 
               
               
                   
               
               
                 The IOP reduction in the test eye was −22.61% from baseline at 3 hours post drug instillation and significant effect persisted up to 6 hours post drug instillation (Table 1). 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Effect of  Ocimum basilicum  (F1) in water loading model 
               
             
          
           
               
                   
                 Test eye 
                 Control eye 
                   
               
               
                 Time 
                 (% Change in 
                 (% Change 
                 % Difference between 
               
               
                 (Minutes) 
                 IOP) 
                 in IOP 
                 test and control eye 
               
               
                   
               
             
          
           
               
                 0 
                 0 
                 0 
                 0 
               
               
                 15 
                 23.39 
                 54.00 
                 −30.61 
               
               
                 30 
                 50.07 
                 80.28 
                 −30.21 
               
               
                 45 
                 66.3 
                 99.89 
                 −33.59 
               
               
                 60 
                 63.58 
                 95.12 
                 −31.54 
               
               
                 75 
                 40.79 
                 68.82 
                 −28.03 
               
               
                 90 
                 24.66 
                 52.46 
                 −27.8 
               
               
                 105 
                 10.69 
                 27.56 
                 −16.87 
               
               
                 120 
                 4.04 
                 17.41 
                 −13.37 
               
               
                   
               
               
                 The peak IOP rise in test eye was 66.3%, 45 minutes post water loading while the same was 99.89% in control eye. The drug prevented rise in IOP during entire period of experiment with peak effect of −33.59% at 45-minute post water loading (Table 2). 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Effect of  Ocimum basilicum  (F1) in steroid induced model 
               
             
          
           
               
                   
                   
                   
                 % Difference 
               
               
                   
                 Test eye (% Change 
                 Control eye (% 
                 between test and 
               
               
                 Time (Hours) 
                 in IOP) 
                 Change in IOP 
                 control eye 
               
               
                   
               
             
          
           
               
                 0 
                 0 
                 0 
                 0 
               
               
                 0.5 
                 −9.8093 
                 −3.56012 
                 −6.24919 
               
               
                 1 
                 −18.2329 
                 −4.49933 
                 −13.7335 
               
               
                 1.5 
                 −19.9482 
                 −5.49952 
                 −14.4487 
               
               
                 2 
                 −24.6492 
                 −5.76607 
                 −18.8831 
               
               
                 2.5 
                 −25.3312 
                 −5.64618 
                 −19.685 
               
               
                 3 
                 −26.2759 
                 −4.99331 
                 −21.2826 
               
               
                 3.5 
                 −24.2935 
                 −4.93426 
                 −19.3592 
               
               
                 4 
                 −21.8965 
                 −4.29147 
                 −17.605 
               
               
                 4.5 
                 −17.2735 
                 −5.72653 
                 −11.547 
               
               
                 5 
                 −12.708 
                 −5.61631 
                 −7.09164 
               
               
                 5.5 
                 −10.9236 
                 −5.17484 
                 −5.74877 
               
               
                 6 
                 −9.45058 
                 −3.86215 
                 −5.58843 
               
               
                 8 
                 −1.43136 
                 −2.09522 
                 0.663862 
               
               
                   
               
               
                 The IOP reduction in the test eye was −26.2759% from baseline at 3 hours post drug instillation and significant effect persisted up to 6 hours post drug instillation (Table 3). 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Effect of  Curcuma longa  (F2) on normotensive rabbit eye 
               
             
          
           
               
                 Time 
                 Test eye (% Change 
                 Control eye (% 
                 % Difference between 
               
               
                 (Hours) 
                 in IOP) 
                 Change in IOP 
                 test and control eye 
               
               
                   
               
             
          
           
               
                 0 
                 0 
                 0 
                 0 
               
               
                 0.5 
                 −18.06 
                 2.06 
                 −20.12 
               
               
                 1 
                 −24.73 
                 −2.32 
                 −22.41 
               
               
                 1.5 
                 −30.07 
                 0.38 
                 −30.45 
               
               
                 2 
                 −31.63 
                 −1.74 
                 −29.89 
               
               
                 2.5 
                 −33.70 
                 −5.51 
                 −28.19 
               
               
                 3 
                 −28.13 
                 −0.41 
                 −27.72 
               
               
                 3.5 
                 −28.90 
                 −0.27 
                 −28.63 
               
               
                 4 
                 −23.39 
                 −0.24 
                 −23.15 
               
               
                 4.5 
                 −16.90 
                 1.39 
                 −18.29 
               
               
                 5 
                 −14.43 
                 0.68 
                 −15.11 
               
               
                 5.5 
                 −9.61 
                 −0.39 
                 −9.23 
               
               
                 6 
                 −3.07 
                 0.01 
                 −3.09 
               
               
                   
               
               
                 The IOP reduction in the test eye was −33.70% from baseline at 2.5 hours post drug instillation and significant effect persisted up to 6 hours post drug instillation (Table 4). 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Effect of  Curcuma longa  (F2) in water loading model 
               
             
          
           
               
                   
                   
                   
                 % Difference 
               
               
                   
                 Test eye (% Change 
                 Control eye (% 
                 between test and 
               
               
                 Time (Minutes) 
                 in IOP) 
                 Change in IOP 
                 control eye 
               
               
                   
               
             
          
           
               
                 0 
                 0 
                 0 
                 0 
               
               
                 15 
                 24.78 
                 55.67 
                 −30.89 
               
               
                 30 
                 50.46 
                 81.14 
                 −30.68 
               
               
                 45 
                 69.33 
                 100.32 
                 −30.99 
               
               
                 60 
                 62.34 
                 95.69 
                 −33.36 
               
               
                 75 
                 38.39 
                 67.86 
                 −29.47 
               
               
                 90 
                 23.50 
                 48.53 
                 −25.03 
               
               
                 105 
                 11.17 
                 24.72 
                 −13.55 
               
               
                 120 
                 −0.22 
                 13.93 
                 −14.15 
               
               
                   
               
               
                 The peak IOP rise in test eye was 69.33%, 45 minutes post water loading while the same was 100.32% in control eye. The drug prevented rise in IOP during entire period of experiment with peak effect of −33.36% at 60 minute post water loading (Table 5) 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Effect of  Curcuma longa  (F2) in steroid induced model 
               
             
          
           
               
                 Time 
                 Test eye (% Change 
                 Control eye (% 
                 % Difference between 
               
               
                 (Hours) 
                 in IOP) 
                 Change in IOP 
                 test and control eye 
               
               
                   
               
             
          
           
               
                 0 
                 0 
                 0 
                 0 
               
               
                 0.5 
                 −14.86 
                 0 
                 −14.86 
               
               
                 1 
                 −22.99 
                 0.48 
                 −23.48 
               
               
                 1.5 
                 −28.97 
                 0.13 
                 −29.09 
               
               
                 2 
                 −32.67 
                 −2.56 
                 −30.11 
               
               
                 2.5 
                 −33.01 
                 −2.81 
                 −30.20 
               
               
                 3 
                 −34.34 
                 −2.81 
                 −31.54 
               
               
                 3.5 
                 −30.78 
                 −2.30 
                 −28.48 
               
               
                 4 
                 −26.53 
                 −1.08 
                 −25.44 
               
               
                 4.5 
                 −22.13 
                 −1.08 
                 −21.05 
               
               
                 5 
                 −17.77 
                 −0.14 
                 −17.63 
               
               
                 5.5 
                 −13.77 
                 −0.14 
                 −13.63 
               
               
                 6 
                 −10.74 
                 −0.14 
                 −10.59 
               
               
                 8 
                 −1.82 
                 −0.92 
                 −0.90 
               
               
                   
               
               
                 The IOP reduction in the test eye was −34.34% from baseline at 3 hours post drug instillation and significant effect persisted up to 6 hours post drug instillation (Table 6). 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Effect of  Solanum nigrum  (F3) on normotensive rabbit eye 
               
             
          
           
               
                 Time 
                 Test eye 
                 Control eye (% 
                 % Difference between 
               
               
                 (Hours) 
                 (% Change in IOP) 
                 Change in IOP 
                 test and control eye 
               
               
                   
               
             
          
           
               
                 0 
                 0 
                 0 
                 0 
               
               
                 0.5 
                 −3.62 
                 1.56 
                 −5.18 
               
               
                 1 
                 −19.61 
                 −0.63 
                 −18.97 
               
               
                 1.5 
                 −22.26 
                 −0.63 
                 −21.63 
               
               
                 2 
                 −23.81 
                 −0.65 
                 −23.16 
               
               
                 2.5 
                 −24.92 
                 −1.41 
                 −23.52 
               
               
                 3 
                 −25.03 
                 −1.07 
                 −23.97 
               
               
                 3.5 
                 −24.69 
                 −1.42 
                 −23.27 
               
               
                 4 
                 −23.31 
                 −1.75 
                 −21.56 
               
               
                 4.5 
                 −18.48 
                 0.09 
                 −18.56 
               
               
                 5 
                 −15.15 
                 0.54 
                 −15.69 
               
               
                 5.5 
                 −11.88 
                 0.54 
                 −12.42 
               
               
                 6 
                 −8.21 
                 0.54 
                 −8.76 
               
               
                 8 
                 −3.86 
                 1.25 
                 −5.11 
               
               
                   
               
               
                 The IOP reduction in the test eye was −25.03% from baseline at 3 hours post drug instillation and significant effect persisted up to 6 hours post drug instillation (Table 7). 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 Effect of  Solanum nigrum  (F3) in water loading model 
               
             
          
           
               
                 Time 
                 Test eye (% Change 
                 Control eye (% 
                 % Difference between 
               
               
                 (Minutes) 
                 in IOP) 
                 Change in IOP 
                 test and control eye 
               
               
                   
               
             
          
           
               
                 0 
                 0 
                 0 
                 0 
               
               
                 15 
                 28.20 
                 55.10 
                 −26.90 
               
               
                 30 
                 51.24 
                 84.23 
                 −32.99 
               
               
                 45 
                 66.70 
                 104.10 
                 −37.40 
               
               
                 60 
                 69.39 
                 99.21 
                 −29.82 
               
               
                 75 
                 39.23 
                 67.91 
                 −28.69 
               
               
                 90 
                 26.36 
                 50.84 
                 −24.47 
               
               
                 105 
                 11.87 
                 26.59 
                 −14.72 
               
               
                 120 
                 4.03 
                 16.12 
                 −12.09 
               
               
                   
               
               
                 The peak IOP rise in test eye was 69.39%, 60 minutes post water loading while the same was 104.10% at 45 minute in control eye. The drug prevented rise in IOP during entire period of experiment with peak effect of −37.40% at 45-minute post water loading (table 8). 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                 Effect of  Solanum nigrum  (F3) in steroid induced model 
               
             
          
           
               
                 Time 
                 Test eye 
                 Control eye (% 
                 % Difference between 
               
               
                 (Hours) 
                 (% Change in IOP) 
                 Change in IOP 
                 test and control eye 
               
               
                   
               
             
          
           
               
                 0 
                 0 
                 0 
                 0 
               
               
                 0.5 
                 −11.62 
                 1.10 
                 −12.72 
               
               
                 1 
                 −31.89 
                 −1.59 
                 −30.30 
               
               
                 1.5 
                 −31.82 
                 −1.96 
                 −29.86 
               
               
                 2 
                 −33.58 
                 −2.70 
                 −30.88 
               
               
                 2.5 
                 −28.52 
                 −2.84 
                 −25.68 
               
               
                 3 
                 −27.39 
                 −3.76 
                 −23.64 
               
               
                 3.5 
                 −25.15 
                 −3.43 
                 −21.73 
               
               
                 4 
                 −25.15 
                 −3.37 
                 −21.78 
               
               
                 4.5 
                 −19.39 
                 −2.84 
                 −16.55 
               
               
                 5 
                 −18.44 
                 −2.97 
                 −15.48 
               
               
                 5.5 
                 −11.25 
                 0.80 
                 −12.05 
               
               
                 6 
                 −9.00 
                 0.80 
                 −9.80 
               
               
                 8 
                 −0.26 
                 2.29 
                 −2.55 
               
               
                   
               
               
                 The IOP reduction in the test eye was −33.58% from baseline at 2 hours post drug instillation and significant effect persisted up to 6 hours post drug instillation (Table 9). 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 10 
               
             
             
               
                   
               
               
                 Effect of  areca catechu  (F4) in normotensive model 
               
             
          
           
               
                 Time 
                 Test eye (% Change 
                 Control eye (% 
                 % Difference Treated 
               
               
                 in hours 
                 in IOP) 
                 Change in IOP 
                 Eye-Control Eye 
               
               
                   
               
             
          
           
               
                 0 
                 0.00 
                 0.00 
                 0.00 
               
               
                 0.5 
                 −13.59 
                 −2.54 
                 −11.05 
               
               
                 1 
                 −17.63 
                 −1.16 
                 −16.47 
               
               
                 1.5 
                 −19.68 
                 −1.94 
                 −17.74 
               
               
                 2 
                 −20.60 
                 −0.75 
                 −19.85 
               
               
                 3 
                 −17.53 
                 −2.58 
                 −14.95 
               
               
                 4 
                 −15.71 
                 −1.23 
                 −14.48 
               
               
                 5 
                 −9.88 
                 −0.48 
                 −9.41 
               
               
                 6 
                 −5.54 
                 −2.96 
                 −2.58 
               
               
                   
               
               
                 The IOP reduction in the test eye was −20.60% from baseline at 2 hours post drug instillation and significant effect persisted up to 6 hours post drug instillation (Table 10). 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 11 
               
             
             
               
                   
               
               
                 Effect of  Areca catechu  (F4) in water loaded model 
               
             
          
           
               
                 Time in 
                 Test eye (% 
                 Control eye (% 
                 % Difference Treated Eye- 
               
               
                 minutes 
                 Change in IOP) 
                 Change in IOP 
                 Control eye 
               
               
                   
               
             
          
           
               
                 0 
                 0.00 
                 0.00 
                 0.00 
               
               
                 15 
                 24.10 
                 46.22 
                 −22.13 
               
               
                 30 
                 41.48 
                 72.98 
                 −31.50 
               
               
                 45 
                 69.10 
                 101.96 
                 −32.87 
               
               
                 60 
                 57.97 
                 84.18 
                 −26.21 
               
               
                 75 
                 42.14 
                 64.31 
                 −22.17 
               
               
                 90 
                 35.27 
                 49.39 
                 −14.12 
               
               
                 120 
                 12.44 
                 15.69 
                 −3.25 
               
               
                   
               
               
                 The peak IOP rise in test eye was 69.10%, 45 minutes post water loading while the same was 101.96% at 45 minute in control eye. The drug prevented rise in IOP during entire period of experiment with peak effect of −32.87% at 45 minute post water loading (Table 11). 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 12 
               
             
             
               
                   
               
               
                 Effect of  areca catechu  (F4) in steroid model 
               
             
          
           
               
                 Time in 
                 Test eye (% Change 
                 Control eye (% 
                 % Difference Treated 
               
               
                 hours 
                 in IOP) 
                 Change in IOP 
                 Eye-Control Eye 
               
               
                   
               
             
          
           
               
                 0 
                 0.00 
                 0.00 
                 0.00 
               
               
                 0.5 
                 −10.92 
                 −0.92 
                 −10.00 
               
               
                 1 
                 −16.18 
                 −0.52 
                 −15.66 
               
               
                 1.5 
                 −19.19 
                 −1.31 
                 −17.87 
               
               
                 2 
                 −25.77 
                 −2.36 
                 −23.41 
               
               
                 3 
                 −22.15 
                 −2.76 
                 −19.40 
               
               
                 4 
                 −16.97 
                 −1.92 
                 −15.04 
               
               
                 5 
                 −12.86 
                 −2.45 
                 −10.41 
               
               
                 6 
                 −5.84 
                 −2.34 
                 −3.50 
               
               
                   
               
               
                 The IOP reduction in the test eye was −25.77% from baseline at 2 hours post drug instillation and significant effect persisted up to 6 hours post drug instillation (Table 12). 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 13 
               
             
             
               
                   
               
               
                 Effect of  Ocimum basilicum ,  Curcuma longa ,  Solanum nigrum   
               
               
                 (F5) in normotensive model 
               
             
          
           
               
                 Time 
                 Test eye (% Change 
                 Control eye (% 
                 % Difference between 
               
               
                 (Hours) 
                 in IOP) 
                 Change in IOP 
                 test and control eye 
               
               
                   
               
             
          
           
               
                 0 
                 0 
                 0 
                 0 
               
               
                 1 
                 −30.01 
                 −0.86 
                 −29.15 
               
               
                 2 
                 −32.03 
                 −0.85 
                 −31.18 
               
               
                 3 
                 −27.46 
                 −0.29 
                 −27.17 
               
               
                 4 
                 −23.77 
                 −0.56 
                 −23.21 
               
               
                 5 
                 −18.89 
                 −0.98 
                 −17.90 
               
               
                 6 
                 −10.49 
                 −0.93 
                 −9.56 
               
               
                 8 
                 −4.88 
                 −0.69 
                 −4.19 
               
               
                 10 
                 −3.74 
                 −0.69 
                 −3.05 
               
               
                   
               
               
                 The IOP reduction in the test eye was −32.03% from baseline at 2 hours post drug instillation and significant effect persisted up to 8 hours post drug instillation (Table 13). 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 14 
               
             
             
               
                   
               
               
                 Comparative data of  curcuma longa ,  Ocimum basilicum  and 
               
               
                   Solanum nigrum  (F1, F2, F3) alone and in combination (F5) in normotensive model 
               
             
          
           
               
                   
                 % Difference 
                 % Difference 
                 % Difference 
                   
               
               
                   
                 between test and 
                 between test and 
                 between test and 
                 % Difference 
               
               
                 Time 
                 control eye 
                 control eye 
                 control eye 
                 between test and 
               
               
                 (Hours) 
                 ( S. nigrum ) 
                 ( O. basilicum ) 
                 ( C. longa   
                 control eye 
               
               
                   
               
             
          
           
               
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 1 
                 −18.97 
                 −15.92 
                 −22.41 
                 −29.15 
               
               
                 2 
                 −23.16 
                 −22.51 
                 −29.89 
                 −31.18 
               
               
                 3 
                 −23.97 
                 −22.49 
                 −27.72 
                 −27.17 
               
               
                 4 
                 −21.56 
                 −20.03 
                 −23.15 
                 −23.21 
               
               
                 5 
                 −15.69 
                 −16.18 
                 −15.11 
                 −17.90 
               
               
                 6 
                 −8.76 
                 −4.0 
                 −3.09 
                 −9.56 
               
               
                   
               
               
                 The maximum peak IOP reduction of −31.18 was observed with instillation of combination of  Ocimum basilicum ,  Curcuma longa  and  Solanum nigrum , (F5) as compared to individual herbs (Table 14). 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 15 
               
             
             
               
                   
               
               
                 Effect of  Ocimum basilicum ,  Curcuma longa , 
               
               
                   Solanum nigrum  (F5) in water loading model 
               
             
          
           
               
                   
                   
                   
                 % Difference 
               
               
                 Time 
                 Test eye (% 
                 Control eye (% 
                 between test and 
               
               
                 (Minutes) 
                 Change in IOP) 
                 Change in IOP 
                 control eye 
               
               
                   
               
             
          
           
               
                 0 
                 0 
                 0 
                 0 
               
               
                 15 
                 20.82 
                 57.20 
                 −36.39 
               
               
                 30 
                 36.24 
                 81.50 
                 −45.26 
               
               
                 45 
                 62.23 
                 102.40 
                 −40.17 
               
               
                 60 
                 58.92 
                 93.56 
                 −34.64 
               
               
                 75 
                 39.54 
                 69.56 
                 −30.03 
               
               
                 90 
                 23.50 
                 50.27 
                 −26.77 
               
               
                 105 
                 8.25 
                 25.85 
                 −17.61 
               
               
                 120 
                 0.96 
                 15.66 
                 −14.70 
               
               
                   
               
               
                 The peak IOP rise in test eye was 62.23%, 45 minutes post water loading while the same was 102.40% in control eye. The drug prevented rise in IOP during entire period of experiment with peak effect of −45.26% at 30 minute post water loading (Table 15). 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 16 
               
             
             
               
                   
               
               
                 Comparative data of  Ocimum basilicum ,  Curcuma longa , 
               
               
                   Solanum nigrum  alone and in combination in water loading model. 
               
             
          
           
               
                   
                 % Difference 
                 % Difference 
                 % Difference 
                 % Difference 
               
               
                   
                 between test and 
                 between test and 
                 between test and 
                 between test and 
               
               
                 Time 
                 control eye 
                 control eye 
                 control eye 
                 control eye 
               
               
                 (Minutes) 
                 ( Solanum nigrum ) 
                 ( O. basilicum ) 
                 ( C. longa ) 
                 (combination) 
               
               
                   
               
             
          
           
               
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 15 
                 −26.90 
                 −30.61 
                 −30.89 
                 −36.39 
               
               
                 30 
                 −32.99 
                 −30.21 
                 −30.68 
                 −45.26 
               
               
                 45 
                 −37.40 
                 −33.59 
                 −30.99 
                 −40.17 
               
               
                 60 
                 −29.82 
                 −31.54 
                 −33.36 
                 −34.64 
               
               
                 75 
                 −28.69 
                 −28.03 
                 −29.47 
                 −30.03 
               
               
                 90 
                 −24.47 
                 −27.8 
                 −25.03 
                 −26.77 
               
               
                 105 
                 −14.72 
                 −16.87 
                 −13.55 
                 −17.61 
               
               
                 120 
                 −12.09 
                 −13.37 
                 −14.15 
                 −14.70 
               
               
                   
               
               
                 The maximum protection against the rise in IOP with a difference of −45.26% between test and control eyes was observed with F5 instillation at 30 minutes post water loading as compared to individual herbs (Table 16). 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 17 
               
             
             
               
                   
               
               
                 Effect of  Ocimum basilicum ,  Curcuma longa , 
               
               
                   Solanum nigrum  (F5) in steroid induced model 
               
             
          
           
               
                   
                   
                   
                 % Difference 
               
               
                 Time 
                 Test eye (% 
                 Control eye (% 
                 between test and 
               
               
                 (Hours) 
                 Change in IOP) 
                 Change in IOP 
                 control eye 
               
               
                   
               
             
          
           
               
                 0 
                 0 
                 0 
                 0 
               
               
                 1 
                 −38.37 
                 −0.86 
                 −37.51 
               
               
                 2 
                 −42.18 
                 −0.85 
                 −41.33 
               
               
                 3 
                 −39.25 
                 −0.29 
                 −38.96 
               
               
                 4 
                 −31.69 
                 −0.56 
                 −31.13 
               
               
                 5 
                 −24.14 
                 −0.98 
                 −23.16 
               
               
                 6 
                 −16.27 
                 −0.93 
                 −15.34 
               
               
                 8 
                 −14.19 
                 −0.69 
                 −13.50 
               
               
                 10 
                 −9.20 
                 −0.69 
                 −8.51 
               
               
                   
               
               
                 The IOP reduction in the test eye was −42.18% from baseline at 2 hours post drug instillation and significant effect persisted up to 10 hours post drug instillation (Table 17). 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 18 
               
             
             
               
                   
               
               
                 Comparative data of  curcuma longa ,  Ocimum basilicum  and 
               
               
                   Solanum nigrum  alone and in combination in Steroid model. 
               
             
          
           
               
                   
                 % Difference 
                 % Difference 
                 % Difference 
                 % Difference 
               
               
                   
                 between test and 
                 between test and 
                 between test and 
                 between test and 
               
               
                 Time 
                 control eye 
                 control eye 
                 control eye 
                 control eye 
               
               
                 (Hours) 
                 ( S. nigrum ) 
                 ( O. basilicum ) 
                 ( C. longa   
                 (Combination) 
               
               
                   
               
             
          
           
               
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 1 
                 −30.30 
                 −13.73 
                 −23.48 
                 −37.51 
               
               
                 2 
                 −30.88 
                 −18.88 
                 −30.11 
                 −41.33 
               
               
                 3 
                 −23.64 
                 −21.28 
                 −31.54 
                 −38.96 
               
               
                 4 
                 −21.78 
                 −17.60 
                 −25.44 
                 −31.13 
               
               
                 5 
                 −15.48 
                 −7.09 
                 −17.63 
                 −23.16 
               
               
                 6 
                 −9.80 
                 −5.56 
                 −10.59 
                 −15.34 
               
               
                   
               
               
                 The maximum peak IOP reduction of −41.33 was observed with instillation of combination of  Ocimum basilicum ,  Curcuma longa  and  Solanum nigrum , (F5) as compared to individual herbs (Table 18). 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 19 
               
             
             
               
                   
               
               
                 Effect of  Ocimum basilicum ,  Curcuma longa , 
               
               
                   Areca catechu  (F6) in normotensive model 
               
             
          
           
               
                   
                   
                   
                 % Difference 
               
               
                 Time 
                 Test eye (% 
                 Control eye (% 
                 between test and 
               
               
                 (Hours) 
                 Change in IOP) 
                 Change in IOP 
                 control eye 
               
               
                   
               
             
          
           
               
                 0 
                 0 
                 0 
                 0 
               
               
                 1 
                 −26.38 
                 −1.60 
                 −24.78 
               
               
                 2 
                 −32.58 
                 −2.12 
                 −30.46 
               
               
                 3 
                 −31.51 
                 −3.16 
                 −28.36 
               
               
                 4 
                 −27.54 
                 −2.85 
                 −24.70 
               
               
                 5 
                 −20.01 
                 −2.15 
                 −17.87 
               
               
                 6 
                 −13.95 
                 −1.01 
                 −12.95 
               
               
                 7 
                 −10.35 
                 −1.79 
                 −8.56 
               
               
                 8 
                 −7.86 
                 −2.13 
                 −5.73 
               
               
                 10 
                 −3.48 
                 −1.40 
                 −2.07 
               
               
                   
               
               
                 The IOP reduction in the test eye was −32.58% from baseline at 2 hours post drug instillation and significant effect persisted up to 8 hours post drug instillation (Table 19). 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 20 
               
             
             
               
                   
               
               
                 Comparative data of  curcuma longa ,  Ocimum basilicum  and 
               
               
                   Areca catechu  alone and in combination in Normotensive model. 
               
             
          
           
               
                   
                 % Difference 
                 % Difference 
                 % Difference 
                 % Difference 
               
               
                   
                 between test and 
                 between test and 
                 between test and 
                 between test and 
               
               
                 Time 
                 control eye 
                 control eye 
                 control eye 
                 control eye 
               
               
                 (Hours) 
                 ( A. catechu ) 
                 ( O. basilicum ) 
                 ( C. longa   
                 (combination) 
               
               
                   
               
             
          
           
               
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 1 
                 −16.47 
                 −15.92 
                 −22.41 
                 −24.78 
               
               
                 2 
                 −19.85 
                 −22.51 
                 −29.89 
                 −30.46 
               
               
                 3 
                 −14.95 
                 −22.49 
                 −27.72 
                 −28.36 
               
               
                 4 
                 −14.48 
                 −20.03 
                 −23.15 
                 −24.70 
               
               
                 5 
                 −9.41 
                 −16.18 
                 −15.11 
                 −17.87 
               
               
                 6 
                 −2.58 
                 −4.0 
                 −3.09 
                 −12.95 
               
               
                   
               
               
                 The maximum peak IOP reduction of −30.46 was observed with instillation of combination of  Ocimum basilicum ,  Curcuma longa  and  Areca catechu  (F6) as compared to individual herbs (Table 20). 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 21 
               
             
             
               
                   
               
               
                 Effect of  Ocimum basilicum ,  Curcuma longa , 
               
               
                   Areca catechu  (F6) in water loading model 
               
             
          
           
               
                   
                   
                   
                 % Difference 
               
               
                 Time 
                 Test eye (% 
                 Control eye (% 
                 between test and 
               
               
                 (Minutes) 
                 Change in IOP) 
                 Change in IOP 
                 control eye 
               
               
                   
               
             
          
           
               
                 0 
                 0 
                 0 
                 0 
               
               
                 15 
                 24.69 
                 57.06 
                 −32.37 
               
               
                 30 
                 36.29 
                 77.04 
                 −40.75 
               
               
                 45 
                 57.48 
                 103.99 
                 −46.51 
               
               
                 60 
                 57.90 
                 93.78 
                 −35.89 
               
               
                 75 
                 45.19 
                 75.29 
                 −30.10 
               
               
                 90 
                 27.11 
                 53.11 
                 −26.01 
               
               
                 120 
                 7.07 
                 27.43 
                 −20.35 
               
               
                   
               
               
                 The peak IOP rise in test eye was 57.90%, 60 minutes post water loading while the same was 103.99% at 45 minute in control eye. The drug prevented rise in IOP during entire period of experiment with peak effect of −46.51% at 45 minute post water loading (Table 21). 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 22 
               
             
             
               
                   
               
               
                 Comparative data of  Ocimum basilicum ,  Curcuma longa , 
               
               
                   Areca catechu  alone and in combination in water loading model 
               
             
          
           
               
                   
                 % Difference 
                 % Difference 
                 % Difference 
                 % Difference 
               
               
                   
                 between test and 
                 between test and 
                 between test and 
                 between test and 
               
               
                 Time 
                 control eye 
                 control eye 
                 control eye 
                 control eye 
               
               
                 (Minutes) 
                 ( Areca catechu ) 
                 ( O. basilicum ) 
                 ( C. longa ) 
                 (combination) 
               
               
                   
               
             
          
           
               
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 15 
                 −22.13 
                 −30.61 
                 −30.89 
                 −32.37 
               
               
                 30 
                 −31.50 
                 −30.21 
                 −30.68 
                 −40.75 
               
               
                 45 
                 −32.87 
                 −33.59 
                 −30.99 
                 −46.51 
               
               
                 60 
                 −26.21 
                 −31.54 
                 −33.36 
                 −35.89 
               
               
                 75 
                 −22.17 
                 −28.03 
                 −29.47 
                 −30.10 
               
               
                 90 
                 −14.12 
                 −27.8 
                 −25.03 
                 −26.01 
               
               
                 120 
                 −3.25 
                 −16.87 
                 −13.55 
                 −20.35 
               
               
                   
               
               
                 The maximum protection against the rise in IOP with a difference of −46.51% between test and control eyes was observed with F6 instillation at 45 minutes post water loading as compared to individual herbs (Table 22). 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 23 
               
             
             
               
                   
               
               
                 Effect of  Ocimum basilicum ,  Curcuma longa , 
               
               
                   Areca catechu  (F6) in steroid induced model 
               
             
          
           
               
                   
                   
                   
                 % Difference 
               
               
                 Time 
                 Test eye (% 
                 Control eye (% 
                 between test and 
               
               
                 (Hours) 
                 Change in IOP) 
                 Change in IOP 
                 control eye 
               
               
                   
               
             
          
           
               
                 0 
                 0 
                 0 
                 0 
               
               
                 1 
                 −38.37 
                 −0.86 
                 −20.00 
               
               
                 2 
                 −42.18 
                 −0.85 
                 −34.2 
               
               
                 3 
                 −39.25 
                 −0.29 
                 −28.7 
               
               
                 4 
                 −31.69 
                 −0.56 
                 −27.00 
               
               
                 5 
                 −24.14 
                 −0.98 
                 −20.5 
               
               
                 6 
                 −16.27 
                 −0.93 
                 −16.6 
               
               
                 8 
                 −14.19 
                 −0.69 
                 −8.3 
               
               
                 10 
                 −9.20 
                 −0.69 
                 −5.2 
               
               
                   
               
               
                 The IOP reduction in the test eye was −42.18% from baseline at 2 hours post drug instillation and significant effect persisted up to 10 hours post drug instillation (Table 23). 
               
             
          
         
       
     
                                                                 TABLE 24                   Comparative data of  curcuma longa (F2),  Ocimum basilicum (F1) and         Areca catechu  (F4)alone and in combination(F6) in Steroid model.                % Difference   % Difference   % Difference   % Difference           between test and   between test and   between test and   between test and       Time   control eye   control eye   control eye   control eye       (Hours)   ( A. catechu )   ( O. basilicum )   ( C. longa     (Combination)                    0   0   0   0   0       1   −15.66   −13.7   −23.48   −20.00       2   −23.41   −18.88   −30.11   −34.2       3   −19.40   −21.28   −31.54   −28.7       4   −15.04   −17.60   −25.44   −27.00       5   −10.41   −7.09   −17.63   −20.5       6   −3.50   −5.59   −10.59   −16.6               The maximum peak IOP reduction of −34.20 was observed with instillation of combination of  Ocimum basilicum ,  Curcuma longa  and  Areca catechu  (F6) as compared to individual herbs (Table 24).            
Results:
 
     From the above results, surprisingly the herbal composition of the present invention comprising synergistic effective amount of  Solanum nigrum, Curcuma longa, Ocimum basilicum  (Formulation-F5) and  Curcuma longa, Ocimum basilicum, Areca catechu  (Formulation-F6) showed enhanced and surprising effects as compared to the individual formulations (F1, F2, F3 and F4). Therefore the said herbal composition (F5 and F6) are working synergistically. 
     Example 1 
     For Formulating the Ophthalmic Drops 
                                                           Quantity per       S.N.   Ingredients   100 ml                                1     Solanum nigrum  extract   250 mg       2     Ocimum basilicum  extract   500 mg       3     Curcuma longa  extract   100 mg       4   Hydroxy propyl methyl cellulose   250 mg       5   Cremophore RH 40   1000 mg        6   Methyl paraben sodium   200 mg       7   Propyl paraben sodium    20 mg       8   Disodium EDTA   100 mg       9   Sodium metabisulphite   100 mg       10   Citric acid   350 mg       11   Disodium hydrogen phosphate   900 mg       12   Sodium chloride   170 mg       13   Water for injection   q.s.                    
Manufacturing Procedure:
         1. Hydroxy propyl methyl cellulose (HPMC) is dissolved in about 50 ml of water under stirring. Cremophore RH 40 is added to this solution and dissolved under stirring. Once it is dissolved completely, then main active ingredient as per above formula (S. No. 1-3) are added to this and dissolved under stirring.   2. The solution is then centrifuged at 5000 rpm for 30 minutes and clear liquid is collected.   3. Methyl and propyl paraben sodium is dissolved in around 10 ml of water and added to clear filtrate of step 2 under stirring.   4. Item number 8 and 9 are then added and dissolved in main bulk under stirring and pH is checked.   5. Finally citric acid and sodium phosphate are added to bulk and volume is made up.   6. The solution is filtered using 0.22 micron filter aseptically and product is filled in 10 ml three piece plastic vial.       

     Example 2 
     For Formulating the Gel 
     
       
         
               
               
               
             
               
               
               
             
           
               
                   
               
               
                   
                   
                 Quantity per 
               
               
                 S.N. 
                 Ingredients 
                 100 ml 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                   Solanum nigrum  extract 
                 250 mg 
               
               
                 2 
                   Ocimum basilicum  extract 
                 500 mg 
               
               
                 3 
                   Curcuma longa  extract 
                 100 mg 
               
               
                 4 
                 Hydroxy propyl methyl cellulose 
                 250 mg 
               
               
                 5 
                 Cremophore RH 40 
                 1000 mg  
               
               
                 6 
                 Methyl paraben sodium 
                 200 mg 
               
               
                 7 
                 Propyl paraben sodium 
                  20 mg 
               
               
                 8 
                 Carbomer 971P 
                 150 mg 
               
               
                 9 
                 Sodium hydroxide 
                 q.s. 
               
               
                 10 
                 Disodium EDTA 
                 100 mg 
               
               
                 11 
                 Sodium metabisulphite 
                 100 mg 
               
               
                 12 
                 Citric acid 
                 350 mg 
               
               
                 13 
                 Disodium hydrogen phosphate 
                 900 mg 
               
               
                 14 
                 Sodium chloride 
                 170 mg 
               
               
                 15 
                 Water for injection 
                 q.s. 
               
               
                   
               
             
          
         
       
         
         
           
             1. Hydroxy propyl methyl cellulose (HPMC) is dissolved in about 50 ml of water under stirring. Cremophore RH 40 is added to this solution and dissolved under stirring. Once it is dissolved completely, then main active ingredient as per above formula (S. No. 1-3) are added to this and dissolved under stirring. 
             2. The solution is then centrifuged at 5000 rpm for 30 minutes and clear liquid is collected. 
             3. Methyl and propyl paraben sodium is dissolved in around 10 ml of water and added to clear filtrate of step 2 under stirring. 
             4. Item number 8 is added under slow stirring, once it is dissolved then pH is increased to about 6.5 with dilute sodium hydroxide solution. Item No. 10 and 11 are then added and dissolved in main bulk under stirring and pH is checked. 
             5. Finally citric acid and sodium phosphate are added to bulk and volume is made up. 
             6. The solution is filtered using 0.22 micron filter aseptically and product is filled in 10 ml three piece plastic vial.