The polyherbal eye drop (Itone™) is a sterile topical formulation that has been found to have pH 5.1 and is hypo-osmolar in nature. It has been claimed to protect eyes from continuous strains, glares of light and various forms of pollution upon its instillation. It has also been indicated as an adjunct in various ophthalmic conditions like conjunctivitis, trachoma, blepharitis, keratitis, corneal ulcers, lentricular opacity, myopia, hypermetropia and dacrocystitis . Experiments so far evaluated using PHF showed several properties viz., lack of any acute, subacute CNS toxicity after oral administration in animals, lack of ocular irritancy in rabbits and prominent antimicrobial activity against Staphylococcus aureus and Klebsiella pneumoniae. In clinical studies, the PHF under study has been found to be effective in trachoma, chronic conjunctivitis , subjective improvement in refractory errors  healing capacity in allergic conjunctivitis , viral conjunctivitis [14, 15] and computer vision syndrome . Although, enough literature is available regarding the above therapeutic uses for the PHF, no systematic study is available to substantiate the claim using controlled experiments. Therefore, the present study was conducted to evaluate antiangiogenic, anticataract, anti-inflammatory, antioxidant and cytotoxic potential of the PHF using several experimental models. As PHF under study was also reported to provide relief from iridocyclitis, early senile lenticular opacity and IOP lowering effect in glaucoma, its intraocular penetration was felt as necessary. Therefore, this study was extended to evaluate the intraocular penetration in the rabbit eyes upon topical application using liquid chromatography coupled tandem mass spectrometry.
Ocular angiogenesis is responsible for the majority of irreversible blindness in the developed world. This debilitating complication affects all age groups and characterizes such diverse and widespread diseases as trachoma, retinopathy of prematurity, diabetic retinopathy, neovascular glaucoma and age-related macular degeneration . Angiogenesis is a tightly regulated process involving the development of new blood vessels from pre-existing blood vessels. During development and normal physiological processes such as wound healing and the menstrual cycle, angiogenesis is regulated by endogenous activators and inhibitors [17, 18]. In pathological settings, such as age-related macular degeneration, rheumatoid arthritis, diabetic retinopathy and tumor growth and metastasis, angiogenesis is critical for disease progression [17, 19].
The present study evaluated the antiangiogenic potential of a PHF using the in ovo chick CAM assay – an assay that is capable of evaluating the action of test substance (plant extracts) on angiogenesis . For this assay, VEGF 50 ng was used as an angiogenesis stimulator and was found to significantly induce proliferation of new blood vessels as compared to the normal group. In this assay, PHF was found to have significant antiangiogenic potential at the studied concentration thereby inhibiting VEGF induced proliferation of new blood vessels.
In order to understand the in vivo significane of the above finding an animal study was carried out using chemical cautery induced corneal neovascularization in rats. Neovascularization has been reported as an important pathologic event during the corneal wound healing process  and VEGF has been reported to play an important role in its pathogenesis . Neovascularization of cornea may cause loss of corneal transperancy and thereby leads to loss of vision . In the present study, topical instillation of the PHF was found to significantly inhibit chemical cautery induced corneal neovascularization as compared to sham treated control. In this assay, bevacizumab, a known anti-VEGF monoclonal antibody was used as a positive control that showed 67% inhibition of the cautery induced corneal neovascularization in comparison to the sham treated group that showed noticeable corneal neovascularization in the cauterized eyes. The antiangiogenic activity observed for PHF was found to be only 18%, thereby concluding that the antiangiogenic activity observed with PHF is mild in comparison to the potential antiangiogenic compounds like bevacizumab.
Interestingly, the present study revealed that the PHF was found to be safe upon application to the chick embryos and there was no toxicity found. Chorio-allantoic membrane assay is also a well recognized method to study ocular toxicity of drugs . It is assumed as acute irritating effects on the small blood vessels and protein membrane are similar to effect induced by same chemical in the eye . Further to the above observation, lack of any cytotoxicity of the studied PHF was confirmed in HeLa cell lines using MTT assay. In this cytotoxicity assay paclitaxel was used as positive control. This study further confirms lack of any ocular toxicity oberved in majority of the above studies .
Inflammatory stimulus is a factor responsible for micro-vascular gowth (neovascularization) during the active pro-angiogenic phase. Therefore, capillary regression is of interest from a clinical perspective . Therapeutic strateties for reducing the vacular growth has been well accepted as a therapeutic option for pathologic angiogenesis in tumors, eye diseases, and inflammation. Anti-inflammatory compounds like COX-2 inhibitors and steroids are reported to have anti-angiogenic potential [27, 28]. Therefore, the present study was extended to carry out anti-inflammatory property of the PHF in the carageenan induced paw edema model of inflammation.
Inflammation is a normal protective responce to tissue injury caused by physical trauma, noxious chemical or microbial agents and involves release of various inflammatory mediators such as leukotrienes and prostaglandins [29–31]. In the present study, anti-inflammatory activity of PHF was tested using carrageenan induced paw edema assay which has been used since time as a standard technique for the screening of anti-inflammatory activity of several herbal extracts [32, 33]. PHF was observed to possess noticeable anti-inflammatory activity, though the activity was comparatively lesser than the positive control (diclofenac). Furthermore, PHF was evaluated for its inhibitory effect on the formation of LTB4 in human WBCs. LTB4 is a known potent inflammatory mediator and has been implicated as a probable cause of chronic ocular inflammation and retinopathy in diabetes  and has also been held responsible for the development and progression of experimental autoimmune uveitis . Our study showed that PHF inhibited the formation of LTB4 in human WBCs while the inhibitory percentage was relatively lower in comparison to zileuton which is a known lipoxygenase inhibitor.
The widespread prevalence of diabetes in developing countries is expected to increase the magnitude of blindness due to cataract and the cellular and molecular mechanisms underlying the pathogenesis of cataract showed the involvement of polyol pathway, advanced glycation end products and oxidative stress . The therapeutic agents that are capable of altering the above events are expected to prevent or delay the progression of cataract in order to exhibit the desired anti-cataract activity e.g. chyavanprash . Considering the major pathways involved in the etiology of cataract such as polyol pathway or sorbitol accumulation pathway, anticataract potential of PHF was evaluated using various pharmacological screening models mimicking the major pathways in cataract. In the developing chick embryo model of cataract, glucocorticoid (hydrocortisone) produced higher incidence (>90%) cataractous changes in lenses within 48 h . Cataract formation is caused by oxidative stresses, probably derived from hydrocortisone effects on the main target organ, the liver and can be prevented by radical scavengers including ascorbic acid and insulin. In this model of hyperglycemia and oxidative changes in lens including glutathione depletion are emphasized as factors for the loss of transparency. The present study observed a delay in the progression of cataract in all three models of cataract as compared to their untreated controls. However, the PHF did not show any noticeable free radical scavenging activity in the DPPH assay thereby demarking the presence of any antioxidant potential at the studied concentration. From this observation, it is evident that the mild anticataract activity observed with PHF could be beyond its antioxidant property or as a cumulative effect of antioxidant compounds after repeated administration.
In order to evaluate the possible intraocular penetration of PHF, a study was conducted by taking the aqueous humor after multiple topical instillations for availability of compounds present in PHF. Information dependent acquision method was develpoed and used for the testing of the availability of compounds in PHF. Furthermore presence of compounds in PHF was confirmed by MRM and used for aqueous humor analysis. This study proved that some of the compounds found in PHF have the propansity to cross the cornea to produce the intraocular effects. Among the penetrated compounds curcumin, rosmarinic acid and quercetin are capable of having anticataract activity as reported previously [39–41] whereby curcumin has also been reported for its antiangiogenic potential . However, further studies with isolated and purified compounds are essential to reveal the extent of their penetration for their correlation with observed activity. As the PHF is a proprietary Ayurvedic formulation having many components with very low concentrations, the mild but significant protective effects observed on inflammation, angiogenesis and cataract can have cumulative and synergestic effects while on repeated topical applications in patients for their obvious therapeutic effect with low or no toxicity as observed in clinical studies.