Decrease in soluble protein content
Decrease in soluble protein content in diabetic lenses compared with those in control lenses in present study could be due to leakage of proteins and insolubilization. T. purpurea treatment could increase the levels of soluble proteins in diabetic lenses may be by preventing cross-linking/aggregation and distribution of soluble proteins. Under conditions of severe oxidative stress, free radical generation leads to protein modification. T. purpurea treatment possessing potent anti-oxidant potential may prevent protein modification, and hence, may be helpful in preventing insolubilization of proteins. This may prevent or delay the development of opacity of lens.
Aldose reductase (AR) is a small monomeric protein belonging to aldo-keto reductase superfamily. AR-derived polyols like sorbitol accumulate in the diabetic ocular lens , which causes osmotic swelling resulting in ionic imbalance, and protein insolubilization leading to cataractogenesis. Osmotic swelling of diabetic lens may render the cells leaky , enhancing loss of GSH accumulated in the lens . Disrupted cell membrane by osmotic stress may also interfere with amino Varenicline Tartrate transport into the lens , and hence biosynthesis of GSH . Moreover, AR reduction of glucose to sorbitol probably contributes to oxidative stress by depleting its cofactor NADPH, which is also required for the regeneration of GSH . Studies suggested that persistent high intracellular glucose concentration-induced superoxide generation inhibits GAPDH activity  which increases the levels of all the glycolytic intermediates located in upstream of GAPDH, finally increasing the first glycolytic metabolite, glucose . Moreover, inhibition of GAPDH is responsible for an increased formation of the AGE-forming compound methylglyoxal . As shown by Chang et al. , methylglyoxal is also responsible for substrate-induced upregulation of AR, which may further facilitate development of diabetic cataract. AR inhibiting activity of investigated plant T. purpurea was evaluated in-vitro using rat lens homogenate. The alcoholic extract as well as flavonoid fraction of the plant have shown significant AR inhibiting activity, which may propose probable mechanism of plant in delaying development of diabetic complications like cataract. Also, docking results suggested that most of the known constituents have a common binding mode in the vicinity of active site of subunit A of aldose reductase, lying between the catalytic amino acid residues, Trp20, Tyr48, Trp111, Phe122, and His110. These compounds showed interactions with several residues near the active site, including Trp20, Lys21, Tyr48, Trp111, Phe122, Pro218 and Ser302, which seems to play a key role in the activity of the enzyme (Table 3). The overall binding of rutin, quercetin and fidarestat (reference standard for docking) in human aldose reductase is illustrated in Fig. 2. Docking results showed that rutin possessed highest docking score of 5.549 and formed five hydrogen bonds (Fig. 3A) with Trp20, Tyr48, Gln49, Lys121 and Pro123 which are important residues for binding of inhibitors. The hydrophobic phenyl ring was found in contact with Phe122, which is an important residues for binding of inhibitors. Quercetin has second highest docking score of 4.43, and formed three hydrogen bonds (Fig. 3B) with Tyr48, Gln49 and Trp111, which are an important residues for binding of inhibitors. Fidarestat as reference molecules formed three hydrogen bonds with Lys21, Tyr48 and Gln49 with a docking score of 3.42. Docking study explored the interaction mechanism, and reasonable binding mode of these natural constituents in the active site of aldose reductase.
Conclusions The present study indicated that T. purpurea possessed significant anti-hyperglycemic activity, as well as anti-oxidant activity in diabetic rats and, also possessed significant in-vitro AR inhibiting activity. Together with the ability to reduce oxidative stress and inhibition of AR, T. purpurea might be beneficial not only in preventing hyperglycemia but also in delaying the development of diabetes induced complications, due to hyperglycemia induced oxidative and osmotic stress.