94 CHAPTER 5 SYNTHESIS AND ANTI CATARACT ACTIVITY OF ALLYLMERCAPTOCAPTOPRIL AGAINST OXIDATIVE STRESS INDUCED EXPERIMENTAL CATARACTOGENESIS: AN IN VITRO AND IN VIVO STUDY 5.1. Introduction In the second experiment as reported in the previous chapter, an anti cataract activity of Captopril against oxidant stress induced cataract was investigated attributed to its antioxidant properties. The antioxidant property of Captopril is greatly influenced by the presence of thiol group in its structure which helps to interact with lens specific enzymes and proteins as well. After absorption, Captopril carried as a disulfide compound in the body which has free radical scavenging potential. The organosulfur compounds present in garlic inhibits lipid peroxidation attributed to their antioxidant and free radical scavenging activity [280]. These pharmacological properties of garlic are due to the presence of organosulfur compound, Allicin, the pungent smelling diallyl disulfide-oxide. Allicin is produced during crushing of garlic by the interaction of Alliin, the non-protein amino acid, with the pyridoxal phosphate containing enzyme, Alliinase. Allicin is present in the form of oil and produced by crushing fresh garlic and it is unstable at room temperature whose activity is decay with time. The Allyl moieties present in Captopril and Allicin can combine covalently through disulfide linkage to form Allylmercaptocaptopril (AMC) [236]. AMC found to have therapeutic benefits of both Captopril and Allicin on blood pressure, measures of
95 glucose and lipid metabolism and markers of renal function when evaluated in the SHROB model of metabolic syndrome by using Sprague-Dawley rats fed a highfructose diet [235]. In view of the antioxidant properties of Captopril and Allicin and since oxidative stress has been implicated in cataractogenesis, Allylmercaptocaptopril might exhibit Anticataractogenic potential. The present invention included method for preparing synthetic Allicin followed by reaction with Captopril to produce AMC for the evaluation of its anti cataract potential in selenite induced cataract model. 5.2. Materials and Methods 5.2.1. Materials Diallyl disulfide and chemicals required for enzyme assay were purchased from Sigma Chemical Company, St. Louis, MO. All other chemicals and reagents used were same as detailed in previous chapter. 5.2.2. Synthesis of Allylmercaptocaptopril Allicin was synthesized by a modified method of Stoll and Seebeck et al.,[281]. Diallyl disulfide (3.24 g, 0.02 mol) was added dropwise in the mixture of 15 ml of acetic acid and 30% hydrogen peroxide (5 ml). During the addition, the reaction flask was kept in ice followed by stirring mixture at room temperature for 1 hour and the 50 ml water was added. The resulting mixture was reduced in volume to half under vacuum at 30 0 C. To the remaining solution, additional 50 ml water was added and then extracted with ethyl ether (3 x 100 ml). The ether extracts were washed with a sodium bicarbonate solution (5%) and then with water. After drying over anhydrous MgSO 4, the ethyl ether was removed under vacuum. The oil was further purified by silica gel column chromatography, employing chloroform as an eluent. The UV absorbing fractions showing a single spot on TLC (silica gel) were collected. The solvent was then removed under vacuum to yield 1.84 g (51 % yield) of pure Allicin.
96 In the second step, Captopril (217 mg, 1 mmol) in 7.5 ml water pre adjusted to ph 5.5 was added dropwise to Allicin (90 mg, 0.5 mmole) dissolved in 3 ml absolute ethanol. The reaction mixture was magnetically stirred for 15-20 minutes at room temperature. Excess Allicin was extracted by ether and the water phase was acidified by the HCl and extracted with ethyl acetate. The organic phase was dried by rotary evaporation, redissolved either in ethanol and dried by speed vacuum centrifugation or redissolved in water and dried by lyophilization. The yield of the reaction was 90%. The reaction product was analyzed by HPLC and its structure was confirmed by IR, NMR and mass spectrometry. IR (KBr) spectra were recorded on a Perkin Elmer FTIR spectrometer (v max in cm -1 ) and 1 H NMR spectra were recorded in CDCl 3 on a Bruker AVIII 500 MHz spectrometer using TMS as internal reference (Chemical shift in δ ppm). Mass spectra were recorded using Agilant HP5937 spectrometer. The purity of synthesized compound was checked by HPLC. The HPLC system consisted of a Shimadzu Class LC-10AT vp and LC- 20AD pumps connected with SPD-10A vp UV-visible detector. The data acquisition was performed by Spincotech 1.7 software. The system was equipped with reverse phase column Gemini C18 (150 mm 4.6 mm i.d., 5 μm) (Phenomenex, Torrance, USA). The mobile phase consisted of 60% methanol in water containing 0.1% formic acid at a flow rate of 0.8 ml/min. The retention time was found to be 8.9 min. AMC was dissolved in 1 ml ethanol and diluted in water to attain the appropriate volume and kept in dark bottles. Fresh solution was prepared daily for administration to animals. The reaction between Captopril and Allicin to form Allylmercaptocaptopril is illustrated in Fig. 5.1. Fig 5.1. Schematic illustration of the chemical reaction between Captopril and Allicin that forms Allylmercaptocaptopril.
97 5.2.3. In vitro phase of the study The experimental procedure and conditions were same as described in the previous chapter. Briefly, transparent lenses were divided equally into three different groups to serve as normal, control and test group. The lenses in the normal group were cultured in DMEM alone. The lenses in the control group were cultured in DMEM plus 100 μm sodium selenite and those in the test group were cultured in the control medium plus 5 mm AMC. The dose of AMC was taken as equimolar dose of Captopril. All lenses were incubated for 24 h at the conditions described earlier. After incubation, lenses were processed for morphological investigation and biochemical parameters like reduced glutathione, malondialdehyde, Ca 2+ -ATPase activity, lens Ca 2+ concentration, lens total protein, water soluble protein level and antioxidant enzymes activity were evaluated according to the procedures described in the previous chapter. 5.2.4. In vivo phase of the study The in vivo phase of the study was carried out by using nine days old Wistar strain rat pups. AMC (50 mg/kg i.p) was given to test group unless and otherwise all the experimental protocols were same as described in the previous chapter. 5.2.5. Statistical analysis All data were expressed as mean ± standard deviation (SD). The groups were compared using one-way ANOVA with post-hoc Dunnett s test using selenite 100 μm group as control and the chi-square test were applied wherever relevant. 5.3. Results 5.3.1. Characterization of Allylmercaptocaptopril The physical characterization and spectral data ( 1 H NMR, IR and Mass) of the synthesized compound was in full agreement with the proposed structure as
98 Allylmercatocaptopril. IR (KBr) v cm -1 : 3459 (OH), 2984 (C-H), 1728 (C=O), 939 (S=S). 1 H NMR (CDCl 3 ) δ (ppm): 10.02 (s, 1H, OH), 3.58 (d, 2H, SCH 2 ), 1.14 (m, 3H, CH 3 ). MS: m/z 289 (M + ). Table 5.1 Physical characterization of Allylmercaptocaptopril Molecular formula C 12 H 19 NO 3 S 2 Molecular weight 289.08 Solubility HPLC Ethanol, Water RT 8.9 minutes % Yield 90% Fig 5.2 Chromatogram of Allylmercaptocaptopril
99 Table 5.2 IR spectral data of Allylmercaptocaptopril Peaks at wavelength (cm -1 ) Functional groups 3459 OH group 2984 C-H stretch 1728 C=O stretch 1446 C-H bend (Methyl) 1375 C-H bend (Methylene) 1095 C-O stretch 939 S=S stretch Fig 5.3 IR spectrum of Allylmercaptocaptopril
100 Table 5.3 1 H NMR spectral data of Allylmercaptocaptopril δ Values (ppm) Absorption position δ - 1.15 d, 3H, CH 3 δ -2.65 m, 1H, CH δ -3.12 d, 2H, CH 2 δ -3.58 d, 2H, CH 2 δ -5.13 d, 2H, CH 2 δ -6.54 δ -10.02 m, 1H, CH s, 1H, COOH Fig 5.4 1 H NMR spectrum of Allylmercaptocaptopril
101 Table 5.4 Mass spectral data of Allylmercaptocaptopril m/z (M + ) m/z (B + ) 289.10 119.35 Fig 5.5 MASS spectrum of Allylmercaptocaptopril
102 5.3.2. In vitro phase of the study 5.3.2.1. Effect on lens morphology As shown in Fig 5.6, after 24 h of incubation in selenite 100 μm, lens became completely opaque (B) as against lenses incubated in DMEM alone (A). Incubation of lenses with Allylmercaptocaptopril (AMC) 5 mm, seem to retard the progression of lens opacification, compared with control group. This is because more number of grid lines are clearly visible in AMC supplemented lenses (C) than in selenite treated lens. Fig. 5.6 Digital image of the rat lenses in the culture media under various conditions. Rat lenses were cultured in (A) normal DMEM, (B) 100 µm sodium selenite and (C) 100 µm sodium selenite + 5 mm AMC exposure. Photographs were taken after 24 h. 5.3.2.2. Effect on biochemical parameters The mean GSH value in the normal lenses was 2.98±0.05 μg/mg of fresh weight of lens. A significant decrease in GSH level was observed in the presence of sodium selenite in the control as opposed to the normal group (P<0.01). In the presence of AMC, there was a significant restoration of GSH level in the treated lenses (P<0.01) as opposed to the control lenses. The mean GSH values in the control and test groups were 1.36±0.01 and 2.28±0.01 μg/mg of fresh weight of lens, respectively. A significant increase in MDA level was found in the control opposed to the normal lenses (1.147±0.02 μmol/g of fresh weight of lens; P<0.01). AMC
103 supplementation significantly protected (P<0.01) the test group lenses from lipid peroxidation; the MDA content was 0.045 ± 0.001 μmol/g of wet weight of lens (Table 5.5). Selenite 100 μm treated lenses showed significantly low concentrations of proteins (total and water soluble proteins) in the lens homogenate (P<0.01) and very high Ca 2+ conc. (P<0.01) compared with normal group having normal lenses (Table 5.5). AMC group had significantly higher concentrations of total lens proteins and water soluble proteins (P<0.01), compared to control group. At the same time, they had lower Ca 2+ conc. (P<0.01) compared to control group. Activity of the membrane ionic pump, Ca 2+ ATPase, was found to be decreased significantly following selenite exposure whereas, treatment with AMC was found to maintain activity close to the normal levels (Fig 5.7). Table 5.5 Levels of various biochemical parameters in Group I, Group II and Group III lenses Parameter Group I Group II Group III GSH (μg/mg wt.) MDA (μmol/g wt.) Calcium (Ca 2+ ) (%wt) Total Protein (mg/mg wt.) Water Soluble Protein (mg/mg wt.) 2.98 ± 0.05* 1.36 ± 0.01 2.28 ± 0.01* 0.061 ± 0.001* 1.147 ± 0.02 0.045 ± 0.001* 0.015 ± 0.004* 0.066 ± 0.002 0.021 ± 0.001* 0.421 ± 0.003* 0.319 ± 0.012 0.368 ± 0.014* 0.312 ± 0.005* 0.192 ± 0.001 0.248 ± 0.004* All values are expressed as mean±sd of five determinations. Group I: Normal, Group II: lenses exposed to sodium selenite only. Group III: lenses exposed to sodium selenite and AMC. Statistically significant difference (*P<0.01) when compared with group II values. GSH: reduced glutathione; MDA: malondialdehyde.
um ip released/h/100 mg protein 104 1.5 1.0 * * 0.5 0.0 Group I Group II Group III Groups Fig. 5.7 Activity of Ca 2+ ATPase in lens. All values are expressed as mean±sd of five determinations. Group I: Normal, Group II: lenses exposed to selenite only. Group III: lenses exposed to selenite and AMC. Statistically significant difference (*P <0.01) when compared with group II values. The effect of 5 mm AMC on different enzymes (SOD, CAT, GPx and GST) is presented in Table 5.6. It was observed that, in presence of selenite stress in group II lenses, antioxidant enzymes were significantly reduced as compared to the normal group. In presence of AMC, there was a significant positive modulation of enzyme activities observed in group III lenses. Table 5.6 Levels of antioxidant enzymes in group I, group II and group III lenses Parameter (IU/mg protein) Group I Group II Group III SOD 2.46 ± 0.14* 0.28 ± 0.03 1.92 ± 0.01* CAT 1.19 ± 0.001* 0.01 ± 0.01 0.89 ± 0.02* GPx 10.73 ± 0.91* 1.07 ± 0.10 8.62 ± 0.17* GST 2.06 ± 0.19* 0.16 ± 0.01 1.28 ± 0.13* All values are expressed as mean±sd of five determinations. Group I: Normal, Group II: lenses exposed to sodium selenite only. Group III: lenses exposed to sodium selenite and AMC. Statistically significant difference (*P<0.01) when compared with group II values. SOD, Superoxide dismutase; CAT, Catalase; GPx, Glutathione peroxidase; GST, Glutathione S-transferase.
105 5.3.3. In vivo phase of the study As shown in Fig 5.8, the lenses of rat pups that had received selenite alone (group A), a mature dense opacity involving the entire lens was observed in all (100%) 10 animals. In contrast, 9 out of 10 (90%) rat pups in group B that had received selenite along with AMC exhibited clear lenses and only 1 animal had initial signs of a posterior subcapsular or nuclear opacity involving tiny scatters lenses. This difference was statistically significant (x 2 [df =1] = 12.93; P<0.01). Fig. 5.8 Photo-slit-lamp images of rat eyes on 16 th day after injection (A) Nuclear cataract in sodium selenite group (B) Clear lens in sodium selenite + AMC treated group. 5.4. Discussion Garlic (Allium sativum) is the oldest of all cultivated plants and is widely used because of its high pharmacological significance. The organosulfur compound, Allicin, a constituent of garlic oil, is responsible for its characteristic odor. Antioxidation and free radicals trapping are part of the mechanisms underlying its wide
106 spectrum of pharmacological activities which includes lowering of blood pressure and insulin [282], improvement of lipid profile [283], anti-atherosclerotic and anticoagulant activity [284, 285]. Cataract, a leading cause of blindness, is a major socioeconomic burden for world population. To date, there is no potent therapeutic agent that can prevent/inhibit the lens from opacification. As a part of better strategic management of cataract, metabolic intervention through natural dietary ingredients is gaining importance in recent times [25]. Based on the previous findings on Captopril as an anti cataractogenic agent, Allylmercaptocaptopril, a covalent conjugate of Allicin with an ACE inhibitor Captopril, should have a greater impact and therapeutic efficacy in correcting oxidative stress induced cataract in animals than captopril alone was predicted. In the present study, biochemical analysis of selenite treated lenses clearly demonstrated a significant depletion of GSH and increased membrane damage as indicated by the levels of MDA, the product of membrane lipid peroxidation and decrease water soluble protein content. Such changes in GSH and MDA levels in presence of selenite have been reported [26]. Restoration of GSH and MDA levels, protection against aggregation and insolubilization of lens proteins and maintenance of lens clarity without doubt establish the protective action of AMC. Moreover, the lower levels of Ca 2+, higher activity of Ca 2+ -ATPase and decreased levels of lipid peroxidation in the lens of the AMC treated group could be attributed to its antioxidant protection against selenite induced oxidative stress. The level of lens antioxidant enzymes were significantly hampered with selenite and positively modulated in the presence of AMC. The data clearly demonstrated that AMC significantly improves the antioxidant defense mechanisms of the normal lens. Based on findings of an in vitro study that AMC attenuates oxidant stress in cataract lenses, it was evaluated against selenite induced cataracts in young rats. AMC significantly protected the lens morphology and clarity: 90% of the eyes were clear in
107 AMC treated group; in contrast, 100% of the control eyes developed dense opacity or nuclear cataract due to selenite treatment. If we compare these results with our previous findings on Captopril, the effect was more pronounced by AMC treatment compared to Captopril. From the current study, it is evident that AMC protects the lens against oxidative stress. The results on selenite induced cataracts in vitro and in vivo not only demonstrate the protective effect of AMC but also indicate that it prevents cataractogenesis by virtue of its antioxidant properties. AMC, therefore, may be useful for prophylaxis or therapy against cataracts. 5.5. Conclusion In conclusion, the study on the evaluation of an anti cataract potential of AMC in experimental animals indicated that it modulates antioxidant parameters in the enucleated eye lenses which is persistent with our previous findings regarding Captopril. The In vivo experimental set up also confirmed its protective effect in rat pups. But both the drugs were tested in the separate experimental phases, so that we could not confirm the comparative efficacy of AMC over Captopril. In this chapter, the persistent effect of AMC as an anti cataract agent with respect to Captopril through its structural modification was confirmed. The next study was planned to compare the efficacy of AMC with Captopril to elucidate the effect of one over another and to discover the exact mechanism of action of AMC which would be different than Captopril.