Modulatory Effects of Decalepis hamiltonii Extract and Its Compounds on the Antioxidant Status of the Aging Rat Brain.

OBJECTIVE: The present study was aimed to investigate the neuroprotective effects of Decalepis hamiltonii (Dh) aqueous root extract and its compounds against age-related oxidative stress (OS) in the discrete regions of the rat brain.
MATERIALS AND METHODS: Male Wistar albino rats of 4- and 22-month-old were divided into control and six supplemented groups. The supplemented groups were orally administered with ellagic acid (EA), 4-hydroxyisophthalic acid (4-HIA), and Dh extract for 30 days.
RESULTS: Age-related decrease in antioxidant enzyme activities was noticed. The hippocampus was found to be more vulnerable to OS as seen by the elevation in the OS markers. Supplementation of the Dh extract, EA, and 4-HIA was found to be effective in up-regulating the antioxidant status. However, the extent of up-regulation was more evident in Dh supplemented animals.
CONCLUSION: Our results suggest that Dh extract and its compounds exhibit neuroprotective effects against age-related OS and can be used as a dietary therapeutic intervention for the treatment of neurological disorders.

INTRODUCTION

Oxidative stress (OS) has been implicated in the aging process and pathophysiology of many neurodegenerative diseases. OS can cause cellular damage and cell death as the reactive oxygen species oxidize vital cellular components such as lipids, proteins, and DNA. Oxidative damage of proteins is one of the hallmarks of aging in biological systems. An increase in the oxidation level of proteins with age has been demonstrated mainly by the determination of the protein carbonyl (PC) derivatives, by analyzing the loss of protein sulfhydryl groups.[1]

The brain is susceptible to OS because of its high content of peroxidizable unsaturated fatty acids, a high rate of oxidative metabolic activity, high levels of free radical-inducing iron/ascorbate, and relatively low levels of antioxidant defense systems.[2] Since the endogenous antioxidant defense systems are not 100% effective, it is plausible to suggest that nutritional antioxidants can be exploited to combat the accumulation of OS over the ever-prolonging human lifespan.

Decalepis hamiltonii (Dh) is a climbing shrub belonging to the family Asclepiadaceae has been used from time immemorial by the folk people for its health benefits.[3] Aqueous root extract is a cocktail of antioxidants such as ellagic acid (EA), 4-hydroxyisophthalic acid (4-HIA), 14-aminotetradecanoic acid, 4 (1-hydroxy methyl-3)-methoxy benzaldehyde, 2,4,8 trihydroxy bicycle octan-3-one.[4] Recent studies from our laboratory have demonstrated that dietary supplementation of Dh extract along with swim exercise helps in attenuating the OS in the aging rat brain.[5] Our studies also demonstrated that 4-HIA and EA are the major phenolic acids which exhibit potent antioxidant activities and inhibits lipid peroxidation (LPO) against AAPH induced OS in rat brain in vitro.[6] Therefore, the present study was hypothesized to investigate the effects of dietary supplementation of Dh extract, EA, and 4-HIA in curtailing the OS in the aging rat brain. The hypothesis was tested by analyzing the antioxidant enzyme activities and markers of OS in the cerebral cortex (CC), hippocampus (HC), and cerebellum (CB) regions of rat brain.

MATERIALS AND METHODS

Chemicals

EA, 4-HIA, epinephrine, reduced glutathione (GSH), GSH reductase, thiobarbituric acid (TBA), t-butyl hydroperoxide, guanidine hydrochloride, 1,1,3,3-tetramethoxy propane (TMP), and nicotinamide adenine dinucleotide phosphate (NADPH) were procured from Sigma (St. Louis, MO, USA). All other chemicals were of analytical grade and solvents were of spectral grade and were procured from Himedia Chemicals (Mumbai).

Preparation of aqueous extract

Tuberous roots of Dh were collected from Savandurga forest, Bengaluru, India. The plant material was identified and deposited in the herbarium of Botany Department, Bangalore University. The fleshy part of the tuberous root was separated, dried at room temperature (RT) and was finely powdered using a grinder. A known (500 g) quantity of powdered sample was soaked in warm water at 50°C and kept on a magnetic stirrer overnight (Remi, India). The extract was filtered through Whatmann No. 1 filter paper (150 mm) and lyophilized (Cleanvac 8 Lyophilizer, Biotron) and stored at 4°C.

Animal maintenance

All animal procedures were approved by the Institutional Animal Ethics Committee, Bengaluru University, Bengaluru, India (Reg. No. 402/CPCSEA, Department of Zoology, Bangalore University). Male albino Wistar rats of 1-month-old were procured from the Central Animal Facility, IISc, Bengaluru and maintained until they were 4- and 22-month-old in a clean rodent room. These animals were placed three/cage in polypropylene cages fitted with stainless steel wire-mesh bottoms, maintained at a temperature of 28 ± 1°C, relative humidity of 77.5 ± 1%, and under daily photoperiods of 12-h light and 12-h dark cycle. They had free access to food (Amruth Feeds, Bengaluru) and tap water. Thirty-five rats of each age group were selected randomly and were segregated into controls (Con, n = 5) and six supplemented groups (i) EA1 (+25 mg), (ii) EA2 (+50 mg), (iii) 4-HIA3 (+30 mg), (iv) 4-HIA4 (+60 mg), (v) Dh5 (+50 mg), and (vi) Dh6 (+100 mg). The sedentary controls remained on a normal diet with supplements of distilled water (d.w.). The animals received daily an oral supplementation of EA, 4-HIA, and Dh extract per kg body weight for a total period of 30 days.

Tissue preparation

The animals were sacrificed under diethyl anesthesia, and the brain tissue was excised, and the CC, HC, and CB regions of the brain were separated, weighed, and homogenized in ice-cold 50 mM phosphate buffer (pH 7.0). The homogenate was used for the estimation of malondialdehyde (MDA), superoxide radical (SOR), and thiols. The homogenate was centrifuged (Plastocrafts, Superspin-RV/FM) at 1000 ×g at 4°C for 10 min. The supernatant obtained was used for antioxidant enzyme assays and PC estimation.

Measurement of antioxidant enzyme activities

Superoxide dismutase (SOD) activity was determined according to the method of Misra and Fridovich.[7] Briefly, tissue supernatant (100 μL) was added to 880 μL carbonate buffer (0.05 M, pH 10.2) and 0.1 mM EDTA. 20 μL of 30 mM epinephrine in 0.05% acetic acid was added to the mixture and absorbance was followed for 5 min at 480 nm in a spectrophotometer (ELICO, SL-210, UV spectrophotometer). The amount of enzyme that results in 50% inhibition of epinephrine autoxidation is defined as one unit.

Catalase (CAT) activity was measured according to the method of Aebi.[8] Briefly, 100 μL of the tissue supernatant with an equal volume of absolute alcohol was incubated for 30 min following which triton X-100 was added. A known volume of this was taken in an equal volume of 0.066 M H2O2 in phosphate buffer, and the decrease in absorbance was measured at 240 nm for a min in a spectrophotometer. An extinction coefficient of 43.6 Mcm−1 was used to determine enzyme activity, one unit of which is equal to the moles of H2O2 degraded/min/mg of protein.

GSH peroxidase (GSH-Px) activity was measured at 37°C by the method of Flohé and Günzler.[9] Briefly, the reaction mixture consisted of 500 μL of phosphate buffer, 100 μL of 0.01 M GSH, 100 μL of 1.5 mM NADPH, and 100 μL of GSH reductase. 100 μL of tissue extract was added to the reaction mixture and incubated at 37°C for 10 min. 50 μL of 12 mM t-butyl hydroperoxide was added to 450 μL of tissue reaction mixture and measured at 340 nm for 180 s in a spectrophotometer. A molar absorptivity of 6. 22 × 103/Mcm was used to determine enzyme activity. One unit of activity is equal to mM NADPH oxidized per min per mg protein.

Measurement of lipid peroxidation

MDA content was measured according to the procedure of Ohkawa et al.[10] using TMP as standard. Briefly, to 100 μL of homogenate, 200 μL of 8.1% SDS, 1.5 mL of 20% acetic acid, 1.5 mL of 0.8% aqueous TBA solution were added, and the solution was made up to 4 mL. The solution was heated on a boiling water bath for 60 min, cooled and 1 mL of d.w. was added. 5 mL butanol and pyridine (15:1) was added, and the mixture was shaken well. The mixture was then centrifuged at 4000 rpm for 10 min. The absorbance of orange layer was read at 532 nm.

Measurement of protein oxidation

PC levels were measured according to the procedure of Levine et al.[11] Briefly, 100 μL of supernatant tissue extract was incubated with 0.5 mL of 10 mM DNPH in 2 M HCl for 60 min in dark. Protein was precipitated using 0.5 mL of 20% TCA and then centrifuged at 10,000 ×g for 3 min at 4°C. The supernatant was discarded, and the pellet was washed with 1:1 ethylacetate/ethanol twice by centrifuging at 3400 ×g for 5 min to remove DNPH. The pellet was dissolved after washing in 1.5 mL of 6 M guanidine hydrochloride in phosphate buffer (pH 6.5). Absorption was read at 370 nm in a spectrophotometer.

Measurement of superoxide radical

SOR was measured according to the method Das et al.[12] Briefly, 200 μL of homogenate was incubated with 80 μL of 0.1% nitroblue tetrazolium (NBT) in an oscillating water bath for 1 h at 37°C. Termination of the assay and extraction of the reduced NBT was carried out by centrifuging the samples for 10 min at 200 ×g then resuspending the pellets with 1 mL of glacial acetic acid. The absorbance was measured at 560 nm and converted to μmoles diformazan using a standard curve generated from nitroblue formazan. Final results were expressed as micromoles diformazan/mg tissue.

Determination of total, protein and nonprotein thiol levels

The thiol groups were determined according to the procedure of Sedlak and Lindsay.[13] For total thiol (T-SH), briefly, aliquots of 250 μL of the tissue homogenate were mixed in 5 mL test tubes with 750 μL of 0.2 M Tris buffer, pH 8.2 and 50 μL of 0.01 M 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB). The mixture was made up to 5 mL with 3950 μL of absolute methanol. A reagent blank and a sample blank were prepared in the same manner. Color developed in 15 min, and the reaction mixture was centrifuged approximately at 3000 ×g at RT for 15 min. The absorbance of the supernatants was read in a spectrophotometer at 412 nm. Molar extinction coefficient at 412 nm was 13,100 Mcm−1 in both T-SH and nonprotein thiol (NP-SH) procedures.

For NP-SH, aliquots of 250 μL of the homogenates were mixed in 5 mL test tubes with 200 μL d.w. and 50 μL of 50% TCA. The test tubes were shaken intermittently for 10 min and centrifuged for 15 min at 3000 ×g. 200 μL of the supernatant was mixed with 400 μL of 0.4 M Tris buffer; pH 8.9, 10 μL DTNB was added. The absorbance was read within 5 min of the addition of DTNB at 412 nm. The protein thiols (P-SHs) groups were calculated by subtracting the NP-SH from T-SHs.

Protein measurement

Total protein content of tissue samples was measured by the method of Lowry et al.[14] using bovine serum albumin as a standard.

Statistical analysis

All the data were expressed as means ± standard error and were analyzed within a two-factor analysis of variance (ANOVA) between groups and regions. When a significant F ratio was found, Tukey's test was used to assess the differences between group means. The statistical analysis was performed using SPSS 20 software package for Windows (Version 22.0. Armonk, NY: IBM Corp.). P < 0.05 was considered statistically significant.

RESULTS

Antioxidant enzymes

The SOD activity significantly decreased with age in all the three regions of the brain. Supplementation of the EA, 4-HIA, and Dh extract resulted in the upregulation of enzyme activity in the CC, HC, and CB regions of the brain in both the age groups over their respective controls. Region-specific changes were evident only in the 4-month-old animals [Table 1].

Table 1

Superoxide dismutase activity in discrete brain regions of control and experimental groups

The CAT activity was significantly enhanced in the 4-HIA and Dh extract supplemented animals in all the regions of the brain. However, in 22-month-old animals, the higher dose of EA supplementation resulted in the enhancement of CAT activity [Table 2]. A noticeable feature was that there was no regional significance in the CAT activity in both the age groups.

Table 2

Catalase activity in discrete brain regions of control and experimental groups

The supplementation of Dh6 (100 mg) extracts significantly enhanced the GSH-Px activity in both the age groups compared to their sedentary controls. An insignificant increase in enzyme activity was noticed in the EA, and 4-HIA supplemented animals. Regional significance was evident in both the age groups [Table 3].

Table 3

Glutathione peroxidase activity in discrete brain regions of control and experimental groups

Oxidative markers

The MDA content, a marker of LPO is increased with age in CC showing the highest content compared to other regions. The MDA content significantly decreased in all the supplemented groups of 4 and 22-month-old animals over their sedentary controls [Figure 1].

Figure 1

The levels of MDA in discrete brain regions of (a) 4- and (b) 22-month-old animals of control and experimental groups. Values are mean ± standard error of 5 animals/group. Significance between group means was analyzed by Tukey's test and statistical significance set at P < 0.05. #Comparison of hippocampus and cerebellum with cerebral cortex. *Comparison of experimental groups with controls

The PC content was measured as a marker of protein oxidation. Age-related increase in the PC content was evident with HC showing the highest content compared to other regions [Figure 2]. The supplementation of EA, 4-HIA, and Dh extract significantly lowered the PC content over their sedentaries.

Figure 2

The levels of protein carbonyl in discrete brain regions of (a) 4- and (b) 22-month-old animals of control and experimental groups. Values are mean ± standard error of 5 animals/group. Significance between group means was analyzed by Tukey's test and statistical significance set at P < 0.05. #Comparison of hippocampus and cerebellum with cerebral cortex. *Comparison of experimental groups with controls

The generation of SOR was significantly higher in the HC in both the age groups. Higher doses of EA and Dh extract supplementation resulted in lower generation of SORs in 4-month-old animals. Whereas in 22-month-old animals, supplementation of 60 mg of 4-HIA and Dh extract significantly inhibited the generation of free radicals [Figure 3].

Figure 3

The levels of superoxide radical in discrete brain regions of (a) 4- and (b) 22-month-old animals of control and experimental groups. Values are mean ± standard error of 5 animals/group. Significance between group means was analyzed by Tukey's test and statistical significance set at P < 0.05. #Comparison of hippocampus and cerebellum with cerebral cortex. *Comparison of experimental groups with controls

Thiols

Table 4 represents T-SH, P-SH, and NP-SH content in 4- and 22-month-old animals. T-SH levels are higher in the Dh supplemented animals compared to EA and 4-HIA supplemented animals in both the age groups. Age-related decrease in NP-SH levels was observed in the CC, HC, and CB regions of 4- and 22-month-old animals. The NP-SH levels were upregulated in Dh6 supplemented 4-month-old animals over their sedentary counterparts. A remarkable feature in the 22-month-old animals is that the levels were significantly increased in all the supplemented groups. The P-SH levels decreased with age, and the levels were higher in the all the supplemented groups.

Table 4

The levels of total thiols, nonprotein thiols, and protein thiols in discrete brain regions of control and experimental groups

DISCUSSION

Free radicals have been suggested to be the most likely candidate responsible for producing the neuronal changes mediating the behavioral deficits in neurodegenerative disorders.[15] Currently, considerable attention has been focused on identifying dietary and medicinal phytochemicals that can inhibit, retard, or reverse the multi-stage pathophysiological events underlying neurological disorders.[16] Recent studies have reported the neuroprotective effects of Dh root extract on mouse brain and Parkinson's disease.[1718] However, in vivo studies on the bioactive compounds present in the Dh extract are not well explored. Therefore, the purpose of the present study was to determine whether Dh extract or individual compounds EA and 4-HIA can attenuate age-related OS in the rat brain. We chose CC, HC, and CB regions in our study as they are critical regions involved in the higher brain functions and varying cellular as well as the regional distribution of antioxidant biochemical defenses.[19]

Antioxidant enzymes are considered to be a primary defense mechanism that protects biological macromolecules from oxidative damage.[20] In the present study, antioxidant enzyme activities were found to be decreased with age. Previous studies have also reported decreased antioxidant enzymes in the aging rat brain.[21] The HC region was found to be more vulnerable to OS compared to CC and CB. The enzyme activities were upregulated in EA, 4-HIA, and Dh supplemented animals. The extent of upregulation was more pronounced in the Dh supplemented animals which may be due to the antioxidant activities of the polyphenols.[22] Previous studies have shown that the supplementation of Dh extract enhances the antioxidant enzyme activities in different tissues.[2324] The percentage increase in enzyme activity was lower in the EA, and 4-HIA supplemented animals which could be attributed to the lesser bioavailability and bioaccessibility of these compounds.

MDA, a marker of LPO was found to be significantly increased with age in the different regions of the brain. The increased LPO in the aged animals may be due to the disruption of lipid membranes leading to a subsequent formation of peroxyl radicals.[25] LPO significantly attenuated in the supplemented animals. This may be due to the scavenging of hydroxyl radicals by the antioxidant compounds. The region-specific changes were evident in both the age groups. These results are in agreement with the finding of Cini and Moretti[26] wherein the cortex had a higher rate of LPO compared to HC.

PC is the most common marker used for assessing the protein oxidation. In the present study, the PC levels were significantly increased with age in the different regions of the brain. The HC showed a higher level of oxidation compared to other two regions. The HC revealed a higher level of oxidation suggesting this region is highly vulnerable to OS. The supplementation of Dh extract and the compounds revealed lower oxidation of proteins indicating their antioxidant potential to scavenge the free radicals. Studies also reported that supplementation of plant extracts decreased the protein oxidation in the rat brain.[2728]

SOR is a major free radical produced during normal aerobic metabolism was found to be increased with age. Our results demonstrated that HC is prone to OS as revealed by higher levels of radical generation. The SOR levels were less in the Dh supplemented animals than the compounds which may be due to the synergistic effects of the compounds in the extract.

Thiols are regarded as the natural reservoir of a reductive capacity of a cell. There is an age-dependent reduction in thiol levels in the different brain regions which indicates the efficiency of S-thiolation as mechanism of antioxidant defense that decreases with age.[29] The decreased levels of thiols may lead to impaired protection of protein sulfhydryl groups on protein oxidation and further, reduced degradation of lipid peroxides may affect the integrity of synaptic plasma membranes thereby leading to neuronal cell death.[29] The supplementation of the extract significantly increased the thiol levels which could be due to the chelation of redox active metals and also by the induction of the enzymes required for GSH synthesis.

CONCLUSION

Our study demonstrated decreased antioxidant status in the different regions of aging rat brain. Dh extract, EA, and 4-HIA were found to be effective in attenuating OS. However, further studies are warranted on the isolation and elucidation of neuroprotective compounds present in the extract. These results suggest that Dh extract and the compounds might be used as a therapeutic strategy for the treatment of neurodegenerative disorders. However, further in vivo studies have to be explored to understand its mechanism of action.

Financial support and sponsorship

Department of Science and Technology, Government of India, Women Scientist -A Scheme (Grant no. SR/WOS-A/LS-88/2011).

Conflicts of interest

There are no conflicts of interest.