Combination of epigallocatechin 3 gallate and curcumin improves d-galactose and normal-aging associated memory impairment in mice

Previously, we observed curcumin improves aging-associated memory impairment in d-galactose (D-gal) and normal-aged (NA) mice. Evidence showed that multiple agents can be used in managing aging-induced memory dysfunction, drawn by the contribution of several pathways. Curcumin and Epigallocatechin 3 gallate (EGCG) combination substantially reduced the oxidative stress that commonly mediates aging. This study examined the combined effect of EGCG and curcumin on memory improvement in two recognized models, D-gal and normal-aged (NA) mice. The co-administration of EGCG and curcumin significantly (p < 0.05) increased retention time detected by passive avoidance (PA) and freezing response determined in contextual fear conditioning (CFC) compared to the discrete administration of EGCG or curcumin. Biochemical studies revealed that the combination of EGCG and curcumin remarkably ameliorated the levels (p < 0.05) of glutathione, superoxide dismutase, catalase, advanced oxidation protein products, nitric oxide, and lipid peroxidation compared to the monotherapy of EGCG or curcumin in mice hippocampi. The behavioral and biochemical studies revealed that the combination of EGCG and curcumin showed better improvement in rescuing aging-associated memory disorders in mice. EGCG and curcumin combination could serve as a better choice in managing aging-related memory disorders.

and hydrogen peroxide (H 2 O 2 ) by galactose oxidase 12 and triggers aging-associated memory loss 13 in several behavioral batteries 14 .Our former study showed that D-gal causes deterioration of retention and fear memory by inducing oxidative overload in mouse hippocampus 15 .
Evidence showed that natural compounds can be the rational choice for treating aging-related memory disorder owing to antioxidants, anti-inflammatory, anti-aging 16 , and safer therapeutic properties 17 .It was reported that Epigallocatechin 3 gallate (EGCG), a natural polyphenolic agent of green tea 18 , improved memory and learning process by modulating the amount of several oxidative markers, including superoxide dismutase (SOD), malondialdehyde (MDA), and nitric oxide 19 .One study showed that EGCG decreased acetylcholinesterase (AChE) activity by ameliorating antioxidants and ROS in dementia rat models 20 .It has also been evident that EGCG improves memory impairment in passive avoidance (PA) 21 and contextual fear conditioning (CFC) 22 in the aging studies.Similarly, another natural compound, curcumin improved retention time in PA and freezing response in CFC tasks in D-gal injected aging animal model 15 .These improvements in behavioral endophenotype appear due to the antioxidant, anti-inflammatory, and anti-senescence 23 properties of curcumin, shown in a previous study 15 .Contrarily, multiple factors accelerate the aging process 24,25 which involves diverse pathways and interactive networks of components at molecular and cellular levels 26 .Therefore, considering curcumin alone to treat memory dysfunction in our previous study 15 certainly missed finding out more substantial protection from aging.
In vitro study showed that curcumin possesses synergistic effects on EGCG 27 .Contrary, limited study has been conducted to find out a suitable adjuvant candidate of EGCG in treating aging-induced memory impairment.This study examined the combined effect of EGCG and curcumin on two widely used animal models, Normal aging and D-gal-administered aging mice, adopting behavioral paradigms, PA and CFC.We also assessed the oxidative stress biomarkers, such as glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), advanced oxidation of protein products (AOPP), nitric oxide (NO), and malondialdehyde (MDA) in the animal hippocampus.Overall, this study has examined the combined effect of EGCG, and curcumin for the very first time on aging-associated memory disorder by behavioral and biochemical tests in mice.
A similar pattern was apparent after 48 h of training (Fig. S1A,B).Two-way ANOVA followed by post hoc Tukey's test demonstrated a significant effect of aging (F 2,91 = 69.16,p < 0.0001), treatment (F 4,91 = 246.58,p < 0.0001) as well as a significant interaction between aging and treatment (F 6,91 = 5.92, p < 0.0001).The combined EGCG and curcumin therapy demonstrated a significant difference in preventing the dropping trend of RT in D-gal (286.5 ± 6.

Effect of EGCG + curcumin on freezing response in contextual fear conditioning task. Effect of EGCG + curcumin on freezing response in conditioning session.
To evaluate the tendency of hyperactivity to a fearful condition, the baseline activity in the first 2 min of the novel environment was detected in conditioning session on day 1 of CFC.This session measured the freezing response (FR) without the application of white noise (CS) and foot shock (US).The baseline activity among the Vehicle, EGCG-Con, Cur-Con, D-gal, NA, EGCG + D-gal, EGCG + NA, Cur + D-gal, Cur + NA, EGCG + Cur + D-gal, EGCG + Cur + NA, Ast + D-gal, Ast + NA groups (Figs. 2, 3) was almost identical.
Effect of EGCG + curcumin on FR in cued test.In the cued trial, the freezing response of mice investigated an altered context in a distinctly shaped chamber.In the presence of Pre-CS application during the first 3 min, the baseline activity among the Vehicle, EGCG-Con, Cur-Con, D-gal, NA, EGCG + D-gal, EGCG + NA, Cur + D-gal, Cur + NA, EGCG + Cur + D-gal, EGCG + Cur + NA, Ast + D-gal, Ast + NA groups (Figs. 4, 5) was approximately identical.

Discussion
Previously we assessed the effect of monotherapy to treat memory dysfunction in D-gal and NA mice, induced by oxidative stress pathway 15 .Contrarily, it is established that interactive systems of molecules, cells, and multiple pathways contribute to the process of aging 26 .Additionally, considering the intricacy of the process of aging, it can be stated that combined therapy may bring a successful treatment strategy by producing substantial protection from memory impairment in the aging population 28 .Therefore, the previous study certainly missed to find out a successful treatment approach by assessing the effect of single therapy on diverse pathology of aging-induced memory impairment.In this current study, we assessed the effects of combination therapy to treat memory impairment in D-gal and NA mice.We found that the combined therapy of EGCG and curcumin exhibits a more substantial improvement of retention and fear memory by ameliorating oxidative stress biomarkers in contrast to their monotherapy in drug and nature-induced aging animals.
Similar to our previous study 15 , the D-gal administered, and NA animals displayed less retention time (RT) and freezing response (FR) in the PA (Fig. 1) and CFC (Figs. 2, 3, 4, 5) tasks, respectively.Studies showed that a large quantity of D-gal generated excessive reactive oxygen species (ROS) 10 by impairing redox homeostasis 12 , commonly observed in the natural progression of aging mice 15 .This high production of ROS induces brainderived neurotrophic factor (BDNF) dysregulation neuroinflammation, and cellular apoptosis, contributing to memory dysfunction 29 .Another study revealed that D-gal caused memory and learning deficit by reducing adult neurogenesis 30 and synaptic protein expressions in the hippocampus 31 , a vital region processing learning, and memory in behavioral tasks 32,33 .Apart from the involvement of hippocampus in memory, a decreased functional connectivity between amygdala and hippocampus and an increased connectivity between amygdala and dorsolateral prefrontal cortex observed in older adults in learning emotion-based memory 34 .Our findings from behavioral studies agree with the previous study 15 , suggesting, D-gal induces natural aging and impairs learning and memory in mice.
The combined EGCG and curcumin therapy in both D-gal and naturally induced aging mice groups showed powerful protection of retention time and freezing response in PA and CFC tasks, respectively (Figs. 1, 2, 3, 4, 5), suggesting better improvement of learning and memory compared to the monotherapy of EGCG or curcumin in both mouse models.Evidence showed that EGCG reversed D-gal administered memory impairment in aging mice 14 .A study demonstrated that EGCG improved cognitive deficits by producing neuroprotective, anti-inflammatory, and molecular effects in mice 35 .EGCG exerts neuroprotective effects by substantiating the functions of antioxidant enzymes and inhibiting the damage of advanced glycation end product in aged animals 14 .It was also evident that EGCG exhibits anti-inflammatory actions by inhibiting the expression of inducible NO synthase (iNOS) and reducing nuclear factor kappa B (NF-κB) and activator protein-1 activities (AP-1) 36 .Moreover, EGCG improved cognitive dysfunction by suppressing the activity of b-secretase 37 , causation of β-amyloid in APP695 expressing neurons 38 , formation of Ab fibrils 39 , and caspase activity in neuronal cells of hippocmapus 40 .
Curcumin produces neuroprotective effects by exerting antioxidant, anti-aging, and anti-neuroinflammation properties 41,42 .A study showed that curcumin protects memory from impairment in D-gal and NA-induced memory impairment 15 by regulating degeneration, proliferation, and senescence of neuronal cells 43 .Additionally, curcumin was found to improve cognitive dysfunction by increasing synaptic density in the AD animal 44 .Apart from these effects, curcumin increased the permeability and bioavailability of EGCG 45 and produces synergistic effects 46 in animals.Therefore, our present study concludes that the combined EGCG and curcumin substantiate the powerful protection via producing potential pharmacological actions in brain aging.Similar to the findings of the previous study 15 , we found a sharply decreased activity of antioxidants including GSH (Fig. 6A,B), SOD (Fig. 6C,D) CAT (Fig. 6E,F) in D-gal treated and NA animals.Moreover, an equivalent level of antioxidant activity was found in both classified mouse models (Fig. 6).GSH reduces H 2 O 2 to form H 2 O by interacting with ROS and RNS directly 47 .Similarly, superoxide dismutase (SOD) accelerates the disproportionation of O .Consequently, the cell stays protected from the damaging effects of H 2 O 2 .These non-enzymatic and enzymatic antioxidant molecules (GSH, SOD, CAT) avert oxidation in the plasma membrane and modulate redox homeostasis 49 .Contrarily, a substantial decline of GSH, SOD, and CAT molecules fails to prevent the uncontrolled production of species containing reactive oxygen (ROS) and nitrogen (RNS) 50 induced by D-gal 51 and NA 52 .On the other hand with the low level of antioxidants, we detected a high level of AOPP (8A and 8B), NO (8C and 8D), and MDA (8E and 8F) in D-gal treated and NA mice, suggesting the emergence of oxidative stress in mice 53 .A high level of oxidized protein signals generated an excess amount of AOPP 53 .A high amount of NO induces more RNS, such as • NO 2 and N 2 O 3 54 .Similarly, an increased level of MDA induces more ROS 55 .Therefore, a large amount of these reactive species (MDA, AOPP, and NO) mediate oxidative stress in aging mice 15 .
In the current study, we noted that combined EGCG and curcumin-treated mice showed powerful protection against the decreased activity of GSH, SOD, CAT (Fig. 6), and increasing level of AOPP, NO, and MDA (Fig. 7) in the brain compared to the monotherapy of EGCG or curcumin.Moreover, combined EGCG and curcumin were comparable to Ast, a standard antioxidant, in modulating the antioxidant level.A study showed that EGCG improved memory dysfunction by elevating the GSH and SOD activity 56 in D-gal-induced aging mice 14 .Another study showed that EGCG decreased hippocampal MDA 56 and cortico-hippocampal ROS and improved memory in AD mice model 57 .These antioxidant activities of EGCG are modulated by binding with several aging and oxidative stress-associated proteins such as KEAP1 58 , BACE1 59 , protein kinase C 60 , p53, Bax, Bcl-XL, and COX 61 .A previous study showed that curcumin improved memory by modulating the level of GSH, SOD, CAT, AOPP, NO, and MDA in aging mice 15 , by interacting with several proteins, such as kelch-like ECH-associated protein 1 (KEAP1), amine oxidase [flavin-containing] A (MAOA), beta-secretase 1 (BACE1), glutathione S-transferase omega-1 (GSTO1), and glutathione S-transferase A1 (GSTA1) 15 .Together previous and present findings suggested that the combined therapy of EGCG and curcumin substantiate greater protection against oxidative stress injury by regulating the oxidative biomarkers and their associated proteins in aging.

Conclusion
We investigated the beneficial effects of EGCG and curcumin on oxidative stress in the two robust aging mice models by performing behavioral and biochemical studies.A combination of EGCG with curcumin exhibits greater protection from aging-related memory impairment by modulating oxidative stress biomarkers.To further substantiate the findings, molecular and cellular studies need to be conducted to examine the regulatory effect of combined EGCG and curcumin on oxidative biomarkers in aging-induced learning impairment.

Animals.
We chose male mice to generate a stable reading in fear-based tasks since a previous study showed that female mice may bring unexpected memory deficits owing to their increasing anxious behavioral endophenotypes modulated by age 62 .We selected Swiss albino strain because of its several advantages including strong learning interest, intricate intelligence in memory paradigms and decreasing variability of experiments in aging study 63 .However, this strain may inherit a rare genetic disorder, albinism, that impairs visual spatial attention 64 required for passive avoidance 65 and contextual fear conditioning 66 .Therefore, we performed visual placing response, demonstrated by W. M. Fox in previous protoco 67 , to assess the visual system before selecting our experimental mice.Briefly, mouse was lowered by suspending its tail toward a metal object while we ensured no contact of vibrissae with the metal object.Mouse, which showed a clear placing reaction by raising its head and extending forelimbs on the metal object, was selected in this study.One hundred four healthy Swiss albino mice were chosen and separated into three age groups, Group-1 (Young adult; 24 mice): 6-8 weeks old, with an average weight of 25 gm; Group-2 (Drug induced aging; 40 mice): 6-8 weeks old, treated with D-gal; and Group-3 (Nature induced aging; 40 mice): 10-12 months of age with an average weight of 40 gm, did not receive D-gal.Eight mice comprised each subgroup of age category.Each mouse was fed ad-libitum.Mice were housed in a 12:12 h light-dark cycle while maintaining 55% ± 15% relative humidity at 25 °C temperature.
Drugs preparation and experimental design.EGCG solution at 0.2 mg/ml concentration was prepared by dissolving it into distilled water 68 .Curcumin was administered at 1 ml per 100 g body weight after suspending in a percentage of 0.25 w/v sodium carboxymethylcellulose 69 .The D-gal solution used to induce aging was prepared before each session of administration by dissolving it into 0.9% of saline 70 .Ast solution at 20 mg/20 ml used as a standard agent to compare the antioxidant effect of combined EGCG and curcumin was prepared by dissolving it into distilled water 71 .We treated and divided one hundred four mice into following subgroups of each age category and assessed the mice using multiple apparatus at the same time by dedicated expert experimenters.
CAT activity was estimated spectrometrically by following the previous method 15,80 .A 1.5 ml reaction mixture consisted of phosphate buffer (1.0 ml; 0.01 M; pH 7.0), homogenized hippocampal tissue (0.1 ml), and H 2 O 2 (0.4 ml of 2 M).After the addition of dichromate-acetic acid reagent (2.0 ml), the mixture (1:3) of potassium dichromate, and glacial acetic acid, the reaction stopped, and the data were expressed in μmol/min/mg protein.AOPP was determined spectrophotometrically by adopting the previous method 15,81,82 .The phosphate-buffered saline (PBS) was used to dilute homogenized hippocampal tissue (50 μl) at a ratio of 1:2.The calibration curve was prepared by using Chloramine T (0-100 mmol/l).Each well was poured with acetic acid (50 μl) and potassium iodide (100 μl; 1.16 M) while PBS was used as a blank.Finally, the absorbance at 340 nm was detected and the data was represented in chloramine units (μmol/ml).The level of NO was detected, using the Griess-Illosvoy reagent, based on the previous method 15,83 .Naphthyl ethylene diamine dihydrochloride (0.1% w/v) was used as a substitute for 1-napthylamine (5%) for modifying Griess-Illosvoy reagent.The PBS was used to dilute NED (1 ml), sulfanilamide (1 ml), phosphate buffer saline (0.5 ml), and homogenized hippocampal tissue at the ratio of 2:8.The incubation was done in a 96-well plate at 25 °C for 15 min 76 .Finally, the absorbances were detected in the spectrophotometer at 540 nm against the blank readings and the data was represented in mmol/mg.The level of MDA was detected using colorimetric analysis while determining TBARS based on the previous protocol 15,84 .The Tris-HCl buffer (pH 7.5) containing homogenized hippocampal tissue (0.1 ml) was treated with 2 ml of TBA (0.37%)-TCA (15%)-HCl (0.25 N) reagent (1:1:1 ratio), later kept in a water bath for 15 min at 70 °C and cooled.The absorbance was determined at 535 nm against the reference blank 85 and finally, the MDA level (nmol/ml) was measured based on a standard curve.

Data analysis.
The GraphPad Prism (version 8.0.1 for windows, GraphPad software, www.graph pad.com) and SPSS software (IBM SPSS 29) was used to execute all analyses.A One-Way ANOVA followed by post hoc Tukey's test detected the effects of groups and a two-Way ANOVA followed by post hoc Tukey's test determined the main effects of aging, compounds, and their interactions.Data presented as mean ± SEM (standard error of the mean) and the difference was considered significant when the p value was less than 0.05.
Ethical approval.The present study protocol was approved by the institutional animal care and use committee (IACUC) of North South University (2020/OR-NSU/IACUC-No.0903,SL no39).The experimental procedures were maintained and executed at the pathogen-free facility according to the NIH Guide for the care and Figure 8. Schematic presentation of whole experimental procedure: the treatment continued for ten weeks.After that, the behavioral parameters were detected using passive avoidance (PA) and contextual fear conditioning (CFC).The biomarkers were measured after completing the behavioral tasks.use of laboratory animals.Maximum efforts were given to reduce animal quantity and ensure their comfort.This study is reported in accordance with ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments).

Limitation
At present we assessed the beneficial effects of EGCG and curcumin on several oxidative biomarkers in one strain of aging animals (male mice only) by performing PA and CFC studies.To further understand the outcomes of aging and the treatment therapies, considering other strains of mice of both male and female, measuring some functional outcomes of increased oxidative stress, such as DNA damage and/or apoptosis and strengthening the validity of behavioral studies by using other reward-based tests, such as radial 8-arm maze or Hebb-Williams maze, and tests require movement, such as the Barnes maze or Morris water maze need to be conducted.

Figure 2 .
Figure 2. Effect of EGCG + Curcumin on the conditioning session (Day 1 of CFC) and context test (Day 2a) of D-gal mice group.The memory was assessed by analyzing the FR.The FR was expressed in percentage (%).Data was presented as mean ± SEM, n = 8 each group; **p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant.

Figure 3 .
Figure 3.Effect of EGCG + Curcumin on the conditioning session (Day 1 of CFC) and context test (Day 2a) of NA mice group.The memory was assessed by analyzing the FR.The FR was expressed in percentage (%).Data was presented as mean ± SEM, n = 8 each group; **p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant.