# Rutin, a Flavonoid That Is a Main Component ofSaussurea involucrata, Attenuates the Senescence Effect in D-Galactose Aging Mouse Model

Authors: Ying-Chen Yang; Hsueh-Yi Lin; Kang-Yi Su; Chien-Hsu Chen; Yung-Lung Yu; Chai-Ching Lin; Sung-Liang Yu; Hong-Young Yan; Kuo-Jung Su; Yi-Lin Sophia Chen
Journal: Evidence-Based Complementary and Alternative Medicine
 (2012)

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## Abstract

Saussurea involucrata (Kar. et Kir.), known as the snow lotus, grows in the Tian Shan and A’er Tai areas of China. It has recently been reported that the ethyl acetate extract of S. involucrata (SI-2) can inhibit proliferation and induce apoptosis in PC-3 human prostate cancer cells. This study investigated the protective effect of ethyl acetate extract of S. involucrata (SI-2) or rutin, a flavonoid extracted from ethyl acetate extract of S. involucrata (SI-2), on D-galactose- (D-gal-) induced brain injury in mice. Administering SI-2 or rutin (30 mg/kg/d and 30 mg/kg/d) for 6 weeks, concomitant with D-gal injection, significantly increased superoxide dismutase and glutathione peroxidase activities and decreased the MDA level in plasma. Furthermore, the result showed that the percentages of cleaved caspase-3 and PARP in the D-gal-treated mice were much higher than those in the control. Pretreatment using SI-2 or rutin decreased the expression of cyclooxygenase-2 via downregulation of NF-kappaB, resulting in a decrease in lipid peroxidation. Furthermore, our results also showed that oral administration of rutin to these mice significantly improved behavioral performance in a step-through passive avoidance task and these results suggest that SI-2 or rutin exerts potent antiaging effects on D-gal in mice via antioxidative mechanisms.


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## Body

## 1. Introduction

Oxidative stress is believed to be a primary factor in neurodegenerative diseases, as well as in the normal process of aging [1–3]. Oxygen-derived free radicals exert detrimental effects including peroxidation of membrane lipids, enzyme inactivation, DNA fragmentation, and activation of apoptosis [4–6]. Exogenous H2O2 can increase oxidative stress and apoptotic cell death by causing mitochondrial dysfunction and activation of caspases. Furthermore, ROS themselves can increase and/or induce cellular cyclooxygenase-2 (COX-2) expression [7–9]. In previous studies, apoptotic cell death induced by exposure to cyanide can be inhibited by selective COX-2 inhibitor [10, 11]. On the other hand, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) act by scavenging the superoxide anion and H2O2 to prevent reactive-oxygen-species- (ROS-) induced damage [12]. Oxidative stress-induced COX-2 expression can also be prevented by free radical scavengers in numerous cell types especially in neurons. Therefore, oxidant stressors may be specific and critical inducers of COX-2 gene expression and natural compounds or extracts that can efficiently reduce the expression of COX-2 may have therapeutic potential for ROS-induced neuronal damage.D-galactose (D-gal) is a reducing sugar that can be metabolized at normal concentration. However, at high levels, D-gal can be converted into aldose and hydroperoxide under the catalysis of galactose oxidase, resulting in the generation of a superoxide anion and oxygen-derived free radicals [13]. D-galactose also reacts readily with the free amines of amino acids in proteins and peptides both in vivo and in vitro to form advanced glycation end products (AGEs). Evidence shows that AGEs can remarkably cause the accumulation of reactive oxygen species (ROS), especially superoxide radicals and hydrogen peroxide. Mice injected with D-gal have been used as an animal model of oxidative stress [14–17]. Recently, oxidative stress mediated DNA damage caused alternative synaptic structure and memory maintenance as well as learning abilities were also demonstrated in mouse models [18–20].Saussurea involucrata (Kar. et Kir.), known as the snow lotus, grows in rocky habitats of mountains 2600 m in elevation or higher in the Tian Shan and A’er Tai areas of China. Because of excessive harvesting of the wild plants used in pharmaceutical preparations and the particularly slow growth ofS. involucrata, the wild population of this plant has been threatened with depletion in recent years. Saussurea involucrata is now almost extinct and has been listed as a second grade national protected wild plant in China [21, 22]. Based on the theories of traditional Chinese medicine, S. involucrata has the effects of warming the kidney, activating “yang,” expelling wind, eliminating dampness, inducing menstruation, and promoting blood circulation. In folk medicine, it is used to treat rheumatoid arthritis, impotence, irregular menses, cough with cold, stomachache, and altitude sickness, among others [23, 24]. It was revealed thatS. involucrata is a potential agent for CAM and investigated its biological mechanism as an antineoplastic agent for androgen-independent prostate cancer patients. It has been demonstrated that treatment of PC-3 cells with ethyl acetate extract of S. involucrata (SI-2) has a highly potent inhibitory effect on the phosphorylation and activation of EGFR and on the AKT and STAT3 pathways [22]. Therefore, these results may offer therapeutic advantages in the treatment and prevention of human prostate cancer. Rutin (3,3′,4′,5,7-pentahydroxyflavone-3-rhamnoglucoside; Figure 1) is a flavonoid of the flavonol type that is found plenty in S. involucrata. It has been reported that rutin has several pharmacological properties, including antioxidant, anticarcinogenic, cytoprotective, antiplatelet, antithrombotic, vasoprotective, and cardioprotective activities [25–30]. Moreover, rutin was found to be a neuroprotective agent [31]. Rutin has demonstrated anticonvulsant activities in pentylenetetrazol models in rats and mice [32] and has also ameliorated ischemic reperfusion injury in the brain [33]. Prolonged supplementation with rutin significantly reversed trimethyltin- (TMT-) induced spatial memory impairment and damage to pyramidal neurons in the hippocampal CA3b region. These effects could be related to the antioxidative effects of rutin [34].Figure 1
Chemical structure of rutin.However, the effect of ethyl acetate extract ofS. involucrata (SI-2) or rutin to attenuate the neural ROS-induced DNA damage in D-gal-treated mice has not been investigated. This study aims to evaluate the antioxidant potential of ethyl acetate extract of S. involucrata (SI-2) and rutin through inhibiting COX-2 expression in D-gal-induced brain DNA damage and explore the mechanisms of its action.


## 2. Materials and Methods

### 2.1. Preparation of Fractions

The wild plant ofS. involucrataused in this study was a gift from Biopure Biotechnology (Changhua, Taiwan). Twenty grams of dried and powdered aerial parts, including flower, ofS. involucratawere extracted with 100 mL of methanol three times under reflux for 2 h, respectively. The methanol extracts (SI-1) were combined, and the solvent was evaporated in vacuo to give a deep brown syrup. The syrup was resuspended in water and then partitioned successively with pentane, ethyl acetate (SI-2), and n-butanol (SI-3) to leave a water layer (SI-4). The solvents were evaporated, respectively, and the residues were used throughout this study.


### 2.2. Reverse-Phase High-Performance Liquid Chromatography (HPLC) Analysis of Flavonoids inS. involucrata

The determination of flavonoids fromS. involucrata was carried out by HPLC with a photo diary detector. The HPLC system consisted of a Shimadzu LC-20AT solvent delivery system, equipped with a SPD-M20A photodiode array detector, set at 270 nm. Samples were injected with SiL-20A autosample to separate on the TSK-Gel ODS-100S column. The column was maintained at an ambient temperature of 25°C. The flow rate of the system was 1.0 mL/min. The mobile phase consisted of solvent A (0.3% formic acid) and solvent B (acetonitrile). The elution profile for A was 0–10 min, with a linear gradient change of 0–5%, 10–40 min, with a linear gradient change to 55%, and maintained for another 10 min with a post runtime to equilibrate the column and for the baseline to return to the normal and initial working conditions.


### 2.3. Chemicals and Reagents

Rutin was dissolved in DMSO to a concentration of 50 mM and stored in −20°C as a master stock solution. Dimethyl sulfoxide (DMSO), 3-(4,5-dimethyl thizol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), 2′,7′-dichlorofluorescein diacetate (H2DCF-DA), Hoechst 33342, thiobarbituric acid (TBA), hydrogen peroxide (H2O2), Trichloroacetic Acid, (TCA), malondialdehyde (MDA), propidium Iodine (PI), and actin antibody were purchased from Sigma Chemical Co. (St. Louis, MO, USA). NuPAGE Bis-Tris Electrophoresis System (precast polyacrylamide mini gel) was purchased from Invitrogen (Carlsbad, CA, USA). COX-2 antibody was purchased from Thermo scientific (Waltham, MA, USA). PARP antibodies and horseradish peroxidase - conjugated anti-mouse or anti-rabbit IgG secondary antibodies were purchased from Cell Signaling (MA, USA). Polyvinyl difluoride (PVDF) membranes, BSA protein assay kit, and western blot chemiluminescence reagent were purchased from Amersham Biosciences (Arlington Heights, IL). Superoxide dismutase activity assay kit was purchased from Biovision (Mountain View, CA). Glutathione peroxidase assay kit was purchased from Cayman Chemical (MI, USA). DNA Fragmentation Assay Kit was purchased from Clontech Laboratories (Mountain View, CA). Nonradioactive Cytoxicity Assay was purchased from Promega (Madison, WI, USA).


### 2.4. Animal Experiments

8–12 weeks old wildtype male C57BL/6 mice were purchased from National Laboratory Animal Center and were housed in Animal Facility of ILan University in Taiwan. The animal study was approved by the review boards of 100–24. Experimental procedures followed the Guidelines of Animal Use and Care of the National Institute of Health. Briefly, mice were housed in a room (23 ± 2°C) maintained on a 12 hr light/dark cycle (lights on at 7 AM) with food and water continuously available. Animals were randomly divided into four groups (control, D-gal administration, rutin administration, and D-gal administration plus rutin 30 mg/kg treatment) D-gal (300 mg/kg) was injected subcutaneously (s.c.) daily into mice for 6 weeks. Rutin (30 mg/kg body weight) was injected subcutaneously (s.c.) 3 h prior to D-gal injection. All control animals were given saline. At the end of administration, mice were performed behavior evaluation or sacrificed on the last day of treatment, and blood, sera, and major organs were immediately collected for histopathological analysis or other molecular experiments.


### 2.5. Intracellular Reactive Oxygen Species Detection

The production of intracellular reactive oxygen species was estimated by using a fluorescent probe, 2′,7′-dichlorofluorescein diacetate (DCFH-DA). DCFH-DA is transported across the cell membrane and hydrolyzed by intracellular esterases to form nonfluorescent 2′,7′-dichlorofluorescein (DCFH), which is then rapidly converted to highly fluorescent 2′,7′-dichlorofluoresecin (DCF) in the presence of reactive oxygen species. The DCF fluorescence intensity is believed to be parallel to the amount of relative oxygen species formed intracellularly. After 24 h treatment with 200μM H2O2, collected cell, and added CH2 DCFDA (final concentration 10 μM) for 60 min at 37°C, cells were washed by PBS for at least three times. The production of reactive oxygen species was measured immediately by Cell lab Quanta SC Flow cytometer (Beckman coulter).


### 2.6. Measurement of MDA Content and Antioxidant Enzyme Activities

The content of MDA was determined using the thiobarbituric acid method. Equal volumes of 0.67% thiobarbituric acid reagent were added to the sample supernatant and boiled for 10 min at 100°C, and cooled; the absorbance of each supernatant was measured at 532 nm. MDA content was calculated by MDA standard. Antioxidant enzyme activities were assayed with superoxide dismutase activity assay kit (BioVision) and Glutathione peroxidase assay kit (Cayman). The assay was in accordance with the manufacturer’s instructions.


### 2.7. Western Blot Analysis

The cells were lysed on ice with 200 mL of lysis buffer (50 mMTris-HCl, pH 7.5, 0.5 MNaCl, 5 mM MgCl2, 0.5% Nonidet P 40, 1 mM phenylmethylsulfonyl fluoridefor, 1 mg/mL pepstatin, and 50 mg/mL leupeptin) and centrifuged at 13,000 g at 4°C for 20 min. The protein concentrations in the supernatants were quantified using a BSA Protein Assay Kit. Electrophoresis was performed on an NuPAGE Bis-Tris Electrophoresis System using 50 mg of reduced protein extract per lane. Resolved proteins were then transferred to polyvinyl difluoride (PVDF) membranes. Filters were blocked with 5% nonfat milk overnight and probed with appropriate dilution of primary antibodies for 1 h at room temperature. Membranes were washed with three times with 0.1% Tween 20 and incubated with HRP-conjugated secondary antibody for 1 h at room temperature. All proteins were detected using Western Lightning Chemiluminescence Reagent Plus and quantified using a densitometer.


### 2.8. Immunohistochemical Staining

All tumor tissues were fixed in 10% formalin at 4°C and then embedded in paraffin. Paraffin sections (5μm) were deparaffinized in xylene and rehydrated through a graded series of ethanol solutions. The sections were incubated with blocking solution (5% nonfat milk powder in phosphate-buffered saline) for 60 min at room temperature, followed by a 4°C overnight incubation with anti-caspase-3-mouse (1/100; Abcam, Cambridge, MA) and anti-PARP-rabbit monoclonal antibodies (1/100; Cell Signaling Technology, Beverly, MA, USA) in blocking solution. Subsequently, the immune complexes were visualized using the LSAB2 system (Dako North America, Carpinteria, CA), and then incubated for 15 min with 0.5 mg/mL diaminobenzidine and 0.03% (v/v) H2O2 in PBS. Finally, sections were counterstained with hematoxylin, mounted, observed under a light microscope at magnifications of ×400, and photographed.


### 2.9. Inhibitory Avoidance Learning Task

Passive one-way avoidance paradigm was used to measure memory retention in the mice. The apparatus consisted of a trough-shaped alley divided by a sliding door which separates an illuminated safe compartment and a dark compartment. A shock generator which produced current was connected to the floor of the dark compartment (UGO Basile, Comerio, VA, Italy). The behavioral task, including the training and testing procedures, was recorded between 8.00 and 18.00 h. One day before the experiment, mice were habituated in the unplug apparatus with the sliding door open for 10 min to adjust to the environment. In the training phase, the mice were placed at the far end of the illuminated compartment facing away from the door. As the mice turned around, the door was closed and, after 1 sec, a 0.7 mA/sec footshock for 3 sec was given. The mice were then removed from the alley and returned to its home cage. The same stimulus was delivered again the next day. The memory retention test was carried out 1 day and 7 days later in the same manner as in the training, but without receiving a shock. Testing was terminated either when mouse entered the dark chamber or after 600 sec without entry. Mice which did not enter the dark compartment and reached the ceiling score of 600 sec were removed from the alley.


### 2.10. Statistical Analysis

Data were shown as mean with standard deviation. The statistical difference was analyzed using Student’st-test for normal distributed values and by nonparametric Mann-Whitney t-test for values of nonnormal distribution. Values of P<0.05 were considered significant. Behavioral data were analyzed with one-way analysis of variance (ANOVA), followed by post-hoc Dunnett’s t test or Newman-Keul’s multiple comparison tests. The q values represent those calculated from separate sets of Newman-Keul’s analysis.


## 2.1. Preparation of Fractions

The wild plant ofS. involucrataused in this study was a gift from Biopure Biotechnology (Changhua, Taiwan). Twenty grams of dried and powdered aerial parts, including flower, ofS. involucratawere extracted with 100 mL of methanol three times under reflux for 2 h, respectively. The methanol extracts (SI-1) were combined, and the solvent was evaporated in vacuo to give a deep brown syrup. The syrup was resuspended in water and then partitioned successively with pentane, ethyl acetate (SI-2), and n-butanol (SI-3) to leave a water layer (SI-4). The solvents were evaporated, respectively, and the residues were used throughout this study.


## 2.2. Reverse-Phase High-Performance Liquid Chromatography (HPLC) Analysis of Flavonoids inS. involucrata

The determination of flavonoids fromS. involucrata was carried out by HPLC with a photo diary detector. The HPLC system consisted of a Shimadzu LC-20AT solvent delivery system, equipped with a SPD-M20A photodiode array detector, set at 270 nm. Samples were injected with SiL-20A autosample to separate on the TSK-Gel ODS-100S column. The column was maintained at an ambient temperature of 25°C. The flow rate of the system was 1.0 mL/min. The mobile phase consisted of solvent A (0.3% formic acid) and solvent B (acetonitrile). The elution profile for A was 0–10 min, with a linear gradient change of 0–5%, 10–40 min, with a linear gradient change to 55%, and maintained for another 10 min with a post runtime to equilibrate the column and for the baseline to return to the normal and initial working conditions.


## 2.3. Chemicals and Reagents

Rutin was dissolved in DMSO to a concentration of 50 mM and stored in −20°C as a master stock solution. Dimethyl sulfoxide (DMSO), 3-(4,5-dimethyl thizol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), 2′,7′-dichlorofluorescein diacetate (H2DCF-DA), Hoechst 33342, thiobarbituric acid (TBA), hydrogen peroxide (H2O2), Trichloroacetic Acid, (TCA), malondialdehyde (MDA), propidium Iodine (PI), and actin antibody were purchased from Sigma Chemical Co. (St. Louis, MO, USA). NuPAGE Bis-Tris Electrophoresis System (precast polyacrylamide mini gel) was purchased from Invitrogen (Carlsbad, CA, USA). COX-2 antibody was purchased from Thermo scientific (Waltham, MA, USA). PARP antibodies and horseradish peroxidase - conjugated anti-mouse or anti-rabbit IgG secondary antibodies were purchased from Cell Signaling (MA, USA). Polyvinyl difluoride (PVDF) membranes, BSA protein assay kit, and western blot chemiluminescence reagent were purchased from Amersham Biosciences (Arlington Heights, IL). Superoxide dismutase activity assay kit was purchased from Biovision (Mountain View, CA). Glutathione peroxidase assay kit was purchased from Cayman Chemical (MI, USA). DNA Fragmentation Assay Kit was purchased from Clontech Laboratories (Mountain View, CA). Nonradioactive Cytoxicity Assay was purchased from Promega (Madison, WI, USA).


## 2.4. Animal Experiments

8–12 weeks old wildtype male C57BL/6 mice were purchased from National Laboratory Animal Center and were housed in Animal Facility of ILan University in Taiwan. The animal study was approved by the review boards of 100–24. Experimental procedures followed the Guidelines of Animal Use and Care of the National Institute of Health. Briefly, mice were housed in a room (23 ± 2°C) maintained on a 12 hr light/dark cycle (lights on at 7 AM) with food and water continuously available. Animals were randomly divided into four groups (control, D-gal administration, rutin administration, and D-gal administration plus rutin 30 mg/kg treatment) D-gal (300 mg/kg) was injected subcutaneously (s.c.) daily into mice for 6 weeks. Rutin (30 mg/kg body weight) was injected subcutaneously (s.c.) 3 h prior to D-gal injection. All control animals were given saline. At the end of administration, mice were performed behavior evaluation or sacrificed on the last day of treatment, and blood, sera, and major organs were immediately collected for histopathological analysis or other molecular experiments.


## 2.5. Intracellular Reactive Oxygen Species Detection

The production of intracellular reactive oxygen species was estimated by using a fluorescent probe, 2′,7′-dichlorofluorescein diacetate (DCFH-DA). DCFH-DA is transported across the cell membrane and hydrolyzed by intracellular esterases to form nonfluorescent 2′,7′-dichlorofluorescein (DCFH), which is then rapidly converted to highly fluorescent 2′,7′-dichlorofluoresecin (DCF) in the presence of reactive oxygen species. The DCF fluorescence intensity is believed to be parallel to the amount of relative oxygen species formed intracellularly. After 24 h treatment with 200μM H2O2, collected cell, and added CH2 DCFDA (final concentration 10 μM) for 60 min at 37°C, cells were washed by PBS for at least three times. The production of reactive oxygen species was measured immediately by Cell lab Quanta SC Flow cytometer (Beckman coulter).


## 2.6. Measurement of MDA Content and Antioxidant Enzyme Activities

The content of MDA was determined using the thiobarbituric acid method. Equal volumes of 0.67% thiobarbituric acid reagent were added to the sample supernatant and boiled for 10 min at 100°C, and cooled; the absorbance of each supernatant was measured at 532 nm. MDA content was calculated by MDA standard. Antioxidant enzyme activities were assayed with superoxide dismutase activity assay kit (BioVision) and Glutathione peroxidase assay kit (Cayman). The assay was in accordance with the manufacturer’s instructions.


## 2.7. Western Blot Analysis

The cells were lysed on ice with 200 mL of lysis buffer (50 mMTris-HCl, pH 7.5, 0.5 MNaCl, 5 mM MgCl2, 0.5% Nonidet P 40, 1 mM phenylmethylsulfonyl fluoridefor, 1 mg/mL pepstatin, and 50 mg/mL leupeptin) and centrifuged at 13,000 g at 4°C for 20 min. The protein concentrations in the supernatants were quantified using a BSA Protein Assay Kit. Electrophoresis was performed on an NuPAGE Bis-Tris Electrophoresis System using 50 mg of reduced protein extract per lane. Resolved proteins were then transferred to polyvinyl difluoride (PVDF) membranes. Filters were blocked with 5% nonfat milk overnight and probed with appropriate dilution of primary antibodies for 1 h at room temperature. Membranes were washed with three times with 0.1% Tween 20 and incubated with HRP-conjugated secondary antibody for 1 h at room temperature. All proteins were detected using Western Lightning Chemiluminescence Reagent Plus and quantified using a densitometer.


## 2.8. Immunohistochemical Staining

All tumor tissues were fixed in 10% formalin at 4°C and then embedded in paraffin. Paraffin sections (5μm) were deparaffinized in xylene and rehydrated through a graded series of ethanol solutions. The sections were incubated with blocking solution (5% nonfat milk powder in phosphate-buffered saline) for 60 min at room temperature, followed by a 4°C overnight incubation with anti-caspase-3-mouse (1/100; Abcam, Cambridge, MA) and anti-PARP-rabbit monoclonal antibodies (1/100; Cell Signaling Technology, Beverly, MA, USA) in blocking solution. Subsequently, the immune complexes were visualized using the LSAB2 system (Dako North America, Carpinteria, CA), and then incubated for 15 min with 0.5 mg/mL diaminobenzidine and 0.03% (v/v) H2O2 in PBS. Finally, sections were counterstained with hematoxylin, mounted, observed under a light microscope at magnifications of ×400, and photographed.


## 2.9. Inhibitory Avoidance Learning Task

Passive one-way avoidance paradigm was used to measure memory retention in the mice. The apparatus consisted of a trough-shaped alley divided by a sliding door which separates an illuminated safe compartment and a dark compartment. A shock generator which produced current was connected to the floor of the dark compartment (UGO Basile, Comerio, VA, Italy). The behavioral task, including the training and testing procedures, was recorded between 8.00 and 18.00 h. One day before the experiment, mice were habituated in the unplug apparatus with the sliding door open for 10 min to adjust to the environment. In the training phase, the mice were placed at the far end of the illuminated compartment facing away from the door. As the mice turned around, the door was closed and, after 1 sec, a 0.7 mA/sec footshock for 3 sec was given. The mice were then removed from the alley and returned to its home cage. The same stimulus was delivered again the next day. The memory retention test was carried out 1 day and 7 days later in the same manner as in the training, but without receiving a shock. Testing was terminated either when mouse entered the dark chamber or after 600 sec without entry. Mice which did not enter the dark compartment and reached the ceiling score of 600 sec were removed from the alley.


## 2.10. Statistical Analysis

Data were shown as mean with standard deviation. The statistical difference was analyzed using Student’st-test for normal distributed values and by nonparametric Mann-Whitney t-test for values of nonnormal distribution. Values of P<0.05 were considered significant. Behavioral data were analyzed with one-way analysis of variance (ANOVA), followed by post-hoc Dunnett’s t test or Newman-Keul’s multiple comparison tests. The q values represent those calculated from separate sets of Newman-Keul’s analysis.


## 3. Results

### 3.1. Histopathological Studies on Effects of Ethyl Acetate Extract of SI-2 or Rutin in the D-gal-Treated Mice Brain

The morphological features of hematoxylin and eosin (H&E) stained brain sections are shown in Figure2(c). Severe structural damages were observed in the brain structure of D-gal-treated mice, as compared to that in the brains of vehicle control mice. We used H&E staining to quantify cell death within the hippocampus and counted the pyknotic nuclei in the H&E section. The result showed that the percentages of pyknotic nuclei in the D-gal-treated mice were much higher than those in the control. Animals with SI-2 or rutin treatment showed a significant decreased damaged cells, suggested that both these compounds can effectively attenuate the hippocampus injury induce by D-gal in mice (Figure 2(c)). To further confirm this phenomenon, mice 42 d after treated with SI-2 or rutin were performed immunohistochemical staining for active caspase-3 and PARP cleaved PARP. Mice with SI-2 or rutin treatment can significantly reduce the expression of active caspase-3 in D-gal injured mice when compared with control ones (Figure 2(a)). Reduced PARP activation also can be confirmed in SI-2 or rutin treatment mice (Figure 2(b)). These results concluded that SI-2 or rutin had neuroprotect effects in D-gal injured animal model.Figure 2
Effect of ethyl acetate extract ofSaussurea involucrata(SI-2) or rutin on neuronal damage analysis in D-galactose-treated (aged) mice. Immunocytochemistry staining shows that cleaved caspase-3 (a) and cleaved PARP (b) in galactose-treated group were significantly increased compared with vehicle-treated group (PBS) and decreased in galatose + SI-2 (SI + D-gal) or rutin (Rutin) treated group  compared with galatose (D-Gal) alone group in the CA1 subfield of hippocampus after 6 weeks of administration. (c) H&E staining for morphologicl analysis.


### 3.2. Ethyl Acetate Extract of SI-2 or Rutin Decreases ROS in the Plasma and Brain of D-gal-Treated Mice

As shown in Figure3, administering D-gal significantly increased ROS by 4.8 times in the mice plasma (Figures 3(a) and 3(b)) and 4.0 times in the mice brain (Figures 3(c) and 3(d)), as compared to that of the control group. The data indicated that oxidative stress in vivo was elevated in the plasma and brain of D-gal-treated mice. SI-2 or rutin + D-gal group could dramatically decrease ROS in the plasma and brain, as compared to the D-gal-treated mice (Figure 3). Pretreatment SI or rutin attenuated the H2O2-induced accumulation of ROS.Effect of ethyl acetate extract ofSaussurea involucrata (SI-2) or rutin on plasma ROS level in D-galactose-treated (aged) mice. The control group received subcutaneous (s.c.) injections of phosphate-buffered saline. The aged group received D-galactose (300 mg/kg, s.c.). SI-2 + G-gal or rutin + G-gal groups received D-galactose (300 mg/kg/day, s.c.) plus SI-2 or rutin (30 mg/kg/day, p.o.). Treatments were administered for 6 weeks. SI-2 + G-gal or rutin + G-gal treatment groups attenuated the aging characteristics of increased ROS level. Data are presented as mean ± standard deviation (SD) (n=3 mice). *P<0.05 versus the aged group.
(a)
(b)
(c)
(d)


### 3.3. Effects of Ethyl Acetate Extract ofS. involucrata (SI-2) or Rutin on the Expression of NF-κB p65, COX-2, and iNOS in the Brain of D-gal-Treated Mice

After treatment with D-gal, the DNA-binding activity of NF-κB p65 in nuclear extract fractions increased, as compared to that of the control. However, this binding activity decreased upon the presence of SI-2 or rutin, as compared to D-gal alone. Furthermore, after treatment with D-gal, the expressions of COX-2 and iNOS were markedly induced (Figure 4(a)). As shown in Figure 4(b), SI-2 or rutin markedly inhibited the expressions of these molecules in the brain of D-gal-treated mice. Upon cotreatment with the SI-2 or rutin, however, the intensities of the COX-2 and iNOS bands significantly reduced, as compared to D-gal treatment alone. Protein expression of the NF-kappa B subunits p65 was downregulated by pretreatment with SI-2 or rutin, indicating that SI-2 or rutin decreased the expression of COX-2 via downregulation of NF-kappa B.Modulation of the cyclooxygenase-2 (COX-2) isozyme and NF-kappa B subunits (RELA and P50) bySaussurea involucrata (SI-2) or rutin pretreatment in D-galactose-treated (aged) mice. SI-2 or rutin pretreatment decreased this protein expression (a). SI-2 or rutin pretreatment also decreased COX-2 protein expression induced by D-galactose (b).
(a)
(b)


### 3.4. Antioxidant Effects of Ethyl Acetate Extract ofS. involucrata (SI-2) or Rutin in the D-gal Aging Model

We measured the activities of T-SOD and GSH-Px and the MDA level in the plasma of mice. The MDA level in D-gal-treated mice was significantly increased, as compared to that in the control group (Figures5(a) and 5(b)). Administering SI-2 or rutin (30 mg/kg/d and 30 mg/kg/d) significantly inhibited this increase. The activities of T-SOD and GSH-Px in D-gal-treated mice were significantly decreased, as compared to those in the control group, and administering SI-2 or rutin (30 mg/kg/d and 30 mg/kg/d) significantly attenuated these decreases (Figures 5(a) and 5(b)).Effect of ethyl acetate extract ofSaussurea involucrata(SI-2) or rutin on plasma MDA level and SOD and GPx activities in D-galactose-treated (aged) mice. The control group received subcutaneous (s.c.) injections of phosphate-buffered saline. The aged group received D-galactose (300 mg/kg, s.c.). SI-2 + G-gal or rutin + G-gal groups received D-galactose (300 mg/kg/day, s.c.) plus SI-2 or rutin (30 mg/kg/day, p.o.). Treatments were administered for 6 weeks. (SI-2) and rutin treatment attenuated the aging characteristics of increased MDA level and downregulated SOD and GPx activities. Data are presented as mean ± standard deviation (SD) (n=6 mice). *P<0.05 versus the aged group.
(a)
(b)


### 3.5. Rutin Prevents D-Galactose-Induced Memory Impairment in Passive One-Way Avoidance Task

In the present study, mice were randomly divided into control, D-gal, rutin, or (rutin + D-galactose) treatments (eachn=10). A footshock was delivered when mice entered a dark chamber from an illuminated chamber. Latency to enter the dark chamber 1 day and 7 days after footshock served as a measure of short-term and long-term memory retention. Cut-off latency was set at 600 s. Before footshock was delivered, all groups of mice spent approximately the same time entering the dark chamber from an illuminated chamber (P>0.05, Figure 6). D-galactose-treated mice showed less retention in the illuminated compartment one day after stimulation (q=4.97, P<0.01, when comparing control versus D-gal-treated groups at Day 1). The concentration of rutin (30 mg/kg) adopted in this study did not cause any effect on memory formation (P>0.05, when comparing control versus rutin groups). When rutin was applied in advance, memory impairment was no longer observed in D-gal-treated mice (q=4.2, P<0.01, when comparing D-gal versus rutin + D-gal groups on Day 1). These results indicated that rutin prevented D-gal-induced short-term memory retrieval impairment in a passive avoidance paradigm. The long-term effect of rutin was assessed 7 days later, demonstrating that D-gal-treated mice still spent less time in the illuminated chamber 7 days after footshock (tD=5.6, P<0.01). Pretreatment of rutin significantly prevented memory retrieval impairment in D-gal-treated mice (q=3.91, P<0.05, when comparing D-gal versus rutin + D-gal groups at Day 7) (Figure 6).Figure 6
Effects of rutin (30 mg/kg) on the step-through latencies in rats. The control group received subcutaneous (s.c.) injections of phosphate-buffered saline. The aged group received D-galactose (300 mg/kg, s.c.). SI-2 + G-gal or rutin + G-gal groups received D-galactose (300 mg/kg/day, s.c.) plus SI-2 or rutin (30 mg/kg/day, p.o.). Treatments were administered for 6 weeks. Retention tests were performed one, two, and seven days after training, *P<0.05, **P<0.01, and compared to the same day control, Tukey-Kramer’s test. Values are expressed as means ± SEM.


## 3.1. Histopathological Studies on Effects of Ethyl Acetate Extract of SI-2 or Rutin in the D-gal-Treated Mice Brain

The morphological features of hematoxylin and eosin (H&E) stained brain sections are shown in Figure2(c). Severe structural damages were observed in the brain structure of D-gal-treated mice, as compared to that in the brains of vehicle control mice. We used H&E staining to quantify cell death within the hippocampus and counted the pyknotic nuclei in the H&E section. The result showed that the percentages of pyknotic nuclei in the D-gal-treated mice were much higher than those in the control. Animals with SI-2 or rutin treatment showed a significant decreased damaged cells, suggested that both these compounds can effectively attenuate the hippocampus injury induce by D-gal in mice (Figure 2(c)). To further confirm this phenomenon, mice 42 d after treated with SI-2 or rutin were performed immunohistochemical staining for active caspase-3 and PARP cleaved PARP. Mice with SI-2 or rutin treatment can significantly reduce the expression of active caspase-3 in D-gal injured mice when compared with control ones (Figure 2(a)). Reduced PARP activation also can be confirmed in SI-2 or rutin treatment mice (Figure 2(b)). These results concluded that SI-2 or rutin had neuroprotect effects in D-gal injured animal model.Figure 2
Effect of ethyl acetate extract ofSaussurea involucrata(SI-2) or rutin on neuronal damage analysis in D-galactose-treated (aged) mice. Immunocytochemistry staining shows that cleaved caspase-3 (a) and cleaved PARP (b) in galactose-treated group were significantly increased compared with vehicle-treated group (PBS) and decreased in galatose + SI-2 (SI + D-gal) or rutin (Rutin) treated group  compared with galatose (D-Gal) alone group in the CA1 subfield of hippocampus after 6 weeks of administration. (c) H&E staining for morphologicl analysis.


## 3.2. Ethyl Acetate Extract of SI-2 or Rutin Decreases ROS in the Plasma and Brain of D-gal-Treated Mice

As shown in Figure3, administering D-gal significantly increased ROS by 4.8 times in the mice plasma (Figures 3(a) and 3(b)) and 4.0 times in the mice brain (Figures 3(c) and 3(d)), as compared to that of the control group. The data indicated that oxidative stress in vivo was elevated in the plasma and brain of D-gal-treated mice. SI-2 or rutin + D-gal group could dramatically decrease ROS in the plasma and brain, as compared to the D-gal-treated mice (Figure 3). Pretreatment SI or rutin attenuated the H2O2-induced accumulation of ROS.Effect of ethyl acetate extract ofSaussurea involucrata (SI-2) or rutin on plasma ROS level in D-galactose-treated (aged) mice. The control group received subcutaneous (s.c.) injections of phosphate-buffered saline. The aged group received D-galactose (300 mg/kg, s.c.). SI-2 + G-gal or rutin + G-gal groups received D-galactose (300 mg/kg/day, s.c.) plus SI-2 or rutin (30 mg/kg/day, p.o.). Treatments were administered for 6 weeks. SI-2 + G-gal or rutin + G-gal treatment groups attenuated the aging characteristics of increased ROS level. Data are presented as mean ± standard deviation (SD) (n=3 mice). *P<0.05 versus the aged group.
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## 3.3. Effects of Ethyl Acetate Extract ofS. involucrata (SI-2) or Rutin on the Expression of NF-κB p65, COX-2, and iNOS in the Brain of D-gal-Treated Mice

After treatment with D-gal, the DNA-binding activity of NF-κB p65 in nuclear extract fractions increased, as compared to that of the control. However, this binding activity decreased upon the presence of SI-2 or rutin, as compared to D-gal alone. Furthermore, after treatment with D-gal, the expressions of COX-2 and iNOS were markedly induced (Figure 4(a)). As shown in Figure 4(b), SI-2 or rutin markedly inhibited the expressions of these molecules in the brain of D-gal-treated mice. Upon cotreatment with the SI-2 or rutin, however, the intensities of the COX-2 and iNOS bands significantly reduced, as compared to D-gal treatment alone. Protein expression of the NF-kappa B subunits p65 was downregulated by pretreatment with SI-2 or rutin, indicating that SI-2 or rutin decreased the expression of COX-2 via downregulation of NF-kappa B.Modulation of the cyclooxygenase-2 (COX-2) isozyme and NF-kappa B subunits (RELA and P50) bySaussurea involucrata (SI-2) or rutin pretreatment in D-galactose-treated (aged) mice. SI-2 or rutin pretreatment decreased this protein expression (a). SI-2 or rutin pretreatment also decreased COX-2 protein expression induced by D-galactose (b).
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## 3.4. Antioxidant Effects of Ethyl Acetate Extract ofS. involucrata (SI-2) or Rutin in the D-gal Aging Model

We measured the activities of T-SOD and GSH-Px and the MDA level in the plasma of mice. The MDA level in D-gal-treated mice was significantly increased, as compared to that in the control group (Figures5(a) and 5(b)). Administering SI-2 or rutin (30 mg/kg/d and 30 mg/kg/d) significantly inhibited this increase. The activities of T-SOD and GSH-Px in D-gal-treated mice were significantly decreased, as compared to those in the control group, and administering SI-2 or rutin (30 mg/kg/d and 30 mg/kg/d) significantly attenuated these decreases (Figures 5(a) and 5(b)).Effect of ethyl acetate extract ofSaussurea involucrata(SI-2) or rutin on plasma MDA level and SOD and GPx activities in D-galactose-treated (aged) mice. The control group received subcutaneous (s.c.) injections of phosphate-buffered saline. The aged group received D-galactose (300 mg/kg, s.c.). SI-2 + G-gal or rutin + G-gal groups received D-galactose (300 mg/kg/day, s.c.) plus SI-2 or rutin (30 mg/kg/day, p.o.). Treatments were administered for 6 weeks. (SI-2) and rutin treatment attenuated the aging characteristics of increased MDA level and downregulated SOD and GPx activities. Data are presented as mean ± standard deviation (SD) (n=6 mice). *P<0.05 versus the aged group.
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## 3.5. Rutin Prevents D-Galactose-Induced Memory Impairment in Passive One-Way Avoidance Task

In the present study, mice were randomly divided into control, D-gal, rutin, or (rutin + D-galactose) treatments (eachn=10). A footshock was delivered when mice entered a dark chamber from an illuminated chamber. Latency to enter the dark chamber 1 day and 7 days after footshock served as a measure of short-term and long-term memory retention. Cut-off latency was set at 600 s. Before footshock was delivered, all groups of mice spent approximately the same time entering the dark chamber from an illuminated chamber (P>0.05, Figure 6). D-galactose-treated mice showed less retention in the illuminated compartment one day after stimulation (q=4.97, P<0.01, when comparing control versus D-gal-treated groups at Day 1). The concentration of rutin (30 mg/kg) adopted in this study did not cause any effect on memory formation (P>0.05, when comparing control versus rutin groups). When rutin was applied in advance, memory impairment was no longer observed in D-gal-treated mice (q=4.2, P<0.01, when comparing D-gal versus rutin + D-gal groups on Day 1). These results indicated that rutin prevented D-gal-induced short-term memory retrieval impairment in a passive avoidance paradigm. The long-term effect of rutin was assessed 7 days later, demonstrating that D-gal-treated mice still spent less time in the illuminated chamber 7 days after footshock (tD=5.6, P<0.01). Pretreatment of rutin significantly prevented memory retrieval impairment in D-gal-treated mice (q=3.91, P<0.05, when comparing D-gal versus rutin + D-gal groups at Day 7) (Figure 6).Figure 6
Effects of rutin (30 mg/kg) on the step-through latencies in rats. The control group received subcutaneous (s.c.) injections of phosphate-buffered saline. The aged group received D-galactose (300 mg/kg, s.c.). SI-2 + G-gal or rutin + G-gal groups received D-galactose (300 mg/kg/day, s.c.) plus SI-2 or rutin (30 mg/kg/day, p.o.). Treatments were administered for 6 weeks. Retention tests were performed one, two, and seven days after training, *P<0.05, **P<0.01, and compared to the same day control, Tukey-Kramer’s test. Values are expressed as means ± SEM.


## 4. Discussion

Results of the present study provide evidence that ethyl acetate extract ofS. involucrata(SI-2) or rutin can exert antiaging effects on D-gal-induced oxidative stress in mice. Concurrent treatment with SI-2 or rutin inhibited intracellular ROS formation. Although a small proportion of H2O2 may be scavenged by cellular antioxidant enzymes, it nonetheless directly induces the oxidation of various intracellular targets, including the fluorescence probe DCFH-DA. When cells were exposed to exogenous H2O2, DCF fluorescence increased significantly. The formation of hydroxyl radicals mediated by intracellular heavy metal ions may also contribute to the increased DCF fluorescence in response to H2O2. Many reports indicate that flavonoids can access intracellular locations, because of their benzylic structures, justifying their ability to attenuate oxidative stress induced by diverse stimuli [35, 36]. The chemical structure of rutin may contribute to its direct antioxidant properties. However, antioxidant activity was also found in other cellular models, and the concentrations of rutin required for antiaging were far lower than those of H2O2 used in our present experiments, suggesting that it may not be a simple stoichiometric interaction.Antioxidant activity of SI-2 or rutin was observed in the present study at dosages of 30 mg/kg/d and 30 mg/kg/d. In our previous studies of H2O2-induced oxidative stress and S. involucrata in SHSY-5Y cultures cells, we have found that SI-2 decreased ROS formation at concentrations of 100 μg/mL. It has been reported that SHSY-5Y cells used in the present study are clonal cells derived from rat neuroblastoma. Treatment using retinoic acid (RA) induces the differentiation of SHSY-5Y cells into a sympathetic neuron-like phenotype [37]. This cell line has been used widely as a model in neurobiologic, neuropharmacologic, and neurotoxicologic studies. Results of the present study provide evidence that SI-2 or rutin can exert neuroprotective effects on H2O2-induced oxidative stress in SHSY-5Y cells. Concurrent treatment with SI-2 or rutin inhibited intracellular ROS formation (data not shown).Results of the present study showed that SI-2 or rutin inhibited the expression of COX-2 and decreased lipid peroxidation. The dual intrinsic enzyme activities of COX-2 catalyze two sequential reactions in the metabolism of arachidonic acid (AA). The COX-2 enzyme possesses cyclooxygenase activity that metabolizes AA to hydroperoxide (PGG2; 9,11-endo-peroxy-15-hydroperoxyprostaglandin) utilizing two oxygen molecules (202). In addition, the COX-2 enzyme also possesses a heme-containing active site that provides peroxidase activity, which requires two electrons (2e−) to become active. The peroxidase reaction converts PGG2 to PGH2 by removing oxygen(s), (Ox), which may be a source of oxygen radicals. Therefore, as more AA is metabolized to PG by COX-2, more electron donors are depleted and more oxygen radicals are generated. The COX-2-dependent production of ROS is likely to be involved in the enhanced lipid peroxidation in H2O2-treated cells. Results in Figure 4 demonstrate that the level of COX-2 in the brain of a D-gal-treated mouse was significantly higher than that in the vehicle control mouse. In various inflammatory events, nitric oxide (NO) may have a decisive function involved with cytokine activation under soft tissue and bone damage conditions [38–40]. Although NO played an important role in the host defense on various pathogens, the overproduction of NO can be harmful and result in septic shock, rheumatoid arthritis, and autoimmune diseases [41]. Therefore, therapeutic agents that inhibit the iNOS may be useful in relieving these inflammatory conditions. Figure 4 shows that the level of iNOS in the brain of the D-gal-treated mouse was significantly higher than that in the vehicle control mice. Expression of iNOS and COX-2 is largely regulated by transcriptional activation. Among these transcription factors, NF-κB, which is a primary transcription factor and regulates various genes, is critical in the inflammation [4, 42]. NF-κB is a redox-sensitive transcription factor that regulates a multitude of inflammatory genes, including cytokines, chemokines, adhesion molecules, and acute phase proteins. Under basal conditions, NF-κB is inactive and prevented from DNA binding and nuclear translocation by tight association in the cytoplasm with inhibitory proteins. Cell activation by a variety of extracellular signals, such as oxidative stress, induces a cascade of events that lead to activating NF-κB then translocating it to the nucleus where it binds to DNA elements in the promoters of numerous proinflammatory gene families [28, 29]. In our study, after treatment with D-gal, the expression of NF-κB p65 in nuclear extract fractions increased, as compared to that of the control (Figure 4). PGE2 is an eicosanoid produced by COX that is usually considered as a potent inflammatory mediator [43]. PGE2 and its signaling pathway has been the target of clinical utility for a variety of disease/pathophysiological conditions, such as kidney failure. However, systemic side effects have limited their clinical utility. The pharmacological activities of PGE2 are mediated through the activation of G-protein coupled receptors, known as the EP receptors. The four subtypes of EP receptors are designated as EP1, EP2, EP3, and EP4. Previous studies have shown that induction of EPs is associated with the expression of proinflammatory enzymes such as iNOS and COX-2 [43].Many studies have shown that flavonoids possess potent antioxidant propertiesin vitro and in vivo. No previous studies have reported on the protective effect of rutin on D-gal-induced aging in mice. To protect cells against oxidative damage induced by ROS, the antioxidant system in the body is activated, and endogenous antioxidant enzymes, such as SOD and GPx, scavenge ROS or prevent their formation. The production of ROS can also be evaluated indirectly by analyzing the level of MDA, a product of free radical-induced lipid peroxidation. Analysis of the number of pyknotic nuclei cells in the hippocampus showed that Rutin had an important protective effect on D-gal-induced cell death (Figure 2(c)). Overall, our findings suggest that rutin can protect mice against oxidative stress injury induced by D-gal and improves impairments in aging mice. It has been reported that administration of rutin at doses of 10 and 100 mg/kg daily for one week prior to training induced a significant increase in memory retrieval, as compared to the control-treated group in the first retention test (one day after training). Rutin at doses of 5, 10, and 100 mg/kg significantly increased the memory retrieval in the second retention test of the passive avoidance paradigm, as compared to the control (two days and one week after training). Furthermore, rutin (10 mg/kg) significantly increased the memory retrieval, as compared to the rutin at doses of 100 mg/kg rats in the third retention test of the passive avoidance paradigm (one week after training) [44]. Our study is the first to demonstrate that six weeks of 30 mg/kg rutin administration daily prior to training results in an increased retrieval of memory in D-gal-treated mice, as observed in the first and second retention tests of the passive avoidance test (Figure 6). This effect was more significant when rutin was administered at a dose of 30 mg/kg, whereas the lower dose of 3 mg/kg had no influence on memory retrieval of D-galactose-induced aging mice (data not shown). In recent years, it has been revealed that a set of ion transport-related genes change their expression in the hippocampus before any detectable memory and learning impairment, thus suggesting that ion transport dysfunction could be crucial in the initiation of D-gal-related brain damage. Additionally, prolonged D-gal administration sequentially affected multiple-pathways, including protein transport and signal transduction, which could play key roles in maintaining the stability of synaptic structures [18]. It is possible that rutin acts by several mechanisms, mediating a potential role in memory retrieval in rats. Further study is necessary to evaluate the effect of rutin on retention of memory and to determine the molecular mechanisms.In conclusion, SI-2 or rutin decreased oxidative stress-induced ROS production and lipid peroxidation and also maintained endogenous antioxidant enzymatic activities, stabilizing mitochondrial function. Although more detailed mechanistic studies are necessary to clarify the mechanisms of antiaging using SI-2 or rutin, these results should encourage further studies to explore the potential antiaging effects of SI-2 or rutin in neurologic diseases.



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*Source: 980276-2012-08-16.xml*