Protective Effects of Cyperus rotundus Extract on Amyloid β-Peptide ( 1-40 )-Induced Memory Impairment in Male Rats . A Behavioral Study

Objectives: The aim of this study was to assess the effectiveness of combination of Nigella sativa and honey (Dosin) in eradication of gastric H. pylori infection. Patients and Methods: Nineteen patients who had positive result for H. pylori infection by Urea breath test (UBT) without the past history of peptic ulcer, gastric cancer or gastrointestinal bleeding, were suggested to receive 1 teaspoon of the mixture of Dosin (6g/ day of N. sativa as ground seeds and 12g/day of honey) three times a day after meals for 2 weeks. The second urea breath test was used to detect the presence of H. pylori 4 weeks after completion of the regimen. Also symptoms of dyspepsia were scored before and after the study and analyzed with Wilcoxon signedrank test. Results: Fourteen patients completed the study. Negative UBT was observed in 57.1% (8/14) of participants after intervention. The median (IQR) of total dyspepsia symptoms was significantly reduced from 5.5 (5-12) to 1 (0-4) (P= 0.005). All patients tolerated Dosin except for one who was excluded due to mild diarrhea. No serious adverse events were reported. Conclusion: Dosin is concluded to be an anti H. pylori and the anti dyspeptic agent. Further studies are recommended to investigate the effect of Dosin plus antibiotics (concurrently or following another) on gastric H. pylori infection. Nigella sativa| Helicobacter pylori| honey| medicine| traditional| phytotherapy Effect of Cyperus rotundus on ischemia-induced brain damage and memory dysfunction in rats Fataneh Hashem Dabaghian, Mehrdad Hashemi 1-Research Institute for Islamic and Complementary Medicine, fataneh.dabaghian@yahoo.com, 2-Genetic Department, Islamic Azad University, Tehran Medical Sciences Branch, Tehran, Iran, hashemi_mehrdad@yahoo.com Objective(s): Global cerebral ischemia-reperfusion injury causes loss of pyramidal cells in CA1 region of hippocampus. In this study, we investigated the possible neuroprotective effects of the ethanol extract of Cyperus rotundus (EECR) on a model of global transient ischemia in rat, by evaluating the pathophysiology of the hippocampal tissue and spatial memory. Materials and Methods: Treatment group (EECR, 100 mg/kg/day) was gavaged from 4 days before, to 3 days after ischemia. Morris water maze test was performed 1 week after ischemia for 4 days. Brain tissue was prepared for Nissl staining. Results: Our data showed no statistical difference between the treatment and ischemia groups in water maze task. So, treatment of ischemia with EECR cannot improve spatial learning and memory. On the contrary EECR ameliorated the CA1 pyramidal cell loss due to transient global ischemia/ reperfusion injury. Conclusion: These results suggest that EECR cannot reduce the ischemiainduced, cognitive impairments seen after transient, global cerebral ischemia but can prevent pyramidal cell loss in CA1 region of hippocampus. Cerebral ischemia| Cyperus rotundus| Morris water maze| Spatial memory Protective Effects of Cyperus rotundus Extract on Amyloid βPeptide (1-40)Induced Memory Impairment in Male Rats. A Behavioral Study Fataneh Hashem Dabaghian, Sara Soleimani Asl, Mehdi Mehdizadeh 1Research Institute for Islamic and Complementary Medicine, fataneh.dabaghian@yahoo.com, 2Anatomy Department, School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran, , 3Cellular and Molecular Research Center, Faculty of Advanced Technologies in Medicine, Department of Anatomy, Iran University Tehran, Iran, Abstract


Introduction
he Alzheimer Disease (AD) is a progressive and irreversible loss of neurons commonly characterized by a gradual decline of memory, language, and cognitive ability (Kumar, Shankar, Reddy, Kumar, & Sumalatha, 2014). Accumulation of Amyloid β (Aβ) in the brain is considered as 1 of the major contributing factors to the development of AD (Citron et al., 1997). Nabeshima and Nitta reported that intraventricular administration of Aβ in the rats resulted in learning and memory impairment accompanied by a decrease in choline acetyltransferase activity, suggesting that accumulation of Aβ is related to cognitive deficiency in AD . Administration of Aβ T leads to changes in Long-Term Potentiation (LTP) in the hippocampus and, consequently, impairs cognition and memory in rodents (Shankar et al., 2008). Stress oxidative, protein oxidation, Reactive Oxygen Species (ROS) formation, and subsequent neuronal death are reported following Aβ deposition generated by an imbalance between ROS and internal antioxidants. Evidence show that appropriate nourishing with external antioxidants could improve brain damage and cognitive function (Khodamoradi, Komaki, Salehi, Shahidi, & Sarihi, 2015;Nezhadi et al., 2011).
Furthermore, the external antioxidants can prevent the detrimental consequences of Aβ and are considered as a promoting approach to neuroprotection in the AD brain Zargooshnia et al., 2014). Cyperus rotundus, as a species of sedge and traditional medicine are widely used worldwide to treat stomach ailments and wounds (Puratchikody, Devi, & Nagalakshmi, 2006;Uddin, Mondal, Shilpi, & Rahman, 2006).
It is shown that the major chemical components of C. rotundus are essential oils, flavonoids, terpenoids, mono and sesquiterpenes (Singh, Kulshrestha, Gupta, & Bhargava, 1970;Sonwa & Knig, 2001). Also, it is shown that hydroalcoholic extract of C. rotundus exhibited powerful free radical scavenging, especially against 1,1-diphenyl-2-picryl-hydrazyl (DPPH) and superoxide anions, as well as a moderate effect on nitric oxide . This extract showed an inhibitory effect against lipid peroxidation, protein oxidation, and glycoxidation (Bahramikia, Ardestani, & Yazdanparast, 2009;Kilani et al., 2008). It appears that C. rotundus contains potent components such as flavonoids that may be potentially useful to modulate the immune cell functions, provoking analgesics, and defending against inflammation and stress oxidative (Dhillon, Singh, Kundra, & Basra, 1993). Malekian and Ghannadi showed that administration of C. rotundus improved the scopolamineinduced learning and memory deficit in mice (Malekian, Rabbani, & Ghannadi, 2012). Due to the common usage of traditional medicine and natural antioxidants, the current study aims at investigating the ameliorating effects of C. rotundus extracts on Aβ (1-40)-induced amnesia.

Animals
Adult male Wistar rats (Pasteur Institute, Tehran, Iran), weighed 250 to 300 g were used in the study. The rats were accommodated the animal house in a 12:12 hours light/dark cycle (light on, 7:00 AM; light off, 7:00 PM) with free access to food and water. A week before the experimental procedure, the animals were habituated to their new environment. The guidelines of the National Institute of Health Guide for Care and Use of Laboratory Animals were performed for all experiments, and approved by the Veterinary Ethics Committee of the Iran University of Medical Sciences, Tehran, Iran.
The animals were randomly classified into the following groups (n=7 each group): The control group which was left undisrupted; and The sham-operated group. The Aβ model group received single bilateral intrahippocampal (IHP) injections of 6 μg Aβ 1-40 . The Cyperus rotundus-treated group received intraperitoneal injection of C. rotundus extract (400 mg/kg) following IHP injection of Aβ 1-40 for 14 days (Hsieh, Peng, Wu, & Wang, 2000).

Preparation of C. rotundus extract
In June 2014, the dried C. rotundus, with herbarium code TARI 12569, was collected from Iranian Institute of Medicinal Plant Field and grounded into coarse powder by electrically driven device and was soaked into aqueous ethanol (80%) for 1 week. It was passed through a Whatman filter paper (a cellulose filter to specify and recognize the materials in the qualitative analytical techniques) and vaporized by a rotary evaporator under the reduced pressure at a maximum of 40°C.The extract was completely dissolved in distilled water and kept at 4°C (Malekianet et al., 2012).

Stereotaxic surgery
The previously established method of stereotaxic surgery was used  In brief, rats were anesthetized by xylazine (10 mg/kg) and ketamine (100 mg/kg), and placed into a stereotaxic device (Stoelting, USA). After retraction of scalp, the area surrounding bregma was cleaned and dried. Relative to the bregma and with the stereotaxic arm at 0°, the coordinates for the dentate gyrus were anterior-posterior (AP): 3.6 mm from bregma; Mediolateral (ML): +2.3 mm from midline; Dorsoventral (DV): 3 mm from skull surface (Figure 1). Aβ solution (6 µL) was bilaterally injected into the region using a 10 µL microsyringe (Hamilton-Reno, NV, USA). Sham operated rats received vehicle solution.

Assessment of spatial memory
Morris Water-Maze (MWM) is a behavioral task to assess hippocampal-related learning, including acquisition and retention of spatial memory and plays an important role in the validation of rodent models for neurocognitive disorders such as the Alzheimer disease (D'Hooge & De Deyn, 2001;Morris, Garrud, Rawlins, & O'Keefe, 1982). Therefore, the protocol used in the current investigation was derived from the previously performed study Soleimani Asl et al. (2013). In brief, a black circular pool filled with water (22±1°C) with an invisible plexiglass platform (located 1 cm below the water) was used. Some constant visual cues such as table, library, and computer were located around the MWM room.
The North, East, South, and West locations were selected to start the training trials. The animals were trained for 3 consecutive days at the same time (10:00 to 12:00 AM). Training included 2 blocks with 4 trials. There was a 5-minute rest between consecutive blocks. During trials, from each of the starting positions, the animals swam up the located hidden platform. The animals were allowed to spend 30 seconds on the platform between the 2 trials. There was a video camera above the pool that recorded escape latency (the time taken to reach the hidden platform) and traveled distance (the length of the swim path). On day 4, a probe trial was performed in which the platform was removed from the pool and each rat was allowed to swim for 60 seconds and percentage of entrance into the target quadrant was recorded.

Statistical analysis
Data were expressed as mean±standard error of the mean (SEM) and processed by commercially available software SPSS version 16. Results were analyzed using repeated measure and 2 way analysis of variance (treatment and training days as the factors). The Turkey multiple comparison test was used to analyze the significance of the differences between the groups, when appropriate. P<0.05 was considered statistically significant.

Results
Effects of Cyperus rotundus on the Aβ (1-40)-induced increase in escape latency in Morris water-maze: A 2-way analysis of variance of escape latency revealed significant effects of treatment [F(3, 4233)=10.63, P<0.001]. In addition, there was no significant interaction between treatment and training days. As shown in Figure 2, escape latency (the time to find the hidden platform) was less in the control group than the other groups. More time to find the hidden platform indicates more intense spatial memory impairment. A post hoc analysis of the 3 training days showed a significant difference between the control and sham-operated groups, and the rats that received Aβ (P<0.001). According to the current study results, the administration of C. rotundus caused significant reduction in escape latency compared with the Aβ-treated group (P<0.05).
Effects of Cyperus rotundus on the Aβ (1-40)-induced increase in traveled distance in Morris water-maze: In accordance with the latency data, treatment had significant effect [F(3, 7080)=9.83, P<0.001]. There was no significant effect in both training days, and also no significant difference was observed in the interaction between training days and treatment. A significant difference in traveled distance was observed between Aβ-treated rats with the control and sham-operated groups (P<0.001) ( Figure  3). Aβ-treated rats that received C. rotundus extract for 7 days showed less traveled distance, in comparison with the Aβ-treated group (P<0.05).
Effects of Cyperus rotundus on the Aβ (1-40)-induced reduction in time spent in target quadrant in Morris watermaze: As shown in Figure 4, a 1-way analysis of variance (ANOVA) of time spent percent in the target quadrant showed that the control group spent more time in target quadrant (26.66±3.33) than the sham-operated (22.66±4.37), Aβ-treated (20±3.1) and C. rotundus (25.8±2.43).

Discussion
The current study aimed at evaluating the protective effects of C. rotundus extract on memory impairment following intrahippocampal injection of Aβ (1-40). The major findings of the current study were: (a) Intrahippocampal injection of Aβ (1-40) resulted in learning impairment; (b) Treatment with C. rotundus extract was protective against Aβ-induced memory impairment. Previously published experimental studies reported that the infusion of Aβ(1-40) into the brain impaired one-trial/day reward learning (Malin et al., 2001), and memory in radial-arm maze task confirmed the current study results (Malin et al., 2001;Tanaka et al., 1998). As memory impairment and degeneration of cholinergic neurons can be observed following the deposition of β-amyloid protein in the brain; it seems that the β-amyloid-treated rats could be used as the animal model for the Alzheimer disease (Nitta, Itoh, Hasegawa, & Nabeshima, 1994).
It seems that due to the low antioxidant and cell membrane lipid, the brain is sensitive to oxidative stress (Butterfield & Lauderback, 2002). Therefore, employment of external antioxidants, such as various spices and herbs, can be 1 of the popular remedial strategies to treat neurological diseases, and recovery of brain damage and cognitive deficiency (Azad, Rasoolijazi, Joghataie, & Soleimani, 2011;Ghahremanitamadon et al., 2014).
Cyperus rotundus is a traditional medicinal plant used against stomach disorders and inflammatory bowel diseases (Jagtap, Shirke, & Phadke, 2004;Uddin et al. 2006).The current study demonstrated that administration of C. rotundus extract could improve Aβ-induced memory impairment. The protective effect of C. rotundus extract on memory can be related to its function of scavenging free radicals . Ardestani et al., showed the preventing effects of C. rotundus on oxidative protein damages by decreasing oxidative stress . Antioxidant activity and metal chelating properties of C. rotundus are shown by Fluorescence Recovery After Photobleaching (FRAP) and Trolox Equivalent Antioxidant Capacity (TEAC) assays . This herbal extract with anti-inflammatory properties is safe in animals (Dang, Parekar, Kamat, Scindia, & Rege, 2010;Seo et al., 2001).
It seems that the antioxidant properties of C. rotundus are due to its phenolic content , which should be studied further. In conclusion, intrahippocampal injection of Aβ induced significant learning deficits in Morris water-maze tasks, and C. rotundus treatment improved Aβ-induced deficiencies. Therefore, it is likely that C. rotundus may be useful to treat patients with impaired memory function.