Effect of Achillea millefolium aqueous extract on memory deficit and anxiety caused by stroke in ovariectomized rats

Introduction One of the main causes of death or incapability in the developed countries is ischemic stroke In fact, progressive aging of the population in these countries has resulted in the incidence of stroke, increasingly (1). The harmful effects of cerebral ischemia include inflammation, apoptosis, oxidative stress, acidosis and necrosis (2-4) Partial paralysis as well as the difficulties with memory, thinking, language, movements (5) and anxiety (6) can be considered as other deficits. Gender differences are important factors in the process of stroke due to the presence or absence of estrogen (7). Different studies have confirmed that an important neuroprotective agent in many models of brain injury like stroke is estrogen (8). Many studied conducted recently proved the claim that estrogens have neuroprotective effects in a variety of focal and global ischemia models (9). Notably, a powerful feminizing hormone such as 17 betaestradiol (E2) in a host of cell and animal models of stroke and neurodegenerative diseases has been also shown to have neuroprotective effects (10). Recently, clinical studies indicated the possibility of occurring some negative health http://www.herbmedpharmacol.com doi: 10.15171/jhp.2019.24


Introduction
One of the main causes of death or incapability in the developed countries is ischemic stroke In fact, progressive aging of the population in these countries has resulted in the incidence of stroke, increasingly (1).The harmful effects of cerebral ischemia include inflammation, apoptosis, oxidative stress, acidosis and necrosis (2)(3)(4) Partial paralysis as well as the difficulties with memory, thinking, language, movements (5) and anxiety (6) can be considered as other deficits.
Gender differences are important factors in the process of stroke due to the presence or absence of estrogen (7).Different studies have confirmed that an important neuroprotective agent in many models of brain injury like stroke is estrogen (8).Many studied conducted recently proved the claim that estrogens have neuroprotective effects in a variety of focal and global ischemia models (9).Notably, a powerful feminizing hormone such as 17 betaestradiol (E2) in a host of cell and animal models of stroke and neurodegenerative diseases has been also shown to have neuroprotective effects (10).Recently, clinical studies indicated the possibility of occurring some negative health Pirzad Jahromi et al consequences following hormone therapies of diseases like breast cancer (11) and endometriosis (12).Therefore, using phytoestrogens, natural nonsteroidal plant-derived compounds with estrogen-like molecules, may alter the concentration of estrogen level (13).Yarrow (Achillea millefolium) is a plant with phytoestrogen properties (14) which belongs to the Asteraceae family growing wild all across the Europe, Asia, North Africa and North America.Notably, it is extensively applied in Italian folk medicine (15).For instance, Italians widely use special teas prepared from the aerial parts of yarrow as a traditional treatment of spasms, menstrual pain, digestive complaints and other ailments (14).Taking the results of epidemiological studies into account, it has been suggested that dietary phytoestrogen intake might have a protective role in hormone-dependent cancers, especially breast cancer (16).Phytoestrogens are able to selectively bind to classical estrogen receptors (ERs) and regulate gene expression mediated by estrogen response elements (17).It should be noted that not only do they bind to ERs, but also exert potent antioxidant activity (17).Therefore, an intensive research has been recently conducted on phytoestrogens (18).The aim of this study was to investigate the effect of A. millefolium extract on memory impairment and anxiety caused by cerebral ischemia with middle cerebral artery occlusion in ovariectomized rats (OVX).

Materials and Methods
In this study, 48 female Wistar rats were used weighting 200 to 250 g.Animals were classified into 6 groups, considering eight animals in each group.They were maintained at 25±2°C under 12-hour light/dark cycle with free access to food.

Preparation of extracts
Achillea millefolium's flowers were used to prepare the extract.So that the branches of the plant from the Herbarium of Isfahan University were purchased (Herbarium approval number is 7757) (19).The tops and leaves of it were separated and washed.Then, they were dried thoroughly in a suitable environment at room's temperature (25°C).After grinding, 3 g of plant powder was added to 150 mL of distilled water with a Soxhlet system.After that, the aqueous extract was separated by a rotary machine (20).The selective dose in this study was 200 mg/kg, based on previous studies (19,21).Due to the fact that the most of rat weighed around 250 g, we converted the mentioned dose per rat (3.5, 7 mg/rat) (19,21).

Ovariectomization (menopause induction)
For anesthesia, animals received intraperitoneally a mixed solution of ketamine and xylazine at a dose of 75 mg/kg and 10 mg/kg, respectively.After that, they were shaved and their abdomen area was sterilized.Then, a gap of 1 to 2 cm was created in abdomen area and the ovarian was removed by a cautery device.The inner and outer layers were then sutured, separately.Finally, the Penicillin was injected into the thigh muscle of the rat at a dose of 0.3 mg /100 g animal's weight and the animal was returned to the cage for surgical recovery (22).
Accurate assessment of ovariectomized rat After 3 days, vaginal smears were prepared from the ovariectomized animals for 6 days and observed under an optical microscope.In this research, lack of ferns represents the true ovariectomy (22).

Creation of ischemia
After anesthetizing the animals using 75 mg/kg of ketamine and 10 mg/kg of xylazine intraperitoneally, the animals were beveled 2 cm between their eyes and ears.After removing the skin and temporalis muscle, the bones were removed and the middle cerebral artery was burned with cautery device.Finally, after returning soft tissues to their place, the skin was sutured (23).
The rats were categorized in different groups as follows: 1. Sham group on which all surgical stages were done without any damage to the ovaries (non-surgical ovariectomy were gavaged with saline for one month without any injury to the middle cerebral artery).2. Control group on which the ovariectomized surgery was performed.After one week, they were gavaged with saline for one month 3. A. millefolium 3.5 group on which the ovariectomized surgery was performed.After one week, the extract of plant was gavaged with 3.5 mg/kg of A. millefolium for 1 month.4. A. millefolium 7 group on which the ovariectomized surgery was performed.After one week, the plant's extract was gavaged with 7 mg/kg of A. millefolium for 1 month.5. Tamoxifen group on which the ovariectomized surgery was performed.After one week, they were gavaged with 100 μg/kg of tamoxifen for 4 weeks.6. A. millefolium 7+ tamoxifen group on which the ovariectomized surgery was performed .After one week, the plant's extract was gavaged with 7 mg/kg of A. millefolium and 100 μg/kg of tamoxifen.

Behavioral test Passive avoidance test
The passive avoidance task was evaluated 1 week after stroke using two-way shuttle box consisting of two near Plexiglas compartments in the same size (30 × 20 × 20 cm) with grid floors covered with stainless steel bars (2 mm) (24).In order to avoid training, each rat was given a 10-minute adaptation period with free access to the light or dark compartment.On the second day and after placing the animals in a shuttle-box, they were placed into the illuminated compartment.Ten seconds later, the sliding door was raised.Initial latency was recorded as learning phase.Entering the dark compartment, the door was closed and the animal's fore and hind paws were exposed to an electrical shock (0.3 mA, 50 Hz) for 3 seconds.Then, the rat was returned to the home cage.24 hours later, the rat was placed into the illuminated compartment to test short-term memory.The sliding door was then raised 5 seconds later.The latency of entering the dark compartment was recorded as memory test (step-through latency).The maximum time considered in this procedure was 300 seconds (25).Statistical analysis Graphs were plotted using SigmaPlot software and data were obtained by SPSS, using significant F-value, oneway analysis of variance (ANOVA), Tukey post hoc test and t test for comparison between groups.P < 0.05 was considered statistically significant between experimental groups.

Results
The effect of A. millefolium on amnesia induced by stroke in the absence and presence of tamoxifen Independent t test showed that stroke destroyed memory formation, compared to sham group (t 12 = 6; P < 0.001).
One-way ANOVA and Tukey post hoc test showed that 100 μg/kg of tamoxifen and 3.5 mg/kg of A. millefolium did not alter amnesia induced by stroke, while A. millefolium at a higher dose (7 mg/kg) restored this phenomenon [F (3, 24) =12, P < 0.01].Moreover, independent t test demonstrated that inactivation of ERs by tamoxifen blocked the restoration effect of A. millefolium on strokeinduced amnesia (t 12 = 2; P<0.05) (Figure 1).
The effect of A. millefolium on anxiogenic-like behavior (%OAT) induced by stroke in the absence and presence of tamoxifen Independent t test showed that stroke decreased OAT% (t 12 = 6; P<0.001, Figure 2A), OAE% (t 12 = 6.1;P < 0.001, Figure 2B) and locomotor activity (t 12 = 6; P < 0.001, Figure 2C).One-way ANOVA and Tukey post hoc test showed that tamoxifen at a dose of 100 μg/kg and A. millefolium at a dose of 3.5 mg/kg did not alter OAT%, OAE% and locomotor activity induced by stroke, while A. millefolium at a higher dose   2C] induced by stroke.Moreover, independent t-test demonstrated that inactivation of ERs by tamoxifen blocked the restoration effect of A. millefolium on OAT% (t 12 = 2; P < 0.05) and locomotor activity (t 12 = 2; P < 0.05) but not OAE (t 12 = 2; P < 0.05) (Figure 2).

Discussion
According to our results, it can be concluded that cerebral ischemia causes cognitive deficits.In a previous study, memory impairment occurred after induction of ischemia (28).In fact, reduced estrogen at menopause leads to a wide range of symptoms such as memory disorders and anxiety (6).Besides that, it is shown that deficits of learning and memory (29), sensation, perception and intellect (30) can be caused by ischemia events.Notably, overproduction of reactive oxygen species in mitochondria occurs as a result of reperfusion after ischemia.In addition, when these radicals consume endogenous antioxidants, a dramatic rise occurs in intracellular reactive oxygen species.It is together with a reaction of cellular macromolecules such as lipids, proteins and nucleic acids which results in oxidative damage of the neurons (31).Previous studies showed that the down-regulation of ERα and ERβ in the cerebral cortex may contribute to the loss of estrogen efficacy against ischemic injury in aged females (32).Estradiol and estrogen-like compounds are powerful neuroprotective agents in vivo and in vitro against apoptotic stimuli including experimental stroke (33).It is now well established that estrogen is a protective factor against focal cerebral ischemia and the global cerebral ischemia (34).It acts as a transcriptional activator through two nuclear receptor isoforms, ERα and ERβ.Both receptors are present in the brain and display overlapping and distinct distributions (35).In vivo, the neuroprotective actions of estrogen in cerebral ischemia have been attributed to the ER subtype activation (36) using ERα and ERβ knockout mice, although both isoforms can be neuroprotective in vitro (37).More investigations on the role of a periodic ER-β agonist regimen demonstrated that reduction of the innate immune response in the brain could help reduce the incidence of global cerebral ischemia and lessen its impact in post-menopausal women (38).Using estrogen increases the risk of catching breast cancer and endometriosis (39).Phytoestrogens can be naturally found in plant-derived compounds, structurally related to the gonadal steroid, 17β-estradiol, which are present in the human diet (40).According to the recent progress in pharmaceuticals, the development of selective ER modulators might bring about lots of estrogen's advantageous effects without suffering from detrimental side effects.However, many women today assume using complementary and alternative therapies beneficial for menopausal symptoms and use high soy diets or soy isoflavone supplements (41).It is needless to say that physiologically achievable doses of isoflavones, behaving as phytoestrogens, are able to simulate some of the neuroprotective effects of estrogens (42).
Achillea millefolium has estrogen-like activity and contains compounds of apigenin and luteolin.Apigenin could stimulate estrogen-receptor pathways and ERs (ERα and ERβ).Luteolin also stimulates the beta receptor (14).
The results of this study demonstrated a decrease in memory deficit and anxiogenic-like behavior of ovariectomized rats which orally received A. millefolium extract (7 mg/kg) during 4 weeks after inducing stroke.According to previous studies, consuming dietary soy containing estrogen-like compounds has neuroprotective effects on brain ischemia (42).In addition, it has been previously shown that genistein is a phytoestrogen compound which is protective to brain against cerebral ischemia through reducing oxidative stress and neuronal degeneration (43).Daidzein as a phytoestrogen compound also exhibits neuroprotective effects on ischemic brain tissue (44).In addition, coumestrol is a phytoestrogen compound which has potential benefits for either prevention or therapeutics use against cerebral ischemia in males (45).In another study, it has been demonstrated that Soy dietary may play a role in the brain injury (46).Soy prevents memory impairment and brain tissue oxidative damage caused by ischemia in OVX rats (47).Therefore, it is determined that A. millefolium extract reduces memory impairment due to having antioxidant and estrogen-like compounds, which is consistent with previous results.In in vitro studies, it has also been shown that pretreatment with dietary levels of Soy phytoestrogens protects primary cortical neurons from ischemic-like injury (48).Apparently, in order to inhibit apoptotic cell death, the isoflavones use the same ER-kinase pathway as estrogen (48).Since plant compounds may also have complications; therefore, it is necessary to do more research on the side effects of these plants.

Conclusion
This study showed that, due to estrogen-like compounds and antioxidant, the gavage of A. millefolium extract could result in the improvement of memory impairments and anxiogenic-like behavior induced by cerebral ischemia caused by the middle cerebral artery occlusion.
Elevated plus maze testThe anxiety was measured 5 days after stroke using an elevated plus maze, a platform with two enclosed and two open arms(26).The elevated plus maze works based on rats' natural detestation of open spaces, which causes behaviors involving the avoidance of open areas through limiting movements to a bounded space or its edges.This apparatus consists of two opposing closed arms (50 × 10 × 40 cm) and two opposing open arms (50 × 10 cm) connected to a center platform (10 × 10 cm).Where these 4 arms intersect, the maze is elevated 50 cm above the floor, with a square platform of 10 × 10 cm.After placing the rats in the center of the maze, their activity were videotaped for 5 minutes.Anxiety reduction in the plus-maze was shown by an increase in the percentage of time weaken in the open arms (%OAT = time in open arms/ total time in open or closed arms ×100), and an increase in the percentage of entries into the open arms (%OAE = entries into open arms/total entries into open or closed arms ×100) (27).