# Extracts or Active Components from Acorus gramineus Aiton for Cognitive Function Impairment: Preclinical Evidence and Possible Mechanisms

Authors: Yan Li; Xi-Le Zhang; Yan-Ran Huang; Yan-Yan Zheng; Guo-Qing Zheng; Li-Ping Zhang
Journal: Oxidative Medicine and Cellular Longevity
 (2020)

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## Abstract

Extracts or active components from Acorus gramineus Aiton (EAAGA) have been clinically used for cognition impairment more than hundreds of years and are still used in modern times in China and elsewhere worldwide. Previous studies reported that EAAGA improves cognition impairment in animal models. Here, we conducted a preclinical systematic review to assess the current evidence of EAAGA for cognition impairment. We searched 7 databases up until June 2019. Methodological quality for each included studies was accessed according to the CAMARADES 10-item checklist. The primary outcome measures were neurobehavioral function scores evaluated by the Morris water maze test, electrical Y-maze test, step-down test, radial eight-arm maze test, and step-through test. The secondary outcome measures were mechanisms of EAAGA for cognition function. Finally, 34 studies involving 1431 animals were identified. The quality score of studies range from 1 to 6, and the median was 3.32. Compared with controls, the results of the meta-analysis indicated EAAGA exerted a significant effect in decreasing the escape latency and error times and in increasing the length of time spent in the platform quadrant and the number of platform crossings representing learning ability and memory function (allP<0.01). The possible mechanisms of EAAGA are largely through anti-inflammatory, antioxidant, antiapoptosis activities, inhibition of neurotoxicity, regulating synaptic plasticity, protecting cerebrovascular, stimulating cholinergic system, and suppressing astrocyte activation. In conclusion, EAAGA exert potential neuroprotective effects in experimental cognition impairment, and EAAGA could be a candidate for cognition impairment treatment and further clinical trials.


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## Body

## 1. Introduction

With the average life expectancy increasing, there is concern about the proportion of cognitive impairment in the global population, which results from degeneration of the brain and very high prevalence in elderly individuals [1]. The World Health Organization estimates that the number of people over the age of 60 will be around 2 billion in 2050, while the number of cognitive impairment patients is expected to rise rapidly along with the aging population worldwide [2, 3]. However, so far, clinical trials have not identified efficacious neuroprotective therapies for cognitive impairment patients [4]. Thus, given the huge translational gap between the animal studies and clinical trials, seeking or developing innovative neuroprotectants is urgently needed.For more than a millennium, traditional Chinese medicine (TCM), a main form of complementary and alternative medicine, has been used in Asian countries, especially in China, Japan, and Korea, to alleviate various symptoms of cognitive deficits and to facilitate learning and memory [5]. Acorus gramineus Aiton (AGA) (record 2322 (http://www.theplantlist.org.)), the dry rhizomes of Acorus gramineus Solander (Shi Changpu), is listed officially in the Chinese Pharmacopoeia and used in oriental medicines for more than hundreds of years to treat neurological disorders. AGA possessed various pharmacological effects on the central nervous system, including neuroprotective effects [6, 7], central inhibitory effects [8], inhibitory effects on excitotoxic neuronal death [9], and stroke [10], and amelioration in learning and memory [5]. AGA may be effective for the improvement of amnesia [9]. AGA contains different extract fractions: volatile oil, composing mainly of β-asarone (63.2–81.2%), and α-asarone (8.8–13.7%) [11], as well as water extract, ethyl ether extract, ethyl acetate extract, N-butanol extract, and the defatted decoction fractions. AGA is often used as a component in some Chinese herbal formulas. Among 75 of the most famous Chinese herbal formulas characterized as improving intelligence both in ancient and modern time in China, more than half contain AGA, such as Kai-Xin-San [12] and Chong-MyungTang [13].Systematic reviews are believed to be preferred; only data that from systematic reviews will be considered as the highest level of medical evidence basis for the levels of evidence from the Centre of Evidence-Based Medicine in Oxford [14]. Preclinical systematic reviews are a powerful approach to analyze and synthesize the results of an intervention from animal data into a useful document that can help to shape further basic research, optimize the experimental studies, and enhance the success rate of future clinical trials [15]. Thus, we conducted a preclinical systematic review to assess the current evidence of extracts or active components from Acorus gramineus Aiton (EAAGA) and active component for animal models of cognitive impairment.


## 2. Materials and Methods

### 2.1. Search Strategies

Experimental studies of EAAGA for cognitive impairment were identified in the databases, including PubMed, Embase, Web of Science, Wanfang database, China National Knowledge Infrastructure (CNKI), CBM, and VIP information database. All searches were performed from inception to June 2019. Studies about assessing the effectiveness of AAGA for improving cognitive function impairment in animals were identified. The search terms were as follows: (Acorus tatarinowii Schott OR Rhizoma acori graminei OR Acorus calamus OR Acorus gramineus Soland OR acorus gramineus aiton OR Acori graminei rhizoma OR Acori tatarinowii rhizoma OR grassleaf sweetfalg Rhizome) AND (cognitive function impairment OR amnesia OR dementia OR Alzheimer’s disease).


### 2.2. Inclusion Criteria

Experimental studies on EAAGA for cognitive impairment models were included, regardless of publication status or animal species, gender, age, and methods of model establishment. The primary outcome measurements were Morris water maze test (MWM test), electric Y-maze test (EY-M test), radial eight-arm maze test (RAM test), Step down test (SD test), and/or Step through test (ST test). The secondary outcome measures were mechanisms of EAAGA for learning and/or memory function.


### 2.3. Exclusion Criteria

Exclusion criteria were prespecified as follows: (1) the article was a review, case report, comment, clinical trial, abstract, or editorial; (2) the article was a clinical orin vitro study; (3) the article was not a research about cognitive impairment model; (4) EAAGA was used as combination; (5) there was no control group; and (6) the article was a duplicate publication.


### 2.4. Data Extraction

The information of each included study was extracted: (1) author and publication year, animal model species, method of anesthesia, and random method; (2) characteristics of animals, including species, sex, animal number, and weight; (3) treatment information from treatment and control groups, including drug, dose, method of treatment, timing for initial treatment, frequency, and duration of treatment; and (4) outcome measures, sample size, and corresponding data including mean value, standard deviation, and intergroup differences. If outcomes were presented at different time points, we extracted data from the last time point. If studies utilized dose gradient of the drug, we extracted data from the highest dose of EAAGA and active component since the dose-response relationship. If the data were incomplete or presented in graphs, we tried to contact the authors for data needed or calculated using relevant software. Information of the mechanism studies of EAAGA and active component for cognitive impairment models among the included articles was extracted.


### 2.5. Quality Assessment

The methodological quality of included studies was evaluated by two independent reviewers using Collaborative Approach to Meta-Analysis and Review of Animal Data from Experimental Studies (CAMARADES) 10-item checklist [16]. For calculating an aggregate quality score, each item of this scale was attributed one point.


### 2.6. Statistical Analysis

Meta-analysis was conducted via RevMan version 5.3. To estimate the effect of EAAGA on cognitive impairment, the random effects model and standard mean difference (SMD) with 95% confidence intervals (CIs) were calculated. Heterogeneity was assessed viaI2 statistics test. If probability value was less than 0.05, the difference was considered statistically significant. In addition, to explore potential sources of high heterogeneity, subgroup analyses were performed according to animal species and models. Difference between groups was determined by partitioning heterogeneity and utilizing the χ2 distribution with degrees of freedom (df).


## 2.1. Search Strategies

Experimental studies of EAAGA for cognitive impairment were identified in the databases, including PubMed, Embase, Web of Science, Wanfang database, China National Knowledge Infrastructure (CNKI), CBM, and VIP information database. All searches were performed from inception to June 2019. Studies about assessing the effectiveness of AAGA for improving cognitive function impairment in animals were identified. The search terms were as follows: (Acorus tatarinowii Schott OR Rhizoma acori graminei OR Acorus calamus OR Acorus gramineus Soland OR acorus gramineus aiton OR Acori graminei rhizoma OR Acori tatarinowii rhizoma OR grassleaf sweetfalg Rhizome) AND (cognitive function impairment OR amnesia OR dementia OR Alzheimer’s disease).


## 2.2. Inclusion Criteria

Experimental studies on EAAGA for cognitive impairment models were included, regardless of publication status or animal species, gender, age, and methods of model establishment. The primary outcome measurements were Morris water maze test (MWM test), electric Y-maze test (EY-M test), radial eight-arm maze test (RAM test), Step down test (SD test), and/or Step through test (ST test). The secondary outcome measures were mechanisms of EAAGA for learning and/or memory function.


## 2.3. Exclusion Criteria

Exclusion criteria were prespecified as follows: (1) the article was a review, case report, comment, clinical trial, abstract, or editorial; (2) the article was a clinical orin vitro study; (3) the article was not a research about cognitive impairment model; (4) EAAGA was used as combination; (5) there was no control group; and (6) the article was a duplicate publication.


## 2.4. Data Extraction

The information of each included study was extracted: (1) author and publication year, animal model species, method of anesthesia, and random method; (2) characteristics of animals, including species, sex, animal number, and weight; (3) treatment information from treatment and control groups, including drug, dose, method of treatment, timing for initial treatment, frequency, and duration of treatment; and (4) outcome measures, sample size, and corresponding data including mean value, standard deviation, and intergroup differences. If outcomes were presented at different time points, we extracted data from the last time point. If studies utilized dose gradient of the drug, we extracted data from the highest dose of EAAGA and active component since the dose-response relationship. If the data were incomplete or presented in graphs, we tried to contact the authors for data needed or calculated using relevant software. Information of the mechanism studies of EAAGA and active component for cognitive impairment models among the included articles was extracted.


## 2.5. Quality Assessment

The methodological quality of included studies was evaluated by two independent reviewers using Collaborative Approach to Meta-Analysis and Review of Animal Data from Experimental Studies (CAMARADES) 10-item checklist [16]. For calculating an aggregate quality score, each item of this scale was attributed one point.


## 2.6. Statistical Analysis

Meta-analysis was conducted via RevMan version 5.3. To estimate the effect of EAAGA on cognitive impairment, the random effects model and standard mean difference (SMD) with 95% confidence intervals (CIs) were calculated. Heterogeneity was assessed viaI2 statistics test. If probability value was less than 0.05, the difference was considered statistically significant. In addition, to explore potential sources of high heterogeneity, subgroup analyses were performed according to animal species and models. Difference between groups was determined by partitioning heterogeneity and utilizing the χ2 distribution with degrees of freedom (df).


## 3. Results

### 3.1. Study Selection

We identified 2368 potentially relevant papers after systematical search from six databases. After removing duplicates, 1887 studies remained. By reading titles and abstracts, 1602 articles were excluded that were reviews, case reports, comments, abstracts, clinical trials, letters, or editorials. After reading the remaining 285 full-text articles, 228 studies were excluded for at least one of following reasons: (1) not an animal study; (2) the article was not a research about cognitive impairment; (3) the study did not access the effects of AGA or active component on the animal model of cognitive impairment; (4) EAAGA was not used as a monotherapy; and (5) lack of control group. Ultimately, 34 eligible articles [5, 6, 10, 11, 17–46] were selected (Figure 1).Figure 1
Flow diagram.


### 3.2. Characteristics of Included Studies

Sixteen studies [5, 6, 10, 11, 17–27, 37] were published in English, and 18 studies were in Chinese between 1999 and 2019. In total, 34 studies with 1431 animals were included. Ten species were referred, including Sprague-Dawley (SD) rat (n=236, 16.49%), Wistar rats (n=130, 9.08%), Kunming mice (n=530, 37.04%), ICR mice (n=236, 16.49%), NIH mice (n=168, 11.74%), AβPP/PS1 double-transgenic mice (n=26, 1.82%); APPswe/PS1dE9 double transgenic mice (n=22, 1.54%), C57BL/6 mice (n=24, 1.68%), senescence-accelerated prone-8 (SAMP8) mice (n=26, 1.82%), and FMR1gene knock mice (n=33, 2.31%). The weight of SD rats ranged from 200 g to 650 g, the weight of Wistar rats used ranged from 30 g to 250 g, and the weight of mice ranged from 17 g to 50 g. Twenty-two studies used male rodents, 1 study used female rodents, 5 study used both female and male rodents, and the remaining 6 studies did not provide gender details. Sodium pentobarbital was used to induce anesthesia in 8 studies, and chloral hydrate was used in 2 studies [20, 21], 1 study [41] used phenytoin sodium, 1 study [17] used CO2, and 1 study [10] used isoflurane, while the remaining 21 studies did not report the type of anesthetics. Cognitive impairment models were induced by lead [17], noise stress [18], LPS [19], amyloid beta 1-42 [11, 21, 26, 28, 29, 37, 41, 46], D-gal plus AlCl3 [22], scopolamine [5, 24, 30, 34–36, 42, 45], ethanol [5, 32, 34–36], sodium nitrite [5, 32], corticosterone [23], Ibotenic acid [25], chronic restraint stress [31], pentobarbital sodium [32], D-galactose [33, 38], AlCl3 [40], streptozotocin (STZ) [43], pent ylenetet razol (PTZ) [44], and NaNO2 [34–36]. As an intervention, fourteen studies [6, 17, 20, 22, 23, 26, 27, 32, 35, 37, 39, 41, 42, 46] used β-asarone, eight studies [18, 19, 21, 24, 33, 38, 40, 44] used α-asarone, three studies [10, 25, 44] utilized AGA, twelve studies [5, 11, 22, 28–32, 35, 36, 43, 45] used essential oil, seven studies [11, 28, 29, 33–36] researched water extract, four studies [11, 28, 29, 32] used defatted decoction, and one study [18] researched ethyl acetate extract. Normal distilled water control was used in 2 studies [17, 33]; Tween 80 control was used in 6 studies [5, 6, 18, 20, 27, 32]; normal saline control was used in 24 studies; 0.5% methylcellulose solution containing 1% Tween 80 control was used in 1 study [24], and 2% propylene glycol containing 2% polyethylene glycol stearate control was used in 1 study [43]. Neurobehavioral function indices as primary outcome measures were carried out by the Morris water maze test (MWM test) (n=28), step-down test (SD test) (n=6), electrical Y-maze test (EY-M test) (n=3), step-through test (ST test) (n=4), and radial eight-arm maze test (RAM test) (n=3). The characteristics of the 34 studies are shown in Table 1.Table 1
Basic characteristics of the included studies.
Study (years)Species (sex,n=experimental/controlgroup)WeightRandom methodModel (method)AnestheticMethod of administrationOutcome index (time)Intergroup differencesExperimental groupControl groupYang et al. [17]SD rats (mix, 7/7)NRNRChronic lead-induced dysmnesia modelCO2β-Asarone (2.5, 10, and 40 mg kg-1, ip); from 9 to 11 weeks old; once daily for 3 weeksDistilled water (same volume, ip); from 9 to 11 weeks old; once daily for 3 weeks(1) MWM test (escape latency)(1)P<0.001(2) MWM test (swimming speed)(2)P>0.05(3) MWM test (time spent in target quadrant)(3)P<0.05(4) MWM test (times crossed the platform)(4)P>0.05(5) Dendritic spine density(5)P<0.001Wei et al., 2013AβPP/PS1 double-transgenic mice (13/13)NRNRAβPP/PS1 double-transgenic miceNRβ-Asarone (7 and 21 mg kg-1, ig); onset the experiment; once daily for 4 monthsTween 80 (same volume, ig); onset the experiment; once daily for 4 months(1) MWM test (escape latency)(1)P<0.001(2) Cell viability(2)P<0.05Sundaramahalingam et al. [18]Wister strain albion rats (male, 6/6)200-220 gNRNoise stress induced memory impairment modelNRα-Asarone (9 mg kg-1, ip); onset the experiment; once daily for 30 dTween 80 (same volume, ip); onset the experiment; once daily for 30 d(1) RAM test (number of errors)(1)P<0.05(2) Hsp 70 mRNA levels(2)P<0.05(3) Ache activity(3)P<0.05(4) SOD/CAT/GPx activity(4)P<0.05(5) VC/VE/GSH levels(5)P<0.05(6) G6PD activity(6)P<0.05Wister strain albion rats (male, 6/6)200-220 gNRNoise stress exposed ratsNREthyl acetate extract (50 mg kg-1, ip); onset the experiment; once daily for 30 dTween 80 (same volume, ip); onset the experiment; once daily for 30 d(1) RAM test (number of errors)(1)P<0.05(2) Hsp 70 mRNA levels(2)P<0.05(3) Ache activity(3)P<0.05(4) SOD/CAT/GPx activity(4)P<0.05(5) VC/VE/GSH levels(5)P<0.05(6) G6PD activity(6)P<0.05Shin et al. [19]C57BL/6 mice (male, 12/12)25-28 gNRLPS-induced cognitive handicap modelNRα-Asarone (7.5, 15, and 30 mg kg-1, ig); 3 days before the LPS injection; once daily for 3 dNormal saline (same volume, ig); 3 days before the LPS injection; once daily for 3 d(1) MWM test (escape latency)(1)P<0.05(2) MWM test (times crossed the platform)(2)P<0.05(3) TNF-α/IL-1β mRNA levels(3)P<0.05(4) CA1 neurons count(4)P<0.05(5) TUNEL-labeled cells count(5)P<0.05(6) BACE1/Iba1 protein expressions(6)P<0.05Ma et al. [11]Six-week-old NIH mice (male, 6/6)20-25 gNRAβ1-42-induced AD modelSodium pentobarbitalWater extract (20 mg g-1, ig); after the first MWM test; once daily for 3 weeksNormal saline; (same volume, ig); after the first MWM test; once daily for 3 weeks(1) MWM test (escape latency)(1)P<0.05(2) Aβ positive cells count(2)P<0.05(3) DCx expression3P<0.05(4) Nestin positive cells count(4)P<0.05Six-week-old NIH mice (male, 6/6)20-25 gNRAβ1-42-induced AD modelSodium pentobarbitalEssential oil (20 mg g-1, ig); after the first MWM test; once daily for 3 weeksNormal saline; (same volume, ig); after the first MWM test; once daily for 3 weeks(1) MWM test (escape latency)(1)P<0.05(2) Aβ positive cells count(2)P<0.05(3) DCx expression(3)P<0.05(4) Nestin positive cells count(4)P<0.05Six-week-old NIH mice (male, 6/6)20-25 gNRAβ1-42-induced AD modelSodium pentobarbitalDefatted decoction (20 mg g-1, ig); after the first MWM test; once daily for 3 weeksNormal saline; (same volume, ig); after the first MWM test; once daily for 3 weeks(1) MWM test (escape latency)(1)P<0.05(2) Aβ positive cells count(2)P<0.05(3) DCx expression(3)P<0.05(4) Nestin positive cells count(4)P<0.05Liu et al. [20]APPswe/PS1dE9 double transgenic mice (male, 11/11)NRNRAPPswe/PS1dE9 double transgenic miceChloral hydrateβ-Asarone (21.2, 42.4, and 84.8 mg kg-1, ig); onset the experiment; once daily for 2.5 monthsTween 80 (same volume, ig); onset the experiment; once daily for 2.5 months(1) MWM test (escape latency)(1)P<0.05(2) MWM test (time spent in target quadrant)(2)P<0.05(3) MWM test (times crossed the platform)(3)P<0.05(4) SYP/GluR1 expression(4)P<0.05Limón et al. [21]Wistar rats (male, 8/8)230–250 gNRAβ1-42-induced AD modelChloral hydrateα-Asarone (10 mg kg-1, i.h.); after injection of amyloid-β; once daily for 16 dNormal saline; (same volume, ig); after injection of amyloid-β; once daily for 16 d(1) RAM test (percentage of correct responses)(1)P<0.001(2) Nitrite levels(2)P<0.05Li et al. [22]Wistar rats (female, 7/7)150–180 gNRD-gal and AlCl3 induced AD modelSodium pentobarbitalβ-Asarone (25, 50 and 100 mg kg-1, i.h.); 28 d after injection of AlCl3 and D-gal; once daily for 14 dNormal saline; (same volume, i.h); after the first MWM test; once daily for 14 d(1) MWM test (escape latency)(1)P<0.05(2) MWM test (time spent in target quadrant)(2)P<0.05(3) MWM test (times crossed the platform)(3)P<0.05(4) MWM test (swimming speed)(4)P>0.05(5) ET-1, eNOS, and APP expression(5)P<0.05(6) Lactic acid and pyruvic acid content(6)P<0.05(7) Na+- K+ ATPase activity(7)P<0.05(8) rCBF(8)P<0.05Zhang et al. [5]Aged Kunming mice (male, 10/10)40-50 gNRAged miceNREssential oil (0.02, 0.04, and 0.08 g kg-1, orally); onset the experiment; once daily for 15 dTween 80 (same volume, orally); onset the experiment; once daily for 15 d(1) SD test (escape latency)(1)P<0.01(2) SD test (number of errors)(2)P<0.05Aged Kunming mice (male, 10/10)40-50 gNRScopolamine-induced dysmnesia modelNREssential oil (0.02, 0.04, and 0.08 g kg-1, orally); onset the experiment; once daily for 15 dTween 80 (same volume, orally); onset the experiment; once daily for 15 d(1) SD test (escape latency)(1)P<0.05(2) SD test (number of errors)(2)P<0.01Aged Kunming mice (male, 10/10)40-50 gNREthanol-induced dysmnesia modelNREssential oil (0.02, 0.04, and 0.08 g kg-1, orally); onset the experiment; once daily for 15 dTween 80 (same volume, orally); onset the experiment; once daily for 15 d(1) SD test (escape latency)(1)P<0.01(2) SD test (number of errors)(2)P<0.01Aged SD rats (male, 10/10)550-650 gNRAged ratsNREssential oil (0.02, 0.04, and 0.08 g kg-1, orally); onset the experiment; once daily for 30 dTween 80 (same volume, orally); onset the experiment; once daily for 30 d(1) EY-M test (number of errors)(1)P<0.01(2) NE, DA and 5-HT level(2)P<0.01(3) AChE activity(3)P<0.01(4)P<0.01Aged SD rats (male, 10/10)550-650 gNRSodium nitrite-induced dysmnesia modelNREssential oil (0.02, 0.04, and 0.08 g kg-1, orally); onset the experiment; once daily for 30 dTween 80 (same volume, orally); onset the experiment; once daily for 30 d(1) EY-M test (number of errors)(1)P<0.05(2)P<0.01Lee et al. [10]SD rats (male, 5/7)250-280 gNRMCAO-induced cognitive impairments modelIsofluraneAGA (100 mg kg-1, po); after occlusion; once daily for 21 dNormal saline; (same volume, i.h); after occlusion; once daily for 21 d(1) MWM test (escape latency)(1)P<0.05(2) Cell density(2)P<0.05Lee et al. [23]SD rats (male, 7/7)200-220 gNRChronic corticosterone-exposed modelSodium pentobarbitalβ-Asarone (50, 100, and 200 mg kg-1, ip); 30 min prior to the CORT; once daily for 21 dNormal saline; (same volume, ip); 30 min prior to the CORT; once daily for 21 d(1) MWM test (swimming speed)(1)P>0.05(2) serum CORT levels(2)P<0.05(3) BDNF and CREB expression(3)P<0.05(4) Bax and Bcl-2 mRNAs expression(4)P<0.05Kumar et al. 2012ICR mice (8/8)NRNRScopolamine-induced amnesic model modeNRα-Asarone (3, 10, and 30 mg kg-1, po); 15 d before scopolamine injection; once daily for 15 d0.5% methylcellulose solution containing 1% Tween 80 (same volume, po); 15 d before scopolamine injection; once daily for 15 d(1) SD test (escape latency)(1)P<0.01(2) AchE activity(2)P<0.001(3) MDA levels(3)P<0.001(4) SOD activity(4)P<0.05Kim et al. [25]SD rats (male, 5/5)260–280 gNRIbotenic acid-induced amnesiaSodium pentobarbitalAGA (100 mg kg-1,ip); after surgery; once daily for 3 weeksSaline (same volume, ip); after surgery; once daily for 3 weeks(1) MWM test (escape latency)(1)P<0.001(2) ChAT positive neurons count(2)P>0.05(3) AchE neurons density(3)P<0.05Geng et al. [26]SD rats (male, 20/20)220-240 gNRAβ1-42-induced AD modelSodium pentobarbitalβ-Asarone (12.5, 25, or 50 mg kg-1, ig); 3 d after Aβ (1-42) hippocampus injection; once daily for 28 dSaline (same volume, ip); 3 d after Aβ (1-42) hippocampus injection; once daily for 28 d(1) MWM test (escape latency)(1)P<0.05(2) MWM test (times crossed the platform)(2)P<0.05(3) Annexin V-positive cells(3)P<0.05(4) Caspase-3 and Caspase-3 mRNA express(4)P<0.05(5) Bcl-2 and Bcl-2 mRNA levels(5)P<0.05(6) Bcl-w, and Bcl-w mRNA express(6)P<0.05(7) P-JNK express(7)P<0.05Chen et al. [27]SAMP8 mice (13/13)NRNRSAMP8 miceNRβ-Asarone (34 mg kg-1, ig); onset the experiment; once daily for 2 monthsTween 80 (same volume, ig); onset the experiment; once daily for 2 months(1) MWM test (number of platform crossing)(1)P<0.05(2) MWM test (escape latency)(2)P<0.05(3) LC3-positive cells(3)P<0.05(4) Beclin express(4)P<0.05(5) 1p62 express(5)P<0.05(6) ROCK1 express(6)P<0.05(7) GAP43, MAP2 and SYN expression(7)P<0.05(8) GAP43, MAP2 and SYN positive cells(8)P<0.05(9) Lipofuscin-positive cells(9)P<0.05Ma et al. [28]NIH mice (male, 6/6)18-20 gNRAβ1-42-induced AD modelSodium pentobarbitalWater extract (0.02 g g-1, ig); after the first MWM test; once daily for 3 weeksNormal saline (same volume, ig) after the first MWM test; once daily for 3 weeks(1) MWM test (escape latency)(1)P<0.05(2) Beta-amyloid IOD(2)P<0.05NIH mice (male, 6/6)18-20 gNRAβ1-42-induced AD modelSodium pentobarbitalDefatted decoction (0.02 g g-1, ig); after the first MWM test; once daily for 3 weeksNormal saline (same volume, ig) after the first MWM test; once daily for 3 weeks(1) MWM test (escape latency)(1)P<0.05(2) Beta-amyloid IOD(2)P<0.05NIH mice (male, 6/6)18-20 gNRAβ1-42-induced AD modelSodium pentobarbitalEssential oil (0.02 g g-1, ig); after the first MWM test; once daily for 3 weeksNormal saline (same volume, ig) after the first MWM test; once daily for 3 weeks(1) MWM test (escape latency)(1)P<0.05(2) Beta-amyloid IOD(2)P<0.05Tian et al. [29]NIH mice (male, 6/6)18-20 gNRAβ1-42-induced AD modelSodium pentobarbitalWater extract (0.02 g g-1, ig); after the first MWM test; once daily for 3 weeksNormal saline (0.2 ml/10 g, ig); after surgery; once daily for 3 weeks(1) MWM test (number of platform crossing)(1)P<0.05(2) NOS activity(2)P<0.05NIH mice (male, 6/6)18- 0 gNRAβ1-42-induced AD modelSodium pentobarbitalDefatted decoction (0.02 g g-1, ig); after the first MWM test; once daily for 3 weeksNormal saline (0.2 ml/10 g, ig); after surgery; once daily for 3 weeks(1) MWM test (number of platform crossing)(1)P>0.05(2) MWM test (time spent in target quadrant)(2)P>0.05(3) NOS activity(3)P<0.05NIH mice (male, 6/6)18-20 gNRAβ1-42-induced AD modelSodium pentobarbitalEssential oil (0.02 g g-1, ig); after the first MWM test; once daily for 3 weeksNormal saline (0.2 ml/10 g, ig); after surgery; once daily for 3 weeks(1) MWM test (number of platform crossing)(1)P<0.05(2) NOS activity(2)P<0.05Zhou et al. [30]SD rats (male, 10/10)250±20gNRScopolamine-induced AD modelNREssential oil (12 g kg-1, ig); onset the experiment; once daily for 21 dNS (same volume, ig); onset the experiment; once daily for 21 d(1) MWM test (escape latency)(1)P<0.01(2) MWM test (number of platform crossing)(2)P<0.01(3) GFAP - positive cells(3)P<0.01(4) SOD content(4)P<0.05(5) MDA content(5)P<0.05Wang GM et al., 2017Kunming mice (mix, 12/12)5-6 weeksNRChronic restraint stress-induced cognitive impairments modeNREssential oil (4.5 g kg-1, ig), onset the experiment; twice daily for 28 dNS (same volume, ig); onset the experiment; twice daily for 28 d(1) MWM test (escape latency)(1)P<0.01(2) MWM test (number of platform crossing)(2)P<0.01(3) Body mass(3)P<0.05(4) Plasma cortisol levels(4)P<0.01Hu et al. [32]Kunming mice (male, 11/11)18-20 gNRSodium nitrite-induced amnesic modelNREssential oil (0.053 g kg-1, ig); 21 d before sodium nitrite injection; once daily for 21 dTween 80 (same volume, ig); 21 d before sodium nitrite injection; once daily for 21 d(1) SD test (escape latency)(1)P<0.05(2) SD test (number of errors)(2)P<0.05Kunming mice (male, 11/11)18-20 gNRSodium nitrite-induced amnesic modelNRDefatted decoction (5 g kg-1, ig); 21 d before sodium nitrite injection; once daily for 21 dTween 80 (same volume, ig); 21 d before sodium nitrite injection; once daily for 21 d(1) SD test (escape latency)(1)P<0.05(2) SD test (number of errors)(2)P<0.05Kunming mice (male, 11/11)18-20 gNRSodium nitrite-induced amnesic modelNRα-Asarone (0.024 g kg-1, ig); 21 d before sodium nitrite injection; once daily for 21 dTween 80 (same volume, ig); 21 d before sodium nitrite injection; once daily for 21 d(1) SD test (escape latency)(1)P<0.05(2) SD test (number of errors)Kunming mice (male, 11/11)18-20 gNRSodium nitrite-induced amnesic modelNRβ-Asarone (0.037 g kg-1, ig); 21 d before sodium nitrite injection; once daily for 21 dTween 80 (same volume, ig); 21 d before sodium nitrite injection; once daily for 21 d(1) SD test (escape latency)(1)P<0.05(2) SD test (number of errors)(2)P<0.01Kunming mice (male, 11/11)18-20 gNREthanol-induced amnesic modelNREssential oil (0.053 g kg-1, ig); 21 d before ethanol injection; once daily for 21 dTween 80 (same volume, ig); 21 d before ethanol injection; once daily for 21 d(1) SD test (escape latency)(1)P<0.05(2) SD test (number of errors)(2)P<0.01Kunming mice (male, 11/11)18-20 gNREthanol-induced amnesic modelNRDefatted decoction (5 g kg-1, ig); 21d before ethanol injection; once daily for 21 dTween 80 (same volume, ig); 21 d before ethanol injection; once daily for 21 d(1) SD test (escape latency)(1)P>0.05(2) SD test (number of errors)(2)P<0.05Kunming mice (male, 11/11)18-20 gNREthanol-induced amnesic modelNRα-Asarone (0.024 g kg-1, ig); 21 d before ethanol injection; once daily for 21 dTween 80 (same volume, ig); 21 d before ethanol injection; once daily for 21 d(1) SD test (escape latency)(1)P<0.01(2) SD test (number of errors)Kunming mice (male, 11/11)18-20 gNREthanol-induced amnesic modelNRβ-Asarone (0.037 g kg-1, ig); 21 d before ethanol injection; once daily for 21 dTween 80 (same volume, ig); 21 d before ethanol injection; once daily for 21 d(1) SD test (escape latency)(1)P<0.05(2) SD test (number of errors)(2)P<0.01Kunming mice (male, 11/11)18-20 gNRSodium pentobarbital-induced amnesic modelNREssential oil (0.053 g kg-1, ig); 21 d before ethanol injection; once daily for 21 dTween 80 (same volume, ig); 21 d before ethanol injection; once daily for 21 d(1) EY-M test (number of errors)(1)P<0.05Kunming mice (male, 11/11)18-20 gNRSodium pentobarbital-induced amnesic modelNRDefatted decoction (5 g kg-1, ig); 21 d before ethanol injection; once daily for 21 dTween 80 (same volume, ig); 21 d before ethanol injection; once daily for 21 d(1) EY-M test (number of errors)(1)P<0.05Kunming mice (male, 11/11)18-20 gNRSodium pentobarbital-induced amnesic modelNRα-Asarone (0.024 g kg-1, ig); 21 d before ethanol injection; once daily for 21 dTween 80 (same volume, ig); 21 d before ethanol injection; once daily for 21 d(1) EY-M test (number of errors)(1)P<0.05Kunming mice (male, 11/11)18-20 gNRSodium pentobarbital-induced amnesic modelNRβ-Asarone (0.037 g kg-1, ig); 21 d before ethanol injection; once daily for 21 dTween 80 (same volume, ig); 21 d before ethanol injection; once daily for 21 d(1) EY-M test (number of errors)(1)P<0.05Chen et al. [33]ICR mice (male, 10/10)18±1gRandom number tableD-gal-induced dementia modelNRWater extract (70, 35, 17.5, or 8.75 mg kg-1, ig); 1 week after D-galactose injection; once daily for 7 weeksDistilled water (same volume, ig); 1 week after D-galactose injection; once daily for 7 weeks(1) MWM test (escape latency)(1)P>0.05(2) MWM test (number of platform crossing)(2)P>0.05(3) MDA levels(3)P>0.05(4) SOD activity(4)P>0.05Gu et al. [34]ICR mice (male, 10/10)19.6±1.5gRandom number tableScopolamine-induced dysmnesia modelNRWater extract (70, 35, 17.5, or 8.75 mg kg-1, ig); onset the experiment; once daily for 2 weeksNS (same volume, ig); onset the experiment; once daily for 2 weeks(1) SD test (escape latency)(1)P<0.01(2) SD test (number of errors)(2)P<0.01ICR mice (male, 10/10)19.6±1.5gRandom number tableNaNO2-induced dysmnesia modelNRWater extract (70, 35, 17.5, or 8.75 mg kg-1, ig); onset the experiment; once daily for 2 weeksNS (same volume, ig); onset the experiment; once daily for 2 weeks(1) SD test (escape latency)(1)P<0.01(2) SD test (number of errors)(2)P<0.05ICR mice (male, 10/10)19.6±1.5gRandom number tableEthanol-induced dysmnesia modelNRWater extract (70, 35, 17.5, or 8.75 mg kg-1, ig); onset the experiment; once daily for 2 weeksNS (same volume, ig); onset the experiment; once daily for 2 weeks(1) ST test (escape latency)(1)P<0.01(2) ST test (number of errors)(2)P<0.01(3) AchE activity(3)P>0.05Wistar rats (male, 10/10)200±25gNRScopolamine-induced dysmnesia modelNRWater extract (35, 17.5, or 8.75 mg kg-1, ig); onset the experiment; once daily for 4 weeksNS (same volume, ig); onset the experiment; once daily for 2 weeks(1) MWM test (escape latency)(1)P<0.01(2) MWM test (number of platform crossing)(2)P<0.01Wu et al., 2004Aged NIH mice (male, 10/10)NRNRAged miceNREssential oil (0. 01075 ml g-1, ig); onset the experiment; twice daily for 10 dNS (same volume, ig); onset the experiment; once daily for 10 d(1) MWM test (escape latency)(1)P<0.05(2) AchE activity(2)P<0.05(3) C-jun express(3)P<0.05Aged NIH mice (male, 10/10)NRNRAged miceNRβ-Asarone (0. 01075 ml g-1, ig); onset the experiment; twice daily for 10 dNS (same volume, ig); onset the experiment; once daily for 10 d(1) MWM test (escape latency)(1)P>0.05(2) MWM test (number of errors)(2)P>0.05(3) AchE activity(3)P>0.05(4) C-jun express(4)P>0.05Aged NIH mice (male, 10/10)NRNRAged miceNRWater extract (0. 01075 ml g-1 g, ig); onset the experiment; twice daily for 10 dNS (same volume, ig); onset the experiment; once daily for 10 d(1) MWM test (escape latency)(1)P>0.05(2) AchE activity(2)P>0.05(3) C-jun express(3)P<0.05Kunming mice (male, 10/10)NRNREthanol-induced dysmnesia modelNRWater extract (0. 01075 ml g-1, ig); onset the experiment; twice daily for 10 dNS (same volume, ig); onset the experiment; once daily for 10 d(1) ST test (escape latency)(1)P<0.05Kunming mice (male, 10/10)NRNRNaNO2-induced dysmnesia modelNREssential oil (0. 01075 ml g-1, ig); onset the experiment; twice daily for 10 dNS (same volume, ig); onset the experiment; once daily for 10 d(1) SD test (escape latency)(1)P>0.05(2) SD test(number of errors)(2)P>0.05Kunming mice (male, 10/10)NRNRNaNO2-induced dysmnesia modelNRβ-Asarone (0. 01075 ml g-1, ig); onset the experiment; twice daily for 10 dNS (same volume, ig); onset the experiment; once daily for 10 d(1) SD test (escape latency)(1)P<0.05(2) SD test (number of errors)(2)P>0.05Kunming mice (male, 10/10)NRNRNaNO2-induced dysmnesia modelNRWater extract (0. 01075 ml g-1, ig); onset the experiment; twice daily for 10 dNS (same volume, ig); onset the experiment; once daily for 10 d(1) SD test (escape latency)(1)P<0.05(2) SD test (number of errors)(2)P>0.05Kunming mice (male, 10/10)NRNRScopolamine-induced dysmnesia modelNREssential oil (0. 01075 ml g-1, ig); onset the experiment; twice daily for 10 dNS (same volume, ig); onset the experiment; once daily for 10 d(1) SD test (escape latency)(1)P<0.05(2) SD test (number of errors)(2)P>0.05Kunming mice (male, 10/10)NRNRScopolamine-induced dysmnesia modelNRβ-Asarone (0. 01075 ml g-1, ig); onset the experiment; twice daily for 10 dNS (same volume, ig); onset the experiment; once daily for 10 d(1) SD test (escape latency)(1)P<0.05(2) SD test (number of errors)(2)P>0.05Kunming mice (male, 10/10)NRNRScopolamine-induced dysmnesia modelNRWater extract (0. 01075 ml g-1, ig); onset the experiment; twice daily for 10 dNS (same volume, ig); onset the experiment; once daily for 10 d(1) SD test (escape latency)(1)P<0.05(2) SD test(number of errors)(2)P>0.05Wen et al., 2009ICR mice (mix, 10/10)20±2gNREthanol-induced dysmnesia modelNRWater extract (3 and 12 g kg-1, ig); onset the experiment; twice daily for 14 dNS (same volume, ig); onset the experiment; twice daily for 14 d(1) ST test (escape latency)(1)P<0.01ICR mice (mix, 10/10)20±2gNRNaNO2-induced dysmnesia modelNREssential oil (3 and 12 g kg-1, ig); onset the experiment; once daily for 14 dNS (same volume, ig); onset the experiment; twice daily for 14 d(2) EY-M test (number of errors)(1)P<0.05ICR mice (mix, 10/10)20±2gNRScopolamine-induced dysmnesia modelNRWater extract (3 and 12 g kg-1, ig); onset the experiment; once daily for 14 dNS (same volume, ig); onset the experiment; twice daily for 14 d(1) SD test (escape latency)(1)P<0.05(2) SD test (number of errors)(2)P<0.05ICR mice (mix, 10/10)20±2gNRScopolamine-induced dysmnesia modelNREssential oil (3 and 12 g kg-1, ig); onset the experiment; once daily for 14 dNS (same volume, ig); onset the experiment; twice daily for 14 d(1) SD test (escape latency)(1)P<0.05(2) SD test (number of errors)(2)P<0.01ICR mice (mix, 10/10)20±2gNRScopolamine-induced dysmnesia modelNRWater extract (3 and 12 g kg-1, ig); onset the experiment; once daily for 14 dNS (same volume, ig); onset the experiment; twice daily for 14 d(1) MWM test (escape latency)(1)P<0.01ICR mice (mix, 10/10)20±2gNRScopolamine-induced dysmnesia modelNREssential oil (3 and 12 g kg-1, ig); onset the experiment; once daily for 14 dNS (same volume, ig); onset the experiment; twice daily for 14 d(1) MWM test (escape latency)(1)P<0.01Yang et al. [37]SD rats (male, 12/12)250±30gNRAβ1-42-induced AD modelNRβ-Asarone (10, 20, and 30 mg kg-1, ig); after the model finished; twice daily for 28 dNS (same volume, ig); after the model finished; once daily for 28 d(1) MWM test (escape latency)(1)P<0.05(2) MWM test (number of platform crossing)(2)P<0.01(3) Astrocyte activity(3)P<0.01Zhou et al. [38]SD rats (male,10/10)200-250 gNRD-gal- and AlCl3-induced AD modelNRα-Asarone (10, 25 mg kg-1, ip); after the after model finished; once daily for 28 dNS (same volume, ip); after the model finished; once daily for 28 d(1) MWM test (number of platform crossing)(1)P<0.01(2) Aβ and tau protein expression(2)P<0.01(3) ACh levels(3)P<0.05(4) AChE levels(4)P>0.05(5) ChAT levels(5)P>0.05Jiang et al., 2007Kunming mice (mix, 10/10)18-20 gNRAlCl3-induced AD modelNRβ-Asarone (1.06, 2.12, and 4.24 mg 100 g-1, ig); after the model finished; once daily for 2 monthsNS (same volume, ig); after the model finished; once daily for 2 months(1) MWM test (number of errors)(1)P<0.01(2) SOD levels(2)P<0.01(3) MAD levels(3)P<0.01Huang et al. [40]FMR1gene knock mice (16/17)17-18 gNRFragile X syndrome modelNRα-Asarone (3, 6, 9, 12, 24 mg kg-1, ip); onset the experiment; once daily for 8 dNS (same volume, ip); onset the experiment; once daily for 8 d(1) SD test (number of errors)(1)P>0.05(2) P-Akt expression(2)P<0.05(3) Akt expression(3)P>0.05Wang BL et al., 2017SD rats (male, 15/15)280±20gRandom number tableAβ1-42-induced AD modelPhenytoin sodiumβ-Asarone (10, 20, and 30 mg kg-1, ig); after the model finished; once daily for 4 weeksNS (same volume, ig); after the model finished; once daily for 4 weeks(1) MWM test (escape latency)(1)P<0.01(2) MWM test (number of platform crossing)(2)P<0.01(3) HIF levels(3)P<0.05Guo et al. [42].Kunming mice (male, 11/11)25±5gRandom block allocation methodScopolamine-induced AD modelNRβ-Asarone (21.2 mg kg-1, ig); after the model finished; once daily for 14 dNS (same volume, ig); after the model finished; once daily for 14 d(1) MWM test (escape latency)(1)P<0.01Jiang et al. [43]Wistar rats (mix, 8/8)250-300 gNRSTZ-induced AD modelNREssential oil (5, 10 and 20 g kg-1, ig); onset the experiment; once daily for 20 dSolvent (same volume, ig); onset the experiment; once daily for 20 d(1) MWM test (escape latency)(1)P<0.01(2) SOD levels(2)P<0.01(3) MAD levels(3)P<0.01Yang et al. [44]Wistar rats (10/10)35±5gNRPTZ-induced epilepsy modelNRα-Asarone (29 mg kg-1, ig); after PTZ injection; twice daily for 7 dNS (same volume, ig); after PTZ injection; twice daily for 7 d(1) MWM test (number of platform crossing)(1)P<0.05(2) MWM test (time spent in target quadrant)(2)P<0.05Wistar rats (10/10)35±5gNRPTZ-induced epilepsy modelNRAGA (2.35 g kg-1, ig); after PTZ injection; twice daily for 7 dNS (same volume, ig); after PTZ injection; twice daily for 7 d(1) MWM test (number of platform crossing)(1)P<0.05(2) MWM test (time spent in target quadrant)(2)P<0.05Wang et al. [45]ICR mice (mix, 10/10)20±2gNRScopolamine-induced dysmnesia modelNREssential oil (100, 150, and 300 mg kg-1, ig); before the experiment; once daily for 7 dNS (same volume, ig); before the experiment; once daily for 7 d(1) MWM test (escape latency)(1)P<0.01(2) MWM test (number of platform crossing)(2)P<0.01(3) MWM test (time spent in target quadrant)(3)P<0.01Ma et al. [46]SD rats (male, 6/6)260-280 gNRAβ1-42-induced AD modelSodium pentobarbitalβ-Asarone (12.5, 25, 50 mg kg-1, ig); after the model finished; once daily for 4 weeksNS (same volume, ig); after the model finished; once daily for 4 weeks(1) MWM test (escape latency)(1)P<0.05(2) GA P-43 mRNA levels(2)P<0.05(3) SYP mRNA levels(3)P<0.05(4) PSD-95 mRNA levelsAch: acetylcholine; AchE: acetylcholinesterase; SD rats: Sprague-Dawley rats; NIH mice: National Institutes of Health mice; SAMP8 mice: senescence-accelerated mouseprone 8 mice; AD: Alzheimer’s disease; AlCl3: aluminum trichloride; ChAT: acetylcholine transferase; D-gal: D-galactose; i.g.: intragastrical injection; i.p.: intraperitoneal injection; i.h.: hypodermic injection; MWM test: Morris water maze test; MCAO: middle cerebral artery occlusion; MDA: malondialdehyde; GSH-PX: glutathione peroxidase; NR: not report; SD test: step down test; STZ: streptozotocin; SOD: superoxide dismute; HIF: hypoxia-inducible factor; GSH-Px: glutathione peroxidase; NE: norepinephrine; 5-TH: 5-hydroxytryptamine; DA: dopamine; SYN/SYN: synaptophysin; NOS: nitric oxide synthase; Bcl-2: B-cell lymphoma/leukemia-2; MAP2: microtubule-associated protein 2; RAM: radial eight-arm maze; EY-M: electric Y-maze; Aβ1-42: amyloid beta 1-42; PTZ: pent ylenetet razol; NS: normal saline.


### 3.3. Study Quality

The quality scores of the 34 included studies varied from 1/10 to 6/10 with the average of 3.32. One study [40] got 1 point; 11 studies [29, 31–36, 38, 39, 42, 44] got 2 points; 9 studies [5, 6, 18, 24, 25, 27, 30, 43, 45] got 3 points; 4 studies got 4 points; 7 studies got 5 points; and 2 studies [20, 37] got 6 points. Thirty-four studies were published. Sixteen studies described control of temperature [6, 10, 17–26, 30, 37, 41, 45]. Random allocation was declared in 28 studies [5, 6, 11, 17, 19–23, 26–28, 30–39, 41–46]; 1 study [42] used random block allocation method, and 2 studies used the method of random digit table [34, 41]. Two studies [23, 37] described the use of blinded assessment of outcome. Thirteen studies did not use anesthetics with significant intrinsic neuroprotective activity, and the remaining 21 studies did not report the type of anesthetics [5, 6, 18, 19, 24, 27, 30–40, 42–45]. Sixteen studies reported compliance with animal welfare regulations [5, 10, 11, 17–22, 24, 27, 28, 37, 41, 43, 45]. Four studies mentioned statement of potential conflict of interests [11, 20, 28, 37]. None of the included studies reported allocation concealment, sample size calculation, and the utilization of animal or model with relevant comorbidities. The quality scores for the included studies are shown in Table 2.Table 2
Quality assessment of included studies.
Study (years)12345678910TotalYang et al. [17]√√√√√5Wei et al., 2013√√√3Sundaramahalingam et al. [18]√√√3Shin et al. [19]√√√√4Ma et al. [11]√√√√√5Liu et al. [20]√√√√√√6Limón et al. [21]√√√√√5Li et al. [22]√√√√√5Zhang et al. [5]√√√3Lee et al. [10]√√√√4Lee et al. [23]√√√√√5Kumar et al., 2012√√√3Kim et al. [25]√√√3Geng et al. [26]√√√√4Chen et al. [27]√√√3Ma et al. [28]√√√√√5Tian et al. [29]√√2Zhou et al. [30]√√√3Wang GM et al., 2017√√2Hu et al. [32]√√2Chen et al. [33]√√2Gu et al. [34]√√2Wu et al., 2004√√2Wen et al., 2009√√2Yang et al. [37]√√√√√√6Zhou et al. [38]√√2Jiang et al., 2007√√2Huang et al. [40]√1Wang BL et al., 2017√√√√√5Guo et al. [42]√√2Jiang et al. [43]√√√3Yang et al. [44]√√2Wang et al. [45]√√√3Ma et al. [46]√√√√41: peer-reviewed publication; 2: statements describing control of temperature; 3: randomization to treatment group; 4: allocation concealment; 5: blinded assessment of outcome; 6: avoidance of anesthetics with known notable intrinsic neuroprotective properties; 7: use of animals with relevant comorbidities; 8: sample size calculation; 9: compliance with animal welfare regulations; 10: declared any potential conflict of interest.


### 3.4. Effectiveness

The Morris water maze test, including the probe test and the spatial test, was conducted in 28 studies [6, 10, 11, 17, 19, 20, 22, 23, 25–31, 33–39, 41–46]. Twenty-seven studies reported the spatial test using the escape latency as an outcome measure. Meta-analysis of 20 studies with 27 comparisons showed EAAGA significantly decreased the escape latency compared with the control (n=490, SMD=−1.09, 95% CI [−1.37 to −0.82], P<0.00001; heterogeneity: χ2=49.48, df=26P=0.004; I2=47%; Figure 2(a)). In the probe test, meta-analysis of 16 studies [17, 19, 20, 22, 26, 27, 29–31, 33, 34, 37, 38, 41, 44, 45] with 19 comparisons showed EAAGA were significant for increasing number of platform crossings (n=398, SMD=1.60, 95% CI [1.25 to 1.94], P<0.00001; heterogeneity: χ2=34.29, df=18P=0.01; I2=47%; Figure 2(b)) compared with controls. Meta-analysis of 6 studies [17, 20, 22, 29, 34, 44] with 7 comparisons showed a significant effect of EAAGA in increasing the length of time spent in platform quadrant compared with control (n=144, SMD=1.78, 95% CI [0.90 to 2.67], P<0.0001; heterogeneity: χ2=22.41, df=6P=0.001; I2=73%). As the values of I2 were greater than 50%, we sequentially omitting each study; two studies [20, 22] were removed and markedly reduced the heterogeneity (n=86, SMD=2.34, 95% CI [1.55 to 3.12], P<0.00001; heterogeneity: χ2=6.57, df=4P=0.16; I2=39%; Figure 2(c)). Two studies [20, 22] used relatively large doses of β-asarone that might have potential toxic effects [47]. Meta-analysis of 3 studies [20, 23, 25] for increasing percentage of time in the platform quadrant (n=44, SMD=4.01, 95% CI [2.86 to 5.15], P<0.00001; heterogeneity: χ2=0.03, df=2P=0.98; I2=0%; Figure 2(d)). Three studies [17, 22, 23] showed there were not a significant difference in improving the swimming velocity compared with controls.Figure 2
The forest plot in Morris water maze test. Effects of EAAGA for decreasing the escape latency (a) in spatial test, increasing crossing numbers (b), increasing exact time (c), and increasing percentage of time (d) in platform quadrant in probe test compared with control group.
(a)(b)(c)(d)The step-down test, including the training test which represents learning ability and retention test which represents memory ability, was conducted in 6 studies [5, 32, 34–36, 40]. Meta-analysis of 5 studies with 19 comparisons showed EAAGA were significant for increasing right reaction latency in the retention test (n=396, SMD=1.15, 95% CI [0.87 to 1.43], P<0.00001; heterogeneity: χ2=28.98, df=18P=0.05; I2=38%; Figure 3(a)) and 1 study [5] for increasing right reaction latency (P<0.05) in the training test. Meta-analysis of 3 studies [32, 35, 36] with 16 comparisons showed EAAGA were significant for decreasing the error times (n=336, SMD=−1.06, 95% CI [−1.30 to −0.83], P<0.00001; heterogeneity: χ2=15.01, df=15P=0.45; I2=0%; Figure 3(b)) in the retention test and 1 study [5] for decreasing the error times (P<0.05) in the training test.Figure 3
The forest plot in Step-down test. Effects of EAAGA for increasing right reaction latency in the retention test (a) and decreasing the error times in the retention test (b) compared with control group.
(a)(b)The electrical Y-maze test was conducted in 3 studies [5, 32, 36]. Meta-analysis of 3 studies showed EAAGA were significant for decreasing error reaction times (n=168, SMD=−1.22, 95% CI [−1.56 to −0.88], P<0.00001; heterogeneity: χ2=3.95, df=7P=0.79; I2=0%; Figure 4).Figure 4
The forest plot in Electrical Y-maze test. Effects of EAAGA for decreasing error reaction times compared with control group.The step-through test was conducted in 4 studies [24, 34–36]. Meta-analysis of 4 studies with 7 comparisons showed EAAGA were significant for decreasing latency in the retention test (n = 134, SMD = 1.26, 95% CI [0.81 to 1.71], P < 0.00001; heterogeneity: χ2 = 8.09, df =6 (P = 0.23); I2=26%; Figure 5(a)) and 2 studies [35, 36] with 5 comparisons showed EAAGA significantly decreased the number of errors in the retention test (n=100, SMD=−1.02, 95% CI [−1.45 to −0.60], P<0.00001; heterogeneity: χ2=1.05, df=4P=0.90; I2=0%; Figure 5(b)) compared with controls.Figure 5
The forest plot in Step-through test. Effects of EAAGA for decreasing latency in the retention test (a) and decreasing the number of errors in the retention test (b) compared with control group.
(a)(b)The eight-arm maze test was conducted in 3 studies [10, 18, 21]. Meta-analysis of 2 studies [10, 21] showed EAAGA were significant for increasing number of correct choices (n=25, SMD=1.15, 95% CI [0.00 to 2.29], P=0.05; heterogeneity: χ2=1.63, df=1P=0.20; I2=38%; Figure 6(a)) and 2 studies [10, 18] with 3comparisons showed EAAGA significantly decreased the number of errors in the training test (n=33, SMD=−2.36, 95% CI [−3.36 to −1.37], P<0.00001; heterogeneity: χ2=1.00, df=2P=0.61; I2=0%; Figure 6(b)) compared with controls.Figure 6
The forest plot in Eight-arm maze test. Effects of EAAGA for increasing correct choices (a) and decreasing the number of errors (b) compared with control group.
(a)(b)


### 3.5. Neuroprotective Mechanisms

The mechanisms of neuroprotection of EAAGA on cognitive impairment were studied in 34 included articles [5, 6, 10, 11, 17–46] as follows: (1) reduction of oxidative reactions by increasing the activity of SOD [30, 35, 39, 41, 43] activity, while decreasing the activity of SOD and AChE [18, 24], decreasing the levels of MDA [24, 30, 33] and nitric oxide [21], decreasing the mRNA levels of hsp 70, increasing the levels of VC, VE, and GSH, and increasing the activity of CAT and G6PD [18]; (2) inhibition of apoptosis by increasing the mRNA levels of Bcl-2, BDNF, CREB [6, 23, 42], Bcl-w and Bcl-2 [26], and c-jun [35], decreasing the mRNA levels of Bax [23], increasing the expression of BDNF, CREB [23], Bcl-w, and Bcl-2 [26], decreasing the expression of caspase-3, p-JNK [26], and BACE1 [19], and preventing cell loss [10], Aβ, and Tau protein [38]; (3) repression of inflammatory reactions by decreasing the expression of TNF-α and IL-1β mRNA levels [19]; (4) repression of autophagy by decreasing LC3, ROCK, and beclin1 expression and increasing p62, GAP43, MAP2, and SYN expression [27]; (5) protection of cerebrovascular by increasing rCBF and the Na-K-ATP activity, decreasing pyruvic acid contents, and decreasing the mRNA levels of ET-1, eNOS, and APP [22]; (6) promotion of cognitive function by increasing the levels of 5-HT, NE, DA, and NE [5] and suppression of astrocyte activation [37]; (7) stimulation of cholinergic system by increasing AchE and ChAT neurons [25]; (8) improvement of memory impairments through regulation of synaptogenesis, which is mediated via Arc/Arg3.1 and Wnt pathway [17]; (9) neuroprotection through damage of Akt pathway [40]; (10) inhibition of neurotoxicity by decreasing the expression of DCx and nestin, decreasing nestin positive cells [11], decreasing Aβ plaques depositions, and decreasing NOS activity [29]; (11) regulation of synaptic plasticity by increasing the expression of SYP and GluR1 [20, 46] and decreasing the expression of GAP-43 and PSD-95 [46]; and (12) inhibition of chronic stress by decreasing plasma cortisol levels [41]. Characteristics of mechanism studies of EAAGA on experimental ischemic stroke are shown in Table 3 and Figure 7.Table 3
Characteristics of mechanism studies of EAAGA on cognition impairment.
Study (years)ModelMethod of administration (experimental group versus control group)ObservationsPossible mechanismsYang et al. [17]Chronic lead-induced dysmnesia modelβ-Asarone versus distilled waterAttenuated memory deficitsArc/Arg3.1 and Wnt pathwayIncreased dendritic spine densityIncreased dendritic spine densityUp-regulated NR2B, Arc/Arg3.1, and Wnt7a protein expressionWei et al., 2013AβPP/PS1 double-transgenic miceβ-Asarone versus Tween 80Improved cognitive functionCaMKII/CREB/Bcl-2 signaling pathwayPrevents PC12 cell and cortical neuron damageInhibition of apoptosisInhibited the apoptosis of PC12 cells and cortical neuronsSundaramahalingam et al. [18]Noise stress induced memory impairment modelα-Asarone versus Tween 80Prevent memory impairmentReduction of oxidative reactionsDecreased hsp 70 mRNA levelsDecreased SOD and AChE activityIncreased CAT and G6PD activityIncreased VC, VE, and GSH levelsShin et al. [19]LPS-induced cognitive handicap modeα-Asarone versus NSAmeliorated memory deficitsRepression of inflammatory reactionsReduced Iba1 protein expressionInhibition of apoptosisReduced TNF-α and IL-1β mRNAReduced BACE1 expressionIncreased CA1 neuronsReduced TUNEL-labeled cellsMa et al. [11]Aβ1-42-induced AD modelWater extract versus NSAmeliorated memory deficitsInhibition of neurotoxicityEssential oil versus NSReduced Aβ positive cellsDefatted decoction versus NSDecreased DCx and nestin expressionDecreased nestin positive cellsLiu et al. [20]APPswe/PS1dE9 double transgenic miceβ-Asarone versus Tween 80Improved the learning and memory abilityRegulation of synaptic plasticityIncreased SYP and GluR1 expressionLimón et al. [21]Aβ-induced AD modelα-Asarone versus NSAmeliorated memory deficitsReduction of oxidative reactionsDecreased NO levelsLi et al. [22]D-gal- and AlCl3-induced AD modelβ-Asarone versus NSImproved the learning and memory abilityProtection of cerebrovascularIncreased rCBF and the activity of Na–K-ATPDecreased pyruvic acid contentsDecreased ET-1, eNOS, and APP mRNA expressionZhang et al. [5]Aged miceEssential oil versus Tween 80Improved cognitive functionIncreased 5-HT, NE, DA, and NE levelsDecreased AChE activityImprovement of cognitive functionScopolamine-induced dysmnesia modelEssential oil versus Tween 80Ethanol-induced dysmnesia modelEssential oil versus Tween 80Aged ratsEssential oil versus Tween 80Sodium nitrite-induced dysmnesia modelEssential oil versus Tween 80Lee et al. [10]MCAO/2 h-induced cognitive impairments modelAGA versus NSAttenuated learning and memory deficitsIncreased cell densityInhibition of apoptosisLee et al. [23]Chronic corticosterone exposedβ-Asarone versus NSImproved cognitive functionIncreased BDNF and CREB expressionIncreased BDNF, CREB, and Bcl-2 mRNAs levelsDecreased Bax mRNAs levelsDecreased serum levels of CORTInhibition of apoptosisKumar et al., 2012Scopolamine-induced amnesic modelα-Asarone versus vehicleImproved cognitive functionIncreased of AchE activityInhibition MDA expression and SOD levelsReduced SOD activityReduction of oxidative reactionsKim et al. [25]Ibotenic acid-induced amnesiaAGA versus NSAmeliorated learning and memory deficitsIncreased ChAT positive neuronsIncreased AchE neuronsStimulation of cholinergic systemGeng et al. [26]Aβ1-42-induced AD rat modelβ-Asarone versus NSAmeliorated learning and memory deficitsIncreased Bcl-2, Bcl-w expressionIncreased Bcl-2 and Bcl-w mRNA levelsDecreased cleavage of caspase-3Reduced caspase-3 mRNA levelsDecreased p-JNK expressionInhibition of apoptosisChen et al. [27]SAMP8 miceβ-Asarone versus NSImproved cognitive functionReduced ROCK, beclin1, and LC3 expressionIncreased p62 expressionIncreased GAP43, MAP2, and SYN expressionIncreased GAP43-, MAP2-, and SYN-positive cellsDecreased lipofuscin-positive cellsReduction of autophagyMa et al. [28]Aβ-induced AD modelWater extract versus NSAmeliorated learning and memory deficitsDecreased Aβ plaques depositionsImprovement of cognitive functionWater extract without oil versus NSEssential oil versus NSTian et al. [29]Aβ-induced AD modelWater extract versus NSAmeliorated learning and memory deficitsDecreased NOS activityInhibition of neurotoxicityDefatted decoction versus NSEssential oil versus NSZhou et al. [30]Scopola-induced AD modelEssential oil versus NSAmeliorated learning and memory deficitsDecreased GFAP expressionDecreased MDA levelsIncreased SOD levelsReduction of oxidative reactionsWang GM et al., 2017Chronic restraint stress-induced cognitive impairments modeEssential oil versus NSAmeliorated learning and memory deficitsIncreased body massDecreased plasma cortisol levelsInhibition of chronic stressHu et al. [32]Sodium nitrite-induced amnesic modelEssential oil versus Tween 80Increased learning and memory deficitsImprovement of cognitive functionDefatted decoction versus Tween 80α-Asarone versus Tween 80β-Asarone versus Tween 80Ethanol-induced amnesic modelEssential oil versus Tween 80Ameliorated learning and memory deficitsImprovement of cognitive functionDefatted decoction versus Tween 80α-Asarone versus Tween 80β-Asarone versus Tween 80Chen et al. [33]D-galactose-induced dementia modelWater extract versus distilled waterAmeliorated memory deficitsDecreased MDA levelsIncreased SOD activityReduction of oxidative reactionsGu et al. [34]Scopolamine-induced dysmnesia miceWater extract versus NSAmeliorated memory deficitsImprovement of cognitive functionNaNO2-induced dysmnesia modelWater extract versus NSAmeliorated memory deficits45% ethanol-induced dysmnesia miceWater extract versus NSAmeliorated memory deficitsThe AchE activity of mice brain was not influencedScopolamine-induced dysmnesia ratWater extract versus NSAmeliorated memory deficitsWu et al., 2004Aged miceEssential oil versus NSAmeliorated memory deficitsDecreased AChE activityIncreased c-jun mRNA levelsInhibition of apoptosisAged miceβ-Asarone versus NSAged miceWater extract versus NSEthanol-induced dysmnesia modelWater extract versus NSNaNO2-induced dysmnesia modelEssential oil versus NSNaNO2-induced dysmnesia modelβ-Asarone versus NSNaNO2-induced dysmnesia modelWater extract versus NSAmeliorated memory deficitsScopolamine-induced dysmnesia modelEssential oil versus NSScopolamine-induced dysmnesia modelEssential oil versus NSScopolamine-induced dysmnesia modelWater extract versus NSWen et al., 2009Ethanol-induced dysmnesia modelWater extract versus NSAmeliorated memory deficitsInhibition of apoptosisNaNO2-induced dysmnesia modelEssential oil versus NSScopolamine-induced dysmnesia modelWater extract versus NSScopolamine-induced dysmnesia modelEssential oil versus NSScopolamine-induced dysmnesia modelWater extract versus NSScopolamine-induced dysmnesia modelEssential oil versus NSYang et al. [37]Aβ1-42-induced AD modelβ-Asarone versus NSImproved cognitive functionInhibited AQP4, IL-1β, and TNF-α expressionDecreased Aβ depositionAlleviated hippocampal damageSuppression of astrocyte activationZhou et al. [38]D-gal- and AlCl3-induced AD modelα-Asarone versus NSImproved cognitive functionDecreased Aβ and Tau protein expressionIncreased ACh expressionInhibition of apoptosisJiang et al. 2007AlCl3-induced AD modelβ-Asarone versus NSImproved cognitive functionIncreased SOD and GSH-Px levelsDecreased MAD levelsReduction of oxidative reactionsHuang et al. [40]Fragile X syndrome modelα-Asarone versus NSImproved cognitive functionDamage of Akt pathwayWang BL et al., 2017Aβ1-42-induced AD modelβ-Asarone versus NSImproved cognitive functionDecreased HIF and MDA levelsIncreased SOD and CAT levelsReduction of oxidative reactionsGuo et al. [42]Scopolamine-induced AD modelβ-Asarone versus NSImproved cognitive functionInhibition of apoptosisJiang et al. [43]STZ-induced AD modelEssential oil versus solventImproved cognitive functionDecreased MDA levelsIncreased SOD levelsReduction of oxidative reactionsYang et al. [44]PTZ-induced epilepsy modelAGA versus NSImproved cognitive functionInhibition of apoptosisPTZ-induced epilepsy modelα-Asarone versus NSImproved cognitive functionInhibition of apoptosisWang et al. [45]Aβ1-42-induced AD modelEssential oil versus NSImproved cognitive functionImprovement of cognitive functionMa et al. [46]D-gal- and AlCl3-induced AD modelβ-Asarone versus NSImproved cognitive functionDecreased GA P-43 mRNA levelsIncreased SYP mRNA levelsDecreased PSD-95 mRNA levelsRegulation of synaptic plasticityAch: acetylcholine; AchE: acetylcholinesterase; AD: Alzheimer’s disease; AlCl3: aluminum trichloride; ChAT: acetylcholine transferase; D-gal: D-galactose; MCAO: middle cerebral artery occlusion; MDA: malondialdehyde; STZ: streptozotocin; SOD: superoxide dismute; HIF: hypoxia-inducible factor; SYN/SYN: synaptophysin; MAP2: microtubule-associated protein 2; Aβ1-42: amyloid beta 1-42; NS: normal saline; PTZ: pent ylenetet razol; GSH-Px: glutathione peroxidase; NE: norepinephrine; 5-TH: 5-hydroxytryptamine; DA: dopamine; NOS: nitric oxide synthase; Bcl-2: B-cell lymphoma/leukemia-2.Figure 7
A schematic representation of possible mechanisms of EAAGA for improving learning and memory function. The possible mechanisms of different active ingredients are as follows: (1) AGA: the dry rhizomes of Acorus gramineus Solander can inhibit apoptosis and stimulate cholinergic system. (2) Essential oil: AGA contains up to 4.86% essential oil, which displayed antioxidation effects by decreasing the levels of MDA and increasing the levels of SOD, exhibited anticytotoxicity effects via decreasing NOS activity, exerted antineurotoxicity effects by decreasing Aβ plaques depositions, and improved cognitive function by decreasing the activity of AChE. (3) β-Asarone: a major component of essential oil (63.2–81.2%) displayed antioxidation effects by decreasing the levels of MDA and HIF, increasing the levels of SOD, CAT, and GSH-Px; exerted antiapoptotic activity through regulating CaMKII/CREB/Bcl-2 signaling pathway and decreasing the levels of Bax mRNAs, caspase-3 mRNA, and JNK; inhibited synaptic loss through reducing ROCK expression; mediated synaptogenesis via Arc/Arg3.1 and Wnt pathway; improved circulation by decreasing the activity of AChE; and exerted antineurotoxicity by decreasing Aβ plaques depositions. (4) α-Asarone: another major component of essential oil (8.8–13.7%) exerted antioxidation effects by increasing CAT, SOD, and GSH-Px.; displayed anti-inflammatory activity through reducing the expression of proinflammatory mediators; improved circulation via decreasing the activity of AChE; and exerted antineurotoxicity by decreasing Aβ plaques depositions. (5) Water extract: displayed antioxidation effects by decreasing the levels of MDA and increasing the levels of SOD, exerted antineurotoxicity by decreasing Aβ plaques depositions; and improved cognitive function by decreasing the activity of AChE. (6) Defatted decoction: exerted antineurotoxicity by decreasing Aβ plaques depositions and displayed anticytotoxicity effects via decreasing the activity of NOS.


## 3.1. Study Selection

We identified 2368 potentially relevant papers after systematical search from six databases. After removing duplicates, 1887 studies remained. By reading titles and abstracts, 1602 articles were excluded that were reviews, case reports, comments, abstracts, clinical trials, letters, or editorials. After reading the remaining 285 full-text articles, 228 studies were excluded for at least one of following reasons: (1) not an animal study; (2) the article was not a research about cognitive impairment; (3) the study did not access the effects of AGA or active component on the animal model of cognitive impairment; (4) EAAGA was not used as a monotherapy; and (5) lack of control group. Ultimately, 34 eligible articles [5, 6, 10, 11, 17–46] were selected (Figure 1).Figure 1
Flow diagram.


## 3.2. Characteristics of Included Studies

Sixteen studies [5, 6, 10, 11, 17–27, 37] were published in English, and 18 studies were in Chinese between 1999 and 2019. In total, 34 studies with 1431 animals were included. Ten species were referred, including Sprague-Dawley (SD) rat (n=236, 16.49%), Wistar rats (n=130, 9.08%), Kunming mice (n=530, 37.04%), ICR mice (n=236, 16.49%), NIH mice (n=168, 11.74%), AβPP/PS1 double-transgenic mice (n=26, 1.82%); APPswe/PS1dE9 double transgenic mice (n=22, 1.54%), C57BL/6 mice (n=24, 1.68%), senescence-accelerated prone-8 (SAMP8) mice (n=26, 1.82%), and FMR1gene knock mice (n=33, 2.31%). The weight of SD rats ranged from 200 g to 650 g, the weight of Wistar rats used ranged from 30 g to 250 g, and the weight of mice ranged from 17 g to 50 g. Twenty-two studies used male rodents, 1 study used female rodents, 5 study used both female and male rodents, and the remaining 6 studies did not provide gender details. Sodium pentobarbital was used to induce anesthesia in 8 studies, and chloral hydrate was used in 2 studies [20, 21], 1 study [41] used phenytoin sodium, 1 study [17] used CO2, and 1 study [10] used isoflurane, while the remaining 21 studies did not report the type of anesthetics. Cognitive impairment models were induced by lead [17], noise stress [18], LPS [19], amyloid beta 1-42 [11, 21, 26, 28, 29, 37, 41, 46], D-gal plus AlCl3 [22], scopolamine [5, 24, 30, 34–36, 42, 45], ethanol [5, 32, 34–36], sodium nitrite [5, 32], corticosterone [23], Ibotenic acid [25], chronic restraint stress [31], pentobarbital sodium [32], D-galactose [33, 38], AlCl3 [40], streptozotocin (STZ) [43], pent ylenetet razol (PTZ) [44], and NaNO2 [34–36]. As an intervention, fourteen studies [6, 17, 20, 22, 23, 26, 27, 32, 35, 37, 39, 41, 42, 46] used β-asarone, eight studies [18, 19, 21, 24, 33, 38, 40, 44] used α-asarone, three studies [10, 25, 44] utilized AGA, twelve studies [5, 11, 22, 28–32, 35, 36, 43, 45] used essential oil, seven studies [11, 28, 29, 33–36] researched water extract, four studies [11, 28, 29, 32] used defatted decoction, and one study [18] researched ethyl acetate extract. Normal distilled water control was used in 2 studies [17, 33]; Tween 80 control was used in 6 studies [5, 6, 18, 20, 27, 32]; normal saline control was used in 24 studies; 0.5% methylcellulose solution containing 1% Tween 80 control was used in 1 study [24], and 2% propylene glycol containing 2% polyethylene glycol stearate control was used in 1 study [43]. Neurobehavioral function indices as primary outcome measures were carried out by the Morris water maze test (MWM test) (n=28), step-down test (SD test) (n=6), electrical Y-maze test (EY-M test) (n=3), step-through test (ST test) (n=4), and radial eight-arm maze test (RAM test) (n=3). The characteristics of the 34 studies are shown in Table 1.Table 1
Basic characteristics of the included studies.
Study (years)Species (sex,n=experimental/controlgroup)WeightRandom methodModel (method)AnestheticMethod of administrationOutcome index (time)Intergroup differencesExperimental groupControl groupYang et al. [17]SD rats (mix, 7/7)NRNRChronic lead-induced dysmnesia modelCO2β-Asarone (2.5, 10, and 40 mg kg-1, ip); from 9 to 11 weeks old; once daily for 3 weeksDistilled water (same volume, ip); from 9 to 11 weeks old; once daily for 3 weeks(1) MWM test (escape latency)(1)P<0.001(2) MWM test (swimming speed)(2)P>0.05(3) MWM test (time spent in target quadrant)(3)P<0.05(4) MWM test (times crossed the platform)(4)P>0.05(5) Dendritic spine density(5)P<0.001Wei et al., 2013AβPP/PS1 double-transgenic mice (13/13)NRNRAβPP/PS1 double-transgenic miceNRβ-Asarone (7 and 21 mg kg-1, ig); onset the experiment; once daily for 4 monthsTween 80 (same volume, ig); onset the experiment; once daily for 4 months(1) MWM test (escape latency)(1)P<0.001(2) Cell viability(2)P<0.05Sundaramahalingam et al. [18]Wister strain albion rats (male, 6/6)200-220 gNRNoise stress induced memory impairment modelNRα-Asarone (9 mg kg-1, ip); onset the experiment; once daily for 30 dTween 80 (same volume, ip); onset the experiment; once daily for 30 d(1) RAM test (number of errors)(1)P<0.05(2) Hsp 70 mRNA levels(2)P<0.05(3) Ache activity(3)P<0.05(4) SOD/CAT/GPx activity(4)P<0.05(5) VC/VE/GSH levels(5)P<0.05(6) G6PD activity(6)P<0.05Wister strain albion rats (male, 6/6)200-220 gNRNoise stress exposed ratsNREthyl acetate extract (50 mg kg-1, ip); onset the experiment; once daily for 30 dTween 80 (same volume, ip); onset the experiment; once daily for 30 d(1) RAM test (number of errors)(1)P<0.05(2) Hsp 70 mRNA levels(2)P<0.05(3) Ache activity(3)P<0.05(4) SOD/CAT/GPx activity(4)P<0.05(5) VC/VE/GSH levels(5)P<0.05(6) G6PD activity(6)P<0.05Shin et al. [19]C57BL/6 mice (male, 12/12)25-28 gNRLPS-induced cognitive handicap modelNRα-Asarone (7.5, 15, and 30 mg kg-1, ig); 3 days before the LPS injection; once daily for 3 dNormal saline (same volume, ig); 3 days before the LPS injection; once daily for 3 d(1) MWM test (escape latency)(1)P<0.05(2) MWM test (times crossed the platform)(2)P<0.05(3) TNF-α/IL-1β mRNA levels(3)P<0.05(4) CA1 neurons count(4)P<0.05(5) TUNEL-labeled cells count(5)P<0.05(6) BACE1/Iba1 protein expressions(6)P<0.05Ma et al. [11]Six-week-old NIH mice (male, 6/6)20-25 gNRAβ1-42-induced AD modelSodium pentobarbitalWater extract (20 mg g-1, ig); after the first MWM test; once daily for 3 weeksNormal saline; (same volume, ig); after the first MWM test; once daily for 3 weeks(1) MWM test (escape latency)(1)P<0.05(2) Aβ positive cells count(2)P<0.05(3) DCx expression3P<0.05(4) Nestin positive cells count(4)P<0.05Six-week-old NIH mice (male, 6/6)20-25 gNRAβ1-42-induced AD modelSodium pentobarbitalEssential oil (20 mg g-1, ig); after the first MWM test; once daily for 3 weeksNormal saline; (same volume, ig); after the first MWM test; once daily for 3 weeks(1) MWM test (escape latency)(1)P<0.05(2) Aβ positive cells count(2)P<0.05(3) DCx expression(3)P<0.05(4) Nestin positive cells count(4)P<0.05Six-week-old NIH mice (male, 6/6)20-25 gNRAβ1-42-induced AD modelSodium pentobarbitalDefatted decoction (20 mg g-1, ig); after the first MWM test; once daily for 3 weeksNormal saline; (same volume, ig); after the first MWM test; once daily for 3 weeks(1) MWM test (escape latency)(1)P<0.05(2) Aβ positive cells count(2)P<0.05(3) DCx expression(3)P<0.05(4) Nestin positive cells count(4)P<0.05Liu et al. [20]APPswe/PS1dE9 double transgenic mice (male, 11/11)NRNRAPPswe/PS1dE9 double transgenic miceChloral hydrateβ-Asarone (21.2, 42.4, and 84.8 mg kg-1, ig); onset the experiment; once daily for 2.5 monthsTween 80 (same volume, ig); onset the experiment; once daily for 2.5 months(1) MWM test (escape latency)(1)P<0.05(2) MWM test (time spent in target quadrant)(2)P<0.05(3) MWM test (times crossed the platform)(3)P<0.05(4) SYP/GluR1 expression(4)P<0.05Limón et al. [21]Wistar rats (male, 8/8)230–250 gNRAβ1-42-induced AD modelChloral hydrateα-Asarone (10 mg kg-1, i.h.); after injection of amyloid-β; once daily for 16 dNormal saline; (same volume, ig); after injection of amyloid-β; once daily for 16 d(1) RAM test (percentage of correct responses)(1)P<0.001(2) Nitrite levels(2)P<0.05Li et al. [22]Wistar rats (female, 7/7)150–180 gNRD-gal and AlCl3 induced AD modelSodium pentobarbitalβ-Asarone (25, 50 and 100 mg kg-1, i.h.); 28 d after injection of AlCl3 and D-gal; once daily for 14 dNormal saline; (same volume, i.h); after the first MWM test; once daily for 14 d(1) MWM test (escape latency)(1)P<0.05(2) MWM test (time spent in target quadrant)(2)P<0.05(3) MWM test (times crossed the platform)(3)P<0.05(4) MWM test (swimming speed)(4)P>0.05(5) ET-1, eNOS, and APP expression(5)P<0.05(6) Lactic acid and pyruvic acid content(6)P<0.05(7) Na+- K+ ATPase activity(7)P<0.05(8) rCBF(8)P<0.05Zhang et al. [5]Aged Kunming mice (male, 10/10)40-50 gNRAged miceNREssential oil (0.02, 0.04, and 0.08 g kg-1, orally); onset the experiment; once daily for 15 dTween 80 (same volume, orally); onset the experiment; once daily for 15 d(1) SD test (escape latency)(1)P<0.01(2) SD test (number of errors)(2)P<0.05Aged Kunming mice (male, 10/10)40-50 gNRScopolamine-induced dysmnesia modelNREssential oil (0.02, 0.04, and 0.08 g kg-1, orally); onset the experiment; once daily for 15 dTween 80 (same volume, orally); onset the experiment; once daily for 15 d(1) SD test (escape latency)(1)P<0.05(2) SD test (number of errors)(2)P<0.01Aged Kunming mice (male, 10/10)40-50 gNREthanol-induced dysmnesia modelNREssential oil (0.02, 0.04, and 0.08 g kg-1, orally); onset the experiment; once daily for 15 dTween 80 (same volume, orally); onset the experiment; once daily for 15 d(1) SD test (escape latency)(1)P<0.01(2) SD test (number of errors)(2)P<0.01Aged SD rats (male, 10/10)550-650 gNRAged ratsNREssential oil (0.02, 0.04, and 0.08 g kg-1, orally); onset the experiment; once daily for 30 dTween 80 (same volume, orally); onset the experiment; once daily for 30 d(1) EY-M test (number of errors)(1)P<0.01(2) NE, DA and 5-HT level(2)P<0.01(3) AChE activity(3)P<0.01(4)P<0.01Aged SD rats (male, 10/10)550-650 gNRSodium nitrite-induced dysmnesia modelNREssential oil (0.02, 0.04, and 0.08 g kg-1, orally); onset the experiment; once daily for 30 dTween 80 (same volume, orally); onset the experiment; once daily for 30 d(1) EY-M test (number of errors)(1)P<0.05(2)P<0.01Lee et al. [10]SD rats (male, 5/7)250-280 gNRMCAO-induced cognitive impairments modelIsofluraneAGA (100 mg kg-1, po); after occlusion; once daily for 21 dNormal saline; (same volume, i.h); after occlusion; once daily for 21 d(1) MWM test (escape latency)(1)P<0.05(2) Cell density(2)P<0.05Lee et al. [23]SD rats (male, 7/7)200-220 gNRChronic corticosterone-exposed modelSodium pentobarbitalβ-Asarone (50, 100, and 200 mg kg-1, ip); 30 min prior to the CORT; once daily for 21 dNormal saline; (same volume, ip); 30 min prior to the CORT; once daily for 21 d(1) MWM test (swimming speed)(1)P>0.05(2) serum CORT levels(2)P<0.05(3) BDNF and CREB expression(3)P<0.05(4) Bax and Bcl-2 mRNAs expression(4)P<0.05Kumar et al. 2012ICR mice (8/8)NRNRScopolamine-induced amnesic model modeNRα-Asarone (3, 10, and 30 mg kg-1, po); 15 d before scopolamine injection; once daily for 15 d0.5% methylcellulose solution containing 1% Tween 80 (same volume, po); 15 d before scopolamine injection; once daily for 15 d(1) SD test (escape latency)(1)P<0.01(2) AchE activity(2)P<0.001(3) MDA levels(3)P<0.001(4) SOD activity(4)P<0.05Kim et al. [25]SD rats (male, 5/5)260–280 gNRIbotenic acid-induced amnesiaSodium pentobarbitalAGA (100 mg kg-1,ip); after surgery; once daily for 3 weeksSaline (same volume, ip); after surgery; once daily for 3 weeks(1) MWM test (escape latency)(1)P<0.001(2) ChAT positive neurons count(2)P>0.05(3) AchE neurons density(3)P<0.05Geng et al. [26]SD rats (male, 20/20)220-240 gNRAβ1-42-induced AD modelSodium pentobarbitalβ-Asarone (12.5, 25, or 50 mg kg-1, ig); 3 d after Aβ (1-42) hippocampus injection; once daily for 28 dSaline (same volume, ip); 3 d after Aβ (1-42) hippocampus injection; once daily for 28 d(1) MWM test (escape latency)(1)P<0.05(2) MWM test (times crossed the platform)(2)P<0.05(3) Annexin V-positive cells(3)P<0.05(4) Caspase-3 and Caspase-3 mRNA express(4)P<0.05(5) Bcl-2 and Bcl-2 mRNA levels(5)P<0.05(6) Bcl-w, and Bcl-w mRNA express(6)P<0.05(7) P-JNK express(7)P<0.05Chen et al. [27]SAMP8 mice (13/13)NRNRSAMP8 miceNRβ-Asarone (34 mg kg-1, ig); onset the experiment; once daily for 2 monthsTween 80 (same volume, ig); onset the experiment; once daily for 2 months(1) MWM test (number of platform crossing)(1)P<0.05(2) MWM test (escape latency)(2)P<0.05(3) LC3-positive cells(3)P<0.05(4) Beclin express(4)P<0.05(5) 1p62 express(5)P<0.05(6) ROCK1 express(6)P<0.05(7) GAP43, MAP2 and SYN expression(7)P<0.05(8) GAP43, MAP2 and SYN positive cells(8)P<0.05(9) Lipofuscin-positive cells(9)P<0.05Ma et al. [28]NIH mice (male, 6/6)18-20 gNRAβ1-42-induced AD modelSodium pentobarbitalWater extract (0.02 g g-1, ig); after the first MWM test; once daily for 3 weeksNormal saline (same volume, ig) after the first MWM test; once daily for 3 weeks(1) MWM test (escape latency)(1)P<0.05(2) Beta-amyloid IOD(2)P<0.05NIH mice (male, 6/6)18-20 gNRAβ1-42-induced AD modelSodium pentobarbitalDefatted decoction (0.02 g g-1, ig); after the first MWM test; once daily for 3 weeksNormal saline (same volume, ig) after the first MWM test; once daily for 3 weeks(1) MWM test (escape latency)(1)P<0.05(2) Beta-amyloid IOD(2)P<0.05NIH mice (male, 6/6)18-20 gNRAβ1-42-induced AD modelSodium pentobarbitalEssential oil (0.02 g g-1, ig); after the first MWM test; once daily for 3 weeksNormal saline (same volume, ig) after the first MWM test; once daily for 3 weeks(1) MWM test (escape latency)(1)P<0.05(2) Beta-amyloid IOD(2)P<0.05Tian et al. [29]NIH mice (male, 6/6)18-20 gNRAβ1-42-induced AD modelSodium pentobarbitalWater extract (0.02 g g-1, ig); after the first MWM test; once daily for 3 weeksNormal saline (0.2 ml/10 g, ig); after surgery; once daily for 3 weeks(1) MWM test (number of platform crossing)(1)P<0.05(2) NOS activity(2)P<0.05NIH mice (male, 6/6)18- 0 gNRAβ1-42-induced AD modelSodium pentobarbitalDefatted decoction (0.02 g g-1, ig); after the first MWM test; once daily for 3 weeksNormal saline (0.2 ml/10 g, ig); after surgery; once daily for 3 weeks(1) MWM test (number of platform crossing)(1)P>0.05(2) MWM test (time spent in target quadrant)(2)P>0.05(3) NOS activity(3)P<0.05NIH mice (male, 6/6)18-20 gNRAβ1-42-induced AD modelSodium pentobarbitalEssential oil (0.02 g g-1, ig); after the first MWM test; once daily for 3 weeksNormal saline (0.2 ml/10 g, ig); after surgery; once daily for 3 weeks(1) MWM test (number of platform crossing)(1)P<0.05(2) NOS activity(2)P<0.05Zhou et al. [30]SD rats (male, 10/10)250±20gNRScopolamine-induced AD modelNREssential oil (12 g kg-1, ig); onset the experiment; once daily for 21 dNS (same volume, ig); onset the experiment; once daily for 21 d(1) MWM test (escape latency)(1)P<0.01(2) MWM test (number of platform crossing)(2)P<0.01(3) GFAP - positive cells(3)P<0.01(4) SOD content(4)P<0.05(5) MDA content(5)P<0.05Wang GM et al., 2017Kunming mice (mix, 12/12)5-6 weeksNRChronic restraint stress-induced cognitive impairments modeNREssential oil (4.5 g kg-1, ig), onset the experiment; twice daily for 28 dNS (same volume, ig); onset the experiment; twice daily for 28 d(1) MWM test (escape latency)(1)P<0.01(2) MWM test (number of platform crossing)(2)P<0.01(3) Body mass(3)P<0.05(4) Plasma cortisol levels(4)P<0.01Hu et al. [32]Kunming mice (male, 11/11)18-20 gNRSodium nitrite-induced amnesic modelNREssential oil (0.053 g kg-1, ig); 21 d before sodium nitrite injection; once daily for 21 dTween 80 (same volume, ig); 21 d before sodium nitrite injection; once daily for 21 d(1) SD test (escape latency)(1)P<0.05(2) SD test (number of errors)(2)P<0.05Kunming mice (male, 11/11)18-20 gNRSodium nitrite-induced amnesic modelNRDefatted decoction (5 g kg-1, ig); 21 d before sodium nitrite injection; once daily for 21 dTween 80 (same volume, ig); 21 d before sodium nitrite injection; once daily for 21 d(1) SD test (escape latency)(1)P<0.05(2) SD test (number of errors)(2)P<0.05Kunming mice (male, 11/11)18-20 gNRSodium nitrite-induced amnesic modelNRα-Asarone (0.024 g kg-1, ig); 21 d before sodium nitrite injection; once daily for 21 dTween 80 (same volume, ig); 21 d before sodium nitrite injection; once daily for 21 d(1) SD test (escape latency)(1)P<0.05(2) SD test (number of errors)Kunming mice (male, 11/11)18-20 gNRSodium nitrite-induced amnesic modelNRβ-Asarone (0.037 g kg-1, ig); 21 d before sodium nitrite injection; once daily for 21 dTween 80 (same volume, ig); 21 d before sodium nitrite injection; once daily for 21 d(1) SD test (escape latency)(1)P<0.05(2) SD test (number of errors)(2)P<0.01Kunming mice (male, 11/11)18-20 gNREthanol-induced amnesic modelNREssential oil (0.053 g kg-1, ig); 21 d before ethanol injection; once daily for 21 dTween 80 (same volume, ig); 21 d before ethanol injection; once daily for 21 d(1) SD test (escape latency)(1)P<0.05(2) SD test (number of errors)(2)P<0.01Kunming mice (male, 11/11)18-20 gNREthanol-induced amnesic modelNRDefatted decoction (5 g kg-1, ig); 21d before ethanol injection; once daily for 21 dTween 80 (same volume, ig); 21 d before ethanol injection; once daily for 21 d(1) SD test (escape latency)(1)P>0.05(2) SD test (number of errors)(2)P<0.05Kunming mice (male, 11/11)18-20 gNREthanol-induced amnesic modelNRα-Asarone (0.024 g kg-1, ig); 21 d before ethanol injection; once daily for 21 dTween 80 (same volume, ig); 21 d before ethanol injection; once daily for 21 d(1) SD test (escape latency)(1)P<0.01(2) SD test (number of errors)Kunming mice (male, 11/11)18-20 gNREthanol-induced amnesic modelNRβ-Asarone (0.037 g kg-1, ig); 21 d before ethanol injection; once daily for 21 dTween 80 (same volume, ig); 21 d before ethanol injection; once daily for 21 d(1) SD test (escape latency)(1)P<0.05(2) SD test (number of errors)(2)P<0.01Kunming mice (male, 11/11)18-20 gNRSodium pentobarbital-induced amnesic modelNREssential oil (0.053 g kg-1, ig); 21 d before ethanol injection; once daily for 21 dTween 80 (same volume, ig); 21 d before ethanol injection; once daily for 21 d(1) EY-M test (number of errors)(1)P<0.05Kunming mice (male, 11/11)18-20 gNRSodium pentobarbital-induced amnesic modelNRDefatted decoction (5 g kg-1, ig); 21 d before ethanol injection; once daily for 21 dTween 80 (same volume, ig); 21 d before ethanol injection; once daily for 21 d(1) EY-M test (number of errors)(1)P<0.05Kunming mice (male, 11/11)18-20 gNRSodium pentobarbital-induced amnesic modelNRα-Asarone (0.024 g kg-1, ig); 21 d before ethanol injection; once daily for 21 dTween 80 (same volume, ig); 21 d before ethanol injection; once daily for 21 d(1) EY-M test (number of errors)(1)P<0.05Kunming mice (male, 11/11)18-20 gNRSodium pentobarbital-induced amnesic modelNRβ-Asarone (0.037 g kg-1, ig); 21 d before ethanol injection; once daily for 21 dTween 80 (same volume, ig); 21 d before ethanol injection; once daily for 21 d(1) EY-M test (number of errors)(1)P<0.05Chen et al. [33]ICR mice (male, 10/10)18±1gRandom number tableD-gal-induced dementia modelNRWater extract (70, 35, 17.5, or 8.75 mg kg-1, ig); 1 week after D-galactose injection; once daily for 7 weeksDistilled water (same volume, ig); 1 week after D-galactose injection; once daily for 7 weeks(1) MWM test (escape latency)(1)P>0.05(2) MWM test (number of platform crossing)(2)P>0.05(3) MDA levels(3)P>0.05(4) SOD activity(4)P>0.05Gu et al. [34]ICR mice (male, 10/10)19.6±1.5gRandom number tableScopolamine-induced dysmnesia modelNRWater extract (70, 35, 17.5, or 8.75 mg kg-1, ig); onset the experiment; once daily for 2 weeksNS (same volume, ig); onset the experiment; once daily for 2 weeks(1) SD test (escape latency)(1)P<0.01(2) SD test (number of errors)(2)P<0.01ICR mice (male, 10/10)19.6±1.5gRandom number tableNaNO2-induced dysmnesia modelNRWater extract (70, 35, 17.5, or 8.75 mg kg-1, ig); onset the experiment; once daily for 2 weeksNS (same volume, ig); onset the experiment; once daily for 2 weeks(1) SD test (escape latency)(1)P<0.01(2) SD test (number of errors)(2)P<0.05ICR mice (male, 10/10)19.6±1.5gRandom number tableEthanol-induced dysmnesia modelNRWater extract (70, 35, 17.5, or 8.75 mg kg-1, ig); onset the experiment; once daily for 2 weeksNS (same volume, ig); onset the experiment; once daily for 2 weeks(1) ST test (escape latency)(1)P<0.01(2) ST test (number of errors)(2)P<0.01(3) AchE activity(3)P>0.05Wistar rats (male, 10/10)200±25gNRScopolamine-induced dysmnesia modelNRWater extract (35, 17.5, or 8.75 mg kg-1, ig); onset the experiment; once daily for 4 weeksNS (same volume, ig); onset the experiment; once daily for 2 weeks(1) MWM test (escape latency)(1)P<0.01(2) MWM test (number of platform crossing)(2)P<0.01Wu et al., 2004Aged NIH mice (male, 10/10)NRNRAged miceNREssential oil (0. 01075 ml g-1, ig); onset the experiment; twice daily for 10 dNS (same volume, ig); onset the experiment; once daily for 10 d(1) MWM test (escape latency)(1)P<0.05(2) AchE activity(2)P<0.05(3) C-jun express(3)P<0.05Aged NIH mice (male, 10/10)NRNRAged miceNRβ-Asarone (0. 01075 ml g-1, ig); onset the experiment; twice daily for 10 dNS (same volume, ig); onset the experiment; once daily for 10 d(1) MWM test (escape latency)(1)P>0.05(2) MWM test (number of errors)(2)P>0.05(3) AchE activity(3)P>0.05(4) C-jun express(4)P>0.05Aged NIH mice (male, 10/10)NRNRAged miceNRWater extract (0. 01075 ml g-1 g, ig); onset the experiment; twice daily for 10 dNS (same volume, ig); onset the experiment; once daily for 10 d(1) MWM test (escape latency)(1)P>0.05(2) AchE activity(2)P>0.05(3) C-jun express(3)P<0.05Kunming mice (male, 10/10)NRNREthanol-induced dysmnesia modelNRWater extract (0. 01075 ml g-1, ig); onset the experiment; twice daily for 10 dNS (same volume, ig); onset the experiment; once daily for 10 d(1) ST test (escape latency)(1)P<0.05Kunming mice (male, 10/10)NRNRNaNO2-induced dysmnesia modelNREssential oil (0. 01075 ml g-1, ig); onset the experiment; twice daily for 10 dNS (same volume, ig); onset the experiment; once daily for 10 d(1) SD test (escape latency)(1)P>0.05(2) SD test(number of errors)(2)P>0.05Kunming mice (male, 10/10)NRNRNaNO2-induced dysmnesia modelNRβ-Asarone (0. 01075 ml g-1, ig); onset the experiment; twice daily for 10 dNS (same volume, ig); onset the experiment; once daily for 10 d(1) SD test (escape latency)(1)P<0.05(2) SD test (number of errors)(2)P>0.05Kunming mice (male, 10/10)NRNRNaNO2-induced dysmnesia modelNRWater extract (0. 01075 ml g-1, ig); onset the experiment; twice daily for 10 dNS (same volume, ig); onset the experiment; once daily for 10 d(1) SD test (escape latency)(1)P<0.05(2) SD test (number of errors)(2)P>0.05Kunming mice (male, 10/10)NRNRScopolamine-induced dysmnesia modelNREssential oil (0. 01075 ml g-1, ig); onset the experiment; twice daily for 10 dNS (same volume, ig); onset the experiment; once daily for 10 d(1) SD test (escape latency)(1)P<0.05(2) SD test (number of errors)(2)P>0.05Kunming mice (male, 10/10)NRNRScopolamine-induced dysmnesia modelNRβ-Asarone (0. 01075 ml g-1, ig); onset the experiment; twice daily for 10 dNS (same volume, ig); onset the experiment; once daily for 10 d(1) SD test (escape latency)(1)P<0.05(2) SD test (number of errors)(2)P>0.05Kunming mice (male, 10/10)NRNRScopolamine-induced dysmnesia modelNRWater extract (0. 01075 ml g-1, ig); onset the experiment; twice daily for 10 dNS (same volume, ig); onset the experiment; once daily for 10 d(1) SD test (escape latency)(1)P<0.05(2) SD test(number of errors)(2)P>0.05Wen et al., 2009ICR mice (mix, 10/10)20±2gNREthanol-induced dysmnesia modelNRWater extract (3 and 12 g kg-1, ig); onset the experiment; twice daily for 14 dNS (same volume, ig); onset the experiment; twice daily for 14 d(1) ST test (escape latency)(1)P<0.01ICR mice (mix, 10/10)20±2gNRNaNO2-induced dysmnesia modelNREssential oil (3 and 12 g kg-1, ig); onset the experiment; once daily for 14 dNS (same volume, ig); onset the experiment; twice daily for 14 d(2) EY-M test (number of errors)(1)P<0.05ICR mice (mix, 10/10)20±2gNRScopolamine-induced dysmnesia modelNRWater extract (3 and 12 g kg-1, ig); onset the experiment; once daily for 14 dNS (same volume, ig); onset the experiment; twice daily for 14 d(1) SD test (escape latency)(1)P<0.05(2) SD test (number of errors)(2)P<0.05ICR mice (mix, 10/10)20±2gNRScopolamine-induced dysmnesia modelNREssential oil (3 and 12 g kg-1, ig); onset the experiment; once daily for 14 dNS (same volume, ig); onset the experiment; twice daily for 14 d(1) SD test (escape latency)(1)P<0.05(2) SD test (number of errors)(2)P<0.01ICR mice (mix, 10/10)20±2gNRScopolamine-induced dysmnesia modelNRWater extract (3 and 12 g kg-1, ig); onset the experiment; once daily for 14 dNS (same volume, ig); onset the experiment; twice daily for 14 d(1) MWM test (escape latency)(1)P<0.01ICR mice (mix, 10/10)20±2gNRScopolamine-induced dysmnesia modelNREssential oil (3 and 12 g kg-1, ig); onset the experiment; once daily for 14 dNS (same volume, ig); onset the experiment; twice daily for 14 d(1) MWM test (escape latency)(1)P<0.01Yang et al. [37]SD rats (male, 12/12)250±30gNRAβ1-42-induced AD modelNRβ-Asarone (10, 20, and 30 mg kg-1, ig); after the model finished; twice daily for 28 dNS (same volume, ig); after the model finished; once daily for 28 d(1) MWM test (escape latency)(1)P<0.05(2) MWM test (number of platform crossing)(2)P<0.01(3) Astrocyte activity(3)P<0.01Zhou et al. [38]SD rats (male,10/10)200-250 gNRD-gal- and AlCl3-induced AD modelNRα-Asarone (10, 25 mg kg-1, ip); after the after model finished; once daily for 28 dNS (same volume, ip); after the model finished; once daily for 28 d(1) MWM test (number of platform crossing)(1)P<0.01(2) Aβ and tau protein expression(2)P<0.01(3) ACh levels(3)P<0.05(4) AChE levels(4)P>0.05(5) ChAT levels(5)P>0.05Jiang et al., 2007Kunming mice (mix, 10/10)18-20 gNRAlCl3-induced AD modelNRβ-Asarone (1.06, 2.12, and 4.24 mg 100 g-1, ig); after the model finished; once daily for 2 monthsNS (same volume, ig); after the model finished; once daily for 2 months(1) MWM test (number of errors)(1)P<0.01(2) SOD levels(2)P<0.01(3) MAD levels(3)P<0.01Huang et al. [40]FMR1gene knock mice (16/17)17-18 gNRFragile X syndrome modelNRα-Asarone (3, 6, 9, 12, 24 mg kg-1, ip); onset the experiment; once daily for 8 dNS (same volume, ip); onset the experiment; once daily for 8 d(1) SD test (number of errors)(1)P>0.05(2) P-Akt expression(2)P<0.05(3) Akt expression(3)P>0.05Wang BL et al., 2017SD rats (male, 15/15)280±20gRandom number tableAβ1-42-induced AD modelPhenytoin sodiumβ-Asarone (10, 20, and 30 mg kg-1, ig); after the model finished; once daily for 4 weeksNS (same volume, ig); after the model finished; once daily for 4 weeks(1) MWM test (escape latency)(1)P<0.01(2) MWM test (number of platform crossing)(2)P<0.01(3) HIF levels(3)P<0.05Guo et al. [42].Kunming mice (male, 11/11)25±5gRandom block allocation methodScopolamine-induced AD modelNRβ-Asarone (21.2 mg kg-1, ig); after the model finished; once daily for 14 dNS (same volume, ig); after the model finished; once daily for 14 d(1) MWM test (escape latency)(1)P<0.01Jiang et al. [43]Wistar rats (mix, 8/8)250-300 gNRSTZ-induced AD modelNREssential oil (5, 10 and 20 g kg-1, ig); onset the experiment; once daily for 20 dSolvent (same volume, ig); onset the experiment; once daily for 20 d(1) MWM test (escape latency)(1)P<0.01(2) SOD levels(2)P<0.01(3) MAD levels(3)P<0.01Yang et al. [44]Wistar rats (10/10)35±5gNRPTZ-induced epilepsy modelNRα-Asarone (29 mg kg-1, ig); after PTZ injection; twice daily for 7 dNS (same volume, ig); after PTZ injection; twice daily for 7 d(1) MWM test (number of platform crossing)(1)P<0.05(2) MWM test (time spent in target quadrant)(2)P<0.05Wistar rats (10/10)35±5gNRPTZ-induced epilepsy modelNRAGA (2.35 g kg-1, ig); after PTZ injection; twice daily for 7 dNS (same volume, ig); after PTZ injection; twice daily for 7 d(1) MWM test (number of platform crossing)(1)P<0.05(2) MWM test (time spent in target quadrant)(2)P<0.05Wang et al. [45]ICR mice (mix, 10/10)20±2gNRScopolamine-induced dysmnesia modelNREssential oil (100, 150, and 300 mg kg-1, ig); before the experiment; once daily for 7 dNS (same volume, ig); before the experiment; once daily for 7 d(1) MWM test (escape latency)(1)P<0.01(2) MWM test (number of platform crossing)(2)P<0.01(3) MWM test (time spent in target quadrant)(3)P<0.01Ma et al. [46]SD rats (male, 6/6)260-280 gNRAβ1-42-induced AD modelSodium pentobarbitalβ-Asarone (12.5, 25, 50 mg kg-1, ig); after the model finished; once daily for 4 weeksNS (same volume, ig); after the model finished; once daily for 4 weeks(1) MWM test (escape latency)(1)P<0.05(2) GA P-43 mRNA levels(2)P<0.05(3) SYP mRNA levels(3)P<0.05(4) PSD-95 mRNA levelsAch: acetylcholine; AchE: acetylcholinesterase; SD rats: Sprague-Dawley rats; NIH mice: National Institutes of Health mice; SAMP8 mice: senescence-accelerated mouseprone 8 mice; AD: Alzheimer’s disease; AlCl3: aluminum trichloride; ChAT: acetylcholine transferase; D-gal: D-galactose; i.g.: intragastrical injection; i.p.: intraperitoneal injection; i.h.: hypodermic injection; MWM test: Morris water maze test; MCAO: middle cerebral artery occlusion; MDA: malondialdehyde; GSH-PX: glutathione peroxidase; NR: not report; SD test: step down test; STZ: streptozotocin; SOD: superoxide dismute; HIF: hypoxia-inducible factor; GSH-Px: glutathione peroxidase; NE: norepinephrine; 5-TH: 5-hydroxytryptamine; DA: dopamine; SYN/SYN: synaptophysin; NOS: nitric oxide synthase; Bcl-2: B-cell lymphoma/leukemia-2; MAP2: microtubule-associated protein 2; RAM: radial eight-arm maze; EY-M: electric Y-maze; Aβ1-42: amyloid beta 1-42; PTZ: pent ylenetet razol; NS: normal saline.


## 3.3. Study Quality

The quality scores of the 34 included studies varied from 1/10 to 6/10 with the average of 3.32. One study [40] got 1 point; 11 studies [29, 31–36, 38, 39, 42, 44] got 2 points; 9 studies [5, 6, 18, 24, 25, 27, 30, 43, 45] got 3 points; 4 studies got 4 points; 7 studies got 5 points; and 2 studies [20, 37] got 6 points. Thirty-four studies were published. Sixteen studies described control of temperature [6, 10, 17–26, 30, 37, 41, 45]. Random allocation was declared in 28 studies [5, 6, 11, 17, 19–23, 26–28, 30–39, 41–46]; 1 study [42] used random block allocation method, and 2 studies used the method of random digit table [34, 41]. Two studies [23, 37] described the use of blinded assessment of outcome. Thirteen studies did not use anesthetics with significant intrinsic neuroprotective activity, and the remaining 21 studies did not report the type of anesthetics [5, 6, 18, 19, 24, 27, 30–40, 42–45]. Sixteen studies reported compliance with animal welfare regulations [5, 10, 11, 17–22, 24, 27, 28, 37, 41, 43, 45]. Four studies mentioned statement of potential conflict of interests [11, 20, 28, 37]. None of the included studies reported allocation concealment, sample size calculation, and the utilization of animal or model with relevant comorbidities. The quality scores for the included studies are shown in Table 2.Table 2
Quality assessment of included studies.
Study (years)12345678910TotalYang et al. [17]√√√√√5Wei et al., 2013√√√3Sundaramahalingam et al. [18]√√√3Shin et al. [19]√√√√4Ma et al. [11]√√√√√5Liu et al. [20]√√√√√√6Limón et al. [21]√√√√√5Li et al. [22]√√√√√5Zhang et al. [5]√√√3Lee et al. [10]√√√√4Lee et al. [23]√√√√√5Kumar et al., 2012√√√3Kim et al. [25]√√√3Geng et al. [26]√√√√4Chen et al. [27]√√√3Ma et al. [28]√√√√√5Tian et al. [29]√√2Zhou et al. [30]√√√3Wang GM et al., 2017√√2Hu et al. [32]√√2Chen et al. [33]√√2Gu et al. [34]√√2Wu et al., 2004√√2Wen et al., 2009√√2Yang et al. [37]√√√√√√6Zhou et al. [38]√√2Jiang et al., 2007√√2Huang et al. [40]√1Wang BL et al., 2017√√√√√5Guo et al. [42]√√2Jiang et al. [43]√√√3Yang et al. [44]√√2Wang et al. [45]√√√3Ma et al. [46]√√√√41: peer-reviewed publication; 2: statements describing control of temperature; 3: randomization to treatment group; 4: allocation concealment; 5: blinded assessment of outcome; 6: avoidance of anesthetics with known notable intrinsic neuroprotective properties; 7: use of animals with relevant comorbidities; 8: sample size calculation; 9: compliance with animal welfare regulations; 10: declared any potential conflict of interest.


## 3.4. Effectiveness

The Morris water maze test, including the probe test and the spatial test, was conducted in 28 studies [6, 10, 11, 17, 19, 20, 22, 23, 25–31, 33–39, 41–46]. Twenty-seven studies reported the spatial test using the escape latency as an outcome measure. Meta-analysis of 20 studies with 27 comparisons showed EAAGA significantly decreased the escape latency compared with the control (n=490, SMD=−1.09, 95% CI [−1.37 to −0.82], P<0.00001; heterogeneity: χ2=49.48, df=26P=0.004; I2=47%; Figure 2(a)). In the probe test, meta-analysis of 16 studies [17, 19, 20, 22, 26, 27, 29–31, 33, 34, 37, 38, 41, 44, 45] with 19 comparisons showed EAAGA were significant for increasing number of platform crossings (n=398, SMD=1.60, 95% CI [1.25 to 1.94], P<0.00001; heterogeneity: χ2=34.29, df=18P=0.01; I2=47%; Figure 2(b)) compared with controls. Meta-analysis of 6 studies [17, 20, 22, 29, 34, 44] with 7 comparisons showed a significant effect of EAAGA in increasing the length of time spent in platform quadrant compared with control (n=144, SMD=1.78, 95% CI [0.90 to 2.67], P<0.0001; heterogeneity: χ2=22.41, df=6P=0.001; I2=73%). As the values of I2 were greater than 50%, we sequentially omitting each study; two studies [20, 22] were removed and markedly reduced the heterogeneity (n=86, SMD=2.34, 95% CI [1.55 to 3.12], P<0.00001; heterogeneity: χ2=6.57, df=4P=0.16; I2=39%; Figure 2(c)). Two studies [20, 22] used relatively large doses of β-asarone that might have potential toxic effects [47]. Meta-analysis of 3 studies [20, 23, 25] for increasing percentage of time in the platform quadrant (n=44, SMD=4.01, 95% CI [2.86 to 5.15], P<0.00001; heterogeneity: χ2=0.03, df=2P=0.98; I2=0%; Figure 2(d)). Three studies [17, 22, 23] showed there were not a significant difference in improving the swimming velocity compared with controls.Figure 2
The forest plot in Morris water maze test. Effects of EAAGA for decreasing the escape latency (a) in spatial test, increasing crossing numbers (b), increasing exact time (c), and increasing percentage of time (d) in platform quadrant in probe test compared with control group.
(a)(b)(c)(d)The step-down test, including the training test which represents learning ability and retention test which represents memory ability, was conducted in 6 studies [5, 32, 34–36, 40]. Meta-analysis of 5 studies with 19 comparisons showed EAAGA were significant for increasing right reaction latency in the retention test (n=396, SMD=1.15, 95% CI [0.87 to 1.43], P<0.00001; heterogeneity: χ2=28.98, df=18P=0.05; I2=38%; Figure 3(a)) and 1 study [5] for increasing right reaction latency (P<0.05) in the training test. Meta-analysis of 3 studies [32, 35, 36] with 16 comparisons showed EAAGA were significant for decreasing the error times (n=336, SMD=−1.06, 95% CI [−1.30 to −0.83], P<0.00001; heterogeneity: χ2=15.01, df=15P=0.45; I2=0%; Figure 3(b)) in the retention test and 1 study [5] for decreasing the error times (P<0.05) in the training test.Figure 3
The forest plot in Step-down test. Effects of EAAGA for increasing right reaction latency in the retention test (a) and decreasing the error times in the retention test (b) compared with control group.
(a)(b)The electrical Y-maze test was conducted in 3 studies [5, 32, 36]. Meta-analysis of 3 studies showed EAAGA were significant for decreasing error reaction times (n=168, SMD=−1.22, 95% CI [−1.56 to −0.88], P<0.00001; heterogeneity: χ2=3.95, df=7P=0.79; I2=0%; Figure 4).Figure 4
The forest plot in Electrical Y-maze test. Effects of EAAGA for decreasing error reaction times compared with control group.The step-through test was conducted in 4 studies [24, 34–36]. Meta-analysis of 4 studies with 7 comparisons showed EAAGA were significant for decreasing latency in the retention test (n = 134, SMD = 1.26, 95% CI [0.81 to 1.71], P < 0.00001; heterogeneity: χ2 = 8.09, df =6 (P = 0.23); I2=26%; Figure 5(a)) and 2 studies [35, 36] with 5 comparisons showed EAAGA significantly decreased the number of errors in the retention test (n=100, SMD=−1.02, 95% CI [−1.45 to −0.60], P<0.00001; heterogeneity: χ2=1.05, df=4P=0.90; I2=0%; Figure 5(b)) compared with controls.Figure 5
The forest plot in Step-through test. Effects of EAAGA for decreasing latency in the retention test (a) and decreasing the number of errors in the retention test (b) compared with control group.
(a)(b)The eight-arm maze test was conducted in 3 studies [10, 18, 21]. Meta-analysis of 2 studies [10, 21] showed EAAGA were significant for increasing number of correct choices (n=25, SMD=1.15, 95% CI [0.00 to 2.29], P=0.05; heterogeneity: χ2=1.63, df=1P=0.20; I2=38%; Figure 6(a)) and 2 studies [10, 18] with 3comparisons showed EAAGA significantly decreased the number of errors in the training test (n=33, SMD=−2.36, 95% CI [−3.36 to −1.37], P<0.00001; heterogeneity: χ2=1.00, df=2P=0.61; I2=0%; Figure 6(b)) compared with controls.Figure 6
The forest plot in Eight-arm maze test. Effects of EAAGA for increasing correct choices (a) and decreasing the number of errors (b) compared with control group.
(a)(b)


## 3.5. Neuroprotective Mechanisms

The mechanisms of neuroprotection of EAAGA on cognitive impairment were studied in 34 included articles [5, 6, 10, 11, 17–46] as follows: (1) reduction of oxidative reactions by increasing the activity of SOD [30, 35, 39, 41, 43] activity, while decreasing the activity of SOD and AChE [18, 24], decreasing the levels of MDA [24, 30, 33] and nitric oxide [21], decreasing the mRNA levels of hsp 70, increasing the levels of VC, VE, and GSH, and increasing the activity of CAT and G6PD [18]; (2) inhibition of apoptosis by increasing the mRNA levels of Bcl-2, BDNF, CREB [6, 23, 42], Bcl-w and Bcl-2 [26], and c-jun [35], decreasing the mRNA levels of Bax [23], increasing the expression of BDNF, CREB [23], Bcl-w, and Bcl-2 [26], decreasing the expression of caspase-3, p-JNK [26], and BACE1 [19], and preventing cell loss [10], Aβ, and Tau protein [38]; (3) repression of inflammatory reactions by decreasing the expression of TNF-α and IL-1β mRNA levels [19]; (4) repression of autophagy by decreasing LC3, ROCK, and beclin1 expression and increasing p62, GAP43, MAP2, and SYN expression [27]; (5) protection of cerebrovascular by increasing rCBF and the Na-K-ATP activity, decreasing pyruvic acid contents, and decreasing the mRNA levels of ET-1, eNOS, and APP [22]; (6) promotion of cognitive function by increasing the levels of 5-HT, NE, DA, and NE [5] and suppression of astrocyte activation [37]; (7) stimulation of cholinergic system by increasing AchE and ChAT neurons [25]; (8) improvement of memory impairments through regulation of synaptogenesis, which is mediated via Arc/Arg3.1 and Wnt pathway [17]; (9) neuroprotection through damage of Akt pathway [40]; (10) inhibition of neurotoxicity by decreasing the expression of DCx and nestin, decreasing nestin positive cells [11], decreasing Aβ plaques depositions, and decreasing NOS activity [29]; (11) regulation of synaptic plasticity by increasing the expression of SYP and GluR1 [20, 46] and decreasing the expression of GAP-43 and PSD-95 [46]; and (12) inhibition of chronic stress by decreasing plasma cortisol levels [41]. Characteristics of mechanism studies of EAAGA on experimental ischemic stroke are shown in Table 3 and Figure 7.Table 3
Characteristics of mechanism studies of EAAGA on cognition impairment.
Study (years)ModelMethod of administration (experimental group versus control group)ObservationsPossible mechanismsYang et al. [17]Chronic lead-induced dysmnesia modelβ-Asarone versus distilled waterAttenuated memory deficitsArc/Arg3.1 and Wnt pathwayIncreased dendritic spine densityIncreased dendritic spine densityUp-regulated NR2B, Arc/Arg3.1, and Wnt7a protein expressionWei et al., 2013AβPP/PS1 double-transgenic miceβ-Asarone versus Tween 80Improved cognitive functionCaMKII/CREB/Bcl-2 signaling pathwayPrevents PC12 cell and cortical neuron damageInhibition of apoptosisInhibited the apoptosis of PC12 cells and cortical neuronsSundaramahalingam et al. [18]Noise stress induced memory impairment modelα-Asarone versus Tween 80Prevent memory impairmentReduction of oxidative reactionsDecreased hsp 70 mRNA levelsDecreased SOD and AChE activityIncreased CAT and G6PD activityIncreased VC, VE, and GSH levelsShin et al. [19]LPS-induced cognitive handicap modeα-Asarone versus NSAmeliorated memory deficitsRepression of inflammatory reactionsReduced Iba1 protein expressionInhibition of apoptosisReduced TNF-α and IL-1β mRNAReduced BACE1 expressionIncreased CA1 neuronsReduced TUNEL-labeled cellsMa et al. [11]Aβ1-42-induced AD modelWater extract versus NSAmeliorated memory deficitsInhibition of neurotoxicityEssential oil versus NSReduced Aβ positive cellsDefatted decoction versus NSDecreased DCx and nestin expressionDecreased nestin positive cellsLiu et al. [20]APPswe/PS1dE9 double transgenic miceβ-Asarone versus Tween 80Improved the learning and memory abilityRegulation of synaptic plasticityIncreased SYP and GluR1 expressionLimón et al. [21]Aβ-induced AD modelα-Asarone versus NSAmeliorated memory deficitsReduction of oxidative reactionsDecreased NO levelsLi et al. [22]D-gal- and AlCl3-induced AD modelβ-Asarone versus NSImproved the learning and memory abilityProtection of cerebrovascularIncreased rCBF and the activity of Na–K-ATPDecreased pyruvic acid contentsDecreased ET-1, eNOS, and APP mRNA expressionZhang et al. [5]Aged miceEssential oil versus Tween 80Improved cognitive functionIncreased 5-HT, NE, DA, and NE levelsDecreased AChE activityImprovement of cognitive functionScopolamine-induced dysmnesia modelEssential oil versus Tween 80Ethanol-induced dysmnesia modelEssential oil versus Tween 80Aged ratsEssential oil versus Tween 80Sodium nitrite-induced dysmnesia modelEssential oil versus Tween 80Lee et al. [10]MCAO/2 h-induced cognitive impairments modelAGA versus NSAttenuated learning and memory deficitsIncreased cell densityInhibition of apoptosisLee et al. [23]Chronic corticosterone exposedβ-Asarone versus NSImproved cognitive functionIncreased BDNF and CREB expressionIncreased BDNF, CREB, and Bcl-2 mRNAs levelsDecreased Bax mRNAs levelsDecreased serum levels of CORTInhibition of apoptosisKumar et al., 2012Scopolamine-induced amnesic modelα-Asarone versus vehicleImproved cognitive functionIncreased of AchE activityInhibition MDA expression and SOD levelsReduced SOD activityReduction of oxidative reactionsKim et al. [25]Ibotenic acid-induced amnesiaAGA versus NSAmeliorated learning and memory deficitsIncreased ChAT positive neuronsIncreased AchE neuronsStimulation of cholinergic systemGeng et al. [26]Aβ1-42-induced AD rat modelβ-Asarone versus NSAmeliorated learning and memory deficitsIncreased Bcl-2, Bcl-w expressionIncreased Bcl-2 and Bcl-w mRNA levelsDecreased cleavage of caspase-3Reduced caspase-3 mRNA levelsDecreased p-JNK expressionInhibition of apoptosisChen et al. [27]SAMP8 miceβ-Asarone versus NSImproved cognitive functionReduced ROCK, beclin1, and LC3 expressionIncreased p62 expressionIncreased GAP43, MAP2, and SYN expressionIncreased GAP43-, MAP2-, and SYN-positive cellsDecreased lipofuscin-positive cellsReduction of autophagyMa et al. [28]Aβ-induced AD modelWater extract versus NSAmeliorated learning and memory deficitsDecreased Aβ plaques depositionsImprovement of cognitive functionWater extract without oil versus NSEssential oil versus NSTian et al. [29]Aβ-induced AD modelWater extract versus NSAmeliorated learning and memory deficitsDecreased NOS activityInhibition of neurotoxicityDefatted decoction versus NSEssential oil versus NSZhou et al. [30]Scopola-induced AD modelEssential oil versus NSAmeliorated learning and memory deficitsDecreased GFAP expressionDecreased MDA levelsIncreased SOD levelsReduction of oxidative reactionsWang GM et al., 2017Chronic restraint stress-induced cognitive impairments modeEssential oil versus NSAmeliorated learning and memory deficitsIncreased body massDecreased plasma cortisol levelsInhibition of chronic stressHu et al. [32]Sodium nitrite-induced amnesic modelEssential oil versus Tween 80Increased learning and memory deficitsImprovement of cognitive functionDefatted decoction versus Tween 80α-Asarone versus Tween 80β-Asarone versus Tween 80Ethanol-induced amnesic modelEssential oil versus Tween 80Ameliorated learning and memory deficitsImprovement of cognitive functionDefatted decoction versus Tween 80α-Asarone versus Tween 80β-Asarone versus Tween 80Chen et al. [33]D-galactose-induced dementia modelWater extract versus distilled waterAmeliorated memory deficitsDecreased MDA levelsIncreased SOD activityReduction of oxidative reactionsGu et al. [34]Scopolamine-induced dysmnesia miceWater extract versus NSAmeliorated memory deficitsImprovement of cognitive functionNaNO2-induced dysmnesia modelWater extract versus NSAmeliorated memory deficits45% ethanol-induced dysmnesia miceWater extract versus NSAmeliorated memory deficitsThe AchE activity of mice brain was not influencedScopolamine-induced dysmnesia ratWater extract versus NSAmeliorated memory deficitsWu et al., 2004Aged miceEssential oil versus NSAmeliorated memory deficitsDecreased AChE activityIncreased c-jun mRNA levelsInhibition of apoptosisAged miceβ-Asarone versus NSAged miceWater extract versus NSEthanol-induced dysmnesia modelWater extract versus NSNaNO2-induced dysmnesia modelEssential oil versus NSNaNO2-induced dysmnesia modelβ-Asarone versus NSNaNO2-induced dysmnesia modelWater extract versus NSAmeliorated memory deficitsScopolamine-induced dysmnesia modelEssential oil versus NSScopolamine-induced dysmnesia modelEssential oil versus NSScopolamine-induced dysmnesia modelWater extract versus NSWen et al., 2009Ethanol-induced dysmnesia modelWater extract versus NSAmeliorated memory deficitsInhibition of apoptosisNaNO2-induced dysmnesia modelEssential oil versus NSScopolamine-induced dysmnesia modelWater extract versus NSScopolamine-induced dysmnesia modelEssential oil versus NSScopolamine-induced dysmnesia modelWater extract versus NSScopolamine-induced dysmnesia modelEssential oil versus NSYang et al. [37]Aβ1-42-induced AD modelβ-Asarone versus NSImproved cognitive functionInhibited AQP4, IL-1β, and TNF-α expressionDecreased Aβ depositionAlleviated hippocampal damageSuppression of astrocyte activationZhou et al. [38]D-gal- and AlCl3-induced AD modelα-Asarone versus NSImproved cognitive functionDecreased Aβ and Tau protein expressionIncreased ACh expressionInhibition of apoptosisJiang et al. 2007AlCl3-induced AD modelβ-Asarone versus NSImproved cognitive functionIncreased SOD and GSH-Px levelsDecreased MAD levelsReduction of oxidative reactionsHuang et al. [40]Fragile X syndrome modelα-Asarone versus NSImproved cognitive functionDamage of Akt pathwayWang BL et al., 2017Aβ1-42-induced AD modelβ-Asarone versus NSImproved cognitive functionDecreased HIF and MDA levelsIncreased SOD and CAT levelsReduction of oxidative reactionsGuo et al. [42]Scopolamine-induced AD modelβ-Asarone versus NSImproved cognitive functionInhibition of apoptosisJiang et al. [43]STZ-induced AD modelEssential oil versus solventImproved cognitive functionDecreased MDA levelsIncreased SOD levelsReduction of oxidative reactionsYang et al. [44]PTZ-induced epilepsy modelAGA versus NSImproved cognitive functionInhibition of apoptosisPTZ-induced epilepsy modelα-Asarone versus NSImproved cognitive functionInhibition of apoptosisWang et al. [45]Aβ1-42-induced AD modelEssential oil versus NSImproved cognitive functionImprovement of cognitive functionMa et al. [46]D-gal- and AlCl3-induced AD modelβ-Asarone versus NSImproved cognitive functionDecreased GA P-43 mRNA levelsIncreased SYP mRNA levelsDecreased PSD-95 mRNA levelsRegulation of synaptic plasticityAch: acetylcholine; AchE: acetylcholinesterase; AD: Alzheimer’s disease; AlCl3: aluminum trichloride; ChAT: acetylcholine transferase; D-gal: D-galactose; MCAO: middle cerebral artery occlusion; MDA: malondialdehyde; STZ: streptozotocin; SOD: superoxide dismute; HIF: hypoxia-inducible factor; SYN/SYN: synaptophysin; MAP2: microtubule-associated protein 2; Aβ1-42: amyloid beta 1-42; NS: normal saline; PTZ: pent ylenetet razol; GSH-Px: glutathione peroxidase; NE: norepinephrine; 5-TH: 5-hydroxytryptamine; DA: dopamine; NOS: nitric oxide synthase; Bcl-2: B-cell lymphoma/leukemia-2.Figure 7
A schematic representation of possible mechanisms of EAAGA for improving learning and memory function. The possible mechanisms of different active ingredients are as follows: (1) AGA: the dry rhizomes of Acorus gramineus Solander can inhibit apoptosis and stimulate cholinergic system. (2) Essential oil: AGA contains up to 4.86% essential oil, which displayed antioxidation effects by decreasing the levels of MDA and increasing the levels of SOD, exhibited anticytotoxicity effects via decreasing NOS activity, exerted antineurotoxicity effects by decreasing Aβ plaques depositions, and improved cognitive function by decreasing the activity of AChE. (3) β-Asarone: a major component of essential oil (63.2–81.2%) displayed antioxidation effects by decreasing the levels of MDA and HIF, increasing the levels of SOD, CAT, and GSH-Px; exerted antiapoptotic activity through regulating CaMKII/CREB/Bcl-2 signaling pathway and decreasing the levels of Bax mRNAs, caspase-3 mRNA, and JNK; inhibited synaptic loss through reducing ROCK expression; mediated synaptogenesis via Arc/Arg3.1 and Wnt pathway; improved circulation by decreasing the activity of AChE; and exerted antineurotoxicity by decreasing Aβ plaques depositions. (4) α-Asarone: another major component of essential oil (8.8–13.7%) exerted antioxidation effects by increasing CAT, SOD, and GSH-Px.; displayed anti-inflammatory activity through reducing the expression of proinflammatory mediators; improved circulation via decreasing the activity of AChE; and exerted antineurotoxicity by decreasing Aβ plaques depositions. (5) Water extract: displayed antioxidation effects by decreasing the levels of MDA and increasing the levels of SOD, exerted antineurotoxicity by decreasing Aβ plaques depositions; and improved cognitive function by decreasing the activity of AChE. (6) Defatted decoction: exerted antineurotoxicity by decreasing Aβ plaques depositions and displayed anticytotoxicity effects via decreasing the activity of NOS.


## 4. Discussion

As far as we know, it is the first preclinical systematic review that determined the efficacy of EAAGA for learning and memory function. In the present study, 34 studies with 1431 animals showed that EAAGA significantly improve learning and memory function, suggesting the potential neuroprotective functions of EAAGA in cognitive function impairment. However, given methodological weaknesses, the overall available evidence from the present study should be interpreted cautiously.Some limitations should be considered while interpreting this study. First, we only searched databases in Chinese and English. The absence of studies published in other languages may cause certain degree selective bias [48]. Second, the methodological quality of included studies showed some inherent drawback. Most of the research had methodological flaws in aspects of blinding, randomization, allocation concealment, sample size calculation, and lacking statement of potential conflict of interests [49, 50]. The studies without adequate sample sizes, allocation concealment, or randomization may result in inflated estimates of treatment efficacy [51, 52]. Lower quality trials could attribute to statistically significant 30–50% exaggeration of treatment efficacy [53]. Third, no study adopted animals with comorbidities, which would have created more relevant models for human pathology [49]. Thereby, the results should be interpreted cautiously.The poor design of animal research hindered the translation of animal research into effective preclinical drug treatments for human disease [54, 55]. Thus, it is necessary to take a rigor experimental design to overcome methodology quality issues for further research. The Animal Research: Reporting of In Vivo Experiments (ARRIVE) [56, 57] is a reporting guideline consisting of a 20-item checklist that provides recommendations on Introduction, Methods, Results, and Discussion which were recommended to be utilized as guidelines when designing and reporting animal research on EAAGA for improving the cognitive function impediment. Meanwhile, many drugs that exerted significant effects in animal researches failed to translate into effective clinical drug treatments [58, 59]. One of the possible reasons is the application of drug doses and the timing of drug administration in animal models that are inapplicable for human disease [55]. In the present study, doses of EAAGA and timing for initial administration in animal models were inconsistent among the 34 included studies. Thus, we suggest further studies to determinate the optimal gradient doses and timing of administration in animal models of cognition impairment.The present study showed that EAAGA had cognitive enhancing effects through different mechanisms as follows: (1) reduction of oxidative reactions by increasing the activity of SOD [30, 35, 39, 41, 43] activity, while decreasing the activity of SOD and AChE [18, 24], decreasing the levels of MDA [24, 30, 33] and nitric oxide [21], decreasing the mRNA levels of hsp 70, increasing the levels of VC, VE and GSH, and increasing the activity of CAT and G6PD [18]; (2) inhibition of apoptosis by increasing the mRNA levels of Bcl-2, BDNF, CREB [6, 23, 42], Bcl-w and Bcl-2 [26], and c-jun [35], decreasing the mRNA levels of Bax [23], increasing the expression of BDNF, CREB [23], Bcl-w, and Bcl-2 [26], decreasing the expression of caspase-3, p-JNK [26], and BACE1 [19], and preventing cell loss [10], Aβ, and Tau protein [38]; (3) repression of inflammatory reactions by decreasing the expression of TNF-α and IL-1β mRNA levels [19]; (4) repression of autophagy by decreasing LC3, ROCK, and beclin1 expression and increasing p62, GAP43, MAP2, and SYN expression [27]; (5) protection of cerebrovascular by increasing rCBF and the Na-K-ATP activity, decreasing pyruvic acid contents, and decreasing the mRNA levels of ET-1, eNOS, and APP [22]; (6) promotion of cognitive function by increasing the levels of 5-HT, NE, DA, and NE [5] and suppression of astrocyte activation [37]; (7) stimulation of cholinergic system by increasing AchE and ChAT neurons [25]; (8) improvement of memory impairments through regulation of synaptogenesis, which is mediated via Arc/Arg3.1 and Wnt pathway [17]; (9) neuroprotection through damage of Akt pathway [40]; (10) inhibition of neurotoxicity by decreasing the expression of DCx and nestin, decreasing nestin positive cells [11], and decreasing Aβ plaques depositions, decreased NOS activity [29]; (11) regulation of synaptic plasticity by increasing the expression of SYP and GluR1 [20, 46] and decreasing the expression of GAP-43 and PSD-95 [46]; and (12) inhibition of chronic stress by decreasing plasma cortisol levels [41]. However, cellular and molecular alteration mechanisms of EAAGA and active components for cognition impairment have not been clearly explored yet, which presented an exciting investigative direction of further studies. All 5 measuring methods for learning and memory ability were used in the 34 included studies, which showed that the measuring methods for cognition impairment were inconsistent. The diverse measuring methods for learning and memory ability need further study.


## 5. Conclusions

Although some factors such as study quality may undermine the validity, EAAGA exert potential neuroprotective effects in cognition impairment. In addition, AGA and active components may be a promising candidate for clinical trials.



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*Source: 6752876-2020-08-25.xml*