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Cognitive Improvement during Treatment for Mild Alzheimer’s Disease with a Chinese Herbal Formula: A Randomized Controlled Trial

  • Yulian Zhang ,

    Contributed equally to this work with: Yulian Zhang, Cuiru Lin

    zhyl220@126.com

    Affiliation Department of Acupuncture and Cerebropathy, Second Affiliated Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin, China

  • Cuiru Lin ,

    Contributed equally to this work with: Yulian Zhang, Cuiru Lin

    Affiliation Department of Acupuncture and Cerebropathy, Second Affiliated Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin, China

  • Linlin Zhang,

    Affiliation Department of Geriatric, Longhua Hospital of Shanghai University of Traditional Chinese Medicine, Tianjin, China

  • Yuanwu Cui,

    Affiliation Department of Acupuncture and Cerebropathy, Second Affiliated Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin, China

  • Yun Gu,

    Affiliation Department of Geriatric, Longhua Hospital of Shanghai University of Traditional Chinese Medicine, Tianjin, China

  • Jiakui Guo,

    Affiliation Department of Acupuncture and Cerebropathy, Second Affiliated Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin, China

  • Di Wu,

    Affiliation Department of Traditional Chinese Medicine, Tianjin Huanhu Hospital, Tianjin, China

  • Qiang Li,

    Affiliation Graduate Institutes, Tianjin University of Traditional Chinese Medicine, Tianjin, China

  • Wanshan Song

    Affiliation Department of Acupuncture and Cerebropathy, Second Affiliated Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin, China

Cognitive Improvement during Treatment for Mild Alzheimer’s Disease with a Chinese Herbal Formula: A Randomized Controlled Trial

  • Yulian Zhang, 
  • Cuiru Lin, 
  • Linlin Zhang, 
  • Yuanwu Cui, 
  • Yun Gu, 
  • Jiakui Guo, 
  • Di Wu, 
  • Qiang Li, 
  • Wanshan Song
PLOS
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Correction

25 Jun 2018: Zhang Y, Lin C, Zhang L, Cui Y, Gu Y, et al. (2018) Correction: Cognitive Improvement during Treatment for Mild Alzheimer's Disease with a Chinese Herbal Formula: A Randomized Controlled Trial. PLOS ONE 13(6): e0199895. https://doi.org/10.1371/journal.pone.0199895 View correction

Abstract

Objectives

To explore the efficacy of Chinese herbal formula compared with donepezil 5mg/day in patients with mild Alzheimer’s disease (AD).

Methods

Patients with mild AD meeting the criteria were randomized into Chinese herbal formula Yishen Huazhuo decoction (YHD) group and donepezil hydrochloride (DH) group during the 24-week trial. The outcomes were measured by ADAS-cog, MMSE, ADL, and NPI with linear mixed-effect models.

Results

144 patients were randomized. The mean scores of ADAS-cog and MMSE in both YHD group and DH group both improved at the end of the 24-week treatment period. The results also revealed that YHD was better at improving the mean scores of ADAS-cog and MMSE than DH. Linear mixed-effect models with repeated measures showed statistical significance in time × group interaction effect of ADAS-cog and also in time × group interaction effect of MMSE. The data showed YHD was superior to DH in improving the scores and long term efficacy.

Conclusions

Our study suggests that Chinese herbal formula YHD is beneficial and effective for cognitive improvement in patients with mild AD and the mechanism might be through reducing amyloid-β (Aβ) plaque deposition in the hippocampus.

Trial Registration

Chinese Clinical Trial Registry ChiCTR-TRC-12002846

Introduction

Alzheimer’s disease (AD), a complex neurodegenerative disease, is the major cause of dementia in the elderly, affecting more than 25 million people worldly [1]. AD is characterized by a progressive and irreversible deterioration of cognitive and functional abilities, leading to complete dependence. The cause for most cases is still unknown, and there exist several hypotheses to explain it, such as genetic heritability [2, 3], cholinergic hypothesis [4], amyloid hypothesis [5], Tau hypothesis [6], and other hypotheses [7, 8].

At present, in Europe, the United States, and Asia, the mainstream medication for AD is typically acetylcholine esterase inhibitor (AchEI), such as donepezil hydrochloride (DH) [9]. Though it has shown significantly clinical efficacy and safety in the treatment of AD, symptomatic improvement is limited in magnitude and duration, after which patients continue to decline [10]. Therefore, new therapeutic agents are essential.

Natural products have been used for medicine for a long time, and much research effort has been made to develop the anti-AD agents from natural sources [11]. Traditional Chinese Medicine (TCM) has a long history of preventing and treating cognitive decline [12] and the efficacy of Chinese herbs to improve cognitive function has been studied in many trials [1316]. Icariin(C33H40O15; molecular weight 676.65) is a flavonoid isolated from Epimedii herba (Yinyanghuo) and it is also the major pharmacological active component of Epimedii herba, a traditional Chinese herb widely used as a tonic and antirheumatic remedy. It has been reported before that icariin has vasodilatory and cardioprotective effects. Recently it was reported that icariin could improve the spatial learning and memory abilities in aluminum-intoxicated rats and decrease the level of amyloid-β (Aβ) in the hippocampus of aluminum-intoxicated rats [17]. Moreover, icariin could improve the learning and memory abilities in Aβ25–35-induced Alzheimer's disease rats through decreasing the production of insoluble fragments of Aβ [18].

Tetramethylpyrazine (TMP, C8H12N2; molecular weight 136.19), a major component of a Chinese herb Ligusticum wallichi Franchat (chuanxiong), has been widely used in the treatment of cerebrovascular diseases. Also, several studies have demonstrated that TMP could improve learning and cognitive function, through inhibiting calcium overload, anti-apoptotic activity, and anti-inflammatory potential [19, 20]. A few reports showed that Astragali Radix(Huangqi) extract could affect brain function. For example, astragalosides, the major components of Astragali Radix, could improve memory in aged mice [21]. Also, extracts of Astragali Radix could ameliorate the memory deficit in mice caused by Ab25–35 [22].

Therefore, our group developed a Chinese herbal formula Yishen Huazhuo decoction(YHD) to treat mild AD, which is composed of Yinyanghuo (Epimedium), Nvzhenzi(Fructus Ligustri Lucidi),Buguzhi (Psoralea fruit), Heshouwu (Radix Polygoni Multiflori), Huangqi (Radix Astragali), Chuanxiong(Ligusticum wallichi Franchat), and Shichangpu(Acorus gramineus). Our previous pharmacological studies have shown that the formula can improve the cognitive function of mice with AD.

Here, we conducted a 24-week, randomized, double-blind, double-dummy, and multicenter trial to explore the efficacy and safety of the Chinese herbal formula in improving cognitive function in patients with mild AD.

Methods and Materials

We did the 24 weeks, randomized, double-blind, double-dummy, DH-controlled and multicenter trial (ChiCTR-TRC-12002846), which was conducted between May 1st, 2011 and May 30th, 2014 in three centers, the Second Affiliated Hospital of Tianjin University of TCM, Tianjin Huanhu Hospital, and Longhua Hospital of Shanghai University of TCM, and was approved by the Ethics Committee of the Second Affiliated Hospital of Tianjin University of TCM in July 26th, 2011(S1 Fig). The trial was performed according to the principles of the Declaration of Helsinki.

We confirm that all ongoing and related trials for this drug/intervention are registered. The trial is funded by Ministry of Science and Technology of China and was one of the 973 Program (2010CB530405). Due to time limitations, we started the recruitment and the registration simultaneously in May, 2011. We tried to register the study on the U.S. Food and Drug Administration (FDA), but failed to get access to it after months of effort. We then decided to register on the Chinese Clinical Trial Registry in March 2012 and after months of complex procedures, we finally succeeded.

Patients

Patients were enrolled from outpatient clinics. Inclusion criteria for this study were: 1) a diagnosis of dementia of the Alzheimer’s type according to the Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV; APA, 1994; provided in S2 Trial Protocol (English)). The diagnosis was confirmed by image test (CT/MRI) and diagnosis procedures among three hospitals were standardized; 2) women or men aged 50–85 years; 3) Hachinski Ischemic Score (HIS) ≤4; 4) Hamilton Depression Rating Scale (HAMD) score of ≤7; 5)written informed consent from all patients (or their legal representatives); 6) Clinical Dementia Rating (CDR) score of 1.

Exclusion criteria included: 1) with vascular dementia or any neurological disorder other than AD that contributed substantially to dementia; 2) with severe heart, liver, kidney and blood system diseases (sinus bradycardia and atrioventricular block, AST and ALT 2 times more than the upper limit of normal values; kidney function tests showing BUN 1.5 times higher than the upper limit of normal values, Cr more than the normal values); 3)allergies or allergic to donepezil hydrochloride and Piperidine Derivatives; 4) the use of any drugs that may affect cognitive function 4 weeks prior to randomization; 5) uncontrolled hypertension; 6) with diseases that may interfere with cognitive tests such as aphasia, hemiplegia, and others(severe visual or hearing loss); 7) with serious complications (asthma and chronic obstructive pulmonary disease(COPD)); 8) participation in another investigational new drug trial; 9) with advanced, severe disease that could interfere with study assessments.

Study design

Due to the differences in appearance of the drugs, dosage, and administration methods between YHD and DH, we used a double-blind and double-dummy design. The simulations were composed of amylum. YHD-simulation, produced and quality controlled by Shenzhen Sanjiu Modern Chinese Medicine Limited Company (Tianjin, China), has the same package, color, smell with YHD; DH-simulation, produced by Eisai China Inc. (Tianjin, China), has the same package, appearance, color, and taste with DH. Patients in both groups should take drugs and simulations simultaneously.

Selection for a treatment group was determined by a computer-generated randomization list under the help of a professional statistician, in a 1:1 ratio in all three centers, and the random number table was sealed in a special envelope. The researchers, drug administrators, and patients were all unaware of the blind design. The evaluators and the statisticians were not involved in the trial.

Drug administration

The TCM formula in our study is YHD, including Yinyanghuo (Epimedium) 10g, Nvzhenzi(Fructus ligustri lucidi) 10g, Buguzhi (Psoralea fruit) 10g, Heshouwu (Radix polygoni multiflori)10g, Huangqi (Radix astragali) 10g, Chuanxiong(Ligusticum wallichi franchat) 6g, and Shichangpu(Acorus gramineus) 6g in one unit(S1 Table). Each herb was provided as herbal concentrate-granules in one bag (the procedure is in S1 Methods and Materials) and quality controlled by Shenzhen Sanjiu Modern Chinese Medicine Limited Company (Tianjin, China). Aristinic acid is not included in YHD decoction. Every patient in YHD group orally took 100 ml of the decoction once a day half an hour after breakfast and DH-simulation 5 mg before sleep for 24 weeks. Patients in DH group orally took DH, provided by Eisai China Inc. (Tianjin, China) 5 mg each day before sleep, and YHD-simulation 100 ml of the decoction half an hour after breakfast for 24 weeks. The lot number of Chinese herbs, DH, and the simulations are provided in S2 Table.

Assessment

The primary outcomes were the Alzheimer’s Disease Assessment Scale-cognitive subscale (Chinese version) (ADAS-cog) and Mini-Mental State Examination (Chinese version) (MMSE) measured at week 0, 12, 24, and 48. The ADAS-cog, a widely used cognitive assessment instrument in AD clinical trials, consists of memory, language, orientation, and praxis assessments. Its scores range from 0 to 70, with higher values indicating higher degree of deficit. MMSE, commonly used in medicine to screen for dementia and to estimate the severity of cognitive impairment, includes simple questions in a number of areas: the time and place of the test, repeating lists of words, language use and comprehension, and basic motor skills. Any score greater than or equal to 27 points (out of 30) indicates a normal cognition. Below this, scores can indicate severe (≤9 points), moderate (10–18 points) or mild (19–24 points) cognitive impairment [23].

The secondary outcome measurements included the Activity of Daily Living Scale (Chinese version) (ADL), a 20-item questionnaire designed to measure the patient’s ability to carry out daily activities such as medication management, food preparation, personal hygiene, and transportation utilization, with each item rating between 0 and 10; and the Neuropsychiatric Inventory (Chinese version) (NPI) to assess the severity of symptoms in 10 behavioral domains which includes delusions, hallucinations, agitation/aggression, dysphoria, anxiety, euphoria, apathy, disinhibition, irritability/lability, and aberrant motor behavior. The total score ranges from 0 to 120, where higher values denote higher severity of symptoms [24, 25].

The safety parameters including spontaneously reported adverse events (AEs) or serious AEs (SAEs), vital signs (temperature, heart rate and blood pressure), physical examination, and laboratory tests were assessed during each visit.

Sample size

According to the reported trial [26], a decrease of 0.67±6.29 (Mean±SD) in ADAS-cog score could be measured after the 24-week treatment by DH 5mg/d. Normally, non-inferiority margin should be less than 1/2 (50%) of the standard deviation [27], thus we set δ as 1/4 (25%) of the standard deviation. With α at 0.05, β = 0.2 (80% power), δ = 1.5, and s = 4, we need 89 patients in each group. Assuming 15% attrition, this inflated the sample size to 103 per group. However, considering the numbers of outpatients in three centers, long term trial, and expenditures, after consulting clinical doctors and professional statistician, we decided to enroll 150 patients, 50 from the Second Affiliated Hospital of Tianjin University of TCM, 30 from the Tianjin Huanhu Hospital, and 70 from the Longhua Hospital of Shanghai University of TCM.

Statistical analysis

Statistical analyses for our trial were performed using the Statistical Package for the Social Sciences (SPSS) v19.0 software. Statistical testing was two-sided and p < 0.05 was considered statistically significant. Descriptive statistics were presented for the baseline characteristics. Chi square test was applied to comparable count data, and independent samples t test to measurement data. Efficacy outcome parameters were analyzed on an intent-to-treat (ITT) basis. The missing data were not disposed. Linear mixed-effect models were used to analyze repeated measures; group and time were entered as fixed effects while group ×time as interaction effect; the measurement indexes were dependent variables and the patients were random variables. The underlying covariance structure was UN covariance structure. Two-independent-samples t test was referred to when the difference met normal distribution.

All AEs were coded using a World Health Organization (WHO)-based dictionary of preferred terms. The numbers (percentages) of patients with at least one AE, at least one serious AE (SAE), and at least one AE leading to discontinuation were summarized irrespective of relationship to treatment. All safety-related observations were summarized using descriptive statistics.

Results

The first patient was screened in May 17th, 2011 and the last patient completed the study in May 30th, 2014. A total of 144 patients were enrolled into the trial (Fig 1), 48 from the Second Affiliated Hospital of Tianjin University of TCM, 24 from the Tianjin Huanhu Hospital, and 72 from the Longhua Hospital of Shanghai University of TCM. There was no statistically significant difference at baseline between the patients in the two groups (Table 1). A total of 104 patients (72.2%) completed the study, 54 (75%) in the YHD group and 50(69.4%) in the DH group.

The major components of YHD decoction

There were eight major categories of compounds in the YHD decoction. Flavones (icariin, epimedin C, ligustroside), anthraquinones (emodin, physcion, emodin-8-o-β-D-glucoside, physcion-8-o-β-D-glucoside), polyhydroxy phenols (2,3,5,4’-tetrahydroxy stilbene-2-o-glycoside TSG), triterpenoid sapnins (oleanolic acid, astragaloside I, astragaloside II, astragaloside III, isoastragaloside I, isoastragaloside II), monoterpenes (linalool), phenylpropanoids (α asarone, β asarone), coumarin (psoralen, isopsoralen), and alkaloids (tetramethylpyrazine TMP) were in the decoction, among which icariin, TSG, β asarone, astragalosides, and TMP were the major components (S2 Fig).

Outcome Measures

The mean±SD scores of both primary and secondary outcomes (including ADAS-cog, MMSE, ADL, and NPI) of the patients in the two groups are shown in Table 2.

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Table 2. Mean efficacy scores at all time points and change at week 24, and 48 (Mean±SD).

https://doi.org/10.1371/journal.pone.0130353.t002

Linear mixed-effect models with repeated measures showed interaction of ADAS-cog and MMSE scores during the 24-week treatment, but no interaction of ADL and NPI. A significant time × group interaction effect was observed in ADAS-cog (F110.186 = 3.164, p = 0.027) (Table 3); the scores in YHD group significantly reduced in week 12 and 24, while the scores of DH group declined in week 24(p = 0.061). The mean scores of ADAS-cog showed a decrease of 3.10±4.55 in YHD group and a decrease of 1.22±4.99 in DH group during the 24 weeks treatment, with statistical significance. The follow-up at week 48 showed that after the treatment, compared with week 24, the scores of ADAS-cog increased 1.45±3.74 in YHD group, but less than the increase of 4.13±8.62 in DH group (p<0.05) (Fig 2).

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Fig 2. Scores of ADAS-cog, MMSE, ADL, NPI in YHD and DH group.

A. ADAS-cog scores in groups of YHD and DH; B. MMSE scores in groups of YHD and DH; C. ADL scores in groups of YHD and DH; D. NPI scores in groups of YHD and DH.

https://doi.org/10.1371/journal.pone.0130353.g002

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Table 3. Linear mixed effect model estimates of fixed effects (ADAS-cog, MMSE, ADL, NPI).

https://doi.org/10.1371/journal.pone.0130353.t003

Both YHD and DH could improve the scores of ADL and NPI, but without statistically significant difference between groups. After dropping the insignificant group × time interaction, there was still no statistically significant difference between groups.

Safety

There were no statistically significant differences between the two groups with respect to the incidence of adverse events. There were 5 REs in YHD group and 6 in the other group (Table 4) and most AEs were mild, most frequent in diarrhea (2) and catching cold (2). Both YHD group and DH group reported a cerebral infarction, but they were not considered to be related to the treatment. Adverse events caused two drop-outs (2.8%) in the DH group and two (2.8%) in the YHD group.

There was no clinically significant change in patients of the two groups in physical examination and vital signs during the study.

Discussion

Donepezil is an established, well-received and effective drug, approved for the treatment of mild to moderate and severe AD [28]. It is available to increase cognitive function as measured by the ADAS-cog [29]. Considering that its subsequent improvement after the treatment has been reported to decrease [30,31], we tried to use Chinese herbal formula to treat mild AD based on the long term use in preventing and treating cognitive decline.

In this study, we assessed the impact of Chinese herbal formula YHD on patients with mild AD compared with DH at 5 mg/day. We speculate that YHD may function in attenuating Aβ neurotoxicity and reducing the Aβ plaque deposition in the hippocampus which is a key pathological event in AD. In this formula, icariin, TSG, β asarone, astragalosides, and TMP are the major components. Icariin, the major component extracted from Epimedium, has been reported to have cardiac protective effects [32], anti-inflammatory effects [33], and even anti-tumor properties [34]. Also, it has been repeatedly shown that icariin has effects on transgenic models of AD. It can attenuate Aβ neurotoxicity [35], reduce the Aβ burden and amyloid plaque deposition in the hippocampus [36], and also has positive effects on cognition [37], learning, and memory [38, 39]. Experimental evidence indicates that β-asarone, the major ingredient of Acorus gramineus, has neuroprotective effects. It can also ameliorate impairment of learning and memory in rats by antagonizing neurotoxicity of Aβ [40, 41]. TMP may be useful in ameliorating microglia-mediated inflammatory by Aβ [42], and studies also suggest it could remarkably enhance learning and memory in AD mice model [43]. TSG has exhibited neuroprotection against brain ischemic injury [44] and can improve cognitive deficits in aged rats [45] according to several studies. Furthermore, the protective effects of TSG on learning and memory deficits in transgenic mice [46], and in Aβ-induced AD mice [47] have been reported.

The results show that YHD can improve cognitive function according to the scores of ADAS-cog, MMSE, ADL, and NPI. Moreover, Chinese herbal formula YHD shows statistically significant superiority over DH 5mg/day in improving the scores of ADAS-cog at all time points during the 24-week treatment, demonstrating evidence of better efficacy. Also, at the follow up in week 48, the scores of YHD measured by ADAS-cog and MMSE show less deterioration than DH, which suggests a possible longer-term symptomatic benefit.

The ADAS-cog is the best suitable for assessing mild-to-moderate stages of dementia [48, 49]. In this study, the ADAS-cog score shows a better improvement in the YHD group than that in the DH group. However, we cannot say that YHD is better than DH in changing cognitive function. Donepezil hydrochloride administrated in our study was at 5 mg/day, an approved dosage for the treatment of mild to moderate AD. It has already been confirmed that the efficacy of donepezil is dose-dependent [5052]. In earlier studies, the ADAS-cog score showed significant improvement at donepezil 23 mg/d or 10 mg/d compared with 5mg/d [5356].

Both YHD and DH are well-tolerated. Cases of cerebral infarction in the YHD group, catching cold in both groups and constipation in DH group are not considered to be related to the drugs. Due to the small sample, our results could not provide significant evidence to prove which drug is better in safety.

It should be noted that our study have some limitations. First, the small sample size leads to underpowered results. Second, our study lack control group with higher dosage of donepezil hydrochloride such as 10mg/day and a placebo control group to exclude the influence from a possible large placebo effect to get further comparison. Third, it is uncertain whether the findings in Han Chinese can be extrapolated to other populations.

Conclusions

Our study suggests that Chinese herbal formula YHD is beneficial and effective for improvement in cognitive deficits in patients with mild AD, and the mechanism might be by reducing the Aβ plaque deposition in the hippocampus. If larger sample size studies could confirm the findings, we could apply the treatment into clinical practice to improve the cognitive deficits for patients with mild AD.

Supporting Information

S1 Fig. Approved file of Ethical Committee.

The trial was approved by the Ethics Committee of the Second Affiliated Hospital of Tianjin University of TCM in July 26, 2011.

https://doi.org/10.1371/journal.pone.0130353.s003

(TIF)

S2 Fig. The main chemicals in the decoction of YHD.

https://doi.org/10.1371/journal.pone.0130353.s004

(TIF)

S1 Methods and Materials. Quality control of the herbal concentrate-granules, Procedure of herbal concentrate-granules, and Direction of herbal concentrate-granules intake.

https://doi.org/10.1371/journal.pone.0130353.s005

(DOC)

S2 Table. Lot number of Chinese herbs, DH, and the simulations.

https://doi.org/10.1371/journal.pone.0130353.s007

(DOCX)

Acknowledgments

We acknowledge the patients and their caregivers taking part in our study.

The study investigators are the following: Zhen Zhou, Jiachun Xu, Haijiao Li, Kai Wang, Kaili Fu, Cheng Li, Xiujuan Wang, He Yuan, Fubin Guo, Xigang Sun, Yingchun Xu, Kai Huang, and Sujing Hang.

Author Contributions

Conceived and designed the experiments: YZ CL LZ. Performed the experiments: LZ YC QL WS. Analyzed the data: JG DW YG. Contributed reagents/materials/analysis tools: JG YC. Wrote the paper: YZ CL. Patient follow-up: DW QL WS. Manuscript revision: DW QL WS.

References

  1. 1. Brookmeyer R, Johnson E, Ziegler-Graham K, Arrighi HM. Forecasting the global burden of Alzheimer’s disease. Alzheimer's & dementia, 2007, 3(3): 186–191.
  2. 2. Gatz M, Reynolds CA, Fratiglioni L, Johansson B, Mortimer J A, Berg S, et al. Role of genes and environments for explaining Alzheimer disease. Arch Gen Psychiatry, 2006,63(2):168–74. pmid:16461860
  3. 3. Waring SC, Rosenberg RN. Genome-wide association studies in Alzheimer disease. Archives of Neurology, 2008,65(3):329–34. pmid:18332245
  4. 4. Francis PT, Palmer AM, Snape M, Wilcock GK. The Cholinergic Hypothesis of Alzheimer's Disease: a Review of Progress. Journal of Neurology, Neurosurgery, and Psychiatry, 1999,66(2):137–47. pmid:10071091
  5. 5. Hardy J, Allsop D. Amyloid Deposition as the Central Event in the Aetiology of Alzheimer's Disease. Trends in Pharmacological Sciences. 1991,12(10):383–88. pmid:1763432
  6. 6. Goedert M, Spillantini MG, Crowther RA. Tau Proteins and Neurofibrillary Degeneration. Brain Pathology (Zurich, Switzerland). 1991,1(4):279–86. pmid:1669718
  7. 7. Itzhaki RF, Wozniak MA. Herpes Simplex Virus Type 1 in Alzheimer's Disease: The Enemy Within. Journal of Alzheimer's Disease. 2008 [Retrieved 5 February 2011],13(4):393–405. pmid:18487848
  8. 8. Shcherbatykh I, Carpenter DO. The Role of Metals in the Etiology of Alzheimer's Disease. Journal of Alzheimer's Disease. 2007,11(2):191–205. pmid:17522444
  9. 9. Feldman H, Gauthier S, Hecker J, Vellas B, Subbiah P, Whalen E, et al. A 24-week, randomized, double-blind study of donepezil in moderate to severe Alzheimer’s disease. Neurology, 2001, 57(4): 613–620. pmid:11524468
  10. 10. Sabbagh M, Cummings J. Progressive cholinergic decline in Alzheimer's Disease: consideration for treatment with donepezil 23 mg in patients with moderate to severe symptomatology. BMC neurology, 2011, 11(1): 21.
  11. 11. Wang Y, Huang L, Tang X, Zhang HY. Retrospect and prospect of active principles from Chinese herbs in the treatment of dementia. Acta Pharmacologica Sinica, 2010, 31(6): 649–664. pmid:20523337
  12. 12. Han Y, Lin L, Xi CT. Treating senile dementia with Traditional Chinese Medicine. Clin Interv Aging, 2007, 2(2):201–208. pmid:18044136
  13. 13. Zhang S J, Xue Z Y. Effect of Western medicine therapy assisted by Ginkgo bilobatablet on vascular cognitive impairment of none dementia. Asian Pacific journal of tropical medicine, 2012, 5(8): 661–664. pmid:22840457
  14. 14. Liu P, Kong M, Liu S, Chen G, Wang P. Effect of reinforcing kidney-essence, removing phlegm, and promoting mental therapy on treating Alzheimer's disease. Journal of Traditional Chinese Medicine, 2013, 33(4): 449–454. pmid:24187864
  15. 15. Bi M, Tong S, Zhang Z, Ma Q, Zhang S, Luo Z, et al. Changes in cerebral glucose metabolism in patients with mild-to-moderate Alzheimer's disease: A pilot study with the Chinese herbal medicine fuzhisan. Neuroscience letters, 2011, 501(1): 35–40. pmid:21741446
  16. 16. Miao Y C, Tian J Z, Shi J, Mao M. Effects of Chinese medicine for tonifying the kidney and resolving phlegm and blood stasis in treating patients with amnestic mild cognitive impairment: a randomized, double-blind and parallel-controlled trial. Zhong xi yi jie he xue bao = Journal of Chinese integrative medicine, 2012, 10(4): 390. pmid:22500712
  17. 17. Luo Y, Nie J, Gong Q H, Lu Y F, Wu Q, Shi JS. Protective effects of icariin against learning and memory deficits induced by aluminium in rats. Clin. Exp. Pharmacol. Physiol. 2007,34(8), 792–795. pmid:17600559
  18. 18. Nie J, Luo Y, Huang X N, Gong Q H, Wu Q, Shi J S. Icariin inhibits beta-amyloid peptide segment 25–35 induced expression of β-secretase in rat hippocampus. European journal of pharmacology, 2010, 626(2): 213–218. pmid:19782061
  19. 19. Fan LH, Wang KZ, Cheng B, Wang C S, Dang X Q. Antiapoptotic and neuroprotective effects of tetramethylpyrazine following spinal cord ischemia in rabbits. BMC Neurosci. 2006,7(1):48–52.
  20. 20. Cheng X R, Zhang L, Hu J J, Sun L, Du G H. Neuroprotective effects of tetramethylpyrazine on hydrogen peroxide-induced apoptosis in PC12 cells. Cell Biol Int, 2007,31:438–43. pmid:17321170
  21. 21. Lei H, Wang B, Li WP, Yang Y, Zhou A W,Chen M Z. Anti-aging effect of astragalosides and its mechanism of action. Acta Pharmacol Sin,2003, 24(3): 230–234. pmid:12617771
  22. 22. Tohda C, Tamura T, Matsuyama S, Komatsu K. Promotion of axonal maturation and prevention of memory loss in mice by extracts of Astragalus mongholicus. British journal of pharmacology, 2006, 149(5): 532–541. pmid:16981006
  23. 23. Mungas D. In-office mental status testing: a practical guide. Geriatrics, 1991, 46 (7): 54–58, 63, 66. pmid:2060803
  24. 24. Cummings J L, Mega M, Gray K, Rosenberg-Thompson S, Carusi D A, Gornbein J. The Neuropsychiatric Inventory comprehensive assessment of psychopathology in dementia. Neurology, 1994, 44(12): 2308. pmid:7991117
  25. 25. Cummings J L, Fairbanks L, Masterman D L. Strategies for analysing behavioural data in clinical trials involving patients with Alzheimer's disease. The International Journal of Neuropsychopharmacology, 1999, 2(1): 59–66. pmid:11281971
  26. 26. Rogers S L, Farlow M R, Doody R S, Mohs R, Friedhoff L T. A 24-week, double-blind, placebo-controlled trial of donepezil in patients with Alzheimer's disease. Neurology, 1998,50(1):136–145. pmid:9443470
  27. 27. Lange S, Freitag G. Choice of delta: requirements and reality—results of a systematic review. Biom J, 2005,47(1):12–27, 99–107. pmid:16395993
  28. 28. Burns A, Rossor M, Hecker J, Gauthier S, Petit H, Moller H J, et al. The Effects of Donepezil in Alzheimer’s Disease—Results from a Multinational Trial. Dementia and geriatric cognitive disorders, 1999, 10(3): 237–244. pmid:10325453
  29. 29. Cummings J L. Alzheimer’s disease. N Engl J Med. 2004, 351(1):56–67. pmid:15229308
  30. 30. Rogers S L, Doody R S, Mohs R C, Friedhoff L T. Donepezil improves cognition and global function in Alzheimer disease: a 15-week, double-blind, placebo-controlled study. Archives of Internal Medicine, 1998, 158(9): 1021–1031. pmid:9588436
  31. 31. Rogers S L, Friedhoff L T. Long-term efficacy and safety of donepezil in the treatment of Alzheimer's disease: an interim analysis of the results of a US multicentre open label extension study. European Neuropsychopharmacology, 1998, 8(1): 67–75. pmid:9452942
  32. 32. Song Y H, Cai H, Gu N, Qian C F, Cao S P, Zhao Z M. Icariin attenuates cardiac remodelling through down-regulating myocardial apoptosis and matrix metalloproteinase activity in rats with congestive heart failure. J Pharm Pharmacol, 2011; 63(4): 541–549. pmid:21401606
  33. 33. Xu CQ, Liu BJ, Wu JF, Xu Y C, Duan X H, Cao Y X, et al. Icariin attenuates LPS-induced acute inflammatory responses: involvement of PI3K/Akt and NF-κB signaling pathway. Eur J Pharmacol, 2010; 642(1–3): 146–153. pmid:20638948
  34. 34. Zhou J, Wu J, Chen X, Fortenbery N, Eksioglu E, Kodumudi K N, et al. Icariin and its derivative, ICT, exert anti-inflammatory, anti-tumor effects, and modulate myeloid derived suppressive cells (MDSCs) functions. Int Immunopharmacol, 2011, 11(7): 887–895.
  35. 35. Zeng K W, Ko H, Yang H O, Wang X M. Icariin attenuates-amyloid-induced neurotoxicity by inhibition of tauprotein hyperphosphorylation in PC12 cells. Neuropharmacology, 2010, 59: 542–550. pmid:20708632
  36. 36. Zhang L, Shen C, Chu J, Zhang R, Li Y, Li L. Icariin Decreases the Expression of APP and BACE-1 and Reduces the β-amyloid Burden in an APP Transgenic Mouse Model of Alzheimer's Disease. International journal of biological sciences, 2014, 10(2): 181–191. pmid:24550686
  37. 37. Wu B, Chen Y, Huang J, Ning Y, Bian Q, Shan Y, et al. Icariin improves cognitive deficits and activates quiescent neural stem cells in aging rats. J. Ethnopharmacol. 2012, 142(3), 746–753. pmid:22687254
  38. 38. Li F, Gong Q H, Wu Q, Lu Y F, Shi J S. Icariin isolated from Epimedium brevicornum Maxim attenuates learning and memory deficits induced by d-galactose in rats. Pharmacol Biochem Behav. 2010, 96(3), 301–305. pmid:20566405
  39. 39. Luo Y, Nie J, Gong Q H, Lu Y F, Wu Q, Shi J S. Protective effects of icariin against learning and memory deficits induced by aluminium in rats. Clin. Exp. Pharmacol. Physiol. 2007, 34(8), 792–795. pmid:17600559
  40. 40. Yu T G, Cheng C L, Ji C L, Xing G, Zhou L, Dong M, et al. Beta-Asarone Improves Cognitive Function by Suppressing Neuronal Apoptosis in the Beta-Amyloid Hippocampus Injection Rats. Biol. Pharm. Bull. 2010,33(5): 836–843. pmid:20460763
  41. 41. Ji C L, Cheng C, Gui H X, Zhou L, Dong M, Geng Y, et al. Beta-Asarone Attenuates Neuronal Apoptosis Induced by Beta Amyloid in Rat Hippocampus. Yakugaku Zasshi, 2010,130(5):737–746. pmid:20460873
  42. 42. Kim M, Kim S, Lee M, Lee J H, Jung W S, Moon S K, et al. Tetramethylpyrazine, a natural alkaloid, attenuates pro-inflammatory mediators induced by amyloid β and interferon-γ in rat brain microglia. European Journal of Pharmacology, 2014,740:504–511. pmid:24975095
  43. 43. Zhao L, Wei MJ, He M, Jin W B, Zhao H S, Yao W F. The effects of tetramethylpyrazine on learning and memory abilities of mice with Alzheimer disease and its possible mechanism. Chin Pharmacol Bull, 2008,24(8):1088–92.
  44. 44. Wang T, Gu J, Wu PF, Wang F, Xiong Z, Yang Y J, et al. Protection by tetrahydroxystilbene glucoside against cerebral ischemia: involvement of JNK, SIRT1, and NF-kappaB pathways and inhibition of intracellular ROS/RNS generation. Free Radic. Biol. Med, 2009,47(3): 229–240. pmid:19272442
  45. 45. Wang R, Tang Y, Feng B, Ye C, Fang L, Zhang L, et al. Changes in hippocampal synapses and learning-memory abilities in age-increasing rats and effects of tetrahydroxystilbene glucoside in aged rats. Neuroscience, 2007, 149(4): 739–746. pmid:17935895
  46. 46. Zhang L, Xing Y, Ye CF, Ai H X, Wei H F, Li L. Learning-memory deficit with aging in APP transgenic mice of Alzheimer’s disease and intervention by using tetrahydroxystilbene glucoside. Behav. Brain Res, 2006,173(2): 246–254. pmid:16901557
  47. 47. Zhou L, Hou Y, Yang Q, Du X, Li M, Yuan M, et al. Tetrahydroxystilbene glucoside improves the learning and memory of amyloid-β(1–42)-injected rats and may be connected to synaptic changes in the hippocampus. Can. J. Physiol. Pharmacol, 2012, 90: 1446–1455. pmid:23181273
  48. 48. Nadkarni NK, Black SE. Cognitive outcomes; in Rockwood K, Gauthier S (eds): Trial Designs and Outcomes in Dementia Therapeutic Research. Abingdon, Taylor & Francis, 2006:85–112. pmid:22081655
  49. 49. Mohs R C, Marin D, Green C R, Davis KL. The Alzheimer’s disease assessment scale: modifications that can enhance its use in future clinical trials. Alzheimer Disease. Birkhäuser Boston, 1997: 407–411.
  50. 50. Whitehead A, Perdomo C, Pratt R D, Birks J, Wilcock G K, Evans J G. Donepezil for the symptomatic treatment of patients with mild to moderate Alzheimer’s disease: a meta-analysis of individual patient data from randomised controlled trials. Int J Geriatr Psychiatry, 2004, 19(7):624–633. pmid:15254918
  51. 51. Homma A, Imai Y, Tago H, Asada T, Shigeta M, Iwamoto T, et al. Donepezil treatment of patients with severe Alzheimer’s disease in a Japanese population: results from a 24-week, double-blind, placebo-controlled, randomized trial. Dement Geriatr Cogn Disord, 2008, 25(5):399–407. pmid:18391486
  52. 52. Nozawa M, Ichimiya Y, Nozawa E, Utumi Y, Sugiyama H, Murayama N, et al. Clinical effects of high oral dose of donepezil for patients with Alzheimer’s disease in Japan. Psychogeriatrics, 2009, 9(2):50–55. pmid:19604325
  53. 53. Sasaki S, Horie Y. The Effects of an Uninterrupted Switch from Donepezil to Galantamine without Dose Titration on Behavioral and Psychological Symptoms of Dementia in Alzheimer's Disease. Dementia and Geriatric Cognitive Disorders Extra, 2014, 4(2): 131–139. pmid:24987402
  54. 54. Sabbagh M, Cummings J, Christensen D, Doody R, Farlow M, Liu L, et al. Evaluating the cognitive effects of donepezil 23 mg/d in moderate and severe Alzheimer’s disease: analysis of effects of baseline features on treatment response. BMC geriatrics, 2013, 13(1): 56.
  55. 55. Yatabe Y, Hashimoto M, Kaneda K, Honda K, Ogawa Y, Yuuki S, et al. Efficacy of increasing donepezil in mild to moderate Alzheimer's disease patients who show a diminished response to 5 mg donepezil: a preliminary study. Psychogeriatrics, 2013, 13(2): 88–93. pmid:23909965
  56. 56. Farlow M R, Salloway S, Tariot P N, Yardley J, Moline M L, Wang Q, et al. Effectiveness and tolerability of high-dose (23 mg/d) versus standard-dose (10 mg/d) donepezil in moderate to severe Alzheimer’s disease: a 24-week, randomized, double-blind study. Clin Ther, 2010, 32(7):1234–1251. pmid:20678673