Efficacy Study of Yi-Zhi-An-Shen Granules For Older Adults With Amnestic Mild Cognitive Impairment: a Randomized, Double-blind, Placebo-controlled Study
Phase: PHASE1
Conditions: Cognitive Dysfunction
Sponsor: Chengdu University of Traditional Chinese Medicine

Study Protocol with SAP 
 Efficacy Study of Yi-Zhi-An-Shen Granules For Older Adults With 
Amnestic Mild Cognitive Impairment: a Randomized, Double-blind, 
Placebo-controlled Study 
Protocol approved by Medical Ethics Review Board of 
Teaching Hospital of Chengdu University of TCM 
April 14, 2018 

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Efficacy Study of Yi-Zhi-An-Shen Granules for Older Adults with 
Amnestic Mild Cognitive Impairment 
Study Protocol with SAP 
 
1. Objectives 
The primary objective of this study is to evaluate the efficacy and safety of Yi-Zhi-An-
Shen Granules (YZASG) in optimizing cognitive performance over time in elderly 
individuals with amnestic mild cognitive impairment (aMCI). Secondly, the 
investigators intend to assess whether YZASG can improve sleep quality among aMCI 
patients. Finally, participants’ fecal genomic DNA will be extracted to analyze the gut 
microbial differences between individuals with aMCI and ones with normal cognition. 
 
2. Design 
This study is a randomized, double-blind, placebo-controlled trial with 16-week 
intervention and 18-month follow-up assessment. The design met the principles of the 
Declaration of Helsinki, and was approved by the Ethics Committee of Teaching 
Hospital of Chengdu University of Traditional Chinese Medicine. Then signed informed 
consents will be obtained from each participant.  
The enrolled participants will be randomly assigned into the YZAS group or the placebo 
group with the allocation ratio of 1:1 using the statistical package. The unique code 
will be assigned to each newly enrolled participant and preserved in the trial 
management board. A statistician expert will act as the coder and be shielded from 
subject recruitment. 
 
3. Methods 
3.1 Participants 

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The investigators anticipate recruiting 80 participants from Teaching hospital of 
Chengdu University of Traditional Chinese Medicine, community, and nursing homes. 
All patients will undergo standard medical examination and neuropsychological testing 
to ensure correct diagnosis of aMCI.  
The inclusion criteria are (1) subject has assigned informed consent to participate in 
the study and continues to give willing consent for participation; (2) age from 60 to 85 
years with a diagnosis of aMCI; (3) educational level of at least 6 years; (4) availability 
of a "study partner" who can assist in completing rating scales for the duration of the 
study; (5) cognitive complaints reported by the subject and confirmed by the "study 
partner"; (6) clinical Dementia Rating (CDR) global score of 0.5, and memory item score 
of 0.5; (7) mini-mental state examination (MMSE) score of 24-30; (8) Diagnostic and 
Statistical Manual of Mental Disorders, Version 5 (DSM-V) criteria of dementia not 
fulfilled 
The exclusion criteria are as follows: (1) has been previously enrolled in this study and 
received the investigational product; (2) has received an investigational product within 
30 days prior to screening; (3) has received disease-modifying therapy; (4) has a known 
allergy to the study drug or any of its constituents; (5) has a history of alcohol abuse 
or alcohol dependency in the 3 years prior to study entry, or is an alcoholic or drug 
addict, as determined by the investigator; (6) has ongoing clinically significant (as 
judged by the investigator), metabolic or any other disease that could currently cause 
impaired memory (e.g., untreated thyroid disease, vitamin or other nutritional 
deficiencies, chronic kidney, or liver disease); (7) memory impairment that can be 
attributed to a disease or condition other than an early phase neurodegenerative 
syndrome; (8) has a parkinsonian movement disorder; (9) use of psychoactive 
medications that would affect the subject's ability to reliably perform neurocognitive 
testing or create uncertainty in distinguishing between the effects of the psychoactive 
medication and the subject's underlying cognitive impairment (e.g., benzodiazepines, 
sedatives, antipsychotics); (10) with history of major recurrent depressive disorder 
(DSM-V) within the last 5 years prior to screening; (11) has a brain tumor or other 

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intracranial lesion, a disturbance of cerebral spinal fluid circulation (e.g., normal 
pressure hydrocephalus), and/or a significant history of head trauma or brain surgery; 
(12) has signs of major cerebrovascular disease, with score of modified Hachinski 
Ischemia Score (mHIS) at more than 4, or as verified by medical history and/or brain 
MRI or CT; (13) has severe visual or hearing impairments that cannot cooperate with 
examinations; (14) has severe digestive system diseases; (15) has received antibiotics 
within 60 days prior to screening. 
Handling of withdrawal and dropout: (1) Voluntarily withdrawal, (2) loss of follow-up, 
(3) poor compliance and presence of severe adverse effects, (4) revealing and 
uncovering blind in urgency, (5) misdiagnosis, (6) using forbidden drugs or treatments 
in the course of the trial, (7) taking no medication during the trial, (8) no evaluable 
records after medication. Reasons for withdrawing participants will be recorded in 
CRFs, and the last data will be included in data analysis. 
3.2 Interventions 
Besides receiving the same basic treatment, including health education, moderate 
aerobic exercise (30-60 minutes per day) and general nutritional support, participants 
assigned to the YZAS group will take Yi-Zhi-An-Shen granule, which is composed of 
YiZhiRen (Alpinia oxyphylla Miq.) - 5g, SanQi (Panax notoginseng) - 3g, ChuanXiong 
(Ligusticum chuanxiong hort) - 10g, ZhiZi (Gardenia jasminoides Ellis) - 10g, YuJin 
(Curcuma longa L.) - 10g, DanZhuYe (Lophatherum gracile) - 10g, while ones in the 
placebo group will take placebo made from starch which appears the same shape, 
color, smell, taste, texture package and Lot Number. Participants will be instructed to 
dissolve granules into 100ml of boiled water and to take the solution orally between 
30℃ to 37 ℃ three times daily for 16 weeks. Each granule is prepared by Sichuan 
Neo-green Pharmaceutical Technology Development Co., Ltd, Sichuan China according 
to the standards of Good Manufactory Practice (GMP). 
80% to 120% of drug usages are eligible for protocol plan. The package, drug name, 
function and indication, usage and dosage, storage condition, valid period and name 

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of the manufacturer will be marked and a tag indicating ‘trial use’ will be attached. 
Drugs must be kept in the appropriate temperature in a dry, cool and shady place. Drug 
administrators should return unused drugs to estimate participant compliance and 
record these in the CRFs. 
3.3 Outcomes 
3.3.1 Primary outcome 
Cognitive decline is measured using the Chinese version of the Alzheimer Disease 
Assessment Scale-Cognitive subscale (ADAS-cog11). The total possible score is 70. A 
score increase indicates greater severity of impairment. The specific hypothesis is that 
the change from baseline to endpoint will be significantly less than that for placebo. 
The ADAS-cog will be assessed at baseline (before intervention), at 16 weeks (the end 
of intervention) and 6, 12, and 18 months after intervention. 
3.3.2 Secondary outcome 
Secondary measures includes the MMSE, MoCA (Changsha Version), CDR, PSQI, ADLs, 
and gut microbiome. 
 The MMSE is an 11-question measure that tests five areas of cognitive function 
(orientation, registration, attention and calculation, recall, and language). The 
maximum score is 30 and a score below 24 is considered abnormal for dementia 
screening. 
 The MoCA (Changsha Version) will also be used to evaluate general cognitive 
function, as it contains visuospatial processing and organizational capability which 
can make up for the shortcoming of the MMSE. The total score for it is 30, with a 
higher number indicating a more intact cognitive function. Meanwhile, the MoCA 
has been shown to be a promising tool to detect MCI and early AD. 
 The CDR will be used as an assistant evaluation for patients’ dementia severity, 
which scored 0-3, with higher scores indicating more severity. It is a semi-
structured interview performed with the patient and caregiver (informant), 

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characterizing six domains of cognitive and functional performance. The CDR sum 
of boxes (CDR-SB) scored 0-18, will also be applied to assess patients’ cognitive 
status, with higher scores indicating worse functioning. 
 The Pittsburgh Sleep Quality Index (PSQI) will be used to assess participants’ 
comprehensive quality of sleep, which involves sleep quality, sleep duration, sleep 
efficiency, sleep disorders, daytime dysfunction, sleeping aids, etc. The total score 
for it is 21, with a higher score indicating a worse sleep quality. 
 ADLs will be assessed including basic activities of daily living (BADL) and 
instrumental activities of daily living (IADL). An individual's BADL will be evaluated 
mainly by the subjects' performance from the perspectives of bathing, dressing, 
grooming, initiation, toileting and feeding, with six items and a sum of scores 
ranging from 0 (normal) to 24 (complete dependence on others). And modified 
Lawton Instrumental Activities of Daily Living Scale will be used to measure the 
IADL of a subject, with eight items and a sum of scores ranging from 0 (normal) to 
32 (complete dependence on others). 
These clinical tests will be administrated by a trained, certified clinician or rater 
experienced in the assessment of patients with cognitive deficits. And the rater who 
will conduct the CDR for a patient cannot complete any other rating scales for the same 
patient, and will be blinded to the results of all other neuropsychological scales. All the 
questionnaires will be assessed at baseline (before intervention), at 8 and 16 weeks 
(during intervention) and 6, 12, and 18 months after intervention. 
 Fecal genomic DNA will be extracted from frozen stools using QIAamp DNA mini 
stool kit (Qiagen, Hiden, Germany), obtained from the patients with aMCI and 15-
20 participants with normal cognition at baseline. After PCR amplification, DNA 
fragments will be sequenced on an Illumina HiSeq 2500 instrument and an 
Illumina HiSeq X instrument for 16SrDNA and metagenomics analyses respectively 
at Biomarker Technologies Co, Ltd (Beijing, China) to analyze the differences in gut 
microbiome between patients with aMCI and individuals with normal cognition. 

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When it is available, the investigators will also assess the change in gut 
microbiome between the treatment group and the placebo group after the 
intervention of YZASG in the same way. 
3.3.3 Other outcomes 
To assess the safety of YZASG compared to placebo in subjects with aMCI, the 
investigators will record the incidence and severity of treatment-emergent adverse 
events (TEAEs), and clinically important changes in safety assessment results (including 
vital signs, weight, clinical laboratory tests, physical and neurological exams, ECGs, and 
MRIs). 
4. Statistical considerations 
4.1 Sample size 
Using pre-intervention and post-intervention scores obtained from Miao et al. [1], 33 
patients are needed for inclusion in the treatment group to achieve a statistical power 
of 90%. Allowing for a maximum dropout rate of 20%, the number of subjects in the 
treatment group has been set to 40 patients with aMCI. With the allocation ratio of 
1:1, 40 subjects will be randomized to the placebo group. 
4.2 Statistic analysis 
Analysis will be conducted by a statistician of the National Clinical Trial Center of 
Chinese Medicine (Chengdu, China), who will be blinded to the whole trial and using 
SAS 9.2 software (Cary, NC, USA) and SPSS 19.0 software (IBM, NY, USA).  
Unless otherwise specified, all mechanism, efficacy and safety analyses will be strictly 
conducted according to the intention-to-treat (ITT) principle and the full analysis set 
(FAS) population, the per-protocol set (PPS) population and the safety set (SS) 
population. Missing values will be replaced by the last observation carried forward 
(LOCF) method. ADAS-cog (including its monomial item) changes from baseline and 
the secondary outcomes will be assessed using an analysis of covariance with 
treatment groups as factors and baseline values as covariates. Standardized mean 

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differences will be used to express effect sizes in SD. The baseline homogeneity of the 
baseline characteristics between the two groups will be analyzed with Fisher’s exact 
test, or 𝒳2 test for categorical measures and with the t test or Wilcoxon rank-sum test 
for continuous measures. The statistical significance is defined as a one-sided P-value 
of <0.05 and 95% confidence interval. 
For the gut microbiome analysis, sequenced data will be interpreted using the 
bioinformatics tools programmed in the Ion Reporter software. QIIME algorithms will 
determine the bacterial diversity within a sample (α diversity) and among all the 
samples (β diversity). Additionally, multivariate data analysis with principal component 
analysis on the diversity indexes and comparisons of genus and species level data will 
be performed to reveal differences in the microbial composition between individuals 
with normal cognition and ones with aMCI. The Benjamini–Hochberg false discovery 
rate adjustment will be used to account for the number of taxa tested in each 
comparison.