Study of the Therapeutic Effects of Naohuan Dan and Idebenone in Treating Mild Cognitive Impairment With Kidney Deficiency and Phlegm Stasis
Conditions: Mild Cognitive Impairment
Sponsor: Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University

Clinical Research Protocol
Project Name: Study of the Therapeutic Effects of Naohuan Dan
and Idebenone in Treating Mild Cognitive Impairment With Kidney
Deficiency and Phlegm Stasis
Bidding unit: Sun Yat-sen Memorial Hospital, Sun Yat-sen
University
Version number: v1.0
Version date: 30/08/2022
Project manager: Qihui Huang

1 Program Summary
This study conducted a retrospective analysis of 64 patients with mild cognitive
impairment of the kidney deficiency phlegm and stasis type who underwent treatment
with Naohuan Dan combined with Idebenone. The patients' cognitive function was
evaluated using the Mini-Mental State Examination (MMSE) and the Montreal
Cognitive Assessment (MoCA) before and after treatment. Daily living abilities were
assessed using the Activities of Daily Living (ADL) scale, depression status was
evaluated using the Geriatric Depression Scale (GDS), and the severity of Traditional
Chinese Medicine (TCM) syndrome was assessed using the TCM diagnostic scale.
Peripheral blood from the patients was also collected for analysis of neuron-specific
enolase (NSE) and inflammatory factors. The aim was to evaluate the safety and
feasibility of using Naohuan Dan combined with Idebenone for the treatment of mild
cognitive impairment of the kidney deficiency phlegm and stasis type.
2 Introduction
Mild cognitive impairment (MCI), a transitional state between aging and dementia,
is clinically characterized by a decline in cognitive function[1], but daily living
abilities remain unaffected or mildly impaired. Previous studies have shown that 50%
of MCI patients will progress to dementia within 3-4 years of onset, and the
proportion of individuals progressing to dementia increases with age and disease
duration[2]. The exact pathogenesis of MCI is not yet fully understood, and there is a
lack of effective clinical treatments[3]. Cognitive impairment in MCI patients can
severely impact their quality of life and burden families and society.
MCI can be divided into amnestic and non-amnestic types, with amnestic MCI
mainly characterized by memory impairment and non-amnestic MCI mainly
characterized by cognitive impairments such as language, visual-spatial recognition,
and executive function[4]. Main neurological and psychiatric symptoms include
anxiety, depression, apathy, and sleep disturbances. A meta-analysis using a
random-effects model showed that the cumulative incidence rate of dementia for MCI

patients aged 65 and above after 2 years was 14.9%. As the aging of the population
accelerates in China and life expectancy gradually increases, the incidence rate of
MCI is on the rise.
MCI belongs to the category of "forgetfulness", "fondness for remembering", and
"dementia" in traditional Chinese medicine, with the pathological basis of marrow
decline, brain consumption, and loss of function of the nerve machinery. It is a
syndrome of virtual and actual deficiency, and the condition is complex. At present,
there are several single herbs in traditional Chinese medicine that can improve mild
cognitive impairment (MCI), such as ginkgo biloba, acorus calamus, ginseng, and
polygala[5], which have effects on inhibiting neuronal apoptosis, promoting neuronal
metabolism, and reducing amyloid deposition, thereby improving cognitive function.
A meta-analysis[6] found that oral administration of Ginkgo biloba leaf extract
preparations for MCI treatment is clinically effective and can effectively prevent the
incidence of dementia, but its safety still needs further exploration. In addition, single
herbs have the drawback of having a single target point and cannot effectively
improve cognitive function in MCI. Chinese medicine compound formulas have
multi-target pharmacological effects and multi-level actions. Haifeng Wang[7] found
that the TCM for invigorating the kidney and activating blood circulation, which is
composed of several Chinese herbs, can improve the executive function and memory
of MCI patients, and its mechanism of action may be related to strengthening the
body's ability to resist oxidative stress and reducing the damage of free radicals to
neurons. Lijing Yang[8] found that the Chuanpu Yizhi Tang compound formula can
reduce the levels of serum Hcy and AchE, improve the cognitive function and daily
life ability of MCI patients, and has good clinical efficacy. In addition, traditional
Chinese medicine techniques such as acupuncture, traditional exercises, and massage
have shown certain efficacy in treating MCI, which is worth further exploration and
promotion.
Based on the above research background, we believe that the combination of
Naohuan Dan combined with Idebenone has great application prospects for the
treatment of Kidney Deficiency Phlegm Stagnation Type MCI. To this end, we have

designed a clinical study to evaluate the efficacy and safety of the combination of
Naohuan Dan combined with Idebenone for the treatment of MCI, by assessing
relevant cognitive function rating scales and TCM syndrome scores. This study aims
to provide valuable information for the integration of TCM and Western medicine in
the treatment of MCI.
3 Research Objectives and Endpoints
3.1 Main purpose
This study evaluated the efficacy of Naohuan Dan combined with Idebenone for
treating MCI patients with Kidney-Deficient and Phlegm-Stasis syndrome. Changes
in cognitive function, daily living abilities, depression levels, and Traditional Chinese
Medicine clinical symptoms were assessed using relevant rating scales, and compared
with the efficacy of Idebenone alone, demonstrating the advantages of combining
traditional Chinese and Western medicine in treatment.
3.2 Main outcome
The primary outcome measure for the study was the Mini-Mental State
Examination (MMSE) scale scores [9], which were used to examine the cognitive
function of the participants before and after therapy. A total score of 23-30 is
considered to indicate normal cognitive function, 17-22 indicates mild cognitive
impairment, 11-16 indicates moderate cognitive impairment, and 10 or below
indicates severe cognitive impairment.
The clinical efficacy was evaluated based on the MMSE scale, and the efficacy was
calculated by comparing the scores before and after the therapy. The following criteria
were used to determine the efficacy: (i) Markedly effective: ≥20% improvement in
MMSE score. (ii) Effective rate: ≥12% improvement in MMSE score. (iii) Ineffective:
<12% improvement in MMSE score[10]. The total efficacy was calculated as the sum
of the markedly effective and effective rates.

3.3 Secondary outcomes
The secondary outcome measures for the study included various scales and
serological indicators. The scales used in the study were the Montreal Cognitive
Assessment (MoCA) scale, the ADL scale, the Geriatric Depression Scale (GDS) and
the TCM syndrome score scale.
The MoCA scale is a widely used screening tool for detecting mild cognitive
impairment. It assesses various cognitive domains such as attention, memory,
visuospatial skills, language, and orientation[11]. The total score on the MoCA scale is
30, and a score of 26 or higher is considered to indicate normal cognitive function.
The ADL scale is used to assess the participants’ ability to perform daily living
activities[12]. The total score on the ADL scale is 100 points.
The GDS scale is a self-report assessment tool used to detect and measure the
severity of depression in older adults[13]. The total score on the GDS scale is 30 points.
The TCM clinical symptoms of the participants were evaluated using the TCM
syndrome score scale[14]. This tool assesses the severity of specific TCM symptoms
and divides them into four grades: none, mild, moderate, and severe, with 0, 2, 4, and
6 points (for main symptoms) or 0, 1, 2, and 3 points (for secondary symptoms),
respectively. The total score was calculated before and after the therapy to determine
the effectiveness of the treatment.
Blood samples were collected from the participants before and after the therapy via
cubital venipuncture. Enzyme-linked immunosorbent assay (ELISA) was used to
determine the serum levels of neuron-specific enolase (NSE), interleukin-8 (IL-8) and
tumor necrosis factor-alpha (TNF-α) using the Reader M3 multi-function microplate
reader.
3.4 Safety Index
Before and after treatment, the patient's elbow vein blood is collected for blood
routine, urine routine, stool routine, and liver and kidney function tests. The patient's
electrocardiogram is also monitored, and any adverse reactions after medication are
observed and treated accordingly.

4 Research Population
4.1 Inclusion criteria
The following inclusion criteria were used: (1) diagnosis in line with Chinese
medicine and Western medicine; (2) suitable for age 55-85 years old, gender is not
limited; (3) a score between 21-26 on Mini-Mental State Examination (MMSE), 1
point for primary school education; (4) agree to take part in this experiment and sign
the informed consent form.
4.2 Exclusion criteria
The following exclusion criteria were used: (1) patients with severely impaired
heart, liver, kidney and other functions; (2) patients with neurological diseases that
affect brain function and cognitive impairment; (3) patients with severe depression
and mental illness; (4) patients with poor compliance and cooperation who failed to
complete the study according to the protocol.
4.3 Lifestyle and Precautions
During the course of treatment, if the patient has comorbidities such as
hypertension, diabetes and hyperlipidemia, their original treatment plan should be
maintained,
and
appropriate
symptom-relieving
treatments
such
as
blood
pressure-lowering, hypoglycemic and lipid-lowering drugs should be provided.
Smoking and drinking should be prohibited during the treatment period, and regular
exercise and a light diet should be maintained.
4.4 Recruitment and Retention Strategies
At the outpatient and ward departments of Traditional Chinese Medicine and
Rehabilitation of Sun Yat-sen Memorial Hospital, eligible MCI patients with the type
of kidney deficiency and phlegm stasis were identified according to the inclusion
criteria. After fully communicating trial-related information with the patients, they

were informed of the possible adverse reactions and risks, and asked to sign an
informed consent form voluntarily and informed about the trial plan.
Retention strategies: (1) Researchers should maintain one-on-one communication
with patients as much as possible. (2) Clear trial-related information should be
provided to patients during the first communication, along with a process calendar,
and patients should be informed of the next appointment time and reminded through
phone calls. (3) Researchers can communicate the current progress with patients at
appropriate times to help them fully recognize their contribution to the research. (4)
Sufficient work should be done to obtain informed consent during the screening phase
and to answer any questions patients may have.
5 Research Design
5.1 Intervention
This retrospective, two-armed, single-center clinical observation evaluates the
combination of Naohuan Dan and Idebenone for the treatment of MCI patients with
Kidney Deficiency and Phlegm Stasis. Eligible patients were randomized using a
random number table into a control group and a treatment group. The control group
was given 30mg of Idebenone tablets three times a day after meals, while the
treatment group was given Naohuan Dan in addition to Idebenone. The main
components of the Naohuan Dan formula are Rehmannia glutinosa 25g, Codonopsis
pilosula 20g, Acorus calamus 10g, Polygala tenuifolia 8g, Eucommia ulmoides 15g,
and Poria cocos 12g, among other ingredients. The herbal granules were provided by
Yifang Pharmaceutical Co., Ltd. The patients in both groups received corresponding
treatments for underlying diseases such as hypertension and diabetes if present. One
dose of herbal granules was administered daily with warm water in the morning and
evening.

Fig. 1 CONSORT flow chart.
5.2 Research Calendar
Program
Screening
period
-7 to -1 day
Enrollment/Base
line Visit 1, Day
1
Study Visit 2
7+/-1 day
Study Visit 3
14+/-1 day
Study Visit 4
21+/-1 day
Study Visit 5
28+/-1 day
Study Visit 6
35+/-1 day
Study Visit 7
42+/-1 day
Study Visit 8
49+/-1 day
Study Visit 9
56+/-1 day
Study Visit 10
63+/-1 day
Study Visit 11
70+/-1 day
Study Visit 12
77+/-1 day
Study Visit 13
84+/-1 day
General
Information
Access
√
Informed
Consent
√
Medical History
√
Random
Grouping
√
Medication
√
√
√
√
√
√
√
√
√
√
√
√
√

5.3 Definition of End of Study
When the last patient who met the inclusion and exclusion criteria completed the
prescribed course of drug treatment, there were no obvious adverse reactions and
events in the last follow-up, and the corresponding data were collected as the end of
the study.
5.4 Statistical Analysis
5.4.1 sample size
This study is a retrospective, two-arm study with a fixed sample size and no strict
hypothesis testing. A total of 64 participants were enrolled in this study.
5.4.2 Data Analysis Set
Full Analysis Set (FAS) analyzed all cases who were enrolled and received at least
The
MMSE
scale
√
√
The MoCA scale
√
√
The ADL scale
√
√
The GDS scale
√
√
The
TCM
Syndrome score
scale
√
√
Lab Test
√
√
Blood
routine,
Urine
routine,
Stool routine and
Liver
and
Kidney function
√
√
Electrocardiogra
m
√
√
Adverse
Events
and Evaluation
√
√
√
√
√
√
√
√
√
√
√
√
√
√
Complete
the
Case
Report
Form
√
√
√
√
√
√
√
√
√
√
√
√
√
√

one dose of the study drug, according to the intention-to-treat (ITT) principle. For
cases where the entire treatment process could not be observed, the last observed data
was carried forward to the final results of the trial (LOCF).
Per-protocol Set (PPS) included all cases that complied with the trial protocol, had
good compliance, did not use any prohibited medication during the trial, and
completed the required case report form. No data imputation was conducted for
missing data. The efficacy of the medication was analyzed for both FAS and PPS.
Safety Analysis Set (SAS) consisted of all enrolled cases who had received at least
one dose of the study drug and had safety records available after medication use. This
dataset was used for safety analysis.
5.4.3 Statistical Analysis
All data were analyzed using SPSS25.0 statistical software. For continuous
variables, median (minimum, maximum) was used for descriptive statistics; mean ±
standard deviation was used if the data followed a normal distribution and had
homogeneous variance. Paired t-test was used for within-group comparisons, and two
independent sample t-tests were used for between-group comparisons. Wilcoxon rank
sum test was used for non-normal distributed data. For categorical variables,
frequency (percentage) was used for descriptive statistics, and rates or composition
ratios were used for comparisons between groups using the χ² test. Rank sum test was
used for ordinal data. A value of P＜0.05 was considered statistically significant.
6 Research Intervention
6.1 Intervention
The intervention was administered as follows: (1) The control group received
Idebenone (Shenzhen Neptunus
Pharmaceutical
Co., Ltd.,
National
standard:
H10970362, Specification: 30mg) orally, at a dose of 30mg per time, three times a day,
after meals for 12 consecutive weeks. (2) The treatment group received Naohuan Dan
combined with Idebenone. The main components of traditional Chinese medicine

were Codonopsis Radix 20g (Batch number: 1031653), Radix Rehmanniae Preparata
25g (Batch number: 1050973), Drynariae Rhizoma 15g (Batch number: 0117293),
Acori Tatarinowii Rhizoma 10g (Batch number: 1021283), Radix Polygaiae 8g (Batch
number: 9086013), Poria Cocos 12g (Batch number: 1051133), etc. The traditional
Chinese medicine granules were provided by Guangdong Yifang Pharmaceutical Co.,
Ltd., at a dose of one per day, delivered with warm water in the morning and evening,
and taken orally for 12 consecutive weeks. (3) Basic and symptomatic treatments
were given to both groups according to the patient's underlying diseases and
complications, such as antihypertensive and hypoglycemic treatments.
6.2 Study Intervention Adherence
Patients who are able to take medication on time and in the correct dosage, attend
regular hospital follow-ups, do not take prohibited drugs, and cooperate with medical
evaluations during the course of treatment are considered to have good compliance.
Methods to improve compliance include: (1) providing medication records and
instructing patients to record medication intake daily, which can be returned during
the next follow-up visit; (2) providing detailed explanations and addressing patient
questions during the first medication distribution, while affirming the patient's
contribution to research; (3) issuing medication in short-term doses and reminding
patients of their next follow-up appointment in advance.
7 Study Intervention Discontinuation/Subject Discontinuation and
Withdrawal
7.1 Study Intervention Discontinuation
The enrolled patients may discontinue treatment at any time for any reason or, in
case of any adverse event, at the discretion of the investigator. Furthermore, the
investigator may stop the treatment of a patient if the patient is deemed unsuitable for
treatment, violates the study protocol, or for management and/or safety reasons.
The subject must discontinue treatment but may continue to be monitored in the

study if any of the following occur: 1) the patient or the patient's family requests
discontinuation of treatment; 2) an intercurrent illness which interferes with further
treatment occurs; 3) the investigator determines that the patient should be withdrawn
from the study; 4) poor compliance by the patient; 5) the investigator believes that
continuing to administer the investigational drug to the subject would place the
subject at unnecessary risk based on the subject's medical condition or personal
situation; 6) completion of the treatment as outlined in the protocol.
7.2 Subject discontinuation/withdrawal from the study
If a patient or their family withdraws their informed consent to participate in the
study, the enrolled patient must be withdrawn from the study. If a patient withdraws
from the study, they will no longer receive treatment or participate in planned visits. If
a patient is lost to follow-up, they must also be withdrawn from the study.
7.3 Lost to follow-up
Timely education and reminders should be provided to enrolled patients. The next
follow-up appointment should be scheduled during each visit, and the patient's contact
information should be recorded to facilitate timely communication, enhance
medication adherence and patient follow-up compliance, and reduce data loss due to
patient loss to follow-up. If there is data loss due to patient loss to follow-up, it should
be excluded from data analysis and appropriately documented.
8 Adverse Events and Unexpected Events
8.1 Adverse event
Adverse events (AE) refer to all adverse medical events that occur in subjects after
receiving the investigational drug. They can manifest as symptoms, signs, diseases, or
laboratory abnormalities, but are not necessarily causally related to the investigational
drug.
The grading of adverse events is as follows:

Grade 1: Mild; asymptomatic or mild symptoms; only clinical or diagnostic
manifestations; no intervention required;
Grade 2: Moderate; requires minimal, partial, or non-invasive intervention; restricts
appropriate age daily activities;
Grade 3: Severe or significant medical events that require drug treatment, but do
not immediately threaten life; hospitalization or prolonged hospitalization; disability;
restricts activities of daily living (ADLs);
Grade 4: Life-threatening consequences; requires urgent intervention;
Grade 5: Death related to AE.
8.2 Serious Adverse Event
Serious adverse events (SAEs) are adverse events that meet at least one of the
following criteria:
1. Results in death, excluding death related to progression of the disease under
investigation.
2. Puts the subject's life at risk ("life-threatening" as defined by the fact that the
event places the subject at risk of death at the time of the AE, not necessarily that
death is a possible outcome if the condition worsens).
3. Results in permanent or significant disability/incapacity.
4. Other medically important events: defined as an event that endangers the subject
or requires medical intervention to prevent any of the above outcomes from
happening.
9 Data collection and management
All patient clinical testing reports in this project strictly adhere to the legally
prescribed national unit of measurement. The patient testing and follow-up records
must be complete, including the date, recorded items, results, and normal value ranges,
among other information, and must be signed by relevant personnel. Clinical case data
and follow-up data are recorded and collected by trained and professional data

collection and management personnel, and reviewed and confirmed for accuracy by
the team leader before being entered into the database.
10 Ethical Requirements
This study follows the "Guidelines for Good Clinical Practice" and "Management
Measures for Clinical Trials Initiated by Researchers in Medical Institutions" (trial)
regulations, as well as the Helsinki Declaration. Before the trial can begin, the study
protocol must be approved by the hospital ethics committee. If revisions to the
protocol are necessary during the study, the updated version must be resubmitted to
the ethics committee for review, and the researcher can proceed only with their
approval.
Every patient who is enrolled in the study must sign an informed consent form.
Copies of the informed consent form and contact information for the researcher and
ethics committee must be provided to patients upon request. This study will collect
clinical data and personal information for scientific research purposes, which may
involve patient privacy. All participants and data analysts involved in the study have
signed confidentiality agreements and will not disclose any patient information or
disease-related information to any unrelated individuals or organizations. The
collected patient data will be managed uniformly to prevent personal privacy from
being leaked.
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