Efficacy and safety of Huatan Qushi Huoxue Fang granules on obese non-alcoholic fatty liver disease: study protocol for a multicenter, randomized, double-blind, placebo-controlled trial

Abstract

Background
The global burden of non-alcoholic fatty liver disease (NAFLD) is parallel to the increasing obesity rates around the world. Phlegm stasis syndrome is a common traditional Chinese medicine syndrome type of obese NAFLD, which is often treated by resolving phlegm, dispelling dampness, and promoting blood circulation. This study mainly explores the clinical efficacy and safety of Huatan Qushi Huoxue Fang (HTQSHXF) granules in the treatment of obese NAFLD.

Methods
This is a multicenter, randomized, double-blind, placebo-controlled clinical trial that will recruit 248 obese NAFLD patients from three hospitals in China. Randomly allocate patients to either the HTQSHXF group or the placebo group in a 1:1 ratio. The intervention phase lasts for 12 weeks. The primary outcome will be the change in relative liver fat content from baseline to week 12 measured by Magnetic resonance proton density fat fraction (MRI-PDFF). The secondary outcomes will be Body fat percentage (BFR), Waist to hip ratio (WHR), Body Mass Index (BMI), Controlled attenuation parameter (CAP), Liver tiffness value (LSM), serum liver function, blood lipids, blood glucose, Free fatty acids (FFA), Cytokeratin 18-M30 (CK18-M30), and Cytokeratin 18-M65 (CK18-M65). The results will be monitored at baseline and 12 weeks of intervention. Adverse events that occur in this study will be promptly managed and recorded.

Discussion
This study will use more recognized quantitative methods to explore the efficacy and safety of HTQSHXF granules in treating obese NAFLD, providing clinical evidence for its translational application.

Trial registration
http://www.chictr.org.cn . Trial number: ChiCTR2200060901. Registered on 14 Jun 2022.

Full text

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Ecacy and safety of Huatan Qushi Huoxue Fang
granules on obese non-alcoholic fatty liver disease:
study protocol for a multicenter, randomized,
double-blind, placebo-controlled trial
Lihui Zhang
The First Aliated Hospital of Henan University of Chinese Medicine
Sutong Liu
The First Aliated Hospital of Henan University of Chinese Medicine
Qing Zhao
The First Aliated Hospital of Henan University of Chinese Medicine
Xiaoyan Liu
The First Aliated Hospital of Henan University of Chinese Medicine
Xuehua Sun
Shuguang Hospital
Tao Wang
Shuguang Hospital
Fenping Li
Shaanxi Provincial Hospital of Traditional Chinese Medicine
Miaoqing Ye
Shaanxi Provincial Hospital of Traditional Chinese Medicine
Minghao Liu
The First Aliated Hospital of Henan University of Chinese Medicine
Wenxia Zhao
The First Aliated Hospital of Henan University of Chinese Medicine
Study protocol
Keywords: Huatan Qushi Huoxue Fang, Randomized controlled trial, Non-alcoholic fatty liver disease,
Obese, Traditional Chinese medicine
Posted Date: September 4th, 2024

Page 2/21
DOI: https://doi.org/10.21203/rs.3.rs-4814797/v1
License:   This work is licensed under a Creative Commons Attribution 4.0 International License. 
Read Full License
Additional Declarations: No competing interests reported.

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Abstract
Background
The global burden of non-alcoholic fatty liver disease (NAFLD) is parallel to the increasing obesity rates
around the world. Phlegm stasis syndrome is a common traditional Chinese medicine syndrome type of
obese NAFLD, which is often treated by resolving phlegm, dispelling dampness, and promoting blood
circulation. This study mainly explores the clinical ecacy and safety of Huatan Qushi Huoxue Fang
(HTQSHXF) granules in the treatment of obese NAFLD.
Methods
This is a multicenter, randomized, double-blind, placebo-controlled clinical trial that will recruit 248 obese
NAFLD patients from three hospitals in China. Randomly allocate patients to either the HTQSHXF group
or the placebo group in a 1:1 ratio. The intervention phase lasts for 12 weeks. The primary outcome will
be the change in relative liver fat content from baseline to week 12 measured by Magnetic resonance
proton density fat fraction (MRI-PDFF). The secondary outcomes will be Body fat percentage (BFR),
Waist to hip ratio (WHR), Body Mass Index (BMI), Controlled attenuation parameter (CAP), Liver tiffness
value (LSM), serum liver function, blood lipids, blood glucose, Free fatty acids (FFA), Cytokeratin 18-M30
(CK18-M30), and Cytokeratin 18-M65 (CK18-M65). The results will be monitored at baseline and 12
weeks of intervention. Adverse events that occur in this study will be promptly managed and recorded.
Discussion
This study will use more recognized quantitative methods to explore the ecacy and safety of HTQSHXF
granules in treating obese NAFLD, providing clinical evidence for its translational application.
Trial registration
http://www.chictr.org.cn . Trial number: ChiCTR2200060901. Registered on 14 Jun 2022.
Background
Non alcoholic fatty liver disease (NAFLD) is a chronic progressive liver disease caused by overnutrition
and insulin resistance in genetically susceptible individuals [1]. Its disease spectrum includes non-
alcoholic fatty liver, non-alcoholic steatohepatitis (NASH), and related brosis and cirrhosis [2]. With the
prevalence of obesity and type 2 diabetes, the global prevalence and incidence rate of NAFLD are
increasing[3]. Moreover, NAFLD and metabolic syndrome are mutually causal, jointly promoting liver
decompensation and the development of malignant tumors such as hepatocellular carcinoma [4].
Therefore, NAFLD has become an increasingly serious global public health issue [5]. Obesity is an

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independent risk factor for NAFLD. Obese and overweight patients have a three fold increased risk of
developing NAFLD compared to non obese or lean individuals [6]. The prevalence of NAFLD among
overweight and obese populations worldwide is 70.0% and 75.3%, respectively [7].
Long term excessive consumption of highly greasy or sweet rened foods or foods with strong avors
can lead to impaired spleen function. Water and grains cannot be effectively converted into essence, qi,
blood, and body uids, leading to the formation of grease and fat [8]. They stop in the fascia cavity with
the ow of qi and blood, resulting in obesity [9]. When grease and fat accumulate in the liver, it can cause
stagnation of liver qi, preventing liver from effectively performing its functions of dredging and regulating
[10]. This leads to the production of pathological products such as phlegm, dampness, and blood stasis.
Phlegm, dampness, and blood stasis intertwine in the liver, further damaging its function [11]. Therefore,
phlegm, dampness, and blood stasis are the main pathological factors of NAFLD. This study recruited
obese NAFLD patients with phlegm stasis syndrome, with the diagnostic criteria for this syndrome based
on the expert consensus on the Chinese medical diagnosis and treatment of nonalcoholic fatty liver
disease (2017) formulated by the Gastroenterology Branch of the China Association of Chinese Medicine
[12].
Traditional Chinese Medicine has certain advantages in the treatment of NAFLD. Previous multi-center
clinical studies have shown that a comprehensive Traditional Chinese Medicine (TCM) treatment plan
based on the use of HTQSHXF granules can signicantly improve clinical symptoms such as abdominal
distension, discomfort in the right hypochondrium, and fatigue in patients with NASH [13]. It also
increases the liver/spleen CT ratio, reduces Body Mass Index (BMI), and decreases serum Alanine
aminotransferase (ALT) and Triglyceride (TG) levels [13]. Experimental studies have shown that
HTQSHXF granules can improve hepatic steatosis and inammatory injury in rat models of NASH,
reduce serum levels of ALT, Aspartate aminotransferase (AST), Total cholesterol (TC), TG, and Fasting
blood glucose (FBG), and lower Free fatty acids (FFA) levels in liver homogenates. The therapeutic
mechanism may be related to the activation of the ADPN/AMPK/ACC and ADPN/AKT/NF-κB signaling
pathways [14, 15]. The herbal drugs of HTQSHXF granules is shown in Table1. However, we have not yet
used a double-blind design to observe the effects of HTQSHXF granules on Magnetic resonance proton
density fat fraction (MRI-PDFF), Controlled attenuation parameter (CAP), and other indicators in NAFLD
patients across multiple medical institutions. This study aims to explore the clinical ecacy and safety
of HTQSHXF granules in obese NAFLD patients using a multi-center, randomized, double-blind design.

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Table 1
Standard formulation of HTQSHXF granules
Pinyin
name Latin scientic
name Amount
(%) TCMH action
Zexie
Alismatis
Rhizoma
24.78 promoting diuresis and eliminating dampness, as
well as regulating qi ow and dissipating blood
stasis
Danshen
Salvia miltiorrhiza
Bunge
12.40 invigorating blood circulation to remove blood
stasis, cooling the blood, and calming the spirit
Yujin
Radix Curcumae
Aromaticae
12.40 invigorating blood ow to alleviate pain, regulating
qi ow, and relieving depression
Haizao
Sargassum
12.40 resolving phlegm, eliminating dampness, softening
hard masses, and eliminating phlegm
Juemingzi
Catsia tora Linn
8.26 clears the liver and improves vision
Shanzha
Crataegus
pinnatida
12.40 eliminates food stagnation and dissipates blood
stasis
Shuifeiji
Silybum
marianum
12.40 clears heat and detoxies
Chaihu
Radix Bupleuri
4.96 soothing the liver and regulating qi, acts as the
guiding herb to introduce the prescription into the
liver meridian
TCMH traditional Chinese medicine herb
Methods
Study design
This is a multicenter, randomized, double-blind, placebo-controlled parallel clinical trial study, aiming to
recruit 248 obese NAFLD patients with phlegm-dampness and blood stasis. 88 cases were allocated to
the First Aliated Hospital of Henan University of TCM, 80 cases to Hepatology Department of
Shuguang Hospital, Shanghai University of TCM, and 80 cases to the Hepatology Department of Shaanxi
Provincial Hospital of CM. Cases in each center were allocated in a 1:1 ratio between the HTQSHXF
group and placebo group. Under the condition of dietary and exercise management, the HTQSHXF group
will take oral HTQSHXF granules, while the placebo group will take oral placebo. The treatment duration
is 12 weeks. The schedule details are listed in Table2. This protocol was approved by the Medical Ethics
Committee of The First Aliated Hospital of Henan University of TCM (approval number: 2022HL-038-
01). The trial protocol (version 1.0, Jun 14, 2022) and was registered in the Chinese Clinical Trial Registry
(ChiCTR2200060901). The owchart of the trial is shown in Fig.1. We present the following article in
accordance with the SPIRIT 2013 reporting checklist [16]. For more details about the checklist, please
see Additional le 1.

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Table 2
, Study procedure table.
Study Period Visit 1 Visit 2
-7 to 0 days 12 weeks (± 3 days)
Data collection at baseline  
Informed consent form × 
Demographic information × 
Life history × 
previous history × 
medical history × 
Inclusion and exclusion
criteria
× 
Ecacy evaluation  
MRI-PDFF × ×
ALT × ×
AST × ×
ALP × ×
GGT × ×
TG × ×
TC × ×
HDL-C × ×
LDL-C × ×
CAP × ×
LSM × ×
FFA × ×
FINS × ×
FBG × ×
HOMA-IR × ×
CK18-M30 × ×

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Study Period Visit 1 Visit 2
-7 to 0 days 12 weeks (± 3 days)
Data collection at baseline  
CK18-M65 × ×
BFR × ×
WHR × ×
BMI × ×
Safety evaluation  
Vital signs × 
Blood routine × ×
Renal function × ×
Electrocardiogram × ×
Other work  
Record adverse events × ×
Evaluation of subject behavior intervention compliance  ×
Recovery and record of study drug  ×
MRI-PDFF Magnetic resonance proton density fat fraction, ALT Alanine aminotransferase, AST Aspartate
aminotransferase, ALP Alkaline phosphatase, GGT Gamma glutamyl transferase, TG Triglyceride, TC
Total cholesterol, HDL-C High-density lipoprotein cholesterol, LDL-C Low-density lipoprotein cholesterol,
CAP Controlled attenuation parameter, LSM liver tiffness value, FFA Free fatty acids, FINS Fasting insulin
FBG Fasting blood glucose, HOMA-IR Homeostasis model assessment of insulin resistance, CK18-M30
Cytokeratin 18-M30, CK18-M65 Cytokeratin 18-M65, BFR Body fat percentage, WHR Waist to hip ratio,
BMI Body mass index.
Eligibility criteria
Diagnostic criteria
Diagnostic criteria for obese NAFLD patients
1.Diagnostic criteria for NAFLD: Formulated in accordance with the Guidelines for Prevention and
Treatment of Non-alcoholic Fatty Liver Disease (2018 Update) established by the Fatty Liver and
Alcoholic Liver Disease Group of the Chinese Society of Hepatology [17].

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(1) The patient had no history of excessive drinking (the weekly consumption of ethanol (alcohol) in the
past 12 months was less than 210g for males and less than 140g for females) and had no other specic
causes of fatty liver.
(2) Imaging manifestations: B-ultrasound, CAP, Computed Tomography (CT), or MRI-PDFF suggest that
the patient has hepatic steatosis.
2. Diagnostic criteria for Obesity: Refer to the " Guideline for primary care of obesity:practice version
(2019) " [18] : BMI ≥ 28kg/m2, or waist circumference ≥ 85cm for men and ≥ 80cm for women.
The diagnosis of obese NAFLD patients must meet both the rst and second criteria mentioned above.
Diagnostic criteria for phlegm stasis syndrome
Based on the syndrome differentiation standards formulated by the Chinese Society of Traditional
Chinese Medicine's Gastroenterology Branch in the "Expert Consensus on the Diagnosis and Treatment
of Nonalcoholic Fatty Liver Disease in Chinese Medicine" (2017), the following is proposed according to
the clinical manifestations of NAFLD patients.
Main symptoms: pain or discomfort in the right ank area.
Secondary symptoms: Obesity, generalized heaviness and lethargy, chest and abdominal fullness, loose
or unformed stool, dullness tongue with possible ecchymosis or petechiae, white and greasy tongue
coating, taut and slippery or deep and sluggish pulse.
Patients need to have the main symptoms and any two of the secondary symptoms to diagnose the
phlegm stasis syndrome.
Inclusion criteria
1. Age: 18–65 years old, no gender limit.
2. Simultaneously meeting the diagnostic criteria for obese NAFLD patients and the diagnostic criteria
for traditional Chinese medicine phlegm stasis syndrome type.
3. 1×ULN < serum ALT ≤ 3×ULN.
4. Fasting blood glucose ≤ 7.0 mmol/L.
5. Sign an informed consent form.
. Have not taken any other drugs for treating NAFLD or obesity, or have discontinued these drugs
before 4 weeks of observation.
Exclusion criteria
Any one of the following should be excluded.

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1. Viral hepatitis, drug-induced liver disease, total parenteral nutrition, hepatolenticular degeneration,
autoimmune liver disease, etc.
2. Patients with severe heart, brain, kidney, hematopoietic system diseases, as well as emotional
disorders such as anxiety and depression, and mental illness.
3. Fatty liver caused by long-term use of glucocorticoids, chlorpromazine, insulin, etc.
4. Type I diabetes patients.
5. Patients with various types of malignant tumors.
. Pregnant and lactating women.
7. Subjects participating in other clinical trials.
Withdrawal criteria and termination criteria
1. If the following situations occur, the researcher will decide whether the patient should withdraw from
the study.
(1) Appearing allergic reactions or serious adverse events.
(2) During the experiment, the patient developed other diseases that affected the ecacy and safety
assessment.
(3) Cases that have to be unblinded during the study due to various reasons.
(4) After randomization, serious violations of inclusion or exclusion criteria were found.
2. The situation which the patient decides to withdraw on their own
(1) Regardless of the reason, if the patient is unwilling or unable to continue the clinical trial and requests
to withdraw from the trial to the supervising physician, the trial will be terminated.
(2) Although the patient did not explicitly request to withdraw from the trial, they will no longer accept
medication and monitoring.
Randomization and blinding
The randomization plan for this study was entrusted to Henan Provincial Evidence Based Medicine
Research Center for Traditional Chinese Medicine. The block length is 8 and the number of blocks is 31.
Adopting a double-blind experimental design. A two-level randomized medication number is generated
by Henan Provincial Evidence Based Medicine Research Center of TCM using statistical software
packages. The rst level is the group corresponding to each case number (Group A, Group B), and the
second level is the treatment corresponding to two groups (HTQSHXF group and placebo group). The
two-level blind bottoms are separately sealed and stored by Henan Province Evidence Based Medicine
Research Center for TCM. Distribute and package drugs according to random grouping codes. Each
coded trial drug has a corresponding emergency letter, which contains a note indicating that the coded
drug belongs to the category of phlegm resolving, dampness dispelling, and blood activating granules or

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placebo, In order to unblind in case of emergency. The envelope is made of thick, opaque kraft paper, and
the number of emergency letters should be the same as the number of participants. The envelope should
indicate clinical study number. The envelope is sent to the research unit along with the packaged drugs.
After the study was completed, the data was locked after blind verication was conducted without any
errors. The staff of Evidence Based Medicine Research Center conducted the rst unblinding, informing
statistical experts of the groups corresponding to each case number with codes A and B, in order to
conduct statistical analysis of all data. After the statistical analysis is completed, write a summary report
and conduct a second unblinding at the clinical summary meeting to announce the exact groups of
Group A and Group B.
Interventions
Both the HTQSHXF group and the placebo group were treated with a diet and exercise regimen as the
basic treatment.
Dietary plan
Developed in accordance with the Expert Consensus on Fasting Therapy in Traditional Chinese Medicine
(2019 Edition) [19].
The dietary principles are formulated based on the Chinese Dietary Guidelines for Residents (2016).
Implement a dietary plan of light fasting for 2 days per week and a 5-day conditioning period. Provide
dietary prescriptions based on the patient's height, weight, and dietary habits, and implement dietary
management. Implement overall calorie control in daily diet. Daily total calorie limit: Men should be
below 25kcal/kg, and women should be below 20kcal/kg. The dietary structure is mainly low fat, protein
rich, and ber rich. Patients must record their diet every day, check in before meals, and upload it to the
WeChat group to provide dietary guidance at any time.
Exercise plan
According to specic situation of the patient, exercise prescriptions should be issued, and the guiding
principles are as follows.
1. During the light fasting period, patients engage in light physical activities, mainly walking, which can
be combined with traditional Chinese medicine techniques such as Tai Chi, Eight Section Brocade,
and Five Animal Play. It is recommended to do about 120 minutes of brisk walking or jogging every
day, which can be divided into 3–4 sessions, with each session lasting about 30 minutes.
2. The conditioning period is mainly characterized by long-term, regular, moderate to low intensity, and
aerobic exercise. The types of sports include brisk walking, jogging, cycling, swimming, aerobics,
rope skipping, etc. Exercise for 30 to 60 minutes each time. Persist at least once a day. At least 5
times a week. Suggest implementing the exercise in the afternoon or evening.
Drug intervention

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HTQSHXF group: HTQSHXF granules (12 g/bag). 2 bag each time, dissolve the particles in 100ml hot
water and drink twice a day. Take it 1 hour after breakfast and dinner every day. Take the medication 6
days a week and stop taking it for 1 day. The treatment will last for 12 weeks.
Placebo group: placebo (HTQSHXF mimetic agent, 12 g/bag. Raw materials are maltodextrin, lemon
yellow pigment, sunset yellow pigment, caramel pigment, lactose, and bittering agent). The
administration method is completely consistent with the HTQSHXF group.
Drug preparation: HTQSHXF granules and corresponding mimetics were prepared by Jiangsu Jiangyin
Tianjiang Pharmaceutical Co., Ltd. batch number: 2205302.
Drug combination: If there is any concomitant medication during the patient's participation in the study,
researchers should record it in detail in the CRF form. It is allowed to undergo necessary treatment under
the guidance of clinical doctors for other diseases or symptoms, but the use of drugs with
hepatoprotective, anti-inammatory, and lipid-lowering effects should be prohibited before the
termination of the trial.
Outcomes
Primary Outcome
MRI-PDFF.
Secondary outcomes
ALT, AST, Alkaline phosphatase (ALP), Gamma glutamyl transferase (GGT), TG, TC, High-density
lipoprotein cholesterol (HDL-C), Low-density lipoprotein cholesterol (LDL-C), CAP, Liver tiffness value
(LSM), FFA, Fasting insulin (FINS), FBG, Homeostasis model assessment of insulin resistance (HOMA-
IR), Cytokeratin 18-M30 (CK18-M30), Cytokeratin 18-M65 (CK18-M65), Body fat percentage (BFR), Waist
to hip ratio (WHR) and BMI.
Safety outcomes
Vital signs, blood routine, renal function, electrocardiogram.
Observation time
Researchers will conduct visits, observations, and records in the 0th and 12 weeks of the study.
Adverse events
Adverse events refer to any symptoms, syndromes, or diseases that occur during the observation period
of clinical research and can affect the patient's health, as well as clinically relevant situations discovered
in the laboratory or other diagnostic processes. The term "adverse event" does not imply a causal
relationship with the investigational drug. Any adverse reactions that occur during the study period will

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be recorded in the "Adverse Event Form" and tracked for investigation. The handling process and results
will be recorded in detail until the laboratory test returns to normal and symptoms and signs disappear.
When adverse reactions are detected, researchers determine the diagnosis and treatment measures
based on the condition, and decide whether to discontinue observation or break the blindness. Ecacy
data that were available prior to blinding could be counted in the ecacy analysis. Otherwise, the case
will be considered as a terminated case and will not be included in the ecacy analysis.
Data collection and registration
All clinical doctors and researchers are required to undergo training and assessment before conducting
this clinical trial. Only after passing the assessment can they participate in this study. The subjects need
to undergo two visits, namely the 0th and 3rd months. The informed consent of the subjects and
information from two visits will be recorded in a paper Case Report Form (CRF) form. The CRF form is
the source le for clinical trial subjects, and researchers in each center should truthfully ll it out and
properly store it according to regulations. After the study is completed, it will be uniformly saved and
managed by the main center. The completed CRF form will be reviewed by the clinical monitor and
handed over to the data administrator. The data management personnel use the online EDC data
program (http://edc.hnzhy.com/portal/) of Henan Province Evidence Based Medicine Research Center
for data entry and management. Data of the subjects will be anonymous, and all collected data will be
kept condential.
Sample size
In this study, the sample size was estimated based on the level of MRI-PDFF, which serves as the primary
ecacy indicator. The calculation formula is as follows:
nE=nC=
According to the literature, after 12 weeks of treatment, 25.9% of patients in the control group (placebo
intervention) had a reduction of more than 30% in MRI-PDFF levels, while 53.3% of patients in the
treatment group (Chinese traditional treatment) experienced a similar reduction [20]. Therefore, it is
anticipated that following 12 weeks of dietary and exercise intervention, 35% of patients will have a
reduction of more than 30% in MRI-PDFF levels. Furthermore, when combined with the treatment of
HTQSHXF granules for 12 weeks, the proportion of patients with a reduction of MRI-PDFF levels
exceeding 30% is expected to reach 57.4%. Assuming α = 0.05, β = 0.10, Zα/2=1.96, Zβ=1.645,
P
E=0.574,
P
C=0.35,
nE
:
nC
=1:1, =(
P
E+
P
C)/2. The calculated sample size required for each group is 99 cases.
Considering a 20% dropout rate, the nal estimated sample size is 248 cases, with 124 cases in each
group.
2
[
Z
√
2−
P
(
1− −
P
)
+
Zβ
√
PE
(1−
PE
)+
PC
(1−
PC
)
]
α
2
(
PE
−
PC
)2
−
P

Page 13/21
Statistical analysis
Entrust a third party to conduct data management and blind review, and be responsible for developing
statistical analysis plans and writing statistical analysis reports. The data statistical analysis was
completed using SPSS 22.0 statistical software. Qualitative data is expressed in terms of rate and
composition ratio. If the quantitative data follows a normal distribution, it should be expressed as mean
± standard deviation; otherwise, the median should be used. When comparing the primary outcome MRI-
PDFF with all secondary outcomes between two groups: If the data conforms to a normal distribution, t-
test will be used (signicance level of 0.05 will be used for inter group homogeneity of variance test, and
t 'test will be used if the variances are not equal); If the data does not follow a normal distribution,
Wilcoxon rank sum test will be used.
P
< 0.05 is considered statistically signicant. All baseline variables
will undergo descriptive analysis.
Quality control
To prevent potential bias in the study, the following measures are taken for quality control:
1. A multicenter, randomized, double-blind design was adopted to address the bias in the selection of
subjects by researchers.
2. In response to the bias in ecacy evaluation, a third-party evaluation is adopted under the premise
of unied evaluation standards, and evaluating physician will conduct the research under blinded
conditions throughout.
3. To address the bias in multicenter study, targeted training for researchers will be provided, and
researcher statements will be signed to strengthen guidance for researchers during the study
process. Designate a xed researcher to undertake the recording of study medical records and the
work of logging in and lling out electronic CRF.
4. To address the bias generated by the subjects, we aim to improve their compliance in the following
aspects: 1) Effective communication: Researchers should conscientiously implement informed
consent to ensure that subjects fully understand the study requirements and cooperate with the
study. 2) Regular education: The researcher designates a dedicated person to conduct regular
WeChat or phone follow-up before important event milestones. 3) Strengthen the diet and exercise
management of subjects: Establish a dedicated WeChat group, where nutritionists develop a diet
and exercise plan, and supervise subjects to clock in, adjust their diet, exercise regularly, undergo
regular follow-up visits, and take medication regularly. 4) The distribution of drugs is managed and
recorded by a xed researcher.
Discussion
There are a total of 18 chemical active ingredients in the HTQSHXF granules, which are quercetin,
chlorogenic acid, silymarin, rutin, ferulic acid, hesperidin, salvianolic acid B, naringenin, kaempferol,
danshensu sodium, hyperoside, quercetin, apigenin, hesperetin, 23-acetyl zeaxanthin C, saikosaponin b2,
saikosaponin b1, and tanshinone IIA [21]. Our previous study has shown that quercetin may improve the

Page 14/21
degree of hepatic steatosis and alleviate liver inammation in NASH rats by regulating the PI3K/AKT/NF-
κB signaling pathway [22]. Existing studies have shown that naringin can improve lipid status and
severity of liver steatosis in obese NAFLD patients [23][24]. Hesperidin can improve liver steatosis, liver
enzymes, metabolism, and inammatory parameters in NAFLD patients. Rutin may improve diabetes
NAFLD by activating Adenosine 5‘-monophosphate (AMP)-activated protein kinase (AMPK) pathway [25].
In addition, chlorogenic acid [26], silymarin [27], ferulic acid [28], salvianolic acid B [29], kaempferol [30],
hyperoside [31] apigenin [32], and tanshinone IIA [33] also have certain therapeutic effects on NAFLD.
Liver tissue pathological biopsy remains the gold standard for diagnosing NAFLD [34]. However, the
invasive nature of liver biopsy and the risk of complications such as bleeding limit its clinical application
[35]. Moreover, the liver tissue samples obtained from liver biopsy can only represent 1/50000 of the liver
volume[36]. MRI-PDFF is an objective non-invasive quantitative imaging method for evaluating the overall
liver fat content. MRI-PDFF ≥ 5% can conrm the diagnosis of fatty liver, while MRI-PDFF ≥ 10% may
indicate moderate to severe fatty liver [37]. A systematic review and meta-analysis involving a total of
636 suspected NAFLD patients who underwent liver biopsy and MRI-PDFF simultaneously found that
MRI-PDFF diagnosed fatty liver (S0 vs. S1-3), moderate to severe fatty liver (S0-1 vs. S2-3), and severe
fatty liver (S0-2 vs. S3) patients with liver biopsy conrmed AUROC (0.98, 0.91, and 0.90, respectively),
sensitivity (0.93, 0.74, 0.74), specicity (0.94, 0.90, 0.87), as well as positive predictive values (16.21,
7.19, 5.89) and negative predictive values (0.08, 0.20, and 0.29). 9, 0.29) are all very high [38]. This
suggests that MRI-PDFF has excellent diagnostic value for liver fat quantication in NAFLD patients, and
has high sensitivity and specicity in distinguishing different degrees of liver fat degeneration. It can be
used as a reference standard for non-invasive quantication of liver fat content and has been
recommended for evaluating changes in liver fat content in clinical trials of new drugs [39][40].
Therefore, this study used non-invasive MRI-PDFF as the main therapeutic indicator to quantitatively
evaluate liver fat fraction.
A multicenter study has been conducted in the past to treat NASH patients with a combination of diet
and exercise based on the formula of HTQSHXF granules. This study included a total of 202 NASH
patients. 101 NASH patients in the treatment group and 101 in the control group received medication
treatment on the basis of health education, diet and exercise. The TCM granules taken by the treatment
group are based on HTQSHXF granules, and are added according to the patient's symptoms based on
syndrome differentiation: patients with spleen deciency and dampness excess symptoms are added
with Codonopsis pilosula, stir fried Atractylodes macrocephala, and raw Coix seed; patients with liver
and gallbladder dampness and heat symptoms are added with Tianjihuang, Yinchen, and lotus leaves;
patients with liver and kidney yin deciency symptoms are added with goji berries, Polygonum
multiorum, and Huai Niugeng. The control group received polyene phosphatidylcholine capsules. The
results showed that on the 90th and 180th day of treatment, the treatment group was superior to the
control group in reducing serum ALT and TG, lowering BMI, and improving liver/spleen CT values in
NASH patients. And it is signicantly better than the control group in improving clinical symptoms such
as epigastric distension, liver discomfort, and fatigue. The above study adopted a multi-center,
randomized, controlled, and superiority design. Despite being a multi-center study, the complexity of

Page 15/21
administering medication to the treatment group made it impossible to implement a double-blind design.
This failure to control for external variables such as subjective biases from researchers and participants
led to various unknown factors inuencing the experimental results, reducing their reliability and validity.
Drawing from the lessons of previous studies with less rigorous designs, this study ensures that the
treatment group only takes HTQSHXF granules without any additional medications. The control group
received the corresponding placebo. This study design adopts a multi-center, randomized, double-blind
approach to enable a more scientic evaluation of the ecacy and safety of the HTQSHXF granules
based on the research results. Despite this, there are still certain limitations in the study design. For
example, the number of research centers is relatively small, with only three units participating. The
treatment duration of 3 months is relatively short. Additionally, the primary ecacy indicator does not
include liver histopathological biopsy. We hope to conduct higher-quality multi-center, randomized,
double-blind clinical studies in the future to provide stronger evidence-based medical support and
guidelines for the treatment of obese NAFLD.
Trial status
This is an ongoing clinical trial. The recruitment time for the rst subject was March 17, 2023. We plan to
complete the recruitment of all subjects by September 2025, and all data will be locked in by December
2025.
Abbreviations

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NAFLD   non-alcoholic fatty liver disease
NASH   non-alcoholic steatohepatitis 
HTQSHXF  Huatan Qushi Huoxue Fang
TCM Traditional Chinese Medicine
SPIRIT Standard Protocol Items: Recommendations for Interventional Trials
MRI-PDFF Magnetic resonance proton density fat fraction
BFR  Body fat percentage
WHR  Waist to hip ratio
BMI    Body Mass Index
CAP    Controlled attenuation parameter
LSM Liver tiffness value
FFA Free fatty acids
CK18-M30 Cytokeratin 18-M30
CK18-M65 Cytokeratin 18-M65
ALT Alanine aminotransferase
AST  Aspartate aminotransferase
ALP  Alkaline phosphatase
GGT Gamma glutamyl transferase
TG  Triglyceride
TC  Total cholesterol
HDL-C High-density lipoprotein cholesterol
LDL-C Low-density lipoprotein cholesterol
FBG  Fasting blood glucose
FINS  Fasting insulin
HOMA-IR Homeostasis model assessment of insulin resistance
CRF  Case Report Form
AMPK  Adenosine 5‘-monophosphate (AMP)-activated protein kinase
Declarations

Page 17/21
Acknowledgements
The authors thanks Henan Provincial Evidence Based Medicine Research Center for Traditional Chinese
Medicine for designing the blinding and random allocation scheme; The authors also thanks the
researchers from the other two centers for their contributions. Finally, the authors would like to thank all
participants for their cooperation.
Author contributions
WZ and ML designed this study. LZ and SL drafted the manuscript and made equal contributions to the
study. QZ, XL, XS, TW, FL, MY participated in the study plan. WZ modied and edited the manuscript. All
authors have read and approved the nal version of the manuscript.
Funding
This work was sponsored by Henan Province's "double rst-class" creation of scientic research in
traditional Chinese medicine (No. STG-ZYX02-202117, No. HSRP-DFCTCM-2023-7-23), National
Traditional Chinese Medicine Clinical Research Base Scientic Research Special Project
(No.2022JDZX098, 2022JDZX114), National Natural Science Foundation of China (No.82205086),
Science and Technology Key Project of Henan Province (No. 232102310438), and The 9th China
Association for Science and Technology Young Talent Support Project (No. 2023QNRC001).The funder
had no role in the design, implementation, analysis, data interpretation, or decision process of submitting
the results of this study.
Availability of data and materials
Not applicable.
Ethics approval and consent to participate
This study will follow the Helsinki Declaration and Good Clinical Practice guidelines, and will require
informed consent from the subjects.This protocol has been reviewed and approved by the Medical
Ethics Committee of the First Aliated Hospital of Henan University of Traditional Chinese Medicine
(Approval Number: 2022HL-038-01).The study results will be published in the form of a paper.
Consent for publication
Not applicable.
Competing interests
The authors declare that the research was conducted in the absence of any commercial or nancial
relationships that could be construed as a potential conict of interest.

Page 18/21
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Figures

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Figure 1
The owchat of the trial
Supplementary Files
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SPIRITitem.docx