Limonene on Regulating Metabolism-related Fatty Liver Disease (MAFLD) and Analysis of TCM Constitution

Study Overview
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Brief Summary
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The prevention and treatment of metabolic-related fatty liver disease (MAFLD) involves many fields in preventive medicine and clinical medicine. So far, western medicine has not yet completed the elucidation of the mechanism of this type of disease, and there is a lack of effective therapeutic drugs.The purpose of this study was to evaluate the effectiveness and safety of limonene capsules (marketed product in China) in the treatment of metabolic-related fatty liver disease and related lipid-lowering mechanisms.

Detailed Description
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In April 2020, in the famous journal Journal of Hepatology in the field of liver disease, an internationally renowned liver disease expert group jointly proposed to replace non-alcoholic fatty liver disease (NAFLD) with metabolic associated fatty liver disease (MAFLD) . The concept of non-alcoholic fatty liver disease (NAFLD) was first proposed by Ludwig in 1980. It specifically refers to the excessive deposition of liver fat without excessive drinking. It is a type of liver that is closely related to insulin resistance and genetic susceptibility. Non-alcoholic fatty liver disease (NAFLD) is China country's largest chronic liver disease and the primary cause of abnormal liver enzymes in health examinations. It can lead to liver disability and death. It is also closely related to a variety of metabolic diseases and the high incidence of colorectal tumors. Western medicine has not yet fully elucidated its mechanism, and no drugs have been officially approved for the clinical treatment of NAFLD. The new MAFLD nomenclature highlights the central role of metabolic factors in causing liver fat deposition in this type of liver disease. Traditional Chinese medicine believes that the abnormal accumulation of fat in the liver of such fatty liver patients is a pathological product of the microscopic loss of water and valley essence. It belongs to phlegm stasis, which blocks the liver collaterals. It coincides with the core of the metabolic etiology of recent liver disease experts. Limonene is widely found in the essential oils of traditional Chinese medicine tangerine peel, green peel and other plants. Its taste is sour, sweet and pungent.It is returned to the liver and gallbladder meridian. It has an aromatic odor effect. A large number of animal and cell experiments in the early stage have shown that limonene can inhibit the differentiation of adipocytes (pre-adipocytes) and promote the apoptosis of mature adipocytes, which is related to the inhibition of fatty acid synthesis. Toxicity load experiments show that limonene has very low toxicity. The accumulation of lipids in the liver of mice has a regulatory effect with significantly reducing the content of liver cholesterol and triglycerides, and also has a certain effect on lipid metabolism disorders, hyperglycemia and other metabolic syndromes. It can alleviate the effects of high-fat diet and N- Efficacy of nitro-L-arginine methyl ester-induced resistance to non-alcoholic fatty liver in rats. The main indication of limonene capsules (marketed product in China) is liver and gallbladder diseases. Traditional Chinese medicine believes that liver and gallbladder are related to each other. This comprehensively shows that limonene capsules are promising to be developed as a pure Chinese medicine product for the safe and effective treatment of MAFLD. However, there has been no clinical evaluation of the clinical efficacy of limonene in the treatment of metabolic-related fatty liver disease. As a typical aromatic Chinese medicine, the mechanism of limonene in the treatment of fatty liver urgently needs to be revealed by modern medicine and molecular biology techniques. This study intends to use a randomized, double-blind, placebo-controlled method to evaluate the effect of limonene on improving the degree of fat infiltration in patients with metabolic-related fatty liver disease (MAFLD), and to evaluate its body mass index BMI, waist circumference, waist-to-hip ratio, subcutaneous fat thickness, fat percentage, changes in metabolic components, safety,etc. The study also intends to use metabonomics, genomics, and molecular biology techniques to study the clinical relationship between metabolites and physiological and pathological changes in patients with liver fat infiltration, and to detect changes in key proteins and lipid components after drug intervention, which is to reveal mechanism on treatment of metabolic-related fatty liver disease by limonene. It is aimed to study the difference in the efficacy of limonene for people with different constitutions of traditional Chinese medicine (TCM) combined with the analysis of the constitution of TCM, and to clarify the modern scientific attributes of TCM therapy. Finally the study will develop a safe and efficient drug treatment technology to control liver fat infiltration , and to promote the development of clinical disciplines in the treatment of metabolic-related fatty liver disease.

Official Title
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Clinical Trial of Limonene on Regulating Metabolism-related Fatty Liver Disease (MAFLD) and Analysis of TCM Constitution

Conditions
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Fatty Liver

Intervention / Treatment
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* Drug: Limonene capsule
* Drug: Limonene capsules(Placebo)


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Clinical diagnosis and imaging diagnosis of MAFLD and BMI index greater than or equal to 23kg/m2； The age of the recruiter is between 18-65 years old; Those who can cooperate with various treatments and data measurements according to the treatment cycle, and can persist in completing the test; Those who accept and are willing to sign the informed consent. Exclusion Criteria: Potential recruiters who meet the inclusion criteria will be excluded if they meet any of the following: Those who routinely take prescription drugs (except regular contraceptive drugs) or those who use auxiliary Chinese and Western drugs to treat non-alcoholic fatty liver; Patients suffering from viral hepatitis, autoimmune hepatitis, hepatolenticular degeneration, hypothyroidism, infection, and biliary tract diseases that lead to abnormal liver function; Patients who have taken the following drugs in the past 4 weeks: hypoglycemic drugs, lipid-lowering drugs (such as statins, fibrates, etc.) and drugs that may affect liver fat content (such as: silybin, ursodeoxycholic acid) , Bicyclic alcohol, phosphatidylcholine and vitamin E, glucocorticoid); Patients with diabetes or those who have undergone bariatric surgery; People who have gained or lost weight by 10 kg or more in the past two months; People who are allergic to limonene capsules; or people who are allergic to citrus foods; people who especially like to eat a lot of citrus foods (daily dosage more than 100 grams); Patients with severe cardiac insufficiency and malignant tumors; Patients who have a history of mental illness and cannot cooperate with this project; Pregnant and lactating women, or women or men who are willing to become pregnant or give birth during the study; Participate in any other clinical trials; Other situations where the researcher thinks it is inappropriate to participate in this research.

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 65 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Treatment
Allocation: Randomized
Intervention Model: Parallel Assignment
Interventional Model Description: Exploratory Clinical Trial；Randomized Controlled Trial
Masking: Triple


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Active Comparator: Limonene capsules(marketed product in China)<br>Limonene capsules(marketed product in China) donate by pharmaceutical company | Drug: Limonene capsule<br>* All the recruiters will be given treatment under the guidance of basic diet. Basic dietary guidelines include high-quality protein and fresh green leafy vegetables. It need to controlled sugar, various sweets and high-calorie foods, frying and other foods with high oil content and foods with high cholesterol content. Limonene capsules group and placebo group were used for treatment. The random number is generated by the central randomization system. All the recruiters were divided into placebo control group and limonene capsule administration group. The numbers will be assigned according to random numbers. In this study, qualified subjects were randomly assigned to the treatment group and the placebo control group at a ratio of 1:1. The drug was administered for 12 weeks, 3 times a day, 5 capsules each time.<br>* Other names: DAN LENING capsule;|
| Placebo Comparator: Limonene capsules(Placebo)<br>Same smell, color and shape as limonene capsules(marketed product in China), without limonene in capsules | Drug: Limonene capsules(Placebo)<br>* Limonene capsules(Placebo)<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Difference of controlled attenuation parameter (CAP) in liver fat | Changes in controlled attenuation parameter (CAP) in liver fat measured with transient elastography between the drug group and the placebo group. | at baseline and twelve weeks after administration | 
| Difference of rate of BMI index | The percent change of BMI index between the drug group and the placebo group. | at baseline, four, twelve weeks after administration | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Difference of waist circumference | The percent change of waist circumference between the drug group and the placebo group. | at baseline, four, twelve weeks after administration | 
| Difference of aspartate transaminase (AST) index | The absolute values of changes in the levels of AST was compared between the two groups. Serum AST content of the patients were detected by an automatic biochemical analyzer before and after treatment. | at baseline, four, twelve weeks after administration | 
| Difference of glutamic transpeptidase (GGT) index | The absolute values of changes in the levels of GGT was compared between the two groups. Serum GGT content of the patients were detected by an automatic biochemical analyzer before and after treatment. | at baseline, four, twelve weeks after administration | 
| Difference of alanine transaminase (ALT) index | The absolute values of changes in the levels of ALT was compared between the two groups. Serum ALT content of the patients were detected by an automatic biochemical analyzer before and after treatment. | at baseline, four, twelve weeks after administration | 
| Difference of total cholesterol (TC) index | The absolute values of changes in the levels of TC was compared between the two groups before and after treatment. Serum TC content of the patients were detected by an automatic biochemical analyzer before and after treatment. | at baseline, twelve weeks after administration | 
| Difference of glycerin trilaurate (TG) index | The absolute values of changes in the levels of TG was compared between the two groups before and after treatment. Serum TG content of the patients were detected by an automatic biochemical analyzer before and after treatment. | at baseline, twelve weeks after administration | 
| Difference of low-density lipoprotein cholesterol (LDL-C) index | The absolute values of changes in the levels of LDLC was compared between the two groups before and after treatment. Serum LDL-C content of the patients were detected by an automatic biochemical analyzer before and after treatment. | at baseline, twelve weeks after administration | 
| Difference of high-density liptein cholesterol（HDL-C）index | The absolute values of changes in the levels of HDL-C was compared between the two groups before and after treatment. Serum HDL-C content of the patients were detected by an automatic biochemical analyzer before and after treatment. | at baseline, twelve weeks after administration | 
| Difference of apolipoprotein E (ApoE) index | The absolute values of changes in the levels of ApoE was compared between the two groups before and after treatment. Serum ApoE content of the patients were detected by an automatic biochemical analyzer before and after treatment. | at baseline, twelve weeks after administration | 
| Difference of insulin index | The absolute values of changes in the levels of insulin was compared between the two groups before and after treatment. | at baseline, twelve weeks after administration | 
| Difference of glycated hemoglobin（GHb）index | The absolute values of changes in the levels of GHb was compared between the two groups before and after treatment. | at baseline, twelve weeks after administration | 
| Difference of blood sugar index | The absolute values of changes in the levels of blood sugar was compared between the two groups before and after treatment. | at baseline, twelve weeks after administration | 
| Difference of the Short Form 36 physical component summary (SF-36 PCS) score | Using the Telephone Interview for the SF-36 PCS score (range, 0 [worst] to 100 [best]). | at baseline, two，four，six，eight，ten weeks, twelve and sixteen weeks after administration | 
| Difference of the Chronic Liver Disease Questionnaire (CLDQ) | Using the Telephone Interview for the CLDQ score (range, 0 [worst] to 100 [best]). | at baseline, two，four，six，eight，ten weeks, twelve and sixteen weeks after administration | 
| Difference of the Traditional Chinese of Medicine ( TCM ) Physique Questionnaire | Using the Telephone Interview for the TCM Physique Questionnaire score (range, 0 [worst] to 100 [best]). | at baseline, two，four，six，eight，ten weeks, twelve and sixteen weeks after administration | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Metabolic associated fatty liver disease (MAFLD), Limonene, Efficacy, Safety