Hepatoprotective effect of Antrodia Cinnamomea mycelia extract in subhealth Japanese adults: a randomized, double-blind, placebo-controlled clinical study

Abstract Antrodia cinnamomea, a unique Taiwanese fungus (mushroom), has demonstrated the hepatoprotective activities in animals with liver injury. Nevertheless, there are few studies reporting the efficacy of the fungus in subhealth subjects (alanine aminotransferase (ALT) levels between 31 and 50 U/L and aspartate aminotransferase (AST) levels ≤ 50 U/L). In this study, we assessed the ameliorating effect of a A. cinnamomea mycelia extract (ACME) on liver health in asymptomatic individuals with marginally high ALT levels. Forty-four eligible Japanese adults were enrolled in this randomized, double-blind, placebo-controlled clinical study and instructed to take an ACME capsule (250 mg of ACME powder) or a placebo capsule daily for 12 weeks. The primary outcomes (i.e. ALT and AST) were analyzed at 0, 4, 8, and 12 weeks. No treatment-related adverse effects were observed throughout this study. In efficacy analyses with the per-protocol (PP) cohort of participants, there were no significant changes in ALT and AST levels within and between groups. However, subgroup analysis showed that ACME could significantly improve the mean ALT level of regular drinkers, consuming alcoholic drinks more than twice a week, after the study in comparison with the result of the placebo group. This exploratory study indicated that the ACME might effectively improve liver health in regular drinkers.


Introduction
Antrodia cinnamomea (also known as Antrodia camphorate, niu-chang-chih) is a basidiomycetous fungus and only grows parasitically on the brown heartwood of the endemic camphor tree (Cinnamomum kanehirae) in Taiwan (Wu et al. 1997;Ao et al. 2009;Geethangili & Tzeng 2011).A. cinnamomea has been used to treat liver diseases, diarrhea, abdominal pain, hypertension, skin itching, and cancers in Chinese folk remedies (Tsai and Liaw 1985;Chen et al. 2001).Recently, pharmacological studies have revelated that A. cinnamomea possesses anti-oxidative, anti-inflammatory, immuno-modulatory, hepatoprotective, anti-tumor, and anti-hepatitis B virus activities c.-Y. hO et al. (Ao et al. 2009;Liu et al. 2007;Rao et al. 2007;Hsiao et al. 2003;Hsu et al. 2005).For example, A. cinnamomea may prevent liver injury, acute hepatitis, and fatty liver in the chemical-inducted rodents (Dai et al. 2003;Liu et al. 2017;Lu et al. 2011;Hsiao et al. 2003;Song and Yen 2003;Kuo et al. 2019).The identified clinical benefits of A. cinnamomea are the treatments for patients with cancers, mild hypertension, and nonalcoholic steatohepatitis as well as improvements in liver function indexes and blood lipids (Chen et al. 2016;Chen et al. 2017;Chiou et al. 2021;Li et al. 2021;Liao et al. 2022;Tsai et al. 2016;Yen et al. 2022).Yen et al. reported that an ethanolic extract of A. cinnamomea mycelia can ameliorate liver function indexes and triglycerides in Korean adults with alcoholic hepatitis (Yen et al. 2022).Chen et al. revealed that a supercritical extraction of A. cinnamomea mycelia helps reduce low-density lipoprotein (LDL) cholesterol in Japanese adults with mildly elevated LDL cholesterol levels (Chen et al. 2017).However, the hepaprotective effect of A. cinnamomea mycelia in individuals with mildly elevated ALT levels is unclear.
Herein, we attempted to investigate whether a A. cinnamomea mycelia extract (ACME) may ameliorate the liver health in subhealth Japanese individuals with ALT levels between 31 and 50 U/L.The ACME has been acknowledged by the U.S. Food and Drug Administration as a new dietary ingredient and has no serious concerns for food safety (U.S. Food and Drug Administration 2020).

Preparation of A. cinnamomea mycelia extract powder
A. cinnamomea mycelia were obtained from A. cinnamomea (BCRC 35396) through solid-state cultivation from Greenyn Biotechnology Co., Ltd.(Taichung, Taiwan). A. cinnamomea mycelia inoculated in the liquid medium (malt extract, dextrose monohydrate, soy peptone) in flasks (shaker) at 24.5 ± 1 °C and 115 ± 5 rpm for 7 days for mass production.The medium (0.06%) were pipetted and spread evenly onto the solid-state medium (cereals) for further cultivation at 24.5 ± 1.0 °C for three months.After cultivation, A. cinnamomea mycelia were collected and pulverized followed by high pressurized water extraction.The sample was dried at 80 ± 2 °C for over 17 h.Finally, the extract powder was stored at ambient conditions.

Ethics
This study was in accordance with the Declaration of Helsinki, the Ethical Guidelines for Medical Research in Humans (Ministry of Education and Science and the Ministry of Health, Japan), and the Guidance of the Ethical Guidelines for Medical Research in Humans (Ministry of Education and Science and the Ministry of Health, Japan).The protocol of the study was approved by the Medical Station Clinic Ethics Committee, Tokyo, Japan (IRB number: 20000022) on May 13, 2021, before initiating the present study.The written informed consent forms were obtained from all participants prior to the study.This study was registered at the University Hospital Medical Information Network Clinical Trials Registry (UMIN-CTR) as UMIN 000044415 and was conducted from June 2021 to October 2021 at the Medical Station Clinic (Tokyo, Japan).

Study design
A total of 328 male and female Japanese adults, aged 20-64 years, who had serum levels of ALT between 31 and 50 U/L and AST levels ≤ 50 U/L, were recruited.Individuals were excluded if they were/had ((i)) test-positive for hepatitis B virus antigen or for anti-hepatitis C virus antibody; ((ii)) unable to conform with test procedures (e.g.neglecting the questionnaire); ((iii)) judged by the medical investigator to be inappropriate as the study participant based on the results of a lifestyle-relevant questionnaire, laboratory examinations, and anthropometric examinations; ((iv)) regularly using a health food containing A. cinnamomea; ((v)) concomitant medication/ dietary supplements which may influence liver functions; ((vi)) with a history of diabetes, hepatic disease, renal disease, heart failure, infectious diseases, or other severe diseases; ((vii)) at risk for allergy relevant to this study, ((viii)) diseases that required regular medication or with a history of a disease that had required medication; ((ix)) participating in another clinical study within one month prior to the present study or expecting to participate in another clinical study after the enrollment; ((x)) currently pregnant, nursing or preparing for pregnancy or breastfeeding during the study period; ((xi)) regular vigorous exercise (e.g.marathon); and ((xii)) irregular lifestyles during the study due to a double-shift duty or night duty.
The study was a randomized, double-blind, and placebo-controlled clinical study.Eligible subjects were randomly allocated to the ACME group or placebo group through a computer-generated randomization plan.Subjects were instructed to take one ACME capsule (250 mg ACME and 50 mg lactose) or one placebo capsule (300 mg lactose with a small quantity of caramel) daily before sleeping, with the aid of 100 mL water, for 12 weeks.Note that the ACME and placebo capsules were sponsored by Greenyn Biotechnology Co, Ltd, Taichung City, Taiwan.250 mg ACME contained 500 μg antrodin A and 75 μg 4.7-dimethoxy-5-methyl-1,3-benzodioxole (DMB).
Antrodin A and DMB content in the ACME was determined by the high-performance liquid chromatography (HPLC) analysis (Figures S1 and S2, Supplementary material).Participants were required to adhere to their daily habits, such as diet pattern, exercise, sleep and work, and drinking frequency.Noncompliance referred to the consumption of < 80% of the total allocated capsules.All non-compliant participants would be excluded from the efficacy assessment.

Anthropometrics and hemodynamics
Height was recorded before the study.Body weight, body-mass index (BMI), systolic and diastolic blood pressures (BPs) were measured at 0, 4, 8, and 12 weeks.

Statistical analysis
All data were presented as mean ± standard deviation (S.D.).The independent samples t-test was used to analyze the differences in demographic and baseline continuous variables as well as the changes of primary outcomes.Pearson's χ 2 -test was used to analyze the categorical variables (e.g.gender).A p-value < 0.05 was considered statistically significant.Statistical analysis was performed using IBM SPSS23 Statistics for Windows (IBM, New York, NY, USA) and Microsoft Excel 2010 (Microsoft, Redmond, WA, USA).

Baseline characteristics
Forty-four participants were enrolled in this study and allocated to either the placebo group (n = 22) or ACME group (n = 22) (Figure 1).All participants accomplished this study and maintained good health (Table 1), but six subjects were excluded from the efficacy analysis due to the following reasons: ((i)) excessive alcohol consumption (n = 1; ACME group); ((ii)) non-treatment-related health issues (n = 2; ACME group); and ((iii)) lifestyle changes (n = 3; placebo group).All participants maintained good health throughout this study.The most common adverse events were side reactions of anti-COVID-19 vaccination (8 in the placebo group and 6 in the ACME group).No treatment-related effects were observed during this study given that unexpected events were mild in intensity and occurred only temporarily.
The compliance rates in the ACME group were in the range of 92.9-100%.There were no significant differences in anthropometric, hemodynamic, and liver function indexes between the placebo and treatment groups (Table 2).

Efficacy assessment with the per-protocol cohort of participants
After the 12-week treatment, the biochemical and hematological parameters were in the normal reference ranges in both groups, and there were no significant differences in these indexes between and within groups (Table 3).Table 4 shows the results of mean liver function indexes at 0, 4, 8, and 12 weeks.The mean ALT and AST levels did not present notable changes in both groups.

Subgroup assessment
We attempted to identify the influence of ACME in regular drinkers, which were defined as the frequency of alcohol consumption is 1-2 days/week or more (Akechi et al. 2006).The mean frequencies of alcohol consumption for the placebo and ACME groups were 13.9 and 16.5 days/month, respectively (Table 5).There were no significant differences in AST and ALT levels between the two groups at baseline.The persistently decreased liver function indexes were shown in the treatment group; by contrast, the trends of the changes in ASL and ALT levels were fluctuating in the placebo group.In comparison with the baseline results, the obvious changes in the AST and ALT levels were observed in the ACME group after the study; especially, there was a significant improvement in the ALT level between groups.

Discussion
It has known that A. cinnamomea is beneficial for liver protection, while few clinical reports revealed the improvement of A. cinnamomea in liver health in subhealth individuals (Chen et al. 2016;Chen et al. 2017;Chiou et al. 2021;Li et al. 2021;   acute alcoholic liver injury and other hepatic disorders in several animal models (Yi et al. 2020;Shie et al. 2016).ALT and AST are well-recognized biomarkers for liver injury (Pratt and Kaplan 2000) and, in particular, ALT activity is the most commonly used variable for assessment of liver disease (Pratt and Kaplan 2000;Craxì and Almasio 1996).No treatment-related effects were found throughout this study.For the per-protocol cohort results, there were no significant decreases in ALT and AST levels in the ACME group after the study.The disparaging outcomes may be due to: (i) the relatively small sample size in this study might limit the statistical power for statistical significance; and (ii) low ALT and AST levels indicated mild liver injury, so the ACME dose used here might be too low to reach a prominent improvement.
On the other hand, in comparison with the placebo group, the mean ALT level of regular drinkers in the ACME group significantly reduced after the study.The result suggests that ACME may relieve the liver injury caused by chronic alcohol use and embody the obvious improvement in liver health in people whose liver bear additional oxidative stress.Ethanol metabolism increases oxidative stress and incurs inflammatory  reactions in the liver, which leads to the onset of steatosis, steatohepatitis, and steatofibrosis (Bakhautdin et al. 2014;Lee et al. 2013;Nitzan & Jia 2019;Yang et al. 2014;Dai et al. 2003).Pharmacological studies have unveiled that A. cinnamomea is a powerful anti-oxidant and helps to reduce oxidative stress induced by alcohol toxicity and prevent alcohol-induced liver damages (Dai et al. 2003;Lu et al. 2011;Liu et al. 2017;Song and Yen 2002;Hsiao et. al. 2003;Mau et al. 2004;Shu and Lung 2008;Huang et al.2010;Liu et al. 2017).Moreover, A. cinnamomea may affect the phosphor-protein kinase (AKT)/phosphor-nuclear factor κB (NF-κB) signal transduction (Liu et al. 2017).
In addition, the safety of the ACME was also verified in our undisclosed clinical investigation (UMIN-CTR ID: UMIN000044645).Fifteen healthy Japanese adults used the ACME with a daily dose of 1,250 mg for 4 weeks, and no adverse events were observed throughout the study.Although we discovered some encouraging results in this research, the limitations of the study design impede the extrapolation of the results to some general scenarios.First, the selected participants for subgroup analysis were not defined before the study.Second, the sample size of subjects is relatively small and the number of female subjects is not sufficient to objectively observe the influence of the ACME in women, so it is hard to make a solid conclusion from the scientific point of view.Moreover, a few subjects received anti-COVID-19 vaccines and had some side effects after the inoculation, while it is challenging to elucidate the impact to this study.We will conduct further investigations to examine the effectiveness of the ACME in subhealth populations and explore the underlying mechanisms and synergistic effect of antrodin A and DMB on liver repair in in-vitro or rodent models.

Conclusion
This pioneering study demonstrated the effectiveness of the A. cinnamomea mycelia extract on improving liver function indexes in regular drinkers.No treatment-related adverse events were observed in this study.Although there are several limitations of the study design, the significant improvement in the mean ALT level in regular drinkers encourages us to conduct further studies to validate its efficacy on preventing alcoholic liver disease.

About the authors
Chun-Yi Ho got her master degree at National Ilan University, Taiwan.She is a senior engineer at Greenyn Biotechnology.She was responsible for this research.
Chen-Meng Kuan received his Ph.D. degree at National Tsing Hua University, Taiwan.He is a R&D manager at Greenyn Biotechnology.His research interest is development of plant-based dietary ingredients.He involved from experimental design to preparation of the publication.
Pang-Kuei Hsu received his Ph.D. degree at National Chung Hsing University, Taiwan.He is vice president at Greenyn Biotechnology.He supervised this study.

Figure 1 .
Figure 1.flow chart for the study participants.

Table 1 .
adverse events reported by subjects in this study.
DMB, on liver function indexes in middle-aged Japanese adults with marginally high serum ALT levels.Antrodin A and DMB are well-defined constituents of A. cinnamomea mycelia and therapeutic potential for various inflammatory diseases, including

Table 2 .
Baseline characteristics for the pp cohort of participants (mean value ± S.D.).

Table 3 .
measured results of the pp cohort of participants after the study (mean value ± S.D.).