TYPE Original Research

PUBLISHED 22 March 2023

DOI 10.3389/fendo.2023.1054674

Clinical efficacy of weight loss

OPEN ACCESS herbal intervention therapy and
EDITED BY
Mikiko Watanabe,
Sapienza University of Rome, Italy
lifestyle modifications on obesity
REVIEWED BY
Ziyi Chen,
and its association with distinct
The Chinese University of Hong Kong,
China
Yingshuai Li,
gut microbiome: A randomized
Beijing University of Chinese Medicine,
China double-blind phase 2 study
*CORRESPONDENCE
Mingsan Miao
[email protected] Ming-Zhuo Cao 1, Chun-Hua Wei 2, Ming-Chun Wen 2,
Xiu-Min Li Ying Song 3, Kamal Srivastava 3,4, Nan Yang 3,4, Yan-Mei Shi 1,
[email protected]
Danna Chung Mingsan Miao 1*, Danna Chung 5* and Xiu-Min Li 3*
[email protected] 1
Academy of Chinese Medical Sciences, Henan University of Chinese Medicine, Zhengzhou, China,
SPECIALTY SECTION
2
Department of Medicine, Wei-en Hospital, Weifang, China, 3 Department of Pathology, Microbiology
This article was submitted to & Immunology, New York Medical College, Valhalla, NY, United States, 4 General Nutraceutical
Obesity, Technology, LLC, Elmsford, NY, United States, 5Healthy Freedom LLC, King of Prussia, PA, United States
a section of the journal
Frontiers in Endocrinology

RECEIVED 27 September 2022

ACCEPTED 21 February 2023 Goals: To assess the efficacy and safety of Chinese Medicine Prescription “W-LHIT”
PUBLISHED 22 March 2023
in subjects with simple obesity, and to explore its potential mechanism of action.
CITATION
Cao M-Z, Wei C-H, Wen M-C, Song Y,
Methods: Thirty-seven patients aged 18 to 60 from Wei-En hospital (Weifang
Srivastava K, Yang N, Shi Y-M, Miao M,
Chung D and Li X-M (2023) City, Shandong, China), participated in a double blinded, placebo-controlled
Clinical efficacy of weight loss herbal study. Subjects were randomly divided into 2 groups, 18 in treatment and 19 in
intervention therapy and lifestyle
modifications on obesity and its placebo group. The treatment group took the “W-LHIT” capsules for two
association with distinct gut microbiome: months, while the control group received placebo capsules. Both groups
A randomized double-blind phase 2 study.
accepted healthy lifestyle education materials. After a 2-month treatment, the
Front. Endocrinol. 14:1054674.
doi: 10.3389/fendo.2023.1054674 placebo group transferred to open-label treatment after unblinding.
COPYRIGHT
© 2023 Cao, Wei, Wen, Song, Srivastava, Results: 72.22% participants in the treatment group lost more than 5% of their
Yang, Shi, Miao, Chung and Li. This is an body weight, compared with 36.84% in the placebo group (p < 0.001). Body
open-access article distributed under the
terms of the Creative Commons Attribution
weight loss and body mass index reduction of the treatment group were also
License (CC BY). The use, distribution or significantly higher than those of the placebo group (p < 0.05). These changes
reproduction in other forums is permitted, were accompanied by increased abundance of Akkermansia muciniphila and
provided the original author(s) and the
copyright owner(s) are credited and that Enterococcus faecium, and decreased abundance of Proteobacteria in gut
the original publication in this journal is microbiota. Furthermore, the treatment group also showed improvement in
cited, in accordance with accepted
academic practice. No use, distribution or
obesity-related comorbidities such as hypertension and elevation of liver
reproduction is permitted which does not enzymes. No serious adverse reactions were found during the study period.
comply with these terms. Weight did not rebound at a follow-up visit 2 months after treatment.

Conclusion: W-LHIT significantly improved body weight and comorbid

conditions without obvious adverse reaction or rebound weight gain. These
effects were associated with increased abundance of probiotics in gut

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Cao et al. 10.3389/fendo.2023.1054674

microbiota. W-LHIT may have a potential for treating obesity in conjunction with
healthy lifestyle modifications.

KEYWORDS

“W-LHIT” capsule, obesity, weight loss, gut microbiome, Akkermansia muciniphila,

lifestyle modifications

Introduction Increasing evidence suggested gut microbiota played a

significant role in the development of obesity. Many clinical or
Obesity has been increasing in prevalence worldwide and is a pre-clinical studies have shown that there are significant differences
serious threat to public health. Over the past 30 years, the in the composition of gut microbiota between healthy versus obese
prevalence of obesity among adults has exceeded 50% in some individuals (10), with decreased diversity and richness in the gut
western developed countries (1). This increase is also observed in microbiome of obese individuals (11). Also, causality between gut
non-western and some developing countries, especially in China, microbiota imbalance and obesity has been demonstrated in animal
where the number of overweight and obese people has been models. Gut microbiota dysbiosis can lead to an increase in
increasing at an alarming rate (2). A surge of related chronic lipopolysaccharide (LPS) in the host’s circulatory system and
diseases is strongly associated with the rising rate in obesity (3), induce chronic and low-level inflammation, thereby playing an
such as type 2 diabetes, hypertension, atherosclerosis, cancers, and important role in the occurrence and development of metabolic
stroke, all of which have been major health threats in China for the diseases such as obesity (12–14). Recently, multiple studies have
past two decades (2). During the ongoing COVID-19 pandemic, confirmed that traditional Chinese medicine can improve obesity in
obesity was found in early studies (4, 5) to be the second strongest animal models by regulating the composition of gut microbiota (13,
predictor of hospitalization and need for mechanical ventilation in 15). Chih-Jung Chang et al. reported that water extract of
the elderly. In obese individuals, there is a reduced quality of life and Ganoderma lucidum could reduce obesity and inflammation in
decline in life expectancy (1). Due to increasing awareness of the high fat diet (HFD) fed mice, which was associated with reversing
risks of obesity, interest in weight loss management has grown HFD-induced gut dysbiosis (as indicated by the reduction of
significantly with calls for effective interventions. Firmicutes-to-Bacteroidetes ratios and endotoxin bearing
Unhealthy eating styles and sedentary behavior and physical Proteobacteria levels) and maintaining intestinal barrier integrity
inactivity have been viewed as the key factors that contribute to (16). Fructus Aurantii extract also showed a similar anti-obesity
obesity. Current guidelines recommend diet, exercise, and behavior effect by modulating the gut microbiota (17). Berberine, the
modification as standard treatments for obesity. However, a study principal component of Coptis chinensis, is known as a potent
found that only 20% of adults who either tried to lose weight or to anti-obesity and lipid lowering agent. Moreover, its anti-obesity
maintain their weight, could both eat fewer calories and complete at effect could be associated with the increased butyrate production in
least 150 minutes of exercise per week (6). Simple exercise and diet the gut and modulation of the gut microbiota (18).
are often challenging for patients attempting to maintain lasting In this study, we aimed to further evaluate the safety and weight
results (7). Due to the risk of serious complications and the issues of loss efficacy of the W-LHIT capsules, in combination with a
nutritional management and treatment follow-up, bariatric surgery standardized lifestyle intervention. In addition, we used 16S
is only applicable to a specific subset of patients, and not widely pyrosequencing technology to evaluate the shift in the
applicable for clinical use (8). Pharmacological intervention can be composition of the gut microbiota before and after W-LHIT
an effective and widely accepted auxiliary method. capsule treatment to explore its potential role in understanding
Weight loss herbal intervention therapy (W-LHIT) is a Chinese W-LHIT capsules in weight loss response.
medicine (CM) prescription, consisting of 5 Chinese herbal
medicines: Ganoderma lucidum, Coptis chinensis, Astragalus
membranaceus, Nelumbo nucifera gaertn and Fructus aurantii. In Methods
our previous studies, we have showed that W-LHIT significantly
and safely reduced the body weight of mice with high-fat diet, and Participants and W-LHIT
normalized their glucose and cholesterol levels, without suppressing capsules preparation
appetite (9). In addition, in our phase I clinical study with 14
patients with simple obesity, the W-LHIT capsules demonstrated Participants who met simple obesity criteria were recruited
clinically significant weight loss (Cao et al. Manuscript submitted). from the Weifang community through WeChat Friends Circle,

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Cao et al. 10.3389/fendo.2023.1054674

advertisement, and local websites. Inclusion criteria were as follows: circumference (cm), blood pressure (Seat systolic blood pressure,
age 18-60 years with a body-mass index (BMI) of 28 - 48 kg/m2, SSBP; seat diastolic blood pressure, SDBP, mmHg) and heart rate
stable body weight within the past 3 months, ability to engage in (sub/min), and they were assessed at the beginning (0 M, one day
physical activity and follow healthy diet guidelines, no before treatment), mid-term (1 M, 30 days after treatment), and
cardiovascular, hepatic, or renal disease. Patients with disease- endpoint (2 M, 60 days after treatment) of the weight loss
induced obesity (such as Cushing’s syndrome or thyroid disease) intervention (please see the supporting information for detailed
were excluded. Exclusion criteria included the above-described methods). The proportion of individuals losing more than 5% of
diseases, psychiatric illness and psychological disorders, baseline weight was also assessed at the end of the intervention,
pregnancy and participants who cannot be clinically observed. including the open trial.
Written informed consent was obtained from all subjects upon
enrollment. The study was approved by the Medical Ethics
Committee of Weifang Wei-En Hospital. Secondary outcomes
W-LHIT capsules were prepared in a GMP facility (Tian-jiang
Pharmaceutical, Jiangsu, China). In our early research, we The Secondary measurements included assessments of fasting
established HPLC fingerprints of individual herbal components, blood glucose, high-sensitivity C-reactive protein (hs-CRP), liver
and monitored the quality of different batches of W-LHIT products function including alanine aminotransferase (ALT), aspartic
by comparing the peak times and intensities of the identified aminotransferase (AST), glutamate transferase (GGT), total
compounds. Berberine was used as a key compound index (9). bilirubin (TBIL), total protein (TP) and albumin (A), renal
Nine capsules are equivalent to the daily crude herbal medicines function including urea nitrogen (BUN), and creatinine (CRE),
dosage for 75 kg individual. The placebo capsule was filled with highly sensitive C - reactive protein (CRP), fasting lipids including
starch with the same weight and color as the formula. cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL-
C) and high-density lipoprotein cholesterol (HDL-C), oral glucose
tolerance test (OGTT; 75-g glucose, glucose, insulin, and C-peptide
Study design concentration changes from 0 to 120 min), and analysis of human
fat composition, bone mineral density, bilateral knee joint, and
Before enrollment, a CT scan of the chest was used to exclude lumbar vertebrae X-ray.
lung disease and a color doppler ultrasound was used to exclude
organic diseases of the heart, liver, and kidneys. Thyroid function,
blood coagulation, and sex hormone lab tests were used to rule out Healthy lifestyle interventions
obesity caused by endogenous diseases.
The design of the study was a randomized double-blind placebo- Healthy lifestyle interventions were carried out throughout
controlled trial. Fourty participants were randomly divided into treatment. All subjects were provided personalized healthy diet
treatment group (treated with W-LHIT capsules) and control guidance according to their living habits, physical condition, and
group (treated with placebo) according to random number table work characteristics. They were also encouraged to adhere to or
with 20 cases in each group. All participants had a light diet and were increase physical exercise based on their physical examination
advised to drink at least 1.5 L of water during the study. W-LHIT was results and their physical condition. However, there was not an
dosed according to body weight, all subjects dosed 9 to 15 capsules obligatory requirement to engage in a strict diet or exercise
daily (3 - 5 capsules a time, before meals, three times a day) for 2 program. All subjects regularly attended classes on the risk of
months and then followed up for an additional 2 months. Other obesity and how to develop a healthy lifestyle. A WeChat group
weight-loss drugs (including weight-loss dietary supplements) were was established to supervise all enrolled subjects. A contracted
discontinued during the treatment and follow up period. If a subject nurse reminded all the subjects to take the medication every day, to
discontinued the study prematurely, the remainder of doses were follow diet and exercise recommendations, and to post recipes for
recalled. Since researchers and subjects were both blinded, W-LHIT meals. Subjects were able to report their diet and exercise every day
capsules and placebo were all distributed by a third-party designated and share their weight loss symptoms and experience in this
distributor. After unblinding, the subjects in control group group forum.
voluntarily participated in another open trial where they were given
a 2-month supply of W-LHIT capsules for treatment, while the active
group stopped W-LHIT. Both groups had their weight checked again Safety assessment
2 months later.
Safety assessment included adverse events and standard
laboratory tests (hematological and biochemical tests). Adverse
Primary outcomes events were recorded by the nurse through the WeChat group. A
physical examination and an exercise cardiopulmonary function
The primary core measurements included the changes in body test (pulmonary function, electrocardiogram, and finger oxygen %)
weight (kg), BMI (kg/m 2 ), hip circumference (cm), waist were performed at enrollment and on the 60th day.

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16S Pacbio sequencing of fecal Statistical analysis

sample DNA
Data were exhibited as the mean with [95% confidence interval]
Fresh fecal samples were collected before and after the treatment (CI),95(n=18 or 19). All statistical analyses were performed using
for the gut microbial analysis. DNA was extracted from the subjects’ one-way ANOVA followed by Bonferroni post hoc by Prism 9
feces using a Power Soil® DNA Isolation kit (MO BIO Laboratories, software (GraphPad Software, Inc, La Jolla, CA). P value smaller
Inc., Carlsbad, CA, USA) according to the manufacturer’s instructions. than 0.05 considered statistically significant.
The bacterial 16S rDNA was amplified by PCR using universal primers
(27F 5′ -AGRGTTTGATYNTGGCTCAG-3′, 1492R 5′ -TASG
GHTACCTTGTTASGACTT-3′). 16S Pacbio sequencing of the PCR Results
products was performed on an Illumina MiSeq platform at Biomarker
Technologies Co, Ltd. (Beijing, China). Weight loss
After using the SMRT Link tool (version 8.0, provided by Pacbio),
then Lima v1.7.0 software to obtain the Barcode-CCS sequence data, 14 women and 26 men who met the screening criteria were
UCHIME v8.1 software to remove the chimera sequence, the recruited. 2 participants withdrew due to personal issues, 1
optimization-CCS sequence was obtained for bioinformatics analysis. participant withdrew due to treatment compliance issues, and 37
Trimmed sequences from each sample were clustered into operational participants (active n=18 vs placebo n=19) completed the trial. As
taxonomic units (OTUs) based on a 97% sequence similarity using shown in Table 1, baseline characteristics at randomization were
USEARCH v10.0 method. The taxonomical analysis was performed by compared between the treatment group and placebo group.
alignment with the Bacterial Silva database (Release132, http:// Weight loss during the study is shown in Figure 1A. From
www.arb-silva.de). Alpha diversity indexes were evaluated based on randomization to the 60th day (2 months), both groups showed
the richness (Chao 1, Ace) and diversity (Shannon index, Simpson sustained weight loss, but mean weight loss and BMI reduction of
index) of these OTUs using Mothur v.1.30 (http://www.mothur.org/). W-LHIT capsule-treated subjects were significantly greater than that of
A PLS-DA analysis (Partial Least Squares Discriminant Analysis) was placebo group, with mean weight reduction of -7.05 [-9.10, -4.99] kg
performed according to the supervised matrix of distance. The linear (- 7.37%, p <0.001) in treatment group, compared to -4.39 [-5.68, -3.09]
discriminant analysis effect size (LEfSe) was conducted for the kg (- 4.78%, p <0.001) in placebo group (p <0.05, Figure 1A). Similar
quantitative analysis of biomarkers (LDA threshold > 4) among each results were obtained for BMI, with significant reduction in both
group. A Metastats analysis was used to identify the most differently groups. The mean BMI in the treatment group decreased by -2.39
abundant taxa between the placebo and treatment groups. A predicted [-3.00, -1.77] kg/m2 (- 7.40%, p <0.001), significantly higher than the
KEGG pathway was also performed using Picrust software to obtain reduction of -1.61 [-2.09, -1.12] kg/m2 (- 4.73%, p <0.001) in the
significant differences in gene function of the flora in the placebo and control group (p <0.05, Figure 1B). Both groups showed a reduced hip
treatment groups. circumference (-7.61cm verse -4.32 cm, p = 0.067, Figure 1C) and waist

TABLE 1 Baseline data.

Treatment (n=18) Control (n=19) P value

Sex (F/M) 6/12 7/12 0.93

Age (y) 39 ± 6.4 39 ± 4.6 0.92

Body Weight (kg) 96 ± 7.5 95 ± 8.7 0.92

BMI 32.25 ± 1.4 34.04 ± 2.5 0.21

Hip Circumference (cm) 115 ± 2.5 116 ± 4.8 0.51

Waist Circumference (cm) 107 ± 4.8 108 ± 6.0 0.78

SDBP (mmHg) 91 ± 5.9 90 ± 5.2 0.98

SSBP (mmHg) 135 ± 9.5 133 ± 8.7 0.76

HR 77 ± 4.6 78 ± 4.0 0.68

HS-CRP (mg/L) 4.5 ± 1.4 4.9 ± 1.5 0.87

Total cholesterol (mmol/l) 5.4 ± 0.5 5.2 ± 0.9 0.80

Triglycerides (mmol/l) 2.0 ± 0.6 3.0 ± 1.1 0.16

HDL cholesterol (mmol/l) 1.1 ± 0.08 1.2 ± 0.15 0.24

LDL cholesterol (mmol/l) 3.3 ± 0.3 3.1 ± 0.4 0.68

F, female; M, male; y, year; BMI, body mass index; SDBP, sitting diastolic blood pressure; SSBP, sitting systolic blood pressure; HR, heart rate, beats per minute; HDL, high-density lipoprotein;
LDL, low-density lipoprotein. Data shown as mean with (±) 95% CI.

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Cao et al. 10.3389/fendo.2023.1054674

circumference (-7.44 cm verse -6.79 cm, p = 0.71, Figure 1D), but decreased by -8.67 [-12.59, -4.64] mmHg (- 9.52%, p <0.001) and
without statistically significant difference between the groups. During -3.21 [-6.47, -0.083] mmHg (- 3.59%, p <0.05), respectively, with a
the study, 72.22% of the subjects in the treatment group lost more than significant difference between the two groups (p <0.05, Figure 2A).
5% of their body weight and 77.78% of the subjects lost at least 5% of The seated systolic blood pressure (SSBP) decreased by -7.99
their BMI, much higher than the control group (36.84% both in body [-13.29, -2.598] mmHg (- 5.88%, p <0.05) and -7.16 [-13.67,
weight and BMI), as shown in Table S1. Encouragingly, the number of -0.6621] mmHg (- 5.33%, p <0.05), in the treatment and placebo
participants in the treatment group whose BMI decreased from obesity groups, respectively. Among the subjects, there were 13 people with
to overweight (5 verse 3) and from severe obesity to obesity (5 verse 3) hypertension, 6 people in the treatment group (5 people at Stage 1
was much higher than that in the control group. hypertension, 1 person at Stage 2), and 7 people in the placebo
The results of the analysis of human fat components were group (6 people at Stage 1, 1 person at Stage 2). There were 5
consistent with the results of the above weight loss. Body fat rate, fat subjects in the treatment group and 2 subjects in the placebo group
weight, fat-free body weight in both groups were significantly whose blood pressure returned to normal after 2 months of
reduced. However, the percentage of skeletal muscle was only treatment. There was no noticeable change in heart rate for all
significantly increased in the treatment group. Moreover, the subjects (Figure 2C).
reduction of body fat rate (- 14.8% vs - 4.56%, p < 0.01), body fat The fasting blood sugar level of subjects in the treatment group
(- 10.0% vs - 3.6%, p < 0.01) and fat-to-muscle ratio (- 12.0% vs - also showed a significant reduction of -0.28 [-0.46, -0.096] mmol/L,
3.33%, p < 0.01) in treatment group was statistically significant p < 0.01), while that of subjects in the placebo group only decreased
compared with that of the placebo group (Table S2). slightly (- 0.1 [-0.17, 0.095] mmol/L, shown in Figure 2D). All
subjects in the treatment group whose blood sugar level exceeded
the threshold (6.1 mmol/L) had varying degrees of blood glucose
Changes in blood pressure, blood sugar, reduction. The blood sugar of the 4 subjects with blood glucose
and blood lipid levels between 6.1-6.7 mmol/L fell below the threshold. In addition,
all the concentration of glucose tolerance, C-peptide, and insulin in
As shown in Figures 2A, B, the blood pressure of both groups the treatment group significantly decreased after W-LHIT
decreased significantly. The seated diastolic blood pressure (SDBP) treatment, and their mean area under the curve (AUC) decreased

A B

C D

FIGURE 1
The Reduction in body weight (A), BMI (B), hip circumference (C), waist circumference (D). The reduction in body weight was significantly greater in
the treatment group than in the placebo group after 2 months (2M). Bars were shown as mean of each group. All intra-group analysis were
performed by using one-way ANOVA followed by bonferroni post hoc, and all inter-group analysis for baseline and treatment effects were
performed by using t test followed by Mann-Whitney (# represents p < 0.05) by Prism 9 software (*, ** and *** represent p < 0.05, p < 0.01 and
p < 0.001). Body mass index, BMI.

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A B

C D

FIGURE 2
Mean Changes in SDBP (A), SSBP (B), HR (C) and Blood sugar levels (D). The reduction in SDBP was significantly greater in the treatment group than
in the placebo group after 2 months (2M). Bars (blue) were shown as mean of each group with 95% CI. All intra-group analyses were performed
using one-way ANOVA followed by Bonferroni post hoc, and inter-group was performed by using t test followed by Mann-Whitney by Prism 9
software, (*, ** and *** represent p < 0.05, p < 0.01 and p < 0.001; # represents p < 0.05, ns represents no significant difference). Seated systolic
blood pressure, SSBP, seated diastolic blood pressure, SDBP, mmHg.

by 5.87%, 10.54% and 32.99% respectively. While the fasting blood After unblinding, 12 of the 19 subjects in the placebo group
glucose and the above indicators in placebo group declined at subsequently completed an additional two months of W-LHIT
individual time points, there was no significant pattern of change capsules treatment. As shown in Figure S1, in the absence of
at most time points (Table 2). healthy lifestyle interventions, only one-third of subjects lost
Additionally, W-LHIT significantly reduced total cholesterol (TC, nearly 5% of their body weight, with a mean weight and BMI loss
Figure 3A, p < 0.01), blood triglycerides (TG, Figure 3B, p < 0.01) and of 3.13 [4.23, 2.03] kg (-3.60%) and 1.13 [1.57, 0.70] kg/m2 (-3.51%).
low-density lipoprotein cholesterol (LDL-C, Figure 3C, p < 0.001). The The reductions in systolic and diastolic blood pressure were
concentrations of TC, TG and LDL-Chol in the treatment group were consistent with those in the treatment group, by 2.49% and
decreased by 3.87%, 19.2% and 12.0%, respectively. There were no 9.80%, respectively (p < 0.001).
noticeable changes in TC, TG, HLD-C and for the placebo
group (Figure 3).
Hs-CRP is positively correlated with atherosclerosis. Subjects in Laboratory safety profile
both treatment and control groups also showed a significant
reduction in their Hs-CRP level, (-73.73% [-60.71%, -83.24%], Most subjects at enrollment had mild to moderate fatty liver,
p <0.001 vs -46.77% [-14.04%, - 79.51%], p <0.01, Figure 4). and 14 subjects had moderate fatty liver. During the treatment, 6
subjects (5 in the treatment group) with moderate fatty liver
improved to mild fatty liver, and 4 subjects with mild fatty liver
Effect on body weight 2 months off in the treatment group returned to normal. Among them in the
double blind placebo-controlled trial treatment group whose glutamyl transferase (GGT, liver function
and open trial index, Figure S2) levels was above the threshold, their GGT levels
decreased significantly after treatment, while that of subjects in the
Two months after the end of treatment, the body weight of all placebo group showed no significant changes.
subjects in the treatment group maintained at the same level after There was no statistically significant difference between the
treatment or slightly decreased (p = 0.45, Figure 5) for the following treatment group and the placebo group in the levels of other liver
two months after completion of therapy. and kidney function indices, including alanine aminotransferase

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TABLE 2 Results of GGT, CRT and ICT in the two group before and after treatment.

Treatment (n=18) Placebo (n=19)

Characteristic Normal values
Baseline Post-treatment Baseline Post-treatment
Fasting 3.90-6.10 5.67 ± 0.8 5.50 ± 0.68 5.58 ± 0.81 5.53 ± 0.77

30 min. 7.78-8.89 9.55 ±1.29 8.98 ± 1.13*** 8.82 ± 1.47 8.58 ± 1.45*

60 min. 7.78-8.89 9.47 ± 2.87 8.96 ± 2.53* 8.32 ± 2.99 8.55 ± 2.91
Glucose (mmol/L)
120 min. 3.90-7.80 7.09 ± 1.59 6.60 ± 1.9** 7.18 ± 2.21 7.03 ± 2.03

180 min. 3.90-6.10 5.2 ± 1.73 4.89 ± 1.38* 5.00 ± 1.17 5.38 ± 1.35

AUC 1379 ± 135.8 1298 ± 130.6 1317 ± 151.7 1324 ± 145.2

Fasting 0.52–4.38 3.72 ± 1.27 3.22 ± 0.86* 3.96 ± 1.37 3.86 ± 1.29

30 min. 3.58–13.2 11.16 ± 4.48 9.89 ± 3.68** 9.5 ± 3.37 9.86 ± 3.7

60 min. 3.58–13.2 12.49 ± 4.05 11.13 ± 2.48* 10.02 ± 2.74 11.24 ± 2.76
C-peptide (ng/mL)
120 min. 1.2–11.3 10.66 ± 3.63 9.46 ± 2.68** 10.24 ± 3.13 10.22 ± 2.54

180 min. 0.38–6.56 6.34 ± 2.76 6.05 ± 2.28* 6.08 ± 2.6 6.75 ± 2.76

AUC 1783 ± 248.6 1595 ± 188.4 1676 ± 188.2 1592 ± 199.4

Fasting 2.3–26.0 21.44 ± 11.01 17.57 ± 7.689* 24.09 ± 14.19 21.99 ± 12.26

30 min. 10.5–61.8 170.33 ± 125.32 126.39 ± 72.06** 120.6 ± 84.15 108.66 ± 74.98*

60 min. 10.5–61.8 155.62 ± 95.62 92.31 ± 55.29*** 104.76 ± 57.01 122.09 ± 62.14
Insulin (mU/mL)
120 min. 1.02– 41.09 112.34 ± 74.74 71.06 ± 39.55** 104.08 ± 63.73 88.59 ± 43.48

180 min. 0.25– 11.53 41.55 ± 33.24 32.03 ± 23.13* 34.98 ± 35.88 40.85 ± 40.40

AUC 20422 ± 5487 13684 ± 3112 15999 ± 3583 15863 ± 4092

Data are shown as mean ± SD. Intra-group analyses were performed by using t test followed by Wilcoxon by Prism 9 software, p value, **< 0.01, ***< 0.001. GTT, Glucose tolerance test; CPRT,
C-peptide release test; IRT, Insulin release test; F.B.S, Fasting blood sugar.

(ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), obtained from the 74 samples through a single molecules real-time
total protein, and albumin. The electrolytes, routine urine, blood sequencing analysis, with an average of 5,647 reads and 82 OTUs per
coagulation, and routine blood of all subjects were within the sample (Table S5). These reads/OTUs were assigned to 11 different
normal range, and there was no significant change (Table S3). phyla (15 class, 27 order, 52 family, 112 genus, and 219 species, and the
principal bacterial phyla of all groups were Firmicutes, Proteobacteria,
and Bacteroides. Main changes in microbial diversity observed before
Clinical adverse reactions and after treatment included the enrichment of Proteobacteria (a slight
increase (29.8% vs 32.8%) in the placebo group versus a slight decrease
Seven subjects reported slight gastrointestinal (GI) reactions (32.3% vs 29.8%) in the treatment group), Verrucomicrobiota (a
(including 5 subjects in the open trail, Table S4), no subjects decrease (1.73% vs 0.7%) in the placebo group but an obvious
reported obvious adverse symptoms during the whole W-LHIT increase (4.47% vs 10.52%) in the treatment group), and
treatment. The GI side effects included decreased hyper appetite, Bacteroidetes (a parallel decrease in both groups, 24.04% vs 20.86%
mild nausea, and increased frequency of stools. All subjects in the placebo group and 22.75% vs 18.31% in the treatment group)
alleviated these side effects quickly by taking W-LHIT after meals (Table S6 and Figure 6B) showed details of the composition on Genus
or reducing the dose. in both group.
The alpha diversity indexes, including Ace and Chao indices,
rarefaction, Shannon-index (Table S4), and rank abundance curve
Characteristics of 16S Pacbio (Figure 6A), indicated that there was similar richness and sufficient
sequencing results sequence coverage in all samples. However, the placebo group had a
significant decrease in reads, OUTs, and the classification of
The effects of W-LHIT on the intestinal microbiota composition microorganisms (family, genus, species), while the treatment
were assessed by sequencing the bacterial 16S rRNA. A total of 417,913 group had a slight increase in OUTs and the classification of
optimization-CCS sequences and 352 OTUs (97% similarity) were microorganisms (phylum, class, order, family, genus, species).

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Cao et al. 10.3389/fendo.2023.1054674

A B

C D

FIGURE 3
W-LHIT significantly reduced total cholesterol (TC), blood triglycerides (TG) and low-density lipoprotein cholesterol (LDL-C). (A) TC; (B) TG; (C) LDL-C;
(D) HDL-C. Bars were shown as mean of each group with 95% CI. All intra-group analyses were performed using one-way ANOVA followed by
Bonferroni post hoc, and inter-group was performed by using t test followed by Mann-Whitney by Prism 9 software (*, ** and *** represent p < 0.05,
p < 0.01, p < 0.001, ns represents no significant difference). TC, Total cholesterol; TG, triglycerides; LDL-C, low-density lipoprotein; and HDL-C, high-
density lipoprotein cholesterol.

FIGURE 4
Hs-CRP level was significantly reduced after the treatment. Bars
were shown as mean of each group with 95% CI. Intra-group
analyses were performed using one-way ANOVA followed by FIGURE 5
Bonferroni post hoc, inter-group analyses were performed by using Weight did not rebound in the treatment group at a follow-up visit 2
t test followed by Mann-Whitney by Prism 9 software (** and *** months after the treatment. Bars were shown as mean of each
represent p < 0.01 and p < 0.001). Hs-CRP, High-sensitivity C- group. The analyses were performed using one-way ANOVA
reactive protein. followed by Bonferroni post hoc by Prism 9 software.

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Cao et al. 10.3389/fendo.2023.1054674

Faecalibacterium_prausnitzii (p = 0.006, metastats) and

A Eubacterium_rectale (p = 0.006, metastats) within the class
Clostridia (p = 0.007, metastats). Furthermore, the analysis of the
KEGG metabolic pathway found that only glycan biosynthesis and
metabolism (Figure 7E) was statistically different between the
treatment group and the placebo group.

Discussion
In recent years, China has ranked first in the world for obesity
and type 2 diabetes (19). Obesity related complications such as type
2 diabetes and hypertension have posed a serious threat to people’s
B health. There has been a growing consensus worldwide on the
importance of obesity treatment not merely to achieve weight loss,
but also to ameliorate adiposity-based complications (20, 21). Along
with lifestyle intervention, W-LHIT capsule treatment resulted in
72.22% of subjects losing more than 5% of their body weight within
2 months, and 78.22% of the subjects to reduce more than 5% of
their BMI. In addition, W-LHIT capsule can also reduce blood
pressure, blood glucose and blood lipid of the subjects. In addition,
W-LHIT capsule treatment also resulted in a greater reduction of
blood pressure, blood sugar and blood fat. In this study, most of the
subjects had impaired glucose tolerance, and about one third of the
subjects were diabetic. In the treatment group, except for one
diabetic subject whose blood glucose and blood lipid levels did
FIGURE 6
not improve during the treatment period, other diabetic subjects
(A) Rank abundance curve. Each curve represents a group; placebo showed significantly reduced levels of cholesterol, low-density
group (WLCB (baseline), WLCA (post treatment)) and treatment
lipoprotein, glucose tolerance, C-peptide release, and insulin
group (WLTB (baseline) and WLTA (post treatment)). (B) PLS-DA
plots based on unweighted unifrac metrics. Each symbol represents release. In the placebo group, diabetic subjects showed little
a sample from the placebo group or treatment group, respectively. improvement in blood glucose and lipid levels. The results of
subjects’ liver and kidney function, and other biochemical
indicators indicate W-LHIT’s favorable safety and tolerance
Differences in microbial structure between profile. These promising results indicate a potential role for W-
the W-LHIT treatment group and the LHIT for addressing weight control & management problems of
placebo group simple obesity patients.
Unhealthy dietary habits and sedentary lifestyle are key
To identify whether W-LHIT-mediated weight loss is associated contributing factors leading to obesity. Therefore, healthy lifestyle
with changes in the gut microbiota, we then profiled the overall interventions (including healthy diet guidance and physical activity)
microbial structure of the placebo and W-LHIT treatment groups. can play a profound role in the treatment process. During the
The PLS-DA results (Figure 6B) based on the weighted Unifrac treatment, the average weight reduction of the placebo group was
distance matrix revealed that the overall structure of the bacteria did 4.58 kg (- 4.9%). For comparison, no healthy lifestyle intervention
not change significantly in either group, and only a small part of the was performed in the open trial. The results showed that the body
microbial structure between the two groups changed significantly weight was only reduced by 3.6% in the open treatment trial, which
(Figures 7A, B and Tables S5, 6). A LEfSe (Line Discriminant was significantly lower than the average weight loss of the treatment
Analysis (LDA) Effect Size) analysis was used to discover the key group with lifestyle interventions (-7.05kg, -7.2%). Despite the
biomarkers (LDA score > 4) of the gut microbiota under W-LHIT insufficient sample size during open-treatment trials, we can still
and placebo treatment, as shown in Figures 7C, D. The enriched see the importance of healthy lifestyle interventions for obesity
phylotypes in the treatment group were the genera Akkermansia (p treatment. There was a limitation that the daily food intake calories
= 0.013, metastats) within the family Akkermansiaceae (p= 0.005, and physical activity for each subject were not quantified during the
metastats) (within the order Verrucomicrobiota (p = 0.04, treatment, and the healthy lifestyle intervention was tailored for
metastats), the species Enterococcus_faecium (p = 0.037, each subject under the guidance of the physician. So, the difference
metastats) within the genera Enterococcus (p = 0.039, metastats). in weight loss of subjects could have possibly been affected
The enriched phylotypes in the placebo group were the species differently. The quality-of-life scale score did not measure changes
Haemophilus_parainfluenzae (p = 0.014, metastats) within the of psychological and physiological functions before and after
genera Haemophilus (p = 0.019, metastats), the species treatment. Improvement of the quality-of-life score could further

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Cao et al. 10.3389/fendo.2023.1054674

A B

C D

FIGURE 7
The structures and compositions of the gut microbiota before and after treatment in the two groups. (A) The Venn diagram of OTUs (species).
(B) The composition of relative abundance on genus. (C) Phylogenetic cladogram of microbial lineage in fecal samples of treatment group and
placebo group, with colors representing the most abundant differences in composition. (D) Key phylotypes of the gut microbiota responding to W-
LHIT treatment. The histogram shows the lineage with LDA value of 4 or higher determined by LEFSe. (E) Key KEGG metabolic pathway responding
to W-LHIT treatment.

contribute to enhancing weight loss (22). In subsequent research, evidences have demonstrated that the occurrence of obesity is strongly
further enhancement of the quality-of-life scale would be included. associated with gut microbiota dysbiosis, which can result in chronic,
As a marker of obesity, the level of hs-CRP is significantly persistent low-grade inflammatory reactions and abnormal lipid
elevated in the obese individual, and positively correlated with BMI metabolism (12, 26, 27). In a review on the profile of the gut
obesity (23). Hs-CRP is synthesized by the liver in response to the microbiota in obese adults, the consistent conclusion was that obese
stimulation of interleukin-6 and tumor necrosis factor-a, indicating individuals (in comparison to leaner individuals) have a greater
a state of inflammation (24). In our study, the hs-CRP level Firmicutes/Bacteroidetes ratio, Fusobacteria, Proteobacteria,
decreased by 72.59% after W-LHIT treatment, which confirmed Mollicutes, Lactobacillus, and reduced Verrucomicrobia (Akkermansia
that managing obesity can help reduce the risk of cardiovascular muciniphila), Faecalibacterium (Prausnitzii), Bacteroides,
disease and comorbidities by inhibiting the inflammatory Methanobrevibacter smithii, Lactobacillus plantarum (27). Our results
mechanism (25). support the trend that there is a negative correlation between
Although there has been no consensus on how changes in the Firmicutes/Bacteroidetes ratio and BMI. After W-LHIT treatment,
composition of gut microbiota can contribute to obesity, cumulative the BMI of both groups decreased, but the Firmicutes/Bacteroidetes

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Cao et al. 10.3389/fendo.2023.1054674

ratio both increased, with the mean ratio increased from 1.95 to 2.53 in and LDL-Chol reduction. The data processed by the unpaired t test
the placebo group and from 1.54 to 1.78 in the treatment group. followed by Mann-Whitney method have significant differences. In
Compared with normal-weight individuals, obese individuals have a the follow-up study, we will further expand the sample size to obtain
significant increase in Proteobacteria and a significant decrease in more meaningful results. Despite these limitations, we found that
Verrucomicrobia. Shin et al. considered that an increased prevalence of WLHT may be of great significance in managing weight loss of
Proteobacteria may be an active feature of metabolic disorders (28, 29). patients by ameliorating microbiome dysbiosis.
We found that the composition of Proteobacteria in 72.9% of the obese In conclusion, our current study assessed the efficacy and safety
subjects exceeded 20% before treatment. The average composition of of W-LHIT capsules in 37 Chinese patients with simple obesity. We
Proteobacteria in the treatment group decreased by about 2.6%, found that W-LHIT significantly reduced the weight of subjects
possibly indicating movement toward normalization of the gut with simple obesity, by greater than 5% of body weight in 72.6% of
microbiota, but in the placebo, group increased by about 3%. One of participants, which was significantly higher than in only 36.6% of
the new generation of probiotic candidates, Verrucomicrobia the placebo group. In addition to weight loss, subjects in the
(Akkermansia muciniphila), can degrade mucin, and is closely related treatment group also had significant improvements in blood
to host health (30). It has been found to enhance the intestinal barrier pressure, blood glucose, and blood lipids. During the 2-month
function and the effects of immunotherapy (31), enhance glucose treatment, 7 subjects reported slight-mild gastrointestinal adverse
tolerance and reduce insulin resistance (32), and moderate reactions. However, with taking the medicine after meal or reducing
inflammatory responses (33, 34), exhibiting beneficial therapeutic the dosage, all the adverse reactions were gradually relieved. In
roles in obesity, type 2 diabetes, atherosclerosis, tumors, and addition, the results of 16S gut microbiota showed that W-LHIT
inflammatory bowel disease (IBD)- related gastrointestinal significantly increased the abundance of akkermansia muciniphila
disturbances (30). Another exciting result in our study was that the and the Firmicutes/Bacteroidetes ratio, and decreased the
abundance of Verrucomicrobia (Akkermansia muciniphila) in the W- abundance of Proteobacteria, facilitating the normalization of gut
LHIT treatment group significantly increased from 4.4% to 10.5%. microbial ecosystem.
Increasing evidence indicates that berberine (key compound index)
target the gut microbiota and reversely modulate the structure and
diversity under pathological conditions, thus exerting poly- Data availability statement
pharmacological effects (18, 35) such as anti-obesity (36), anti-
hyperlipidemia (37), anti-diabetes (38). The raw data supporting the conclusions of this article will be
Coptis chinensis is the sovereign medicine in W-LHIT made available by the authors, without undue reservation.
prescription. However, 2 subjects in the treatment group
occasionally experienced mild gastrointestinal adverse events due to
the large oral dose (9-15 capsules) of W-LHIT and the bitter taste of Ethics statement
its main components (Coptis chinensis). Coptis chinensis has been
used for thousand years in China safely at its therapeutic dose to treat The studies involving human participants were reviewed and
various inflammatory disorders and related diseases, such as diarrhea, approved by Wei-En hospital. The patients/participants provided
vomiting, abdominal distention, high fever coma, toothache, diabetes their written informed consent to participate in this study. Written
and eczema (39), and it is highly safe at its therapeutic dose. Linn et al. informed consent was obtained from the individual(s) for the
found 20 patients administered with Coptis chinensis at a daily dose of publication of any potentially identifiable images or data included
3 g for 1055 patient-days without any organ toxicity or electrolyte in this article.
imbalance (40). Our follow-up study will refine the ingredients
further and enteric coating should be another good way to address
this issue. Coptis chinensis is cold in nature (39), so, patients with Author contributions
weak spleen and stomach should use it with caution.
This study still has limitations. Firstly, no lean mass subjects C-HW, M-CW, and Y-MS were significantly involved in
were enrolled in this trial, the concern should be addressed in our conducting experiments. M-ZC, YS, KS, and NY were involved in
follow-up study. Secondly, weight loss is a long-term and arduous data analysis. M-ZC was significantly involved in manuscript
work for subjects with sever obesity. So, only two months treatment preparation. X-ML, DC, and M-SM were significantly involved in
is insufficient, and subjects with severe obesity need to receive study design, data interpretation, and manuscript revision.
longer intervention duration. Thirdly, this is a single-center study, All authors contributed to the article and approved the
limited by the small sample size, and some efficacy indicators submitted version.
between the treatment and control groups are not statistically
significant, including the hip circumference (p =0.09) and waist
circumference (p = 0.7). Due to the small sample size, several results Funding
of the two groups failed the normality test (alpha=0.05), so, the data
processed by the 2-way ANOVA method shows that there is no This manuscript was supported by Healthy Freedom LLC and
statistical difference between them in weight loss, SDBP reduction Henan University of Chinese Medicine.

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Cao et al. 10.3389/fendo.2023.1054674

Acknowledgments Publisher’s note

We thank Henry Ehrlich for helping us revise the writing of All claims expressed in this article are solely those of the authors
the manuscript. and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
Conflict of interest
claim that may be made by its manufacturer, is not guaranteed or
This study shared the US Patent No: US20160296573A1 Weight endorsed by the publisher.
loss formulations, methods, and compositions based on Traditional
Chinese Medicine by X-ML, DC, and NY. Author DC was
employed by Healthy Freedom LLC. Authors NY and K.S are Supplementary material
members of General Nutraceutical Technology LLC.
The remaining authors declare that the research was conducted The Supplementary Material for this article can be found online at:
in the absence of any commercial or financial relationships that https://www.frontiersin.org/articles/10.3389/fendo.2023.1054674/
could be construed as a potential conflict of interest. full#supplementary-material

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