Journal of Functional Foods 97 (2022) 105256

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Journal of Functional Foods

journal homepage: www.elsevier.com/locate/jff

Corosolic acid improves glucose and insulin responses in middle-aged men

with impaired fasting glucose: A randomized, double-blinded,
placebo-controlled crossover trial
Masanobu Hibi a, *, Yuji Matsui b, Sachiko Niwa b, Sachiko Oishi a, Aya Yanagimoto a,
Takahiro Ono c, Tohru Yamaguchi b
a
Biological Science Research Laboratories, Kao Corporation, 2-1-3 Bunka, Sumida, Tokyo 131-8501, Japan
b
Health & Wellness Products Research Laboratories, Kao Corporation, 2-1-3 Bunka, Sumida, Tokyo 131-8501, Japan
c
Ueno-Asagao Clinic, 2-7-5, Higashiueno, Taito-ku, Tokyo 110-0015, Japan

A R T I C L E I N F O A B S T R A C T

Keywords: Corosolic acid (CA) improves glucose metabolism in diabetics and prediabetics, but its effects in healthy subjects
Banaba extracts with borderline hyperglycemia are unclear. Non-diabetic middle-aged men (n = 14) with impaired fasting
Hyperglycemia glucose tolerance (mean ± SD; age, 51.7 ± 9.0 years; fasting blood glucose, 6.0 ± 0.4 mmol/L) underwent an
Liver insulin sensitivity
oral glucose tolerance test (OGTT) after taking 1 mg/d CA or placebo for 2 weeks in a randomized double-blind
Impaired fasting glucose
crossover trial. In the 13 subjects who completed the study, the incremental area under the curve (iAUC) of
Oral glucose tolerance test
Pentacyclic triterpene plasma glucose after the OGTT was significantly lower in the CA condition than in the placebo condition (p =
0.028). The AUC and iAUC of the serum insulin and C-peptide concentrations, and liver insulin sensitivity values
were also significantly lower in the CA condition. Glucagon, glucagon-like peptide-1, gastric inhibitory peptide,
and non-esterified fatty acid levels did not differ significantly between conditions. CA may improve glucose
tolerance in both healthy and prediabetic subjects.
Trial registration: UMIN-CTR; http://www.umin.ac.jp/; Registration No. UMIN000034130.

1. Introduction therapies, diabetes remains a persistent global health challenge (Nathan

Incompetent production and use of insulin by the body leads to type
2 diabetes, which presents as fasting and postprandial hyperglycemia,
and is a major public health burden affecting 460 million people
worldwide. The number of individuals diagnosed with diabetes has
increased 4-fold in the past 30 years, and includes 31 million in the
United States and 4.9 million in Japan (Centers for Disease Control and
Prevention, 2020; International Diabetes Federation, 2019; Ministry of
Health, 2021). The World Health Organization (WHO) defines impaired
fasting glucose as a 2-h oral glucose tolerance test (OGTT) result < 7.8
mmol/L (140 mg/dL) and a fasting glucose level of 6.1–6.9 mmol/L (
mg/dL) (World Health Organization, 2012). The American Diabetes
Association’s Diabetes Care Guidelines apply the same thresholds used
by the WHO for impaired glucose tolerance, but the cutoff for impaired
fasting glucose is lower (fasting glucose; 5.6–6.9 mmol/L or mg/dL)
(American Diabetes Association, 2006). Despite significant efforts to
normalize blood glucose levels through dietary and pharmacologic
et al., 2007). Treatment of prediabetes by improving insulin resistance
and postprandial blood glucose control through dietary changes and
increased physical activity is critical toward delaying or preventing the
progression to type 2 diabetes (Cavalot et al., 2006; Ceriello, 2005;
Nathan et al., 2007).
Banaba (Lagerstroemia speciosa L.) leaf extracts have anti-diabetic
properties and are used as medicine in Southeast Asia, especially in
the Philippines (Garcia, 1940). In animal experiments, aqueous Banaba
leaf extract reduces blood glucose levels (Kakuda et al., 1996; Stohs,
Miller, & Kaats, 2012). The Banaba leaf contains a natural pentacyclic
triterpene, corosolic acid (CA, 2α-hydroxyursolic acid), that activates
cellular glucose transport and increases cellular glucose uptake, thereby
lowering blood glucose concentrations (Miura et al., 2004). The appli­
cation of CA attenuates cardiomyocyte hypertrophy in vitro by acti­
vating autophagy (Wang et al., 2019) and in mice, CA exhibits anti-
inflammatory and anti-arthritic activity (Kim et al., 2016; Kuraoka-
Oliveira et al., 2020).

* Corresponding author.
E-mail address: hibi.masanobu@kao.com (M. Hibi).

https://doi.org/10.1016/j.jff.2022.105256
Received 12 March 2022; Received in revised form 2 September 2022; Accepted 8 September 2022
Available online 19 September 2022
1756-4646/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
M. Hibi et al.
Preliminary clinical studies were performed to examine the safety
and antidiabetic activity of CA or banaba extracts. The safety and effi­
cacy of CA or banaba extracts were reviewed by Stohs et al. (2012). In a
study of 12 subjects with fasting blood glucose levels greater than 110
mg/dL that were given 10 mg of CA as a banaba extract daily for 2
weeks, consumption of banaba extract reduced fasting and postprandial
blood glucose levels by 12 %, with no side effects observed during or
after the study (Tsuchibe et al., 2006). Another study of 15 subjects
treated with once daily 100-mg tablets of a water-soluble banaba extract
for up to 1 year demonstrated significant improvements in glucose
tolerance and HbA1c levels after 6 months and 1 year of treatment with
banaba extract (Ikeda et al., 2002). The banaba extract was confirmed to
not cause hypoglycemia, and adverse effects or changes in hematologic
and biochemical characteristics were not observed during the 1-year
study period (Ikeda et al., 2002). A dose-dependent comparative study
of 10 individuals with type 2 diabetes revealed that CA at a dose of 0.48
mg/d for 2 weeks reduced fasting blood glucose levels by 30 % (Judy
et al., 2003). Fukushima et al. (2006) studied 31 subjects (16 men and
15 women) with higher blood glucose levels (fasting blood glucose
levels of 6.1–7.8 mmol/L or 110–125 mg/dL) and found that 10 mg/
d CA for 1 week improved postprandial glucose levels after a 75-g OGTT.
The effects of low doses (~1 mg/d) of CA, however, are unclear, espe­
cially in healthy subjects with normoglycemia to hyperglycemia
(Nathan et al., 2007; “Report of the Expert Committee on the Diagnosis
and Classification of Diabetes Mellitus,” 1997). Further clinical trials
evaluating the efficacy of CA as a potential treatment for hyperglycemia
are thus needed.
We hypothesized that intake of 1 mg/d CA for 2 weeks would
improve glucose and insulin responses to OGTT and insulin sensitivity in
prediabetic subjects. In the present study, we examined whether
continuous intake of CA for 2 weeks improved blood glucose levels after
75-g glucose loading. In addition, we examined insulin, glucagon, and
gastrointestinal hormone responses; insulin sensitivity indices; β-cell
secretory function indices; and hepatic insulin resistance indices.
2. Materials and methods
2.1. Subjects
Middle-aged men with impaired glucose function were recruited via
websites according to the following inclusion criteria: (1) age between
25 and 65 years, (2) fasting blood glucose from 5.6 to 6.9 mmol/L,and
HbA1c ≤ 6.4 %; and exclusion criteria: (1) history of diabetes, hepatic
dysfunction, kidney disease, or other serious disease, (2) current
smoking history, (3) current alcohol use ≥ 30 g/d, (4) shift- and night-
workers, and (5) individuals judged as unsuitable for participation in
the study by the study physician. The menstrual cycle is reported to
affect glucose metabolism (Yeung et al., 2010), and because the short
intervention period of the present study made it impractical to adjust for
the menstrual cycle, only male participants were included. This trial was
approved by the ethics review committee of Kao Corporation (Tokyo,
Japan, Approval date: August 23, 2018, Approval #T159-180720) and
Ueno Oriental Clinic (Tokyo, Japan, Approval date: August 30, 2018,
Approval #2018–08-27), and conducted in accordance with the tenets
of the Declaration of Helsinki. The trial was conducted from September
2018 to December 2018, and complied with the study protocol. Subjects
were provided details of the trial both verbally and in writing, and all
subjects provided written informed consent to participate. This trial was
registered with the University Hospital Medical Information Network
(UMIN; https://www.umin.ac.jp/; Registration No. UMIN000034130).
2.2. Study design
This randomized, double-blind, placebo-controlled, cross-over study
included two 2-week intervention periods with a 2-week wash-out
period. The intervention involved providing subjects with a powdered
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Journal of Functional Foods 97 (2022) 105256
beverage containing 1 mg CA or placebo (PL) daily for 2 weeks, and the
conditions were then switched after a 2-week wash-out period. The
sample size, selected on the basis of the results of a previous study
(Tsuchibe, Kataumi, Mori, & Mori, 2006), was set to 14 per group to
achieve a detection power of 0.8 with a significance level of 5 % for
postprandial glucose levels and a predictive dropout rate of 5 %. All
tasks related to the trial, including subject recruitment and randomiza­
tion, were performed by TES Co. ltd. (Tokyo, Japan). An independent
researcher allocated the subjects by stratified randomization according
to their fasting glucose levels and age using the Excel spreadsheet pro­
gram (Microsoft Excel , Microsoft Corp., Redmond, WA, USA). During
the intervention period, subjects were instructed to consume a test
beverage containing either 1 mg CA or PL for 14 days while maintaining
their usual diet and physical activity. Subjects recorded the details of all
meals on a dietary recording form for 3 days to confirm that their eating
and exercise habits were not altered. The subjects were asked to avoid
strenuous exercise, and alcohol and caffeine consumption for 24 h prior
to beginning each intervention period. The restrictions on food intake
and exercise were removed during the wash-out period, and subjects
were encouraged to return to their normal lifestyle. The day before the
measurements, the subjects were instructed to eat pre-packaged meals
provided by the study coordinator (total energy content: 2412 kcal/d,
14 E% from protein, 25 E% from fat, 61 E% from carbohydrate) and to
go to bed before midnight without ingesting any food or beverages other
than water after 9:00 pm. Baseline measurements were obtained
immediately after the subjects ingested a test meal following overnight
fasting for at least 12 h. After the 2-week intervention, the subjects were
instructed to arrive at the outpatient unit (Ueno Asagao Clinic, Tokyo,
Japan) before 8:00 am, where they remained until 12:00 pm. Body
weight, body fat percentage (body fat scale, model TF-780, Tanita Corp.,
Tokyo, Japan), blood pressure (digital blood pressure monitor, model
HEM-1000, Omron Corp., Tokyo, Japan), and body temperature were
measured, followed by a 75-g OGTT (Trelan-G75; AY Pharmaceuticals
Co., ltd. Tokyo, Japan) starting at 09:00 on day 14. Blood was obtained
by venous sampling under fasting conditions (0 h) and at 0.5, 1, 1.5, 2,
and 4 h postprandially. The outcome measurements were glucose, in­
sulin, glucagon, C-peptide, glucagon-like peptide-1 (GLP-1), glucose-
dependent insulinotropic polypeptide (GIP), non-esterified fatty acids
(NEFA), ketone bodies, and triglyceride concentration. Low-density li­
poprotein cholesterol, high-density lipoprotein cholesterol, and total
cholesterol concentrations were measured under fasting conditions.
2.3. Test beverages
The following test beverages were prepared at Kao Corporation. The
active beverage was prepared using extract obtained from the leaf of
L. speciose (CA), which was purified with hydrous ethanol from Tokiwa
Phytochemical Co., ltd. (Chiba, Japan), and a placebo beverage without
CA (0 mg of CA). CA, as the L. speciose leaf extracts, was concentrated to
a level greater than 18.0 %. CA in the active beverage was quantified by
high-performance liquid chromatography, as reported previously (Judy
et al., 2003) and determined to contain 1.0 mg/drink. Both beverages
were prepared to have an indistinguishable appearance and flavor.
2.4. Appetite questionnaire
Appetite (hunger, fullness, prospective food consumption, and
satiety) was assessed by having the subjects complete a 100-mm visual
analogue scale (VAS)-based questionnaire every hour beginning on the
morning of day 14 after the OGTT (Flint, Raben, Blundell, & Astrup,
2000; Nagai et al., 2012). The study coordinator provided verbal guid­
ance to all subjects on how to complete the questionnaire. The study
coordinator recorded the responses as the distance from the left end of
the VAS measured using a ruler.
M. Hibi et al.
2.5. Analytical procedure for blood samples
Plasma and serum samples obtained from subjects in a fasting state
and after the OGTT were stored at − 80 ◦ C until analysis. Glucagon,
active GLP-1, and total GIP levels were measured using enzyme-linked
immunosorbent assay kits (glucagon: Mercodia, Uppsala, Sweden;
active GLP-1: Immune Biology Laboratories, Gunma, Japan; total GIP:
Merck Millipore, Darmstadt, Germany). All other items were analyzed at
LSI Medience, Co., ltd. (Tokyo, Japan). Total area under the curve (AUC)
and incremental AUC (iAUC) for glucose, insulin, glucagon, C-peptide,
GLP-1, and GIP were calculated using the trapezoidal rule. Fasting in­
sulin resistance was assessed by a quantitative insulin sensitivity check
index (Katz et al., 2000) and the homeostasis model assessment of in­
sulin resistance (HOMA-IR) (Matthews et al., 1985). The Matsuda index
of whole-body insulin sensitivity (Matsuda & DeFronzo, 1999) and
under dynamic conditions with oral glucose insulin sensitivity (Mari,
Pacini, Murphy, Ludvik, & Nolan, 2001), which describes glucose
clearance during glucose loading, was calculated from measurements
obtained after the OGTT. Indices of insulin secretion, homeostasis model
assessment β-cell function (HOMA-β) (Katz et al., 2000), insulinogenic
index (Abdul-Ghani, Williams, DeFronzo, & Stern, 2007), and modified
β-cell function index (Zhang et al., 2018) were calculated using glucose
and insulin data from the 75-g OGTT, as previously described. Hepatic-
specific insulin resistance indices (Liver-IR) were calculated as previ­
ously described (Vangipurapu et al., 2011).
Fig. 1. Flow diagram of the trial.
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Journal of Functional Foods 97 (2022) 105256
2.6. Statistical analysis
Data are presented as mean ± standard error unless otherwise indi­
cated. The AUCs and iAUCs were calculated according to the trapezoidal
rule, and maximum concentrations (Cmax) for the primary and sec­
ondary outcome measures were obtained from measurements at each
time-point. The following statistical comparisons were performed be­
tween the CA and PL conditions. With respect to the AUC, iAUC, and
Cmax, differences between the CA and PL conditions were determined
for each subject, and comparisons between the treatments were per­
formed using a paired t-test to evaluate the effects of the interventions.
Time series data were analyzed using a mixed-effects model. An analysis
of covariance (ANCOVA) model was used to assess treatment differ­
ences, with treatment-condition, time, and treatment-condition × time
interaction as fixed effects. An unstructured covariance structure was
applied to treat the serial correlation, and empirical variances
(EMPIRICAL option) were used to estimate the fixed effects. The sig­
nificance of the fixed effects was evaluated, and treatments were
compared at each time-point. In all analyses, the statistical tests were
double-sided with a significance level of 5 %. Significant differences
were tested using the following statistical analysis software: SAS version
9.4 (SAS Institute Inc., Cary, NC, USA).
3. Results
3.1. Subjects
A total of 14 male subjects were recruited through websites and
M. Hibi et al. Journal of Functional Foods 97 (2022) 105256

completed the study between September 2018 and November 2018
(Fig. 1). One subject committed a major protocol violation in glycemic
control prior to the test day and was excluded before the key open.
Accordingly, data from 13 subjects (mean age ± SD, 52 ± 9 years; mean
BMI ± SD, 23.5 ± 2.3 kg/m2; mean glucose ± SD, 6.0 ± 0.4 mmol/L;
mean HbA1c ± SD, 5.6 ± 0.3 %) who completed the trial were analyzed.
Physical parameters and blood pressure were within normal ranges for
all subjects.
3.2. Fasting glucose and lipids
Anthropometric and fasting blood glucose and lipid parameters after
each 2-week intervention period are shown in Table 1. Body weight,
body fat, and fasting blood glucose levels were not significantly altered
after 2 weeks of CA intake compared with PL. Diastolic blood pressure
was significantly decreased by CA intake, but all individual changes
were within normal ranges. CA intake significantly increased 3-hydroxy­
butyric acid and total ketone bodies. No adverse events were reported.
3.3. Glucose and insulin response
Glucose, insulin, C-peptide, and glucagon as assessed up to the end of
the OGTT (240 min after drinking the 75-g glucose solution) after the 2-
week intervention periods are shown in Fig. 2. Postprandial glucose
levels were compared between CA and PL treatments using ANCOVA,
which showed a significant main effect of treatment (p = 0.022), but no
time effect and treatment × time interaction (p = 0.130 and p = 0.231,
respectively; Fig. 2a). Postprandial glucose concentrations were signif­
icantly lower in the CA condition than in the PL condition at 30 min (p =
0.024). Although the AUC and Cmax of glucose revealed no significant
difference between conditions (p = 0.090 and p = 0.186), the iAUC for
glucose was significantly lower in the CA condition (p = 0.028, Table 2).
ANCOVA revealed no significant treatment effect between the CA and PL
conditions on the change in the insulin concentration (p = 0.057), but
there was a time effect and treatment × time interaction (p = 0.039 and
p = 0.044, respectively; Fig. 2b). Postprandial insulin concentrations
were significantly lower in the CA condition than in the PL condition at
120 min (p = 0.033). The AUC, iAUC, and Cmax of insulin were
significantly lower in the CA condition (p = 0.044, p = 0.010 and p =
0.039, respectively; Table 2). The insulin sensitivity indices are shown in
Table 2. Although the HOMA-IR, QUICKI, Matsuda index, and OGIS did
not differ significantly between the CA and PL conditions (p = 0.257, p
= 0.377, p = 0.344, p = 0.380, and p = 0.128, respectively), the Liver-IR
was significantly decreased after the 2-week CA treatment compared
with the 2-week PL treatment (p = 0.021). The insulin secretion indices
are shown in Table 2. The HOMA-β and insulinogenic index did not
differ significantly between the CA and PL conditions (p = 0.196 and p
= 0.715, respectively), but the modified β-cell function index (MBCI)
was significantly increased after the 2-week CA treatment compared
with the 2-week PL treatment (p = 0.047).
Comparison between the CA and PL treatments using ANCOVA
revealed a significant main effect of time and treatment for the change in
C-peptide (p = 0.026 and p = 0.014, Fig. 2c), but no significant treat­
ment × time interaction (p = 0.123). Postprandial C-peptide concen­
trations were significantly lower in the CA condition than in the PL
condition at 120 min (p = 0.032). The AUC and iAUC of C-peptide was
significantly lower in the CA condition (p = 0.037 and p = 0.003,
respectively; Table 2). Although ANCOVA showed no main treatment
effect or treatment × time interaction of the intervention on glucagon
fluctuations (p = 0.912 and p = 0.140, respectively), or a significant
effect of effect of time (p = 0.938 and p = 0.263, respectively), there was
a significant time effect (p = 0.038; Fig. 2d). The AUC, iAUC, and Cmax
of glucagon did not differ significantly between the CA and PL condi­
tions (p = 0.891, p = 0.624, and p = 0.365, respectively; Table 2).
3.4. Ketone bodies, NEFA, and gastric hormones
Fluctuations in GLP-1 and GIP up to the end of the OGTT did not
differ significantly between the CA and PL treatments. Analysis of
temporal data using ANCOVA revealed no main effect of the interven­
tion and time × treatment interaction on GLP-1 and GIP (Fig. 3a, b).
NEFA and ketone bodies up to the end of the OGTT did not differ
significantly between the CA and PL treatments. ANOVA revealed no
main effect of the intervention on NEFA and ketone bodies (p = 0.398
and p = 0.269, respectively; Fig. 3c, d).
3.5. Appetite

Table 1 Data regarding appetite (hunger, fullness, prospective food con­

Anthropometric and fasting blood lipid parameters after 2-week intervention sumption, and satiety) during the 4 h of the OGTT are summarized in
periods. Table 3. The AUC for hunger, fullness, prospective food consumption,
PL CA p and satiety did not differ significantly between the CA and PL conditions
values (p = 0.729, p = 0.764, p = 0.409, and p = 0.085, respectively).
Weight (kg) 70.6 ± 2.2 70.6 ± 2.2 0.951

BMI (kg/m2) 23.7 ± 0.7 23.7 ± 0.7 0.933 4. Discussion

Body fat (%) 22.4 ± 1.3 22.2 ± 1.4 0.553
SBP (mmHg) 118 ± 3 116 ± 4 0.691 In the present study, we examined the effects of CA on glucose and
DBP (mmHg) 74 ± 2 71 ± 3 0.025
Glucose (mmol/L) 6.0 ± 0.2 6.0 ± 0.2 0.888
insulin responses in an OGTT, and perceived appetite in middle-aged
Insulin (mU/L) 6.5 ± 0.9 7.8 ± 1.5 0.185 healthy adult males with impaired fasting glucose. The iAUCs of the
C-peptide (ng/mLF) 1.3 ± 0.1 1.5 ± 0.2 0.272 plasma glucose response, and the AUCs and iAUCs of the serum insulin
Glucagon (pmol/L) 5.7 ± 0.9 5.9 ± 1.1 0.699 response in the OGTT indicated that, compared with daily intake of PL,
TG (mg/dL) 181 97 178 104 0.777
daily intake of 1 mg of CA for 2 weeks had beneficial effects on glucose
± ±
Total-C (mg/dL) 233 ± 13 233 ± 13 0.975
HDL-C (mg/dL) 56 ± 4 57 ± 4 0.846 and insulin responses. Postprandial glucose changes after OGTT showed
LDL-C (mg/dL) 150 ± 12 148 ± 11 0.628 a significant treatment main effect, and revealed a significant treatment
NEFA (mmol/L) 0.47 ± 0.04 0.47 ± 0.03 1.000 × time interaction. Although similar changes were also observed for C-
Total ketone bodies (mmol/ 63.8 ± 8.4 78.8 ± 13.1 0.044 peptide, no changes were observed for glucagon, gastrointestinal hor­
L)
3-hydroxybutyric acid 43.6 ± 6.2 55.6 ± 9.8 0.033
mones such as GLP-1 and GIP, NEFA, or appetite variables. Moreover, as
(mmol/L) a liver insulin sensitivity index, the Liver-IR was significantly decreased
Acetoacetic acid (mmol/L) 20.2 ± 2.5 23.2 ± 3.4 0.135 after the 2-week daily CA treatment. The present results extend previous
1)
Data are expressed as mean ± SE., P values are calculated by paired t tests. findings that CA intake has beneficial effects on glucose and insulin
BMI, body mass index; CA, corosolic acid; DBP, diastolic blood pressure; HDL-C, responses following OGTT.
high density lipoprotein cholesterol; LDL-C, low density lipoprotein cholesterol; Postprandial glucose may be a better marker than fasting glucose for
NEFA, Non-esterified fatty acids; PL, placebo; TG, triglyceride; Total-C, Total early detection of disturbances in glucose metabolism, as non-diabetic
cholesterol; SBP, systolic blood pressure. and diabetic patients spend most of the day in a fasting or

4
M. Hibi et al.
Fig. 2. Fasting and postprandial concentrations of glucose (a), insulin (b), C-peptide (c), and glucagon (d) were measured for 4 h after intake of a 75-g glucose
solution (OGTT) following daily intake of CA or PL for 2 weeks. Values are means and standard errors represented by bidirectional bars. White circles indicate PL
treatment and black circles indicate CA treatment. * Mean values were significantly different from that of the PL condition at the same time (paired t-test). CA,
corosolic acid; OGTT, oral glucose tolerance test; PL, placebo.
postprandial state. Postprandial glucose is also a strong predictor of
mortality or cardiovascular disorders (Bonora & Tuomilehto, 2011).
Previous studies described the importance of postprandial hyperglyce­
mia in the pathogenesis of glucose intolerance (Suzuki et al., 2003). In
the post-absorptive state, many factors positively and negatively regu­
late blood glucose levels, and the combination of these factors de­
termines blood glucose levels. We observed that the glucose iAUC
decreased by 18 % and the insulin iAUC decreased by 15 % during a 4-h
OGTT after intake of CA compared with PL. At 30 min after OGTT, blood
glucose levels were lower in the CA condition than in the PL condition, a
finding that explains the lower insulin secretion at 120 min. Further­
more, the decreased C-peptide response after CA intake as well as the
insulin changes robustly support the beneficial effects of CA. Previous
chronic and repeated ingestion studies of high-dose CA (~10 mg/d) in
subjects with diabetes or borderline diabetes also demonstrated effects
on fasting blood glucose (Judy et al., 2003), and postprandial blood
glucose after OGTT (Fukushima et al., 2006; Tsuchibe et al., 2006). The
findings from the present double-blind, placebo-controlled, cross-over
study demonstrate that intake of 1 mg/d CA can improve not only
blood glucose levels but also insulin secretion after glucose loading
without direct glucose absorption inhibition or incretin activity in sub­
jects with impaired fasting glucose tolerance. A significant increase in
fasting ketone bodies was observed with continuous intake of CA, sug­
gesting an enhancement of beneficial fat metabolism with continuous
CA intake. The present results support the interpretation that the
5
Journal of Functional Foods 97 (2022) 105256
improvement in postprandial glucose concentrations after continuous
CA intake is independent of the inhibition of carbohydrate degradation
in the gastrointestinal tract or the increase in insulin secretion based on
incretins such as GLP-1 and GIP actions during OGTT.
The mechanisms underlying the effect of CA intake to reduce post­
prandial glucose and insulin secretion are not fully elucidated, but the
improvements in the Liver-IR and the increase in fasting ketone bodies
after CA intake provide clues suggesting improved liver fat oxidation
and insulin resistance. An animal study reported that mice fed 0.023 %
CA for 9 weeks with a high fat diet exhibited a 23 %, 41 %, and 22 %
decrease in fasting plasma concentrations of glucose, insulin, and tri­
glycerides, respectively, as well as a 10 % reduction in body weight and
a 15 % reduction in total fat mass (Yamada et al., 2008b). Dietary CA
also increased the expression of peroxisome proliferator-activated re­
ceptor alpha (PPARα) in the liver and PPARγ in white adipose tissue,
providing a mechanistic explanation for the reduction in body weight
and hepatic adiposity. Another study examined the mechanism of action
of CA on glycogenesis in rat liver using perfused liver and isolated he­
patocytes. CA (20–100 mM) increased fructose-2,6 diphosphate forma­
tion and decreased gluconeogenesis in a dose-dependent manner by
decreasing cyclic AMP levels and inhibiting protein kinase A activity
(Yamada et al., 2008a). Furthermore, CA increased glucokinase activity
without affecting glucose-6-phosphatase activity, suggesting increased
glycolysis. These findings provide new mechanistic information on the
anti-diabetic effects of CA. The major cause of hyperglycemia in diabetic
M. Hibi et al. Journal of Functional Foods 97 (2022) 105256

Table 2
Fasting and postprandial glucose, insulin, C-peptide, and glucagon concentrations and indices of insulin sensitivity during the OGTT after the 2-week interventions.1)
PL CA p values
Glucose
AUC (mmol/L*h) 34.8 ± 1.5 32.7 ± 1.6 0.090

Cmax (mmol/L) 12.3 ± 0.5 11.9 ± 0.6 0.186

iAUC (mmol/L*h) 10.8 ± 1.1 8.8 ± 1.2 0.028
Insulin
AUC (mU/L*h) 162.5 ± 19.1 146.2 ± 16.9 0.044
Cmax (mU/L) 75.1 ± 8.9 66.9 ± 8.9 0.039
iAUC (mU/L*h) 136.7 ± 16.8 115.1 ± 12.9 0.010
C-peptide
AUC (ng/ml*h) 21.9 ± 1.6 20.4 ± 1.7 0.037
Cmax (ng/ml) 8.5 ± 0.6 7.9 ± 0.8 0.183
iAUC (ng/ml*h) 16.5 ± 1.3 14.5 ± 1.3 0.003
Glucagon
AUC (pmol/L*h) 17.5 ± 3.1 17.3 ± 3.5 0.891
Cmax (pmol/L) 9.8 ± 1.6 9.0 ± 1.7 0.365
iAUC (pmol/L*h) − 5.4 ± 10.3 − 6.2 ± 9.2 0.624
Insulin sensitivity indices
HOMA-IR 1.7 ± 0.2 2.1 ± 0.5 0.257
QUICKI 0.36 ± 0.01 0.36 ± 0.01 0.377
Matsuda index 4.9 ± 0.7 5.2 ± 0.7 0.344
OGIS 364 ± 12 373 ± 11 0.380
Hepatic insulin sensitivity indices
Liver-IR 1.38 ± 0.04 1.36 ± 0.04 0.021
Insulin secretion and β-cell function indices
HOMA-β 53.2 ± 7.8 63.3 ± 10.7 0.196
Insulinogenic index 0.64 ± 0.11 0.70 ± 0.17 0.715
MBCI 2.85 ± 0.40 3.89 ± 0.74 0.047
1)
Data are expressed as mean ± SE. P-values were examined between CA and PL treatments using a paired t-test. AUC, area under the curve; CA, corosolic acid;
Cmax, maximum concentration; HOMA-IR, homeostasis model assessment insulin resistance index; HOMA-β, homeostasis model assessment β-cell function; iAUC,
increment of area under the curve; Liver-IR, liver insulin resistance; MBCI, modified β-cell function index; QUICKI, quantitative insulin sensitivity check index; OGIS,
oral glucose insulin sensitivity; PL, placebo.

patients is thought to be excessive glucose production in the liver,
making inhibition of excessive hepatic glucose production a potential
target for treating glucose metabolism disorders (Bogardus, Lillioja,
Howard, Reaven, & Mott, 1984; Stumvoll, Nurjhan, Perriello, Dailey, &
Gerich, 1995). Recent experiments in HepG2 cells and zebrafish
revealed that CA has the potential to regulate hepatic phosphoenol­
pyruvate carboxykinase (PEPCK), which is the rate-limiting enzyme for
gluconeogenesis in the liver (Xu et al., 2019). Transcriptional activity of
PEPCK is regulated by glucagon, glucocorticoids, and hormones
involved in glucose homeostasis such as glucagon and glucocorticoids,
and is suppressed by insulin (Tang et al., 2018). This effect of CA is
probably due to the downregulation of PEPCK and other genes involved
in glucose metabolism, type-2 diabetes-related oxidative stress, and
inflammation. The improved liver fat oxidation and insulin resistance
following CA intake may be related to the inhibition of PEPCK. Thus,
clinical trials with further analysis of individual organs, including the
liver, muscle, pancreas, and adipose tissue, are needed to elucidate the
mechanisms underlying the effects of CA to lower the blood glucose.
The present findings demonstrate the potential of CA intake to
improve glucose metabolism in borderline pre-diabetics with deterio­
rating glucose metabolism. An important strength of this study is the
high rate of adherence to the intervention and measurements of not only
glucose and insulin, but also incretins during the OGTT. A limitation of
this study is the small number of participants, and the analysis was
conducted with the per protocol set due to a protocol violation. Only
male participants were included in the present study due to reported
effects of the female menstrual cycle on glucose metabolism (Yeung
et al., 2010), as well as the short intervention period, which did not
permit adjustments for the menstrual cycle. Therefore, it is necessary to
consider not only the CA intake but also sex when evaluating the effect
on postprandial glucose metabolism in prediabetic participants. In
addition, because of the short intervention period, there was no differ­
ence in fasting insulin sensitivity (HOMA-IR) between the 2 conditions,
suggesting that the 2 conditions were similar with respect to the glucose
tolerance status at the time of the OGTT. Further large-scale, long-term
intervention studies are needed to address these issues.
In conclusion, this study investigated the effect of CA intake on
glucose and insulin responses in a OGTT to analyze the effect of CA
intake on diabetes prevention in more detail. CA intake improved
glucose metabolism, especially postprandial insulin sensitivity, in
healthy subjects with borderline hyperglycemia, but the mechanism
remains unclear. This was an exploratory study and additional large-
scale studies must be performed to confirm the results of the present
study and determine the mechanism of the CA-mediated improvement
of glucose metabolism.
5. Author contributions statement
MH designed the study; YM, SO, and AY conducted the study; TO
supervised the study; MH, YM, and TY analyzed the data; TY and SO
performed the statistical analyses; SN prepared the test beverage; MH,
YM, SO, and AY wrote the manuscript; NO critically reviewed the
manuscript; All authors provided critical conceptual input, analyzed and
interpreted data, and critically revised the report.
6. Ethics statement
This trial was approved by the ethics review committee of Kao
Corporation (Tokyo, Japan, Approval date: August 23, 2018, Approval
#T159-180720) and Ueno Oriental Clinic (Tokyo, Japan, Approval date:
August 30, 2018, Approval #2018–08-27), and conducted in accordance
with the tenets of the Declaration of Helsinki. The trial was conducted
from September 2018 to December 2018, and complied with the study
protocol. Subjects were provided details of the trial both verbally and in
writing, and all subjects provided written informed consent. This trial
was registered with the University Hospital Medical Information
Network (UMIN; https://www.umin.ac.jp/; Registration No.
UMIN000034130).

6
M. Hibi et al. Journal of Functional Foods 97 (2022) 105256

Fig. 3. Fasting and postprandial concentrations of GLP-1 (a), GIP (b), NEFA (c), and ketone bodies (d) were measured for 4 h after intake of a 75-g glucose solution
(OGTT) following daily intake of CA or PL for 2 weeks. Values are means and standard errors represented by bidirectional bars. White circles indicate PL treatment
and black circles indicate CA treatment. * Mean values were significantly different from that of PL condition at the same time (paired t-test). GIP, glucose-dependent
insulinotropic polypeptide; GLP-1, glucagon-like peptide-1; CA, corosolic acid; OGTT, oral glucose tolerance test; PL, placebo.

References
Table 3
Appetite variable scores calculated as AUC during OGTT.1) Abdul-Ghani, M. A., Williams, K., DeFronzo, R. A., & Stern, M. (2007). What is the best
PL CA p values predictor of future type 2 diabetes? Diabetes Care, 30(6), 1544–1548. https://doi.
org/10.2337/dc06-1331
Hunger (mm⋅h) 21.3 ± 1.6 20.7 ± 2.0 0.729 American Diabetes Association. (2006). Diagnosis and classification of diabetes mellitus.
Fullness (mm⋅h) 12.8 ± 1.4 12.4 ± 1.6 0.764 Diabetes Care, 29, S43–S48.
Prospective to eat (mm⋅h) 25.8 ± 1.2 24.8 ± 1.4 0.409 Bogardus, C., Lillioja, S., Howard, B. V., Reaven, G., & Mott, D. (1984). Relationships
Satiety (mm⋅h) 18.9 ± 2.2 16.9 ± 2.2 0.085 between insulin secretion, insulin action, and fasting plasma glucose concentration
in nondiabetic and noninsulin-dependent diabetic subjects. The Journal of Clinical
1)
Data are expressed as mean ± SE. P values were calculated by paired t tests. Investigation, 74(4), 1238–1246. https://doi.org/10.1172/jci111533
AUC, area under the curve; CA, corosolic acid; PL, placebo; OGTT, oral glucose Bonora, E., & Tuomilehto, J. (2011). The pros and cons of diagnosing diabetes with A1C.
tolerance test. Diabetes Care, 34 Suppl 2(Suppl 2), S184–S190. https://doi.org/10.2337/dc11-s216
Cavalot, F., Petrelli, A., Traversa, M., Bonomo, K., Fiora, E., Conti, M., et al. (2006).
Postprandial blood glucose is a stronger predictor of cardiovascular events than
Declaration of Competing Interest fasting blood glucose in type 2 diabetes mellitus, particularly in women: Lessons
from the San Luigi Gonzaga Diabetes Study. The Journal of Clinical Endocrinology and
Metabolism, 91(3), 813–819. https://doi.org/10.1210/jc.2005-1005
The authors declare that they have no known competing financial Centers for Disease Control and Prevention. (2020). National Diabetes Statistics Report,
interests or personal relationships that could have appeared to influence 2020. Retrieved from https://www.cdc.gov/diabetes/pdfs/data/statistics/nat
ional-diabetes-statistics-report.pdf.
the work reported in this paper.
Ceriello, A. (2005). Postprandial hyperglycemia and diabetes complications: Is it time to
treat? Diabetes, 54(1), 1–7. https://doi.org/10.2337/diabetes.54.1.1
Data availability Flint, A., Raben, A., Blundell, J. E., & Astrup, A. (2000). Reproducibility, power and
validity of visual analogue scales in assessment of appetite sensations in single test
meal studies. International Journal of Obesity and Related Metabolic Disorders: Journal
The data that has been used is confidential. of the International Association for the Study of Obesity., 24(1), 38–48. https://doi.org/
10.1038/sj.ijo.0801083
Acknowledgements Fukushima, M., Matsuyama, F., Ueda, N., Egawa, K., Takemoto, J., Kajimoto, Y., et al.
(2006). Effect of corosolic acid on postchallenge plasma glucose levels. Diabetes
Research and Clinical Practice, 73(2), 174–177. https://doi.org/10.1016/j.
This study was conducted with funds provided by Kao Corporation. diabres.2006.01.010
We would like to thank all the volunteers who participated in this study.

7
M. Hibi et al.
Garcia, F. (1940). On the hypoglycemic effect of decoction of Lagerstroemia speciosa
leaves (banaba) administered orally. Journal of the Philippine Medical Association, 20,
395–402.
Ikeda, Y., Noguchi, M., Kishi, S., Masuda, K., Kusumoto, A., Zeida, M., et al. (2002).
Blood glucose controlling effects and safety of single and long-term administration
on the extract of banaba leaves. Journal Nutrition and Food, 5, 41–53.
International Diabetes Federation. (2019). IDF Diabetes Atlas, 9th edition. Retrieved from
https://diabetesatlas.org/atlas/ninth-edition/.
Judy, W. V., Hari, S. P., Stogsdill, W. W., Judy, J. S., Naguib, Y. M., & Passwater, R.
(2003). Antidiabetic activity of a standardized extract (Glucosol) from Lagerstroemia
speciosa leaves in Type II diabetics. A dose-dependence study. Journal of
Ethnopharmacology, 87(1), 115–117. https://doi.org/10.1016/s0378-8741(03)
00122-3
Kakuda, T., Sakane, I., Takihara, T., Ozaki, Y., Takeuchi, H., & Kuroyanagi, M. (1996).
Hypoglycemic effect of extracts from Lagerstroemia speciosa L. leaves in genetically
diabetic KK-AY mice. Bioscience, Biotechnology, and Biochemistry, 60(2), 204–208.
https://doi.org/10.1271/bbb.60.204
Katz, A., Nambi, S. S., Mather, K., Baron, A. D., Follmann, D. A., Sullivan, G., et al.
(2000). Quantitative insulin sensitivity check index: A simple, accurate method for
assessing insulin sensitivity in humans. The Journal of Clinical Endocrinology and
Metabolism, 85(7), 2402–2410. https://doi.org/10.1210/jcem.85.7.6661
Kim, S. J., Cha, J. Y., Kang, H. S., Lee, J. H., Lee, J. Y., Park, J. H., et al. (2016). Corosolic
acid ameliorates acute inflammation through inhibition of IRAK-1 phosphorylation
in macrophages. BMB Reports, 49(5), 276–281. https://doi.org/10.5483/
bmbrep.2016.49.5.241
Kuraoka-Oliveira Â, M., Radai, J. A. S., Leitão, M. M., Lima Cardoso, C. A., Silva-Filho, S.
E., & Leite Kassuya, C. A. (2020). Anti-inflammatory and anti-arthritic activity in
extract from the leaves of Eriobotrya japonica. Journal of Ethnopharmacology, 249,
112418. doi: 10.1016/j.jep.2019.112418.
Mari, A., Pacini, G., Murphy, E., Ludvik, B., & Nolan, J. J. (2001). A model-based method
for assessing insulin sensitivity from the oral glucose tolerance test. Diabetes Care, 24
(3), 539–548. https://doi.org/10.2337/diacare.24.3.539
Matsuda, M., & DeFronzo, R. A. (1999). Insulin sensitivity indices obtained from oral
glucose tolerance testing: Comparison with the euglycemic insulin clamp. Diabetes
Care, 22(9), 1462–1470. https://doi.org/10.2337/diacare.22.9.1462
Matthews, D. R., Hosker, J. P., Rudenski, A. S., Naylor, B. A., Treacher, D. F., &
Turner, R. C. (1985). Homeostasis model assessment: Insulin resistance and beta-cell
function from fasting plasma glucose and insulin concentrations in man. Diabetologia,
28(7), 412–419. https://doi.org/10.1007/bf00280883
Ministry of Health, L. a. W. o. J. (2021). The National Health and Nutrition Survey in
Japan. Retrieved from https://www.e-stat.go.jp/stat-search/files?
page=1&toukei=00450171&tstat=000001041744.
Miura, T., Itoh, Y., Kaneko, T., Ueda, N., Ishida, T., Fukushima, M., et al. (2004).
Corosolic acid induces GLUT4 translocation in genetically type 2 diabetic mice.
Biological & Pharmaceutical Bulletin, 27(7), 1103–1105. https://doi.org/10.1248/
bpb.27.1103
Nagai, N., Hibi, T., Yamaguchi, T., Yoji, K., Kobayashi, S., & Shima, M. (2012). The
making of the Japanese edition appetite question paper using the visual analog scale
(visual analogue scales: VAS) and reproducibility, examination of the validity.
Journal of Japan Society for the Study of Obesity, 18(1), 39-51.
Journal of Functional Foods 97 (2022) 105256
Nathan, D. M., Davidson, M. B., DeFronzo, R. A., Heine, R. J., Henry, R. R., Pratley, R.,
et al. (2007). Impaired fasting glucose and impaired glucose tolerance: Implications
for care. Diabetes Care, 30(3), 753–759. https://doi.org/10.2337/dc07-9920
Report of the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus.
Diabetes Care, 20(7), (1997), 1183–1197. https://doi.org/10.2337/
diacare.20.7.1183
Stohs, S. J., Miller, H., & Kaats, G. R. (2012). A review of the efficacy and safety of
banaba (Lagerstroemia speciosa L.) and corosolic acid. Phytotherapy Research: PTR,
26(3), 317–324. https://doi.org/10.1002/ptr.3664
Stumvoll, M., Nurjhan, N., Perriello, G., Dailey, G., & Gerich, J. E. (1995). Metabolic
effects of metformin in non-insulin-dependent diabetes mellitus. The New England
Journal of Medicine, 333(9), 550–554. https://doi.org/10.1056/
nejm199508313330903
Suzuki, H., Fukushima, M., Usami, M., Ikeda, M., Taniguchi, A., Nakai, Y., et al. (2003).
Factors responsible for development from normal glucose tolerance to isolated
postchallenge hyperglycemia. Diabetes Care, 26(4), 1211–1215. https://doi.org/
10.2337/diacare.26.4.1211
Tang, Y., Zhang, Y., Wang, C., Sun, Z., Li, L., Cheng, S., et al. (2018). Overexpression of
PCK1 gene antagonizes hepatocellular carcinoma through the activation of
gluconeogenesis and suppression of glycolysis pathways. Cellular Physiology and
Biochemistry : International Journal of Experimental Cellular Physiology, Biochemistry,
and Pharmacology., 47(1), 344–355. https://doi.org/10.1159/000489811
Tsuchibe, S., Kataumi, S., Mori, M., & Mori, H. (2006). An inhibitory effect on the
increase in the postprandial blood glucose by Banaba extract capsule enriched
corosolic acid. Journal for the Integrated Study of Dietary Habits, 17(3), 255–259.
Vangipurapu, J., Stančáková, A., Kuulasmaa, T., Paananen, J., Kuusisto, J.,
Ferrannini, E., et al. (2011). A novel surrogate index for hepatic insulin resistance.
Diabetologia, 54(3), 540–543. https://doi.org/10.1007/s00125-010-1966-7
Yamada, K., Hogokowa, M., Fujimoto, S., Fujiwara, H., Fujita, Y., Harada, N., et al.
(2008a). Effect of corosolic acid on gluconeogenesis in rat liver. Diabetes Research &
Clinical Practice, 80, 48–55.
Yamada, K., Hosokawa, M., Yamada, C., Watanabe, R., Fujimoto, S., Fujiwara, H., et al.
(2008b). Dietary corosolic acid ameliorates obesity and hepatic steatosis in KK-Ay
mice. Biological & Pharmaceutical Bulletin, 31, 651–655.
Yeung, E., Zhang, C., Mumford, S., Munford, S., Ye, A., Trevisan, M., et al. (2010).
Longitudinal study of insulin resistance and sex hormones over the menstrual cycle:
The BioCycle Study. Journal of Clinical Endocrinolgy & Metabolism, 95, 5435–5442.
https://doi.org/10.1210/jc.2010-0702
Wang, Z. P., Shen, D., Che, Y., Jin, Y. G., Wang, S. S., Wu, Q. Q., et al. (2019). Corosolic
acid ameliorates cardiac hypertrophy via regulating autophagy. Bioscience Reports,
39(12), BSR20191860. doi: 10.1042/bsr20191860.
World Health Organization. (2012). Prevention and control of noncommunicable diseases:
Guidelines for primary health care in low-resource settings. Geneva: World Health
Organization. Available from: Https://www.ncbi.nlm.nih.gov/books/NBK148622/.
Xu, S., Wang, G., Peng, W., Xu, Y., Zhang, Y., Ge, Y., et al. (2019). Corosolic acid isolated
from Eriobotrya japonica leaves reduces glucose level in human hepatocellular
carcinoma cells, zebrafish and rats. Scientific Reports, 9(1), 4388. https://doi.org/
10.1038/s41598-019-40934-7
Zhang, W. Q., Tian, Y., Chen, X. M., Wang, L. F., Chen, C. C., & Qiu, C. M. (2018).
Liraglutide ameliorates beta-cell function, alleviates oxidative stress and inhibits low
grade inflammation in young patients with new-onset type 2 diabetes. Diabetology &
Metabolic Syndrome, 10, 91. https://doi.org/10.1186/s13098-018-0392-8