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RESEARCH ARTICLE
Mulberry-extract improves glucose tolerance
and decreases insulin concentrations in
normoglycaemic adults: Results of a
randomised double-blind placebo-controlled
study
Mark Lown
1☯
*, Richard Fuller
1☯
, Helen Lightowler
2☯
, Ann Fraser
2☯
, Andrew Gallagher
3☯
,
Beth Stuart
1☯
, Christopher Byrne
4,5☯
, George Lewith
1☯
1Primary Care & Population Sciences, Faculty of Medicine, University of Southampton, Aldermoor Health
Centre, Southampton, United Kingdom, 2Functional Food Centre, Oxford Brooks University, Gipsy Lane
Campus, Oxford, United Kingdom, 3Chief Operating Officer, Phynova Group Ltd, 16 Fenlock Court, Long
Hanborough, United Kingdom, 4Nutrition and Metabolism, Faculty of Medicine, University of Southampton
and University Hospitals Southampton, United Kingdom, 5Southampton National Institute for Health
Research, Biomedical Research Centre, University Hospital Southampton, Southampton, United Kingdom
☯These authors contributed equally to this work.
*m.lown@soton.ac.uk
Abstract
Background
High sugar and refined carbohydrate intake is associated with weight gain, increased inci-
dence of diabetes and is linked with increased cardiovascular mortality. Reducing the health
impact of poor quality carbohydrate intake is a public health priority. Reducose, a proprietary
mulberry leaf extract (ME), may reduce blood glucose responses following dietary carbohy-
drate intake by reducing absorption of glucose from the gut.
Methods
A double-blind, randomised, repeat measure, phase 2 crossover design was used to study
the glycaemic and insulinaemic response to one reference product and three test products
at the Functional Food Centre, Oxford Brooks University, UK. Participants; 37 adults aged
19–59 years with a BMI 20kg/m
2
and 30kg/m
2
. The objective was to determine the
effect of three doses of mulberry-extract (Reducose) versus placebo on blood glucose and
insulin responses when co-administered with 50g maltodextrin in normoglycaemic healthy
adults. We also report the gastrointestinal tolerability of the mulberry extract.
Results
Thirty-seven participants completed the study: The difference in the positive Incremental
Area Under the Curve (pIAUC) (glucose (mmol / L x h)) for half, normal and double dose ME
compared with placebo was -6.1% (-18.2%, 5.9%; p = 0.316), -14.0% (-26.0%, -2.0%; p =
0.022) and -22.0% (-33.9%, -10.0%; p<0.001) respectively. The difference in the pIAUC
PLOS ONE | DOI:10.1371/journal.pone.0172239 February 22, 2017 1 / 14
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OPEN ACCESS
Citation: Lown M, Fuller R, Lightowler H, Fraser A,
Gallagher A, Stuart B, et al. (2017) Mulberry-extract
improves glucose tolerance and decreases insulin
concentrations in normoglycaemic adults: Results
of a randomised double-blind placebo-controlled
study. PLoS ONE 12(2): e0172239. doi:10.1371/
journal.pone.0172239
Editor: Stephen L Atkin, Weill Cornell Medical
College Qatar, QATAR
Received: September 6, 2016
Accepted: January 21, 2017
Published: February 22, 2017
Copyright: ©2017 Lown et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Funding: The work has been funded by a
Technology Strategy Board/Innovate UK grant
(101726). CB is supported in part by the
Southampton National Institute for Health
Research Biomedical Research Centre. The funders
had no role in study design, data collection and
(insulin (mIU / L x h)) for half, normal and double dose ME compared with placebo was
-9.7% (-25.8%, 6.3%; p = 0.234), -23.8% (-39.9%, -7.8%; p = 0.004) and -24.7% (-40.8%,
-8.6%; p = 0.003) respectively. There were no statistically significant differences between
any of the 4 groups in the odds of experiencing one or more gastrointestinal symptoms (nau-
sea, abdominal cramping, distension or flatulence).
Conclusions
Mulberry leaf extract significantly reduces total blood glucose rise after ingestion of malto-
dextrin over 120 minutes. The pattern of effect demonstrates a classical dose response
curve with significant effects over placebo. Importantly, total insulin rises were also signifi-
cantly suppressed over the same time-period. There were no statistically significant differ-
ences between any of the treatment groups (including placebo) in the odds of experiencing
one or more gastrointestinal symptoms. Mulberry extract may have multiple modes of action
and further studies are necessary to evaluate ME as a potential target for the prevention of
type 2 diabetes and the regulation of dysglycaemia.
Introduction
Excess calorie intake including those from sugar and carbohydrates along with inactivity can
make a significant contribution to becoming overweight [1,2] and thus increase the risk of
developing Type 2 diabetes mellitus (T2DM) [3,4]. In 2013 a large long-term European study
investigating the effect of diet on health [5] found an association between the amount of sugary
soft drinks people consumed and their risk of T2DM. In the study, weight gain had a large
effect on diabetes risk and sugary drinks had a small effect on diabetes risk even after Body
Mass Index (BMI) was corrected for [5]. The global rise in T2DM is linked to the metabolic
syndrome (dyslipidemia, hypertension, insulin resistance), and obesity is thought to be one of
the greatest risk factors for metabolic syndrome and T2DM [6]. Dietary sugars and carbohy-
drates play a significant role as calories from these foods promote fat storage and hunger [7]. A
recently completed review of nutrition and its impact on T2DM concluded that dietary restric-
tion of carbohydrate intake is the single most effective approach to manage T2DM [8]. It is
estimated that more than 1 in 17 people in the UK have diabetes (diagnosed or undiagnosed)
[9] and thus reducing the health impact of poor quality carbohydrate intake is a public health
priority. Herbal agents could be effective in reducing post-prandial blood glucose in combina-
tion with carbohydrate restriction [10]. Indeed, the history of the widely prescribed agent Met-
formin (dimethylbiguanide) can be traced back to the use of Galega officinalis Linn as a herbal
medicine in medieval Europe [11].
Mulberry (Morus alba) leaves have been used in traditional Chinese medicine (TCM) for
several millennia and its use was first recorded in around 500AD in the Divine Husbandman’s
Classic of the Materia Medica [12]. In the Grand Materia Medica, it states "if the juice (of the
herb) is decocted and used as a tea substitute it can stop wasting and thirsting disorder.”
Reports have shown that the leaves are nutritious and non-toxic [13]. The Chinese Ministry of
Health and the Taiwanese Bureau of Food Safety recognise Morus alba leaves as both a food
and a medicine [14]. Mulberry leaf extracts (ME) have a history of safe ‘traditional’ use for nor-
malizing post-prandial blood glucose, and it is thought that iminosugars such as 1-deoxynojiri-
mycin (DNJ), a reversible, competitive natural α-glucosidase inhibitor, are the main active
Mulberry-extract improves glucose tolerance and decreases insulin concentrations in healthy adults
PLOS ONE | DOI:10.1371/journal.pone.0172239 February 22, 2017 2 / 14
analysis, decision to publish, or preparation of the
manuscript.
Competing interests: Andrew Gallagher is Chief
Operating Officer at Phynova, the developers of
Reducose. He was involved in the development of
the product but was not involved in the collection,
analysis and interpretation of data. Phynova did not
fund the study. The funder did not provide salaries
support in the form of salaries for any of the
authors. The funding provided by the grant was
simply to write the protocol and supervise the data
collection and data interpretation and write the
paper. The funding was paid into the research
funds at the University of Southampton and was
not used to pay any direct salary or to make any
personal payments to any of the Southampton
University affiliated authors. The remaining authors
have declared that no competing interests exist.
The commercial affiliation does not alter the
authors’ adherence to all PLOS ONE policies on
sharing data and materials.
components responsible for the activities [10]. ME 1000-fold diluted has also been shown to
inhibit absorption of sucrase, maltase, isomaltase, trehalase and lactase (by 96%, 95%, 99%,
44% and 38% respectively) [10]. ME also contains gallic acid and may have additional anti-dia-
betic effects via translocation of the GLUT4 receptor [15]. As ME inhibits the absorption of
carbohydrates from the intestine, GI side effects are possible.
Previous research has suggested that ME could significantly reduce the peak blood glucose
levels and insulin response levels [16,17], providing protection to blood glucose metabolic
function of healthy and hyperglycemic subjects [18]. Long-term administration of ME pro-
duced a dose-dependent decrease in body weight and hepatic lipid accumulation [19], stimu-
lated skeletal muscle 5’-AMP-activated protein kinase activity acutely without changing the
intracellular energy status [20], suppressed the elevation of postprandial blood glucose and
cholesterol in humans [16] and exhibited potential hypoglycemic and hypolipidemic effects in
patients with diabetes [21]. ME has been shown to suppress postprandial glucose and insulin
in healthy human subjects when added to confections in a small study with ten healthy females
[22]. Sucrose and starch absorption was inhibited and they were subsequently fermented by
intestinal microbiota which could lead to an additional beneficial prebiotic effect [22].
While Mulberry tea has been shown to suppress the postprandial rise of blood glucose levels
after 90 minutes of its consumption in T2DM subjects [23] the interpretation of the clinical
relevance of the effects of ME has been challenging due to limitations including study design
and small numbers of subjects [10,16,17,21–23]. High quality, double blind placebo controlled
trials are therefore required to determine the effects of ME on glucose tolerance and to ascer-
tain its potential as a target for further investigation for the prevention of T2DM and regula-
tion of dysglycaemia. We aimed to investigate the effects of ME in healthy volunteers with a
high quality placebo controlled clinical trial in the UK.
Materials and methods
Study design
The primary outcome of the study was to test the effect of three doses of mulberry-extract
(250mg Reducose containing 12.5mg DNJ), half (125mg Reducose containing 6.75mg DNJ)
and double (500mg Reducose containing 25mg DNJ) the normal dose of a proprietary water
extract of mulberry leaves standardized to contain 5% DNJ (Reducose), versus placebo, on
blood glucose (pIAUC for glucose concentration over 120 minutes) when co-administered
with 50g maltodextrin in normoglycaemic healthy adults. Secondary outcomes were to test the
insulin response (pIAUC for insulin concentration over 120 minutes) and gastrointestinal tol-
erability of the mulberry extract using normal, half and double the normal dose of ME and pla-
cebo. Maltodextrin is a dietary starch with a high glycaemic index and is commonly added to
many foods and beverages. The exact dosage regime investigated was determined by a series of
initial phase 1 studies carried out on normal healthy subjects by Phynova, the company that
owns and produces Reducose. A double-blind, randomised, repeat measure, crossover design
trial was used to study the glycaemic response (GR) and insulinaemic response (IR) to three
products: one reference product and three test products. Participants acted as their own con-
trols. The trial was conducted at the Functional Food Centre at Oxford Brookes University.
The Centre is internationally renowned for its work on GR with extensive publications and
their procedure for glycaemic index testing is based on well-established FAO/WHO guide-
lines. Ethical approval for the study was obtained from the Oxford Brookes University
Research Ethics Committee (UREC Registration No: 140806 for glycaemic response (2014);
UREC Registration No: 110594 for insulaemic response (2012)). The exclusion criteria of the
Mulberry-extract improves glucose tolerance and decreases insulin concentrations in healthy adults
PLOS ONE | DOI:10.1371/journal.pone.0172239 February 22, 2017 3 / 14
MULBERRY trial are listed in Table 1. The Study design, rationale and methodology have
been previously described in detail [24].
Trial registration
ISRCTN: ISRCTN 14597438
Recruitment
Participants were recruited following local advertisements. All participants were given full
details of the study protocol and the opportunity to ask questions. They subsequently gave
written informed consent prior to participation and were paid £10 per visit, on completion of
all four visits. This was determined as an appropriate amount to cover travel costs and the time
spent during each visit. The trial was registered on 21/04/2015 and the first patient recruited
on 22/04/2015. The last patient was followed up and the study completed on 29/08/2015. The
authors confirm that all ongoing and related trials for this intervention are registered.
Mulberry leaf extract
Reducose is a mulberry leaf extract standardised to contain 5% (+/- 10%, i.e. 4.5%-5.5%)
1-deoxynojirimycin (DNJ). Batch-to-batch consistency is maintained through a quality con-
trol (QC) process that starts with the raw material to ensure the leaves contain a minimum
required DNJ content. Production yields batches with >5% DNJ and the content is standard-
ised through batch blending and dilution with excipients. All batches are subjected to rigorous
QC during manufacturing and each batch is quantitatively (HPLC-ELSD) assayed for DNJ
and qualitatively fingerprinted using HPTLC. All batches undergo routine quality control to
ensure contaminant levels (heavy metals, microbes) are within the European pharmacopoeia
limit. The exact dosage regime investigated was determined by a series of initial phase 1 studies
Table 1. Exclusion criteria of the mulberry trial.
Exclusion Criteria
1. Aged <18 or >60 years
2. Pregnant or lactating
3. Body mass index (BMI) <20kg/m
2
and >30kg/m
2
4. Fasting blood glucose value >6.1 mmol/l
5. Any known food allergy or intolerance including mulberry extract
6. Medical condition(s) or medication(s) known to affect glucose regulation or appetite and/or influence
digestion and absorption of nutrients
7. Known history of diabetes mellitus (Type I/II) or the use of antihyperglycaemic drugs or insulin to treat
diabetes and related conditions
8. Use steroids, protease inhibitors or antipsychotics (all of which have major effects on glucose
metabolism and body fat distribution)
9. Current oral hypoglycaemic use
10. Symptomatic IBS
11. History of renal or liver diseases
12. History of clotting or bleeding disorders
13. Taken antibiotics in last 3 weeks prior to screening
14. Taking daily medications or dietary supplements that are not suitable for the study in the opinion of the
PI
15. Anaemia
16. Subject to a major medical or surgical event requiring hospitalization within the preceding 3 months
17. Current participation in another clinical study.
doi:10.1371/journal.pone.0172239.t001
Mulberry-extract improves glucose tolerance and decreases insulin concentrations in healthy adults
PLOS ONE | DOI:10.1371/journal.pone.0172239 February 22, 2017 4 / 14
carried out on normal healthy subjects by Phynova, the company that owns and produces
Reducose.
Randomisation
Participants and investigators were blinded. Participants were assigned a participant number
according to their chronological order of enrolment in the study. The allocated participant
number was used to identify the participants and their corresponding intervention sequence.
Four products were tested in this study—one placebo reference product (four capsules con-
taining 125mg microcrystalline cellulose) and three test products containing different doses of
mulberry extract (test product groups received either 1, 2, or 4 capsules containing 125mg ME,
with either 3, 2, or 0 placebo capsules respectively so that participants always took 4 capsules).
Each test/reference product was co-administered with 50g maltodextrin dissolved in 250ml
water.
The reference product and test products were administered to participants in a randomised,
repeated measures design. All volunteers received the reference product and test products in
random order on (four) separate days, with at least a two-day gap between measurements to
minimise carry over effects. DNJ has a relatively short half-life in vivo of approximately 2
hours (when measured in rats using hydrophilic interaction chromatography coupled to a
mass spectrometric detector [25]).
Study procedures
On the day prior to a test, participants were asked to restrict their intake of alcohol and caf-
feine-containing drinks and to restrict their participation in intense physical activity (for
example, long periods at the gym, excessive swimming, running, aerobics). Participants were
also told not to eat or drink after 10.00 pm the night before a test, although water was allowed
in moderation. Participants were studied in the morning after an overnight fast. Anthropomet-
ric measurements (height, weight and BMI) were taken before any products were consumed.
Body composition measurements (Fat Mass (FM), Fat-Free Mass (FFM)) were taken using the
Tanita BC-418MA segmental body composition analyser. Participants consumed the products
at a comfortable pace, within 5 minutes and the reference product and test products were
served with 50g maltodextrin dissolved in 250 ml water.
Participants remained sedentary during each test session and did not consume any addi-
tional food or fluid. They were instructed to record stool consistency for the first bowel move-
ment after their visit and the frequency and intensity of gastro intestinal symptoms for 0–24
hours after the study product consumption. Gastrointestinal symptoms were measured via
questionnaire for 24 hours following each study visit. Subjects used a 5-point scale to rate stool
consistency for each bowel movement for 0–24 h after the study product consumption. The
five-point scale includes: 1 = watery, 2 = loose/mushy, 3 = soft, 4 = formed, 5 = hard. Fre-
quency and intensity were recorded using a 10-centimeter (cm) line scale (0 representing
“Absent” for frequency and “Usual” for intensity; 10 representing “More than usual” for fre-
quency and “Severe” for intensity).
Laboratory measurements
The glycaemic response method used was adapted from that described by Brouns et al [26]
and was carried out in accordance with the ISO 26642:2010 standards. Blood measurements
were taken at -5 min and 0 min before consumption of the reference product/test products
and the baseline value taken as a mean of these two values. Further blood measurements were
taken at 15, 30, 45, 60, 90 and 120 minutes after the start. Blood glucose was measured using
Mulberry-extract improves glucose tolerance and decreases insulin concentrations in healthy adults
PLOS ONE | DOI:10.1371/journal.pone.0172239 February 22, 2017 5 / 14
the HemoCue Glucose 201+ analyser (HemoCue Ltd). The same time points were used for
determining insulin levels. At each test time point, 300 μL of capillary blood (from finger
pricks) was obtained using the Unistik 3 single-use lancing device (Owen Mumford, Wood-
stock, UK) and collected into chilled Microvette capillary blood collection tubes treated with
di Potassium EDTA (CB 300 K2E; Sarstedt Ltd., Leicester, UK). The Microvette tubes were
centrifuged and 200 μL of the supernatant plasma obtained. Insulin concentrations in the
plasma samples were determined by electrochemiluminescence immunoassay using an auto-
mated analyzer (Cobas E411; Roche diagnostics, Burgess Hill, UK). The Cobas system is a reli-
able method of plasma insulin determination. Sufficient blood was taken to enable a second
set of analysis to be performed at every time point (if the first analysis failed) and there was no
missing data. The second sample was used for two participants due to faulty equipment but
only one data value at each time point was obtained in all subjects.
Sample size
A recent unpublished phase 1 study in 12 healthy individuals age 18–25 using 250mg ME dose
showed a reduction in the glycaemic index of maltodextrin by 58% when compared to placebo.
We estimated a sample size of n = 30 participants would provide over 90% power to detect a
similar size of effect. Being more conservative and allowing for a smaller difference to be
detected in the lower concentration doses, 30 participants would still allow at least 80% power
to detect a difference of 25% in the positive Incremental Area Under the Curve (pIAUC). In
order to account for a potential loss to follow up, and the possibility that our sample size may
be inaccurate as it is based on a small pilot sample we aimed to recruit 40 participants.
Statistical analyses
We calculated the positive incremental area under the curve for the 4 study products and com-
pared using repeated measures ANOVA to determine whether there was a statistically signifi-
cant difference in the primary outcome (glucose response over 120 minutes) and in the
secondary outcome measures (insulin response over 120 minutes and gastrointestinal side
effects). Repeated measures ANOVA were used to compare treatments across time-points, rec-
ognising that responses were clustered within individual participants. For binary outcomes,
results are expressed as proportions and repeated measures logistic regression was used (Stata’s
xtlogit command). All analyses were carried out in Stata v12.1. The presence/absence of gastro-
intestinal symptoms in the 24 hours following the study visit was assessed using logistic regres-
sion models.
Results
Of 40 randomised subjects, three participants dropped out (one found the study day too long,
and the study was closed before two other participants could complete the remaining visits).
Recruitment began in April 2015 and the study was closed at the end of August 2015 with 37
participants having completed all four visits. Fig 1 depicts the trial flow diagram.
37 participants completed the study and the baseline characteristics are shown in Table 2.
Positive incremental area under the curve was calculated for all glucose and insulin measure-
ments from baseline to 120 minutes in accordance with FAO/WHO’s ‘Joint Guidelines on gly-
caemic index testing of foods’ and the International Standard ‘ISO 26642/2010:Food Products–
determination of the glycaemic index (GI) and recommendation for food classification’.
Mulberry-extract improves glucose tolerance and decreases insulin concentrations in healthy adults
PLOS ONE | DOI:10.1371/journal.pone.0172239 February 22, 2017 6 / 14
Positive incremental area under the curve–Glucose
As shown in Table 3, there are significant differences in the positive incremental area under
the curve between treatments. Compared to the placebo dose, the positive incremental area
Fig 1. Mulberry study CONSORT diagram.
doi:10.1371/journal.pone.0172239.g001
Table 2. Baseline characteristics of the study population.
Characteristic Male Female Total sample
Female 25/37 (67.6%)
Age 27.17 (7.51) 30.40 (12.24) 29.35 (10.93)
Height (cm) 173.08 (6.49) 164.40 (6.28) 167.22 (7.49)
Weight (kg) 70.74 (7.35) 61.37 (6.98) 64.41 (8.29)
BMI 23.61 (2.09) 22.71 (2.34) 23.00 (2.27)
Waist circumference (cm) 81.72 (4.99) 76.46 (6.52) 78.17 (6.50)
Hip circumference (cm) 99.30 (4.00) 99.20 (6.65) 99.24 (5.86)
FM(%) 15.02 (4.44) 28.94 (5.46) 24.43 (8.34)
FM (kg) 10.65 (3.53) 18.06 (5.19) 15.65 (5.84)
FFM(%) 84.98 (4.44) 71.06 (5.46) 75.57 (8.34)
FFM(kg) 60.09 (6.91) 43.31 (3.18) 48.75 (9.21)
Unless otherwise stated, data are means (SD), (FM–Fat Mass, FFM–Fat-Free Mass).
doi:10.1371/journal.pone.0172239.t002
Mulberry-extract improves glucose tolerance and decreases insulin concentrations in healthy adults
PLOS ONE | DOI:10.1371/journal.pone.0172239 February 22, 2017 7 / 14
under the curve was significantly lower in the 250mg and 500mg doses. The pIAUC for the
125mg dose was not significantly different from placebo. The 500mg dose also had an area
under the curve 0.44 mmol / L x h (95% CI -0.78, -0.11) lower than the 125mg dose. This was
statistically significant (p = 0.010). None of the other pairwise comparisons were statistically
significant. The average glycaemic response for the four groups is shown in Fig 2.
Subgroups
Two planned subgroup analyses were to be carried out. Although not powered to detect statis-
tically significant differences within subgroups, exploratory analysis could help to determine
whether there is any signal to support hypotheses that differential effects would be observed in
those aged over 50 years and in those with a BMI greater than 25 kg/m
2
. There were only two
individuals aged >50 years and therefore this subgroup analysis was not carried out. Similarly,
there were no participants with a BMI >25 kg/m
2
.
Table 3. Positive incremental area under the curve for glucose.
Positive incremental area under the curve (mmol
/ L x h)
Difference compared to placebo (mmol /
L x h)
Placebo 2.81 (1.19)
125 mg 2.64 (1.35) -0.17 (-0.51, 0.16; p = 0.316)
250 mg 2.42 (1.27) -0.393 (-0.73, -0.06; p = 0.022)
500 mg 2.19 (0.99) -0.62 (-0.95, -0.01; p<0.001)
Difference compared to placebo calculated using repeated measures ANOVA model
doi:10.1371/journal.pone.0172239.t003
Fig 2. Mean plasma glucose concentrations according to group during the maltodextrin tolerance test.
doi:10.1371/journal.pone.0172239.g002
Mulberry-extract improves glucose tolerance and decreases insulin concentrations in healthy adults
PLOS ONE | DOI:10.1371/journal.pone.0172239 February 22, 2017 8 / 14
Positive incremental area under the curve–Insulin
As shown in Table 4, the placebo group had significantly higher pIAUC than the 250mg or
500mg treatments. There were no other statistically significant differences at the 5% level. Fig
3shows the average insulin response of the groups.
Gastrointestinal symptoms
Table 5 below sets out the proportions experiencing any gastrointestinal symptoms. These
were recorded as nausea, abdominal cramping, distension or flatulence. The proportions
Table 4. Positive incremental area under the curve for insulin.
Positive incremental area under the curve (mIU /
L x h)
Difference compared to placebo (mIU / L
x h)
Placebo 59.9 (48.5)
125mg 54.1 (34.5) -5.83 (-15.5, 3.8; p = 0.234)
250mg 45.6 (22.9) -14.3 (-23.9, -4.6; p = 0.004)
500mg 45.1 (26.5) -14.8 (-24.4, -5.2; p = 0.003)
Difference compared to placebo calculated using repeated measures ANOVA model
doi:10.1371/journal.pone.0172239.t004
Fig 3. Mean plasma insulin concentration according to group during the maltodextrin tolerance test.
doi:10.1371/journal.pone.0172239.g003
Table 5. Side effects experienced by placebo / ME dosage.
Proportion experiencing one or
more gastrointestinal symptoms
Proportion
experiencing nausea
Proportion experiencing
abdominal cramping
Proportion
experiencing
distension
Proportion
experiencing
flatulence
placebo 21/37 (56.8%) 6/37 (16.2%) 7/37 (18.9%) 15/37 (40.5%) 18/37 (48.6%)
125mg 23/37 (62.2%) 8/37 (21.6%) 7/37 (18.9%) 9/37 (24.3%) 16/37 (43.2%)
250mg 20/37 (54.0%) 6/37 (16.2%) 8/37 (21.6%) 13/37 (35.1%) 17/37 (45.9%)
500mg 20/37 (54.0%) 4/37 (10.8%) 8/37 (21.6%) 12/37 (32.4%) 19/37 (51.4%)
doi:10.1371/journal.pone.0172239.t005
Mulberry-extract improves glucose tolerance and decreases insulin concentrations in healthy adults
PLOS ONE | DOI:10.1371/journal.pone.0172239 February 22, 2017 9 / 14
experiencing each symptom are also recorded in Table 5 for descriptive purposes. There were
no statistically significant differences between any of the treatment groups in the odds of
experiencing one or more gastrointestinal symptoms through repeated measures logistic
regression.
Discussion
In this randomised, double-blind, placebo-controlled phase 2 dose ranging trial, carried out in
healthy normoglycaemic individuals, we have shown that ME can decrease total glucose and
insulin rises without significant side effects. Moreover, Reducose, a proprietary mulberry leaf
extract demonstrates a classical dose response curve with significant effects over placebo.
Importantly, we did not find any significant differences between the treatment groups in the
odds of experiencing one or more gastrointestinal symptoms. We did not observe an increased
incidence of gastrointestinal side effects from ME with increasing dose and no subjects
dropped out of the study due to side effects. Furthermore, a previous study using ME three
times daily for twelve weeks also reported no adverse events [27].
In a crossover trial it is important to ensure that there was no carry over effects. In addition
to animal data on the short half-life of DNJ of approximately two hours [25], we performed
analysis using the trial data. We calculated carry-over effects using the omnibus test (a measure
reflecting the degree to which the study design allows the treatment effects to be estimated
independently of the carryover effects) and we found no evidence of a carryover effect in the
trial (F = 1.04, p = 0.377). We also tested for a treatment by period interaction and the terms
were not significant. However, the trial may not have been powered to detect carry-over
effects.
A particular finding from this study was that the ME did not appear to affect the average
glucose or insulin responses until 30 minutes after ingestion. Other studies using ME have
shown a reduction in glucose and insulin responses occurring more rapidly after ingestion
when ME was not encapsulated [22]. The capsule material used in this study was hydroxypro-
pyl methylcellulose (HPMC) and in vitro studies have shown that this capsule material can
impact (and significantly lengthen the) disintegration and dissolution behaviour of plant
extracts [28]. It is possible that the choice of capsule material led to a delay in the release of the
active contents and a reduction in effect size.
Mulberry leaf extracts (ME) have a long history of safe and side-effect free use. It is thought
that iminosugars such as 1-deoxynojirimycin (DNJ), a reversible, competitive natural α-gluco-
sidase inhibitor, are the main active components [10] and therefore ME may have a similar
mode of action to acarbose [29]. Acarbose can be an adjunct to diet and exercise as monother-
apy when other oral antidiabetic agents are contraindicated, or in any combination of oral
antidiabetic drugs and insulin in the management of type 2 diabetes mellitus. Acarbose has
been shown to reduce HbA
1C
and the results of several large trials evaluating cardiovascular
outcomes are awaited [30]. Gastrointestinal side effects are the main limiting factor in the clin-
ical use of acarbose, leading to high rates of non-compliance and discontinuation [30]. Gastro-
intestinal side effects are also common and can be problematic occurrences with other antidia-
betic agents such as metformin [31].
Previous research has demonstrated that Mulberry leaf extracts (ME) can reduce postpran-
dial glucose and insulin levels [16] but the clinical interpretation of many trials have been lim-
ited by poor study design and small numbers of subjects. In addition to the proposed direct
effect of ME on α-glucosidase (amongst other enzymes) and on sugar and carbohydrate
absorption, the ability of ME to reduce insulin rises is important in that whole-body glucose
uptake progressively increases with higher rates of systemic insulin concentrations [32,33].
Mulberry-extract improves glucose tolerance and decreases insulin concentrations in healthy adults
PLOS ONE | DOI:10.1371/journal.pone.0172239 February 22, 2017 10 / 14
Indeed, suppression of insulin secretion (without dietary or exercise intervention) may lead to
loss of body weight and fat mass [34]. Long-term administration of ME has produced a dose-
dependent decrease in body weight and hepatic lipid accumulation in mice [19].
ME contains several herbal glycoproteins and in addition to α-glucosidase inhibition, in
vitro studies have demonstrated the presence of fagomine in ME which may be responsible for
enhanced insulin sensitivity to glucose metabolism [23]. ME has also been shown to produce
hypolipidemic effects in patients with diabetes [21]. Interestingly, α-glucosidase inhibitors
augment incretin hormone secretion and thus, enhanced β-cell function could, in part, explain
these beneficial effects on glucose homeostasis. By altering gut microbiota flora, α-glucosidase
inhibitors could also exert beneficial effects on glucose tolerance [35].
The enzyme binding kinetics of ME require further elucidation in relation to its potential
pragmatic efficacy including its activity during the consumption of complex carbohydrates
along with fats, which may delay gastric emptying, as may varied eating patterns such as snack-
ing. Long-term trials are needed to investigate the safety and impact of ME on long-term glu-
cose tolerance. Glucose-lowering agents show ethnic variations and future work should
include assessment in more ethnically diverse populations.
Limitations
We only evaluated the short-term effects of ME using single doses and longer administration
and follow-up periods would be required to determine if there is a sustained effect or other
potential side effects. We also used a test carbohydrate in fasting individuals and did not evalu-
ate the pragmatic effects of ME with carbohydrates mixed with fats and proteins. The subjects
in the study were not on medications which may impact on the efficacy of ME such as proton
pump inhibitors or other agents disrupting stomach pH or gastric emptying. Although the use
of capillary blood glucose has been validated and is recommended for determining glycaemic
responses (ISO 26642: 2010(E)), there is less evidence for the robustness of capillary insulin.
We did however observe a high degree of correlation between respective glucose and insulin
responses suggesting that capillary insulin could be a valid measure. Although we have demon-
strated that ME can reduce glucose and insulin rises in healthy volunteers with non-impaired
glucose homeostasis, the results should be interpreted with caution regarding dysglycaemia.
Conclusion
We have demonstrated that ME substantially reduces the increase in plasma glucose after
ingestion of maltodextrin over 120 minutes. The pattern of effect demonstrates a classical dose
response curve with significant effects over placebo. Importantly, total insulin rises were also
significantly suppressed over the same period. There were no statistically significant differ-
ences between any of the treatment groups in the odds of experiencing one or more gastroin-
testinal symptoms indicating that ME is well tolerated. Mulberry extract may have multiple
modes of action and further studies are necessary to evaluate the potential of ME for the pre-
vention of type 2 diabetes and regulation of dysglycaemia.
Supporting information
S1 Checklist.
(DOC)
S1 Protocol.
(PDF)
Mulberry-extract improves glucose tolerance and decreases insulin concentrations in healthy adults
PLOS ONE | DOI:10.1371/journal.pone.0172239 February 22, 2017 11 / 14
S1 Dataset.
(XLSX)
Acknowledgments
The work has been funded by a Technology Strategy Board/Innovate UK grant.
CDB is supported in part by the Southampton National Institute for Health Research Bio-
medical Research Centre.
Author Contributions
Conceptualization: ML RF HL AF AG BS CB GL.
Formal analysis: ML BS.
Funding acquisition: RF GL AG.
Investigation: HL AF.
Methodology: ML RF HL AF AG BS CB GL.
Project administration: ML RF GL.
Resources: ML AF AG BS.
Supervision: ML RF CB GL.
Validation: ML RF HL AF AG BS CB GL.
Visualization: ML GL BS.
Writing – original draft: ML RF HL AF AG BS CB GL.
Writing – review & editing: ML RF HL AF AG BS CB GL.
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