1. The study characterized an apple polyphenol extract standardized to 5% phloridzin and evaluated its effects on glucose transport. 2. In vitro, the extract showed a dose-dependent inhibition of up to 60% on glucose uptake by cells, comparable to known inhibitors. 3. In mice, the extract had a lower acute toxicity than green tea extract when administered orally or intraperitoneally. 4. The results suggest the extract may help regulate blood glucose levels and have potential benefits for managing diabetes and body weight control through inhibition of intestinal glucose transport. Further human studies are needed to confirm these effects.

1. Polyphenolic apple extract:
characterisation, safety and potential effect on human
glucose metabolism
MATTHIEU BESNARD2*, DENIS MEGARD2, ISABELLE ROUSSEAU2,
MARIA CLEOFÉ ZARAGOZÁ1, NÚRIA MARTINEZ1, MARIA TERESA MITJAVILA1, CLAUDE INISAN2
*Corresponding author
1. Department of Physiology, Faculty of Biology, University of Barcelona
Avda. Diagonal 645, Barcelona, 08028, Spain
2. Innovation and Development Division, Diana Naturals, Phytonutriance, La Gare, BP15, Antrain, 35560, France
ABSTRACT: Apples have been considered for their positive impact on health. Among the numerous compounds contained on apple
one of them, a polyphenol called phloridzin, specific of the Rosaceae family, is well known to inhibit glucose transport. As this pure
compound is well documented to inhibit the active glucose transport (SGLT) in the intestine, we decided to produce a total apple
polyphenolic extract, with a guaranteed content of 5 percent phloridzin, and to study its action on facilitative glucose transport GLUTn in
vitro. Then we have demonstrated a good effective glucose uptake reduction of The Polyphenolic Apple extract by inhibition on the
facilitative glucose transport GLUTn, together with a less acute toxicity than green tea extract, showing its potential effect on body
weight control, or on disorders caused by diabetes.
INTRODUCTION Milford, USA) was used to quantify polyphenols content in the apple
extract. Chromatographic separation was carried out on a Waters
Polyphenols
Apples have always been known for their positive impact on health, XTerraRP18 column (3x150 mm, 3.5µm) at 40°C with a binary solvent
leading to the famous adage 'one apple a day keeps the doctor system of acidified water (TFA 0.015 percent) (solvent A) and
away'. Among the documented health benefits of regular consumption acetonitrile (solvent B). Gradient : 100 percent A 2 min, 3 percent B 3
of apple (1, 2, 3), we can mention antioxidant activity, inhibition of min, isocratic till 30 min, 16 percent B 48 min, isocratic till 65 min, 40
cancer cell proliferation, improvement of the blood sugar balance, percent B 85 min, 100 percent B 100 min, followed by washing and
anti-oxidative effect, hypotensive effect, anti-allergic activity, and even reconditioning column prior to next injection. Samples are redissolved
some skin protective and deodorizing properties. Among the in a methanol/water 2/1 v/v mixture and filtrate through 0.45µm filter
numerous compounds contained on apple, that may be responsible prior to injection of 15µL onto the HPLC system.
for this impressive set of benefits, the famous and broad family of
polyphenols are paramount. One of them, phloridzin, is a specific ORAC determination
one, found quite exclusively within the Rosaceae family and Fluorescein is a naturally fluorescing molecule, which is very sensitive
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especially in apple (4). This compound is well known to inhibit to free radicals. Free radicals generated by AAPH (2,2'-Azobis (2-
glucose transport and has been widely used at high concentration to AmidinoPropane) diHydrochloride) in the reaction mixture react with
induce glycosurea (5) and thus, we decided to make a total apple the antioxidants from the plant extract. Once all radical scavengers
polyphenolic extract, Polyphenolic Apple Extract (patents EP1243586 are consumed, the free radicals destroy fluorescein, and the
and EP1338270), with a guaranteed content of 5 percent phloridzin, fluorescence vanishes. The quantification is made by measuring the
dedicated to glucose transport management. And, as the inhibition of area "under the curve", hence taking into account both the inhibition
the active glucose transport (SGLT) in the intestine by the pure time and the inhibition percentage. Results are compared to the
n
phloridzin is well documented, the objective of this study was to TROLOX figures, and expressed as micromole TEQ per gramme.
July/Aug 2008
evaluate the effect of our extract on the facilitative glucose transport Analyses are run in duplicates.
GLUTn in vitro. After a complete characterisation of our apple extract,
and a toxicity study performed with mice, we will show how a global Acute toxicity study
polyphenolic apple extract, containing phloridzin, acts as an inhibitor Swiss male/female mice (26-30 g) were provided by Harlan
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of the facilitative glucose transport GLUTn in vitro. Interfauna Ibérica (Barcelona, Spain) and were kept at 22 ± 2 ºC with
55 ± 10 percent relative humidity and controlled lighting (12 h
light/dark cycle) throughout the study. Mice were fed with standard
MATERIALS AND METHODS feedstuff and had water ad libitum. The procedures and animal care
complied with European Union guidelines.
Reagents For acute toxicity test we followed the Up & Down method (6)
Green Tea extract was obtained from ZC ltd (China). Serum free according to OCDE 425 guidelines. The Polyphenolic Apple extract
cellular medium for cellular culture, phloridzin, and all other and green tea extract were resuspended in water and orally
chemicals, solvents, and reagents were commercially available from administered by intragastric canula, or by intraperitoneal route.
Sigma (St Louis, MO, USA). Animals were observed over a period of 7 days and the approximate
lethal doses 50 (LD ) or doses by which half of the animals died, was
50
Characterisation of the standardized extract established. The highest dose used was 3000 mg/kg. An Irwin test (7)
General composition of the extract was characterised by standardized was performed at 1 h and 5 h after administration of the extracts to
techniques: proteins (DUMAS), fats (98/64/CE) carbohydrates (HPLC see whether motor activity, respiratory capacity, temperature,
+ Electrochemistry), minerals (71/250/CE) and total dietary fibres circulatory system, behaviour, neurological or other alterations were
(AOAC 991.43). detected. Animals were then observed for one week
Polyphenols and phloridzine determination: HPLC Glucose uptake assay
analysis with a photodiode array The cellular model used in the study was the Ishikawa Var 1
HPLC (600E multisolvent delivery system, 717 plus auto sampler, 996 endometrial cell line whose genes code for GLUT1 to GLUT4
photodiode array detector and Empower pro manager system, receptors, GLUT1 being the major one (8, 1).
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2. Cells were suspended in a serum free cellular medium to minimize Acute toxicity study
polyphenols/proteins interactions. The LD of Polyphenolic Apple extract was compared to the toxicity of
50
The reaction was initiated by adding radio - labelled 2 - deoxy - a green tea extract. The LD by oral route of The Polyphenolic Apple
50
glucose to solutions containing the product to be tested. By this extract in mice of both sexes was over 3000 mg/kg (Table 2) and
method, only the actual uptake of glucose by the cells was measured green tea extract was more toxic (Table 3) The intraperitoneal route
(9, 10). A negative control was realized by incubating the cells without gave a higher acute toxicity. However, The Polyphenolic Apple extract
any glucose absorption inhibitor. The level of absorption in this was also less toxic (LD between 1250 and 2000 mg/kg, Table 2) than
50
experiment delivers the 100 percent absorption base line. the green tea extract (LD between 75 and 125 mg/kg, Table 3).
50
Two known natural inhibitors of the GLUTn transporters were used as
positive controls and genistein (MW 270,24) at concentrations
ranging from 1 to 100 µM, i.e. 0.27 to 27 µg/ml (10, 11). Polyphenolic
apple extract was used at concentrations of 5 µg/ml to 500 µg/ml, i.e.
0.25 µg/ml to 25 µg/ml phloridzin equivalent.
The Ishikawa cells were incubated during 30 minutes, at 37°C, pH Table 2. Acute toxicity test of the extract 1 Polyphenolic Apple Extract
7.2, 5 percent CO in a solution containing the products to be tested
2
and radio - labelled 2 - deoxy - glucose. After contact, cells were
carefully washed to remove the radioactive traces of the medium and
lysed to determine the specific internal radioactivity of the cells.
RESULTS Table 3. Acute toxicity test of the extract 2 Commercial Green tea
extract
Characterisation of the standardized extract
It is a total polyphenolic extract of apple whose composition is given The Irwin test did not show important alterations in the parameters
in Table 1 and Figure 1. The green tea extract composition is given by observed. A slight paralysis and lose of reflexes were detected at the
Polyphenols
the technical data sheet from the supplier (Table 1). highest doses of each extract by intraperitoneal route, that
disappeared with time. The highest doses of green tea extract
administered by both routes induced shivering and convulsions and
macroscopic observations indicated that no organs were affected.
The Polyphenolic Apple extract can be considered non toxic by oral
route whereas commercial green tea extract is more toxic than The
Polyphenolic Apple extract by intraperitoneal route.
Glucose Uptake Assay
Results were expressed in inhibition percentage of the glucose
uptake versus the negative control (cells incubated without inhibitor).
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Both genistein and The Polyphenolic Apple extract exhibit, in vitro, a
clear and dose related inhibition of the glucose uptake: almost 30
percent inhibition with 2,7µg/ml genistein and more than 60 percent
inhibition with the Polyphenolic Apple at 2,5µg/ml eq phloridzin,
corresponding to 50µg/ml crude extract (Figure 2). It can be expected
from the previous evidence that the decrease in the blood glucose
level consecutive to the ingestion of phloridzin prior to a sugar rich
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July/Aug 2008
diet, could reduce the amount of calories uptake by the organism.
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vol 19 n 4
Table 1. Composition of the standardized Polyphenolic Apple extract
and the green tea extract
Figure 2. Estimation of Polyphenolic Apple Extract and genistein
effect on glucose uptake in vitro at 5, 50, 150 and 500 µg/ml
corresponding to 0.25, 2.5, 7.5 and 25 µg/ml phloridzin and 0.27, 2.7,
8.1 and 27 µg/ml, respectively
DISCUSSION
Phloridzin (Figure 3) is a specific and
unique polyphenol of the plants family
Figure 1. Typical HPLC profile of The Polyphenolic Apple Extract to which the apple tree belongs Figure 3. Phloridzin structure
17

3. (Rosaceae), is well known to inhibit the glucose transport and has secreted into the digestive tract hydrolyse the food into smaller
been widely used at high concentration to induce glycosurea molecules. One of these small molecules is glucose, a major
(elimination of the glucose through the urinary tract). As a component of our dietary carbohydrates. After crossing the intestine
consequence of this specific glucose transport inhibition, the impact epithelial wall, glucose is found in the blood flow with a consequent
of phloridzin on diabetes has been documented. In a review, elevation of the so called glycaemia. This elevation triggers a
Ehrenkranz et al. (2005) have observed that among many beneficial secretion of the hormone insulin by the β cells of the Langerhans
effects, the main pharmacological action of the phloridzin was the islets located in the pancreas. In case of diabetes disorder, the above
production of a renal glycosuria, to block the glucose (and galactose) process does not work, either because there is no or too little insulin
absorption through the brush border cells of the small bowel, and to secretion (type I diabetes) or because the cells do not respond to the
improve the insulin sensitivity after glucose induced toxicity (type II insulin signal (insulin - resistance type II diabetes). There are many
diabetes). In 2005, Song et al. have shown that women eating one possible causes to both kind of diabetes: genetic background, auto -
apple or more a day had a 28 percent lower risk of developing a type immune disorder, metabolism disorder, bad nutritional practices,
II diabetes. The consumption of apple has been demonstrated as obesity, …). Diabetes has potential long term complications that can
being the prime factor leading to a lower incidence of type II diabetes. affect the kidneys, eyes, heart, blood vessels and nerves leading to
The most likely candidate responsible for this beneficial impact daily annoyance.
appears to be phloridzin. The current in vitro study demonstrated first that the phloridzin apple
The glucose, one moiety of the common saccharose, is the main fuel extract showed a lower acute toxicity than the green tea extract, well
of the body. During the digestion, molecules of glucose are produced known for its health benefits associated with its low toxicity and
by the digestive enzymes from the various foods we have ingested, frequently used in dietary supplements and a strong inhibition on the
and are made available at the lumen border of the intestinal cells. To facilitative glucose transport. The inhibition delivered by the extract
reach our blood flow, and consequently feed the various organs of the can reach 60 percent, meaning that only 40 percent of the glucose is
body, they have to be absorbed by active and facilitative transport. absorbed by the cells (Figure 2). It may be expected from these
The active transport, called SGLT, is sodium dependant and results that the phloridzin apple extract should be an efficient glucose
consumes energy to transport the glucose, in the gradient direction or absorption modulator in the human digestive tract. Consequently, the
in the opposite direction. There are two kinds of SGLT receptors regulation of the glucose balance in the blood should be improved
Polyphenols
mainly located in the intestine level and in the kidney microtubules with a potential positive effect on the post ingestion (reduction of the
(5). The facilitative transport, called GLUTn, is ubiquitous and glucose peak and consecutive insulinic shock) on healthy people, and
transports glucose in the direction of the gradient, with a very high in the regulation of the glycaemia in population suffering of type I and
stereo selectivity. At least 12 isoforms of the ubiquitous GLUTn type II diabetes
receptors are known and expressed selectively at specific locations of In conclusion, the good effective glucose uptake reduction by the
the organism. The GLUT1 is found almost in the entire body (8, 13). polyphenolic apple extract inhibiting the facilitative glucose transport
The inhibition of the glucose transport by phoridzin is well GLUTn, together with a less acute toxicity than the green tea extract,
documented by many studies, both in vitro (14-16) and on animals showed its potential effect on body weight control, and on disorders
(17-19). And, as the inhibition of the active glucose transport (SGLT) caused by diabetes. Further studies have to be conducted on human
in the intestine by phloridzin is well documented, we decided to study model in order to confirm in vitro results and bibliographic data.
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the inhibition of the GLUTn transporters by the apple extract
standardized in phloridzin (5 percent).
It can be deduced from the previous evidence that the decrease in ABREVIATIONS USED
the blood glucose level consecutive to the ingestion of phloridzin prior
to a sugar rich diet, could reduce the amount of calories uptake by the SGLT: active glucose transport
organism. It is generally accepted that an exceedingly sugar rich diet GLUTn: facilitative glucose transport
leads to a poor body weight control. The glucose which is not stored b.w.: body weight
in the liver as a potential energy source or which is not LD : Lethal Dose 50 percent
n
50
July/Aug 2008
straightforwardly consumed to produce
energy in the cells is taken up by the
adipocytes with a consequent
development of the fat tissues. The
n
inhibition of the glucose uptake to
vol 19 n 4
reduce the blood sugar can be
assumed as having a direct effect on
body weight control.
Interestingly, it has been suggested
that the phloridzin may play a role in
the size reduction of the white
adipocytes size and consequently
generate a body weight loss. The
mechanism of this effect is yet unclear
but could be claimed as an indirect
effect on body weight.
The diabetes is a common endocrine
disorder characterised primarily by a
consistent, or quasi, permanent
elevated blood glucose
(hyperglycaemia). Many causes can
lead to a high blood concentration.
Diabetes are correctly divided into two
major subgroups: type I and type II
diabetes.
During the digestion, the enzymes
18

4. ACKNOWLEDGMENT
We thank Dr Stuart Milligan from Kings College for glucose
absorption inhition study.
The study was made with the Phytonutriance® polyphenolic apple
extract Appl'In™.
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