U.S. patent application number 13/739747 was filed with the patent office on 2013-05-23 for composition and method for reducing post-prandial blood glucose.
This patent application is currently assigned to KEMIN FOODS, L.C.. The applicant listed for this patent is Kemin Foods, L.C.. Invention is credited to Rod Ausich, Zoraida DeFreitas, Jerry Newman, Andrew Shao, Fayad Sheabar.
Application Number | 20130129847 13/739747 |
Document ID | / |
Family ID | 33309930 |
Filed Date | 2013-05-23 |
United States Patent
Application |
20130129847 |
Kind Code |
A1 |
Ausich; Rod ; et
al. |
May 23, 2013 |
Composition and Method for Reducing Post-Prandial Blood Glucose
Abstract
A nutritional intervention composition for reducing
post-prandial blood glucose levels in humans, including between
about 0.1 mg and about 10 mg of a proteinase inhibitor that is
administered prior to the meal. The composition is effective for
treating or ameliorating the effects of hyperglycemia and Type II
diabetes. The composition also is effective in combating obesity.
The proteinase inhibitor is preferably isolated from plant
material, such as potatoes, soy, and beans. Potato proteinase
inhibitor II and soybean Bowman-Birk inhibitor are included in the
group of effective proteinase inhibitors.
Inventors: |
Ausich; Rod; (Des Moines,
IA) ; Shao; Andrew; (Pittsburgh, PA) ; Newman;
Jerry; (O'Fallon, MO) ; DeFreitas; Zoraida;
(Polk City, IA) ; Sheabar; Fayad; (Midlothian,
VA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Kemin Foods, L.C.; |
Des Moines |
IA |
US |
|
|
Assignee: |
KEMIN FOODS, L.C.
Des Moines
IA
|
Family ID: |
33309930 |
Appl. No.: |
13/739747 |
Filed: |
January 11, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10426678 |
Apr 30, 2003 |
8377877 |
|
|
13739747 |
|
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|
09900555 |
Jul 6, 2001 |
6767566 |
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10426678 |
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Current U.S.
Class: |
424/757 ;
424/725 |
Current CPC
Class: |
A23L 33/105 20160801;
A23V 2002/00 20130101; A23V 2002/00 20130101; A61K 38/56 20130101;
A61K 36/81 20130101; A23V 2200/328 20130101; A61K 36/48 20130101;
A23V 2250/54 20130101 |
Class at
Publication: |
424/757 ;
424/725 |
International
Class: |
A61K 36/48 20060101
A61K036/48; A61K 36/81 20060101 A61K036/81 |
Claims
1. A nutritional intervention composition for reducing
post-prandial blood glucose in humans, comprising between about 0.1
mg and about 500 mg of a proteinase inhibitor administered prior to
the meal.
2. A nutritional intervention composition as defined in claim 1
wherein said composition is taken 1 to 30 minutes before the
meal.
3. A nutritional intervention composition as defined in claim 1,
wherein the proteinase inhibitor is isolated from plant
material.
4. A nutritional intervention composition as defined in claim 3,
wherein the proteinase inhibitor is derived from the group of
plants consisting of potatoes, soy and beans.
5. A nutritional intervention composition as defined in claim 1,
wherein the proteinase inhibitor is proteinase inhibitor II
isolated from potatoes.
6. A nutritional intervention composition as defined in claim 1,
wherein the proteinase inhibitor is Bowman-Birk inhibitor isolated
from soybeans.
7. A nutritional intervention composition as defined in claim 1,
wherein the proteinase inhibitor reduces the initial blood glucose
spike.
8. A nutritional intervention composition as defined in claim 1,
wherein the proteinase inhibitor reduces the area under the curve
blood glucose.
9. A nutritional intervention composition as defined in claim 8,
wherein the area under the curve blood glucose is measured over a
time period of up to 4 hours after the meal.
10. A nutritional intervention composition as defined in claim 1,
wherein the proteinase inhibitor reduces the initial blood glucose
spike by between about 5 percent and about 30 percent.
11. A nutritional intervention composition as defined in claim 1,
wherein the proteinase inhibitor reduces the area under the curve
blood glucose over a period of about 3.5 hours following the meal
by between about 5 percent and about 40 percent.
12. A nutritional intervention composition as defined in claim 1,
wherein the proteinase inhibitor is potato proteinase inhibitor II
administered orally in a dose of between about 0.5 and about 500
mg.
Description
[0001] This application is a divisional application of U.S. patent
application Ser. No. 10/426,678, filed on Apr. 30, 2003, which
claims priority to U.S. patent application Ser. No. 09/900,555,
filed on Jul. 6, 2001, which issued as U.S. Pat. No. 6,767,566 on
Jul. 27, 2004.
BACKGROUND OF THE INVENTION
[0002] The invention relates to compositions for reducing
post-prandial blood glucose in humans and, more specifically, to a
proteinase inhibitor that delays gastric emptying and reduces
post-prandial glycemia which may be beneficial in combating obesity
and Type II diabetes.
[0003] Regulation of body weight depends on genetic as well as
physiologic and lifestyle factors that are known to influence
energy balance, such as diet, appetite control, metabolism, and
physical activity (Aronne, L. J. (2001) J Clin Psychiatry 62,
13-22; Fernandez-Lopez, J. A., Remesar, X., Foz, M. & Alemany,
M. (2002) Drugs 62, 915-44). Despite measures to combat obesity and
an increased awareness of the associated co-morbidities, the
condition has become an epidemic, with nearly 60% of Americans
classified as overweight or obese (Visscher, T. L. & Seidell,
J. C. (2001) Annu Rev Public Health 22, 355-75). Since the gene
pool has not changed, researchers believe the culprit is primarily
due to a combination of environmental and lifestyle influences. A
focus on dietary fat as a leading cause of obesity the last several
decades has been successful in reducing overall fat intake by
Americans (from 40% to just over 30% of total calories, from the
1960's to present), but has done little to stave the rise in
obesity rates (Lichtenstein, A. H., Kennedy, E., Barrier, P.,
Danford, D., Ernst, N. D., Grundy, S. M., Leveille, G. A., Van
Horn, L., Williams, C. L. & Booth, S. L. (1998) Nutr Rev 56,
S3-19; discussion 519-28).
[0004] Corresponding with this profound rise in obesity incidence,
a similar rise in the consumption of foods higher in processed and
refined carbohydrates has been observed (Grundy, S. M. (1998) Am J
Clin Nutr 67, 563S-72S), along with an increased incidence of type
II diabetes (Disdier-Flores, O. M., Rodriguez-Lugo, L. A.,
Perez-Perdomo, R. & Perez-Cardona, C. M. (2001) P R Health Sci
J20, 123-30; Felber, J. P. & Golay, A. (2002) Int J Obes Relat
Metab Disord 26 Suppl 2, S39-45). These events have led researchers
to question the effect of dietary fat on body fat accumulation, and
suggest that dietary factors other than fat consumption play an
important role in body weight regulation (Willett, W. C. (1998) Am
J Clin Nutr 67, 556S-562S; Willett, W. C. (2002) Obes Rev 3,
59-68). Evidence now exists suggesting that chronic glycemia can
lead to increased fat synthesis and storage, and may contribute
significantly to the development of obesity and other chronic
diseases such as diabetes and cardiovascular disease (Jenkins, D.
J., Kendall, C. W., Augustin, L. S., Franceschi, S., Hamidi, M.,
Marchie, A., Jenkins, A. L. & Axelsen, M. (2002) Am J Clin Nutr
76, 266S-73S; Ludwig, D. S. (2002) JAMA 287, 2414-23; Leeds, A. R.
(2002) Am J Clin Nutr 76, 286S-9S). Concerns over safety and
efficacy of many anti-obesity products have limited their
usefulness. Therefore, developments of natural, safe, and effective
nutraceutical and/or medications that can help treat or prevent
obesity are essential to mitigate this public health crisis.
[0005] Both soybeans and potatoes are sources of proteinase
inhibitors (PI's), proteins that have been hypothesized to enhance
the release of cholecystokinin (CCK), one of several gut peptides
that regulate gastric emptying and satiety in humans (Liddle, R. A.
(1995) Am J Physiol 269, G319-27; Beglinger, C. (1994) Ann N Y Acad
Sci 713, 219-25; Beglinger, C. (2002) Curr Opin Investig Drugs 3,
587-8). Delayed gastric emptying, in turn, has been shown to result
in a decreased rate of glucose absorption, and lower post-prandial
glucose levels (Lefebvre, P. J. & Scheen, A. J. (1999) Eur J
Clin Invest 29 Suppl 2, 1-6). Proteinase inhibitor II (PI2) is a
naturally occurring 21 kDa dimer and potent trypsin and
chymotrypsin inhibitor present in white potatoes (Melville, J. C.
& Ryan, C. A. (1972) J Biol Chem 247, 3445-53; Bryant, J.,
Green, T. R., Gurusaddaiah, T. & Ryan, C. A. (1976)
Biochemistry 15, 3418-24). Previous studies using large doses of
highly pure PI2 demonstrated increased CCK release and satiety in
humans (Peikin, S. R., Springer, C. J., Dockray, G. J., Blundell,
J. E., Hill, A. J., Calam, J. & Ryan, C. A. (1987)
Gastroenterology 92, A1570; Hill, A. J., Peikin, S. R., Ryan, C. A.
& Blundell, J. E. (1990) Physiol Behav 48, 241-6; Schwartz, J.
G., Guan, D., Green, G. M. & Phillips, W. T. (1994) Diabetes
Care 17, 255-62). In addition, oral administration of PI2 at high
doses in a liquid form has been shown to reduce both post-prandial
glucose and insulin levels in humans (Schwartz, et al., supra),
supporting the use of PI2 as both a promising hunger management
tool and an effective agent to reduce post-prandial glycemia
experienced by the body.
[0006] The development of an efficient proprietary commercial
process providing an extract from potatoes containing PI2 has
increased the availability of this compound. It was hypothesized
that administration of PI2 extract as a nutraceutical ingredient in
a low dose, encapsulated form, prior to a meal, might reduce
post-prandial glucose levels. This could have important
implications for the use of PI2 as part of a diet to help maintain
healthy blood sugar levels and reduce the propensity for weight
gain.
SUMMARY OF THE INVENTION
[0007] The invention consists of a method for reducing
post-prandial glycogen levels in the blood of humans by the oral
administration of a proteinase inhibitor or a combination of
proteinase inhibitors. The proteinase inhibitor or combination is
administered prior to the ingestion of a meal and reduces not only
the initial rise in blood glucose following a meal (A Glucose or
AG) but also the integrated area under the blood glucose curve
(AUC) following a meal. The proteinase inhibitor(s) is effective
for helping to maintain healthy blood sugar levels and for treating
persons, such as those with Type II diabetes, which have adverse
health effects due to hyperglycemia. Further, the proteinase
inhibitor(s) is expected to reduce the propensity for weight gain
by reducing the glycemia experienced by the body.
[0008] Proteinase inhibitors which exhibit the property include
potato proteinase inhibitor II and soybean Bowman-Birk inhibitor,
although other proteinase inhibitors with similar amino acid
sequences and with similar proteinase inhibition properties may be
used. While single proteinase inhibitors have been shown to be
effective, combinations of two or more distinct proteinase
inhibitors may also be used.
[0009] In a preferred embodiment, a proteinase inhibitor product
isolated from potatoes is administered orally prior to a meal. The
potato proteinase inhibitor extract contains between about 15% and
about 25% by weight PI2 and also contains other proteins, including
Bowman-Birk inhibitor. The potato proteinase inhibitor extract is
present in an amount between about 1 mg and about 1000 mg per dose,
and preferably between about 5 mg and about 100 mg per dose, and
most preferably between about 7.5 mg and about 30 mg per dose. The
potato proteinase inhibitor is effective to reduce the blood
glucose spike following a meal by between about 5% and about 30%
and the AUC glucose by between about 5% and about 40%. Another
preferred proteinase inhibitor is Bowman-Birk inhibitor, which is
typically isolated from soybeans. The Bowman-Birk inhibitor is
present in an amount between about 0.1 mg and about 5.0 mg per
dose, and preferably between about 0.5 mg and about 2.0 mg per
dose. The Bowman-Birk inhibitor is effective to reduce the blood
glucose spike following a meal by between about 10% and about 25%
and the AUC glucose by between about 5% and about 30%.
[0010] It is an object of the present invention to reduce
post-prandial glycemia in humans by the oral administration of one
or more proteinase inhibitors prior to a meal.
[0011] It is a further object of the invention to reduce the
initial blood glucose spike following a meal by the oral
administration of one or more proteinase inhibitors prior to the
meal.
[0012] It is another object of the invention to reduce the total
area under the curve blood glucose following a meal by the oral
administration of one or more proteinase inhibitors prior to the
meal.
[0013] It is yet a further object of the invention to treat
hyperglycemia by the oral administration of one or more proteinase
inhibitors.
[0014] It is yet another object of the invention to prevent obesity
by the oral administration of one or more proteinase
inhibitors.
[0015] Yet a further object of the invention is to combat Type II
diabetes through the administration of one or more proteinase
inhibitors either alone or in combination with other medications
that are used in combating diabetes.
[0016] These and other objects of the invention will be understood
by those skilled in the art upon a review of this specification,
the associated figures and the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIGS. 1a and 1b are HPLC chromatograms of the potato PI2
extract used in the experiments and an authentic PI2 standard,
respectively.
[0018] FIG. 2 is a photograph of an SDS PAGE of the potato PI2
extract used in the experiments and an authentic PI2 standard.
[0019] FIG. 3 is a graph showing the effect of an increasing PI2
dose on post-prandial integrated area under the blood glucose curve
(AUC) after a test meal.
[0020] FIG. 4 is a graph showing the effect of an increasing PI2
dose on the initial rise in blood glucose above the baseline (A
Glucose) thirty minutes after a test meal.
[0021] FIG. 5 is a schematic diagram of the effects of chronic
consumption of a high glycemic load.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0022] The composition for reducing post-prandial blood glucose
levels in humans is based on a proteinase inhibitor that delays
gastric emptying and reduces post-prandial glycemia which may be
beneficial in combating obesity and in the therapeutic treatment of
patients suffering from hyperglycemia. The proteinase inhibitor is
believed to enhance the release of cholecystokinin (CCK), a peptide
which regulates gastric emptying. The preferred proteinase
inhibitors include potato proteinase inhibitor II and Bowman-Birk
inhibitor. In particular, a proteinase inhibitor extracted from
potatoes and available commercially from Kemin Consumer Care, L.C.,
Des Moines, Iowa, under the trademark Bioffect.TM. was used in some
of the examples. Bioffect.TM. is also available in tablets
formulated to contain 15 mg per dose and sold under the trademark
Satise.TM..
[0023] The invention is based on the surprising result that
proteinase inhibitors administered orally before a meal have the
effect of reducing the initial post-prandial glucose spike and also
reduce the total integrated area under the curve blood glucose over
more than three hours after a meal. Also surprising is that the
proteinase inhibitors are effective when administered in a dose in
the less than ten milligram range.
Example 1
Methods
Subjects
[0024] Twenty-six men and 13 women, mean age 35 years (range 23-61
years) with a mean body mass index of 27 (range 23-32) participated
in the study. Sample size was based on the study by Schwartz et al.
who showed significant decreases in mean post-prandial glucose in
six type II diabetic subjects following ingestion of a
glucose/protein shake in the presence and absence of a high dose of
PI2 (1.5 g). All subjects gave informed consent before the study
began, and could withdraw at any time.
Study Design
[0025] Subjects were randomly allocated to receive placebo and two
of the three following doses: 7.5, 15, or 30 mg PI2 extract. On
each study day subjects arrived at 8.00 AM after a 10 hour fast.
They were given breakfast and 500 ml of water to drink throughout
the morning, but ate nothing further until the test meal. Height
and weight of all subjects were recorded during their first visit.
Three and a half hours after breakfast the first blood glucose
measurement was made, and subjects were given the experimental
capsule and 500 ml of water. Thirty minutes later the test lunch
was served. As soon as each subject completed the meal, the timing
for post-prandial glucose measurements began. Subjects recorded any
adverse reactions at fifteen minute intervals for 200 minutes after
eating the meal.
Test Meal
[0026] On each test day subjects were fed a breakfast of granola,
skim milk, and orange juice that contained 330 kilocalories derived
from 67 g of carbohydrate, 2.5 g of fat, and 12 g of protein. No
other food was permitted until the test meal, which was consumed at
noon on the test day. The test meal was Chicken Teriyaki (Boston
Market) and contained no potato products. The nutritional content
of the test meal is set out in Table 1. All subjects consumed all
meals in their entirety.
TABLE-US-00001 TABLE 1 Characteristics of the test meal Lunch Test
Meal Energy (kcal) 460 Fat (g) 11 Carbohydrate (g) 53 Protein (g)
27 Number of participants taking meal challenge after 39 placebo
Average AUC for placebo participants (SD) 2196.6 .+-. 1567.2
Glucose Measurements
[0027] Finger-prick capillary blood samples were taken 30 minutes
before the test meal (Baseline), and 30, 60, 90, and 120 minutes
post-prandially. Glucose measurements were made with a Dex
glucometer, Model #3952E (Bayer Pharmaceuticals), in accordance
with the manufacturer's instructions.
Proteinase Inhibitor
[0028] PI2 extract was provided by Kemin Consumer Care, L.C. (Des
Moines, Iowa), and was supplied in 00 gelatin capsules containing
7.5, 15, or 30 mg, respectively. A mixture of dextrose and whey
protein was used to bring all capsules to a uniform weight and
volume and also served as a placebo. The doses in the present study
were chosen based on previous studies demonstrating efficacy at 30
mg in liquid form (Spiegel, T. A., Hubert, C. & Peikin, S. R.
(1999) University of Medicine and Dentistry of New Jersey;
Vasselli, J. R., Greenfield, D., Schwartz, L. & Heymsfield, S.
B. (1999) Obesity Research Center, St. Luke's-Roosevelt Hospital
Center, Columbia University), and 7.5 mg (Gary Green, University of
Texas, San Antonio, 1996, 1997, unpublished data). The active
material was produced from a single lot of potatoes (Russet
Nuggets; Kemin lot 87289C, approximately 244.39 mg PI2
extract/kg).
Measurement of PI2
[0029] RP-HPLC: Formulation of the active doses was based on
quantitation by high performance liquid chromatography (HPLC).
Reversed-phase HPLC (RP-HPLC) analyses were performed on a Hewlett
Packard Model 1100 equipped with a diode array detector using a
Microsorb C-18, 5 .mu.m particle size, 300 Angstrom pore size,
4.6.times.250 mm (Varian Analytical Instruments, Walnut Creek, CA).
The chromatographic conditions were as follows: Isocratic elution
for five minutes of 80% of 0.1% TFA in H.sub.2O (20% of 1% TFA in
acetonitrile). Gradient from 80-30% of 0.1% TFA in H.sub.2O (20-70%
of 1% TFA in acetonitrile) for 34 minutes. Gradient from 30-0% of
0.1% TFA in H.sub.2O (30-100% of 1% TFA in acetonitrile) for 4
minutes. Flow rate was 1 ml/min for all gradients, and the column
temperature was maintained at 30.degree. C. Integration of the HPLC
peak area provided the relative concentration of each sample (mg/g
solids).
[0030] SDS-PAGE: To further characterize the PI2 extract, samples
were analyzed by gel electrophoresis. SDS gels were prepared as 4%
stacking, 15% resolving with 1.5 M Tris, 0.5 M Tris, 10% SDS, 30%
ammonium persulfate, TEMED, and 40% Acryl/Bis. Wells were loaded
with pre-stained marker, PI2 standard, and PI2 extract. A current
of 80 volts was applied for 1.5 hours. Gels were then stained with
Coomasie blue staining. Pure PI2 standard was obtained by
sequential RP-HPLC followed by gel filtration chromatography.
Western blot using a rabbit polyclonal antibody developed by Kemin
Foods, L.C. against PI2 protein, was used to further determine the
identity of the major protein in the potato PI2 extract used in the
current study.
Calculations and Statistical Analysis
[0031] The difference between the 30-minute post-prandial and
baseline blood glucose values was calculated for each subject visit
(A glucose). The integrated area under the blood glucose-time curve
(AUC) after each test meal was calculated using the pre-meal value
as the baseline, and integrating the area from 0 to approximately
120 minutes after the meal. Repeated measure analysis of variance
was used to test for significant differences between areas. The
research design involved repeated measures, so the PROC MIXED
function in SAS was used, as this allows a more general
specification of the covariance matrix of the dependent variable,
and allows random factors of both the model and error terms to be
correlated (Hongsen, Z. (2001) Proceedings of the 12th Annual
Conference of the Midwest SAS Users Group, 132-140). All subjects
received placebo on one visit, but only two of the three possible
active treatments during the other visits, so an incomplete block
design was used to evaluate the relative effectiveness of the
doses. The strategy described by Wolfinger (Wolfinger, R. D. (1993)
Communications in Statistics, Simulation, and Computation 22,
1079-1106) was followed to select an appropriate
variance-covariance structure for the ANOVA test. The Akaike's
Information Criterion was used to select the appropriate
variance-covariance structure for the model. Chi-square analysis
was used to evaluate data obtained as discrete variables with
p<0.05 considered to be significant.
Results
[0032] Doses of active PI2 extract were quantified by RP-HPLC. The
integrated peak representing the PI2 extract co-eluted with a pure
authentic PI2 standard, indicating that PI2 is contained in the
extract and that it is the major protein (FIGS. 1a and 1b). Results
of gel electrophoresis further confirm the findings of the analysis
by RP-HPLC and show that the PI2 in the extract is likely present
as a monomer with a molecular weight of approximately .about.12 kDa
(FIG. 2). MALDI MS analysis of the purified PI2 protein
demonstrated that this protein has a molecular weight of 12 kDa.
Western blots of the separated proteins using a rabbit polyclonal
antibody for PI2 protein demonstrated that the major protein band
separated by SDS-PAGE is PI2. The actual amount of PI2 protein
present in a given extract could vary and ranges from 17-20%. The
PI2 extract was also characterized for its trypsin and chymotrypsin
inhibition activity using an in vitro assay demonstrating both
trypsin and chymotrypsin inhibition. PI2 extract product contained
a ratio of 0.9-1.7:1 units of trypsin: chymotrypsin inhibition
activity, respectively.
[0033] The volunteers in the present study consumed 120 test meals.
Forty placebo doses were administered, along with 27 of each of the
7.5 mg and 15 mg doses, and 26 of the 30 mg dose, respectively (one
individual declined to provide blood samples and was included in
determination of adverse events monitoring). Table 1 shows the
nutrient value of the test meal and the mean glucose AUC following
placebo. We first examined the effect of PI2 extract on AUC; the
repeated measure ANOVA model used for this analysis showed a
statistically significant effect of the experimental treatment
(f=3.3, p<0.05) but no statistically significant difference
between the experimental blocks. Subjects given a dose of 7.5 mg
PI2 extract before the test meal experienced no significant
reduction in post-prandial glucose compared to placebo. The AUC of
subjects receiving both 15 and 30 mg PI2 extract prior to the test
meal was significantly reduced compared to placebo, but there was
no significant difference in post-prandial AUC between the two
higher doses (FIG. 3). The decrease in AUC for 15 and 30 mg was
29.8% and 24.5% respectively, each compared to placebo. There was a
significant reduction in glucose at both the 15 mg and 30 mg dose
levels compared to placebo, but there was no significant difference
in glucose between the two higher doses (FIG. 4). The decrease in
glucose for the 15 and 30 mg doses was 25% and 20% respectively,
each compared to placebo.
[0034] Feeding 120 test meals resulted in 14 reports of an adverse
reaction from subjects. These are summarized in Table 2.
TABLE-US-00002 TABLE 2 Subjects recording adverse effects after
eating a test meal preceded by PI2 extract Gastrointestinal
Chi-square PI2 Extract Dose (mg) Symptoms Headache Total (vs.
Placebo) 0 3 -- 3 -- 7.5 5 1 6 0.19 15 -- 2 2 1.76 30 3 -- 3
0.23
Gastrointestinal symptoms included nausea, cramping and diarrhea.
Differences in occurrence rates of adverse reactions between the
treatments and the placebo were not significant (p>0.05, Chi
square). Subjects experiencing symptoms rated them as mild, and
frequently they were noted at only one of the recording times.
Discussion
[0035] The drastic rise in obesity rates over the past 10 years has
been accompanied by diets resulting in chronic glycemia and hyper
secretion of insulin (Grundy; Wolever, T. M. & Bolognesi, C.
(1996) J Nutr 126, 2807-12). This, in turn, initiates a cascade of
metabolic and physiologic events resulting in decreased lipolysis,
increased de novo lipogenesis, and faster onset of hunger and
subsequent food intake (Jenkins; Ludwig). Rapid and drastic
excursions in blood sugar may not only contribute to obesity but
other chronic diseases, including diabetes and cardiovascular
disease (as summarized in FIG. 5 and in Ludwig).
[0036] Accordingly, lowering the glycemic load experienced by the
body by diet or other means may be an effective way to reduce the
post-prandial glycemia that can lead to weight gain and obesity.
Findings of the present study suggest that it is possible to lower
the glycemic load experienced by the body by ingesting a supplement
containing a low dose of PI2 extract prior to a meal. Doses of
either 15 mg or 30 mg taken 30 minutes before a test meal
significantly reduced the subsequent rise in blood glucose (FIGS. 3
and 4). A dose of 7.5 mg had no significant effect, indicating that
under these test conditions the lowest effective dose lies between
7.5 and 15 mg. This study was limited to acute observations, and
the effect of chronic oral administration of PI2 extract on blood
sugar levels remains to be studied. However, this study is unique
because PI2 has not previously been administered in solid form in
an encapsulated supplement prior to the meal, and because a solid
mixed meal was used for the first time. In addition, the dose used
was substantially lower and less pure than that previously
reported, and a larger cohort of subjects was studied. A dose of
1.5 g PI2 (90-100% pure) by column chromatography (Clarence Ryan,
Washington State University, Pullman, Wash.), administered in
liquid form was used in two previous studies; in one study PI2 was
added to soup and fed 8 minutes before a test meal, and in the
other it was incorporated in a test beverage (Hill et al.; Schwartz
et al.). In neither case was it encapsulated. Other differences
include the size and glycemic index of the test meals and potential
variations in the PI2 dose bioactivity. We found a mean reduction
in post-prandial blood glucose AUC of 29.8% with a 15 mg dose of
PI2 extract and 24.5% decrease with a 30 mg active dose (FIG. 3).
Schwartz et al. reported a comparable 24.5% reduction in AUC after
feeding 1.5 g PI2 with a liquid glucose and protein beverage
administered to diabetics. While the dose of PI2 administered in
that study was apparently 100-fold larger, we cannot be sure that
it was of the same specific activity as used in our current study.
Therefore, it is unclear whether larger doses of PI2 extract would
evoke a greater response.
[0037] Inspection of the responses of individual subjects to
placebo or active dose reveals that 9 subjects experienced no
reduction in glycemia with either of the two dose levels of PI2
extract administered (non-responders). There was no significant
effect of BMI, age, or fasting blood glucose on responsiveness.
Among the 9 non-responders from the initial study, 8 were male and
one was female, although this difference was not significant
(p=0.07, Chi-square).
[0038] The notion of lowering the glycemic load to reduce or
maintain weight is supported by both animal and human studies.
Normal rats fed isocaloric diets differing dramatically in terms of
glycemic load, experience large differences in post-prandial
glycemia and insulin response (Kabir, M., Rizkalla, S. W., Champ,
M., Luo, J., Boillot, J., Bruzzo, F. & Slama, G. (1998) J Nutr
128, 35-43; Kabir, M., Rizkalla, S. W., Quignard-Boulange, A.,
Guerre-Millo, M., Boillot, J., Ardouin, B., Luo, J. & Slama, G.
(1998) J Nutr 128, 1878-83). Maintaining rats on these diets for
weeks at a time results in drastic differences in glucose and lipid
metabolism. The levels of fatty acid synthase and de novo
lipogenesis, as well as adipocyte size, are higher in rats
consuming a high vs. low glycemic load diet (Kabir et al. 35-43;
Kabir et al. 1878-83). These data provide evidence at the cellular
and metabolic level, that drastic elevations in blood sugar caused
by exposing the body to a high glycemic load results in increased
fat accumulation over a relatively short window of time. Consistent
with this are results from long term research showing that adult
rats fed isocaloric diets evoking chronic hyperglycemia gain a
significant amount of weight while those fed a diet with moderate
glycemia maintain their weight (Pawlak, D. B., Denyer, G. S. &
Brand-Miller, J. C. (2000) Proc Nutr Soc Aust 24, 215).
[0039] Weight loss studies in humans suggest that reducing the
glycemia experienced by the body is an effective means to reduce
and maintain weight. Subjects consuming an isocaloric diets
consisting of low glycemic index foods lose more weight or maintain
their relative to those consuming high glycemic index foods
(Slabber, M., Barnard, H. C., Kuyl, J. M., Dannhauser, A. &
Schall, R. (1994) Am J Clin Nutr 60, 48-53; Wolever, T. M.,
Jenkins, D. J., Vuksan, V., Jenkins, A. L., Wong, G. S. &
Josse, R. G. (1992) Diabetes Care 15, 562-4; Clapp, J. R. (1997)
Arch Gynecol Obstet 261, 101-107). These findings suggest that
manipulation of the glycemic load, in these cases by consuming low
glycemic load diets, can effectively stimulate weight loss and/or
prevent weight gain. Combined with the results from the present
study, these support the hypothesis that PI2 extract can serve as
an effective nutraceutical to lower the glycemia experienced by the
body, and may help promote weight loss and reduce the propensity
for weight gain.
[0040] PI2 extract is proposed to exert its effect on post-prandial
glucose by enhancing the release of a well characterized peptide
hormone, CCK, which is naturally secreted into the blood stream by
enteroendocrine cells in response to a meal (Crawley, J. N. &
Corwin, R. L. (1994) Peptides 15, 731-55). CCK acts on various
target tissues throughout the body including the gastrointestinal
tract, where it delays gastric emptying leading to feelings of
fullness, and the brain leading to feelings of satiety. Although
not measured in the present study, previous studies in the late
1980's and 1990's demonstrated that large doses of purified PI2
enhance the release of CCK (Peikin et al.; Schwartz et al.) delay
gastric emptying time (Schwartz et al.), and decrease energy intake
(Hill et al.) in humans. These were followed by studies using a
lower dose of less pure PI2 extract which demonstrated reduced
hunger and increased fullness ratings (Spiegel et al.; Vasselli et
al.) (summarized in Table 3). These findings are consistent with
the well established fact that PI's are potent stimulators of CCK
release in rats (Liddle).
TABLE-US-00003 TABLE 3 Summary of PI2 Clinical Trials Study
Institution Dose Form Outcome Spiegel et al. Columbia University 30
mg Liquid Significant decrease in 1999 PI2 (pre-meal hunger
ratings; increase extract shake) in fullness ratings; 2 kg weight
loss Vasselli et al. Robert Wood Johnson 30 mg Liquid Significant
decrease in 1999 Medical School, PI2 (pre-meal hunger ratings;
increase University of Medicine extract shake) in fullness ratings
and Dentistry of New Jersey Schwartz et al. University of Texas,
San 1500 mg Liquid Significant increase in 1994 Antonio PI2 (shake)
plasma CCK; delayed gastric emptying; decreased blood sugar Hill et
al. 1990 University of Leeds, U.K 1500 mg Liquid Significant
decrease in PI2 (pre-meal food consumption soup) Peikin et al.
Robert Wood Johnson 1000 mg Liquid Significant increase in 1987
Medical School, PI2 (shake) plasma CCK levels University of
Medicine and Dentistry of New Jersey Green, 1996-1997* University
of Texas, San 7.5-100 mg Liquid Doses as low as 7.5 mg Antonio PI2
(shake) delayed gastric emptying and reduced blood sugar levels
*Unpublished data
[0041] PI2 is a pH, heat, and salt stable protein (Bryant et al.),
allowing it to be effective when administered orally, and making it
unique among plant PI's. The extract used in the present study
contains PI2 (FIGS. 1 and 2), and is derived from white potatoes
using a method generally as described in U.S. patent application
Ser. No. 09/900,555, incorporated herein by this reference.
Although normally present in potatoes as a dimer, the PI2 separated
from our extract appears to be in the monomeric form. The pure PI2
possess trypsin and chymotrypsin inhibition activities of 1.4 and
3.6 inhibition unites and the PI2 extract possess relative trypsin
and chymotrypsin inhibition activities of 22 and 13 inhibition
units. The added beneficial effect of reducing post-prandial
glycemia makes PI2 extract a unique and promising
nutraceutical.
[0042] Some studies involving the direct infusion of CCK have
reported minor adverse side effects such as headache, nausea, and
diarrhea (Crawley, J. N. & Corwin, R. L. (1994) Peptides 15,
731-55; Pi-Sunyer, X., Kissileff, H. R., Thornton, J. & Smith,
G. P. (1982) Physiol Behav 29, 627-30). For this reason we
questioned participants specifically about these effects which may
ultimately have prompted reporting of events that would otherwise
have gone un-noticed. Although there are a number of reports in the
literature demonstrating morphological changes in the pancreas as a
result of long term exposure to extremely high doses of natural and
synthetic PI's in rodents, similar studies in pigs and primates are
not associated with such effects (Struthers, B. J., MacDonald, J.
R., Dahlgren, R. R. & Hopkins, D. T. (1983) J Nutr 113, 86-97;
Harwood, J. P., Ausman, L. M., King, N. W., Sehgal, P. K.,
Nicolosi, R. J., Liener, I. E., Donatucci, D. & Tarcza, J.
(1986) Adv Exp Med Biol 199, 223-37; Garthoff, L. H., Henderson, G.
R., Sager, A. O., Sobotka, T. J., Gaines, D. W., O'Donnell, M. W.,
Jr., Chi, R., Chirtel, S. J., Barton, C. N., Brown, L. H., Hines,
F. A., Solomon, T., Turkleson, J., Berry, D., Dick, H., Wilson, F.
& Khan, M. A. (2002) Food Chem Toxicol 40, 501-16; Garthoff, L.
H., Henderson, G. R., Sager, A. O., Sobotka, T. J., O'Dell, R.,
Thorpe, C. W., Trotter, W. J., Bruce, V. R., Dallas, H. L., Poelma,
P. L., Solomon, H. M., Bier, J. W., O'Donnell, M. W., Jr., Chi, R.
K., Chirtel, S. J., Barton, C. N., Brown, L. H., Frattali, V. P.
& Khan, M. A. (2002) Food Chem Toxicol 40, 487-500). Such
effects have yet to be observed in humans using PI's from natural
sources. Furthermore, previous studies using PI2 have not
demonstrated any side effects with doses many times that used in
this study (Peikin et al.; Schwartz et al.). Side effects noted by
our subjects were mild and inconsistent, and caused no withdrawals
from the study. No increasing dose response was noted for any of
these effects and the rate of occurrence was not different between
placebo and treatment. If persistent use of PI2 extract were
contemplated we may see additional mild side effects, although it
is equally possible that tolerance to undesired effects would
develop over time.
[0043] In conclusion, we have demonstrated in the largest
randomized controlled clinical trial to date that a low dose of PI2
extract prior to a standardized meal reduces significantly
post-prandial glycemia in the majority of healthy subjects.
Additional studies will be required to ascertain long term effects
of this supplement on blood glucose, appetite and body weight.
While a mechanism of action has been proposed, it will be important
to confirm this hypothesis in future studies addressing changes in
serum CCK, insulin, and the like. Such studies could be
instrumental in applying PI2 to the clinical problems of obesity
and diabetes.
Example 2
[0044] To better understand if the trypsin/chymotrypsin inhibiting
activity of the PI2 protein was related to the glucose response to
the potato proteinase inhibitor extract, a preparation that
purified the PI2 fraction from the potato proteinase inhibitor
extract (abbreviated pPI2) was tested along side a preparation of
Bowman-Birk inhibitor after meal challenge. The Bowman-Birk
inhibitor used was obtained from Sigma-Aldritch and had a stated
purity of greater than 80%. Bowman-Birk inhibitor has similar
enzyme inhibiting properties as pPI2. The meal challenge was
conducted at a breakfast meal instead of a lunch meal, as in the
prior study, and consisted of 390 kcal with 100 kcal from fat and
53 g carbohydrate and was provided to individuals who had been
fasting for at least 10 hours. Each participant made two visits to
the research center and underwent two meal challenges--one for the
placebo and one for an active (15 mg pPI2 or 0.8 mg Bowman-Birk
inhibitor) and these treatments were provided in a double-blinded
format. The randomization scheme also prevented the participants or
the study personnel from knowing at which visit the placebo was
given until the code was broken.
[0045] In this study we found that among healthy volunteers, both
pPI2 and Bowman-Birk inhibitor decreased the post-prandial glucose
spike. The data is summarized in Table 4. For example G was
decreased by 25.5% compared to placebo among the 10 individuals
taking pPI2. This difference (from a mean G of 52.1 mg/dl+15.9 sd
for the placebo dose versus 38.8+34.6 sd for those taking the pPI2)
was evaluated by one-tailed, paired student's t test and yielded
p=0.065. The Bowman-Birk inhibitor also inhibited the post-prandial
glucose spike with a decrease in G of 42.4%. The absolute decrease
was from 47.9 mg/dl.+-.22.6 sd for the placebo treatments compared
to 27.6 mg/dl.+-.21.6 sd for the Bowman-Birk inhibitor treatment.
This was significant by student's t test with p=0.04.
[0046] Neither pPI2 nor Bowman-Birk inhibitor showed statistically
significant differences in AUC, although the trend was toward
absolute decreases in this parameter of 17% and 11.5% for both pPI2
and Bowman-Birk inhibitor, respectively.
[0047] In the earlier study we did discover that some individuals
seemed to be unresponsive to pPI2. Again, we found this to be the
case with pPI2. Three individuals were identified as non-responders
with pPI2 and two individuals were unresponsive to Bowman-Birk
inhibitor. A non-responder was defined as an individual who did not
have a lower absolute G after treatment with the active test
material than with the placebo.
[0048] Thus far we are supporting the hypothesis that the PI2
protein in the potato proteinase inhibitor extract and the
trypsin/chymotrypsin inhibiting activity may be related to
modulation of post-prandial glucose since the relative
concentration of this activity in the pPI2 showed comparable
glucose modulating activity as with the original potato proteinase
inhibitor extract and the Bowman-Birk inhibitor which also contains
an inhibitor or inhibitors of trypsin and chymotrypsin showed
glucose modulating activity.
TABLE-US-00004 TABLE 4 Comparison of purified PI2 and Bowman-Birk
Inhibitor on G and AUC Purified PI2 Product Bowman-Birk Inhibitor
Parameter Placebo Active Placebo Active Mean G .+-. Study 1 52.1
.+-. 15.9 38.8 .+-. 34.6 47.9 .+-. 22.6 27.6 .+-. 21.6 SD *p =
0.065 *p = 0.04 Mean G .+-. Study 2 55.1 .+-. 21.3 45.7 .+-. 21.7
49.8 .+-. 13.9 45 .+-. 21.8 SD *p = 0.058 *p > 0.05 Mean AUC
.+-. Study 1 2279 .+-. 1187 2747 .+-. 1325 2015 .+-. 1702 1783 .+-.
1935 SD *p = 0.15 *p = 0.32 Mean AUC .+-. Study 2 2496.9 .+-.
1878.1 2153.6 .+-. 2181.3 SD *p = 0.229 *one tailed paired t test
(versus placebo)
[0049] Although the invention has been described with respect to a
preferred embodiment thereof, it is to be also understood that it
is not to be so limited since changes and modifications can be made
therein which are within the full intended scope of this invention
as defined by the appended claims.
* * * * *