Blueberry paste treatment and prophylaxis effect with experimental pancreatic diabetes

Approved by                                                   Approved by
A.P. Levitsky,                                                 I.A. Musonova
Professor, Corresponding Member,                   General Manager    
Stomatology Institute,                                    SIA "Odessa Biotechnology"
Academy of Science, Ukraine                           03.01.2012.
03.01.2012

 

REPORT


on studying blueberry paste treatment and prophylaxis effect with experimental pancreatic diabetes 
(under contract № 067A concluded on 01.11.2011 with TEKMASH Institute)


A.P. Levitsky, Doctor of Biology, professor, Corresponding Member of Academy of Science, Scientific Adviser;
O.A. Makarenko, Candidate of Biology, Head of Biochemistry Laboratory at Stomatology Institute, Research Executor


Odessa 2012


Contents
1. Research theoretical justification
2. Materials and methods
3. Blueberry paste effect on blood sugar levels in diabetes rats
4. Blueberry paste effect on microbial contamination levels in diabetes rats
5. Blueberry paste effect on nonspecific immunity in diabetes rats
6. Blueberry paste effect on peroxidation in diabetes rats
7. Conclusion
8. References

 

1. Research theoretical justification
Recently pancreatic diabetes (PD) has become a most serious problem in medicine. The number of diabetes patients in Ukraine has been constantly growing, amounting to 1.2 millions at present [E.V. Titovich, 2009].
By 2020 the projected PD patients in the world will amount to about 350 million [Blumgarden, 2000; Zimmet, 2002].
What makes pancreatic diabetes especially dangerous is the complications it causes in cardiovascular and nervous systems, in eyesight, kidneys [Dedov at al., 2001]. Diabetes patients die of malignant tumors 8 times more often [Balabolkin, 2000].
Present-day concepts of PD pathogenesis is based on the idea of glucose toxicity, according to which high glucose concentration in blood predetermines the nature of all pathological processes: lipid and protein intensive peroxidation, enhanced glucose polyol exchange, protein glycation activation, stress development [Tsiselsky, Levitsky, 2004].
Lately our laboratory has substantiated another concept of PD pathogenesis, namely: dysbiotic concept [Tsiselsky, 2011]. According to it hyperglycernia produced by PD causes dysbacteriosis which promotes conditionally pathogenic bacteria concentration, thus increasing the amount of microbial toxins, intestinal endotoxin (lipopolysaccharide) being of special importance [Yakovlev, 2003]. It possesses certain toxic properties which several million times exceed glucose toxicity and can reduce immunity and start inflammatory process [Levitsky et al., 2010].
Proceeding from this dysbiotic concept and from other non-infectious diseases, we have suggested to use prebiotics [Levitsky et al., 2008] and polyphenols [ Levitsky, Tsiselsky, 2010] for treating and preventing PD.
Our previous report (under contract with TEKMASH Institute) testifies to great amounts of polyphenols represented by bioflavonoids, chlorogenic acid and other compounds of polyphenolic nature in LiQberry blueberry paste. Besides, their availability is more that twice higher than that of fresh blueberries.
Blueberry paste treatment and prophylaxis effect has been confirmed with experimental toxic hepatitis caused by intestinal dysbiosis [Levitsky et al., 2009].
This research has been aimed at determining blueberry paste treatment and prophylaxis effect with experimental pancreatic diabetes of type I.
The basic pathogenetic characteristics considered are:
- blood sugar level;
- microbial contamination level;
- nonspecific immunity;
- lipid peroxidation.

2. Materials and methods
Blueberry paste as a preserved product (LiQberry TM) has been made using the customer's original technology (hydrothermodynamic technology) by SIPE TEKMASH Institute. According to the previous tests, polyphenol compounds in the paste amounted to 280-600 mg/100 g, chlorogenic acid (CGA) amounted to 1600 mg/100 g, which makes about 10% paste dry weight [S.B. Osipenko, 2001, S.B. Osipenko et al, 2002].
The paste biological effect has been tested on 49 white rats of Vistar line (males, 260±12 g live weight). Pancreatic diabetes of type I has been produced by a single intraperitoneal introduction of alloxan, the dose being 100 mg/kg, previously diluted in 0.1 M citrate buffer pH 6.2.
Starting from the day of alloxan introduction, the rats were fed blueberry paste in their ration. The paste was mixed with flour and added to the ration so that the blueberry dose equaled 1-2 g/kg per rat daily.
The rats were killed under thiopental anaesthetic by blood-letting from the heart on the 7th and 14th days of the experiment. Homogenates of small intestines and liver mucous membrane as well as blood serum have been used for urease analysis which serves as a marker of microbial contamination level [Levitsky et al, 2007], for lysozyme analysis which testifies to nonspecific immunity level [Levitsky, 2005] and for malonic dialdehyde (MD) content analysis which indicates lipid peroxidation intensity and testifies to inflammation [Stalnaia and Garishvili, 1977; Levitsky et al, 2010]. Blood sugar level has been determined by glucose content in blood serum [Goriachkovsky, 2005].


3. Blueberry paste effect on blood sugar levels in diabetes rats
Blood sugar levels or, to be more exact, glucose concentrations in blood serum are given in table 1. The data testify to the fact that on producing PD of type I by alloxan, hyperglycemia develops gradually and exceeds the standard rate on the 14th day. Blueberry paste dosing decreases the hyperglycemia level but only in case the hyperglycemia is intractable. We have not been able to establish the interrelation between blueberry paste sugar-decreasing effect and the chosen doses (1 - 2 g/kg).
Table 1. Blueberry paste effect on glucose content in blood serum of alloxan diabetes rats

Group n Glucose, mmol/l
no blueberry 1 g/kg blueberry 2 g/kg blueberry
 1 Intact (standard) 7 6.2±0.26 - -
2 Diabetes, 7 days 21 6.89±0.32
p>0.05
6.74±0.31
p>0.05
p1>0.5
6.72±0.24
p>0.05
p1>0.5
3 Diabetes, 14 days 21 7.34±0.31
P<0.05
6.28±0.25
p>0.3
p1<0.05
6.41±0.24
p>0.2
p1<0.05


Notes
1. p - confidence factor to compare with standard
2. p1 - confidence factor to compare with "no blueberry" group


4. Blueberry paste effect on microbial contamination levels in diabetes rats
Inoculation method commonly used for determining microbial contamination is not quite reliable as most microbes (up to 98% !) do not grow on standard nutrient mediums [Levitsky et al, 2007].
We have suggested an express-method to determine microbial contamination by urease level, this enzyme being produced by numerous microbe species but not by human or animal body cells [Levitsky et al, 2006].
Urease level determination in three biological mediums: blood serum, small intestine mucous membrane and the liver, has yielded the results given in tables 2-3, respectively. As it has been expected, the greatest microbial contamination is observed in small intestine mucous membrane, less contaminated is the liver, while blood serum contamination is quite insignificant (20 times lower!).


Table 2. Blueberry paste effect on urease level in blood serum of alloxan diabetes rats

Group n Urease
 no paste 1 g/kg paste 2 g/kg paste 
 1 Intact (standard) 7 0.16±0.02 - -
2 Diabetes, 7 days 21 0.19±0.02
p>0.1
0.16±0.01
p = 1
p1>0.05
0.14±0.01
p>0.05
p1<0.05
3 Diabetes, 14 days 21 0.21±0.02
p>0.05
0.14±0.01
p>0.1
p1<0.05
0.14±0.01
p>0.05
p1<0.05

Notes
1. p - confidence factor to compare with standard
2. p1 - confidence factor to compare with "no blueberry" group

Table 3. Blueberry paste effect on urease level in small intestine mucous membrane of alloxan diabetes rats

Group n Urease
no paste 1 g/kg paste 2 g/kg paste
1 Intact (standard) 7 3.40±0.11 - -
2 Diabetes, 7 days 21 3.89±0.10
p<0.05
3.42±0.16
p>0.6
p1<0.05
3.33±0.14
p>0.4
p1<0.05
3 Diabetes, 14 days  21 4.10±0.12
p<0.01
3.82±0.12
p<0.05
p1>0.05
3.70±0.13
p>0.05
p1<0.05

Notes
1. p - confidence factor to compare with standard
2. p1 - confidence factor to compare with "no blueberry" group

Table 4. Blueberry paste effect on urease level in the liver of alloxan diabetes rats

Group n Urease
no paste 1 g/kg paste 2 g/kg paste 
1  Intact (standard) 7 3.19±0.16 - -
2 Diabetes, 7 days 21 4.06±0.21
p<0.05
3.63±0.20
p >0.05
p1>0.1
3.46±0.30
p>0.3
p1>0.05
3 Diabetes, 14 days  21 4.35±0.23
P<0.01
3.40±0.20
p>0.3
p1<0.05
3.21±0.25
p>0.8
p1<0.01

Notes
1. p - confidence factor to compare with standard
2. p1 - confidence factor to compare with "no blueberry" group


As alloxan diabetes develops, urease activity in all groups increases depending on the diabetes duration period.
Blueberry paste evidently decreases urease activity, the dose of 2 g/kg being more effective than 1 g/kg, and restores the small intestine microflora completely.
The data obtained testify convincingly to the ability of blueberry paste biologically active substances to decrease microbial contamination of human body under PD.
But as dysbacteriosis is so essential to PD complications [Tsiselsky, 2011], we may come to the conclusion that blueberry paste can contribute significantly to diabetes complications prevention.
These data agree with those of blueberry paste therapeutic effect on experimental hepatitis and intestinal dysbacteriosis [Levitsky et al, 2009].


5. Blueberry paste effect on nonspecific immunity in diabetes rats
Nonspecific antimicrobial immunity in human and animal body depends on a number of factors, lysozyme enzyme being the most essential [Levitsky, 2005].
Lysozyme activity is different in blood serum, small intestine mucous membrane and the liver of diabetes rats, which is shown in tables 5 - 7.
Table 5. Blueberry paste effect on lysozyme activity in blood serum of alloxan diabetes rats

№  Group  Lysozyme, unit/l
 no blueberry 1 g/kg blueberry  2 g/kg blueberry 
1  Intact (standard) 7 72 ± 6 ;-  -
2 Diabetes, 7 days 21 50 ± 5
р<0,05
53 ± 2
р<0,05
р1>0,2
57 ± 4
р<0,05
р1>0,3
3  Diabetes, 14 days 21 38 ± 6
р<0,01
52 ± 4
р<0,05
р1>0,05
58 ± 3
р<0,05
р1<0,05

Notes
1. p - confidence factor to compare with standard
2. p1 - confidence factor to compare with "no blueberry" group

 

Table 6. Blueberry paste effect on lysozyme activity in small intestine mucous membrane of alloxan diabetes rats

Group n Lysozyme, unit/l
no blueberry 1 g/kg blueberry  2 g/kg blueberry 
1  Intact (standard) 7 183 ± 5  -  -
2 Diabetes, 7 days 21 138 ± 4
р<0,001
148 ± 2
р<0,01
р1<0,05
155 ± 5
р<0,01
р1<0,05
3  Diabetes, 14 days  21 86 ± 7
р<0,001
142 ± 2
р<0,01
р1<0,01
163 ± 3
р<0,05
р1<0,001

Notes
1. p - confidence factor to compare with standard
2. p1 - confidence factor to compare with "no blueberry" group

Table 7. Blueberry paste effect on lysozyme activity in the liver of alloxan diabetes rats

Group n Lysozyme, unit/l
no blueberry 1 g/kg blueberry  2 g/kg blueberry 
1 Intact (standard) 7 125 ± 10 - -
2 Diabetes, 7 days 21 63 ± 4
р<0,001
61 ± 4
р<0,001
р1>0,6
68 ± 3
р<0,001
р1>0,3
3 Diabetes, 14 days 21 38 ± 2
р<0,001
66 ± 2
р<0,001
р1<0,001
79 ± 2
р<0,01
р1<0,001

Notes
1. p - confidence factor to compare with standard
2. p1 - confidence factor to compare with "no blueberry" group

As the data show, lysozyme activity is considerably decreased in all biological mediums studied, this decrease being dependent on the disease length.
Blueberry paste helps to increase lysozyme activity but not up to the standard. It should be noted that the dose of 2 g/kg is more effective than 1 g/kg.
Thus, decreased lysozyne activity testifies to a considerably weaker nonspecific immunity under PD, and it is just this that can cause microbial contamination growth and bacterial poisoning.
As blueberry paste strengthens immunity, it can also eliminate dysbacteriosis phenomena under PD.

6. Blueberry paste effect on peroxidation in diabetes rats
Any pathologic processes in the body are accompanied by higher free radical oxidation which promotes the formation of oxygen active forms (OAF). On the one hand, such forms have a positive antimicrobial effect, but on the other hand, they cause peroxidation of proteins, lipids and membranes formed by them [Balabolkin, 2006].
Malonic dialdehyde (MDA) serves as a biochemical marker of lipid (or it would be more accurate to say, unsaturated fatty acid) peroxidation, its level testifying to the prooxidant process intensity in the body.
Tables 8-10 show MDA content in three biological mediums (blood serum, small intestine mucous membrane and liver).
Table 8. Blueberry paste effect on MDA content in blood serum of alloxan diabetes rats

Group MDA, mmol/l
no blueberry 1 g/kg blueberry  2 g/kg blueberry 
1 Intact (standard) 7 0,39 ± 0,01 ;- -
2 Diabetes, 7 days 21 0,55 ± 0,01
р<0,001
0,45 ± 0,01
р<0,01
р1<0,001
0,41 ± 0,01
р>0,05
р1<0,001
3 Diabetes, 14 days 21 0,59 ± 0,01
р<0,001
0,45 ± 0,01
р<0,01
р1<0,001
0,41 ± 0,01
р>0,05
р1<0,001

Notes
1. p - confidence factor to compare with standard
2. p1 - confidence factor to compare with "no blueberry" group

Table 9. Blueberry paste effect on MDA content in small intestine mucous membrane of alloxan diabetes rats

Group n MDA, mmol/l
no blueberry 1 g/kg blueberry  2 g/kg blueberry 
1 Intact (standard) 7 5,68 ± 0,10 - -
2 Diabetes, 7 days 21 7,25 ± 0,07
р<0,001
6,26 ± 0,08
р<0,001
р1<0,001
5,39 ± 0,07
р<0,05
р1<0,001
3 Diabetes, 14 days 21 8,93 ± 0,09
р<0,001
6,12 ± 0,08
р<0,01
р1<0,001
4,21 ± 0,09
р<0,001
р1<0,001

Notes
1. p - confidence factor to compare with standard
2. p1 - confidence factor to compare with "no blueberry" group

Table 10. Blueberry paste effect on MDA content in the liver of alloxan diabetes rats

Group n MDA, mmol/l
no blueberry 1 g/kg blueberry  2 g/kg blueberry 
1 Intact (standard) 7 31,00±0,70  -  -
2 Diabetes, 7 days 21 36,70±0,58
р<0,001
28,17±0,41
р<0,05
р1<0,001
30,03±0,25
р>0,05
р1<0,001
3 Diabetes, 14 days  21 39,74±0,41
р<0,001
33,99±0,46
р<0,05
р1<0,001
36,04±0,19
р<0,01
р1<0,01

Notes
p - confidence factor to compare with standard
p1 - confidence factor to compare with "no blueberry" group

As the data show, the greatest lipid peroxidation takes place in the liver (31.0±0.7 mmol/kg), the smallest one being in blood serum (0.39±0.01 mmol/kg). In all cases we can observe increased MDA content, especially in the small intestine mucous membrane (57% greater on the 14th day of disease).
Introduction of blueberry paste causes considerable drop in MDA content, the greatest drop being observed in small intestine mucous membrane (more than twice on the 14th day, the dose being 2 g/kg).
Blueberry paste antioxidant effect depends on the dose and increases substantially with the dose increased twice.
Thus, greater MDA content under PD testifies to greater intensity of peroxidation process, which is considered an important pathogenetic factor in diabetes complications development [Balabolkin, 2006]. Lower levels of MDA content and, consequently, of peroxidation due to blueberry paste introduction attest its positive effect in treating diabetes.

Conclusion


The investigation performed gives grounds for the conclusion that dysbiosis caused by pancreatic diabetes starts proinflammatory reactions (IL-1, IL-6, TNFα cytokines participating) caused by microbial toxins (mainly lipopolysaccharide), exhausts nonspecific immunity, which is evidenced by lower lysozyme level, and, at the same time, it activates the peroxidation processes, which is confirmed by higher MDA content.
Biologically active substances in the blueberry paste (mainly polyphenols and chlorogenic acid especially) influence positively the processes provoked by microbial toxins in three ways, namely: blueberry paste polyphenols inhibit microbes growth (this is confirmed by decreased urease activity), restore nonspecific immunity (this is confirmed by increased lysozyme activity) and decrease considerably peroxidation intensity, lower MDA level testifying to it.
As is obvious from the foregoing, LiQberry paste is an effective remedy for diabetes and can be recommended as part of a patient's diet, being a preventive and therapeutic agent which necessitates further profound clinical research.
It should be noted that the therapeutic action of blueberry paste can be highly useful in treating other infectious and noninfectious diseases.

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