Download a formulation containing silk protein

A comparative study of Morning FreshTM (a formulation containing silk protein) in
prevention of alcohol hangover using an animal model
Radhakrishna Pallipadi*, Vidya Murugesan and Bharat Tandon
Sericare, Division of Healthline Private Limited, Bangalore, India -560064
*Email:[email protected]
Abstract:
Alcohol hangover is characterized by the combination of unpleasant physical and mental symptoms that occur after
a bout of heavy alcohol drinking. The acetaldehyde intermediate formed during metabolism of alcohol is believed
to be majorly responsible for hangover effects.
TM
The present study was designed for comparative evaluation of efficacy of Morning Fresh (MFH), a formulation
containing silk protein with glycine, serine (both amino acids predominantly found in silk protein) and antioxidants
(part of MFH), on the reduction of alcohol induced hangover in Sprague Dawley rats. Efficacy in managing
hangover syndrome was indirectly evaluated based on NAD/NADH ratio, ADH level, and blood ethanol level. MFH
was significantly superior to serine, glycine and antioxidant formulation, when compared to ethanol control, with
respect to decreasing NAD/NADH ratios, increasing ADH activity, decreasing blood ethanol concentration at two
hour post treatment.
Introduction:
The toxic effects of alcohol are directly related to the level of alcohol and its immediate metabolite acetaldehyde in
the plasma. Three important steps involved in understanding effects of alcohol after consumption is: its absorption
in the stomach and the small intestine, its distribution to other organs and its elimination by metabolism. It has
been established that practically all the alcohol absorbed is metabolized in the liver through oxidative process
1
mainly catalyzed by alcohol dehydrogenase (ADH), leading to toxic acetaldehyde. Acetaldehyde so formed gets
2
quickly oxidized to non-toxic acetate by mitochondrial acetaldehyde dehydrogenase (MADH) action. Physical
symptoms of a hangover include fatigue, headache, increased sensitivity to light and sound, redness of the eyes,
muscle aches, and thirst, tremor and sweating. Mental symptoms include dizziness and possible mood
3
disturbances, especially depression, and irritability. The acetaldehyde intermediate formed during metabolism of
alcohol is believed to be majorly responsible for hangover effects. Understanding pharmacokinetics of alcohol
metabolism clearly indicates that formation of acetaldehyde being rate determining, factors affecting this stage
need to be manipulated to arrive at quicker formation and elimination of acetaldehyde.
The turnover rate of ADH primarily depends on availability of nicotinamide adenine dinucleotide (NAD) in its
oxidized form NAD+. Formation of NAD+, in turn depends upon supply of reduced form NADH and resultant
equilibrium shift in the redox system. The supply of NADH from cytoplasm into mitochondria happens through
carrier oxalacetic acid originating from amino acids like serine and aspargine. Another bio-mechanism operative in
the liver is free-radical scavenging system involving glutathione peroxidase derived from tripeptide glutathione, a
4
combination of glutamic acid, cystine and lysine. There is also a report indicating role for amino acid glycine
2+
5
effectively blocking Ca in Kupffer cells and thus hastening repairing of alcohol induced liver injury. Yet, another
amino acid alanine is reported to promote alcohol metabolism in the liver thus causing rapid reduction of
6
concentration of alcohol in the blood. In addition, the role of anti-oxidant and free radical scavenging molecules in
liver protection is very well known which would further help in protecting the liver from the harmful effects caused
7
by ethanol. There are several patents and publications related to this subject. These findings involve use of
combination of vitamins like ascorbic acid, thiamine, vitamin B12, & folic acid, amino acid L- cysteine or its
derivative cysteic acid, L-glutamine, flavones and combination of amino acids with organic acids like fumaric acid &
succinic acid. There is a publication on protective effect of silk protein, sericin peptide against alcohol induced liver
4
injury in mice. Keeping some of these points in view we thought of using a formulation MFH containing silk
protein containing key amino acids as discussed above, like serine, lysine, glycine, alanine, aspargine, glutamic acid,
cystine along with antioxidants like ascorbic acid and mulberry leaf extract in order to assess alcohol hangover
reduction effect in comparison to amino acids like serine, glycine found in silk protein and antioxidants, similar in
concentration and composition to MFH using a rat model.
Materials & Methods:
MFH and antioxidant formulation containing combination of ascorbic acid and mulberry extract was provided by
Healthline Private limited, and glycine, serine and ethyl alcohol obtained from reputed chemical manufacturers
and prepared in suitable dilution using purified water. Rats from strain Sprague Dawley was obtained from In vivo
biosciences, Bangalore. The animal study and subsequent biochemical parameters measurement, statistical
analysis and final report was prepared by Vipragen Biosciences, Mysore (Institutional ethics Committee
8,9,10
registration number 1683/RO/c/13/CPCSEA) as per the standard protocols.
Animals were dosed orally with
the test items as shown in the experimental design (Refer Table 1). Blood samples (0.4 mL) was collected in 2 mL
Eppendorf tubes containing 0.010 mL (10 uL) of 10% K2EDTA through retro orbital plexus puncture under
0
Isoflurane anaesthesia at 0 min, 30 min, 1h, and 2h post administration. Plasma was harvested and stored at -20 C.
Experimental Design: Table 1
Group
1
2
Test compounds
Control
Ethanol treatment
3
Ethanol & serine
4
Ethanol & glycine
5
6
Ethanol &
antioxidants
Ethanol & MFH
Number of animals per group is 4.
Treatment (Dose: ml/kg)
Vehicle-Purified water 20 ml
47.5% ethanol 6 ml + Purified water 14 ml
47.5% ethanol 6 ml immediately followed by serine administration
(0.47g in 14ml purified water)
47.5% ethanol at 6 ml immediately followed by glycine administration
(0.21g in 14 ml purified water)
47.5% ethanol at 6 ml immediately followed by antioxidants
administration (0.06g in 14 ml purified water)
47.5% ethanol at 6ml immediately followed by MFH 14 ml
11.12
Determination of NAD/NADH levels
The reaction mixture containing 40 µM NAD, 10 µL rat plasma and 750 µM Tris-HCl (pH 9.0) buffer was incubated
at 25°C and absorbance was measured at both 260 nm for NAD and 340 nm for NADH at 0 minute and 15 minutes.
The concentration of NAD and NADH were estimated at 15 min using calibration curves for NAD and NADH. The
NAD/NADH ratio was calculated by considering ratio for control animal as 1.0. (Ref, figure 1 and table 2)
13.14
Determination of alcohol dehydrogenase (ADH)
The reaction mixture (90 µl) containing 50 µM NDMA, 40 µM NAD +, 750 µM Tris-HCl (pH 9.0) and 10 µl of plasma
0
was incubated at 25 C and absorbance was recorded at 0 minute and 30 minutes at 440 nm. The change in the
absorbance was used to calculate ADH activity using a NDMA calibration curve. (Ref, figure 2 and table 3)
15
Determination of blood ethanol concentration
0
The reaction mixture containing 40 µM NAD, 10 µl plasma in 750 µM Tris-HCl (pH 9.0) was incubated at 25 C, and
absorbance was recorded at 340 nm at 0 and 5 minutes. A calibration curve was constructed for ethanol by adding
40 µM NAD to 50 µl of ADH (300 U/ml) in presence of various concentration of ethanol and absorbance was
measured at 340 nm at 0 and 5 min. (Ref, figure 3 and table 4)
Ethanol (mg/dL) =A340 Sample * concentration of ethanol standard
A340 Standard
Present assay was based on the NADH to determine the ethanol concentration in the blood. As the vehicle control
animals will also show some basal level of NADH production, the basal level NADH values of vehicle control were
deducted from all the treatment groups respectively and constructed/tabulated the graphs and tables.
Data compilation and statistical analysis:
Statistical analysis was performed using Graphpad Prism. The mean (± SD) values for each concentration were
subjected to one-way analysis of variance (ANOVA) followed by Dunnett's Multiple Comparison Test. P <0.05 were
chosen as the criterion for statistical significance.
Results:
Measurement of NAD/NADH levels:
Hours
Table 2: Total NAD/NADH (μ M) in different groups
Ethanol
Ethanol &
Ethanol &
Ethanol &
control
serine
glycine
antioxidants
Control
*#
3.24±0.22
*
2.29±0.23
0.5
1.00±0.15
1.0
1.00±0.16
*
#
2.32±0.28
#
1.29±0.44
#
*
2.61±0.31
*
1.43±0.03
#
#
2.54±0.54
*
1.37±0.43
#
Ethanol &
MFH
*
1.95±0.33
*
*
1.16±0.23
*#
1.06±0.18
*
*
Total NAD/NADH(μ M)
2.0
1.00±0.17
2.90±0.43
2.91±0.28
2.40±0.49
2.03±0.28
Results are expressed as mean ± SD.
*
#
P <0.05 compared to ethanol control; P <0.05 compared to ethanol & MFH
3.5
3
2.5
2
1.5
1
0.5
0
Control
Ethanol control
Ethanol & serine
Ethanol & glycine
Ethanol & antioxidants
0.5
1
2
Ethanol & MFH
Hours
Fig. 1 NAD/NADH Ratio in different groups
Determination of alcohol dehydrogenase (ADH):
Table 3: ADH levels (μ mol/ml) in different groups
Ethanol
Ethanol &
Ethanol &
Ethanol &
control
serine
glycine
antioxidants
Hours
Control
0.5
12.14±2.42
#
19.74±4.06
1.0
13.75±4.70
#
18.03±4.38
#
#
32.81±4.34
31.9±2.89
#
27.28±4.75
30.82±8.07
#
*
*
*
28.23±11.16
28.70±5.65
#
Ethanol &
MFH
*
40.18±5.41
*
35.43±6.66
*
2.0
14.22±5.97 15.37±11.03
25.16±9.07
17.42±5.90
25.43±2.62
30.28±1.99
*
#
Results are expressed as mean ± SD. P <0.05 compared to ethanol control; P <0.05 compared to
ethanol & MFH
ADH levels μmol/ml
50
40
30
20
10
0
Control
Ethanol control
Ethanol & serine
Ethanol & glycine
0.5
1
Ethanol & antioxidants
2
Ethanol & MFH
Hours
Fig. 2 ADH levels in different groups
Blood ethanol concentration:
Ethanol concentration
mg/dL
Table 4: Ethanol concentration (mg/dL) in different groups
Ethanol
Ethanol &
Ethanol &
Ethanol &
Ethanol &
Hours
Control
control
serine
glycine
antioxidants
MFH
*#
#
*
*
0.5
23.31±2.87
229.1±47.26
133±15.55
151.6±43.5
167.3±59.03
112.8±40.5
*#
#
*
*
*
1.0
29.66±8.24
238.3±34.56
214.5±103.6
125.2±39.67
136.7±39.99
134.5±24.39
*
#
*
2.0
30.76±24.95
288.4±129.3
223.4±109.1
279.7±61.76
243.4±68.18
128.6±10.21
*
#
Results are expressed as mean ± SD. P <0.05 compared to ethanol control P <0.05 compared to ethanol & MFH
350
300
250
200
150
100
50
0
Control
Ethanol control
Ethanol & serine
Ethanol & glycine
Ethanol & antioxidants
0.5
1
2
Ethanol & MFH
Hours
Fig. 3 Ethanol concentration in different groups
Discussion:
In accordance with literature cited earlier, free amino acid treatment of glycine showed significant decrease in the
total mean NAD/NADH ratios compared to ethanol control at 1 hour post treatment, significant increase in ADH
values compared to control at 0.5 hour post treatment and exhibited significant decrease in blood alcohol level at
1 hour post treatment. Similarly, serine showed significant decrease in NAD/NADH ratios at 0.5 and 1 hour post
treatment, significant increase in ADH values at 0.5 hour post treatment and significant decrease in blood alcohol
level at 0.5 hour post treatment. These results confirms bio-chemical role of serine and glycine for faster oxidation
of alcohol in the system, but also indicates its limitation of short term activity. Though antioxidants showed a
significant decrease in the mean NAD/NADH ratios compared to ethanol control in all the time points, there was
no significant increase in ADH level throughout. Antioxidants showed significant decrease in blood alcohol level
only at 1 hour post treatment. MFH containing proteins having glycine, serine, alanine, aspargine, lysine and
glutamic acid & anti-oxidants showed a significant decrease in the mean NAD/NADH ratios, significant increase in
ADH values compared to ethanol control at 0.5, 1 and 2 hour post treatment. MFH also showed a significant
decrease in blood alcohol level in all the time points. These findings confirms requirement of slow release of active
amino acids for longer duration effect, presence of synergistic effects resulting from various amino acids and antioxidants in bringing about significant reduction in alcohol hangover effect. It also shows that silk proteins are
better than individual amino acids for sustained management of alcohol hangover syndrome.
Conclusions:
In the rat model of ethanol induced hangover, MFH was significantly superior to serine, glycine and antioxidants,
when compared to ethanol control, with respect to decreasing NAD/NADH ratios, increasing ADH activity,
decreasing blood ethanol concentration from 0.5 to 2 hour post treatment.
Note: The findings reported in this paper is part of patent applications filed with competent authorities.
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