Current Research in Food Science 1 (2019) 8–16

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Current Research in Food Science

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Research Paper

Influence of food commodities on hangover based on alcohol

dehydrogenase and aldehyde dehydrogenase activities
Shraddha Srinivasan, Kriti Kumari Dubey *, Rekha S. Singhal
Food Engineering and Technology Department, Institute of Chemical Technology, Matunga, Mumbai 400019, India

A R T I C L E I N F O A B S T R A C T

Keywords: Alcohol consumption often leads to hangover, a condition characterized by several symptoms, characteristically
Fruit juice blends headache, nausea, fatigue and drowsiness. Hangover may be alleviated by altering the rate of alcohol metabolism
Alcohol dehydrogenase (ADH) and facilitating elimination of acetaldehyde by affecting the activity of alcohol dehydrogenase (ADH) and/or
Aldehyde dehydrogenase (ALDH)
aldehyde dehydrogenase (ALDH) enzymes. In the present study, several food commodities like fruits, vegetables,
Anti-hangover formulation
Kinetics
cereals, pulses, dairy products, spices and other miscellaneous products (ascorbic acid, cocoa sample, tea, coffee,
egg yolk and date samples) were investigated for their effect on the in vitro activities of the enzymes and their
antioxidant properties. Of the many screened food commodities, few showed an increase in the activity of either
one or both the enzymes, ADH and ALDH. Studies showed no correlation between ADH and ALDH enzyme ac-
tivities and antioxidant property of the selected food commodities for anti-hangover effect. Further, an anti-
hangover (AHO) product was developed using pear (65%), sweet lime (25%) and coconut water (10%) and,
validated for in vitro ADH and ALDH enzyme activities. AHO product was found to enhance ADH and ALDH
activities by 23.31% and 70.02%, respectively.

1. Introduction dinucleotide (NADþ) serves as the intermediate electron carrier for ADH,
and it gets reduced to nicotinamide adenine dinucleotide-reduced
Hangover or veisalgia is the term that refers to the psychological and (NADH). The increase in the NADH causes oxidative stress in the liver
physiological effects following alcohol consumption. It is characterized which also contributes to the symptoms exhibited during the phase of
by an unpleasant and uneasy feeling that includes, but is not limited to, hangover. Acetaldehyde built-up has been attributed as major causative
headache, fatigue, drowsiness, nausea, and in some cases, vomiting factor of hangover (Zakhari, 2006). The alleles of the ADH and ALDH
(Swift and Davidson, 1998). The symptoms set in when the concentration form the genetic basis for the manifestation of hangover. The genetic
of ethanol in the blood reaches zero, implying that it is the metabolism or effect is predominantly seen among the racial populations (Cederbaum,
the metabolite that leads to this effect. The effects of hangover are 2012; Quertemont, 2004). The isozymes of ADH and ALDH have
attributed to several causes, the major ones being due to i) the direct different affinity for their substrates, and thus the genetic makeup plays a
effects of alcohol, ii) metabolism of alcohol, iii) other non-alcohol factors major role in determining the rate at which an individual will eliminate
such as the presence of congeners (biologically active molecules present ethanol from the system.
in alcohol, apart from ethanol, for example, methanol or ethyl formate), Hangover poses a considerable threat at the individual level, where
and iv) genetic makeup. Ethanol consumption affects specific organs or along with the occurrence of unpleasantness there is also a risk of health
systems such as the brain, gastrointestinal tract, liver and immune system issues that might impact everyday functioning. It also affects the econ-
(Swift and Davidson, 1998) manifesting as symptoms that are charac- omy due to a decrease in the productive work caused by sleep depriva-
teristic of a typical hangover. tion, loss of attentiveness and alertness (Cederbaum, 2012). Several
During metabolism of ethanol, it gets broken down either through the natural compounds like 6-gingerol (Takahashi et al., 2010), dehy-
oxidative or non-oxidative pathway (Zakhari, 2006). During oxidative droevodiamine (Wang et al., 2016a), ginsenosides (Kim et al., 2011),
metabolism of alcohol to acetate, ethanol is converted to acetaldehyde by linolenic acids (Lee et al., 2014a) among many others, have been rec-
the hepatic alcohol dehydrogenase (ADH). Nicotinamide adenine ommended as a cure for hangover. Researchers have investigated and

* Corresponding author.
E-mail address: Kritibiotech@gmail.com (K.K. Dubey).

https://doi.org/10.1016/j.crfs.2019.09.001
2665-9271/© 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
S. Srinivasan et al. Current Research in Food Science 1 (2019) 8–16

identified natural components (such as polyphenols) from various food properties were aimed at so as to test for their effect on the hepatic en-
sources such as fruits, mushroom, and herbs to alleviate hangover zymes and also to check for their antioxidant activity.
symptoms (Zhao et al., 2017; Zhang et al., 2016; Kim, 2016; Bajpai et al.,
2016). These natural food sources are reported to function effectively by 2.3.2. Dairy products
exhibiting positive effects on the hepatic enzymes, i.e. by either Extracts of milk and cheddar cheese were obtained by following the
enhancing the activity of ALDH and/or ADH and thereby assisting in protocol as per Pritchard (Pritchard et al., 2010) with modifications.
aldehyde and alcohol clearance from the system, respectively. A herbal Briefly, Cheddar cheese was homogenized in a ratio of 1:3 w/v with
mixture “DTS 20” containing Viscum album L. (40%), Lycium chinense L. deionized water and was subjected to magnetic agitation at 40  C for 1 h.
(30%), Inonotus obliquus (20%), and Acanthopanax senticosus H. (10%) The contents were then centrifuged at 4250 g at 4  C for 30 min. The
has also been studied and found to reduce the oxidative stress and plasma supernatant was filtered through Whatman filter paper (No. 42) followed
alcohol concentrations (Hong, 2015). by membrane filtration (pore size 0.22 μm). Diluted samples (1:1 v/v) of
While pharmaceutics have also gained momentum in the search to buttermilk and commercial probiotic product were used after filtration
find a cure for hangover, the identification of natural compounds will through a membrane of pore size 0.45 μm. Samples were stored at 20  C
target the implementation of dietary modifications to ensure better re- in deep freezer and used for assays within two days.
sults. This research work aims at studying the influence of food com-
modities on ADH and ALDH enzyme activities and their kinetic 2.3.3. Miscellaneous
properties that could further help to identify specific components to Ascorbic acid was used at the concentration of 2 mg/mL for various
formulate an effective natural AHO product. Few studies have observed assays. Cocoa sample (1:100) was prepared in 80% methanol solution
that sugars like glucose and fructose do alleviate hangover by facilitating with stirring (1 h) followed by solvent evaporation and resuspension in
alcohol detoxification. However, these do not result through the action of deionized water. Tea and coffee (0.1 g) were brewed with 8 mL boiling
the hepatic enzymes (ADH and ALDH) and are a separate entity for deionized water for 2 min (Floegel et al., 2011) and filtered through
research scope. Whatman filter paper and membrane filtration (0.45 μm membrane). For
egg yolk, sample preparation was done as per the method reported by Su
2. Materials and methods et al. (2015). Egg yolk was separated from egg white and beaten with
addition of 95% ethanol in 1:10 ratio. The contents were then mixed in a
2.1. Sample procurement magnetic stirrer at 65  C for 1 h and filtered, the process was repeated
twice. The ethanol fractions were pooled and ethanol was evaporated.
The fruits, vegetables, cereals, pulses, dairy products, spices and other Deionized water was added to the residue and the sample was assayed
miscellaneous products were purchased from the local market of Mumbai within two days. Date sample was prepared by measuring 1 g of seedless
city, India. The food commodities used are listed in Table 1. dates and mixing it in 10 mL of deionized water for 48 h under stirring
conditions. The contents were then mixed in a mixer and centrifuged at
2.2. Chemicals and reagents 4000 rpm (Remi Centrifuge, REMI- CRP-30, Mumbai, India) at 4  C for
20 min. The supernatant was collected further and used for assays
2,2 Diphenyl-1-picrylhydrazyl (DPPH), ADH and ALDH were pro- (Zangiabadi et al., 2011). All the processed samples were stored at
cured from Sigma-Aldrich, Bangalore, India. Bovine serum albumin 20  C until the assays were performed.
(BSA), β-mercaptoethanol, potassium chloride, gallic acid, β-nicotin-
amide adenine dinucleotide (NADþ) and NADH were purchased from
2.4. Enzymatic assays for screening of food commodities
HiMedia Laboratories, Mumbai, India. Ethanol, methanol, and acetal-
dehyde were purchased from Merck Ltd, Mumbai, India.
2.4.1. Determination of ADH activity
The ADH activity was determined according to the protocol reported
2.3. Preparation of samples
by Lee et al. (2012) with slight modifications. The following solutions
were prepared: 1 M Tris HCl (adjusted to pH 7.5 with 1M HCl at 25  C),
2.3.1. Fruits, vegetables, spices and cereals
freshly prepared 3 mM β NADþ, 12.5 mM ethanol solution, 0.5% Triton X
The fruits and vegetables were washed, cleaned and dried. The fruit/
vegetable juices were extracted by grinding/pulping them in a mixer/ Table 1
grinder and filtered through Whatman filter No. 2 as samples for further List of food commodities screened for AHO formulation.
experiments. Spices were ground to size 0.25 mm and extracted in a 1%
Fruits
(w/v) solution of 60% methanol using magnetic stirrer for 1 h. The ex- Carambola/starfruit (Averrhoa carambola), orange (Citrus sinensis L.),
tracts were filtered through Whatman No. 1 filter paper, dried and gooseberry (Phyllanthus emblica), black grapes (Vitis amurensis), green grapes
resuspended in deionized water at a concentration of 100 mg/mL (Sub- (Vitis vinifera), pitahaya/dragonfruit (Hylocereus undatus), pear (Pyrus communis),
baraj et al., 2016). Cereals and pulses samples were prepared by Apple (Malus pumila), sweet lime (Citrus limetta), mango (Mangifera indica),
kiwifruit (Actinidia deliciosa), pineapple (Ananas comosus), papaya (Carica papaya),
following the method as per Meneses et al. (2013) with some modifica-
lemon (Citrus limon L.), watermelon (Citrullus lanatus), pomegranate (Punica
tions. Cereals and pulses were milled to a particle size of 0.25 mm and granatum)
stored in refrigerator prior to sample preparation. The sample was pre- Vegetables
pared by taking 10% w/v solution of the cereals or pulses in 60% Garlic (Allium sativum), cucumber (Cucumis sativus), coriander (Coriandrum sativum),
fenugreek leaves (Trigonella foenum-graecum), spinach (Spinacia oleracea),
methanol and subjected to magnetic agitation for 30 min at 60  C. The
bitter gourd/bitter melon (Momordica charantia), carrot (Daucus carota), Onion
obtained extracts were then filtered, dried and resuspended in a similar (Allium cepa), tomato (Solanum lycopersicum)
fashion as the spice samples. All the fruits, vegetables, spices and cereal Cereals
samples were stored at 20  C and used for various assays within two Sorghum (Sorghum sp. bicolor), oats (Avena sativa), wheat (Triticum aestivum), maize
days. Fruits and vegetables were used directly as juices since the con- (Zea mays), peanuts (Arachis hypogaea), millet (Pennisetum glaucum)
Spices
stituents are generally homogenized and most of the bioactive com-
Black pepper, cassia, cinnamon, cloves, ginger, cumin (jeera), mace, nutmeg, turmeric
pounds are available for the reaction. However, for spices the Milk Products
components need to be extracted using suitable solvents so as to make Butter-milk, cheese, milk, a commercial probiotic drink
them available for the reaction. Similarly, dairy products were diluted in Miscellaneous
Ascorbic acid, black tea, cocoa, coconut water, coffee, dates, egg white egg yolk,
order to prevent interference from particulate matter. In the current
fenugreek seeds, green tea
study, extraction of the components that would also exhibit antioxidant

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S. Srinivasan et al. Current Research in Food Science 1 (2019) 8–16

100, 100 mM Tris HCl buffer with 0.02% (w/v) BSA at pH 8.0, 20 U/mL 3. Theory/calculation
ADH (prepared in buffer) and 1 mL of the test sample extract. The in vitro
activity of ADH was measured as NADH formed, determined spectro- An AHO product was developed based on the effect of the sub-
photometrically at 340 nm using an ELISA plate reader (BioTek ELx808, stances on ADH activity, ALDH activity and organoleptic accept-
Mumbai, India). The reaction was started by addition of ethanol to a ability. D-optimal mixture design was applied to find the optimal
pre-incubated reaction mixture containing the remaining solutions and response for any mixture of the selected substances, and to obtain
made-up to a final volume of 300 μL. The reaction was carried out at the influence on the response of the combination of substances.
25  C and pH of 7.5. The control reaction was carried out in the absence Percentage of components; pear (A), sweet lime (B) and coconut
of any test sample. A blank reaction (in the absence of the substrate) was water (C) were the three independent parameters. The dependent
also measured and values were corrected with respect to the baseline in variables selected as the response was overall average acceptability
order to eliminate the effect of any artefacts. score given by the sensory panel comprising of 15 panellists (8 fe-
males and 7 males in the age group of 22–28 years). The scoring
2.4.2. Determination of ALDH activity was based on 9-point hedonic scale as follows: 9-like extremely; 8-
The ALDH activity was also determined according to the protocol like very much; 7-like moderately; 6-like slightly; 5-neither like nor
mentioned by Lee et al. (2012) with slight modifications. The following dislike; 4-dislike slightly; 3-dislike moderately; 2-dislike very much;
solutions were prepared: 1 M Tris HCl (adjusted to pH 8.0 with 1M HCl at 1-dislike extremely.
25  C), freshly prepared 3 mM β NADþ, 100 mM acetaldehyde solution, The statistical software package Design Expert 7.0 (Stat-Ease Inc.,
3 M KCl, freshly prepared 1 M β-mercaptoethanol solution, 100 mM Tris Minneapolis, MN, USA), was used for the experimental design and data
HCl buffer with 0.02% (w/v) BSA at pH 8.0, 0.1 U/mL ALDH (prepared in analysis. All the analyses were carried out in triplicates and the data
buffer) and 1 mL of the test sample extract. The in vitro activity of ALDH generated has been reported in terms of mean  standard deviation (SD).
was measured as NADH formed, determined spectrophotometrically at Data were assessed by ANOVA (Analysis of Variance) to determine the
340 nm using an ELISA plate reader (BioTek ELx808, Mumbai, India). significance of difference observed in the samples. The significance for
The reaction was started by addition of acetaldehyde to a reaction the mean difference for all the data generated and reported was
mixture of the other solutions and made-up to a final volume of 300 μL. compared using Duncan's multiple comparison test (p < 0.05).
The reaction was carried out at 25  C and pH of 8.0. The control reaction
was carried out in the absence of any test sample. A blank reaction (in the 4. Results and discussion
absence of the substrate) was also measured and values were corrected
with respect to the baseline in order to eliminate the effect of any arte- Food samples necessarily enhancing the ADH and/or ALDH are
facts. The percent activity of ADH and ALDH was calculated by the suitable for AHO formulation.
formula.

ðNADH formed in sample reaction  NADH formed in control reactionÞ

Percent enzyme activity ¼
NADH formed in sample reaction

2.5. Antioxidant potential 4.1. Effect of fruits and vegetables on alcohol dehydrogenase (ADH)
activity and aldehyde dehydrogenase (ALDH) activity
Antioxidant property of a substance was assayed by the 2,2-Diphenyl-
1-picrylhydrazyl radical (DPPH) scavenging assay (Ramadan et al., Fruits and vegetables that decrease the ALDH activity are not suit-
2008) using a spectrophotometer at 517 nm. Gallic acid was used as a able as an AHO substance. The effect of fruits and vegetables on the
standard. The percentage inhibition was calculated using the formula percent difference in activities of ADH and ALDH is shown in Table 2.
given. Grapes, dragon fruit and gooseberry did not significantly affect the
activity of ADH while starfruit and orange significantly decreased the
ðOD of control  OD of sampleÞ activity of ADH. In the present work, the high percentage activity dif-
Percent radical scavenging activity ¼
OD of control ference between the watermelon sample reaction and control reaction
The antioxidant capacities for the samples were reported as gallic acid indicates a positive effect of watermelon in enhancing the activity of
equivalent (GAE). ADH by 67.22%. Grapes did not alter the ALDH activity, while goose-
berry decreased it. Studies performed by Zhang et al. (2016) also
confirmed the ability of starfruit to decrease the ADH activity, though
2.6. Kinetic studies
not very significantly. A commercially available AHO product “Par-
tySmart” containing grapes and gooseberry as ingredients has been
The activation kinetics of the samples that positively affected the
reported to have a positive effect on both ADH and ALDH (Pordie,
activity of ADH and ALDH were studied and the apparent Km value was
2015). Wang et al. (2016b) observed fresh orange to decrease the ADH
calculated.
activity. Similarly, mango pulp has also been reported to enhance the
ADH activity in rats by 129.76% (Kim et al., 2011). However, Zhang
2.7. Correlation between antioxidant property and enzyme activity
et al. (2016) did not find any significant difference in the ADH activity
by watermelon and lemon, Actinidia chinensis (closely related to kiwi
The correlation between ADH activity, ALDH activity and antioxidant
fruit). While the difference in the reported results from that of the
property of the samples were determined by plotting a scatter plot and
present study can be attributed to variations in the samples used, it
calculating the R2 values.

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S. Srinivasan et al. Current Research in Food Science 1 (2019) 8–16

Table 2 activity of ALDH, while the other samples such as buttermilk, cheese and
Effect of fruits and vegetables on ADH and ALDH activities. a commercial prebiotic drink showed an increase in the activity of the
Food commodities ADH activity (%) ALDH activity (%) ALDH. In addition to the ability to enhance the activity of the enzyme,
this effect could be attributed to the presence of microorganisms in the
Fruits
Malus pumila (apple) 28.06  0.95i 76.15  1.46b samples that are able to produce ALDH and thereby enhance the rate of
Mangifera indica (mango) 31.74  1.65i 61.97  1.08c product formation. Lactobacillus sp. fermented cream cheese has been
Citrullus lanatus (watermelon) 67.22  2.81m 57.39  1.38c reported as an AHO product due to its ability to increase the rate of
Carica papaya (papaya) 46.50  3.73k 50.55  2.24d acetaldehyde metabolism (Konkit et al., 2016). Moreover, the presence of
Vitis amurensis (black grapes) 1.36  0.26de 33.44  2.03fg
Phyllanthus emblica (gooseberry) 2.73  0.22d 31.42  2.20g
several functional peptides in these fermented products may contribute
Ananas comosus (pineapple) 39.28  2.98j 30.35  3.06g to the modified effect on the enzymes.
Punica granatum (pomegranate) 67.97  6.95m 22.48  4.75h All the cereal samples decreased ADH and ALDH activities.
Actinidia deliciosa (kiwi fruit) 38.80  6.29j 20.09  4.48h While maize and wheat had no significant effect on the activity of
Citrus limon (L.) (lemon) 47.39  8.76k 19.59  4.12h
ALDH, the other samples analysed, showed a decrease in the activity of
Hylocereus undatus (dragon fruit) 4.57  0.38e 3.06  1.44j
Vitis vinifera (green grapes) 3.02  0.16def 0.20  0.02j ALDH. This suggests that it is not advisable to include cereals and
Citrus sinensis (L.) (orange) 8.54  1.91c 15.48  5.16l pulses in the diet post consumption of alcohol until the hangover has
Averrhoa carambola (starfruit) 13.39  1.18ab 22.76  9.26i subsided.
Citrus limetta (sweet lime) 29.64  7.36i 33.47  1.38n Among the spice samples, mace, turmeric and ginger increased ADH
Pyrus sp. (pear) 22.11  3.21h 90.98  1.96p
Vegetables
activity. However, a commercial AHO product with turmeric as its
Allium sativum (garlic) 44.11  4.32a 42.23  2.72e principal component showed a decrease in the activity of ADH. The
Cucumis sativus (cucumber) 18.37  2.26b 87.25  1.55p product also contained Vitamin C as one of its ingredients. The activity of
Coriandrum sativum (coriander) 12.36  0.70c 58.38  12.20b ALDH was not significantly altered by cumin. Other spice samples
Trigonella foenum-graecum 2.62  0.58def 6.43  1.53ij
showed a significant decrease in the activity of ALDH. The commercial
(fenugreek leaves)
Spinacia oleracea (spinach) 6.10  0.72f 38.94  2.27ef AHO product was found to decrease the ALDH activity. So far, research
Momordica charantia 14.42  1.87g 1.59  0.16j work on effect of spices on ADH and ALDH have not been elaborately
(bitter gourd/bitter melon) carried out and hence further studies (especially in vivo) need to be un-
Daucus carota (carrot) 29.33  2.45i 10.76  3.55i dertaken to establish these results.
Allium cepa (Onion) 48.25  3.51k 2.24  0.90k
Among the miscellaneous samples, fenugreek seeds, egg white, green
Solanum lycopersicum (tomato) 57.25  2.79l 41.19  6.37
Control 0.00def 0.00  0.00jk tea, black tea, and dates enhanced the activity of ADH. Cocoa and
Commercial anti-hangover product 11.64  1.49c 82.50  0.57a

Values are mean  SD of three determinants. Table 3

Different alphabet in superscript represent that the values are significantly Effect of dairy products, cereals, pulses, spices and other miscellaneous food
different (p < 0.05). products on ADH and ALDH activity.
Food commodities ADH activity (%) ALDH activity (%)

could also be that in vitro and in vivo studies have yielded different Dairy products
results. Milk 10.75  2.04i 3.93  0.55
Buttermilk 4.01  2.15j 17.50  0.97r
Pear showed the highest positive effect on ALDH activity at 90.98%.
A commercial probiotic drink 4.04  1.96j 24.23  3.14s
Orange, starfruit, and sweet lime also enhanced the ALDH activity Cheddar cheese 97.31  6.25p 18.63  2.34r
significantly by 15.48%, 22.76% and 33.47%, respectively. The potential Cereals and pulses
use of pear to alleviate hangover has been reported in previous studies Avena sativa (oats) 24.83  1.53de 76.81  1.15d
based on their effect on the activity of the enzyme (Lee et al., 2012). Arachis hypogaea (peanuts) 15.06  1.44gh 66.41  3.78e
Pennisetum glaucum (millet) 1.74  0.58jk 47.13  3.50h
Enhanced ALDH activity of pear facilitates in faster elimination of acet- Sorghum sp. (sorghum) 26.70  4.24d 32.16  3.11jk
aldehyde and thus it serves as a suitable AHO. Zhang et al. (2016) re- Zea mays (maize) 14.38  2.01ghi 0.51  0.13op
ported that orange and starfruit showed a decrease in the activity of Triticum sp. (wheat) 17.71  1.13fg 0.32  0.21opq
ALDH (by 11.81% and 61.95%, respectively) while dragon fruit did not Spices
Pepper 67.41  2.95b 34.08  1.67ijk
significantly affect the ALDH activity. Wang et al. (2016b) also reported
Cloves 67.15  2.06b 60.81  5.58f
fresh orange juice to decrease the ALDH activity. This variation could be Nutmeg 46.21  2.87c 97.89  7.81a
due to the differences in the sample nature and sample preparation Cumin 23.56  0.80de 3.54  2.23
methods. The presence of polyphenols in the fruits has been reported to Cinnamon 21.38  1.88ef 61.16  2.02f
enhance the activity of ADH and ALDH (Lee et al., 2012). However, the Cassia 18.13  2.01fg 21.06  4.61n
Mace 13.26  1.67m 82.48  4.09c
exact mechanism is yet to be elucidated. Turmeric 10.73  2.28m 23.79  1.28mn
Among the vegetable samples analysed, bitter gourd and carrot Ginger 20.04  1.41n 35.96  1.44ij
showed increase in ADH activities and decrease in ALDH activities. To- Miscellaneous
mato and cucumber samples showed an increase in the activity of ALDH Vitamin C 88.77  0.65a 88.97  3.97b
Coffee 42.75  3.42c 53.44  2.85g
(41.19% and 87.25%). Investigators have reported that heat treated
Egg yolk 13.15  1.41hi 38.93  6.78i
cucumber had the ability to enhance the activity of ALDH and ADH Coconut water 0.23  0.01jkl 13.95  1.36r
(Bajpai et al., 2016). Contrary to this result, our work showed a signifi- Cocoa 0.75  0.31kl 29.04  2.08kl
cant decrease of ADH activity by cucumber. This could be due to dif- Black tea 2.87  0.20l 5.37  0.26q
ferences in the variety as well sample preparation methods. Dates 13.55  1.15m 26.77  2.26lm
Green tea 11.56  1.46m 2.64  0.36pq
Egg white 25.50  1.41 3.47  1.20
4.2. Effect of dairy products, cereals, pulses, spices and other miscellaneous Fenugreek seeds 86.79  2.04p 62.40  2.50ef
food products on ADH and ALDH activity Control 0.00kl 0.00  0.00opq
Commercial anti-hangover product 11.64  1.49hi 82.50  0.57c
The effect of dairy products, pulses, spices, and other miscellaneous Values are mean  SD of three determinants.
food products on ADH and ALDH activities are shown in Table 3. Dairy Different alphabet in superscript represent that the values are significantly
products like milk showed a marginal but significant decrease in the different (p < 0.05).

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S. Srinivasan et al. Current Research in Food Science 1 (2019) 8–16

coconut water did not significantly alter the activity of ADH. Ascorbic consumption could slower the rate of ethanol elimination, and hence it
acid significantly decreased the activity of the ADH to a very large should not be preferred during hangover. Egg white showed enhanced
magnitude. Most of the commercial products are marketed based on their ADH activity, while egg yolk showed a decrease in the ADH activity.
antioxidant property and these are often rich in minerals and also contain Black tea did not significantly affect the ADH activity (2.87%) while
a mixture of components such as polyphenols, flavonoids and other green tea showed a marginal increase in ADH activity (11.56%). These
bioactive components. In the current study, the AHO product tested did results for green tea and black tea are in agreement with Wang et al.
not show any positive effect on both the hepatic enzymes (ADH and (2016b). It was interesting to note that both green and black tea did not
ALDH). Thus, it is possible that there exists another mechanism of action significantly affect the activity of ALDH. The difference in the results
for this product. A substantial decrease in the ADH and ALDH activities exhibited by green and black tea may be probably due to the difference in
due to coffee indicates that consumption of coffee post alcohol their polyphenols and mineral contents.

100
80
60
40
20
0
-100 -50 -20 0 50 100 150

-40
-60
-80
-100 A

100
80
60
40
20
0
-20 0 20 40 60 80 100 120

-40
-60
-80
-100 B

150

100

50

0
0 20 40 60 80 100 120
-50

-100 C

Fig. 1. A) Correlation between alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activity, B) radical scavenging effect and ADH activity, and C)
radical scavenging effect and ALDH activity.

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Fenugreek seeds increased the activity of ADH significantly to 86.79%. resulting in prolonged hangover. It was observed that dates increased the
However, when fenugreek leaves were tested for their effect, no significant activity of ADH but decreased the activity of ALDH. Samples that were able
difference in the activities of ADH and ALDH was observed. Coconut water to enhance ADH and ALDH or only ALDH will be able to prevent acetal-
was found to enhance the activity of ALDH (13.95%), whereas egg white dehyde build-up, and thus may serve as an AHO.
did not show any significant effect on both ADH and ALDH activities. Other
samples decreased the activity of ALDH significantly. Coffee decreased the 4.3. Correlation between antioxidant property and enzyme activity
ALDH activity by a large magnitude; hence it is not advisable to consume
coffee post alcohol intake as it might lead to acetaldehyde build-up Selected food commodities were evaluated for antioxidant potential

Control Pear Linear (Control) Linear (Pear)

y = 211.7x + 16.911 y = 215.4x + 31.578
500

400

300
1/Vo (min-1)

200

100

0
-1 -0.5 0 0.5 1 1.5 2 2.5
-100
1/[S] (mM-1) C

Fig. 2. Lineweaver-Burk plot of A) aldehyde dehydrogenase (ALDH) catalyzed oxidation of ethanol in the absence (control) and presence of a modifier (sweet lime),
and B) alcohol dehydrogenase (ADH) catalyzed oxidation of ethanol, in the absence (control) and presence of a modifier (sweet lime) C) aldehyde dehydrogenase
(ALDH) catalyzed oxidation of ethanol in the absence (control) and presence of a modifier (pear), and D) alcohol dehydrogenase (ADH) catalyzed oxidation of ethanol,
in the absence (control) and presence of a modifier (pear) E) aldehyde dehydrogenase (ALDH) catalyzed oxidation of ethanol in the absence (control) and presence of a
modifier (coconut), and F) alcohol dehydrogenase (ADH) catalyzed oxidation of ethanol, in the absence (control) and presence of a modifier (coconut).

13
S. Srinivasan et al. Current Research in Food Science 1 (2019) 8–16

control pear Linear (control) Linear (pear)

y = 529.42x + 99.831 y = 326.49x + 54.269
700

600

500

400
1/Vo (min-1)

300

200

100

0
-0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 1.2
-100

-200 D
1/[S] (μM-1)
Control Coconut water Linear (Control) Linear (Coconut water)
y = 211.7x + 16.911 y = 224.44x + 20.408

500

400

300
1/Vo (min-1)

200

100

0
-1 -0.5 0 0.5 1 1.5 2 2.5
-100
1/[S] (mM-1)
E

control coconut water Linear (control) Linear (coconut water)

y = 529.42x + 99.831

y = 123.41x + 21.156
700
600
500
400
1/Vo (min-1)

300
200
100
0
-0.6 -0.4 -0.2 -100 0 0.2 0.4 0.6 0.8 1 1.2
F
-200
1/[S] (μM-1)

Fig. 2. (continued).

which is reported in supplementary file (Table S1-S6). The correlation to their antioxidant activity. A herbal formulation, "DTS20" has been
between ADH activity, ALDH activity and percent inhibition was deter- shown to prevent gastric mucous damage and also accelerate the meta-
mined from the correlation graphs (Fig. 1.). It is evident that there was no bolism of alcohol (Hong, 2015). Hence, it has been widely considered
correlation between ADH, ALDH, and antioxidant activities. The corre- that compounds having antioxidant property would have the ability to
lation between the hepatic enzymes, (ADH and ALDH) and the antioxi- enhance the activity of ADH and ALDH. However, from the results pre-
dant property was also found to be invalid. It has been reported sented in Fig. 1 (A,B,C), there were hardly any correlations between the
previously that a substance known to have high antioxidant activity can hepatic enzymes (ADH and ALDH) as well as between their activities and
serve as an AHO as it will be able to reduce the oxidative stress induced in the antioxidant properties of the samples. These results emphasize the
the body upon alcohol consumption. Asparagus officinalis (Kim et al., need to study the effect of food commodities on ADH and ALDH activities
2009) and red ginseng (Lee et al., 2014b) have been reported to have the and further use them to screen for suitable AHO ingredients rather than
ability to exert a positive effect on the activity of the hepatic enzymes due relying solely on the antioxidant properties of substances.

14
S. Srinivasan et al. Current Research in Food Science 1 (2019) 8–16

4.4. Effect of selected food constituents on the kinetics of ADH and ALDH viz. sweet lime, pear, and coconut water, were selected for the AHO
beverage formulation. Mango and watermelon were not selected for the
On the basis of results on enhancement of ADH and/or ALDH ac- beverage formulation despite their ability to enhance the activity of ADH,
tivities obtained in section 2.0, pear, sweet lime and coconut were neither of them enhanced the activity of ALDH. This suggests that while
selected for their effect on Km of these enzymes. The presence of sweet these fruit juices can facilitate metabolism of alcohol to acetaldehyde,
lime reduced the Km value for ADH (control) and ALDH (control) from they would not metabolize it further to acetic acid, both of which would
12.52 to 6.61 mM (Fig. 2A) and 5.30 μM to 2.62 μM (Fig. 2B), result in acetaldehyde build-up, and hence retention of hangover. The
respectively. In case of pear juice, the apparent Km value of ADH upper and lower limits for all three components were set according to
reduced from 12.52 mM to 6.82 mM (Fig. 2C), but did not alter the Km preliminary trials (based on sensory analysis)which were set as pear
of ALDH (Fig. 2D), while that of the control was 12.52 mM (Fig. 2C&D). (25%–65%), sweet lime (25–65%), and coconut water (10–25%). Sixteen
Coconut water showed a very marginal decrease in Km value for ADH formulations were generated with different composition of each ingre-
(Fig. 2E) and almost no change in the Km for ALDH activity (Fig. 2F). dient and evaluated randomly in terms of sensory perception (Fig. 3.).
The decrease in the Km value, correlated with the increase in ADH ac- The formulation containing comprising of pear (65% v/v), sweet lime
tivity of pear and sweet lime but not with coconut water. Similarly, the (25% v/v), and coconut water (10% v/v) showed best acceptability,
increase in activity of ALDH by pear and coconut could not be explained which was then compared with commercially available AHO (Table S7).
by the kinetic parameters, suggesting that there could be other mech- While the formulated AHO had a higher overall acceptability (7.8), it did
anisms that cause this effect. not differ much in terms of appearance and aroma with other formula-
Similarly, other food commodities were also tested for their effect tions in this set of experiments. The formulated AHO could enhance the
on kinetic parameters (data not shown) and it was observed that the activity of ADH by 23.31% and that of ALDH by 70.02%, thus validating
increase in enzyme activities could be correlated to kinetic parameters its use as an AHO product. These findings are in accordance with the
only for few cases suggesting other mechanisms through which the other investigators who have reported effective use of Korean pear in
activity of the enzyme may have altered. A few studies have reported alleviating hangover (Lee et al., 2013).
the inhibitory effect of certain food components on the kinetics of Recently, a variety of anti-hangover (AHO) products have been
ALDH. Durian fruit has shown to have a mixed type of inhibitory ac- launched into the market. “PartySmart” is a formulation containing
tion on yeast ALDH (Maninang et al., 2009), while quercetin has been grapes, gooseberry, date palm, Phyllanthus amarus, green chiretta
shown to be a non-competitive inhibitor for ALDH (Bhuiya et al., (Andrographis paniculata), and chicory, which is reported to significantly
2017). Further studies to ascertain the role of specific molecules in the reduce several of the hangover symptoms, and prevent formation of
food commodities that have activated ADH and ALDH evaluated in this acetaldehyde adducts. As a result, rapid elimination of acetaldehyde from
work need to be carried out. the blood occurs (Ramakrishna et al., 2005). While the exact mechanism
of the action by the product has not been elucidated, the study suggested
4.5. Formulation of anti-hangover (AHO) beverage the antioxidant property of the various ingredients in addition to the
positive effect on ADH and ALDH by few ingredients in the product
Anti-hangover effect can be expected when there is an increase in the (Venkataranganna et al., 2008). Similarly, a product named “Oh!K”
ADH and/or ALDH activities. Further, the rate of conversion of acetal- containing turmeric, ginger, black pepper, green tea extracts has also
dehyde to other metabolites must be higher than the rate of conversion of been formulated. Studies have shown that this drink is an effective
alcohol to acetaldehyde. The results obtained in this study provide an remedy to treat alcoholic hangover by replenishing the body with the
insight into the dietary components that can be used to prepare an AHO vital nutrients that are lost as a result of hangover (Gopi et al., 2014).
formulation. Another product, “DotShot” containing curcumin as the principle
A beverage formulation is suitable among the various food types. component and other vital electrolytes, has also been reported to
Hence preliminary efforts were made to incorporate fruit (sweet lime, enhance the ALDH activity and thereby assist in the breakdown of
pear, and coconut water) and vegetable (cucumber and tomato) juices in acetaldehyde (Harisha, 2018). "LIVitup" is an AHO tablet containing
a beverage formulation. However, all formulations containing vegetable kalmegh ghan, a mixture of kalmegh (Andrographis paniculata) and neem
juices were not acceptable by the sensory panel. Hence three fruit juices, (Azadirachta indica) leaves which prevents the hangover symptoms by

Design-Expert® Sof tw are

overall acceptability
7.8

6.2 8.1

X1 = A: pear
overall acceptability

7.5
X2 = B: sw eet lime
X3 = C: cocnut w ater
6.9

6.3

5.7

A (65.000)
B (25.000)
C (50.000)

C (10.000)
A (25.000)

B (65.000)

Fig. 3. Response surface plot showing the effect of concentration of different components (pear, sweet lime, and coconut) on the overall acceptability of the anti-
hangover (AHO) product.

15
S. Srinivasan et al. Current Research in Food Science 1 (2019) 8–16

reducing the acetaldehyde build-up in the body after alcohol consump- Kim, S., Cho, S.K., Min, T., Kim, Y., Yang, S., Kim, H., Hyun, S., Kim, H., Kim, Y., Choi, H.,
2011. Ameliorating effects of mango (Mangifera indica L.) fruit on plasma ethanol
tion. The fruit juice blend developed as AHO formulation in the present
level in a mouse model assessed with 1H-NMR based metabolic profiling. J. Clin.
study is a simple, effective, economical and ready-to-prepare alternative Biochem. Nutr. 48, 214–221.
to these formulations with good sensory appeal. Konkit, M., Choi, W.J., Kim, W., 2016. Aldehyde dehydrogenase activity in Lactococcus
chungangensis: application in cream cheese to reduce aldehyde in alcohol metabolism.
J. Dairy Sci. 99, 1–7.
5. Conclusion Lee, H., Isse, T., Kawamoto, T., Woo, H., Kim, A.K., Park, J.Y., Yang, M., 2012. Effects and
action mechanisms of Korean pear (Pyrus pyrifolia cv. Shingo) on alcohol
This study analyzed the effect of certain common food commodities detoxification. Phyther. Res. 26, 1753–1758.
Lee, H.S., Isse, T., Kawamoto, T., Baik, H.W., Park, J.Y., Yang, M., 2013. Effect of Korean
on hangover through in vitro studies by enhancing either alcohol dehy- pear (Pyrus pyrifolia cv. Shingo) juice on hangover severity following alcohol
drogenase and aldehyde dehydrogenase activities, or at least the alde- consumption. Food Chem. Toxicol. 58, 101–106.
hyde dehydrogenase activity, to formulate an anti-hangover product. A Lee, D.I., Kim, S.T., Lee, D.H., Yu, J.M., Jang, S.K., Joo, S.S., 2014. Ginsenoside-free
molecules from steam-dried ginseng berry promote ethanol metabolism: an
beverage made from a blend of sweet lime, pear, and coconut water could alternative choice for an alcohol hangover. J. Food Sci. 79, 1323–1330.
be used to overcome hangover. No correlation between the antioxidant Lee, M., Kwak, J.H., Jeon, G., Lee, J., Seo, J., Lee, H., Lee, J.H., 2014. Red ginseng relieves
activity and the activity of alcohol dehydrogenase and aldehyde dehy- the effects of alcohol consumption and hangover symptoms in healthy men: a
randomized crossover study. Food Funct. 5, 528–534.
drogenase were seen. This dispels the common belief that an antioxidant Maninang, J.S., Lizada, M.C.C., Gemma, H., 2009. Inhibition of aldehyde
could serve as an anti-hangover product. The consumption of this dehydrogenase enzyme by durian (Durio zibethinus Murray) fruit extract. Food
beverage with cheese, cucumber, and tomatoes may further alleviate the Chem. 117, 352–355.
Meneses, N.G.T., Martins, S., Teixeira, J.A., Mussatto, S.I., 2013. Influence of extraction
hangover symptoms.
solvents on the recovery of antioxidant phenolic compounds from brewer's spent
grains. Separ. Purif. Technol. 108, 152–158.
Conflict of interest Pordie, L., 2015. Hangover free! the social and material trajectories of PartySmart.
Anthropol. Med. 22, 34–48.
Pritchard, S.R., Phillips, M., Kailasapathy, K., 2010. Identification of bioactive peptides in
There is no conflict of interest with any individual or organization. commercial Cheddar cheese. Food Res. Int. 43, 1545–1548.
Quertemont, E., 2004. Genetic polymorphism in ethanol metabolism: acetaldehyde
Acknowledgement contribution to alcohol abuse and alcoholism. Mol. Psychiatry 9, 570–581.
Ramadan, M.F., Osman, A.O.M., El-Akad, H.M., 2008. Food ingredients total antioxidant
potential of juices and beverages screening by DPPH in vitro assay. Dtsch. Lebensm.-
This work was supported by Department of Biotechnology (DBT), Rundsch. 104, 235–239.
Government of India and Department of Science and Technology (DST), Ramakrishna, S., Manu, M.B., Kolhapure, S.A., 2005. Evaluation of the efficacy and safety
of ‘PartySmart’ in the prevention of alcohol-induced hangover: a prospective,
SERB (PDF/2017/001108), Government of India. randomized, double blind, comparative, phase III clinical trial alcohol-induced
hangover is a poorly understood physiological manifestation of the biochemical
Appendix A. Supplementary data effects of alcoholic beverages on the body. Indian J. Clin. Pract. 15, 23–35.
Su, Y., Tian, Y., Yan, R., Wang, C., Niu, F., Yang, Y., 2015. Study on a novel process for the
separation of phospholipids, triacylglycerol and cholesterrol from egg yolk. J. Food
Supplementary data to this article can be found online at https://do Sci. Technol. 52, 4586–4592.
i.org/10.1016/j.crfs.2019.09.001. Subbaraj, D.K., Baskaran, A., Sureka, I., Velmurugan, M., Emelda, E.A.J.,
Sathiyamurthy, K., 2016. Bioactive potential of plants and spices extracts against
human bacterial pathogens. IOSR J. Pharm. Biol. Sci. 11, 2278–3008.
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