Potentiality of CSN1S2 Protein from Goat Milk for the Management of

Anxiety

Abstract

Objective: The aim of the present study was to evaluate the anxiolytic activity of CSN1S2
protein isolated from goat milk in mice using various experimental animal models.

Method: The elevated plus maze test (EPM), open field test (OFT) and light and dark test
(LDT) were used to assess the anxiolytic activity of the goat milk CSN1S2 protein (500 and
750 mg/kg, p.o.) in mice. In addition, attempts have been conducted to explore the level of
brain monoamine neurotransmitters like GABA and serotonin using flame flourimeter.

Result: The results from the models followed in the present study were interpreted and
observed that the goat milk CSN1S2 protein significantly (p<0.001) exhibited anxiolytic
activity at both the doses of 500 and 750 mg/kg while the results were more significant at the
dose 750 mg/kg. An increase in the brain GABA and decrease in brain serotonin
concentration supported the anxiolytic activity of CSN1S2 protein.

Conclusion: The results of the present study suggest that goat milk CSN1S2 protein may
possess anxiolytic activity by the elevated brain GABA and demoted brain serotonin
concentration which provides scientific evidence for its traditional claim.

Keywords: Anxiolytic, gross behaviour, goat milk, CSN1S2 protein, GABA, serotonin.

Introduction

In the present modernized lifestyle, anxiety is one of the most prevailing psychiatric
disorders which affect people of different age groups worldwide. Anxiety is unpleasant
feeling of stress, discomfort, tension, insomnia, worried thoughts, and physical changes like
elevated blood pressure, sweating, difficulty in breathing and increased heart rate along with
substantial disability, including educational and professional impairment, which also has a
negative influence on quality of life [1-2]. It is evident that psychotropic drugs are one type
of pharmacotherapy for anxiety disorders, but their effectiveness is constrained by their
adverse effects, dietary restrictions, and drug interactions like the frequent benzodiazepines
causes cognitive impairments, addictions, psychomotor impairments, anterograde amnesia,
confusion, aggressive behaviour, physical depletion [3]. These are some of the aspects that
have prompted numerous researchers to investigate novel compounds derived from nature in
the hope of discovering new anxiolytic treatments with no or fewer undesirable side effects.
In our previous study and other scientific investigations, it was evident that goat milk was
found to reduce the risks of gastrointestinal disorders, diabetes and many other chronic
diseases [4-7]. Literature shows that goat milk can reduce the anxiety-like behaviour and also
helps in brain development along with its traditional use to manage anxiety [8-9].
Additionally, goat milk has a high concentration of bioactive peptides derived from the
proteins α-CN-S1, α-CN-S2 (CSN1S2), β-casein (CSN2) and κ-casein (CSN3) [10] and
casein account for most of the protein in milk approximately 80 percent of the total protein
content that may have multiple functions [10]. In the present study the most prominent
protein fraction of goat milk CSN1S2 has been considered to explore its antianxiety
potentiality. As it is well known that anxiety is mediated by imbalance of brain
neurotransmitters mainly GABA and serotonin which remain the main targets for most of the
anxiety agents to modulate and give relief from anxiety [11]. It was found that goat milk has
high levels of GABA (gamma-aminobutyric acid) and CSN1S2 is a protein which may be a
source for its synthesis [12]. Keeping in view of the medicinal importance of goat milk and
other evidence to support the research, an attempt has been carried out to investigate the
antianxiety activity of its CSN1S2 protein fraction along with the estimation of its effect on
the brain bioamine concentrations mainly GABA and serotonin.

Materials and Method

Isolation of CSN1S2

The CSN1S2 protein was isolated from 250 ml goat milk sample purchased from a local
breeder of Greater Noida. The milk was heated to a temperature of 40˚C and then glacial
acetic acid was added drop wise (5 mL) while stirring until precipitates are formed. The
caprine CSN1S2 protein was purified by standard method. The protein precipitation was then
separated by filtration through a nylon mesh. The obtained protein was subjected to chemical
tests for its chemical nature and then stored at -20 °C till further chemical test to confirm its
pharmacological studies [13].

Experimental animals

Swiss albino mice (25-30 gm; of either sex) were used for the present research. The animals
were procured from the from the animal facility of Noida Institute of Engineering and
Technology (Pharmacy Institute), Greater Noida. The exploratory animals were housed in
standard polypropylene limits at (27.00±1.00)oC temperature with 12-hour light and 12-hour
diminish cycles along with relative humidity 55%–65%. Enough food, as well as water and
libitum, was given. Prior to the trial, the animals were given a seven-day adaptation period to
the laboratory conditions. Following the guidelines for animal experimentation, the protocol
for the present research was approved by the Institutional Animals Ethics Committee
(IAEC/NIET/2020/01/08), CPCSEA Reg No: 1845/Re/S/16/CPCSEA.

Gross behavioural study

The gross behavioural modification in mice due to the administration of CSN1S2 was
conducted for observing information of their effect on CNS of mice. Their effect on the
reflexes of mice like righting, pinna and corneal reflexes was observed carefully. At the same
time, the effect of CSN1S2 on the muscle tone, motor activity, traction test, stereotyped
behaviour and awareness were evaluated to get maximum information of behavioural effects
of CSN1S2 in mice. Individual behaviour was observed at various time periods to get
appropriate result of these drinks on CNS response [ref].

Assessment of anxiolytic activity

The anxiolytic activity was examined by using the elevated plus maze (EPM) test, open field
test (OFT), light and dark test (L and DT). The animals were divided into four groups, with
each group consisting of six mice. Group 1 received vehicle (normal saline, 0.9%); group 2
received diazepam (1 mg/kg; i.p.); groups 3 and 4 received goat milk CSN1S2 protein (500
and 750 mg/kg) orally (p.o.).

Elevated plus maze (EPM) test: The elevated plus maze apparatus has been utilized for the
assessment of anxiolytic property of a moiety. Its four arms, two open arms (50x10 cm) and
two close arms (50x10x40 with an open roof, are connected to a central square. This
apparatus is raised 50 cm above from the ground. The mice were treated with saline,
diazepam (1 mg/kg) and CSN1S2 protein (500 and 750 mg/kg) once daily for 7 days before
the experiment. In this test, the mice were placed gently in the central square facing one of
the open arms. The number of entries and average time spent in both arms was recorded for
duration of 5 minutes [14].

Open field test (OFT): This apparatus consisted of a wooden box (60 × 60 × 60 cm). The
open field arena is divided into 16 squares (15x15 cm): the four inner squares in the centre
and 12 squares in the periphery along the walls. The open field arena was illuminated with a
40-W lamp, focusing on the field from a height of about 75–100 cm. After treatment with
saline, diazepam (1 mg/kg) and CSN1S2 protein (500 and 750 mg/kg) once daily for 7 days
the mice were placed gently in one of the corner squares and the number of squares crossed,
time spent in the central square and number of rearing in the central square were observed for
5 minutes [15].

Light and dark test (LDT): The light and dark test apparatus consisted of open top wooden
box. Two distinct chambers, a black chamber (25 cm long × 35 cm wide × 35 cm deep),
painted black and made dark by covering its top with black plywood, and a bright chamber
(25 cm long × 35 cm wide × 35 cm deep), painted white and brightly illuminated with 40-W
white light source, were placed 25 cm above the open box. The two chambers were
connected through a small open doorway, (7.5 cm long × 5 cm wide) situated on the floor
level at the centre of the partition. The mice were placed individually in centre of the light
box after 7 days of treatment once daily and the time spent in light chamber, number of
entries in bright chamber, number of rearing in bright chamber and number of rearing in dark
chamber were observed for 5 min. [16]

Estimation of Neurotransmitters in mice brain

Estimation of GABA: Twenty-four mice after the induction of anxiety by EPM were carried
forward to estimate their effect on brain GABA level as they have already treated with
CSN1S2 protein. Brain tissue samples were separately homogenized in 10 volumes of 0.01 M
HCl using a glass homogenizer. The reagents used in the assay were 0.05 M glutamic acid in
0.2 M sodium phosphate buffer, pH 6.4, 14 mM ninhydrin in 0.5M sodium carbonate buffer,
pH 9.9-10 and copper tartrate reagent consisting of 1.6g Na2CO3, 329 mg tartaric acid and
300 mg CuSO4 × 5H2O, all made up in 1 litre of distilled water. 0.25 ml of homogenate was
diluted with 0.25 ml of 0.01 M HCl and 0.5 ml of 10% trichloroacetic acid. This last reagent
was used to precipitate the proteins. After the samples were centrifuged, 100 µl aliquots of
the supernatant were added to 15 µl of glutamate solution and 200 µl of the ninhydrin
solution. This mixture was incubated at 60ºC for 30 minutes and allowed to cool before the
addition of 5 ml copper tartrate reagent. The determination of endogenous GABA
concentration is based on a fluorimetric assay that depends on the formation of a fluorescent
product from the reaction between GABA and ninhydrin at alkaline pH and in the presence of
glutamate and then read at λ 380 nm-λ 460 nm [17].
Estimation of Serotonin: Twenty-four mice were divided into four groups, control group
(Normal saline, 0.9%), standard group (Diazepam, 1mg/kg), test group-I (CSN1S2 protein,
500mg/Kg) and test group-II (CSN1S2 protein, 750mg/kg) the same manner and were
subjected to induce anxiety by using EPM. As mentioned above, the brain of individual mice
was completely dissected after anesthetising with isoflurane. At 0oC, brain tissues were
homogenized in a teflon tissue homogenizer with 0.1N HCl. Homogenized tissue was placed
on ice for 10 minutes before being centrifuged at 14,000 rpm for 15 minutes at 0°C. 1 ml
10% zinc sulphate and 0.5 ml 1N NaOH were added to the clear supernatant solutions. The
tubes were shaken for 5 minutes before being centrifuged at 2,500 rpm for 20 minutes. A
quartz cuvette holding 0.3 ml of 2N HCL was filled with 1 ml of the clear supernatant
solution. The intensity of the fluorescence in the resulting solutions was determined by the
fluorometer at the wavelength [excitation (nm) / fluorescence (nm)] 290/550 when the
opening of the slits was 5/3 (Excitation/Emission) and the sensitivity of the instrument was
0.3 [18].

Analysis of obtained data

All the results were expressed as mean ± SEM and the data were analysed using one-way
analysis of variance (ANOVA) followed by Dunnett’s “t” test. A P value of p<0.05 was
considered as the level of significance. Whereas p<0.001 was considered as highest
significance.

Results

Gross behavioral study

The gross behaviour estimation showed that the test extracts have less effect on reflexes, the
animals were not observed to be alert. The CSN1S2 was found to have less effect on muscle
relaxant property and so on catalepsy in mice suggesting its antianxiety effect on Central
Nervous System (CNS) comparable to the standard drug diazepam. The test CSN1S2 protein
were found to increase the stereotyped behaviour i.e., increase in the rearing, sniffing and
grooming activities. CSN1S2 protein from the present gross behaviour study illustrates that it
is certainly effective against anxiety.

Effect of CSN1S2 on EPM

The saline-treated mice (Normal control) had spent more time in closed arm and showed less
entries in open arm compared to closed arm of the maze during 5 min. Animal treated with
diazepam (Standard) showed significant (p<0.001) increase in the open arms entries as well
as time spent in open arm. Oral administration of CSN1S2 (500 and 750 mg/kg) exhibited
significant (p<0.05 and p<0.001 respectively) increase in the number of open arm entries and
time spent in open arm but CSN1S2 at the dose of 750 mg/kg showed better anxiolytic
activity as compared to the CSN1S2 dose 500 mg/kg (Table 1).

Table 1: Anxiolytic activity of CSN1S2 in elevated plus maze test.

Treatment Number of entries Average time spent

Group

Close arms Open arms Close arms Open arms

Saline 4.16±0.30 2.16±0.16 268.33±2.17 17.33±0.33

Diazepam 9.17±0.30* 7.00±0.36** 195.83±2.82** 91.67±3.904*

CSN1S2-500 5.83±0.307** 3.17±0.30** 229.83±2.857** 53.83±1.641**

CSN1S2-750 7.66±0.33** 6.50±0.22* 205.50±2.24** 78.83±2.03*

Data represent in mean ± SEM, n = 6 mice per group, Statistical analysis by One-way
ANOVA followed by Dunnett’s Multiple Comparison test. P value = p<0.05*, p<0.001** as
compared to control group.

Effect of CSN1S2 on open field test

During the open field test (OFT), the control group animals were observed to spent
maximum time in the central square. Whereas diazepam and CSN1S2 (500 and 750 mg/kg;
p.o.) treated mice showed significant increase in the number of squares crossed, number of
rearing in central square and time spent in central square during 5-min interval as compared
to saline treated control group. Whereas the test CSN1S2 showed more significant (p˂ 0.001)
result at 750 mg/kg than that of the animals treated with 500 mg/kg of CSN1S2 protein
(p˂0.05) (Table 2).

Table 2: Anxiolytic activity of CSN1S2 in Open field test

Treatment Total no. of squares Time spent in No. of rearing in central

Group crossed central square square

Saline 53.16±1.424 12.00±0.577 1.50±0.223

 Diazepam 34.33±1.229** 149.00±2.250** 7.833±0.307*

CSN1S2-500 53.50±1.176* 88.166±1.815* 2.50±0.223**

CSN1S2-750 37.66±1.68** 139.50±6.25** 4.50±0.22*

Data represent in mean ± SEM, n = 6 mice per group, Statistical analysis by One-way
ANOVA followed by Dunnett’s Multiple Comparison test. P value = p<0.05*, p<0.001** as
compared to control group.

Effect of CSN1S2 on light and dark test

In the present model the animals of control group were observed to remain in light chamber
most of the time to avoid anxiety. On the other hand, in case of the animals treated with
CSN1S2 (500 and 750 mg/kg) increased the number of entries into the dark chamber with
more time spent in dark chamber and rearing (Table 3) reflecting its dose-dependent
inhibition of anxiety behaviour which was well compared to that of the standard drug
diazepam. However, as compared to the control group, the anxiolytic activity of CSN1S2 was
observed to be more significant (p˂ 0.001) at the higher dose of 750 mg/kg (18.833±0.30).

Table 3: Anxiolytic activity of CSN1S2 in light and dark test

Treatment Time spent in No. of entries in No. of rearing No. of rearing

Group light chamber dark chamber in bright in dark
chamber

Saline 14.00±0.36 2.33±0.21 1.33±0.21 11.66±0.421

Diazepam 30.83±0.47** 3.66±0.21* 6.166±0.30* 21.166±0.60*

CSN1S2-500 25.00±0.36** 2.00±0.25** 3.83±0.30** 14.16±0.47*

CSN1S2-750 27.83±0.30* 2.66±0.21* 5.33±0.33* 18.833±0.30**

Data represent in mean ± SEM, n = 6 mice per group, Statistical analysis by One-way
ANOVA followed by Dunnett’s Multiple Comparison test. P value = p<0.05*, p<0.001** as
compared to control group.

Effect of CSN1S2 on GABA levels

From the biochemical estimation it has been observed that in control group the brain GABA
level (mean±S.E.) were reduced than that of the normal GABA level (mean±S.E.). Whereas,
in CSN1S2 treated groups the brain tissues of the animals exhibited significant (p<0.001)
increased GABA level by bringing it towards the normal brain level. The CSN1S2 protein at
dose of 750 mg/kg was observed to possess maximum enhancement in the GABA
concentration (mean±S.E.) as compared to that of the control group. However, CSN1S2
protein at dose 500 mg/kg was also observed to increase the GABA level (mean±S.E.) but
not as much as the higher dose as shown in (Fig. 1).

Figure 1: Estimation of brain GABA level

Figure 1: Estimation of the effect of CSN1S2 on brain GABA concentration. The values are
represented as mean ± S.E.M; (n=6). The values were compared with the control group. Significant
variation against control was estimated by Dunnett’s test and p<0.001considered as extremely
significant. The results in CSN1S2 treated groups are well comparable to that of the standard group.

Effect of CSN1S2 on Serotonin levels

In the present study, the brain serotonin level increased in control group (mean±S.E.) and was
found to be higher than that of the normal brain serotonin concentration (108 µg/gm of brain
tissue) [22]. The brain of mice, treated with both doses of CSN1S2 (mean±S.E. and
mean±S.E. respectively) exhibited reduction in the concentration of serotonin significantly
(p<0.001) as compared to that of the control group animals. However, CSN1S2 at dose of
750 mg/kg was having more effective inhibitory effect on brain serotonin level as shown in
the figure 2. The potentiality of the CSN1S2 to reduce the brain serotonin level was observed
to be well comparable to that of diazepam (mean±S.E.).
 Fig.2. Estimation of brain serotonin level

Figure 2: Estimation of the effect of CSN1S2 on brain serotonin concentration. The values are
represented as mean ± S.E.M; (n=6). The values were compared with the control group. Significant
variation against control was estimated by Dunnett’s test and p<0.001considered as extremely
significant. The results in CSN1S2 treated groups are well comparable to that of the standard group.

Discussion

The result of current work showed that CSN1S2 protein given to mice via the oral route had a
substantial anxiolytic effect in three well-established anxiety animal models: EPM, OFT, and
LDT [19-21]. Anxious behaviour in mice was considerably reduced in animal study,
indicating that anxiety in mice was reduced following treatment with the CSN1S2 protein.

The results of our study on the elevated plus maze after treatment with goat milk CSN1S2
protein revealed anxiolytic activity, as there was significant attenuation of anxiety-like
behaviour (increased time spent in open arm and number of entries) in EPM, which is one of
the most representative indices of anxiolytic activity [22]. The overall arm entries are a
measure of changes in anxiety or general activity, and time spent on the central platform
appears to be related to decision making and/or risk evaluation [23].

In the open field test, the statistical analysis of the data obtained from the experiment
supported the anxiolytic activity of CSN1S2 protein, as its effect shows a significant increase
in the time spent in Central Square, number of central area rearings, and number of squares
crossed, as compared to the control group, indicating its anxiolytic effect.

In the light/dark test, the anxiolytic effect was also observed. Despite the transition parameter
being strongly reliant on locomotor activity, the number of transitions between the bright and
dark chamber, the time spent in the bright chamber as well as number of rearing in bright
chamber are recognised as anxiety indices in this test [24]. The time spent in the bright
chamber was increased in mice treated with CSN1S2 protein, as were the number of entries
and rearings, indicating anxiolytic activity.

From the brain monoamine estimation of GABA and serotonin, it showed an increase in the
GABA levels and reduction in the level of serotonin as compared to the control group which
indicates the anxiolytic activity of CSN1S2 protein.

Conclusion

In conclusion, it was found that goat milk CSN1S2 protein certainly possess anxiolytic
activity at both the doses (500 and 750 mg/kg) while the results were more significant at the
dose 750 mg/kg. This can be a basis for further development of functional foods having
anxiolytic activity with other health benefits. This shows that goat milk protein has many
beneficial effects on brain activity and can help in the management of anxiety disorders. It
can be used as food supplement with the treatment of anxiety. The research also supports
further studies on casein from goat milk for its better formulation and clinical trials that it can
be used in pharmaceutical industries.

Acknowledgement

We are very thankful to Noida Institute of Engineering and Technology (Pharmacy institute)
for providing this opportunity to work on this project.

Conflict of interest

The author declares that there is no conflict of interest among the authors of the article.

Funding
There is no supporting funding to report.
Ethical approval
In the present research the animal study was approved by the Institutional Animals Ethics
Committee as mentioned under the section of experimental animals and it is not applicable
for any other ethical approval as no clinical study was conducted.

Reference
1. Tripathi KD, Essentials of Medical Pharmacology, Jaypee Brothers and Medical
Publishers (P) Ltd, New Delhi, 2003, 399-402.
2. Kasper S. Social phobia: the nature of the disorder. Journal of affective disorders.
1998 Sep 1;50:S3-9.
3. Kumar S, Sharma A. Apigenin: The Anxiolytic Constituent of Turnera
aphrodisiaca. Pharmaceutical biology. 2006 Jan 1;44(2):84-90.
4. SHRUTI DHASMANA*1 , SANJITA DAS1 , SHIVANI SHRIVASTAVA1 , IRFAN KHAN1 ,
KUMARI RENU SINGH, Goat Milk: A Potent Nutraceutical 1
5. Protective effect of CSN1S2 protein of goat milk on ileum microstructure and
inflammation in rat-CFA-induced rheumatoid arthritis. Asian Pacific Journal of
Tropical Disease. 2015 Jul 1;5(7):564-8.
6. Ribeiro AC, Ribeiro SD. Specialty products made from goat milk. Small
Ruminant Research. 2010 Apr 1;89(2-3):225-33.
7. Fatchiyah F, Rohmah RN, Triprisila LF, Ohta T, Meidinna HN. The Caprine
Casein-Alpha-S2 Protein Modulates the Molecular Mechanism 2 of Insulin Signal
Transduction in Type2 Diabetes Rat. Acta Biochimica Polonica. 2020 Sep
14;67(3):401-8.
8. Soares JK, de Melo AP, Medeiros MC, Queiroga RC, Bomfim MA, Santiago EC,
Guedes RC. Anxiety behavior is reduced, and physical growth is improved in the
progeny of rat dams that consumed lipids from goat milk: an elevated plus maze
analysis. Neuroscience letters. 2013 Sep 27;552:25-9.
9. Joung JY, Song JG, Kim HW, Oh NS. Protective Effects of Milk Casein on the
Brain Function and Behavior in a Mouse Model of Chronic Stress. Journal of
Agricultural and Food Chemistry. 2021 Jan 26;69(6):1936-41.
10. Fatchiyah F, Raharjo SJ, Dewi FR. Virtual selectivity peptides of CSN1S2 protein
of local goat Ethawah breeds milk modulate biological mechanism of Calmodulin.
International Journal of Pharma and Bio Sciences. 2015;6(2).
11. LaLonde L. Neural Pathways of Anxiety. Brain Matters. 2019 Nov 18;1(1):8-9.
12. Limon A, Gallegos-Perez JL, Reyes-Ruiz JM, Aljohi MA, Alshanqeeti AS, Miledi
R. The endogenous GABA bioactivity of camel, bovine, goat and human milks.
Food chemistry. 2014 Feb 15;145:481-7.
13. Setiawan B, Kania N, Ohta T, Agustina V. Safe Dosage of Caprine CSN1S2
Protein from Fresh Local Goat Milk for Rats Evaluated by Acute and Sub-Chronic
 Toxicity Testing. Journal of Mathematical & Fundamental Sciences. 2017 Jan
1;49(1)
14. Doukkali Z, Kamal R. Anti-anxiety effects of Mercurialis annua aqueous extract
in the elevated plus maze test. J. Pharmacol. Rep. 2016;1:1-5.
15. Thippeswamy BS, Mishra B, Veerapur VP, Gupta G. Anxiolytic activity of
Nymphaea alba Linn. in mice as experimental models of anxiety. Indian journal of
pharmacology. 2011 Feb;43(1):50.
16. Ganatra TH, Joshi UH, Patel MN, Desai TR, Tirgar PR. Study of sedative,
anxiolytic, CNS-depressant and skeletal muscle relaxant effects of methanolic
extract of Hibiscus rosa-sinensis on laboratory animals. Journal of Pharmaceutical
Sciences and Research. 2011 Apr 1;3(4):1146.
17. Arsene AL, Aramă C, Mitrea N, Cristea AN. Experimental assessment of cerebral
monoaminergic status in a murine model of behavior. Farmacia. 2009 Jan
1;57(4):492-9.
18. Das S, Sasmal D, Basu SP. Evaluation of CNS depressant activity of different
plant parts of Nyctanthes arbortristis linn. Indian journal of pharmaceutical
sciences. 2008 Nov;70(6):803.
19. Lister RG. The use of a plus-maze to measure anxiety in the mouse.
Psychopharmacology. 1987 Jun;92(2):180-5.
20. Belzung C, Griebel G. Measuring normal and pathological anxiety-like behaviour
in mice: a review. Behavioural brain research. 2001 Nov 8;125(1-2):141-9.
21. Walf AA, Frye CA. The use of the elevated plus maze as an assay of anxiety-
related behavior in rodents. Nature protocols. 2007 Feb;2(2):322-8.
22. Lister RG. Ethologically-based animal models of anxiety disorders. Pharmacology
& therapeutics. 1990 Jan 1;46(3):321-40.
23. File SE. Factors controlling measures of anxiety and responses to novelty in the
mouse. Behavioural brain research. 2001 Nov 8;125(1-2):151-7.
24. Bourin M, Hascoët M. The mouse light/dark box test. European journal of
pharmacology. 2003 Feb 28;463(1-3):55-65.