Effect of Fish Syrup – a rich Source of Amino Acids on Anxiety and
Depression
Askari Beguma, Sarwar Aqleem Hasana, Omer Mukhtar Tarara, Samee Haidera,
Syed Abdul Alia, Zeba Hisamb, Rana Qamarb, Iftikhar Ahmedb, Muhammad
Masroorb, Masood Hameed Khanb and Inam Rasoolc
aPCSIR
Laboratories Complex, Karachi,
b
Civil Hospital, Karachi,
cBaqai
University Hospital, Karachi
Abstract
In order to investigate the effect of fish syrup on anxious and depress human subjects a
collaborative study was designed. 59 cases of anxiety and depression were selected from
out patient department (OPD) of Civil Hospital, Karachi and treated systematically. These
cases carried severe to moderate symptoms of anxious and depress conditions. The patients
were evaluated for 4 weeks and examined by Hamilton Anxiety and Depression Scale. The
results revealed that about 95% depress cases and about 85% cases with features of anxiety
showed marked improvement. Somatic symptom like disturb sleep was converted into
sound sleep, fatigue, aches and pains were disappeared. Appetite was improved. Patients
gained energy for doing their work as long as they were given Fish Syrup. Maximum pretreatment Hamilton Score for Anxiety and Depression was found 38 and 29 respectively at
the commencement of study, which after treatment was reduced down to 2 in some of the
cases. It is evident from the data that besides established treatment, fish syrup is helpful for
treatment of depression and anxiety and may be used as an alternative psychiatric
medication.
Key Words: Anxiety, Depression, soluble Fish Protein Hydrolysate, Essential, nonessential, minor amino acid
INTRODUCTION
Depression is an emotional disability that leads to distress and seriously damages
individual’s abilities (Khawaja and Bryden, 2006). Likewise generalized anxiety disorder is
a chronic, disabling mental illness badly affecting the body functioning. Although common
and treatable, but about 65–70% of depression remains undiagnosed and untreated (Fieve,
1
1989). The reasons are many but one of the big factors is ignorance. Only fifty percent
people with the disorder, consult an expert. In developing countries mostly patients are seen
and treated by primary care physicians, who may be unfamiliar with the condition.
The brain biochemistry and mood behaviour are intricately linked. Neurotransmitters are
natural chemicals that facilitate communication between brain cells and govern our
emotions, memory, mood, behaviour, learning abilities and sleep pattern. Brain is a
chemical factory that produces the neurotransmitters by using nutrients namely amino
acids, vitamins, minerals. These nutrients are safest and most effective for increasing the
brain amine levels (Slagle, 1992). Orthomolecular psychiatric therapy is the practice of
preventing and treating mental disease by the provision of the optimum molecular
environment for the brain, especially the optimum concentrations of natural substances,
normally present in the human body (Pauling, 1968). Research has confirmed the
effectiveness of using just a few targeted amino acid precursors to increase the key
neurotransmitters, thereby eliminating depression, stress, anxiety and craving for food and
drugs (Ross, 1999; Ross, 2002). Specific amino acid supplementation taken on a daily basis
helps to feel more mentally balanced and provides us with an overall sense of well being. It
has also been studied that countries with greater per capita rates of seafood consumption
have lower rates of major depression, bipolar depression, post-partum depression and
mortality from homicide (Hallahan and Garland, 2005).
At PCSIR Laboratories Complex, Karachi, high protein fish syrup has been developed by
using soluble fish protein hydrolysate through enzymatic hydrolysis (Askari and Qadri,
1998). This syrup is a rich pool of highly soluble essential and free amino acids as well as
important trace and major minerals augmented with some B vitamins particularly B1, B6
and B12 which are considered nature’s antidotes to stress. The addition of further
permissible food ingredients i.e. stabilizer, emulsifier, colour and flavour have converted it
into a palatable syrup. A collaborative study was conducted on subjects of anxiety and
depression at Civil Hospital Karachi under the supervision of physicians and psychiatrists
to determine the efficacy of fish syrup.
2
MATERIALS and METHODS
Enzyme: Fresh papaya juice from Carica papaya, extracted from raw papaya having
activity 20,000-25,000 IU/L at 85oC (Askari; 1998)
Extraction of Papaya Juice. Fresh unripe papaya were purchased from local market,
washed, cut into small pieces and juice was extracted in electric juicer MJ-280 N (National
Matsushita), filtered through a cotton plug to remove fine pulp. The clear juice was kept at
- 20 C before the experiment. The yield of juice obtained was about 50% (v/w) of the
unripe papaya i.e. 1 kg raw papaya yielded about 400-500 mL of juice (Askari; 1998).
Measurement of Proteolytic Activity. Proteolytic was measured by (Cheftal; 1971).
Units of proteolytic activity. One protease unit was defined as the amount of enzyme which
will hydrolyse casein in order to produce a colour (Folin Ciocalteaeu) equivalent to one
micro mole of tyrosine ml-1 Minute-1 under the assay condition
Substrate: Sardines, Sardinella sendensis selected as substrate.
Proximate chemical composition of sardines during the course of four different fishing
seasons showed seasonal variation (A.B Munshi; 2005)
Ingredients used for making Fish Syrup,
Cane sugar, (sucrose 99.9%) citric acid, gum tragacanth, butylated hydroxy toluene (BHT),
sodium benzoate, potassium sorbate, as preservative, colour; Chocolate brown powder (H7334) and Apple green powder (H-9195). Flavour, Orange (Sweet No.1) and Orange
emulsion (J-6209) Bush Boak & Allen, Thiamine hydrochloride, Pyridoxin hydrochloride,
Cyanocobalamine. All ingredients used are food grade and purchased from local market.
Preparation of Fish Syrup
Process Steps
(a)
Preparation of soluble Fish Protein Hydrolysate (SFH) (Figure 1)
(b)
Preparation of Fish Syrup from Sardine Fish Hydrolysate (SFH) (Figure II)
3
Methods and patients selection for clinical trials
Adult patients between ages 18-60 years of age, both males and females attending medical
outpatient department with features of anxiety and depression judged by HAD scale were
selected. Cut off point was 8. In these patients clinical and biochemical screening was done
including CP, ESR, FBS and lipid profile to exclude patients with concomitant organic
diseases. Physical examination included blood pressure, pulse, height, weight and BMI.
Patients with known diseases like diabetes mellitus and with neurological disorders were
excluded from the study. Previously diagnosed patients of anxiety and depression were not
inducted as they were already on their medications and it was not ethically possible to stop
their treatment, to wait for their effects to subside and then to put them on new treatment
regimen of amino acids. Patients on antipsychotic, antidepressants or anxiolytic drug
therapy were also dropped from participants list. New patients with somatic complaints
were selected through the medical OPD.
Patient’s selection
All 59 patients fulfilling the inclusion criteria were selected for the study and were divided
into two groups of subjects and control by following a randomized chart. One group
comprising on 30 subjects were given Fish Syrup, and other group with 29 subjects was
kept on placebo. In these patients, treatment was started with 10 ml Fish Syrup and Placebo
with instructions to be taken twice daily before breakfast and dinner. Each of the patients
was given the appointment to visit after 2 weeks. These patients were reevaluated on the
same scale after 2 weeks. The evaluation continued fortnightly for 4 weeks for each patient.
At the end of 4 weeks all the patients were reevaluated and tested again for CP, ESR, FBS
and lipid profile after stopping the drug. Finally the results were analyzed by using SPSS
for statistical data.
Analytical procedure
The fish syrup was subjected to determination of amino acids composition using an amino
acid analyzer according to the method of Schuster (1988). The amino acids were assayed
using ion-exchange chromatography after hydrolizing with 6 M HCl for 24h at 110°C.
Methionine and cystine were analyzed by using formic acid protection prior to acid
hydrolysis (Song et al., 2003). The dry residues were then dissolved in a known quantity of
4
citrate buffer (2.2 pH) and filtered to obtain a clean solution of the hydrolysate. An aliquot
of it was injected into the column (Shim-pack ISC-07/S1504 Na) of the HPLC based amino
acid analyzer (RF-10AXL, Shimadzu Corporation, Tokyo, Japan) equipped with
fluorescence detector (FLD-6A). Sodium hypochlorite and o-phthalaldehyde solutions were
used as reaction solutions. Tryptophane was determined by simultaneous Tryptophane
system by alkali hydrolysis using the same column Shim-pack ISC-07/S 1504 NA, as for
other amino acids
The minerals were determined on Hitachi Z-8000 Atomic Polarization Zeeman
Absorption Spectrophotometer. Phosphorous was determined according to (Pearson 1976).
Vitamins of B group were determined using HPLC system (Agilent Technologies, 2007).
Results and Discussion
Fish Syrup is a prime source of all essential, non essential and minor amino acids (Table 1).
These amino acids play a significant role in human body (Slagle, 1992; Ross, 2002).
Taurine a sulphur containing minor amino acids is present in appreciable amount in Fish
Syrup, helps to promotes healthy brain function (Urquhart N; 1974), act as anti anxiety
agent (HIERD; 2004) Minerals present in the fish syrup (Table 1) act as a catalyst for the
conversion of amino acids into neurotransmitters with B vitamins which participate as
coenzymes, necessary for all amino acid metabolism. The vitamins B1, B6 and B12 which
are generally considered the nerve relaxants are added in fish syrup according to
recommended daily allowance (RDA). These help the brain to communicate with the
nervous system and the body through neurotransmitters.
Among all the participant patients the feature of anxiety was common and 39 of them have
feature of depression together with anxiety (Table 2). Depression and anxiety were
diagnosed by asking the patients a series of questions by using Hospital Anxiety and
Depression Scale (HAD Scale) (Aronson, 2002) and by Hamilton Depression and Anxiety
Scale (Hedlund and Vieweg, 1979). On screening by Hamilton scale for depression, many
patients admitted that they had committed self harm. Studies have shown a significant link
between suicidal and aggressive behavior with a low concentration of a chemical 5-hydroxy
indole acetic acid (serotonin) in the spinal fluid (Feiger, 1983). Suicides and violent acts are
closely associated with deficiencies of serotonin, which is produced by amino acid
5
tryptophane and vitamin B6 (Weissbach et al., 1957). Serotonin is the powerful hormone
which influences the sleep pattern and mood. Depletion of trypotophane can spur an
increase in depressed mood, particularly in individuals sensitive to effective disorders
(Young et al., 1985; Benkelfat et al., 1994; Smith et al., 1997). The fish are a rich source of
a beneficial amino acid, taurine. It is essential for neuronal growth in human body (Chen et
al., 1998) and associated with control of emotional disturbances such as depression (Mauri
et al., 1998) and anxiety (Chen et al., 2004).
Statistics of total 59 cases as depicted in Table 2 shows maximum pretreatment Hamilton
score for anxiety among these patients was 38, while minimum score after treatment came
down to 2 in some of the patients. Whereas, maximum pretreatment Hamilton score for
depression among these patients was 29 and minimum score after treatment came down to
as low as 3. A decrease of 45-50% in Hamilton Rating Scale for depression and anxiety
after treatment with fish syrup was observed. At the initiation of the study no patients was
judged under normal category by Hamilton Anxiety Scale. But after completing the study
about 66.7% cases improved and scored normal range, whereas out of 12 subjects with mild
anxiety, 6 cases improved and showed positive clinical response. At the end of 4 weeks
trial an overall 86% of the patients treated with fish syrup had significantly improved
(Table 3). This result is in accordance with the study of Bronson and Whitcomb (1996) who
got positive results with amino acids supplements in 3-4 weeks, regularly prescribed amino
acids as preventive medicine for the patients under the treatment. The study reveals that in
treatment group a decrease of 8.07 percentile point on Hamilton Anxiety Scale was
recorded in anxious patients (Fig. 3) whereas in depress patients after fish syrup treatment,
a decline of 7.19 percentile points was noted (Fig. 4). It results in a significant improvement
of 43.5% in anxious group and 46.3% in depress group on Hamilton Anxiety Scale.
Out of 39 patients suffering from depression, 20 were given fish syrup and rest were on
placebo. At the commencement of the study there was no subject lying under normal
category and after treatment with fish syrup 15 subjects improved and scored normal range.
Whereas 4 of 8 patients were improved from mild depression and 1 of 5 subjects attained
normal score from moderate depression and out of 6 subjects all improved from severe
category to normal. An overall 95% improvement including mild subjects was attained
6
among the depression group (Table 4). Improvement was clearly observed in all categories
but mild cases could get normal range if the treatment would be continued for further 4
weeks. All the patients getting fish syrup had many of their symptoms relieved and top of
the list were disturb sleep converted into sound sleep, appetite was improved, muscle
tension and aches and pain disappeared and patients were more interested in doing work
and found energy in themselves as long as they were on fish syrup but later, on stopping the
fish syrup, they started returning to OPD with same complaints.
Laboratory investigations carried out before and after treatment showed no significant
difference (Table 5) as the subjects under study were not physically weak. However, the
investigations had to be done in order to exclude any concomitant disease at the time of
selection for the study.
Conclusion:
The patients with somatic complaints were selected through the medical OPD. Observation
and results showed short-term improvement by amino acid on anxiety and depression. To
confirm long term efficacy, an other study will be required. In the present study some of
the most severe cases get Placebo instead of Fish Syrup as the study was kept double blind
through out the study period. After this preliminary study, it was concluded that more
elaborate clinical trials with known severe cases of anxiety and depression may be treated
with fish Syrup against tricyclic, SSRI and MOI are expected to give some spectacular
results.
7
Table-1
Composition of Fish Syrup
Each 5ml (1 tsp.) Contains:
Soluble Fish Protein
Essential Amino Acids
500mg
(in mg)
Lysine
53.0 ± 0.15
Leucine
45.0 ± 0.12
Isoleucine
35.0 ± 0.10
Valine
46.5 ± 0.13
Threonine
20.0 ± 0.09
Phenylalanine
25.5 ± 0.11
Tyrosine
22.5 ± 0.08
Methionine
14.9 ± 0.05
Tryptopha
6.9 ± 0.05
Cystine
4.25 ± 0.03
Non Essential Amino acids
Glutamic acid
55.5 ± 0.13
Alanine
43.5 ± 0.11
Aspartic acid
39.0 ± 0.10
Glycine
31.0 ± 0.08
Proline
23.5 ± 0.06
Arginine
25.65 ± 0.08
Serine
21.8 ± 0.05
Histidine
12.55 ± 0.03
Minor Amino Acids
Taurine
91.15 ± 0.17
Major Minerals
Sodium
14.33 ± 0.11
Potassium
11.27 ± 0.91
Calcium
10.20 ± 0.07
Phosphorus
6.50 ± 0.05
Magnesium
2.50 ± 0.01
8
Trace Minerals
Iron
4.0mg ± 0.008
Zinc
13.4mg ± 0.1
Maganese
15.7mcg ± 0.03
Copper
52.0mcg ± 0.002
Selenium
20.2 mcg ± 0.003
Cobalt
0.84 mcg ± 0.001
Vitamins
Vitamin B1 (Thaiamine HCI)
0.7mg ± 0.001
Vitamin B6 (Pyridoine HCI)
1.5mg ± 0.002
Vitamin B12 (Cyanocobalmine)
0.7 mcg ± 0.001
CONTRA INDICATION
None-no side effect, it is a food supplement can be used daily
DOSAGE & ADMINISTRATION
 Adults:
02 teaspoons twice a day
 Children (5-10):
1 tsp. twice a day
 Children (1-5):
½ tsp. twice a day
Table 2
Pre treatment anxiety and depression scores of the subjects
Pre treatment
No of patients with anxiety
59
Anxiety score
20.78(10-38)
No of patients with depression
39
Depression score
17.00(9-29)
Hospital Anxiety And Depression (HAD)
Scale Rating
24.22 (15-38)
9
Figure 3
HAMILTON ANXIETY SCALE
Treatment periods
2nd Follow up
1st Follow up
Pre-treatment
0
5
10
15
20
25
Score
PLACEBO GROUP
Figure 4
FISH SYRUP GROUP
HAMILTON DEPRESSION SCALE
Treatment periods
2nd Follow up
1st Follow up
Pre-treatment
0
5
10
15
20
25
Score
PLACEBO GROUP
FISH SYRUP GROUP
10
Table 3
Category
Evaluation of anxious subjects under treatment through Hamilton
Anxiety Scale
Pre
Ist Follow up 2nd Follow up
% Before %
After
Treatment
Treatment
Treatment
Normal
0
7
20
0
66.7
Mild
12
16
06
40
20.0
Moderate
12
07
04
40
13.33
0
0
20
0
Moderate to 6
Severe
Table 4
Category
Evaluation of depress subjects under treatment through Hamilton
Depression Scale
Pre
Ist
2nd
% Before %
After
Treatment Follow up
Follow up
Treatment
Treatment
Normal
0
10
15
0
75
Mild
9
6
4
45
20
Moderate
5
3
1
25
5
Severe
6
1
0
30
0
Table 5:
Pre and post treatment blood chemistry indicators data
Blood biochemistry
Pre treatment
Post treatment
ESR
9.5 (2-30)
9.0(2-28)
Hb g/L
107 (91-136)
106(90-138)
TLC
6303 (3800-9800)
6370 (3800-9100)
Platelets
228833 (133000-378000)
224467 (135000-325000)
FBS mg/dl
90.7 (69-105)
93.4 (84-100)
Cholesterol mg/dl
162 (90-212)
159.8 (88-211)
Triglycerides mg/dl
111.6 (55-200)
107.9 (64-198)
11
Reference:
A.B. Munshi, S.A. Ali and S. Shakir, 2005. Seasonal Variations in Biochemical
Composition of Sardines and Mullet from Pakistani Waters, Jour. Chem. Soc. Pak.
vol .27, no. 2,
Agilent Technologies. 2007. LC and LC/MAS In: The Essential Chromatography and
Spectroscopy Catalog, 2007-2008 ed, p. 656, Agilent Technologies, Mississauga,
Ontario.
Askari, B., Qadri, R.B. 1998. Studies on the proteolytic activity of papaya juice. Pak J Sci
Ind. Res, 41(3):151-155.
Benkelfat, C., Ellenbogen, M.A., Dean, P., Palmour, R.M., Young, S.N. 1994. Moodlowering effect of tryptophan depletion enhanced susceptibility in young men at
genetic risk for major affective disorders. Arch Gen Psychiatry, 51(9): 687-697.
Bronson, P., Whitcomb, H. 1996. Anxiety and depression, skip the Valium, hold the
Prozac. Alter Med Digest, 29: 49-52.
Chen, S.W., Kong, W.X., Zhang, Y.J., Li, Y.L., Mi, X.J., Mu, X.S. 2004. Possible
anxiolytic effects of taurine in the mouse elevated plus-maze. Life Sciences,
75:1503-1511.
Chen, X., Pan, Z., Liu, D., Han, X. 1998. Effect of taurine on human fetal neuron cells:
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Fieve RR. 1989. Mood swing. Bantam Books.
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Hedlund, J.L., Vieweg, B.W. 1979. The Hamilton rating scale for depression, adapted from
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Khawaja, N., Bryden, K. 2006. The development and psychometric investigation of the
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Mauri, M., Ferrara, A., Boscati, L., Bravin, S., Zamberlan, F., Alecci, M., Invernizzi, G.
1998. Plasma and platelet amino acid concentrations in patients affected by major
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Pauling, L. 1968. Orthomolecular Psychiatry Science, 160:265-271.
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Ross, J. 1999. The Diet cure: The 8 step program to rebalance your body chemistry and end
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from http://wwwwellbeingjournalcom retrieved on July 25, 2010.
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depression with amino acids and vitamins supplements. St. Martin's Press, New
York.
Smith, K.A., Fairburn, C.G., Cowen, P.J. 1997. Relapse of depression after rapid depletion
of tryptophan. Lancet, 349(9056); 915-919.
Song G.L., Li D.F., Piao X.S., Chi F., Wang T.J. 2003 Comparison of amino acids
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F473-F474 2004
13
Fig. 1: Flow chart showing production of Sardine Fish Hydrolysate (SFH)
Sardines
Eviscerated & Washed
Minced Homogenized with Water
in the ratio 1:2
Enzyme (Papaya Juice) added
at 83  2ºC
Hydrolysed for 2.5 hrs.
(pH6)
Enzyme inactivated at
temperature 90ºC
pH of Hydrolysed mixture
adjusted at 4.7 with citric acid
Filtered through Filtration Units
Sludge Contains Undigested
protein bones & scales
(Clear filtrate)
Soluble Hydrolysate
Concentrated under vacuum
Spray Drying
Converted into Fish Syrup,
Sardine Fish Hydrolysate
(SFH) Powder
14
Fig. II: Production of Fish Syrup, from Sardine Fish Hydrolysate (SFH)
Warmed concentrated hydrolysate to 60C
Adjusted pH of hydrolysate at 4.0
with citric acid
Added Sugar & gum tragacanth with stirring
Added other ingredients
Homogenized
Cooled
Added colour flavour & vitamins
Homogenized
Filtered
Fish syrup with 10% protein
15
NOVELTY/ NEW INFORMATION TO SCIENCE REPORTED IN THE PAPER
Novelty of the process is solubilization of fish protein by crude papaya juice as a source of
proteolytic enzyme, extracted from raw papaya, having optimum activity at higher
temperatures from 80 – 85 ºC and can easily be adjusted in the environment of Pakistan.
The use of crude papaya juice as proteolytic enzyme is an economical / feasible source for
commercial production of protein hydrolysates. The economic or cost considerations are
also important when using protein as ingredients. Protein hydrolysates play a significant
role in lowering the caloric content of foods, increasing the protein level and balancing the
amino acid profile.
High Protein Fish Syrup, food supplement has been prepared by using soluble fish protein
hydrolysate having major and trace minerals. Presence of soluble natural proteins, short
peptides complete amino acid profile with some minor amino acids like taurine make the
preparation novel and unique. Further augmentation with some specific B vitamins like B1,
B6 and B12, which are considered antidotes to stress enhance its efficacy, exert soothing
effect and helps to alleviate the crippling feeling of depression in natural healthy way which
has been proved by efficacy study conducted on patients of anxiety and depression at Civil
Hospital Karachi under the supervision of Doctors of Medicine and psychiatry.
16
Here are two refrees from Pakistan.
1.
Dr. Ghulam Mueen-ud-Din
Assistant Professor,
Department of Food Sci. & Technology
University of Sargodha,
Sargodha- Pakistan
Mobile +923006631122
Office +92489230316
E.mail: gmd_ft@yahoo.com
2.
Dr. Shahid Mahmood Rana
Assistant Professor
Food & Nutrition
Institute of Home and Food Science
G.C. University, Faisalabad-38000
Pakistan
Cell # +923458281615
E.mail: sm_ft1615@yahoo.com
Two refrees outside Pakistan
1.
Dr. Nuzhat Huma
Reeding University UK
drnuzhathuma@gmail.com
2.
Prof. Dr. Lin Hong
Food Safety Laboratory
College of Food Science & Technology
Ocean University of China
Qingdao, China
E.mail: linhong@ouc.edu.cn
17