December 2010, Volume 1, No.

2
International Journal of Chemical and Environmental Engineering

An exploratory study on possible preparations that

may be formulated from Tahong Shells collected
from Cavite City, Philippines
Judilynn N. Solidum
Chair and Associate Professor, Department of Pharmaceutical Chemistry,
College of Pharmacy, University of the Philippines, Manila,
Corners Taft Avenue and Pedro Gil, Manila,
(02)5262273
Corresponding author :graloheus@yahoo.com

Abstract
Cavite, Philippines is home of inexpensive shell foods, so tahong samples were taken there. Shells of tahong attract vectors of
diseases where these wastes are dumped due to foul odor. Exploring the protein and chitin/chitosan contents of tahong shells will help
provide the vertebra for its recycling which will aide lessen shell wastes, vectors of diseases, and also be pivotal in the discovery of
relevant medicinal and non-medicinal formulations.
Qualitative tests were used to screen the presence or absence of proteins, chitin/chitosan from tahong shells. Quantitative analysis
using Flameless Atomic Absorption Spectrophotometry was used to show the presence of lead in the extracted chitin/chitosan and the
de-leading ability of 20% sodium chloride to help reduce lead from the substances of interest. Skin patch and scratch tests were used
in guinea pigs to show dermatologic safety of powdered chitin/chitosan.
Tahong shells contain proteins but the pasty substance produced from these proteins was not comparable to commercial paste. Chitin
and chitosan were extracted from the tahong shells as evidenced by osazone crystal formations at 25 minutes. The powdered chitin
and chitosan contains 3.1427 and 4.0171 ppm lead respectively, which fall below the standard recommended by the United States
Pharmacopoeia for Nutritional Supplements of 10 ppm. Sodium chloride reduced the lead in chitin and chitosan at 24.91 and 23.47%
respectively. The primary irritation index for both chitin and chitosan was zero showing that it is a non-irritant substance which can be
used for dermatologic products. Chitin was able to remove 55.83%, 32.69% and 28.19% lead from sample numbers 1(10%),2(30%)
and 3(50%) lead acetate solution respectively. Chitosan was able to remove 44.79%, 32.40%, and 46.882% lead from sample numbers
1 (10%),2(30%), and 3(50%) lead acetate solution respectively.
Studies on the improvement of the production of paste is suggested. Quantitative analysis of proteins, chitin and chitosan is
recommended for systemic pharmacologic preparations. Refinement of dermatologic preparations is suggested.

Keywords: Tahong shell,,chitin, chitosan, glucosamine., lead

1. Introduction
A.Background and Significance of the Study
like the horseshoe crabs ,tahong have exoskeletons
(hard outer shell) that gives protection from predators.
Chitins, a tightly interwoven strand of cellulose-like
material serve as glue that holds the shell together [1].
It is a tough yet flexible polysaccharide essential to
crustaceans and insects as it is part of its exoskeletons.
Chitin is also present in the cell walls of some fungi [2].
As chitin is present in the shells of horseshoe crabs,
then it may also be present in tahong shells.
The earth approximates a closed system.
Energy is exchanged but not matter. As we increase the
number of people in the world, the amount of waste
products increase as well. The reduction, reuse and
recycling of waste accomplishes two goals namely: It
reduces the amount of waste that must be managed, and
it conserves resources [3]. Wastes like that of tahong
shells, invite insects and rodents to swarm in its
disposal site. These are vectors of diseases. Increase in
its number will likely increase the incidence of diseases
in nearby areas. With the recycling of tahong shells,
possible health problems may be prevented.
In 1970, EPA ordered fishing companies to
stop dumping shells of seafoods (lobsters and crabs in
particular) into the ocean. Since then studies related to
chitin started. With the prospect that chitin is present in
tahong shells, recycling of the said waste may prove
fruitful. Chitin was discovered to be useful in cleaning
water sources of organic contaminants, minerals, metals
and oils. Chitin and its derivative chitosan is natural,
 non-toxic, non-allergenic, anti-microbial and
biodegradable [4]. Chitin may be used as part of
moisturizers, face, hand and body creams. It is also an
antifungal agent.
Traditional Chinese Herbal medicines used
cicada (cicada insect, chan) skins in their formulations.
These skins contain chitin which in turn contains
glucosamine and acetylglucosamine. It was described
that cicada showed sedative, antipyretic,
anticonvulsive, anti-tetanus activities. It was said to be
effective in treating vocal polyps [5], acute nephritis
[6], allergic skin disease, Bell’s palsy, and cataract [7].
Addition of cicada to formulae at a range of 1.5 to 6
grams was said to be effective to infants’ food digestion
and helps prevent night crying in infants.. This
discovery became the basis of most Chinese herbal
preparations of incorporating large dosage of cicada for
pediatric patients [8].
Other than chitin and chitosan, tahong shells
being mussels may contain proteins. Researches have
shown that mussels contain proteins essential for
underwater adhesion. Research efforts are geared
toward identifying the gene responsible for the
adhesive proteins, environmental factors that may
influence protein production and strategies for
producing natural adhesives similar to the mussel
adhesive proteins. It is envisioned that such adhesive
could bind materials like glass, plastics, metals, and
wood to materials like bones, teeth, biological
organisms and other chemicals or molecules [9].
To reiterate, with the prospect of chitin,
chitosan and proteins in tahong shells, recycling of the
mussel exoskeleton may prove fruitful. The initiative
will help not only in the decline of the shell wastes,
vectors of diseases swarming in certain areas, but may
also be pivotal in the discovery of relevant medicinal
and non-medicinal formulations. This study is hoped to
also catalyze similar researches on other material
wastes to open doors in producing important needs of
man from wastes. Man will be able to conserve
untouched resources by using wastes as substitute
resources.
1.1. Conceptual Framework
INPUT
Collection and preparation of tahong shells
THROUGHPUT
Chemical screening of tahong shell ( protein,
glucosamine);. FAAS of lead levels in tahong shells and
water after treatment with tahong shells; preparation of
applicable products and skin scratch and patch Tests
103
OUTPUT
Presence of varied important proteins and glucosamine and
C. Problems
formulation of applicable products
1.2. Statement of the problems
1. Can tahong shells be recycled into something
useful?
2. Are proteins present in tahong shells?
3. Are chitin/ chitosan present in tahong shells?
4. Can formulations with non-medicinal and with
possible medicinal or cosmetic values be
prepared?
5. Are the preparations with possible medicinal
value safe for use by man?
1.3. Objectives of the Study
General Objective
This study aims to recycle tahong shells to
formulations with non-medicinal and with possible
medicinal properties.
Specific Objectives
This study aimed to:
1. Identify the presence or absence of proteins in
tahong shells.
2. Identify the presence or absence glucosamine in
extracted chitin/chitosan from tahong shells.
3. Formulate preparations with possible medicinal
and non-medicinal values from tahong shells.
4. Assess the safety of tahong shell preparations
using skin scratch and patch test and Flameless
Atomic Absorption Spectrophotometry (FAAS)
for lead levels.
1.4. Scope and limitation of the study
The research will look into the possible different
protein components in tahong shells. Such protein
components may be used for the preparation of glue.
The study will also look into the presence or absence of
glucosamine from extracted chitin and chitosan as
possible active component to powders and deleading
agent to waste water. The safety of the preparations
with possible medicinal property will be tested
dermatologically to guinea pigs and lead levels
assessed using FAAS.
2. Materials and Methods
2.1. Research Design
This research followed the descriptive-
quantitative type of study. Surveys, qualitative
chemical tests, and graded skin scratch and patch
analyses were undertaken to complete the research.
Preparations with possible medicinal and non-
medicinal properties were explored.
 2.2. Location
The study included areas near Longos junction in
Cavite where vending of tahong and disposal of its
shells are common and popular.
2.3. Sample Collection
The tahong shells wiere collected from vendors in
the chosen sampling area.
2.4. Sample Preparation
The tahong shells were washed with running
water for ten minutes, damped with a clean cloth and
pulverized using mortar and pestle. Twenty percent
aqueous and thirty percent ethanolic extracts were used
for the chemical screening tests. For the quantitative
analysis 10%tahong shell aqueous solution was
prepared.
2.5. Extraction of chitin, chitosan and proteins from
tahong shells
To 100 g of pulverized tahong shells, 50 ml of 1-
10% sodium hydroxide solution is added. The solution
was heated from 80 to 100 C in a water bath to remove
the proteins. The solution was decanted. The liquid
portion was dried over low fire using water bath.
To the de-proteinized residue, 50 ml of 1-10%
hydrochloric acid was added at room temperature to
demineralize the tahong shells. The solution was
decanted. The liquid portion was discarded. If the
residue remaining is colored, 1% sodium hypochlorite
solution was used to decolorize the chitin.
To produce chitosan, the same procedure as above
was followed except that 50% sodium hydroxide was
used employing 90-120C temperature [adapted 10].
2.6. Qualitative tests for proteins
The protein residue from the extraction procedure
above was taken up by water for the chemical analysis
using color reactions. The following were the tests
utilized-Ninhydrin reaction, Biuret test,Xanthoproteic
test, Millon,s test,.Hopkins-Cole reaction,.Lead Acetae
reaction, and Nitroprusside reaction [11].
2.7. Qualitative Analysis for Chitin/Chitosan
Phenyl hydrazine Reaction
To 300 mg of phenyl hydrazine mixture 5 ml of
the test sample solution was added, shaken well, and
heated on a boiling water bath for one-half to three-
quarters of an hour. The tube was allowed to cool
slowly (not under the tap) and examined the crystals
microscopically. An insoluble yellow osazone crystal
of glucosamine is formed within 25 minutes [11].
2.8. Safety Assessment of Cosmetic
For the evaluation of the safety of powdered
tahong shells the skin patch and scratch tests were
performed using male and female guinea pigs from 24
104
to 72 hour observation periods [13]. The scoring
criteria for the evaluation of skin reactions are given in
Table 1 below.
Table 1 Evaluation Of Skin Reactions (Scoring Criteria) [12,13]
Erythema and Eschar Formation Scores
No erythema 0
Very slight erythema 1
Well defined erythema 2
Moderate erythema 3
Severe erythema 4
Total Possible Erythema Score 4
Edema Formation Scores
No edema 0
Very slight edema (barely perceptible) 1
Slight edema (edges of area well defined by 2
definite raising) 3
Moderate edema (raised approximately 1 mm) 4
Severe edema (raised more than 1 mm and 4
extending beyond area of exposure)
Total Possible Edema Score
2.9. Analysis of lead levels of tahong shells using FAAS
Five grams of the sample was digested in 10ml
concentrated nitric acid in an open glass container
overnight, at room temperature, and the next day at 80
C for 5 hours. It was cooled to room temperature, and
the volume was adjusted to 50 ml with distilled water
and then analyzed using flameless atomic absorption
spectroscopy
2.10. Possible Formulations
2.10.1. Tahong glue (Pandikitahong)
Measured 500 ml of protein extracted from tahong
was placed into a 1000 ml beaker to which 100 ml of
vinegar
(5% acetic acid) was added and stirred well. The
mixture was warmed in a hot plate with continuous
stirring. About 50 ml of water was added and 5 g of
baking soda (sodium bicarbonate) to neutralize any
remaining vinegar and was stirred well. The product
was tested by pasting together two pieces of white
paper. [adapted 15]
2.10.2. Tahong powder (TahPow)
To the deproteinated tahong shells, 50 ml of 1-
10% hydrochloric acid was added at room temperature
to demineralize the tahong shells. The solution was
decanted., The liquid portion was discarded. 1%
sodium hypochlorite solution was used to decolorize
the chitin. The residue was thoroughly washed with
distilled water until washing was neutral to pH paper.
The residue was air dried and powdered using 80 mesh
sieves. [16]
The resultant powder may be used as dusting or as
face powder after it has been perfumed and/or
medicated.
 Table.2, Chemical Tests For Proteins
compound which is unsubstituted in the 3,5 position
Chemical Tests Actual like tyrosine, phenol and thymol. Glyoxylic acid
Theoretical Results
(Qualitative) Results (CH(OH)2COOH reaction or Hopkins-Cole reaction is
Ninhydrin test Blue colored solution Deep blue due to the presence of tryptophan. Nitrates, chlorates,
purple
colored
nitrites or excess of chlorides interfere with the reaction
solution while copper sulfate increases its sensitivity. The
Biuret’s test Purplish violet or Red-violet nitroprusside reaction is due to the presence of
pinkish violet solution tinged creatinine the anhydride form of creatine or
with blue precipitate solution with
aqua blue
methylguanidinoacetic acid [11].
precipitate The extracted proteins from the tahong shells
Xanthoproteic test White to yellow Yellow showed amber coloration. Upon treatment for the
precipitate that dissolves solution with possible formation of glue or paste, a substance with
and results to yellow reddish tinge moderate sticky consistency was produced. From 35.3g
colored solution that
deepens to orange and 13.1g extracted proteins from the chitin and
Millon’s test White that turns to red Cherry red chitosan of 100g tahong shells each, 21.5g and 7.1g
precipitate or red colored colored pasty substance was recovered respectively. Using
solution solution papers to test for its adhesive property, it was observed
Hopkin Cole’s test Reddish violet ring Purple
junction
that the glue produced from the tahong shells is not
Nitroprusside test Ruby red color to yellow Purple tinge comparable to commercial pastes.
to yellow As chitin and chitosan contain acetyl glucosamine
2.10.3 Water De-leading agent and glucosamine, phenylhydrazine reaction was used to
Five grams of extracted chitin and chitosan (of test for osazone crystal formation. Yellow osazones
which lead levels were measured before the test) were were formed within 25 minutes which were viewed
placed in 50mL distilled water with known lead under the microscope. The time of formation in minutes
concentration (10%, 30% and 50% lead acetate of osazones confirm the substance present eg mannose
solution) measured through FAAS. After twelve hours, 1-5, fructose 2 ,glucose 5, xylose 7, arabinose 10, galactose
the amount of lead in the water was determined using 20, glucosamine 25, and sucrose 30. Extracted chitin
FAAS. and chitosan from tahong shells contain 3.1427 and
4.0171 ppm of lead. The concentrations are below the
limit recommended by the United States
3. Results and Discussion Pharmacopoeia (USP) for Nutritional Supplements
The results for the qualitative tests for proteins which is 10 ppm [17]. Lead is said to be one of the
showed that tahong shells contain proteins (Table 2). deadliest heavy metals which is capable of impairing
The ninhydrin or triketohydrindene hydrate reaction several organs. The most heavily affected of which is
gives positive results in general to proteins, peptones, the central nervous system [18] thus its level in the
peptides, amino acids and even primary amines above said substances were evaluated. Because of its
including ammonia. The amino acids react with the prospective use in non-medicinal and medicinal
ninhydrin compound (C6H4.(CO)3.H20 to yield carbon formulations, lead levels in extracted chitin and
dioxide, ammonia and usually but not always an chitosan from tahong shells were analyzed and are
aldehyde containing one less carbon atom than the within standard limits (Tables 3 and 4).
original amino acid. For the Biuret’s test, copper sulfate
was added in excess such that the aqua blue precipitate Table .3 Lead Levels (Ppm) In Extracted Chitin From Tahong Shells
of copper hydroxide obscured the red-violet tinge Standard
formed by the solution. The result is indicative of Chitin Concentration
deviation
proteoses and peptones. This test is given by substances
which molecules contain two carbamyl (-CONH2) Trial 1 4.1920
groups joined directly or through a single atom of Trial 2 2.2683
nitrogen or carbon.Substances which contain –CSNH2, Trial 3 2.7929
-C (NH) NH2, or -CH2NHt2 also responds to this test.
Average 3.1427 0.0006
Proteins give positive result because of the presence of
–CONH groups in the molecule.
Table 4. Lead Levels In (Ppm) In Extracted Chitosan From Tahong Shelss
The end result according to Schiff, of Biuret’s
test is dependent on the formation of cuppripotassium Standard
Chitosan Concentration
biuret or biuret potassium cupric hydroxide. The deviation
Xanthoproteic test result is due to the presence of Trial 1 4.8916
phenyl group (-C6H5) in the protein molecule Trial 2 4.3669
exemplified by tyrosine and tryptophan. The Millon’s
reaction is due to the presence of hydroxyphenyl group, Trial 3 2.9678
-C6H4OH in the protein molecule and any phenolic Average 4.0171 0.0006
105
 Deleading chitin and chitosan however will 8). Chitin was able to remove 55.83%, 32.69% and
lower its lead component. Use of 20% sodium chloride 28.19% lead from sample numbers 1(10%),2(30%) and
caused 24.91 and 23.47% deleading of chitin and 3(50%) lead acetate solution respectively. Chitosan was
chitosan respectively(Tables 5 and 6). In a previous able to remove 44.79%, 32.40%, and 46.882% lead
study, it was shown that 10% sodium chloride deleaded from sample numbers 1 (10%),2(30%), and 3(50%)
malunggay leaves 8.4992%. Sodium chloride is a cheap lead acetate solution respectively.
and practical chemical that has the ability to delead
other materials. Table.8, I Mean Lead Concentration (Ppm) Of Water With
Known Lead Amounts After De-Leading With Chitin And
Chitosan
Table .5, Lead Levels (Ppm) After Deleading Chitin Using 20% lead in
Sodium Chloride lead in sample
lead in sample sample
Lead levels after de-
No before after de-
Lead levels of after washing leading
Percent deleading leading
Chitin chitin upon with 10% with chitin
deleaded with chitosan
extraction sodium 1 136.1962 60.1556 75.1959
chloride
Trial 1 3.8795 2.6567
2 469.7394 316.1892 317.5386
Trial 2 2.1335 1.8976
Trial 3 2.5687 1.8897
Average 2.8606 2.148 24.91% 3 720.7013 517.4834 382.8209

Table.6, Lead Levels (Ppm) After Deleading Chitosan Using 20%

Sodium Chloride
Lead levels Lead levels 4. Conclusions
of chitin after washing Percent Tahong shells may be recycled into something
Chitin
upon with 10% deleaded useful. Using qualitative chemical tests it was shown
extraction sodium chloride that Tahong shells contain proteins, tyrosine,
Trial 1 3.9784 2.9567
Trial 2 4.1269 3.3832
tryptophan, creatinine, peptones and proteoses. A pasty
Trial 3 3.9788 2.9086 substance was produced from these proteins though its
Avrag: 4.0280 3.0828 23.47% strength was not comparable to commercial paste or
The extracted, deleaded chitin and chitosan glue. Chitin and chitosan were extracted from the
were powdered using 80 mesh sieve. It was prepared as tahong shells as evidenced by osazone crystal
such for the preliminary development of dusting and formations at 25 minutes and observed under the
face powder. The powdered chitin and chitosan were microscope. The powdered chitin and chitosan contains
tested for dermatologic safety. There were no erythema 3.1427 and 4.0171 ppm lead respectively, which fall
nor edema observed on the 24th and the 72nd hour on below the standard recommended by the United States
male and female guinea pigs. It therefore showed zero Pharmacopoeia (USP) for Nutritional Supplements
(0) primary irritation index. The primary irritation which is 10 ppm. De-leading with 20% sodium chloride
index of tested samples with the following rates: 0-8, 9- reduced the lead in chitin and chitosan at 24.91 and
29, 30-59, 60-80, above 81 mean same as control or 23.47% respectively. The primary irritation index for
non-irritant, mild, moderate, strong and severe irritant both chitin and chitosan is zero (0) which makes it a
respectively [12]. Thus the powdered chitin and non-irritant substance potential for the formulation of
chitosan are non-irritant and are safe for dermatologic dermatologic products like dusting powder and face
usage (Table 7). powder. Chitin and chitosan from tahnong shells were
shown to remove lead from lead contaminated water
Table .7, Evaluation Of Skin Reactions To Chitin And Chitosan
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