1

Chapter 2

Review of Related Literature and Studies

This chapter presents the overview of the related literature and studies that

helped the researchers all through the entirety of the study.

Foreign Literature

In Histology and Cell Biology: An Introduction to Pathology

(Kierszenbaum & Tres, 2012) stated that glycosaminoglycans (GAG) are a part of

a proteoglycan that makes up the extracellular matrix in man. GAGs establish

links of proteoglycans with cell surface components, growth factor and other

ECM component. It is also made up of polymers of disaccharides with sulfate

residues. Each type of GAGs aggregate to a core protein to form proteoglycan

then linked to hyaluronan.

Molluscs foot have various locomotion, it may be an attachment to a

substratum or a combination of a function. It is usually a ventral, sole-like

structure in which waves of muscular contraction effect a creeping locomotion. Its

mucus secretion is a natural aid to adhesion or as its form of gliding in its cilia

described by Hickman, et al. (2011).

In blood analysis with additives in obtaining the separation of plasma and

serum by centrifugation, the plasma contains fibrinogen which lacks in the serum.

A mucoitin polysulfuric acid, heparin, is an effective anticoagulant that could

 2

work safely in humans. The action of antithrombin III accelerates in neutralizing

the formation of fibrin from fibrinogen in the presence of heparin. In an average

of 30 minutes, clotting time with gel separator may clot the blood and tubes with

thrombin will clot in 5 minutes (McPherson & Pincus, 2011).

Acton (2013) also stated that new compounds are obtained which show

the best anti-thrombotic activity and a bleeding potential lower than that of any

other heparin like glycosaminoglycan. Particularly, it has been found that new

glycosaminoglycans have a very high anti-thrombin activity and a bleeding

potential lower than the heparin obtained. New glycosaminoglycan is having

improved and selective anti-thrombin activity, useful as specific coagulation-

controlling and anti-thrombotic agents.

Golden Apple Snails are considered to be the world’s worst invasive

species by ISC or Invasive Species Compendium. P. canaliculata breeds only on

summer. “Locally, variation in reproductive regime may be related to local

climatic variatin, especially availability of water. In humid tropical Southeast Asia

range the controlled environment of a rice paddy, P. canaliculata can grow and

breed year round as long as water is present” (Anonymous, 2015)

Mohan (2002) stated, “over half (1.2 to 1.6 million hectares) of rice fields

in the Philippines are infested with the golden apple snail. The damage and

economic loss have been devastating. Snails consume the base of rice seedlings

and feed on new transplants. The costs of controlling the snail, replanting, and
 3

rice yield loss all account for economic loss. Beyond these costs are the costs

associated with damaging the ecosystem. Snails destroy plants and affect the food

web. They may easily out-complete native species.”

Snail slime is made up of many different enzymes and components such as

proteins, lipids, cellulose, and calcium which plays a vital role in blood clot

activation. Calcium is not found primarily in the snail’s body but is found more

on the feet of the snail where slime is produced. (Gomot 1997)

Local Literature

When P. canaliculata was first discovered in the Philippines, they figured

that it can be a source of income in the country especially in the food industry

since it was high in protein. It was soon referred to as the “Golden Kuhol”. The

Philippines wanted to pursue this as a source of income in the food industry, the

authorities failed to take good care of it and failed to manage where these snails

would go. They failed to monitor its cleanliness —these golden kuhols, then,

escaped and multiplied in the creeks and polluted water areas making it unclean

for eating, as per stated in the apple snail website by Cagauan and Joshi (2002).

The apple snail website described the golden apple snail as a major plant

pest mostly seen in farms. It feeds on varied types of plants such as algae, azolla,

duckn weed, water, hyacinth, rice seedlings, etc., and also feeds on decomposing

organic matter (Anonymous, Invasive species Compendium, 2005)

 4

The feeding habits of the golden snails were also described as highly

generalist and voracious macrophytophagous herbivore. Its diet varies on its age

and size and they sometimes eat brine shrimps and other frozen foods, or dead

fish and insects. It needs calcium to maintain the strength of its shell (Blog Spot).

Golden apple snails eat young and emerging rice plants by cutting the rice

stem at the base and destroying the whole plant. Golden apple snails are

distinguished by their color and size, having a muddy brown shell and golden

pinkish or orange-yellow flesh. They are bigger and lighter in color compared to

the native snails. If no control measure is taken, they could deal damage up to

50% yield loss for the rice farmers. (Rice Knowledge Bank)

Foreign Studies

A study said that P. canaliculata species has become highly damaging

invasive species which affected several agriculture and fisheries as well as public

health. It also included P. canaliculata as an intermediate host for

Angiostrongylus canaliculata, a nematode, that led to the occurrence of human

eosinophilic meningitis in China. Genomic divergence plays an important role in

the adaptive nature of P. canaliculata but some genomic factors pertaining to

competition and displacement are unknown (Mu, et al., 2015).

Angiostrongylus cantonensis, also known as rat lung worm can be

transmitted to man through slugs or snails which are considered to be their

 5

intermediate host. It causes mengitis in humans. The infection can be transmitted

to humans if they consume a raw or under-cooked snails or slugs (Cowie, 2013).

A study was also conducted by exposing snails for three months and found

that Cu, had a highest concentration found in the gills and highest concentrations

of Cr, Zn and Fe found in the digestive gland of the snail by Kruatruchue,

Sumritdee, Pokethitiyook, and Singhakaew (2010).

A study by Sueffert, Burela & Martin (2010) discussed that the snails in

the field were active during winter season at which temperature was 13-15°C.

Most of the snails in the field were inactive but not in deep lethargic stage. At

temperatures between 10 and 30°C the snails were observed active and increased

feeding and at temperature constant above 10°C, it spends its time crawling.

While temperature above 30 °C activity was decreased but no heat stroke was

observed when temperature was raised to 36.2 °C.

The study by Tsumuki, Izumi, and Wada (2009) stated that most snails

will not survive in very low temperatures reaching to 0oC. Although some

survived reaching to 0oC, injuries in the mantle of the snails were observed.They

also found the concentrations of glycerol in the digestive gland and the

concentration of glucose in the posterior chamber of the kidney were higher in

cold habituate snails than those of non-acclaimed ones which mean there is an

alteration in the carbohydrate metabolic pathways which are altered in the snails

during cold acclimation.

 6

Pomacea canaliculata has different uses because of its characteristics

according to one article. It is used as a dietary supplement because of its high

protein content. However, the industries failed to distribute it because the

expenses were too high. As time passed by, studies proved that a golden snail

contains a parasite that may be harmful for humans—it contained a rat-worm

parasite that can be acquired through uncooked snail. For that reason, P.

canaliculata was removed from the market and released to the wild (U.S

Department of State: Archive, 2009).

Ozawa & Makino (1987 as cited by Takeichi, Hirai and Yusa, 2007)

“reported aggregation of immature snails in containers under homogenous

environments, although they did not discuss the mechanism of aggregation…both

attraction by water-born pheromones and trail-following behaiour are known as

mechanisms of aggregation of gastropods…they are attracted by water-borne

chemicals (pheromones) released from conspecific; the function of pheromones is

related to reproduction; individuals follow mucus trails of conspecific; and they

distinguish the direction of the trails.

According to Sri Harti et al., they stated that the snail slime has

antibacterial and anti-inflammatory effects and therefore the proliferation phase

will heal wounds immediately. The content of the snail slime is supposed to be

the most influential fibroblast proliferation is heparin sulfate which assists in

blood clotting process and fibroblast proliferation. They stated in their work that
 7

the results obtained proved that the snail’s slime is potential and an effective

remedy for in vivo wound healing. The snail’s slime contains many active

substances that aids in wound healing such as heparin sulfate, and calcium which

plays a major role in hemostasis or clotting of blood to prevent further bleeding in

wounds.

Polyphosphates are linear polymers of inorganic phosphate that are

abundant in the acidocalcisomes of prokaryotes and unicellular organisms as well

as in the dense granules of human platelets. Polyphosphates modulate haemostasis

by: (1) triggering clotting via the contact pathway; (2) accelerating the activation

of coagulation factor V (a key cofactor in blood clotting) and (3) causing fibrin to

form clots whose fibrils are thicker and more resistant to fibrinolysis. (Yun &

Morrissey, 2009)

Plastic tubes require clot activators that use either intrinsic or extrinsic

pathways to ensure rapid and dense clot formation. Clot activation by the intrinsic

pathway is surface-dependent and a greater density of activating surface sites

speeds clotting time. Siliceous substances (e.g., glass, silica, kaolin, bentonite,

diatomaceous earth) accelerate clot formation through contact activation but

particulate clot activators work relatively slowly (30 to 60 minutes) (Bowen &

Remaley, 2014)

Local Studies
 8

Methods used for extraction of slimes for protein analysis was made on

the journal Endosymbiotic and Host Proteases in the Digestive Tract of the

Invasive Snail Pomacea canaliculata: Diversity, Origin and Characterization

(Godoy, Castro-Vasquez, & Vegal, 2013). They first removed the shell, then

aspirated of the crop and style sac contents with the use of syringe while the

coiled gut was collected by gentle squeezing of the sectioned gut.

It was concluded in one article that Golden kuhol (P. canaliculata) is an

effective alternative protein source. It was also stated that there is no significance

between the commercial protein agar and the Golden kuhol medium as protein

source for bacterial growth (2011).

Golden kuhol has many and varied uses since its first discovery as a

source of diet or meal. It is a very invasive alien species of snail that can be

parasitic to plants on farms because of its feeding mechanisms. Snail species are

also said to secrete slime or mucus secretion either for motion and lubrication or

for mating purposes and reproduction but the amount of its secretion can be

affected by the environment and several factors like temperature. In accordance to

the species livelihood feeding on rice plants and other polyphytophagous plants, it

can be a definitive host for Angiostrongylus cantonensis which can cause human

eosinophilic meningitis. Presently, many studies are conducted for the knowledge

of knowing about other certain uses of golden kuhol instead of treating it as a pest
 9

to farm lands. Also, extraction methods should be implied in conducting the study

for full collection of the slime.

 10

Chapter 3

Method and Procedure

In this chapter, the methodologies used in the experiments are described.

This chapter has also included the research design, the tools used in

identifying the accuracy and reliability of the instrument, along with the

locale where the samples were supplied.

Research Design

A quantitative methodology was followed in the study. The researchers

had included specific tests and experimentation for the gathering of data

and/or results. A tabulation table was used to include trials and tests

conducted to the variables. This study had gathered information through

experimentation and tabulation of results.

A tabulation table was used in order to follow procedures and certain

results released.

Research Instrumentation

The researchers used three charts to check the experiments; these served

as the checklist for the experiments that was conducted. The first chart is the

physical testing for the slime of the Pomacea canaliculata. Under the

physical testing are the pH, color, odor and appearance. For the testing of pH,

the researchers used a pH meter to determine the pH level of the slime, if it

 11

was acidic or basic. The pH of the slime is a factor that can affect the reaction

of the slime to the blood. Slime’s color may vary but according to some

articles, most of the color seen is yellow. The researchers collected enough

slime and compared the color to different shades until the researchers came

up with a conclusion on the odor, color and shade.

Data Gathering Procedures

The researchers used different procedures and techniques determining

the activity of Golden Apple Snail (Pomacea canaliculata) as an effective

clot activator in Clinical laboratory.

1. Collection, Preparation and Storage of samples

1.1. Blood Sample

The researchers collected 5.0 ml of whole blood from qualified patients

through screening.

1.2. Slime Sample

The slime samples were obtained from different rice fields in Biñan

Laguna. The slime sample was brought to the National Museum for

authentication. It was placed in a cooler half filled with water and food and a

temperature ranging 20-30 degrees Celsius. The slime was collected by

steaming under low heat of 75o-250oC. 250-300 snails was collected and used

in the study.
 12

2. Procedure

2.1. Physical Testing

The slime from Golden Apple Snail was tested physically to determine its

pH, color, odor, and appearance.

2.2. Chemical Testing

The slime had undergone certain chemical tests, tested for its chemical

component.

2.2.1 Carbohydrate testing

2.2.1.1 Molisch’s test - In a test tube, 2 ml of the test carbohydrate solution and 2

drops of α-naphthol solution was added. They carefully inclined the tube and

poured drop wise concentrated H2SO4, using a dropper, along the sides of the

tube. They observed the violet colour at the junction of the two liquids.

2.2.1.2 Iodine test- Add 2 drops of iodine solution to about 2 mL of the carbohydrate

containing test solution. A blue-black color was observed which was

indicative of the presence of polysaccharides.

2.2.2 Protein determination

2.2.2.1 General Testing for proteins

2.2.2.1.1 Reaction with Heat. 5.0mL of slime extract was placed in a test tube and was

heated in boiling water bath for a few minutes. Formation of precipitation

was observed.
 13

2.2.2.1.2 Reaction with salts of heavy metals. Add a drop of 1% silver nitrate at a time

for slime extract. Observe. Repeat the procedures with 5% mercuric chloride.

2.2.2.1.3 Reaction with alkaloidal reagents. To 2.0 mL of slime extract in a test tube,

add 1.0 mL of saturated picric acid solution. Repeat the procedure using

tannic acid, trichloroacetic acid and tannic acid.

3. Experimental trials

The researchers performed 5 trials with slime of varying amounts of 10

uL, 25 uL, 50 uL, 100 uL, 350 uL and a constant volume of blood which is

5mL.

4. Parallel Testing

4.1. Standing of blood with slime at a varying ratio in comparison to the control

tube, which is the Serum Separator Tube measured by the time clotting.

4.2. Pipet 10 uL, 25 uL, 50 uL, 100 uL, 350 uL of slime extracted into a set of test

tubes and add 5 ml of blood from random patients; based on the normal ratio

of blood to anticoagulant which is 1:9, the researchers started at the ratio of

1:1. Stand and observe until the blood has clotted. Centrifuge the clotted

blood at 3500 for 5 minutes. Each test and control plasma should be

performed in triplicate. The results showed which blood clotted.

 14

Chapter 4

Presentation, Analysis and Interpretation of Data

This chapter covers the determination the clotting ability of the extract

from the Golden Apple Snail or Pomacea canaliculata. Several tests were done to

prove the effectiveness of the said extract. The tests conducted were Physical and

Chemical testing to check for the viability of the extract and for the confirmation

of the extracts constituent. Experimental tests on human blood were the basis of

the parallel testing conducted in comparison to the commercially prepared Serum

Separator tube.

1. Identification Test for Slime Extract

1.1 Physical Tests. Results were obtained through observations and

assessment using human senses.

Table 4.1 Result of Physical Testing of the Slime Extract

Physical Testing Results

Appearance Clear liquid

Color Yellowish

Odor Pungent

pH 8.47
 15

1.2 Chemical Tests. The slime extract was examined by its chemical

composition through the following tests: Iodine Test, Molisch Test,

Biuret Test, and General Testing for Proteins

Table 4.2 Results of the Chemical Properties of the Slime Extract

Chemical Test Result

Carbohydrate Test

Molisch Test Formation of violet ring at

junction indicating a positive
result.

Iodine Test Formation of yellow orange

color indicating a negative
result

Protein Test

Biuret Production of blue precipitate

indicating a positive result

Reaction with Heat Production of white precipitate

indicating a positive result

Reaction with Salts of Heavy Metals

a. Silver Nitrate Formation of brown precipitate

which indicates a negative
result

b. Mercuric Chloride Formation of powdery white

 16

precipitate which indicates a

positive result

Reaction with Alkaloidal Reagents

a. Saturated Picric Acid Formation of yellow precipitate

which indicates a positive
result

b. Tannic Acid Formation of dirty white or

flesh precipitate which
indicates a positive result

c. Trichloroacetic acid Formation of white precipitate

which indicates a positive
result

2. Parallel Test

2.1 The Determination of Clot Activating Property of Slime Extract

The researchers collected a total of fifteen (15) milliliters of blood

from three (3) volunteers and were applied to increasing concentrations of

slime (10 uL, 25 uL, 50 uL, 100 uL, 350 uL)

Table 4.3 Results of the Clotting Activity of the Increasing

Concentration of Slime
 17

Contr 10uL 25uL 50uL 100uL 350uL 555uL 714uL 5mL

ol

1 7min 8.48m 7.23m 5.15m 5.45m 4.30m 4.37m 4.26m 7.25m

in in in in in in in in

7.05m 8.38m 7.15m 6.16m 5.38m 4.15m 4.22m 4.24m 6.35m

in in in in in in in in in

7.22m 8.52m 7.38m 6.23m 5.49m 4.43m 4.30m 4.32m 6.49m

in in in in in in in in in

2 6.34m 8.15m 6.58m 5.45m 5.15m 4.18m 4.13m 4.05m 9.21m

in in in in in in in in in

6.45m 8.35m 6.51m 5.58m 5.50m 4.29m 4.31m 4.10m 9.15m

in in in in in in in in in

7.05m 8.56m 7.28m 6.13m 5.28m 4.38m 4.28m 4.07m 8.56m

in in in in in in in in in
 18

3 6.58m 8.38m 7.12m 5.38m 6.09m 4.45m 4.45m 5.01m 7.56m

in in in in in in in in in

7.10m 8.45m 7.18m 5.42m 6.15m 4.25m 4.12m 4.45m 8.10m

in in in in in in in in in

7.12m 8.42m 7.30m 5.28m 5.30m 4.33m 4.27m 4.28m 7.42m

in in in in in in in in in

Figure 4.1 Clotting Time Activity of Slime Extract in Increasing

Concentration (1/3 Triplication)

Clotting Time Activity of Slime

Extract
10
Control
Time (min)

5 10 uL
25 uL
0
50 uL
Trial 1 Trial 2 Trial 3

Figure 4.2 Clotting Time Activity of Slime Extract in Increasing

Concentration (2/3 Triplication)

 19

Clotting Time Activity of Slime

Extract
10 Control

Time (min)
10 uL
5
25 uL
50 uL
0
Trial 1 Trial 2 Trial 3 100 uL

Figure 4.3 Clotting Time of Slime Extract in Increasing

Concentration (3/3 Triplication)

Clotting Time Activitiy of Slime

Extract
10 Control
Time (min)

10 uL
5
25 uL
50 uL
0
Trial 1 Trial 2 Trial 3 100 uL

The control showed normal clotting time activity of the patients’ blood

since there is no extract or chemical added.

For the experimental group, the concentration of slime specifically 10 uL

exhibited a longer time of clotting activity compared to the control. While,

experimental values 50 uL, 100 uL, and 350 uL exhibited faster rates of clotting
 20

activity on human blood than the control value and the experimental value, i.e.,

1.25 uL exhibited similar rates of clotting activity with the control value.

1. Statistical Treatment of Values

Table 4.4 Clotting Time Activity

Concentr Mean Standar Interpretation

ation d
Deviatio
n
Serum 6.88 0.33 Control value
Separator
Tube
10 uL 8.41 0.12 Slightly prolonged clotting time

25 uL 7.08 0.31 Slightly prolonged clotting time with

slightly higher mean value

50 uL 5.64 0.42 Slightly lower mean value and faster

 21

clotting activity

100 uL 5.53 0.42 Slightly lower mean value and faster

rate of clotting activity

350 uL 4.31 0.10 Lower mean value and faster rate of

clotting activity

555 uL 4.27 0.11 Lower mean value and faster rate of

clotting activity

714 uL 4.31 0.29 Lower mean value and faster rate of

clotting activity

5 mL 7.79 1.05 Prolonged clotting activity and slightly

higher mean value

Table 4.4 showed the control value to have the clotting time mean

value of 6.88 min, the experimental dose 10 uL has a longer clotting time

activity because of its mean value of 8.41 min, experimental dose 25 uL

has a slightly prolonged clotting time activity with the control value with

its mean value of 7.08, experimental doses 50 and 100 uL have slightly

faster clotting time activity with the mean value of 5.64 and 5.53

respectively, experimental dose 350 uL has a faster clotting time activity

compared to the controlled value with the mean value of 4.31 min,

experimental dose 555 uL has a lower mean value of 4.27 min compared

to the control which gives a faster clotting activity, experimental dose 714

has a lower mean value compared to the control with a mean value of 4.31
 22

min which gives a faster clotting activity and the experimental dose 5 mL

has a prolonged clotting activity with its mean value of 7.79 min.

Table 4.5 ANOVA treatment for the Values

ANOVA TABLE

Source df SS MS F P- Significance

value

Treatments 5 93.532 18.706 212.4878 1.0000 P=1.0000>0.5

Error 48 4.226 0.088 Not

Significant
Total 53 97.758

The ANOVA table showed a P-value of 1.0000 and is more than

0.05 indicating a not significant difference which denotes its comparability

to 5.53, the control value.

 23

Chapter 5

Summary, Conclusions and Recommendations

This chapter presents the summary of findings, relevant conclusions and

recommendations drawn by the researchers from the results obtained in the study.

Summary of Findings

1. Physical Properties of Slime Extract from Pomacea canaliculata

The data obtained from the physical testing of slime shows that the slime

is yellowish in color, has a mousy odor, a pH level of 8.47 and has an appearance

of a clear liquid.

2. Chemical Properties of Slime Extract from Pomacea canaliculata

Different tests were done for the identification of carbohydrate and protein

structure of the snail slime. The general tests for carbohydrates, Molisch’s test

gave a positive result. Iodine test for the identification of starch gave a negative

result.

For protein determination, a production of blue precipitate indicated a

positive result for Biuret. When heated, there was a production of white

precipitate which also indicates a positive result. There were two test made to

identify the reaction of slime with salts of heavy metals. First was by using Silver

nitrate in which a formation of brown precipitate indicated a negative result, and

another test was by using Mercuric Chloride where a formation of a powdery

 24

white precipitate indicated a positive result. In determining the reaction with

Alkaloidal reagents, three tests were involved namely: Saturated Picric acid,

Tannic acid and Trichloroacetic acid. Results obtained showed a formation of a

yellow precipitate, formation of dirty white or flesh precipitate, and a formation of

white precipitate, respectively. All results obtained indicated a positive result.

3. Evaluation of Clotting Activity of the Slime Extract of Pomacea

canaliculata and Serum Separator Tube

Among the experimental doses applied to 5 mL of blood, 50 uL, 100 uL, and 350

uL of slime extract exhibited faster rates of clotting time activity compared to the

control tube sample.

Conclusion

Based on the results, there was a progressive rate of clotting time activity

of the experimental doses i.e., 50 uL, 100 uL and 350 uL.

It is therefore concluded that the slime is not as effective as the

commercially prepared clot activator tubes is rejected.

It is also concluded that the slime has no clot activating property is

rejected.

Due to the result shown on Chapter 4, Table 4.5, having the P-value as

1.0000 therefore makes a not significant result on the varying doses of slime to
 25

blood ratio. Thus reveals that the hypothesis stated as, “There is no significance

between the varying blood to slime ratio” is accepted.

Recommendations

Based on the summary of findings and conclusions, the researchers

recommended the following:

1. Further examine the clot activating property of slime extract.

2. Isolate the specific component of the slime extract to be used as a clot

activator on the clinical laboratory.

3. Test the capacity of the snail extract as a possible antithrombotic agent

used in an additive tube in the clinical laboratory.

4. Further explore the antimicrobial property of the slime extract.

 26

APPENDIX A
Certificate of Authentication
 27

APPENDIX B
Slime Extraction & Physical Test
 28

APPENDIX C
Chemical Test
 29

APPENDIX D
Parallel Test
(Slime and Blood)