Banerjee

The Effect of Salicin on Enzyme Elastase

By: Sumona Banerjee
Niles North High School

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Table of Contents

Title......1

Table of Contents..2

Acknowledgements.......3

Purpose......4

Hypothesis....5

Variables......6

Review of Literature....7

Materials.........18

Procedure....19

Results.........25

Data Analysis.....33

Discussion......36

Experimental Error.....38

Conclusion..40

Impact.....42

Reference List.........43

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Acknowledgments

I would like to thank Mrs. Camel, Mr. Gaynes, & Mr. Margolis for supporting and mentoring me
extensively throughout this project. I would also like to thank Mrs. Camel, Mrs. Posnock and Mr.
Thielsen for staying many hours in the lab so that I could finish conducting research. Also, I
would like to thank Mrs. France for helping me statistically analyze my data.

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Purpose
The purpose of this project is to determine if salicin, produced from willow tree bark, has the

ability to inhibit enzymatic elastase activity and eventually be developed into another method of

treatment for emphysema.

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Hypothesis

If elastase (enzyme) reacted with different concentrations of elastin (substrate) is treated with

salicin, then the concentration of elastin with no enzyme will be the same as elastin reacting with

both the enzyme and salicin. This is because salicin has anti inflammatory properties that will be

able to inhibit elastase from breaking down elastin and changing its concentration.

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Variables
Independent Variable: Concentration of substrate put into each assay well at the beginning of
the experiment.

Dependent Variable : Absorbance at 405 nm

Control Group: Wells with inhibitor included in the kit (elastatinal)

Negative Control: Wells without any inhibitor or enzyme

Constants: Amount of salicin, amount of distilled water, amount of elastase, etc.

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Review of Literature

Heart disease and cancer are known to be two of the leading causes of death around the

world. (CDC, 2014) But what about COPD? Many people do not know that Chronic Obstructive

Pulmonary Disease is the third leading cause of death in America. Approximately 12 million

people in the United States have been diagnosed with COPD, and many more may be affected

but unaware. (American Lung Association COPD Fact Sheet, 2014) But why does the common

man not know about COPD? It is equally as deadly as known diseases such as cancer, yet it

remains relatively unstudied. In order to direct the publics attention to this topic, it is extremely

important to research and study this condition to find an effective method for treatment.

Chronic Obstructive Pulmonary Disease: Emphysema and Chronic Bronchitis

Chronic Obstructive Pulmonary Disease, otherwise known as COPD, consists of two

separate diseases: emphysema and chronic bronchitis. Chronic bronchitis is caused by the

buildup of dust in the bronchial tubes. When someone breathes in, they take in dust particles and

other things along with the air. These particles cause irritation within the lungs. Fortunately, the

airways of the lungs have a special mucus lining to capture this dust and filter it out. However,

when a persons bronchial tubes are chronically irritated, they stay inflamed and the dust does

not filter out, making it hard to exhale. This irritation also impairs the cilia, hairs in the bronchial

tube, thus resulting in the thickening of the mucus. (COPD, n.d.) Symptoms of chronic bronchitis

are wheezing, cough, dyspnea and phlegm. Phlegm is the liquid hypersecretion by the mucous

membrane. Emphysema occurs when the alveolar sacs lose elasticity. The alveolar sacs are tiny

sacs within the lungs that allow oxygen and carbon dioxide to move within the lungs and

bloodstream. (Alveoli Function, n.d.) When someone normally breathes the air goes down the

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lungs and ends up in alveoli. The alveoli helps make the largest possible surface area so that

oxygen can move through the lungs and into the bloodstream. When the air sacks begin to

inflame they become weak and less flexible, thus causing their surface area to reduce. This also

results in the inadequate exchange of fresh oxygen. A collapsed lung, heart problems and large

holes in the lungs are some complications that can be a result of emphysema. The symptoms of

this disease are greatly similar to chronic bronchitis. (COPD, n.d.)

Figure 1: Spirometer Diagram

Furthermore, there are several ways to test for COPD. Spirometry is the most common

and effective method to diagnose emphysema, chronic bronchitis and other lung diseases such as

asthma, pulmonary fibrosis etc. This test measures the volume and the speed of the air that can

be exhaled. When one is being tested by spirometry, their nose is clipped and they are asked to

take in a full breath and then seal their lips around the mouthpiece of the spirometer. Nose clip

and mouthpiece are shown in figure 1. They are then told to blow out as fast and as much air as

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they can until their lungs are empty. (Seheult, 2014) The amount of total air exhaled into the

spirometer chamber, or the forced vital capacity (FVC), is then measured. Since COPD is an

obstructive disease it will impede the persons ability to release air instantaneously. So, after this

step has finished, the patient is then asked to take another deep breath and blow into the

spirometer one again. However, this time, the amount of air exhaled will only be measured for

one second. This will determine the forced expiratory volume in one second (FEV1). Finally in

order to determine the persons precise lung functionality, the FEV1 is divided by the FVC to

find the FEV1/FVC ratio. If the measurement determined is less than 0.7, then this is an

indication of a lung complication and possibly COPD. Spirometry can also assess if treatment is

opening up the airways. This is called reversibility. These readings tend to improve if the

airways become wider after medication. After the person has been given a bronchodilator, the

severity of COPD is determined by reversibility strictly based on the FEV1.

A. Mild COPD - FEV1 is 80% or more of the predicted value. This effectively means that
someone with mild COPD can have normal spirometry after bronchodilator medication.
B. Moderate COPD - FEV1 is 50-79% of the predicted value after a bronchodilator.
C. Severe COPD - FEV1 is 30-49% of the predicted value after a bronchodilator.
D. Very severe COPD - FEV1 is less than 30% of the predicted value after a bronchodilator.
(Spirometry NW Medicine, n.d.)

A diagram of the spirometer is shown above in Figure 1. (Nuffield Foundation, n.d.)

Another way to diagnose COPD is through the arterial blood gas test. This test measures

the levels of oxygen and carbon dioxide in the blood from an artery. To perform this test, blood

samples are taken from either the radial artery in the wrist, femoral artery in the groin, or the

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brachial artery in the arm. Abnormal results may be due to COPD. (Seheult, 2014) Normal

results of this test include:

A. Partial pressure of oxygen (PaO2): 75 - 100 mmHg

B. Partial pressure of carbon dioxide (PaCO2): 38 - 42 mmHg
C. Arterial blood pH: 7.38 - 7.42
D. Oxygen saturation (SaO2): 94 - 100%
E. Bicarbonate - (HCO3): 22 - 28 mEq/L

Note: mEq/L = milliequivalents per liter; mmHg = millimeters of mercury (Hadjiliadis, 2014)

To elaborate on the causes of COPD, according to a long term study done by

researchers in Sweden, smoking is recognised as the most important causative factor of it.

From this study, it was reported that approximately fifty percent of smokers eventually

develop COPD. Cigarette smoke contains several harmful toxins such as tar, arsenic, benzene

and many more that affect lung functionality. Long-term exposure to these toxins can lead to

high levels of abnormal lung irritation, causing COPD. The alveoli in the lungs also inflame

as a response to the toxins in cigarette smoke (Eur Respir, 2006). Total deaths from COPD

are projected to increase by more than 30% in the next 10 years without interventions to cut

risks, particularly exposure to tobacco smoke (WHO, 2013).

Unfortunately, there is no effective cure for both chronic bronchitis or emphysema,

but to relieve symptoms, doctors prescribe bronchodilator medications, steroids or antibiotics.

They also suggest that the patient to look into oxygen therapy, surgery or pulmonary

rehabilitation. A BiPaP or an endotracheal tube are often also given to improve ventilation

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while sleeping. These management options are beneficial because they also improve exercise

ability, sleep and the cognitive performance of the patient. (Celli, 2004)

Pathophysiology of Emphysema

To expand on emphysema, the lungs are a large part of the respiratory system. Their main

job is to maintain fresh air and get rid of waste gases in the body. Oxygen is needed for every

cell in the body. When we breathe in air, oxygen travels through the windpipe, or the trachea,

and into the lungs. The trachea divides into two main parts called the bronchial tubes. The

bronchial tubes subdivide into each lobe of the lungs and consist of bronchioles and alveoli. The

alveolus is a small anatomical structure that regulates gas exchange. The alveoli are made up of

elastic fibers called elastin. Elastin allow the alveoli to stretch as they fill with air when breathing

in. They then spring back during breathing out in order to discharge of carbon dioxide. Every

human contains about four hundred and eighty million alveoli. The alveolus has a huge surface

area. If all the alveoli in the human body were to be stretched out, they would take up as much

space as a tennis court. (Ochs, 2003) However, during COPD, the inflammation of the alveoli

occurs and they then shrink to having the surface area similar to the size of a table tennis court.

This causes the airway to become collapsible and the air cannot get out of the lungs. The lungs

become extremely unventilated. Inflammation may also cause walls between the air sacs to

deteriorate. (Khan, 2010) As shown in figure 2, alveoli lose the different chambers within them.

The walls are not thick and without shape. (Unknown, n.d.) Also, alveoli that have been distorted

by emphysema contain a large amount of dead space which fill up with CO2.

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Figure 2: Normal vs Alveoli with Emphysema

Furthermore, elastin, which maintains the surface area of the alveoli, is constantly being

broken down by elastase, a protease. A protease is an enzyme that regulates many bodily

functions. Alpha-1 antitrypsin, which is a protease inhibitor, inhibits elastase and allows for more

elastin, therefore regulating the shape of the alveoli. When inflammation occurs, elastase is

hypersecreted. Neutrophils, which are a type of white blood cell, produce too much elastase for

the alpha-1 antitrypsin inhibitor to manage. This results in the breakdown of elastin and

consequently, the impairment of the alveoli. Also, elastin maintains equilibrium of the chests

tendency to expand and the lungs tendency to collapse in. When elastin is broken down, this

equilibrium is set off and the chest begins to become abnormally bigger; thus, the total lung

capacity rises even though lungs are not well ventilated. (Khan, 2010)

In a study done by Cecile Onclinx et al., it was determined that distances between

alveolar duct walls in rats increased when injected with elastase, displaying that they had a large

area of dead space. In this experiment, there were three groups of rats that were categorized by

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either having one tracheal injection of 300 IU of elastase, two tracheal injections of the same

amount, or having a tracheal injection of 200 L of sterile normal saline. The group with the

highest amount of elastase induced had a significantly higher mean interwall distance (MIWD)

than the other two groups. The researchers concluded that this could have been caused by the

inflammation of the alveoli. When the inflammation occurred, the elastin broke down and this

resulted in the increased amount of dead space. To add to this study, they also tested the static

and dynamic compliance of the same rat lungs. Static and dynamic compliance measure the

lungs ability to stretch and expand. A great increase or decrease in compliance equates stiffer

lungs. Emphysema may be associated with an increase in the compliance, due to the loss of

alveolar and elastic tissue. (Martin, n.d.) The results of this experiment showed that the groups

injected with elastase had a significantly higher dynamic and static compliance, but there was not

any difference between the group injected once and the group injected twice. (Onclinx, 2006)

Enzymatic Inhibition

As stated before in this paper, an enzyme is a protein that speeds up a reaction by

providing an alternative path to overcoming activation energy. The substance in which the

enzyme acts upon is called the substrate. The selective qualities of an enzyme is called

specificity and the specific area in which the enzyme and substrate binds and reacts at is called

the active site. Enzymes bind temporarily to one or more reactant and when doing so they lower

the amount of energy needed thus speed up the reaction. (Pal, n.d.) In figure 3, the black line (Ea)

indicates the route a reaction would take without the help of an enzyme. The yellow line (Ea1)

indicates the route of a reaction in the presence of an enzyme. The amount of energy needed for

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Ea is
much more an the energy needed for Ea1. This shows the significance of an enzyme in a

reaction. However, sometimes the presence of an enzyme can be beneficial and other times it can

be detrimental. In the case of emphysema, the enzyme elastase speeds up the amount of elastin

being broken down, thus it causes the weak ventilation in the alveoli. To stop the action of an

enzyme, an inhibitor is needed.

Figure 3: Importance of Enzyme Diagram

An enzyme inhibitor is a substance that interacts in some way with the enzyme to

prevent it from working in the normal manner. (Ophardt, 2003) There are a variety of inhibitors

which include: nonspecific, irreversible, reversible - competitive and noncompetitive.

Nonspecific inhibition includes any physical or chemical action that can denature the entire

enzyme, and thus it is irreversible. An example of a nonspecific inhibitor can be extremely high

or low temperatures, excess amounts of acids or bases or a strong substance that had the ability

to drastically affect an enzyme. A specific inhibitor affects a specific enzyme. The inhibitor only

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has the ability to inhibit or slow down one enzyme. As stated earlier in this paper, alpha-1

antitrypsin only has the ability to inhibit elastase, thus it is a specific inhibitor. A competitive

inhibitor is a compound which has a close chemical structure to the enzymes substrate. The

inhibitor competes for the same active site as the substrate. Furthermore, the inhibitor may

interact with the enzyme at the active site and prevent any substrate-enzyme interaction. The

competitive inhibitor is, however, reversible if the substrate concentration is higher than the

inhibitor concentration. Lastly, a noncompetitive inhibitor is a substance that interacts with an

enzyme in a location other than its active site. The goal of a noncompetitive inhibitor is to

change the shape of the active site by changing the shape of the enzyme, so that consequently,

the substrate can no longer interact with the enzyme to give a reaction. Noncompetitive

inhibitors are usually reversible for the same reasons as a competitive inhibitor. (Ophardt, 2003)

Willow Tree Bark: Salicin

The use of willow bark dates back thousands of years to when Hippocrates and other

rising philosophers were coming up with new ideas and concepts. In 400 BC, patients were

advised to chew on the bark to reduce fever and inflammation. Willow bark has also been used

throughout history in China and Europe to treat pain, headaches and inflammatory diseases.

(Ehrlich, 2013) Physicians of ancient Greece, including Dioscorides, who wrote the precursor to

all modern pharmacopeias, prescribed willow for its analgesic and anti-inflammatory properties.

(Hedner, 1998) Today, we have determined that the chemical that gives willow bark these

properties is salicin.

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Salicin was first used to produce acetylsalicylic acid, which is most commonly known as

aspirin. Aspirin has the ability to act as an anticoagulant, a painkiller and it can be characterized

as a non-steroidal anti-inflammatory drug (NSAID). (Nordqvist, 2014) NSAIDs help to reduce

fever and relieve pain caused by headaches, muscle aches and stiffness. They also reduce

inflammation and swelling. (Pain Relievers, n.d.) Salicin is very similar to aspirin and some

studies show salicin being compared to aspirin for reducing pain and inflammation but at a much

lower level. Salicin also does not create unwanted side effects associated with aspirin, such as

gastric upset. (What is Salicin?, n.d.)

Moreover, from muscle and tendon to bone and ligament inflammation, the basic

characteristics of it remains the same. It can be defined by pain, heat, redness and swelling.

(Maroon et al, 2010) Osteoarthritis has recently been characterized as an inflammatory disease

due to the presence of synovitis. (Sokolove, 2013) Synovitis is the inflammation of the joint

lining called the synovium. (Synovitis, n.d.) In a study done by Schmid B, the efficiency of

salicins anti inflammatory and analgesic properties were tested on patients with osteoarthritis.

Seventy eight patients were divided into two groups, placebo or active treatment. The dimension

of pain, amount of muscle stiffness and physical functions were measured prior to treatment. The

active treatment group received 240 mg of salicin per day for two weeks. After this time period,

the dimension of pain, amount of muscle stiffness and physical functions were measured again

and compared to the initial scores. The pain score in the active treatment group was reduced by

14% from the baseline level after two weeks compared to an increase of 2% in the placebo

group. The amount of muscle stiffness also changed. Due to salicin previously producing a

NSAID and because salicin is characterized as anti inflammatory, scientists concluded this

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change was because the amount of inflammation in the joints decreased in the active treatment

group. (Schmid, 2001)

Furthermore, the anti inflammatory properties of salicin were shown in a study done by

Nirmal Verma et al. Mice suffering from inflammatory bowel disease (IBD) were observed after

seven days of salicin and dextran sulfate sodium administrations. Gut flora is a vital

microorganism that live in the digestive tracts of animals and humans. When IBD occurs the

amount of gut flora decreases and with no treatment, the bacteria can completely wipe out.

(Sisson, 2010) Through histological examinations, it was indicated that salicin had allowed for

the suppression of edema and mucosal damage. Also, it was observed that salicin had prevented

the loss of gut flora in the mice. Therefore, scientists concluded that salicin has an anti

inflammatory effect at the colorectal site and it may have a therapeutic value in ameliorating

inflammation during IBD. (Verma et al, 2013) If salicin has been successful in reducing

inflammation in the colon, it would be beneficial to test if it has the same ability with

emphysema. Furthermore, if an experiment is conducted and salicin is proven to have the ability

to inhibit elastase then it would also be important to find out, specifically, what type of

enzymatic inhibition does it perform.

In conclusion, emphysema is a progressive disease, mainly caused by smoking. Although

it has been studied by researchers, it still has very few effective methods for treatment. The

hypersecretion of elastase can be very deadly and can cause the inflammation of the elastic fibers

in the alveoli. Consequently, the lungs become extremely unventilated and clogged. However,

with the success of several anti inflammatory substances inhibiting elastase, the future of

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inhibition of elastase as a treatment looks bright. Based on previous studies, it has been shown

that the willow bark salicin has many anti inflammatory properties, thus it may have the ability

of inhibiting elastase, and therefore treating emphysema.

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Materials:
Neutrophil elastase colorimetric drug discovery kit (Contains elastase, substrate,
calibration standard, control inhibitor, buffer, 96-Well Microplate.)
D-( - ) Salicin 5g
Pipets capable of pipetting .1-200 ul accuracy
Pipet Tips capable of pipetting .1-200 ul accuracy
Pipets capable of pipetting .02-20 ml accuracy
Ice bucket
Glass test tubes
Distilled Water
Gloves
Beaker
Graduated Cylinder
Scoopula
Glass stir rod
Wax paper
Goggles

Equipment:
Incubator
Absorbance Microplate reader
Absorbance Microplate reader filter 405 nm
Scale
Centrifuge
Electronic Pipet

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Procedure

Free Trial: Finding Most Effective Concentration of Salicin:

1) Make 1000 uM stock solution with 0.028g of Salicin diluted in 100 mL of distilled water.

2) Prepare the following dilutions for the following concentrations of salicin with the stock

solution. Prepare each in a separate test tube.

Salicin Concentration (uM) Volume of 1 mM Stock (ml) Volume of Water (ml)

500 10 10

250 5 15

100 2 18

50 1 19

10 .2 19.8

1 .02 19.98

3) Defrost Neutrophil Elastase Colorimetric Drug Discovery Kit components and hold on ice

until use. Briefly centrifuge all vials. Minimize the time that any kit component is thawed.

4) Dilute 8 uL of substrate BML-P213-9090 with 72 uL of assay buffer in a test tube. Label

test tube substrate. A few minutes prior to start of assay, warm to reaction temperature (37oC).

5) Dilute 1.6 uL of neutrophil elastase enzyme with 158.4 uL of assay buffer in a test tube.

Label test tube enzyme. A few minutes prior to start of assay, warm to reaction temperature

(37oC).

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6) Dilute 1 uL of inhibitor elastatinal BML-PI103-9090 with 39 uL of assay buffer in a test

tube. Label test tube KI (kit inhibitor). A few minutes prior to start of assay, warm to reaction

temperature (37oC).

7) 96 Well Plate Diagram Shown Below. Add the specified amounts of each solution to the

corresponding well. Each wells total volume is 95 uL.

10 11

A 20 uL of Salicin with concentration of 20 uL of Salicin with

500 uM concentration of 500 uM
10 uL of elastase solution 10 uL of elastase solution
65 uL of assay buffer 65 uL of assay buffer

B 20 uL of Salicin with concentration of 20 uL of Salicin with

250 uM concentration of 250 uM
10 uL of elastase solution 10 uL of elastase solution
65 uL of assay buffer 65 uL of assay buffer

C 20 uL of Salicin with concentration of 20 uL of Salicin with

100 uM concentration of 100 uM
10 uL of elastase solution 10 uL of elastase solution
65 uL of assay buffer 65 uL of assay buffer

D 20 uL of Salicin with concentration of 20 uL of Salicin with

50 uM concentration of 50 uM
10 uL of elastase solution 10 uL of elastase solution
65 uL of assay buffer 65 uL of assay buffer

E 20 uL of Salicin with concentration of 20 uL of Salicin with

10 uM concentration of 10 uM
10 uL of elastase solution 10 uL of elastase solution
65 uL of assay buffer 65 uL of assay buffer

F 20 uL of Salicin with concentration of 20 uL of Salicin with

1 uM concentration of 1 uM
10 uL of elastase solution 10 uL of elastase solution
65 uL of assay buffer 65 uL of assay buffer

G Add the 20 uL of KI solution made Add the 20 uL of KI solution

in step 4 made in step 4
10 uL of elastase solution 10 uL of elastase solution
65 uL of assay buffer 65 uL of assay buffer

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H 10 uL of elastase solution 10 uL of elastase solution

85 uL of assay buffer 85 uL of assay buffer

8) Incubate plate for 30 minutes at reaction temperature (37oC) to allow inhibitor\enzyme

interaction.

9) Start assay by the addition of 5 uL BML-P213-9090 substrate (diluted and equilibrated to

reaction temperature in step 4).

10) Continuously read plates at A405nm in absorbance microplate reader. Record data at 1 min.

time intervals for 10 mins.

11) Remove assay plate from absorbance microplate reader. Clean well plate and test tubes.

Testing Effectiveness of Salicin on Different Concentrations of Substrate:

1) Defrost Neutrophil Elastase Colorimetric Drug Discovery Kit components and hold on ice

until use. Briefly centrifuge all vials. Minimize the time that any kit component is thawed.

2) Prepare the following solutions with substrate BML-P213-9090 and the assay buffer

included in the kit. A few minutes prior to start of assay, warm all solutions to reaction

temperature (37oC).

Substrate Substrate (uL) Buffer (uL) Total Volume (uL)

Concentration (M)

0.00173 (A) 2.6 27.4 30

0.00186 (B) 2.8 27.2 30

0.00199 (C) 3 27 30

0.00213 (D) 3.2 26.8 30

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3) Dilute 2 uL of inhibitor (elastatinal BML-PI103-9090) with 78 uL of assay buffer

BML-KI581 in separate test tube. Label test tube KI for kit inhibitor. Warm to reaction

temperature (37oC)

4) Dilute 2.2 ul neutrophil elastase enzyme in 197.8 ul assay buffer. 10 ul per well. (Make sure

not to add enzyme to blanks! Follow well diagram.) Warm to reaction temperature.

5) Make 1000 uM stock solution with 0.028 grams of Salicin diluted in 100 mL of distilled

water.

6) From stock solution prepare the following dilution

Salicin Concentration Volume of 1 mM Stock (ml) Volume of Water (ml)

1 uM 0.02 19.98

7) 96 Well Plate Diagram Shown Below. Pipet the following amounts of the prepared solutions

to the corresponding wells. (Columns 9-12 are not shown because they are not used.)

Key: B - Blank
C - Control
IE - Inhibitor Elastatinal
TI- Test Inhibitor (Salicin)
X - Well Not Used

1 2 3 4

A B B B B
-95 uL assay buffer -95 uL assay buffer -95 uL assay buffer -95 uL assay buffer
-0 uL neutrophil -0 uL neutrophil -0 uL neutrophil -0 uL neutrophil
elastase elastase elastase elastase
-0 uL elastatinal -0 uL elastatinal -0 uL elastatinal -0 uL elastatinal
-0 uL salicin -0 uL salicin -0 uL salicin -0 uL salicin

B C C C C
-85 uL assay buffer -85 uL assay buffer -85 uL assay buffer -85 uL assay buffer
-10 uL elastase -10 uL elastase -10 uL elastase -10 uL elastase
-0 uL elastatinal -0 uL elastatinal -0 uL elastatinal -0 uL elastatinal
-0 uL salicin -0 uL salicin -0 uL salicin -0 uL salicin

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C TI TI TI TI
-65 uL assay buffer -65 uL assay buffer -65 uL assay buffer -65 uL assay buffer
-10 uL elastase -10 uL elastase -10 uL elastase -10 uL elastase
-0 uL elastatinal -0 uL elastatinal -0 uL elastatinal -0 uL elastatinal
-20 uL salicin -20 uL salicin -20 uL salicin -20 uL salicin

D TI TI TI TI
-65 uL assay buffer -65 uL assay buffer -65 uL assay buffer -65 uL assay buffer
-10 uL elastase -10 uL elastase -10 uL elastase -10 uL elastase
-0 uL elastatinal -0 uL elastatinal -0 uL elastatinal -0 uL elastatinal
-20 uL salicin -20 uL salicin -20 uL salicin -20 uL salicin

E TI TI TI TI
-65 uL assay buffer -65 uL assay buffer -65 uL assay buffer -65 uL assay buffer
-10 uL elastase -10 uL elastase -10 uL elastase -10 uL elastase
-0 uL elastatinal -0 uL elastatinal -0 uL elastatinal -0 uL elastatinal
-20 uL salicin -20 uL salicin -20 uL salicin -20 uL salicin

F IE IE IE IE
-65 uL assay buffer -65 uL assay buffer -65 uL assay buffer -65 uL assay buffer
-10 uL elastase -10 uL elastase -10 uL elastase -10 uL elastase
-20 uL elastatinal -20 uL elastatinal -20 uL elastatinal -20 uL elastatinal
-0 uL salicin -0 uL salicin -0 uL salicin -0 uL salicin

G X X X X

H X X X X

8) Incubate plate for 30 minutes at reaction temperature (37oC) to allow inhibitor\enzyme

interaction

9) Start assay by the addition of 5 uL (various concentrations diluted and equilibrated in step 2)

of the correct concentrations of substrate to the corresponding wells. Follow well diagram below.

Total volume of all wells should be 100 uL.

1 2 3 4

A 5 uL of substrate 5 uL of substrate 5 uL of substrate 5 uL of substrate

concentration A concentration B concentration C concentration D

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B 5 uL of substrate 5 uL of substrate 5 uL of substrate 5 uL of substrate

concentration A concentration B concentration C concentration D

C 5 uL of substrate 5 uL of substrate 5 uL of substrate 5 uL of substrate

concentration A concentration B concentration C concentration D

D 5 uL of substrate 5 uL of substrate 5 uL of substrate 5 uL of substrate

concentration A concentration B concentration C concentration D

E 5 uL of substrate 5 uL of substrate 5 uL of substrate 5 uL of substrate

concentration A concentration B concentration C concentration D

F 5 uL of substrate 5 uL of substrate 5 uL of substrate 5 uL of substrate

concentration A concentration B concentration C concentration D

G X X X X

H X X X X

10) Continuously read plates at A405nm in absorbance microplate reader. Record data at 1 min.

time intervals for 10 minutes.

11) Clean lab area and materials and dispose of any used pipets.

12) Perform data analysis

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Results

Neg Elastatinal 500uM 250uM 100uM 50uM 10uM 1uM

Control

Trial 1 1.081818 0.87272727 0.554545 0.790909 0.618181 0.690909 0.72727 0.95091

Trial 2 2.254545 1.82727273 0.6 0.872727 1.063636 0.645455 1.19091 1.69091

Average 1.668182 1.34999979 0.577273 0.831818 0.840909 0.668182 0.95909 1.32091

Units: OD\min

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Substrate Blank Control Trial 1 with Trial 2 with Trial 3 with Elastatinal
Concentration Salicin Salicin Salicin Trial

.00173 (A) 0.52818182 0.39090909 0.509090909 0.509090909 0.518181818 0.518181818

.00186 (B) 0.536363636 0.450505051 0.5363636364 0.5354545455 0.5181818182 0.5354545454

.00199 (C) 0.554545455 0.495959596 0.5545454545 0.5436363636 0.5545454545 0.5323232323

.00213 (D) 0.681818182 0.532323232 0.6747474747 0.6818181818 0.6818181818 0.6272727273

Units: OD\min

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Average Blanks vs. Average Elastatinal (All Substrate Concentrations)

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Average Blanks vs. Average Salicin (All Substrate Concentrations)

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Average Control vs. Average Elastatinal (All Substrate Concentrations)

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Average Control vs. Average Salicin (All Substrate Concentrations)

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Data Analysis

For determining the most effective concentration of salicin on constant concentrations of

salicin, it was necessary to compare them to two control groups, the negative control, which

contained no inhibitor, and the elastatinal trial. During trial one, 500 uM showed the least

absorbance of color, while 1 uM showed the most. The 250 uM trial absorbed .2363636 OD\min

more than 500 uM. 100 uM absorbed more than 250 uM and 10 uM absorbed more than 100uM.

50 uM absorbed less than 100 uM, 250 uM, 10 uM, and 1 uM but it absorbed similarly to 500

uM. 1 uM showed the most similarity to the elastatinal trial. There was an increase in absorbance

from 500 uM to 1 uM, except for the data for 50 uM. To validate these results, another trial was

conducted. The data in trial 2 showed the same trend as trial one. Due to 1 uM of salicin having a

similar absorbance as elastase inhibiting elastatinal, it was chosen as the most effective

concentration.

After establishing 1uM to be the most effective concentration of salicin, it was used to

determine its efficiency in inhibiting elastase from breaking down different concentrations of the

substrate. For .00173 M (A) concentration of the substrate the control showed a 0.39091 OD\min

absorbance and the blank showed a 0.52818 absorbance. The average absorbance with salicin

was 0.51212 and for elastatinal it was 0.51818. Both salicin and elastatinal absorbed more than

the control and similarly to the blank. On average, salicin absorbed about 0.00970 less than the

blank and 0.17576 more than the control. Elastatinal absorbed 0.00364 less than the blank and

0.12424 more than the control. The average difference in absorbance between salicin and

elastatinal was 0.00606 OD\min.

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For 0.00186 M (B) concentration of the substrate the control showed a 0.53636 OD\min

absorbance and the blank showed a 0.45051 absorbance. Wells treated with salicin showed an

average absorbance of 0.52999 and the absorbance with elastatinal was 0.53545. For this

concentration too, both salicin and elastatinal absorbed more than the control and similarly to the

blank. Wells with elastatinal absorbed 0.08495 more than the control and 0.00009 less than the

blank. On average, wells with salicin absorbed 0.07798 more than the control and 0.00609 less

than the blank. The average difference in absorbance between salicin and elastatinal was 0.00546

OD\min.

For 0.00199 M (C) concentration of the substrate, the control absorbed 0.49596 OD\min

and the blank absorbed 0.55455. For this concentration, as well, both salicin and elastatinal

absorbed more than the control and similarly to the blank. Wells treated with salicin absorbed

0.55091 OD\min on average, and with elastatinal 0.53232 was absorbed. Wells with elastatinal

absorbed 0.03636 more than the control and 0.02222 less than the blank. On average, wells with

salicin absorbed 0.05494 more than the control and 0.01091 less than the blank. The average

difference in absorbance between salicin and elastatinal was 0.01859 OD\min.

Lastly, for 0.00213 M (D) concentration of the substrate, the control absorbed 0.53232

OD\min and the blank absorbed 0.68182. On average, wells treated with salicin absorbed

0.67946 OD\min and wells with elastatinal absorbed 0.62727. For this concentration, as well,

both salicin and elastatinal absorbed more than the control and similarly to the blank. Wells

treated with salicin absorbed, on average, 0.14713 more than the control and 0.00236 less than

the blank Wells with elastatinal absorbed 0.09495 more than the control and 0.05455 less than

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the blank. The average difference in absorbance between salicin and elastatinal was 0.05219

OD\min.

Based off of the graphs indicating the correlation between the averages of all four

substrate concentrations, it was shown that the average of the blanks versus the average of the

elastatinal trials had a correlation coefficient of 0.98. The blanks with salicin had a correlation

coefficient of 1.00. The controls and elastatinal had a 0.62 correlation coefficient and the

controls with salicin has a 0.70 correlation coefficient.

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Discussion

Several things can be concluded from the data collected. In order to determine the

efficiency of salicin inhibiting elastase, it was first necessary to determine which concentration

of salicin was the most effective, if any, in inhibiting elastase. Since the wells with 500 uM and

50 uM of salicin showed about the same absorbance, it was not clear if the inconsistency was due

to because the concentrations from 500 to 50 uM of salicin were denaturing the enzyme.

Nevertheless, the results also showed that the absorbance of 1 uM and the control inhibitor,

elastatinal, were about the same. This meant that salicin of 1uM was inhibiting elastase as much

as elastatinal was. Since elastatinal has already been proven as elastase inhibiting, the 1 uM of

salicin was chosen to be most effective. This concentration was used to test the efficiency of

salicin on different concentrations of the substrate.

For this test, the data showed that salicin was able to inhibit elastase as well as elastatinal

was. The blanks only contained the substrate and buffer with no enzyme. Thus, the data for the

blanks showed the absorbance of the substrate before it was broken down by the enzyme. The

control wells contained the enzyme, substrate and buffer. Thus, this data showed the absorbance

of the substrate after being broken down by the enzyme. A lower absorbance meant that there

was more substrate in the well and more substrate can be explained by the enzyme breaking

down the substrate. Wells treated with salicin absorbed similarly to the blank. This meant that

salicin was able to maintain the substrates concentration for all four concentrations tested. The

data also showed that salicin was able to inhibit elastase from breaking down the substrate and

consequently decreasing the amount of absorbance. The correlational graphs showed that there

was a correlation coefficient of 1.00 for the blanks and salicin. The closer the correlation is to 1,

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the stronger the relationship is. This meant that salicin and the blanks were very closely

correlated and other points can easily be predicted using the graphs best fit line. Salicin and the

blanks were more correlated than elastatinal the the blanks. This meant that the absorbance of the

wells with salicin and the blanks were more similar than elastatinal and the blanks. Compared to

elastatinal, salicin inhibited elastase better for the substrate concentrations C and D, the higher

two concentrations. On average, however, salicin and elastatinal inhibited elastase very similarly.

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 Banerjee

Experimental Error

There were several areas that error could have occurred. It was hard to see and verify if

the correct amounts were going into each well since microliters is such a small unit. It was

difficult to verify if small amounts such as 0.4 uL were properly transferring from the pipet tips

into the wells. If these small measurements were not transferring correctly, then this may have

caused an overall difference in the amount of absorbance. For example, if 0.1 uL of the substrate

did not transfer properly from the pipet tip to the well, then a lower absorbance would have

showed up for that well. To avoid this error in the future, the use of a pipet that says how much it

has pipetted and released would be helpful to verify the correct transfer of material.

Also, when measuring 0.028 grams of salcin for the stock solution, the actual amounts

deviated a small amount from 0.028. This may have caused the solutions to be either a higher or

lower molar concentration than indicated. In addition, the salicin solutions were made 8 hours

prior to testing. Although, it is not known for sure, this 8 hour waiting period may have

diminished the effects of salicin. Thus, if the solutions were made immediately prior to testing,

then the effects may have been a bit stronger. In order to eliminate this error in the future,

solutions should be made immediately before testing to make sure that the effects of salicin are

not being diminished by a waiting period.

Another source of error may have occurred when the vials in the Neutrophil Elastase

Colorimetric Drug Discovery Kit were being defrosted. For each trial, there wasnt a set time

indicated that the components would defrost until. The components were placed in the incubator

at 37oC and they were checked on periodically to see if they felt warm enough to test with.

Consequently, for each trial the amount of defrosting time may have been different. This may

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have resulted in the components being more active in some trials than others. In order to make

sure that all the components are functioning at the same level for all trials, in the future it would

be necessary to indicate how much time the components should defrost for.

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Conclusion

The world today is filled with abundant sources of technology, science and ideas.

However, even with so many resources, so many problems are left unattended. Chronic

Obstructive Pulmonary Disease is the third leading cause of death in America, yet it is relatively

left unstudied. Emphysema, a branch of COPD, is a progressive, inflammatory disease, mainly

caused by smoking. With 1 in every 5 adults smoking, it is necessary to find effective methods of

treatment for emphysema. (ACS, 2014) The purpose of this experiment was to determine if

salicin, a natural anti inflammatory substance, has the ability to inhibit elastase and eventually be

used as another method of treatment for emphysema. The hypothesis was that if elastase reacted

with different concentrations of elastin, is treated with salicin, then the concentration of elastin

with no enzyme will be the same as elastin reacting with both the enzyme and salicin. This is

because salicin has anti inflammatory properties that should be able to inhibit elastase from

breaking down elastin and changing its concentration.

In order to conduct this experiment, the most effective concentration of salicin, if any,

had to be determined. For this part of the experiment, concentrations of salicin ranging from 500

uM to 1 uM were tested on constant amounts of the substrate and compared to a negative control

and a control inhibitor of elastase. The data showed that the wells with 1uM of salicin had a

similar absorbance to the wells with the control inhibitor, elastatinal. Due to this finding, the

1uM concentration of salicin was chosen to be the most effective and it was used on the next part

of the experiment. The second part of this experiment consisted of 1uM of salicin being tested on

different concentrations of the substrate. The different concentrations signified how the substrate

would be broken down in different stages of emphysema in real life. In a higher stage of

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emphysema, there would be a higher concentration of the substrate. For this part, the data

showed that salicin had maintained the concentration of the substrate. The wells without the

enzyme and just the substrate had very similar absorbances in comparison to the wells with the

enzyme treated with salicin. This applied for all four concentrations of the substrate. In

comparison to elastatinal, salicin inhibited elastase better in the higher two concentrations of the

substrate. Overall, however, salicin and elastatinal inhibited elastase very similarly.

In conclusion, the hypothesis was supported. It was predicted that salicin would be able

to inhibit elastase and keep the concentration of elastin with no enzyme and elastin reacting with

both the enzyme and salicin the same. The average difference between the blanks and the wells

treated with salicin was 0.00727 OD\min. This inhibition can be explained by salicins anti

inflammatory properties. It was able to act as a specific inhibitor and prevent any

substrate-enzyme interaction. Due to this finding, salicin would be effective in treating

emphysema. Although future research must be conducted, it is shown that salicin inhibited

elastase better than elastatinal in the higher two concentrations of the substrate. Since the higher

concentrations signified higher degrees of emphysema, it may be concluded that salicin would be

more effective in treating higher degrees of emphysema.

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 Banerjee

Impact

We can use this information that was concluded from this experiment by testing salicins

effects on the lungs of a COPD patient. If successful, salicin would be an effective, cheap and

abundant source to treat emphysema with. Unlike other forms of treatment, salicin would be

natural.

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 Banerjee

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