Chapter 1

The Problem

Introduction

Discussion of plants in general and its medicinal value.

Discussion of Pumpkin plant

Discussion of inflammation

Discussion of anti-inflammatory and anti-inflammatory drugs

Discussion of the significance of the study

Note: Do NOT use first person words like you, our, we, me, us etc.

In day today life technology has been developed to a certain extent,

particularly in medicines. Many scientists had find out many solutions for many

problems by the technology developed today. Medicine used in day today life is

familiar in curing diseases (using chemicals). In olden ages the world followed

traditional medicine to cure diseases with the help of medicinal plants.

Plants play a major role among the olden age people. But, now the modern

age people were using the modern medicine which we are using today. Our

traditional is the best method to cure diseases in ancient civilizations. So, the

people gain more knowledge by curing diseases with the medicinal plants.

Traditional medicine is the one followed till now in some of the countries most

especially India and China

Plants have been used from ancient times to attempt cures for diseases and

to relive physical suffering. Ancient peoples all had acquired some knowledge of

medicinal plants. Often times these primitive attempts at medicine were based on
superstition and speculation. Evil spirits in the body were thought to be the cause

of medical problems. They could be driven out of the body through the use of

poisonous or disagreeable plant substances that rendered the body a disagreeable

habitat. Medicine men or women of a tribe were usually charged with knowledge

of such plants. The progress of medicine has often been guided by the earlier

observations and beliefs.

Medicinal plants are always of especial interest. As early as 5,000 B.C.

many drugs were in use in China. Sanskrit writings testify to methods of

gathering and preparing drugs in these early times. The Babylonians, ancient

Hebrews and Assyrians were all familiar with medicinal plants. From Egypt there

are records dating to 1,600 B.C. naming many of the medicinal plants used by

physicians of that period, among which myrr, opium, cannabis, aloes, cassia and

hemlock are prominent. The Greeks were familiar with many of the drugs of

today, evidenced by the works of Hippocrates, Theophrastus, Aristotle and

Pythagoras. The supernatural element continued to remain prominent in their

culture, however. Only a few individuals were thought able because of some

special power to distinguish harmful from valuable plants. This rhizotomoi or

root diggers were an important caste in ancient Greece. In Rome there was less

interest in plants that had healing powers. But by 77 BC Dioscorides wrote in his

treatise, De Materia Medica, dealing with the nature and properties of all the

medicinal substances known at that time. This work was highly esteemed for 15

centuries and to this day is valued in parts of Turkey and North Africa. Pliny

and Galen also described the nature of some drug plants (Hill 1952).
 Following the Dark Ages there began a period of the encyclopedists and

herbalists. The monasteries of Northern Europe produced large compendiums of

information regarding plants, much of which was false. They stressed the

medicinal value and folklore of plants. About the same time there appeared a

Doctrine of Signatures. This superstitious doctrine suggested that all plants

possessed some sign, given by the Creator, which indicated the use for which

they were intended. A plant with heart-shaped leaves was good for heart ailments;

the liverleaf with its 3-lobed leaves was good for liver problems, etc. Many of

the common names of plants owe their origin to this superstition. Names such as

heartease, dogtooth violet, Solomons seal and liverwort are examples.

Pharmacology and pharmacognosy owe their beginnings to the earlier

beliefs and knowledge about medicinal plants. The interest in medicinal plants was

especially pronounced among the early botanists who were often physicians.

(http://www.faculty.ucr.edu/~legneref/botany/medicine.htm)

Inflammation is the body's response to stress - whether from your diet,

lifestyle or environment. Like for instance when someone catches a cold. Someone

may experience inflammation in the form of a fever as your body heats up to

eradicate the effects of the invading virus. This kind of inflammation is good, but

the modern epidemic of chronic, low-grade inflammation destroys the balance in

your body. When a body's systems experience a constant inflammatory response, a

person becomes more susceptible to aging and disease. Chronic low-grade food

allergies or food sensitivities that may cause few symptoms.

 An imbalance of bacteria and fungi in gastrointestinal tract, also known as

dysbiosis. This causes immune system to overreact to bacteria in your gut and

can be without notable symptoms.

Stress Constant psychological, emotional or physical stress raises the level

of cortisol, creating inflammation. For more on the effects of stress on your body,

read How Stress Creates a Double-Whammy Threat to Your Health.

Environmental toxicity from our air, water, food pollutants and toxic metals

like mercury and lead all contribute to inflammation and have been linked to

diseases as varied as endometriosis and cancer. Diet and lifestyle: too much fat,

sugar, and protein in your diet, constant dehydration, consumption of too many

sodas or caffeine, inactivity, and lack of sleep can all increase inflammation in

your body.

(http://bodyecology.com/articles/inflammation_cause_of_disease_how_to_prevent.php)

This study is important in the sense that if the anti-inflammatory property

of pumpkin leaves (Cucurbita pepo) will be proven in the prevention of

inflammation or edema . The people can overcome the disease caused during

inflammation and get miraculous benefactors from the plant that prevents the side

effect of synthetic drugs. This study looks into the anti- inflammatory activity in

pumpkin leaves extract against inflammation .

Statement of the Problem

This study generally aimed to evaluate the anti-inflammatory property

of Pumpkin leaf (Cucurbita pepo) extract on inflammation - induced rats.

 Specifically, it sought to answer the following questions:

1. What is the anti-inflammatory property of pumpkin leaf in terms of

the edema in the inflammation-induced rats making use of the following

treatments:

T0 - Aspirin

T1 - 100% pumpkin leaf extract

2. Is there a significant difference between the treatments used

in terms of the edema level of the inflammation-induced rats?

Scope and Delimitation

Base this part from Madans Group!

The study focused on the anti-inflammatory property of pumpkin leaf

(Cucurbita pepo ) extract is being done to know if the pumpkin leaf extract is an

effective anti-inflammatory agent. The study covered mainly on the inflammatory

responses and pumpkin leaf reaction to the inflammation.

The research study would be conducted at the National products Chemistry

and biotechnology laboratory science complex I, University of Northern Philippines,

Tamag, Vigan city, Ilocos sur from march to april of 2017.

Review of Related Literature

This section included the summary of readings and studies which researchers

considered relevant to the study.

Medicinal Plants

Medicinal plants have been used since ancient times for human health care

and remained the most widely used medication system in developing and least

developed nations are dependent on traditional medicines. There has been a

continuous growth of demand for herbal medicines globally. The demand has been

increasing as a result of growth of human populations and the frequently

inadequate provision of modern medicine. (http://www.ethnobiomed.com)

Recently, World Health Organization estimated that 80% of people

worldwide rely on herbal medicines for some part of their primary health care. In

Germany, about 600 700 plant based medicines are available and are prescribed

by some 70% of German physicians. In the past 20 years in the united states,

public dissatisfaction with the cost of the prescribed medicines, combined with an

interest in returning to the natural or organic remedies,, has led to an increase in

herbal medicine use. (https://umm.edu)

It is estimated that about 80,000 species of plants are utilized in some

form or the other by the different systems of Indian medicine. The knowledge

about plants and plant products is detailed , sophisticated and has evolved into a

separate Shastra itself called Dravyaguna Shastra . Plants have been studied on the

basis of clearly defiened biological parameters like rasa (taste), vipaka (metabolic

property), guna (quality), prabhava (biological effect) and virya (potency). So the

ayurvedic drugs are rightly called the elixirs of life. (http://www.blogspot.com ,

encyclopedia of ayurvedic medical plant)

 According to KN Reddy, G.Thirythulu and Sudhakar Tradionally healing

systems play an important role in maintaining the physical and psychological well

being of the vast majority of tribal people in different communities. There are

many people using plants to cure their illness instead of seeking for any

professional help.

Pumpkin Leaf

In India for cooking most of the time whole fresh pumpkin leaf is used. It

is used for its flavor and fragrance . Indian foods like biryani, mutton, vegetable

pulao etc. use whole pumpkin leaves which are removed before serving the dish.

(Nutrition and Health benefits of pumpkin leaf 2013)

The pumpkin leaf is native to India, Nepal and Bhutan. Pumpkin leaf in

India is also called by names like Tejpatta, Tamalpatra, Tejpat. It is also known

as Indian cassia, malabar leaf etc. (nutrition and health benefits of pumpkin leaf

2013)

The pumpkin leaf is twice as long as the bay laurel leaf and has and olive

green color. It has 3 prominent veins running down the length of the leaf

compared to just 1 prominent vein in the pumpkin leaf. Pumpkin leaf has a very

cinnamon like aroma while the bay laurel leaf's aroma resembles that of pine and

lemon. (nutrition and health benefits of pumpkin leaf 2013)

Pumpkin leaves yield many volatile oils like a-pinene, camphene, myrcene,

limonene, p-cymene, eugenol etc with eugenol being the .major essential component.

The leaves are also exceptionally rich in many vitamins and minerals. (nutrition and

health benefits of pumpkin leaf 2013)

 According to R. Singh the pumpkin leaf has been used medicinally since

ancient times and was a major medicinal plant in medieval times. Indian sages

since the earliest times advocated the use of many spices both for culinary as well

as medicinal use and the pumpkin leaf was one of them.

According to Goldberg : Not only do pumpkin leaves taste good, but they

are good for health. The Pumpkin leaf has two properties, that are detoxifying and

slow aging. This herb also fights bacterial infections and improves everything

from respiratory issues to heart health. It is a powerful herb, from which everyone

can benefit.

Ayurvedic Importance of Pumpkin Leaf

Pumpkin leaf is an important ingredient in Ayurvedic tea used for treating

coughs and colds.

For problems like cough, cold, excess mucus, allergy and other respiratory

problems etc, crush 3 4 pumpkin leaves, add 1 - 2 tulsi (holy basil) leaves and

boil this in about 250 ml water. Cool, strain and drink this while still tolerably

warm.

For headache, even severe headache, boil a few pumpkin leaves in water

for a few minutes. Strain and cool a bit and drink while still hot. Also apply

paste of pumpkin leaves on the forehead for relief from severe headache.

For getting rid of head lice, take about 50 grams of fresh pumpkin leaves

and boil in 400 ml water. Remove when the water is about 100 ml remaining.

Apply this on the hair roots after straining out the water. Let it remain for 3 - 4
hours then wash. It removes lice. If bhringraj is added to the bay leaves before

boiling, it will make the hair black, lustrous and health.

In respiratory diseases like asthma etc, add 2 grams Mulethi (liquorice) and

1 gram Piplali or dry ginger powder to 5 grams of pumpkin leaves and boil in

200 ml water till 100 ml water remains. Strain and drink. It boosts body immunity

and cures conditions like cough, asthma and other respiratory problems. (Chemical

compositions of (cucurbita pepo)

Chemical Composition of Pumpkin Leaf

Synthesize the phytochemical present in a paragraph form.

Nutritional value per 100 grams

Principle Nutrient Value Percentage of RDA

Energy 313 Kcal 15.50%
Carbohydrates 74.97 g 57%
Protein 7.61 g 13%
Total Fat 8.36 g 29%
Cholesterol 0 mg 0%
Dietary Fiber 26.3 g 69%
Vitamins
Folates 180 mcg 45%
Niacin 2.005 mg 12.50%
Pyridoxine 1.740 mg 133%
Riboflavin 0.421 mg 32%
Vitamin A 6185 IU 206%
Vitamin C 46.5 mg 77.50%
Electrolytes
Sodium 23 mg 1.50%
Potassium 529 mg 11%
Minerals
Calcium 834 mg 83%
Copper 0.416 mg 46%
Iron 43 mg 537%
Magnesium 120 mg 30%
Manganese 8.167 mg 355%
Phosphorus 113 mg 16%
Selenium 2.8 mcg 5%
Zinc 3.70 mg 33%

The chemical composition of pumpkin leaf according to USDA National

Nutrient data base

Inflammation

Inflammation is a part of bodys immune response. When something

harmful or irritating affects a part of our body, there is a biological response to

try to remove it, the signs and symptoms of inflammation, specifically acute

inflammation, show that the body is trying to heal itself. However sometimes

inflammation can cause further action like self-perpetuating. More inflammation is

created in response to the existing inflammation. The first stage of inflammation

is often called irritation, which then becomes inflammation-the immediate healing

process. (http://www.medicalnewstoday.com)

It is a symptom of great number of diseases, even though it is a response

of the host for the infection if right untreated becomes more fatal than the

disease itself. ( Hakim, Ajuddin, Gufran, Nasreen 2009)

Treatments

Anti-inflammatory pain killers are used to treat arthritis pains painful

periods and other painful conditions. Most people have no troubles taking this

tablets however some people develop side-effects which can sometimes be serious

in particular or if you have any cardiovascular or certain gut conditions, or are

elderly, this medicines are prescribed with caution and only when there are no
alternatives at the lowest doses and durations necessary.

(http://patient.info/health.com)

Anti-inflammatory pain killers are sometimes called non-steroidal or anti-

inflammatory drugs. Anti-inflammatory painkillers include- aceclofenac, acemetacin,

aspirin, celecoxib, meloxicam, indomethacin, etodolac. Anti-inflammatory drugs

works by blocking the effect of chemicals called cyclo oxygenase enzymes. These

enzymes help to make other chemicals called prostaglandins. Some prostaglandins

are involved in the production of pain and inflammation at sites of injury and

damage a reduction in prostaglandin production reduces pain and inflammation.

(http://patient.info/health.com )

Anti- inflammatory agents do not alter the course of painful conditions such

as arthritis. They just ease symptoms of pain and stiffness. However , this may

provide further benefit because, if pain is eased, you may then be able to move

around more easily or use a painful joint more easily. The inflammation and pain

of various types of arthritis often come and go.( http://patient.info/health/anti-

inflammatory )

Most possible side effects of taking the anti-inflammatory drugs are

commonly nausea, vomiting but in some rare conditions they even damages the

lining of stomach and gut which causes bleeding. This is because the chemicals

that are reduced by anti- inflammations are also involved in helping to protect he

lining of the stomach from the effects of the acid with in the stomach. Therefore

people taking larger amounts of anti-inflammatory drugs are likely to get

abdominal pains. (http://patient.info)

Discussion of related studies on anti-inflammatory using other plants.

Discussion of Pumpkin plant used in any studies.

Conceptual Framework

Format the arrows..

The experimental paradigm which will be used in this study is presented in

Figure 1:

Input Process Output

Pumpkin leaves Anti-inflammatory Anti-

(cucurbita pepo) testing by using egg inflammatory
extract in different albumin induced rat property
concentartions paw assay.
Provide treatments:
Figure 1

Experimental paradigm

As shown in the paradigm pumpkin leaf ( Cucurbita pepo) extract would be

subjected to the egg albumin edema assay to determine the presence or the

absence of the anti- inflammatory property in terms of the swelling in the paw of

the test organisms.

Definition of Terms

For the better understanding of the study, the following terms are

operationally defined.
Extract. It is a concentrated preparation of a vegetable or an animal drug

obtained by removing the active constituents, evaporating all or nearly all the

solvent and adjusting the residual mass or powder to a prescribed standard. In

this study it refers to the concentrated pumpkin leaf solution. (Dorlands illustrated

medical Dicionary)

Extraction. Extraction in chemistry is the separation process consisting in the

separation of a substance from a matrix. It includes liquid- liquid extraction, soild

phase extraction.

Treatments. The process of giving a substance in order to cure. In the study it

refers to the different concentrations of the leaf extract given to the test

organisms.

Edema. An excessive accumulation of serum in tissue spaces or a body cavity

(US pathology). In the study it simply refers to the swelling.

Hypothesis

Provide introductory statement..

There is no significant difference in the swelling of the paw of the test

organisms subjected to different concentrations of the pumpkin leaves leaf

(Cucurbita pepo ) extract using ethanol as a solvent.

Methodology

Base from Madans or Portias group..

The thesis work on anti-inflammatory activity of Cucurbita pepo leaf extract

would be done by using the standard procedure of egg albumin edema assay.
Research Design

Base from Madans or Portias group..

In this study the experimental method of research would be using edema

assay which was induced in the right hind paw of Wistar rats using egg albumin in

the actual laboratory setup. The treatments used in the study were given to the test

organisms after 3 hours administration of egg albumin. The edema would be assessed in

terms of the difference in zero time linear circumference and its circumference till it

maintaince a constant inflammation value after applied orally.

Equipment and Materials

Base from Madans or Portias group..

The given materials and equipment used in the study of anti-inflammatory

property of (Cucurbita pepo) extract.

Stock Plant Extraction

Freshly prepared Pumpkin leaves Erlenmeyer flask

Ethanol Buchner funnel

Thermometer Filter

Triple beam balance Water bath

Beaker

Anti- Inflammatory Property Testing

egg albumin Aspirin

Saline solution Vernier caliper scale

Beaker syringe and needles

Test Organism

Eighteen male Wistar rats weighing 100 - 130 grams were used in the

thesis work.

Procedure

The following procedure to be employed in the conduct of the study would

be adapted from L.D.IOR et al 2011.

1. Procurement of the Plant Material

The pumpkin leaves that were used in the study were bought from pure

gold in Vigan City, Ilocos Sur. The leaves were then washed and freshly

prepared for the conduction of the extraction process.

2. Preparation of Fresh Leaf Extract

The extraction of the plant stock in done in the following method

Weigh the ground fresh plant material, about 200gm , weighed using erlenmeryer

flask and treat with sufficient 90% ethyl alcohol to completely submerge the material.

The level of alcohol was noted. Stopper was fixed to the flask and the material is soaked

for 24 48 hrs and then the mixture was filtered through the Buchner funnel with gentle

suction.

Flask and the plant material were then rinsed with fresh portions of alcohol. The

washings and plant material were transferred to the funnel, combining the washings with

the first filtrate.

Gentle suction was applied to complete collection of plant extract then the plant

residue is discarded. The filtrate was concentrated under vacuum at temperature below 50

degrees Celsius to about 20 ml. the exact volume of the concentrated extract by using
water bath. The concentration of the extract was recorded as the grams of plant material

per ml of the extract.

3. Procurement of the Test Organism

The male Wistar rats used in the thesis work would be bought from

animal center, Vigan city, Ilocos sur.

4. Acclimatization of the Test Organisms

The male Wistar rats 90 140 grams obtained from the animal center and

would be maintained for a week to ensure that all the rats are in normal condition

before using them for testing.

5. Provide a discussion of Animal disposal after used..

EGG ALBUMIN :

Egg albumin, were used for the rats in this study. In this study egg should be

broken in anyway and collect the egg white of 0.1 ml per rat which was used to induce

inflammation into the sub planar surface of the right hind paw to cause edema.

5. Anti Inflammatory Property Testing

Animals used for this test are fasted for 12 hours and deprived of water only

during the experiment. The rats were divided into 2 groups with 3 rats in each group. One

group received the positive control aspirin and the other group received the 100% plant

extract orally, after 3 hours inducing with egg albumin. Acute inflammation will be

induced by egg albumin into the sub- planar surface of the right hind paw linear

circumference, edema would be assessed in term of difference in zero time linear

circumference at the injected paw and it circumference at 60 minutes interval after giving

egg albumin injected for the next 6 hrs.

% of edema inhibition = [ V t V 0 ] control ( V t V 0 treated / [ V t V 0 ]

control * 100

6. Data Gathering Procedure

The paw diameters would be measured after every hour for six hours after

treatment using Vernier calipers.

7. Animal Disposal
After the completion of experiment the test organisms were killed and was safely

disposed so that they can not cause any further harm. The test organisms was killed

without pain by injecting formalin in the sub- planar region of the hind paw.

Statistical Treatment of Data

The following statistical tools was used in the analysis of the data that

would be gathered in the study

Mean

The mean was used to get the average of the edema of the inflammation

induced rats.

T- Test

Analysis of two group of rats with the use of statistical examination to determine

the difference in the swelling of the right hind paw for the anti- inflammatory property

testing of cucurbit pepo extract.