Camellia Sinensis Versus Synthesised Penicillin Antibiotics Biology Essay

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This experiment was designed to investigate and compare the effectiveness of Green Tea Camellia Sinensis or tea bush as the selected natural antibacterial agent and Phenoxymethylpenicillin as the selected conventional antibiotic against bacteria Staphylococcus Aureus, thus determining which one is the most effective in combating bacteria. The activity of the antibacterial agents against bacterial growth was represented using the clear zone around the paper discs soaked in various kinds of tea extract. The paper discs were placed in the bacterial lawn. After 24 hours of incubation, the area of the clear zones around the paper discs soaked in tea extract was measured. The statistical t-test showed that the area of clear zone around the paper discs soaked with Phenoxymethylpenicillin is significantly larger than Green Tea Camellia Sinensis. The results support the hypothesis; Phenoxymethylpenicillin is more effective compared to Green Tea Camellia Sinensis, since the larger the zone of inhibition, the more effective the compound which act as antimicrobial properties.

Key words- Green Tea Camellia Sinensis, Antimicrobial properties, Inhibition zone, Staphylococcus Aureus.

Research and Rationale

The aim of this present work is to conduct a bioassay to verify the comparison between effectiveness of Green Tea and penicillin thus determine which one is more effective in combating bacteria, in this case Staphylococcus Aureus is used in this experiment.

Bioassay is the determination of the relative strength of a substance by comparing its effect on a test organism with that of a standard preparation. In this case, Green Tea is the newly believed substance that can bring potential effect towards life as it has the same beneficial purpose as a medicine to be used in the future.

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Introduction

Camellia Sinensis or tea bush is a Chinese species of tea plant whose leaves and the leaf buds are collected and be used to produce Chinese tea. It belongs to a genus of flowering plants in the family Theaceae. There are a quite number of tea types that can be extracted from this species namely White Tea, Green Tea, Oolong Tea, Pu-Erh Tea and Black Tea.

Camellia Sinensis's chemistry value is complex, which contains polyphenols, alkaloids, amino acids, glucides, proteins, volatile compounds, minerals and trace elements. The most biologically active group in tea components are the polyphenols that has antioxidative, antimutagenic and anticarcinogenic effects. Regular consumption of tea can bring daily requirements that we need in some of these elements. Due to the importance of minerals in tea like Camellia Sinensis, several studies had been conducted to find whether Camellia Sinensis can be used as future pharmaceutical medicine, replacing semi-synthetic antibiotics.

Penicillin is a group of thousands of antibiotics derived from penicillum fungi. All penicillin are beta-lactam antibiotics and used in the treatment of bacterial infections caused by Gram-positive organisms such as Staphylococcus Aureus. Beta-lactam antibiotics work by inhibiting the formation of peptidoglycan cross-links in the bacterial cell wall. It binds to the enzyme that links the peptidoglycan molecules in the bacteria. Enzymes that hydrolyse the cross-links continue to function, weakens the cell wall of the bacterium. This means, the presence of the antibiotics causes cytolysis or death due to osmotic pressure being acted upon the bacteria. Furthermore, the build-up of peptidoglycan precursors can cause activation of the bacterial cell wall hydrolases and autolysins to eventually further digest the bacterium's existing peptidoglycan.

With the advance of further experiments, the usage of penicillin as an ideal medicine is tremendously decreasing. This is because for the past decades since its introduction in medicinal field, most Gram-positive bacteria has been developing its own resistance against penicillin, by producing different types of peptidoglycan structure that can't be affected by

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beta-lactam, making the antibiotic failed to destroy existence bacteria. Due to such effect, the natural and active ingredients of penicillin are derived and chemically developed, modified to form semi-synthetic penicillin which can be more effective against the bacteria. These semi-synthetic penicillin work on the principle of selective toxicity, restrict or kill the infecting bacteria without harming the host.

Meanwhile, the extracts of Green Tea from Camellia Sinensis has been proven can disrupting a specific stage of the bacterial DNA replication process. Therefore, it disrupts the Gram-positive Staphylococcus Aureus from reproducing its DNA to synthesis cell wall and division of the cell. Camellia Sinensis also proven can reverse antibiotic resistant such as methicillin and penicillin against Gram-positive Staphylococcus Aureus. This means, Camellia Sinensis can be a huge conversion element that eventually assimilated in the defence mechanism with antibiotics.

Hence, this experiment is aimed to compare how effective Green Tea of the Camellia Sinensis as a naturally derived antibacterial agent, compared to synthetically manufactured antibiotic against bacterial infection. In this experiment, Staphylococcus Aureus, a Gram-positive bacterium was it is one of the infectious disease-causing common skin infections, but may also cause pneumonia and meningitis.

The results from this study can be used to show that synthetic manufactured antibiotic may still be effective in the future compare to using natural food resources. There are some people who take plant extract with antimicrobial properties alone to cure disease. As they do so, they may develop allergic reactions with synthetically manufactured antibiotics, since they are not getting used to take on modern medicine. Some believe that by consuming plant extract would be just enough to combat bacterial infection. It is also shows that plant extract can be consumed daily as a prevention of bacterial infection, but is ineffective once the disease growing worse, instead taking medicinal antibiotics can cure a hundred times better.

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Experimental Hypothesis

There are significant differences between the effectiveness of antimicrobial properties in Phenoxymethylpenicillin and extract of green tea in Camellia Sinensis. Phenoxymethylpenicillin effectively inhibits the Staphylococcus Aureus growth compared to Green Tea.

Null Hypothesis

There is no significant difference between the effectiveness of antimicrobial properties in Phenoxymethylpenicillin and Green Tea.

Variables

Manipulated Variable : Types of antibacterial agent. In this case, we use Green Tea

and Phenoxymethylpenicillin as the two samples.

Responding Variable : Area of clear zone around paper discs soaked in respective

antimicrobial agents. We use ruler to measure the radius of

the inhibition zone and using mathematical method to

calculate the area.

Fixed Variables : Size of filter paper discs, temperature in incubation, thickness

and composition of agar, type and amount of bacteria plated,

and concentration of antibacterial agents.

Apparatus

Petri Dishes, Label Stickers, 200µl micropipette, sterilised filter paper discs, pairs of sterilised forceps, pestle and mortar, weighing balance, Bunsen burner, Sterile Swabs, Volumetric Flask, Small Beaker, Marker Pen and Ruler.

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Materials

1% of Phenoxymethylpenicillin, Weighed Green Tea extract (Camellia Sinensis), 70% Ethanol, Sterilised Distilled Water, Staphylococcus Aureus in a sample bottle, Molten Nutrient Agar in a Volumetric Flask, Antiseptic Solution (Dettol).

Experimental Planning

A trial experiment was conducted to seek for which plant extract and selected antibiotics have the most marked effect on bacterial growth. Four Petri dishes were used. Eight sterilised filter paper discs were soaked in about 4.0g of plant extract using pestle and mortar. Four paper discs were soaked with 1% of selected antibiotics respectively and three in sterile distilled water. The first Petri dish is used to place the first four paper discs soaked with plant extract while the other four in the second Petri dish. The Third Petri dish is used to place paper discs soaked with selected antibiotics and the final Petri dish for the paper discs soaked with distilled water. Before that, bacterium has been added into the agar plate with known volume.

Antibacterial Agents

Materials

Mean Diameter of clear zone (mm)

Naturally Derived

Green Tea (Camellia Sinensis)

21.5

Ginger

13.0

Onion

17.0

Fennel

12.0

Cinnamon

14.0

Chilli

12.0

Cumin

14.0

Turmeric

17.0

Synthetically Manufactured

Phenoxymethylpenicillin

48.0

Carbenicillin

26.0

Streptomycin

21.0

Ampicillin

32.0

Table 1: The diameter of clear zones with respect to antimicrobial agents

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Based on the results, it is shown that Green Tea (Camellia Sinensis) and Phenoxymethylpenicillin had the most marked effect against bacterial growth. Therefore, in this experiment, they were chosen.

Two methods were tested which the disk diffusion method and bacterial colony which determines which is the most suitable method to carry out the experiment. Three Petri dishes were used. Disk diffusion method is a method where a disc paper is dipped into a solution and placed on top of the bacterial lawn. The inhibition zone will be recorded surrounding the paper discs. Bacterial colony is a method to count for the white dots on the bacterial lawn.

Methods

Green Tea (Camellia Sinensis)

Phenoxymethylpenicillin

Disk Diffusion

Diameter of Inhibition Zone was at 21.5mm

Diameter of Inhibition Zone was at 49.0mm

Bacterial Colony

1 bacterial colony was found

Agar was clear

Table 2: Results for the different methods used to determine the effect of antibiotics

Based on the results obtained, it is clear that disk diffusion method will be selected as a method used to obtain significant results as seen in the table 2, where significant results are obtained in the Inhibition Zones for Green Tea (Camellia Sinensis) and Phenoxymethylpenicillin as to compare with the bacterial colony method. It inhibits the growth of bacteria in the lawn, thus there are no colonies seen in the agar.

A test is conducted to find the best concentration needed for both samples to react with the bacteria and obtain significant results at the end. The concentrations were varied using 0.1% and 1.0% of chemical compound and one Petri dish is used. 1 ml of 1.0% Phenoxymethylpenicillin and 1 ml of 100% Camellia Sinensis Green Tea Extract, (which is approximately 1% as potent as Phenoxymethylpenicillin in its antibiotic properties) was prepared. Another preparation will involve 1ml of 0.1% Phenoxymethylpenicillin and 1ml of 10% Camellia Sinensis Green Tea Extract (which is approximately 0.1% as potent as penicillin in its antibiotic properties). Green Tea extract weighing 5.05g was used, as the "average daily dosage of fresh tea recommended per day is 1 teaspoon (5 grams) which has >800mg of polyphenols to obtain noticeable benefit"[,].

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Percentage Concentration (%)

Mean Diameter Of Inhibition Zone (mm)

Green Tea Polyphenols

0.1

0.0

1.0

21.0

Table 3: Diameters of Inhibition Zones with respect to different concentration

The result above recommends that 1% concentration will be just enough to obtain significant results compared to 0.1%, in which the Green Tea Extract has no effect at all.

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Actual Experimental Procedures

Before experiment begins, hands must be washed with sanitizer hand wash prepared. The working area was sprayed thoroughly with the disinfectant spray of 1% ethanol. By using a paper towel, it was then wiped after leaving it for 10 minutes.

All the apparatus and materials needed were prepared and sterilised in this experiment, which includes the experimental table with 70% ethanol to prevent any bacterial activity. The table is then wiped cleanly with a sanitised tissue paper.

About 20ml sterilised culture medium was poured into the labelled Petri dish.

Then 200µl of Staphylococcus Aureus was taken using a micropipette and was being added drop by drop into the culture medium in separate places until it was finished.

The culture medium was swirled on the table with the shape of digit 8 to allow the bacterium to spread evenly in the agar and it was left aside to cool. The medium then solidified.

Procedure 3 to 5 is repeated with another 19 Petri dishes, 10 Petri dishes were for penicillin and another 10 Petri dishes were for Green Tea Camellia Sinensis Extract.

About 5.05g of Green Tea Camellia Sinensis harden leaf extract was weighed and placed in mortar together with 10ml of 70% ethanol to inhibit any bacterial activity while crushing it into powder before diluting it with 100ml of distilled water.

The solution will consist of 100% extract, equivalent to 1% of Phenoxymethylpenicillin, which was prepared and diluted with 100ml of distilled water as well.

A sterilised paper disc was then dipped into the Phenoxymethylpenicillin solution and placed using a sterilised forceps on top of the medium. The procedure was repeated with another 9 sterilised paper discs placed into 9 different Petri dishes.

Procedure 9 is repeated with Green Tea Camellia Sinensis Extract solution. 10 paper discs are dipped into the solution and placed using a sterilised forceps into 10 different Petri dishes.

The cover of all 10 Petri dishes that was test for Phenoxymethylpenicillin were labelled as PEN while the other 10 that tests for Green Tea Camellia Sinensis extract were labelled as CGT.

All the Petri dishes were placed inverted in the incubator at the temperature of 30oC for 24 hours.

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At the end of 24 hours, all the Petri dishes were removed from the incubator.

The inhibition zone around each paper disc was observed without opening the cover.

The diameter of the inhibition zone including the diameter of paper disc, 7mm was measured using a ruler vertically, horizontally and diagonally and the mean value was obtained.

The area of the inhibition zone was determined by using this formula:

Ï€ Ã- Diameter Ã- Diameter

4

The data of diameters and areas of were recorded in Table 1.1 and Table 1.2. A graph of mean area of inhibition zone against antibacterial agents was plotted.

A t-test was used to statistically analyse the data.

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Risk Assessment

Aseptic technique was used from the start until the end of the experiment. This is to inhibit the introduction of surrounding bacteria that can cause contamination and may affect the result of the experiment. This is the reason why the experimental table is sprayed with 70% ethanol before and after the experiment. Hands were washed with sanitizer hand wash before handling sterilised apparatus such as the forceps and the paper discs. This is to avoid the materials handled during experiment from being contaminated. Gloves were worn and hands were washed after the experiment to ensure that we didn't spread Staphylococcus Aureus bacteria into the surrounding; especially that this type of bacteria may cause skin infections and can harm other students. The apparatus were also sterilised to prevent the bacteria from being introduced into the Petri dish and affect the result obtained. Petri dish cover was lifted as slight as possible when transferring the bacteria using a micropipette, pouring the cultural medium and placing the paper discs. This is to prevent the bacteria Staphylococcus Aureus from escaping the surrounding while preventing surrounding bacteria from getting into the dish as well. The mouths of the flask containing the cultural medium and the sample bottle containing Staphylococcus Aureus were flamed before proceed with the next step. This is to ensure that there will be no contamination due to any other bacteria. After using the micropipettes, the pipette tip is then disposed into the biohazard disposal container containing disinfectant solution to avoid bacteria from spreading. After the experiment, the Petri dishes were sent for autoclaving before disposal.

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Experimental Results

Petri Dish

Diameter Of Inhibition Zone (mm)

Camellia Sinensis

Phenoxymethylpenicillin

1st

2nd

3rd

Mean

1st

2nd

3rd

A

21.0

21.0

22.0

21.3

49.0

48.0

48.0

B

21.0

21.0

21.0

21.0

48.0

47.0

48.0

C

22.0

21.0

22.0

21.7

49.0

49.0

47.0

D

22.0

23.0

20.0

21.7

48.0

48.0

48.0

E

23.0

20.0

21.0

21.3

48.0

48.0

48.0

F

21.0

21.0

22.0

21.3

47.0

47.0

47.0

G

23.0

21.0

23.0

22.3

46.0

48.0

47.0

H

23.0

23.0

23.0

23.0

47.0

48.0

49.0

I

20.0

22.0

21.0

21.0

49.0

47.0

47.0

J

22.0

21.0

23.0

22.0

49.0

49.0

48.0

Table 1.1: Diameters of clear zones in both Camellia Sinensis and Phenoxymethylpenicillin (mm)

Petri Dish

Area Of Inhibition Zone (mm2)

Camellia Sinensis

A

356.4

B

346.5

C

370.0

D

370.0

E

356.5

F

356.5

G

390.7

H

415.6

I

346.5

J

380.3

Mean

368.9

Table 1.2: Areas of inhibition zones of Camellia Sinensis and Phenoxymethylpenicillin (mm2)

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Mean Area of Inhibition Zone against Antibacterial Agents

Graph 1.3: Bar Chart of mean area of inhibition zones against antibacterial agents (mm2).

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Experimental Pictures

Picture 1.4: The inhibition zone seen on one of the Petri dishes tested for Green Tea

Camellia Sinensis.

Picture 1.5: The inhibition zone seen on one of the Petri dishes tested for

Phenoxymethylpenicillin.

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Statistical Analysis

The inhibition zone around the disc shown between the two antibacterial agents is significantly different where phenoxymenthylpenicillin is wider than the

Camellia Sinensis. The calculated t-value is 97.58, and it is significant as it exceeds far from the tabulated t-value which is 2.093 as p < 0.05 and d.f. = 19, based on the table. Therefore, the experimental hypothesis is accepted and the null hypothesis is rejected.

Formula

Phenoxymethylpenicillin

Camellia Sinensis

∑x

18007.0

3689.0

n

10

10

x̄=∑x/n

1800.7

368.9

∑d2=∑(x-x̄)2

15120.92

4255.8

s2=∑d2/n-1

1680.1022

472.8667

t= | x̄1- x̄2|

__________

√ (s12/n1) + (s22/n2)

t= |1800.7-368.9|

______________________

√ (1680.1022/10) + (472.8667/10)

= 97.58

Table 1.6: Calculation for the t-test in this experiment

Where x̄1 = mean sample 1 (Phenoxymethylpenicillin)

x̄2 = mean sample 2 (Camellia Sinensis)

n1 = number of subjects in sample 1

n2 = number of subjects in sample 2

s12 = variance sample 1

s22 = variance sample 2

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Data Analysis and Discussion

Based on the table given, the mean area for Phenoxymethylpenicillin is being calculated and it is 1431.8mm2 larger than Green Tea extract. In percentage, the difference is 79.5%, which explains the effectiveness of semi-synthetic antibiotic that is better than natural antibacterial agent in eliminating bacteria. Error bars are displayed in the bar chart graph to show the overall distribution of the data. The upper error bar for Green Tea Camellia Sinensis does not exceed the range of value within error bar of Phenoxymethylpenicillin. Hence, the mean area value between the two varies significantly.

Both solutions of Phenoxymethylpenicillin and Camellia Sinensis diffuse out of the disc into the culture medium and create an area to prevent the growth of bacteria. The inhibition zone will appear if the antibacterial agents were successfully preventing the growth of the bacteria in that particular area. The effectiveness is higher around the disc and it reduces slowly as it became further from the disc. Hence, the size of inhibition zone can determine the effectiveness of each agent. The larger the area of the inhibition zone, the more effective the agent against bacteria growth. Distilled water acts as a control of the experiment and shown no inhibition zone at the end.

In general, the existence of antimicrobial properties of Green Tea Camellia Sinensis is largely more effective than any other kind of tea with the same Camellia Sinensis species. This is due to the highly percentage of polyphenols in green tea compare to any other teas. A research by A.B Sharangi, the Department of Spices and Plantation Crops, Faculty of Horticulture at West Bengal, India stated that instead being antioxidant, there are hydroxyl hydrogens in the molecular structure of Green Tea polyphenols that can end the chain reaction of excessive free radicals which results in pathological changes in the human body. The comparison made shown between two most effective antimicrobial agents: Green Tea and black Tea of the same Camellia Sinensis species gives the overall view.

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Table 1.7: Comparisons between Green and Black Teas

Source: 6T6V-4VDY7XV-1-1&_cdi=5040&_user=6533825&_pii=S096399690900012X&_origin=search&_zone=rslt_list_item&_coverDate=07/31/2009&_sk=999579994&wchp=dGLbVzb-zSkzV&md5=bae3e1c955d17c199a48937a08e1fd42&ie=/sdarticle.pdf"http://www.sciencedirect.com.ezaccess.library.uitm.edu.my/science/sdarticle.pdf, as also seen in http://www.green-tea-benefit.com , downloaded 29 March 2011.

In this experiment, the effectiveness of polyphenols as antimicrobial substances present in Green Tea Camellia Sinensis as the natural antibiotic is tested with Phenoxymthylpenicillin as the pharmaceutical antibiotic. Phenoxymethylpenicillin and Green Tea Camellia Sinensis were used as both showed the most marked effect after conducting the trial experiment. Both are broad spectrum antibiotics which mean they work on a wide range of bacteria.

Both inhibit the growth of bacteria Staphylococcus Aureus and produce inhibition zones on the nutrient medium. It disturbs the ability of bacteria to form cell walls. The cell walls of bacteria are vital for their survival. They keep unwanted substances from entering their cells and stop the contents of their cells from leaking out. Phenoxymethylpenicillin impairs the bonds that hold the bacterial cell wall together. This allows holes to appear in the cell walls and kills the bacteria. In Green Tea Camellia Sinensis, the presence of polyphenols acts as a chain reaction inhibitor by inhibiting the formation of protein synthesis further in bacteria.

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However, the size of the inhibition zone differs mainly because the presence of polyphenols in Green Tea Camellia Sinensis is impure compared to phenoxymethylpenicillin which was derived and synthesised to have the original antibiotic molecules. Active ingredient should be extracted to show optimum inhibition towards bacterial growth. Phenoxymethylpenicillin is an orally active form of penicillin which is then developed to form semi-synthetic antibiotic. These chemical changes are made antibiotic last longer and more effective.

Another research conducted by Yun-Seok Cho, Neal L Schiller and Kye-Heon Oh at University of California has shown that Green Tea Polyphenols have prevented the growth of Methicillin Resistant Staphylococcus Aureus (MRSA), as they conducting the experiment to find the effectiveness of Green Tea polyphenols as an antibacterial agent. The number of inhibition zone with Green Tea Polyphenols (TPP) and without it as shown in the figure proves that the presence of Polyphenols will have effect towards the growth of MRSA.

Picture 1.8: The inhibition zone without Tea Polyphenols or TPP (left) and with TPP (right) against Methicillin-Resistant Staphylococcus Aureus.

Source: <http://www.springerlink.com.ezaccess.library.uitm.edu.my/content/0727266410782514 /fulltext.pdf>, Downloaded 29 March 2011.

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Evaluation

The cultural medium was poured at about 50oC as it was not too hot to be handled and to minimise the effect of condensation in the Petri dish. The agar was disallowed to cool too much as it will start to solidify at around 40oC. One disc soaked in sterile distilled water was placed in each Petri Dish, to use it as the control of the experiment. Three discs were placed far apart from each other in a Petri dish and not too close to the side of the Petri dish, so that the growth of bacteria and the inhibition zone around the discs can be clearly seen. The Petri dishes were left for 24 hours as the rate of bacteria growth is very fast.

There is variation in the results and the results may differ slightly as expected. One, it is the bacteria that might have not spread evenly although the Petri dishes were swirled. It can be due to the contamination of the air while drying the discs in an open Petri dish. Another cause for the variation is the irregular zones that are not supposed to exist in the Petri dish, which may lead to unreliable results in measuring area of the inhibition zone. To minimise this, the diameter of the inhibition zone is measured three times in three different sides to find the mean diameter. A large sample of 10 Petri dishes is used in each antibacterial agent to minimise error of the results and decrease the possibility of inconsistent data. The Green Tea Camellia Sinensis was dried and the antibiotic molecules might be lost through dehydration. Apart from that, the diameter was recorded to a precision of 10 mm as the accuracy of plastic ruler used has the calibration of 0.1cm. There is no ruler with more markings on it to measure the diameter more precisely. If there is any measurement used especially for measuring of the inhibition zone, it will be much helpful. Phenoxymethylpenicillin is used as the pharmaceutical antibiotic as the Green Tea Camellia Sinensis extract are unavailable as pills. The experiment could be modified by using pestle mortar to turn both into fine powders and dissolve it in distilled water to form a solution. The results would be more reliable as its concentration can be measured directly. Besides that, Phenoxymethylpenicillin and Green Tea Camellia Sinensis can be mixed and the synergistic effects on the bacteria, thus determining efficiency as well.

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Conclusion

Synthetically manufactured antibiotics, in this case phenoxymethylpenicillin, are significantly more effective in combating bacteria than Green Tea Camellia Sinensis, which contains polyphenol in it to mark its antimicrobial effect. This was determined by the significantly larger area of inhibition zones of Phenoxymethylpenicillin than Green Tea Camellia Sinensis. Null hypothesis can be rejected and experimental hypothesis is accepted.

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Source Evaluation

Source 1 is an informational website that is defined around the world, therefore it is reliable. Source 2 is a Wikipedia about information regarding Camellia Sinensis, therefore the information can be trusted as it gives accurate definition. Source 3 is an original paper being downloaded from www.springerlink.com. It consists of a research team lead by Márcia Reto, Maria Eduardo Figueira, Helder Mota Filipe and Cristina M. M. Almeida, published online on 27th September 2007. The information is reliable, factual and can be used as a reference in this subject as they are research professionals. Source 4 is an internet encyclopaedia giving true information about penicillin and its uses,. Therefore, the information can be trusted. Source 5 comes officially from U.S National Library of Medicine, National Institutes of Health. The information is reliable as it is lead by professional researches. Source 6 is an informational site giving information based in the author's book. Therefore, the information can be trusted. Source 7 is a book created by Lester A. Mitscher and Victoria Dolby in 1998, Lester held a PhD doctorate in Medical Chemistry, and thus gives reliable information about this subject. Source 8 is a research paper that referred based on other trusted sources; therefore the information is reliable and can be trusted as well. Source 9 is an article related to Medicinal and Potential advantages of Green Tea, lead by professional researcher by Food Research International. So, the source found in Science Direct is reliable and the subject has been done in research. Finaly, source 10 gives medical information about the subject and the website is lead by Dr Roger Henderson, and Christine Webber, which gives valuable information regarding the subject from international researcher.

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