This experiment was designed to look into and compare the effectivity of Green Tea Camellia Sinensis or tea shrub as the selected natural antibacterial agent and Phenoxymethylpenicillin as the selected conventional antibiotic against bacteriums Staphylococcus Aureus, therefore finding which one is the most effectual in battling bacteriums. The activity of the antibacterial agents against bacterial growing was represented utilizing the clear zone around the paper phonograph record soaked in assorted sorts of tea infusion. The paper phonograph record were placed in the bacterial lawn. After 24 hours of incubation, the country of the clear zones around the paper phonograph record soaked in tea infusion was measured. The statistical t-test showed that the country of clear zone around the paper phonograph record soaked with Phenoxymethylpenicillin is significantly larger than Green Tea Camellia Sinensis. The consequences support the hypothesis ; Phenoxymethylpenicillin is more effectual compared to Green Tea Camellia Sinensis, since the larger the zone of suppression, the more effectual the compound which act as antimicrobic belongingss.
Key words- Green Tea Camellia Sinensis, Antimicrobial belongingss, Inhibition zone, Staphylococcus Aureus.
Research and Rationale
The purpose of this present work is to carry on a bio-assay to verify the comparing between effectivity of Green Tea and penicillin therefore determine which one is more effectual in battling bacteriums, in this instance Staphylococcus Aureus is used in this experiment.
Bioassay is the finding of the comparative strength of a substance by comparing its consequence on a trial being with that of a standard readying. In this instance, Green Tea is the freshly believed substance that can convey possible consequence towards life as it has the same good intent as a medical specialty to be used in the hereafter.
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Camellia Sinensis or tea shrub is a Chinese species of tea works whose foliages and the foliage buds are collected and be used to bring forth Chinese tea. It belongs to a genus of blooming workss in the household Theaceae. There are a quite figure of tea types that can be extracted from this species viz. White Tea, Green Tea, Oolong Tea, Pu-Erh Tea and Black Tea.
Camellia Sinensis ‘s chemical science value is complex, which contains polyphenols, alkaloids, aminic acids, glucides, proteins, volatile compounds, minerals and hint elements. The most biologically active group in tea constituents are the polyphenols that has antioxidative, antimutagenic and anticarcinogenic effects. Regular ingestion of tea can convey day-to-day demands that we need in some of these elements. Due to the importance of minerals in tea like Camellia Sinensis, several surveies had been conducted to happen whether Camellia Sinensis can be used as future pharmaceutical medical specialty, replacing semi-synthetic antibiotics.
Penicillin is a group of 1000s of antibiotics derived from penicillum Fungis. All penicillin are beta-lactam antibiotics and used in the intervention of bacterial infections caused by Gram-positive beings such as Staphylococcus Aureus. Beta-lactam antibiotics work by suppressing the formation of peptidoglycan cross-links in the bacterial cell wall. It binds to the enzyme that links the peptidoglycan molecules in the bacterium. Enzymes that hydrolyse the cross-links continue to map, weakens the cell wall of the bacteria. This means, the presence of the antibiotics causes cytolysis or decease due to osmotic force per unit area being acted upon the bacterium. Furthermore, the build-up of peptidoglycan precursors can do activation of the bacterial cell wall hydrolases and autolysins to finally farther digest the bacteria ‘s bing peptidoglycan.
With the progress of farther experiments, the use of penicillin as an ideal medical specialty is enormously diminishing. This is because for the past decennaries since its debut in medicative field, most Gram-positive bacterium has been developing its ain opposition against penicillin, by bring forthing different types of peptidoglycan construction that ca n’t be affected by
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beta-lactam, doing the antibiotic failed to destruct being bacterium. Due to such consequence, the natural and active ingredients of penicillin are derived and chemically developed, modified to organize semi-synthetic penicillin which can be more effectual against the bacterium. These semi-synthetic penicillin work on the rule of selective toxicity, restrict or kill the infecting bacterium without harming the host.
Meanwhile, the infusions of Green Tea from Camellia Sinensis has been proven can interrupting a specific phase of the bacterial DNA reproduction procedure. Therefore, it disrupts the Gram-positive Staphylococcus Aureus from reproducing its Deoxyribonucleic acid to synthesis cell wall and division of the cell. Camellia Sinensis besides proven can change by reversal antibiotic resistant such as methicillin and penicillin against Gram-positive Staphylococcus Aureus. This means, Camellia Sinensis can be a immense transition component that finally assimilated in the defense mechanism mechanism with antibiotics.
Therefore, this experiment is aimed to compare how effectual Green Tea of the Camellia Sinensis as a of course derived antibacterial agent, compared to synthetically manufactured antibiotic against bacterial infection. In this experiment, Staphylococcus Aureus, a Gram-positive bacteria was it is one of the infective disease-causing common tegument infections, but may besides do pneumonia and meningitis.
The consequences from this survey can be used to demo that man-made manufactured antibiotic may still be effectual in the hereafter comparison to utilizing natural nutrient resources. There are some people who take works infusion with antimicrobic belongingss entirely to bring around disease. As they do so, they may develop allergic reactions with synthetically manufactured antibiotics, since they are non acquiring used to take on modern medical specialty. Some believe that by devouring works infusion would be merely plenty to battle bacterial infection. It is besides shows that works infusion can be consumed daily as a bar of bacterial infection, but is uneffective one time the disease turning worse, alternatively taking medicative antibiotics can bring around a hundred times better.
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There are important differences between the effectivity of antimicrobic belongingss in Phenoxymethylpenicillin and infusion of green tea in Camellia Sinensis. Phenoxymethylpenicillin efficaciously inhibits the Staphylococcus Aureus growing compared to Green Tea.
There is no important difference between the effectivity of antimicrobic belongingss in Phenoxymethylpenicillin and Green Tea.
Manipulated Variable: Types of antibacterial agent. In this instance, we use Green Tea
and Phenoxymethylpenicillin as the two samples.
Reacting Variable: Area of clear zone around paper phonograph record soaked in respective
antimicrobic agents. We use swayer to mensurate the radius of
the suppression zone and utilizing mathematical method to
cipher the country.
Fixed Variables: Size of filter paper phonograph record, temperature in incubation, thickness
and composing of agar, type and sum of bacteriums plated,
and concentration of antibacterial agents.
Petri Dishes, Label Stickers, 200Aµl micropipette, sterilised filter paper phonograph record, braces of sterilized forceps, stamp and howitzer, weighing balance, Bunsen burner, Sterile Swabs, Volumetric Flask, Small Beaker, Marker Pen and Ruler.
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1 % of Phenoxymethylpenicillin, Weighed Green Tea infusion ( Camellia Sinensis ) , 70 % Ethanol, Sterilised Distilled Water, Staphylococcus Aureus in a sample bottle, Molten Nutrient Agar in a Volumetric Flask, Antiseptic Solution ( Dettol ) .
A test experiment was conducted to seek for which works infusion and selected antibiotics have the most pronounced consequence on bacterial growing. Four Petri dishes were used. Eight sterilised filter paper phonograph record were soaked in approximately 4.0g of works infusion utilizing pestle and howitzer. Four paper phonograph record were soaked with 1 % of selected antibiotics severally and three in sterile distilled H2O. The first Petri dish is used to put the first four paper phonograph record soaked with works infusion while the other four in the 2nd Petri dish. The Third Petri dish is used to put paper phonograph record soaked with selected antibiotics and the concluding Petri dish for the paper phonograph record soaked with distilled H2O. Before that, bacteria has been added into the agar home base with known volume.
Mean Diameter of clear zone ( millimeter )
Green Tea ( Camellia Sinensis )
Table 1: The diameter of clear zones with regard to antimicrobic agents
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Based on the consequences, it is shown that Green Tea ( Camellia Sinensis ) and Phenoxymethylpenicillin had the most pronounced consequence against bacterial growing. Therefore, in this experiment, they were chosen.
Two methods were tested which the disc diffusion method and bacterial settlement which determines which is the most suited method to transport 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 suppression zone will be recorded environing the paper phonograph record. Bacterial settlement is a method to number for the white points on the bacterial lawn.
Green Tea ( Camellia Sinensis )
Diameter of Inhibition Zone was at 21.5mm
Diameter of Inhibition Zone was at 49.0mm
1 bacterial settlement was found
Agar was clear
Table 2: Consequences for the different methods used to find the consequence of antibiotics
Based on the consequences obtained, it is clear that disc diffusion method will be selected as a method used to obtain important consequences as seen in the tabular array 2, where important consequences are obtained in the Inhibition Zones for Green Tea ( Camellia Sinensis ) and Phenoxymethylpenicillin as to compare with the bacterial settlement method. It inhibits the growing of bacteriums in the lawn, therefore there are no settlements seen in the agar.
A trial is conducted to happen the best concentration needed for both samples to respond with the bacteriums and obtain important consequences at the terminal. The concentrations were varied utilizing 0.1 % and 1.0 % of chemical compound and one Petri dish is used. 1 milliliter of 1.0 % Phenoxymethylpenicillin and 1 milliliter of 100 % Camellia Sinensis Green Tea Extract, ( which is about 1 % every bit powerful as Phenoxymethylpenicillin in its antibiotic belongingss ) was prepared. Another readying will affect 1ml of 0.1 % Phenoxymethylpenicillin and 1ml of 10 % Camellia Sinensis Green Tea Extract ( which is about 0.1 % every bit powerful as penicillin in its antibiotic belongingss ) . Green Tea infusion weighing 5.05g was used, as the “ mean day-to-day dose of fresh tea recommended per twenty-four hours is 1 teaspoon ( 5 gms ) which has & gt ; 800mg of polyphenols to obtain noticeable benefit ” [ , ] .
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Percentage Concentration ( % )
Mean Diameter Of Inhibition Zone ( millimeter )
Green Tea Polyphenols
Table 3: Diameters of Inhibition Zones with regard to different concentration
The consequence above recommends that 1 % concentration will be merely plenty to obtain important consequences compared to 0.1 % , in which the Green Tea Extract has no consequence at all.
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Actual Experimental Procedures
Before experiment Begins, custodies must be washed with sanitizer manus wash prepared. The working country was sprayed exhaustively with the bactericidal spray of 1 % ethyl alcohol. By utilizing a paper towel, it was so wiped after go forthing it for 10 proceedingss.
All the setup and stuffs needed were prepared and sterilised in this experiment, which includes the experimental tabular array with 70 % ethyl alcohol to forestall any bacterial activity. The tabular array is so wiped flawlessly with a sanitized tissue paper.
About 20ml sterilized civilization medium was poured into the labeled Petri dish.
Then 200Aµl of Staphylococcus Aureus was taken utilizing a micropipette and was being added bead by bead into the civilization medium in separate topographic points until it was finished.
The civilization medium was swirled on the tabular array with the form of digit 8 to let the bacteria to distribute equally in the agar and it was left aside to chill. The medium so 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 foliage infusion was weighed and placed in howitzer together with 10ml of 70 % ethyl alcohol to suppress any bacterial activity while oppressing it into pulverization before thining it with 100ml of distilled H2O.
The solution will dwell of 100 % infusion, tantamount to 1 % of Phenoxymethylpenicillin, which was prepared and diluted with 100ml of distilled H2O every bit good.
A sterilized paper phonograph record was so dipped into the Phenoxymethylpenicillin solution and placed utilizing a sterilized forceps on top of the medium. The process was repeated with another 9 sterilised paper phonograph record placed into 9 different Petri dishes.
Procedure 9 is repeated with Green Tea Camellia Sinensis Extract solution. 10 paper phonograph record are dipped into the solution and placed utilizing a sterilized forceps into 10 different Petri dishes.
The screen of all 10 Petri dishes that was trial for Phenoxymethylpenicillin were labelled as PEN while the other 10 that trials for Green Tea Camellia Sinensis infusion were labelled as CGT.
All the Petri dishes were placed inverted in the brooder at the temperature of 30oC for 24 hours.
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At the terminal of 24 hours, all the Petri dishes were removed from the brooder.
The suppression zone around each paper phonograph record was observed without opening the screen.
The diameter of the suppression zone including the diameter of paper phonograph record, 7mm was measured utilizing a swayer vertically, horizontally and diagonally and the average value was obtained.
The country of the suppression zone was determined by utilizing this expression:
Iˆ A- Diameter A- Diameter
The information of diameters and countries of were recorded in Table 1.1 and Table 1.2. A graph of average country of suppression zone against antibacterial agents was plotted.
A t-test was used to statistically analyze the information.
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Aseptic technique was used from the start until the terminal of the experiment. This is to suppress the debut of environing bacteriums that can do taint and may impact the consequence of the experiment. This is the ground why the experimental tabular array is sprayed with 70 % ethyl alcohol before and after the experiment. Handss were washed with sanitizer manus wash before managing sterilised setup such as the forceps and the paper phonograph record. This is to avoid the stuffs handled during experiment from being contaminated. Baseball gloves were worn and custodies were washed after the experiment to guarantee that we did n’t distribute Staphylococcus Aureus bacterium into the surrounding ; particularly that this type of bacteriums may do skin infections and can harm other pupils. The setup were besides sterilised to forestall the bacterium from being introduced into the Petri dish and impact the consequence obtained. Petri dish screen was lifted every bit little as possible when reassigning the bacterium utilizing a micropipette, pouring the cultural medium and puting the paper phonograph record. This is to forestall the bacteriums Staphylococcus Aureus from get awaying the environing while forestalling environing bacteriums from acquiring into the dish every bit good. The oral cavities of the flask incorporating the cultural medium and the sample bottle incorporating Staphylococcus Aureus were flamed before proceed with the following measure. This is to guarantee that there will be no taint due to any other bacteriums. After utilizing the micropipettes, the pipette tip is so disposed into the biohazard disposal container incorporating bactericidal solution to avoid bacteriums from distributing. After the experiment, the Petri dishes were sent for autoclaving before disposal.
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Diameter Of Inhibition Zone ( millimeter )
Table 1.1: Diameters of clear zones in both Camellia Sinensis and Phenoxymethylpenicillin ( millimeter )
Area Of Inhibition Zone ( mm2 )
Table 1.2: Areas of suppression 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 average country of suppression zones against antibacterial agents ( mm2 ) .
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Picture 1.4: The suppression zone seen on one of the Petri dishes tested for Green Tea
Picture 1.5: The suppression zone seen on one of the Petri dishes tested for
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The suppression zone around the phonograph record shown between the two antibacterial agents is significantly different where phenoxymenthylpenicillin is wider than the
Camellia Sinensis. The deliberate t-value is 97.58, and it is important as it exceeds far from the tabulated t-value which is 2.093 as P & lt ; 0.05 and d.f. = 19, based on the tabular array. Therefore, the experimental hypothesis is accepted and the void hypothesis is rejected.
a?‘d2=a?‘ ( x-xI„ ) 2
t= | xI„1- xI„2|
a?s ( s12/n1 ) + ( s22/n2 )
a?s ( 1680.1022/10 ) + ( 472.8667/10 )
Table 1.6: Calculation for the t-test in this experiment
Where xI„1 = average sample 1 ( Phenoxymethylpenicillin )
xI„2 = average sample 2 ( Camellia Sinensis )
n1 = figure of topics in sample 1
n2 = figure of topics in sample 2
s12 = discrepancy sample 1
s22 = discrepancy sample 2
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Data Analysis and Discussion
Based on the tabular array given, the average country for Phenoxymethylpenicillin is being calculated and it is 1431.8mm2 larger than Green Tea infusion. In per centum, the difference is 79.5 % , which explains the effectivity of semi-synthetic antibiotic that is better than natural antibacterial agent in extinguishing bacteriums. Mistake bars are displayed in the saloon chart graph to demo the overall distribution of the information. The upper mistake saloon for Green Tea Camellia Sinensis does non transcend the scope of value within error saloon of Phenoxymethylpenicillin. Hence, the average country value between the two varies significantly.
Both solutions of Phenoxymethylpenicillin and Camellia Sinensis diffuse out of the phonograph record into the civilization medium and make an country to forestall the growing of bacteriums. The suppression zone will look if the antibacterial agents were successfully forestalling the growing of the bacteriums in that peculiar country. The effectivity is higher around the phonograph record and it reduces easy as it became further from the phonograph record. Hence, the size of suppression zone can find the effectivity of each agent. The larger the country of the suppression zone, the more effectual the agent against bacteriums growing. Distilled H2O Acts of the Apostless as a control of the experiment and shown no suppression zone at the terminal.
In general, the being of antimicrobic belongingss of Green Tea Camellia Sinensis is mostly more effectual than any other sort of tea with the same Camellia Sinensis species. This is due to the extremely per centum of polyphenols in green tea comparison 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 alternatively being antioxidant, there are hydroxyl Hs in the molecular construction of Green Tea polyphenols that can stop the concatenation reaction of inordinate free groups which consequences in pathological alterations in the human organic structure. The comparing made shown between two most effectual antimicrobic agents: Green Tea and black Tea of the same Camellia Sinensis species gives the overall position.
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Table 1.7: Comparisons between Green and Black Teas
Beginning: 6T6V-4VDY7XV-1-1 & A ; _cdi=5040 & A ; _user=6533825 & A ; _pii=S096399690900012X & A ; _origin=search & A ; _zone=rslt_list_item & A ; _coverDate=07/31/2009 & A ; _sk=999579994 & A ; wchp=dGLbVzb-zSkzV & A ; md5=bae3e1c955d17c199a48937a08e1fd42 & A ; ie=/sdarticle.pdf ” hypertext transfer protocol: //www.sciencedirect.com.ezaccess.library.uitm.edu.my/science/sdarticle.pdf, as besides seen in hypertext transfer protocol: //www.green-tea-benefit.com, downloaded 29 March 2011.
In this experiment, the effectivity of polyphenols as antimicrobic 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 pronounced consequence after carry oning the test experiment. Both are wide spectrum antibiotics which mean they work on a broad scope of bacteriums.
Both inhibit the growing of bacteriums Staphylococcus Aureus and bring forth suppression zones on the alimentary medium. It disturbs the ability of bacteriums to organize cell walls. The cell walls of bacteriums are critical for their endurance. They keep unwanted substances from come ining their cells and halt the contents of their cells from leaking out. Phenoxymethylpenicillin impairs the bonds that hold the bacterial cell wall together. This allows holes to look in the cell walls and kills the bacterium. In Green Tea Camellia Sinensis, the presence of polyphenols Acts of the Apostless as a concatenation reaction inhibitor by suppressing the formation of protein synthesis further in bacteriums.
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However, the size of the suppression zone differs chiefly because the presence of polyphenols in Green Tea Camellia Sinensis is impure compared to phenoxymethylpenicillin which was derived and synthesised to hold the original antibiotic molecules. Active ingredient should be extracted to demo optimal suppression towards bacterial growing. Phenoxymethylpenicillin is an orally activeA signifier ofA penicillin which is so developed to organize semi-synthetic antibiotic. These chemical alterations are made antibiotic last longer and more effectual.
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 growing of Methicillin Resistant Staphylococcus Aureus ( MRSA ) , as they carry oning the experiment to happen the effectivity of Green Tea polyphenols as an antibacterial agent. The figure of suppression zone with Green Tea Polyphenols ( TPP ) and without it as shown in the figure proves that the presence of Polyphenols will hold consequence towards the growing of MRSA.
Picture 1.8: The suppression zone without Tea Polyphenols or TPP ( left ) and with TPP ( right ) against Methicillin-Resistant Staphylococcus Aureus.
Beginning: & lt ; hypertext transfer protocol: //www.springerlink.com.ezaccess.library.uitm.edu.my/content/0727266410782514 /fulltext.pdf & gt ; , Downloaded 29 March 2011.
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The cultural medium was poured at approximately 50oC as it was non excessively hot to be handled and to understate the consequence of condensation in the Petri dish. The agar was disallowed to chill excessively much as it will get down to solidify at around 40oC. One phonograph record soaked in sterile distilled H2O was placed in each Petri Dish, to utilize it as the control of the experiment. Three phonograph record were placed far apart from each other in a Petri dish and non excessively near to the side of the Petri dish, so that the growing of bacteriums and the suppression zone around the phonograph record can be clearly seen. The Petri dishes were left for 24 hours as the rate of bacteriums growing is really fast.
There is fluctuation in the consequences and the consequences may differ somewhat as expected. One, it is the bacteriums that might hold non spread equally although the Petri dishes were swirled. It can be due to the taint of the air while drying the phonograph record in an unfastened Petri dish. Another cause for the fluctuation is the irregular zones that are non supposed to be in the Petri dish, which may take to undependable consequences in mensurating country of the suppression zone. To understate this, the diameter of the suppression zone is measured three times in three different sides to happen the average diameter. A big sample of 10 Petri dishes is used in each antibacterial agent to understate mistake of the consequences and diminish the possibility of inconsistent informations. The Green Tea Camellia Sinensis was dried and the antibiotic molecules might be lost through desiccation. Apart from that, the diameter was recorded to a preciseness of 10 millimeter as the truth of fictile swayer used has the standardization of 0.1cm. There is no swayer with more markers on it to mensurate the diameter more exactly. If there is any measuring used particularly for mensurating of the suppression zone, it will be much helpful. Phenoxymethylpenicillin is used as the pharmaceutical antibiotic as the Green Tea Camellia Sinensis infusion are unavailable as pills. The experiment could be modified by utilizing pestle howitzer to turn both into all right pulverizations and fade out it in distilled H2O to organize a solution. The consequences would be more dependable as its concentration can be measured straight. Besides that, Phenoxymethylpenicillin and Green Tea Camellia Sinensis can be assorted and the interactive effects on the bacteriums, therefore finding efficiency every bit good.
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Synthetically manufactured antibiotics, in this instance phenoxymethylpenicillin, are significantly more effectual in battling bacteriums than Green Tea Camellia Sinensis, which contains polyphenol in it to tag its antimicrobic consequence. This was determined by the significantly larger country of suppression zones of Phenoxymethylpenicillin than Green Tea Camellia Sinensis. Null hypothesis can be rejected and experimental hypothesis is accepted.
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Beginning 1 is an informational web site that is defined around the universe, therefore it is dependable. Beginning 2 is a Wikipedia about information sing Camellia Sinensis, hence the information can be trusted as it gives accurate definition. Beginning 3 is an original paper being downloaded from www.springerlink.com. It consists of a research squad lead by Marcia Reto, A Maria Eduardo Figueira, A Helder Mota FilipeA andA Cristina M. M. Almeida, published online on 27th September 2007. The information is dependable, factual and can be used as a mention in this topic as they are research professionals. Beginning 4 is an internet encyclopedia giving true information about penicillin and its utilizations, . Therefore, the information can be trusted. Source 5 comes officially from U.S National Library of Medicine, National Institutes of Health. The information is dependable as it is lead by professional researches. Beginning 6 is an informational site giving information based in the writer ‘s book. Therefore, the information can be trusted. Beginning 7 is a book created by Lester A. Mitscher and Victoria Dolby in 1998, Lester held a PhD doctor’s degree in Medical Chemistry, and therefore gives dependable information about this topic. Beginning 8 is a research paper that referred based on other sure beginnings ; hence the information is dependable and can be trusted every bit good. Beginning 9 is an article related to Medicinal and Potential advantages of Green Tea, lead by professional research worker by Food Research International. So, the beginning found in Science Direct is dependable and the topic has been done in research. Finaly, beginning 10 gives medical information about the topic and the web site is lead by Dr Roger Henderson, and Christine Webber, which gives valuable information sing the topic from international research worker.