Piper Betle Is One Of The Popular Plants Biology Essay

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In Malaysia, Piper betle is one of the popular plants that has been used by the women in classical area to wash their genital area as well as Mimosa pudica. Since urethra is located just next to the vagina, I have been wondering if both plants also can prevent the UTI from occurring, especially in women. However, this practice has not yet been proven scientifically. This triggers me to do the research on 'which extracts between ethanol extract of Piper betle, ethanol extract of Mimosa pudica and mixture of both ethanol extracts is the most effective in preventing Urinary Tract Infections by inhibiting the growth of Escherichia coli and how does the effect of different concentration of these extracts on the growth of Escherichia coli?'Extended essay, which is one of the components of International Baccalaureate, has provided me the opportunity to do so.

Urinary Tract Infection (UTI) is an infection involving the urinary tract. Urinary tract basically consists of a few structures that allow the urine to pass through before being excreted from the body which are kidneys, ureters, bladder and urethra. Any part of this system can easily become infected. UTI is most common among women compared to men. There was a research that shown 40% of women have UTI at some time in their life, compared to men, which is only about 12%.

Urine in the body is normally sterile. However, an infection may occur when bacteria get into the urine and begin to grow. The infection usually begins at the opening of the urethra and moves upward into the urinary tract. Escherichia coli, (E. coli) is the most common bacteria that contribute to 90% of the UTI [1] . These bacteria normally live in the bowel (colon) or around the anus. They can travel from the area around the anus to the opening of the urethra which is commonly causes by poor hygiene of a person and sexual intercourse. Usually, the flushing action of urine helps to keep infections to a minimum in urinary tract, but then, if there are too many bacteria especially E. coli, urinating may not be enough to stop their spread. As the bacteria are being introduced to the bladder they can proliferate and cause an infection. The bacteria can spread further as the bacteria move up from the bladder via ureters. If they reach kidney, they can cause kidney infection, which could lead to serious condition if not treat promptly.

Picture 1: The female urinary tract

1.3 Escherichia coli

Escherichia coli is a gram negative bacteria. Virulent strains of Escherichia coli cause UTI. Escherichia coli has cell wall, that forms a protective layer which prevents damage from outside and also bursting if internal pressure is high. [2] One way to kill and inhibit the growth of these bacteria is by disrupting the cell wall of the bacteria. Antimicrobial may act as inhibitors of essential enzymes or disruptors of cell wall. [3] 

Picture 2: Structure of Escherichia coli

1.4 Background information of Piper betle

Piper betle is a native of central and eastern Malaysia. It spread at a very clear date throughout tropical Asia and later to Madagascar and East Africa. It has a diuretic property, which helps in easing urination. Other than that, it can also give instant relief from headache and helps in stopping bleedings. An analysis of the betel leaf shows it consists of 85.4% moisture, 3.1% protein, 0.8% fat, 2.3% mineral, 2.3% fibre and 6.1% carbohydrates per 100 grams. Some of its minerals and vitamin contents are niacin and riboflavin. Niacin plays an important role in the digestive system as well as in having healthy skin. Meanwhile riboflavin involves in vital metabolic processes in the body and is necessary for normal cell function, growth, and energy production. Recent studies have shown that betel leaves also contain tannins, sugar and diastases as well as an essential oil which can act as urinary antiseptic. It contains a phenol called chavicol which has powerful antiseptic properties. [4] 

Picture 3: Piper betle

1.5 Background information of Mimosa pudica

Mimosa pudica belongs to Mimosaceae family. It is commonly found in the South America and Central America. Mimosa pudica has been known as the creeping annual herb and its fasten plant movement. Internally, Mimosa pudica is used in vast range of diseases. It arrests the bleeding as well as boosts the healing process. It is very useful in curing diarrhea, amoebic dysentery, bleeding piles, urinary infections. Its leaves get fold inward and droop and reopen after some minutes when they are touched. An adrenaline like substance has been identified in the leaf extract. The roots of Mimosa pudica are bitter, acrid, cooling, alexipharmic and vulneraryin nature. The decoction of the roots renders excellent results in urinary ailments, especially, vesicular calculi. [5] The root extract contains ash, calcium oxalate crystals and mimosine. Mimosine is one of the active compound in Mimosa pudica that can inhibit the growth and protein synthesis in some microorganisms. The root of the plant is considered useful to relieve astma, diarrhoea and urinary complaints. Its anti-inflammatory activity reduces inflammation and helps to control secondary infections. [6] 

Picture 4: Mimosa pudica

HYPOTHESIS

The mixture of Piper betle and Mimosa pudica extract has higher effectiveness to prevent urinary tract infection by inhibiting the growth of Escherichia coli.

This is because the amount of chemical substances that contains in the extracts is various compared to another 2 types of extracts. All of these substances would cooperate with each other and further inhibit the growth of Escherichia coli.

The higher the concentration of extracts, the higher the effectiveness to inhibit the growth of Escherichia coli.

Higher concentration of extract would contain higher amount of chemical substances to inhibit the Escherichia coli growth. As for that, the antimicrobacterial properties would be most significant in higher extract concentration compared to the lower one. Therefore, growth of Escherichia coli will be most inhibited when highest amount of extract used.

3.0 METHODS DEVELOPMENT AND PLANNING

In order to conduct this experiment, I have chosen disc diffusion technique. All of the plant materials that needed; Piper betle and Mimosa pudica are being collected from my housing area, which located at Jerantut, Pahang. All of the plant materials are then being dried, so that it will be easier for the extraction of the plant materials and also to make the weighing process more accurate as dried leaves are said do not contain water in them. The experiment was conducted in University Putra Malaysia (UPM) Microbiology Laboratory for 5 days. Due to time constrain, I decided to make ethanol extraction from the plant materials that I have obtained, as it is an easier yet effective way for extraction. For the tested bacteria in this experiment, I have chosen Escherichia coli because this is the most common bacteria that cause UTI. The bacteria are obtained from the UPM Microbiology Laboratory.

On the first day, I started up by preparing a Nutrient broth [7] and Nutrient agar [8] solution. Next, I gathered all of the apparatus and materials needed, to be autoclaved for about 10-15 minutes. After autoclaving, I transferred the Nutrient agar into Petri dishes to be used in the disc diffusion assays. Nutrient agar is chosen because it suitable for Escherichia coli to grow. Then, I prepared the initial step of making the plant extraction which is crushing the plant materials into small pieces and immersed it into ethanol for 48 hours to ensure that all of the chemical compounds in the plants will dissolve in the ethanol.

On the second day, the stock of tested bacteria, Escherichia coli were subcultured into Nutrient Agar using strip method and incubated for 37°C in 24 hours. Then, on the third day, a colony is picked and transferred into Nutrient broth and be incubated once again for 37°C in 24 hours. Later, I continued the preparation of the crude extract of the plants - crude extract of Piper betle, crude extract of Mimosa pudica and mixture of crude extract of Piper betle and Mimosa pudica - and varied the final concentrations of the extract by dilution process which are 10 mg/ml, 20mg/ml, 30 mg/ml, 40 mg/ml and 50 mg/ml. I have to prepare the discs extract. 96 sterile filter paper discs (Whatman AA, 6 mm) are labeled and impregnated with the extracts under laminar flow by using micropipette according to the type and concentration of extract that they are referring to.

On the fourth day, after all the preparations for the discs diffusion have been ready - discs extract, Nutrient Agar in Petri dishes- now I have reached the last part of the experiment which is the disc diffusion test. The antibacterial activity is interpreted from the size of all of the diameters of zone inhibition measured to the nearest millimeter (mm) using Vernier caliper as observed from the clear zones surrounding the discs. The mean value is calculated from the six inhibition zones.

During the experiment, there were a few precautions that I have to take to ensure the accuracy of the result obtained as well as to minimize or prevent any contamination of the samples that would affect the result. First of all, any apparatus and materials that have already been autoclaved cannot be opened until they are ready to be used. Plus, all materials have to be sterilized with 70% ethanol before entering the laminar flow. This is strictly implied during culture of Escherichia coli, preparation of disc extract and disc diffusion test which were needed to be implemented under laminar flow. The laminar flow itself also has to be sterilized before it is being used.

4.0 APPARATUS AND MATERIALS

4.1 List of Apparatus

Table 1: The apparatus together with their quantities needed in the experiment.

Apparatus

Quantity

1. Electrical balance

1

2. Blender

1

3. Stopwatch

1

4. Filter tube

1

5. 250 ml round-bottom conical flask

3

6. 500 ml conical flask

3

7. Pestle and mortar

1

8. 100 ml measuring cylinder

1

9. 50 ml measuring cylinder

1

10. Rotary evaporator

1

11. Spatula

1

12. Small spatula

3

13. Petri dishes

40

14.Microtubes

30

15. Syringe

15

16. Sterile 0.22 µm membrane filter

15

17. Inoculating loop

1

18. Incubator

1

19. 100 ml bottle

1

20. 500 ml bottle

1

21. 250 ml bottle

2

22. Autoclave

1

23. Laminar flow

1

24. Micropipette

1

25. Forceps

1

26. Vernier caliper

1

4.2 List of Materials

Table 2: The materials together with their quantities needed in the experiment.

Materials

Quantity

1. Piper betle leaves

75 g

2. Mimosa pudica plants

75 g

3. Sealing film (Parafilm)

-

4. Filter paper

-

5. Sterile distilled water

-

6. Nutrient broth powder

0.4 g

7. Nutrient agar powder

14 g

8. Labeling tape

-

9. Autoclave tape

-

10. Marker

1

11. 70% ethanol

-

12. Ethanol

750 ml

13. Plastic packaging

1

14. Sterile filter paper discs (Whatman AA, 6 mm)

96

15. Sterile cotton bud

33

16. Bunsen burner

1

5.0 VARIABLES

Dependent

The diameter of inhibition zone, mm

The diameter of inhibition zones is measured by using Vernier caliper.

Independent

Type of extracts

Type of extracts is varied - Piper betle extract, Mimosa pudica extract and mixture of Piper betle and Mimosa pudica extract.

Concentration of each type of extract.

Concentrations of all the three extracts are varied by - 10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml and 50 mg/ml

Constant

Type of bacterial that causes urinary tract infection

The same bacterium is used; Escherichia coli for all trials so that the effectiveness of those extracts against the bacteria can be compared easily.

Surrounding temperature

Surrounding temperature is being kept constant at room temperature, 27°C by conducting the experiment at the same time in the same laboratory. Plus, the surrounding temperature is also being monitored by using thermometer for every 30 minutes.

pH of the extracts

The pH of the extracts is taken, to ensure its uniformity. Different pH will affect the effectiveness of those extracts. The pH is measured by using pH meter. The pH of Piper betle extract, Mimosa pudica extract and mixture of Piper betle and Mimosa pudica extract are 6.7, 6.1 and 6.8 respectively.

Volume of the extract used

The volume of the extract for each disc is kept constant, 100 µl which measured by using micropipette.

6.0 RESEARCH METHODOLOGY

A. Preparation of crude extracts [9] of Piper betle and Mimosa pudica

50 g of Piper betle leaves are measured by using electrical balance.

By using blender, the measured mass of leaves are crushed into small pieces and blended by adding 200 ml of ethanol to make the blending process easier.

The blended Piper betle material is immersed in 50 ml ethanol for 48 hours at room temperature in 500 ml conical flask and the mouth of the flask is sealed with sealing film.

After 48 hours, the mixture is then filtered.

The filtrate is poured into a 250 ml round conical flask and concentrated under reduced pressure using a rotary evaporator until a crude extract is obtained.

The resulting crude extract obtained is transferred into Petri dishes by using spatula and the mass of the extract is then measured by using electrical balance.

The crude extract is dissolved in sterile distilled water to final concentrations of 10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml and 50 mg/ml in microtubes

The diluted extract is the sterilized by passing through a 0.22 µm membrane filter and transferred into another microtube.

The same procedures (steps 1-8) are repeated by replacing the Piper betle with Mimosa pudica (taking the whole parts of the plant; leaves, stems and roots).

B. Preparation of mixture of crude extracts of Piper betle and Mimosa pudica

25 g of Piper betle leaves and 25 g of Mimosa pudica plants are measured by using electrical balance and are crushed into small pieces and blended together by adding 200 ml of ethanol to make the blending process easier.

The blended mixture is immersed in 50 ml ethanol for 48 hours at room temperature in 500 ml conical flask.

The same procedures are repeated (steps 4-8) for the mixture of Piper betle and Mimosa pudica extract to get the crude extract in different final concentrations.

C. Culture of Escherichia coli and preparation of Nutrient Broth

The stocked test microorganism, Escherichia coli is subcultured to Nutrient agar by streaking method and is incubated at 37°C in incubator for 24 hours.

0.4 g of Nutrient broth powder is diluted into 50 ml of sterile distilled water into a 100 ml bottle and mixed well. The bottle is labeled with NB and is autoclaved.

After incubation of Escherichia coli, the colonies are picked up and transferred into the Nutrient broth.

The colonies in the Nutrient broth then incubated at 37°C in incubator for 24 hours once again to grow the bacteria.

D. Preparation of Nutrient Agar

Nutrient agar is prepared in the plates as the media for the test microorganisms. 14 g of nutrient agar powder is diluted into 500 ml of sterile distilled water into a 500 ml bottle.

The bottle is labeled with NA and is autoclaved.

Under laminar flow, the 32 Petri dishes are labeled with type and concentration of extract. Lines dividing into 3 parts are drawn on each base of Petri dish

2 Petri dishes are allocated for each type and concentration of extract.

The other 2 Petri dishes are labeled with distilled water for the controlled experiments.

The dilution of Nutrient agar is poured evenly into each labeled dish until it reaches half of the dish. The dishes are left for about 30 minutes for the agar to solidify.

After 30 minutes, the dishes are closed with their lids and then stacked together in a plastic packaging. The plastic is tied tightly and then is left in a fridge.

E. Preparation of discs extract.

96 sterile filter paper discs (Whatman AA, 6 mm) are labeled by writing the name and concentration of extract on it. 6 discs are allocated for each type and concentration of extract.

The discs are arranged in Petri dishes according to their concentration.

The discs then impregnated with 100 µl of each of the extracts under laminar flow by using micropipette following the type and concentration of extract that they are referring to.

For the controlled experiment, 6 discs are impregnated with sterile distilled water.

The extract discs then are left to dry under the laminar flow cabinet overnight.

F. Disc Diffusion Test.

50µl of Escherichia coli (Part C) is transferred by using micropipette from the Nutrient Broth to the Nutrient Agar (Part D).

The bacterial inoculum from the Nutrient broth is spread evenly onto the surface each of the Nutrient agar plates using a sterile cotton bud. (the mouth of the NB bottle is flamed each time transferring the broth into the dishes)

The extract discs are positioned on the inoculated agar surface respectively to the label on the dishes by using forceps, 3 discs per dish. (the forceps used is flamed each time new disc extract is taken) Each extract is assayed in triplicate.

2 dishes are allocated for controlled experiment where 3 discs that have been impregnated with sterile distilled water are placed in each dishes.

All the plates were incubated for 24 hours at 37° C.

The antibacterial activity is interpreted from the size of all of the diameters of zone inhibition measured to the nearest millimeter (mm) using Vernier caliper as observed from the clear zones surrounding the discs.

The mean value is calculated from the three inhibition zone.

Precautions

Each material is ensured to be sterilized with 70% ethanol before entering the laminar flow.

When using the laminar flow, it is also needed to be sterilized using 70% ethanol

When the apparatus have been autoclaved, do not open the foil until it is ready to be used.

7.0 DATA COLLECTION

7.1 Quantitative Data

Mass of Resulting Crude Extract

Table 3: The mass of resulting crude Piper betle extract, Mimosa pudica extract and mixture of Piper betle and Mimosa pudica extract.

Type of Extract

Initial Mass of Raw Material, g (±0.01g)

Mass of Resulting Crude Extract, g (±0.01g)

Piper betle

Mimosa pudica

Piper betle

50.00

-

1.12

Mimosa pudica

-

50.00

1.56

Mixture of Piper betle and Mimosa pudica

25.00

25.00

1.22

Diameter of Inhibition Zone

Extract 1: Piper betle

Table 4: The diameter of Inhibition Zone for various concentrations of Piper betle extract.

Concentration of Extract, mg/ml

Diameter of Inhibition Zone, mm (±0.05 mm)

Inhibition Zone in Petri Dish 1

Inhibition Zone in Petri Dish 2

1

2

3

1

2

3

10

11.30

12.10

12.50

12.10

10.50

11.60

20

19.30

18.10

19.90

17.10

18.50

19.60

30

25.10

26.80

24.50

29.00

27.60

25.90

40

34.10

40.10

30.30

37.10

39.40

31.80

50

46.60

42.10

45.10

41.30

43.20

48.90

Extract 2: Mimosa pudica

Table 5: The diameter of Inhibition Zone for various concentrations of Mimosa pudica extract.

Concentration of Extract, mg/ml

Diameter of Inhibition Zone, mm (±0.05 mm)

Inhibition Zone in Petri Dish 1

Inhibition Zone in Petri Dish 2

1

2

3

1

2

3

10

10.90

9.10

9.90

9.80

10.20

9.70

20

12.60

13.50

13.50

14.60

14.20

15.10

30

19.00

13.90

18.90

15.20

17.80

14.90

40

20.70

26.40

20.50

25.60

21.60

22.60

50

29.80

37.40

30.60

33.10

31.10

30.50

Extract 3: Mixture of Piper betle and Mimosa pudica

Table 6: The diameter of Inhibition Zone for various concentrations of mixture of Piper betle and Mimosa pudica extract.

Concentration of Extract, mg/ml

Diameter of Inhibition Zone, mm (±0.05 mm)

Inhibition Zone in Petri Dish 1

Inhibition Zone in Petri Dish 2

1

2

3

1

2

3

10

13.80

14.70

14.90

15.60

14.20

15.50

20

23.70

23.50

25.70

24.90

25.80

25.20

30

35.10

37.10

35.50

30.10

38.00

39.00

40

40.70

43.60

44.70

45.10

44.00

45.00

50

54.00

54.30

49.90

50.20

53.10

47.90

Controlled Experiment: Sterile Distilled Water

Table 7: The diameter of Inhibition Zone for sterile distilled water discs.

Diameter of Inhibition Zone, mm (±0.05 mm)

Inhibition Zone in Petri Dish 1

Inhibition Zone in Petri Dish 2

1

2

3

1

2

3

0.00

0.00

0.00

0.00

0.00

0.00

7.2 Qualitative Data

1. The Piper betle crude extract was dark green in colour.

2. The Mimosa pudica crude extract was dark greenish brown in colour.

3. The mixture of both Piper betle and Mimosa pudica was dark greenish brown in colour.

4. All three types of extract shows the largest inhibition zones for extract

concentration of 50 mg/ml, and smallest inhibition zones for extract concentration of 10 mg/ml.

8.0 ANALYSIS

8.1 Data Processing

A. Diameter of Inhibition Zone According to the Concentration of Extracts

Concentration: 10 mg/ ml

Table 8: The diameter of inhibition zone for concentration of 10 mg/ ml of P.betle extract, M. pudica extract and mixture of P. betle and M.pudica extract.

Type of Extract

Diameter of Inhibition Zone, mm (±0.05 mm)

Inhibition Zone in Petri Dish 1

Inhibition Zone in Petri Dish 2

1

2

3

1

2

3

Piper betle

11.30

12.10

12.50

12.10

10.50

11.60

Mimosa pudica

10.90

9.10

9.90

9.80

10.20

9.70

Mixture of Piper betle and Mimosa pudica

13.80

14.70

14.90

15.60

14.20

15.50

Diagram 1: The scatter diagram shows the diameter of inhibition zone by P.betle extract, M.pudica extract and mixture between P.betle and M.pudica extract with the concentration of 10 mg/ml in 6 trials.

Concentration: 20 mg/ ml

Table 9: The diameter of inhibition zone for concentration of 20 mg/ ml of P.betle extract, M. pudica extract and mixture of P. betle and M.pudica extract.

Type of Extract

Diameter of Inhibition Zone, mm (±0.05 mm)

Inhibition Zone in Petri Dish 1

Inhibition Zone in Petri Dish 2

1

2

3

1

2

3

Piper betle

19.30

18.10

19.90

17.10

18.50

19.60

Mimosa pudica

12.60

13.50

13.50

14.60

14.20

15.10

Mixture of Piper betle and Mimosa pudica

23.70

23.50

25.70

24.90

25.80

25.20

Diagram 2: The scatter diagram shows the diameter of inhibition zone by P.betle extract, M.pudica extract and mixture between P.betle and M.pudica extract with the concentration of 20 mg/ml in 6 trials.

Concentration: 30 mg/ ml

Table 10: The diameter of inhibition zone for concentration of 30 mg/ ml of P.betle extract, M. pudica extract and mixture of P. betle and M.pudica extract.

Type of Extract

Diameter of Inhibition Zone, mm (±0.05 mm)

Inhibition Zone in Petri Dish 1

Inhibition Zone in Petri Dish 2

1

2

3

1

2

3

Piper betle

25.10

26.80

24.50

29.00

27.60

25.90

Mimosa pudica

19.00

13.90

18.90

15.20

17.80

14.90

Mixture of Piper betle and Mimosa pudica

35.10

37.10

35.50

30.10

38.00

39.00

Diagram 3: The scatter diagram shows the diameter of inhibition zone by P.betle extract, M.pudica extract and mixture between P.betle and M.pudica extract with the concentration of 30 mg/ml in 6 trials.

Concentration: 40 mg/ ml

Table 11: The diameter of inhibition zone for concentration of 40 mg/ ml of P.betle extract, M. pudica extract and mixture of P. betle and M.pudica extract.

Type of Extract

Diameter of Inhibition Zone, mm (±0.05 mm)

Inhibition Zone in Petri Dish 1

Inhibition Zone in Petri Dish 2

1

2

3

1

2

3

Piper betle

34.10

40.10

30.30

37.10

39.40

31.80

Mimosa pudica

20.70

26.40

20.50

25.60

21.60

22.60

Mixture of Piper betle and Mimosa pudica

40.70

43.60

44.70

45.10

44.00

45.00

Diagram 4: The scatter diagram shows the diameter of inhibition zone by P.betle extract, M.pudica extract and mixture between P.betle and M.pudica extract with the concentration of 40 mg/ml in 6 trials.

Concentration: 50 mg/ ml

Table 12: The diameter of inhibition zone for concentration of 50 mg/ ml of P.betle extract, M. pudica extract and mixture of P. betle and M.pudica extract.

Type of Extract

Diameter of Inhibition Zone, mm (±0.05 mm)

Inhibition Zone in Petri Dish 1

Inhibition Zone in Petri Dish 2

1

2

3

1

2

3

Piper betle

46.60

42.10

45.10

41.30

43.20

48.90

Mimosa pudica

29.80

37.40

30.60

33.10

31.10

30.50

Mixture of Piper betle and Mimosa pudica

54.00

54.30

49.90

50.20

53.10

47.90

Diagram 5: The scatter diagram shows the diameter of inhibition zone by P.betle extract, M.pudica extract and mixture between P.betle and M.pudica extract with the concentration of 50 mg/ml in 6 trials.

B. Mean of the 6 diameter of the inhibition zones for each type and concentration of extracts

Data obtained are calculated by using the formula below;

Mean, mm

= Total diameter of all 6 inhibition zones for each type and concentration of extract

6

Table 13: Mean diameter of inhibition zones for each type and concentration of extract.

Type of Extract

Concentration of Extract,

Mg/ ml

Mean of the Inhibition Zone, mm (±0.05 mm)

Piper betle

10

11.70

20

18.80

30

24.50

40

35.50

50

44.50

Mimosa pudica

10

9.90

20

13.90

30

19.90

40

22.90

50

32.10

Mixture of Piper betle and Mimosa pudica

10

14.80

20

24.80

30

35.80

40

43.90

50

51.60

Distilled Water

-

0.00

Diagram 6: The graph shows the mean diameter of inhibition zone for different

concentration of P. betle extract, M.pudica extract and the mixture of both

extract.

8.2 Statistical Analysis [10] 

8.2.1 The significance testing between data from Piper betle and Mimosa pudica Extracts.

Let D be the different in effectiveness in inhibiting E. coli growth between P. betle and M. pudica extracts.

Table 14: The difference between the effectiveness of P. betle and M. pudica extracts.

Concentration of Extract, mg/ml

 

10

 20

30 

40 

 50

Type of Extract

P. betle

11.70

18.80

24.50

35.50

44.50

M. pudica

9.90

13.90

19.90

22.90

32.10

D (P.betle - M.pudica)

1.80

4.90

4.60

12.60

12.40

∑D = 36.3 ∑D² = 360.93 n = 5

ÄŽ = 36.3

5

= 7.26

σD² = 5 360.93 - (7.26)²

4 5

= 4.9344²

H0 : µD = 0 (the effectiveness of both P.betle and M.pudica extracts are the same)

H1: µD > 0 (P.betle extract is more effective than M.pudica extract)

If H0 is true, Ď ~ N 0, 4.9344²

5

(rejection region)Performing one-tailed test at 5% significance level, reject H0 if t > 2.1318

2.1318 3.2900

t = 7.26 - 0

4.9344

√5

= 3.2900

Since t > 2.1318, H0 is rejected in favour of H1.

Therefore, at 5% significance level, there is enough significant evidence that Piper betle extract has better effectiveness in inhibiting the growth of E. coli than Mimosa pudica extract.

8.2.2 The significance testing between data from mixture of Piper betle and Mimosa pudica and Piper betle Extracts.

Let D be the different in effectiveness in inhibiting E. coli growth between mixture of P. betle and M. pudica and P. betle extracts.

Table 15: The difference in effectiveness between the mixture of P.betle and M.pudica and P.betle extracts.

Concentration of Extract, mg/ml

 

10

 20

30 

40 

 50

Type of Extract

Mixture of P. betle and M. pudica

14.80

24.80

35.80

43.90

51.60

P. betle

11.70

18.80

24.50

35.50

44.50

D (Mixture - P.betle)

3.10

6.00

11.30

8.40

7.10

∑D = 35.9 ∑D² = 294.27 n = 5

ÄŽ = 35.9

5

= 7.18

σD² = 5 294.27- (7.18)²

4 5

= 3.0211²

H0 : µD = 0 (the effectiveness of both P.betle and Mixture of P. betle and M. pudica extracts are the same)

H1: µD > 0 (Mixture of P. betle and M. pudica extract is more effective than P.betle extract)

If H0 is true, Ď ~ N 0, 3.0211²

5

Performing one-tailed test at 5% significance level, reject H0 if t > 2.1318

(rejection region)

2.1318 5.3143

t = 7.18 - 0

3.0211

√5

= 5.3143

Since t > 2.1318, H0 is rejected in favour of H1.

Therefore, at 5% significance level, there is enough significant evidence that mixture of Piper betle and Mimosa pudica extract has better effectiveness in inhibiting the growth of E. coli than Piper betle extract.

8.2.3 The significance testing between data from mixture of Piper betle and Mimosa pudica amd Mimosa pudica Extracts.

Let D be the different in effectiveness in inhibiting E. coli growth between M. pudica and mixture of P. betle and M. pudica extracts.

Table 16: The difference in effectiveness between mixture of P.betle and M.pudica and M. pudica extracts.

Concentration of Extract, mg/ml

 

10

 20

30 

40 

 50

Type of Extract

Mixture of P. betle and M. pudica

14.80

24.80

35.80

43.90

51.60

M. pudica

9.90

13.90

19.90

22.90

32.10

D (Mixture - M.pudica)

4.90

10.90

15.90

21.00

19.50

∑D = 72.2 ∑D² = 1216.88 n = 5

ÄŽ = 72.2

5

= 14.44

σD² = 5 1216.88- (14.44)²

4 5

= 6.6014²

H0 : µD = 0 (the effectiveness of both M.pudica and Mixture of P. betle and M. pudica extracts are the same)

H1: µD > 0 (Mixture of P. betle and M. pudica extract is more effective than M.pudica extract)

If H0 is true, Ď ~ N 0, 6.6014²

5

Performing one-tailed test at 5% significance level, reject H0 if t > 2.1318

(rejection region)

2.1318 4.8912

t = 14.44 - 0

6.6014

√5

= 4.8912

Since t > 2.1318, H0 is rejected in favour of H1.

Therefore, at 5% significance level, there is enough significant evidence that mixture of Piper betle and Mimosa pudica extract has better effectiveness in inhibiting the growth of E. coli than Mimosa pudica extract.

9.0 Discussion

From the research obtained, all ethanol extracts which are Piper betle extract, Mimosa pudica extract and mixture of both Piper betle and Mimosa pudica extract showed that they can inhibit the growth of Escherichia coli. This is probably due to the presence of the chemical compounds such as tannins and mimosine in Piper betle and Mimosa pudica respectively which could hinder the growth of Escherichia coli.

Tannins is one of the chemical compounds in Piper betle that may help in inhibiting the growth of Escherichia coli. Tannins are basically derived from simple phenolic acids like gallic acid or ellagic acid and when heated they give away pyrogallol. Pyrogallol is also known as hepatotoxic and has antiseptic as well as caustic properties. [11] 

Tannins may destroy and kill the bacteria by disrupting the cell wall which may interfere with the life cycle of the bacteria [12] .

Mimosine is one of the active compounds in Mimosa pudica. Mimosine is a free amino acid produced in a large quantity in the leaves, seeds, and other parts of the Mimosa pudica plant. It can inhibit the growth and protein synthesis in some microorganisms. Mimosine inhibits DNA synthesis by preventing the formation of the replication fork and inhibiting deoxyribonucleotide metabolism. This free amino acid is known to chelate metals, bind pyridoxal phosphate, and inhibit the enzymes tyrosine decarboxylase, tyrosinase, and ribonucleotide reductase. [13] 

Maybe all those compounds help in preventing the growth of Escherichia coli by inhibition of protein synthesis that are needed for the Escherichia coli growth and degradation of bacterial cell wall by cell wall inhibitors. Cell wall inhibitors prevent synthesis of peptidoglycan which is major component of the bacteria cell wall or interrupt peptide side chains which are linkage between peptidoglycan. Moreover, the presence of ethanol in those extracts further enhance the disruption of cell wall of Escherichia coli, as the ethanol helps to wash away the outer layer of lipoposaccharide.

The result also shows that as the concentration of extract increases, the diameter of the inhibition zones also increase. For Piper betle extract, the mean inhibition zone for concentration of 10 mg/ml is 11.7 mm which increases to 44.5 mm for the concentration of 50 mg/ml. For Mimosa pudica extract, the mean inhibition zone for concentration of 10 mg/ml is 9.9 mm which increases to 32.1 mm for the concentration of 50 mg/ml. For mixture of Piper betle and Mimosa pudica extract, the mean inhibition zone for concentration of 10 mg/ml is 14.8 mm which increases to 51.6 mm for the concentration of 50 mg/ml. Possible reason for this is that as the concentration of the extract increases, the concentration of the chemical compounds found in the extract also increase. Therefore as the extract concentrations increases, the ability to inhibit the growth of Escherichia coli also increases.

Besides that, the result also shows that mixture of Piper betle and Mimosa pudica extract has the largest mean diameter of the inhibition zones for all concentration, followed by Piper betle extract and Mimosa pudica extract. This is proven by the significance testing between the data from the mixture of Piper betle and Mimosa pudica extract compared to the data from the Piper betle extract and Mimosa pudica extract. This shows that this extract has the highest effectiveness in inhibiting the growth of Escherichia coli. While Piper betle extract has moderate effectives and Mimosa pudica has the lowest effectiveness to inhibit the growth of Escherichia coli. This is because the chemical substances that contains in the mixture of Piper betle and Mimosa pudica extracts is various compared to the other two types of extracts. The mixture of the substances may have increase the effectiveness of the extract to inhibit the growth of Escherichia coli.

EVALUATION, LIMITATION AND SUGGESTION

There were limitations in the course of the research study apart from doing the experiment. First of all, the variables used to manipulate the experiment were too little in number. Apart from the concentration and the type of extract, the type of bacteria can also be varied, by using other bacteria that could cause UTI else than Escherichia coli such as Staphylococcus saprophyticus and Proteus mirabilis [14] . From this, we can see the actual effectiveness of the Piper betle and Mimosa pudica in preventing UTI.

The other limitation in this experiment was the type of extract of the Piper betle and Mimosa pudica was only ethanol extract. Those extracts can be obtained through other type of extract such as water, chloroform or acetone. Different types of extract may have different effects on the bacteria inhibition.

During the streaking process of the inoculums bacteria, there might be some contamination on the agar plate that caused inconstant inhibition zones diameter around the extract disc. This limitation could be minimized during the preparation of nutrient agar. The media containing agar should be boiled and stirred constantly without burning [15] , so that any undesired microorganism could be eliminated. Then the agar is allowed to cool immediately. Other than that, an obvious limitation during the experiment were time and place for the experiment constraint. Because the experiment was to be done during school holidays, the time was limited towards a certain extent that I could only manipulate only the concentration of the extract and not other factors. As the college did not have appropriate apparatus and materials, the Universiti Putra Malaysia was really a big help in granting the request of proving the materials and apparatus for the research to be done. But due to the place was very far, the experiment manipulation could not be done appropriately than it should be.

CONCLUSION

The research proves that different types of ethanol extract, Piper betle extract, Mimosa pudica extract and mixture between Piper betle and Mimosa pudica extract and different concentrations of each type of extract, 10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml and 50 mg/ml can have different effects on the growth of bacteria Escherichia coli which is indicated by the diameter of the inhibition zone produced around the discs of each extract of each concentration in the discs diffusion method.

The mixture of Piper betle and Mimosa pudica extract is proven to be the most effective in preventing the growth of Escherichia coli compared to Piper betle extract and Mimosa pudica extract. This is further proven by the statistical analysis where the effectiveness between the extracts is compared at 5% significance level. Thus from the experiment, the mixture of Piper betle and Mimosa pudica can be said to have more potential to become antimicrobial agent in preventing UTI, but further investigation using other bacteria must be carried out to know the real effectiveness.

It is also proven that as the concentration of the extracts increases, the effectiveness in inhibiting the growth of Escherichia coli also increases. Further investigation is also suggested to be carried to find out more on the active chemical compounds responsible in inhibiting the growth of Escherichia coli because it is not possible to determine the actual chemical compounds from this experiment.

Therefore, the hypotheses are accepted.

For further research, it is suggested to investigate the significant concentration of the mixture of Piper betle and Mimosa pudica extract to inhibit the growth of bacteria of at least 50% of the Escherichia coli population. Besides, the antimicrobacterial compounds of those extracts such as essential oils can also be investigated.

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