Antibacterial Activity of Zingiber officinale against three Common Upper Respiratory Tract Pathogens

INTRODUCTION

Ginger (Zingiberofficinale) is either the scraped or unscraped rhizome of Zingiberofficinale(family Zingiberaceae)1. It is grown in many parts of the world including Uganda of which Mpigi district is the highest source of exported ginger in the country. It is known as ÔÇ£entangawuuziÔÇØ in luganda and is an important component of cough and flu crude herbal preparations and is now used in approved remedies such as ÔÇÿkabutiÔÇÖ

Streptococcus pneumoniae or pneumococcus is a Gram-positive, alpha hemolytic diplococcus bacterium and a member of the genus Streptococcus2. It is a significant human pathogen with seven sero-types (6A, 6B, 9V, 14, 19A, 19F, 23F). S.pneumoniae remains the commonest cause of pneumonia and is normally found in the nasopharynx of 5-10% of healthy adults, and 20-40% of healthy children3.

Hemophilus influenza is a non-motile Gram-negative coccobacillus of serotypes; un-encapsulated strains and the encapsulated strains like Hib3. Hib first described by Richard Pfeiffer in 1892 during an influenza pandemic is generally aerobic, but can grow as a facultative anaerobe. It was mistakenly considered to be the cause of the common flu until the viral etiology was discovered. However, William Barry emphasized that indeed H. influenza complicates the viral illness and remains the primary cause of ear and sinus infection3. The World Health Organization (WHO), published that Hemophilus influenza Type b, or Hib, is a bacterium estimated to be responsible for some three million serious illnesses and an estimated 386000 deaths per year, chiefly through Meningitis and Pneumonia4.

Streptococcus pyogenes (Group A streptococcus) is a Gram-positive, non-motile, non-spore forming coccus that occurs in chains or in pairs of cells. Individual cells are round-to-void cocci, 0.6-1.0 micrometer in diameter2. William Barry, again emphasized that it is the most important bacterial pathogen in the upper respiratory tract infections and its infections typically begin in the throat or skin for example Pharyngitis (ÔÇ£strep throatÔÇØ)3

Trease G.E. found out that Ginger was a good carminative and stimulant1 whereas Mowrey and Claysonadded that the powdered Ginger could be a more effective antiemetic than Dramamine5. They emphasized that it could ameliorate the effects of motion sickness in the gastro intestinal tract itself. This meant that Ginger only acted locally in the gastro intestinal tract (GIT) leaving a question; whether it possessed any antimicrobial activity in addition to its other indications in the GIT since the effects of motion sickness were similar to those of a GIT infection.

This was demonstrated by Wood C.D, who in his research claimed that the gingerols found in Zingiber officianale (ginger) have analgesic, sedative, antipyretic, antibacterial and gastrointestinal tract motility effects6. He emphasized that ginger has the capacity to eliminate harmful bacteria, such as Escherichia coli, responsible for most of the diarrhea, especially in children and since ginger eases both diarrheoa and constipation, it should have impact on the growth of Bacillus cereus which mainly cases diarrheoa and nausea. This research was however focused on gastro intestinal infections leaving the question: Does ginger possess any antibacterial activity against the common upper respiratory tract pathogens?

A study by Akoachere J.F. showed that ethanolic extracts of Ginger exhibited antibacterial activity against respiratory pathogens (S. pneumoniae, S. pyogenes and H. influenza)7. However, aqueous extracts are the most commonly used in local preparations yet his study focused on only ethanolic extracts.

Though he did not focus on pathogens common to the respiratory tract, Reginald A. Onyeagba, in his study, established that the aqueous and ethanolic extracts of Ginger when used singly exhibited antimicrobial activity against his test species (Helicobacter pylori, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli)8. He concurred with a study by Hiral Chandarana that showed that although all his test spices (Ginger, Manoginger and Tumeric) had antimicrobial activity against Escherichia coli, Ginger posed an even higher antimicrobial activity against staphylococcus aureus one of the causes of Respiratory Tract Infections9. However, his study was not completely focused on respiratory tract pathogens. This left the question whether aqueous extracts of Ginger possessed antibacterial activity against the Upper respiratory tract pathogens (S.pyogenes, S.pneumoniae and H. influenza)

In view of the above literature, research was needed to determine the anti-bacterial spectrum of ethanolic and most importantly aqueous Ginger extracts against the common respiratory tract pathogens: Haemophilus influenza, Streptococcus pneumoniae and Streptococcus pyogenes. 

MATERIALS AND METHODS

Apparatus

Water bath, Test tubes, Petri dishes, Centrifuge, Rotary evaporator, Autoclave, Filter papers, Distiller, Balance scale, Hot air oven, Beakers, Inoculation wire loop, Mortar and pestle and Agar plate

Chemicals

Plant extracts, Penicillin G, Sulphuric acid, Barium chloride, Distilled water, DMSO and Agar (sheep blood agar and chocolate agar)

Organisms used

Haemophilus influenza ATCC 49247, S. Pyogenes ATCC 19615 and S. Pneumoniae ATCC 49619

Collection and identification of plant materials

The fresh plant material of Ginger (Zingiber officinale) was obtained from Makonzi estate, a commercial farm in Mubende district, Uganda.The rhizome was identified and the species confirmed by a competent taxonomist with the Department of Pharmacy, Makerere University

Extraction of the active ingredients

The ginger rhizomes were washed with clean sterile distilled water and allowed to air-dry for one hour. Then the outer covering of the ginger was manually peeled off and the ginger washed again and extracted using the following procedures:

1. 2000g of fresh ginger were crashed and dried in the sun for 7 days

2. The dried rhizome was then crashed in a mortar and pestle to form a fine powder

3. 200g of the ginger powder was macerated in cold distilled water for 72hours. The liquids obtained were mixed, filtered using a sterile muslin cloth and then kept in a refrigerator overnight to allow the colloidal particles to settle down, the liquid was decanted off for recovery of the extract.

3. 200g of dried Ginger powder was macerated in hot water in a closed giant vacuum flask for 72hours. The liquids obtained were mixed and then kept in a refrigerator overnight to allow the colloidal particles to settle down, the liquid was decanted off for recovery of the extract.

4. 200g of fried Ginger was macerated in 95% ethanol for 72hours. The liquids obtained were mixed and clarified by filtration.

Recovery of the extracts

1. The ethanolic liquid was transferred to a rotary evaporator to evaporate off the ethanol leaving behind a brown oily semisolid which was kept in a clean sterile conical flask till use.

2. The hot water and cold water liquids were freeze dried and the extracts were sealed in water proof polyethene bag till use.

Culture of microorganisms

Heamophilus influenza grows on Chocolate agar whileStreptococcus pyogenes and Streptococcus pneumoniae grow on blood agar medium. Blood agar contains Muller Hinton (75%) and sheep blood (25%)2. Chocolate agar is heated blood agar.

Preparation of the agar medium

1. 17.4g of Muller Hinton were mixed with 500mls of water in a conical flask. The resultant solution was autoclaved for 15 minutes to sterilize it and then heated in a water bath at 37Ôö¼ÔûæC till ready for pouring into the agar plates. 25mls were measured off and were replaced by 25mls of sheep blood agar.

2. The flask was then shaken to ensure thorough mixing and the agar was poured to a depth of 4mm in each of the 18 sterile standard agar plates.

3. The remaining Muller Hinton/sheep blood mixture was heated in a water bath at 37Ôö¼ÔûæC for 15 minutes to form chocolate agar which was also poured to a depth of 4mm in 9 sterile agar plates.

4. The agar in the plates was given time to set and the plates were then incubated at 37Ôö¼ÔûæC for 24hours.

Preparation of McFarland standard

1. Exactly 0.5 McFarland equivalent turbidity standards was prepared by adding 0.6ml of 1% barium chloride solution (BaCl2.2H2O) to 99.4ml of 1% sulphuric acid solution (H2SO4) and mixed thoroughly.

2. A small volume of the turbid solution was transferred to a capped tube of the same type that was used to prepare the test and control innocula. This was then stored in the dark at room temperature (25Ôö¼ÔûæC). Exactly 0.5 McFarland gives an equivalent approximate density of bacteria 1x10-8 cfu.2

Inoculum preparation by direct colony suspension method

A small volume of normal saline was poured inside a test tube to which general colonies of the test organisms, taken directly from the plate were emulsified and the suspension was adjusted to match the 0.5 McFarlandÔÇÖs standard which has a similar appearance of an overnight broth culture by adding normal saline8

Antimicrobial screening test

1. A glass dropper was used to add 0.02mls of the suspension to an already prepared medium.

2. A sterile cotton swab was used to spread by streaking the organisms all over the surface of the medium and was allowed to dry for about 5 minutes. Cups of 5mm in diameter were made in the agar using sterile cork borer.

3. Two drops of DMSO were added to 2 grams of each extract and then 5mls distilled water to attain the required initial concentration.

4. Five graded dilutions of 0.5 fold of the plant extracts were prepared with the original concentration of the extract as the first that is 400mgml-1, 200mgml-1, 100mgml-1, 50mgml-1, and 25mgml-1, the sixth dilution was of two drops of DMSO mixed with 5mls of distilled water and it contained 0.0mgml-1 of the extracts to act as the negative control. The dilutions were put in six different test tubes for each extract and were placed in a test tube rack.

5. 6Ôö¼├üg/ml of penicillin G was prepared alongside to act as the positive control.

6. Exactly 0.02ml of each concentration was introduced into each hole on the medium and was allowed to stand on the bench for about one hour for proper diffusion. It was thereafter incubated at 37Ôö¼ÔûæC for 24hrs

7. The diameters of the zones of inhibition of the different concentrations of extracts on the organisms were measured using a calibrated ruler

MIC by Agar Dilution Method

1. Serial two- fold dilutions of the ethanolic extract were prepared in test tubes (highest 300,000Ôö¼├üg/ml and lowest 125Ôö¼├üg/ml) alongside which similar serial dilutions were done for Penicillin G such that the highest concentration was 3.84Ôö¼├üg/ml and the lowest was 0.03Ôö¼├üg/ml

2. 1ml of each dilution was poured in agar plate and it was mixed with 19mls of the agar medium. The mixture was left to set and the plates were then incubated at 37Ôö¼ÔûæC for 24hours (to exclude the contaminated) after which the organisms were inoculated onto the agar medium.

3. The plates were re-incubated again for 24hours and then observed for growth.

RESULTS

The results are summarized in table 1-5 below.

Table 1: Yield of the extracts

 

Extract

Weight

%yield

Hot water

6.11g

3.056

Cold water

6.65g

3.320

Ethanolic

9.51

4.760

 

 

 

 

 

 

DISCUSSION OF RESULTS

Extraction using ethanol yielded more extract than extraction using water because constituents of ginger are soluble in ethanol than they are in water.

The result of this study indicated that the ethanolic extract of ginger was the only one with biologic activity and it depended on the concentration with the widest zone of inhibition as 16mm.

This study correlates with the study by AkoachereJF that showed that the ethanolic ginger extract indeed possessed activity against Streptococcus pneumoniae, Streptococcus pyogenes, and Hemophilus influenza7. However, this study has contradicted with his MIC results that ranged from 0.0003microg/ml to 0.7microg/ml. when the extracts were tested on S. pyogenes, S. pneumoniae, H. influenza the widest zone of inhibition was obtained with S. pyogenes with MIC values ranging from 3125mcg/ml for S. pyogenes to 6259mcg/ml for S.pneumoniae. The high MIC values could be attributed to the fact that the extract used had not been standardized to obtain the pure active ingredient. The differences in the zones of inhibition and MICs are not exactly known but are probably due to the susceptibility of each test organisms to the ethanolic Ginger extracts


 

Sensitivity patterns of streptococcus pneumoniae, streptococcus pyogenes and Heamophilus influenza to the aqueous and ethanolic extracts

Table 2: Sensitivity pattern of Streptococcus pneumoniae, Streptococcus pyogenes and Heamophilus influenza to the ethanolic extract

Concentration of extract in mgml-1

Diameter of Zone of Inhibition in mm

 

S. pyogenes

H. influenza

S. pneumoniae

400

16

14

12

200

12

10

N1

100

N1

N1

N1

50

N1

N1

N1

25

N1

N1

N1

Penicillin

47

46

46

DMSO + Distilled water

N1

N1

N1

 

NI=No Inhibition

Table 3: Sensitivity pattern of Streptococcus pneumoniae, Streptococcus pyogenes and Hemophilus influenza to Hot water extract

 

Concentration of Extract in mgml-1

Diameter of zone of inhibition in mm

 

S. pyrogenes

H. influenza

S. pneumoniae

400

NI

NI

NI

200

NI

NI

NI

100

NI

NI

NI

50

NI

NI

NI

25

NI

NI

NI

Penicillin

47

46

47

DMSO + Distilled water

NI

NI

NI

 

NI=No inhibition

Table 4: Sensitivity pattern of Streptococcus pneumoniae, Streptococcus pyogenes and Hemophilus influenza to cold water extract

 

Concentration of extract in mgml-1

Diameter of Zone of Inhibition in mm

 

S.pyogenes

H. influenza

S. pneumonia

400

NI

NI

NI

200

NI

NI

NI

100

NI

NI

NI

50

NI

NI

NI

25

NI

NI

NI

Penicillin

47

45

47

DMSO + Distilled water

NI

NI

NI

 

NI means No inhibition

Table 5: Minimum inhibitory concentration (MIC) for the ethanolic extract of ginger and penicillin

 

Organisms 

MIC for ethanolic Ginger extract 

MIC for penicillin 

S. pyogenes

3125

0.06

H. influenza

6150

1.92

S. pneumonia

6250

0.06

 


The concentrations of hot water extracts used did not inhibit the growth of all the test organisms. This may be explained by the fact that the antimicrobial substances in ginger, which are mainly phenolic compounds, are destroyed by heat from the hot-water which might have raised the temperature of the extracts hence deactivating them 10.

In general, active ingredients in spice plants are heat labile; hence all the spices lose their biologic activity within 20 minutes at 100Ôö¼ÔûæC10. Secondly, it is most likely that since most constituents of ginger are oils, the active ingredients are hydrophobic and therefore they were not extracted. The concentrations of cold water extracts used like the hot water extracts, did not at all exhibit antibacterial activity. The reason for this could not be ascertained but factors such as insolubility of active ingredients in the water extracts, concentration of the extract, rate of diffusion of extract, depth of culture medium and density of medium were speculated.

The pure solvent (2 drops of DMSO + 5MLS OF WATER) used to dissolve the extract acted as the negative control and it exhibited no biologic activity when used alone this means that the antibacterial activity of the extracts was attributed to the extracts alonewhereasPenicillinGactedasthepositivecontrol.

CONCLUSION

Ginger contained compounds with antibacterial activity against the common upper respiratory tract pathogen. However based on the study, this may not have be the rationale for its use in local herbal medicine preparations. This is because high concentrations are needed for it to exhibit antibacterial activity if used initscrudeformand secondly because the extraction done using water (as commonly used in traditional/local extraction) did not yield extracts with antibacterial effect.

RECOMMENDATIONS

A study should be carried out using aqueous extracts with higher concentrations. The extracts should not be stored for a long time to exclude the fact that they could lose their viability with time

REFERENCES

1.Trease, George Edward and William Evans, Pharmacognsy. London: BailliereTindall& Cox 1983.

2. Monica Chessbrough, District Laboratory Practice In Tropical Countries. Part 2. Cambridge University Press, United Kingdom, 2000: 222.

3.Allan Denver Russell, William Barry Hugo, pharmaceutical microbiology, Macmillan publishing co. inc 1977 pg 134 to 149

4. WHO 1999: World Health Organization. WHO Monographs on Selected Medicinal Plants, Volume 1. Geneva (CHE): World Health Organization; 1999.

5. Mowrey DB, Clayson DE. Motion sickness, ginger, and psychophysics. Lancet 1982; 1(8273): 655-657.

6. Wood C.D, Comparison of efficacy of ginger with various antimicrobial sickness drugs. Clinical Research Practices and Drug Regulatory Affairs, 1988; 6(2): 129-136.

7. Akoachere JF et al East African Medical Journal 2002

8. Isu NR, Onyeagba RA. Basic Practicals in Microbiology.2nd edition.Fasmen Communication, Okigwe, 2002: 25-45

9. HiralChandarana 2004

10. Chen HC, Chang MD, Chang TJ. Antibacterial properties of some spice plants before and after heat treatment. Pubmed 1985; 18(3): 190-195