Abstract:The study ofantimicrobial properties of Piper betel leaf which are cultivated in India .TheMethanolic extract of dried leaf were testedfor antibacterial and antifungal properties against pathogenic microorganismssuch as Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli andCandida albican. Methanolic extract showed effective zone of inhibition againstthe used pathogens.
25mm against Staphylococcus aureus followed by the zone of16mm against Pseudomonas aeruginosa followed by the zone of 20mm againstEscherichia coli and 17mm against Candida albican by using agar well diffusionmethod. Antibiogram was also be done against these pathogenic bacteria by usingantibiotic ciprofloxacin, Penicillin, Tetracycline, streptomycin, Erythromycinfor antibacterial and Amphotericin B, Fluconazole, Itraconazol for antifungalpathogens. Key words: Piperbetel leaf, Antibacterial properties, Pathogens, Methanolic extract, agar welldiffusion, Antibiogram. Introduction: Antibiotics arealso called as antibacterial, and are a type of antimicrobial drug used in thetreatment and prevention of bacterial and fungal infection. They may eitherkill or inhibit the growth of bacteria. Sometimes the term antibiotics are usedto refer any substance used against microbes.
Antibiotics revolutionizedmedicine in 20th century. Using antibiotics to eliminate the infection produceadverse effect to host organs, tissues and cells. The effect produced by theantimicrobial agents can be prevented or cure with herbal medicines. Herbalmedicines are safe, and will overcome the resistance produced by pathogens.
Some medicinal herbs have antibacterial and antifungal properties which will beuseful for the clinical use (1,3). Herbal medicines are a traditional method offighting against pathogens. The herbal medicines or medicinal plants have been usedsince time immemorial in Indian villages for the treatment of uncountablediseases (2). Piper betel Leafbelongs to family Piperaceae commonly known as Pan i.e. the Black Pepper familyit is traditionally used in India, China, Thailand (7,8). The Piper betel leafis green in color and has aromatic smell, it is bitter in taste and it is heartshaped. It is largely distributed in tropical and subtropical region of theworld.
For mouth freshener the betel leaf is the best freshener that can betaken after lunch or dinner (9). It hasgreat capacity to increase are physical health and mental function. In English itis called betel, betel vine and betel piper and in Hindi it is call pan (8).Nutritionalcomposition of fresh betel leaf: S.
No. Constituents Approximate composition 1 Water 85-90% 2 Protein 3-3.5% 3 Fat 0.4-1.0% 4 Mineral 2.3-3.3% 5 Fiber 2.3% 6 Chlorophyll 0.
01-0.25% 7 Carbohydrate 0.5-6.
10% 8 Nicotinic acid 0.63-0.89mg/100g 9 Vitamin C 0.005-0.01% 10 Vitamin A 1.9-2.
9mg/100g 11 Thiamine 10-70mg/100g 12 Riboflavin 1.9-30mg/100g 13 Tannin 0.1-1.
3% 14 Nitrogen 2.0-7.0% 15 Phosphorus 0.05-0.
6% 16 Potassium 1.1-4.6% 17 Calcium 0.2-0.
5% 18 Iron 0.005-0.007% 19 Iodine 3.4mg/100g 20 Essential Oil 0.08-0.2% 21 Energy 44 kcal/100g Contents of Betelleaves: Betel leavescontain good minerals and vitamins and also contain huge number of bioactivemolecules. Betel leaf has oil, sugar, vitamin c, and starch.
Betel leaf has phenol that is called aschavicol that have properties of reducing central nervous system (7, 9).Traditional uses ofBetel leaves:Betel leaf istraditionally known to be useful for the treatment of various diseases like badbreath, boils and abscesses, conjunctivitis, constipation, headache, hysteria,itches, mastitis, mastoiditis, leucorrhoea, otorrhoea, ringworm, swelling ofgum, rheumatism, abrasion, cuts and injuries , wound and inflamation etc asfolk medicine while the root is known for its female contraceptive effects(2,6).Anti-inflammatoryeffects:Piper betal leafpossess anti-inflamatory effects in various animal models of studies withvarious inflamogens. Betal leaf used as a common home remedy for infalmmationin the oral cavity (7).Antioxidanteffects:The contents ofPiper betal leaf extract increased the cellular antioxidants and mediate thechemopreventive effects at least in part. Three varities of Piper betal leafshowed antioxidant effects whenevaluated by in vitro systems such as DPPH radical scavenging, superoxideradical scavenging, hydroxyl radical scavenging and prevention of lipidperoxidation (7,9). AntimicrobialActivity:The antimicrobialactivity of leaves towards bacteria in mouth i.
e. Streptococcus viridans, Staphylococcus aureus and Streptococcusmutans and heals many bacterial diseases and also show antimicrobial activityagainst various obligate oral anaerobes. The methanolic extract was moreeffective than other extracts in inhibating the microbial organisms. Piperbetal leaf is most active antimicrobial plant (3,7,9). Antifungalactivity: The extract ofPiper betel L., (Piperaceae) is used from which the Hydroxychavicol, isolatedfrom the chloroform extraction to isolate the antifungal activity agaist theselected fungi. Hydroxychavicol compound can be used as an antifungal agents toexhibated the antifugal activity and also used to treat topical infections aswell as gargle mouthwash against oral Candida infections (7). Anti-diabeticactivities: Methanolic extractof Piper betal leaf possess hypoglycaemic activity when tested in normoglycaemic rats using hotwater and cold water extracts (9).
Role of betel leafextract on thyroid function:The effect of betalleaf extract depend on the thyroid hormones concentrations lipid peroxidation(LPO) and on the activities of superoxide dismutase (SOD) and catalase (CAT).Administration of betel leaf extract exhibited a dual role, dependingon thedifferent doses. While the lowest dose decreased thyroxine (T4) and increasedserum triiodothyronine (T3) concentrations, reverse effects were observed attwo higher doses. Lipid peroxidation (LPO) increased with higher doses anddecreased in superoxide dismutase (SOD) and catalase (CAT) activities. However, most of these effects were reversed with lowest dose. Betel leaf can be bothstimulatory and inhibitory to thyroid function, particularly for T3 generationand lipid peroxidation in male mice, depending on the amount consumed (7). Materials and Methods.
Sample:Piper betal leaf (Sanchi pan)Apparatus requirement:Plates, Flask, Tubes, Pipette, Bunsen burner, Match box, Wire loop,weight machine, oven, nutrient agar, sabouraud dextrose agar.Orgainsms:· Pseudomonasaeruginosa· Staphylooccusaureus· Escherichia coli· Candida albicans.Pseudomonas aeruginosa:P.aeruginosa is a gram negative organism an a rod shaped bacterium. Itcause diseases in animal, plants as well as humans. It occur especially inpatients with compromised immune system.
It is commonly found in soil, waterand moist enviroment.Staphylococcus aureus:S.aureus is a Gram positive bacteria and it is a round shaped bacterium. It is ommnonly found in skin, noseand respiratory track. It is the leading cause of skin and soft tissueinfections such as abscesses, furuncles and cellulitis.The infection of S.aureusare not serious but it can serious infection such as pneumonia, blood streaminfection, bone and joint infections.
Escherichia coli:Escherichia coli is agram nagative organism, it is a rod shapedbacteria. Ecoli found in food, environment and intestine of humans and animals.It is harmless and beneficial floral of gut.
By eating contaminated food ordrinking water so some strains of Ecoli can cause diarrhea.Candida albicans:Candida albicans is an opportunistic yeast and it is a member of human gut flora, itdoes’nt exist outside the body. It is commonly found in gastrointestinal trackand in mucous membranes such as the vagina, mouth of healthy adults, or rectum.But it can be pathogenic in immunocompromised patients.
Agars:Nutrient agarSabouraud dextrose agarNutrient agar:It is a basic growth medium used for the routine cultivation of nonfastidious organism. It is useful because it doesn’t lose consistency andremain in solid state evev at high temperature. We can easily observe bacterialgrowth because of its clear surface. Different types of organism can easilygrow on nutrient agar but some bacteria cannot grow on this.Composition:0.5% peptone0.3% beef extract/yeast extract1.5% agar0.
5% NaClDistilled waterpH 6.8 at 25CSabouraud dextrose agar:SDA media is used to cultivating pathogenic fungi, and alsodetermining the microbial and fungal content of cosmetics. Dextrose is thefermentable carbohydrate incorporated in high amount of energy and carbonsource. Peptone mixture give vitamins, minerals, nitrogen and amino acidessential for growth. The concentration of dextrose is high and acidic pH make this medium suitable for fungi.
Composition:10gm peptone 40gm Dextrose15gm AgarDistilled waterpH adjust to 5.6 at 250 C Protocol:First day session: First clean your table top and surrounding with alcohol dipped cotton. First clean your table top andsurrounding with alcohol dipped cotton. Leave flame open for 5 minute to make your environment clear. Dried leaves in hot air oven for 5 minutes. Take a leaves and washed with distilled water. Take 2gm of powdered dried sample.
Soaked 2gm of dried powder sample in 80ml methanol. Kept in dark place for 4 days until the secondary metabolites get dissolved. Take 80ml methanol. Now grinder the dried leaves and makes a powder.
Fourth day session: After 4 days filter the filtrate in petriplates by the help of What’s man filter paper No.1. Now the dried metabolite extract was dissolved in DMSO (Dimethyl Sulfoxide). Nutrient agar and SDA agar plates were spreaded with 60 microliter of bacterial culture to check the antimicrobial activity. Make 2 wells of 5mm diameter on each plate with the help of sterile borer. Plates were incubated at 37 degree foe 24 hours. ON next day note the results.
Now these wells were filled with 60 microliter of Methanolic extract and Streptomycin antibiotic for antibiogram. Take 3 nutrient agar and 1 Sabouraud dextrose agar plates and label with each organism. Kept the petriplates in hot air oven at 50 degree so that methanol gets evaporated. Fifth day session:On fifth day we perform Antibiogram analysis of tested organisms: First clean your table top and surrounding with alcohol dipped cotton. Take 3 nutrient agar plates and label the each organism. Place the plates in incubator at 37 degree for 24 hours. Now place the antibiotics on each plate in order to check the effectiveness. Leave flame open for 5 minute to make your environment clean.
On next day note the results. Observation:Antibacterial susceptibility assay of Methanolic extract of piper betelleaf against Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coliand Candida albican. TEST ORGANISM ZONE OF INHIBATION BY STREPTOMYCIN (in mm) ZONE OF INHIBATION BY SAMPLE (in mm) Pseudomonas aeruginosa 29mm 16mm Staphylococcus aureus 35mm 25mm Escherichia coli 30mm 20mm Candida albicans ______ 17mm Candida albican Escherichia coli Pseudomonas aeruginosa Staphylococcus aureus Antibiogram analysis: Antibiotics Pseudomonas aeruginosa Staphylococcus aureus Escherichia coli Streptomycin 18mm 14mm 16mm Tetracycline 4mm 5mm ____ Penicillin No zone No zone No zone Erythromycin No zone 10mm 25mm Ciprofloxacin 30mm 29mm 27mm Results:1 sample of Piper betel leaf has been taken from the market.The sample was preceded for the analysis of antibacterial and antifungalactivity of extract of Piper betel leaf. The sample first dried and then makepowder of dried leaves of 2gm were soaked in 80ml methanol and incubated for 4days. After 4 days filter the extract in plate and dried the extract in hot airoven so methanol get evaporated.
Now dried metabolites dissolved in DMSO tomake the extract. Now the organism was cultured on nutrient agar and onsabouraud dextrose agar. Make wells by the help of borer and filled the wellswith extract after one day incubation the zone of inhibition observed against Pseudomonasaeruginosa, Staphylococcus aureus, Escherichia coli and candida albican.
Pseudomonas aeruginosa give 16mm diameter of zone, Staphylococcus aureus give25mm diameter of zone, Escherichia coli give 20mm diameter of zone and Candidaalbican give 17mm diameter of zone. The result of Antibiogram against thetested organism showed that Pseudomonas aeruginosa inhibit Ciprofloxacin andgive 30mm zone by streptomycin 18mm and by tetracycline 4mm zone.Staphylococcus aureus inhibit Ciprofloxacin and give 29mm zone, by Streptomycin14mm zone, Erythromycin 10mm zone and by Tetracycline 5mm zone. Escherichiacoli inhibit Ciprofloxacin and give 16mm zone, by Streptomycin 16mm zone and byerythromycin give 25mm zone. Discussion: Piper betel leaf is a herbal medicine and it is medicallyvaluable. It is the best alternative antibiotic that is available against theorganisms. It is in green color with aromatic smell and bitter in taste.
Formouth freshener the betel leaf is the best freshener that can be taken afterlunch or dinner.Methanolic extract of Piper betel leaves were prepared thatcan be used for antimicrobial studies in the research work.Agar well diffusion method of Kirby borer is used to observethe antibacterial and antifungal properties of Piper betel leaf extract againstthe pathogenic organisms.The methanolic extract of Piper betel leaf has goodantibacterial properties against Staphylococcus aureus that showed zone ofinhibition (25mm) and Escherichia coli showed (20mm) zone of inhibition.Ciprofloxacin antibiotic that give large zone of inhibitionagainst Pseudomonas aeruginosa (30mm), against Staphylococcus aureus (29mm) andEscherichia coli (27mm) zone. Streptomycin is the second antibiotic that can give largezone and inhibit by these organism against Pseudomonas (18mm), againstStaphylococcus aureus (14mm) and against Escherichia coli (16mm).
Piper betel leaf can be a good source of herbal medicineespecially in methanol as a solvent. The methanolic extract can be effectiveagainst the above bacteria.