Journal of Chemical, Biological and Physical Sciences An International Peer Review E-3 Journal of...

May 2013- July 2013, Vol. 3, No. 3; 1886-1899. E- ISSN: 2249 –1929

Journal of Chemical, Biological and Physical Sciences An International Peer Review E-3 Journal of Sciences

Available online atwww.jcbsc.org

Section B: Biological Science

CODEN (USA): JCBPAT Research article

1886 J. Chem. Bio. Phy. Sci. Sec. B 2013, Vol.3, No.3; 1886-1899

Studies on Antimicrobial Properties of Vegetable, Fruit & Spice Peels and Shells

#Suvro Saha, ^Uma Ghosh and *John Barnabas

*#Post Graduate-Department of Microbiology, *Centre for R &D in Life Sciences; Microbiology Research Laboratory, Dayananda Sagar Institutions, Dr.C.D.Sagar Centre for Life Sciences,

Shavige Malleshwara Hills, Kumaraswamy Layout, Bangalore-560078, India

^Food Technology and Biochemical Engineering, Jadavpur University, Kolkata-700032, India

Received: 27 May 2013; Revised: 18 June 2013; Accepted: 21 June 2013

Abstract: The current study examines the extraction and assay of antimicrobial metabolites from peels and shells of vegetables, fruits and spices. Peels and shells are primarily discarded into environment as agro waste, nevertheless in our studies we initiate that these may well be explored for therapeutics, which could be economic, eco-friendly, presumed not to cause side effects as they originate from a natural source and therefore also benefit the waste management. Peels and shells were dried, powdered, followed by extraction with solvents such as acetone, methanol, hot water and cold water. Our findings report the antimicrobial effect of ten peels and shells against six species of bacteria and three species of fungi. The significance of our work also goes to show that all peels and shells except for the orange peels were probably the earliest investigation of this sort to survey such wastes for antimicrobial properties. Our findings demonstrate that, maximum antibacterial and antifungal action were experiential to be found by cold-water extraction of jackfruit against Proteus vulgaris and Aspergillus niger respectively

Key words: Antimicrobials, peels, shells, vegetables, fruits.

Studies on…. Suvro Saha et.al.

1887 J. Chem. Bio. Phy. Sci. Sec. B; 2013, Vol.3, No.3; 1886-1899.

INTRODUCTION

The antibacterial properties of plant extracts have been a searing area for investigators of microbiology, pharmacology and related fields. Although there is a great sum of progress in containing infectious diseases and the development of new drugs, diseases caused by various pathogenic microorganisms are still contemplated to be a major threat to public health1. These pathogens have become resistant to many traditionally used antimicrobial agents. In the past few decades it is observed that there is a dramatic increase in microbial resistance to antimicrobial agents, therefore this has led the scientific community to ascertain new antimicrobials and consequently to formulate new drugs. A global problem of mortality due to drug-resistant infections and related illnesses is found on the rise at an alarming rate. An estimated twenty five thousand patients succumb to death as a result of antibiotic resistant in Europe each year and embody a cost of $1·85 billion3and in the USA an estimated $20 billion a year4 alone and so in the Asian countries and the other parts of the world. It is thought that the antimicrobial resistance is one of the three greatest threats to human health by The World Health Organization. The nosocomial infections are thought to be more severe worldwide meritoriously escape the effects of ratified antibacterial drugs5-7. This nature of antibiotic pipeline struggle may actually alter the exercise of medicine as it is known.

It is comprehended that the operational life span of any antibiotic is limited, for this reason there is growing interest of clinical microbiologists for antimicrobial plant extracts as these phytochemicals may find their way into the store of antimicrobial drugs recommended by physicians, several of which have already being tested on humans. It is exciting to note that, two or three antibiotics resulting from microorganisms are propelled each year8. Of late, the plant compounds their extracts are relishing boundless popularity and the sales of these medicines increase enormously. Factually, plants offer a natural source of creativeness for novel drug compounds, as plant derived medicines have made huge impacts on human health and well-being. Over fifty percent of the western drugs are understood to have derived from plant materials 9 moreover these antibiotics made from these sources have not been found to cause side effects and are also found to be less expensive10,11.

Peels and shells of vegetables, fruits and spices are discarded as garbage and a great amount of such wastes are produced during industrial processing. Most of the time these wastes are utilized as biofertilizers but using this agro waste therapeutically is an innovative concept which is gradually gaining acceptance moreover, these are novel, natural, eco-friendly and biodegradable. These highly economic sources for deriving antimicrobials, can be used in prevention of diseases caused by pathogenic microbes. It was estimated that only citrus waste amount was to approximately 350,000 tons per year. This is probably a new alternative way of waste management. The current work focuses on the extraction and assay of antimicrobial components from peels and shells of vegetables, fruits

and spices. Peels and shells: orange (Citrus sinensis) peels, stone apple (Aeglem armelos) shells, coconut (Cocos nucifera) shells, pea (Pisum sativum) peels, jack fruit (Artocarpus heterophyllus) peels, Banana Inflorescence peel, pumpkin (Cucurbit apepol) peels, watermelon (Citrullus lanatus) peels, black cardamom (Amomum subulatum) peels, nutmeg (Myristica fragrans) shells. Microbial Cultures: Escherichia coli, Klebsiella pneumonia, Staphylococcus aureus, Bacillus subtilis, Proteus vulgaris, Lactococcus lactis, Aspergillus niger, Candida albicans, Sporotrichum pruinosum.

MATERIALS AND METHODS

Plant Materials: Fresh samples of fruits, spices and nuts were obtained from industrial wastes. They were thoroughly washed with tap water. After air-drying, the peels were separated and cut into small



pieces. In case of coconut and stone apple the shells were neatly shaven. Then these peels and shells were set aside for drying in sunlight for 24 hours and shifted to an hot air oven at 400C

Microbial Strains & Culture Preparation :

Bacterial Strains used: Staphylococcus aureus, Proteus vulgaris, Lactococcus lactis, Bacillus subtilis, Escherichia coli, Klebsiella pneumonia.

Fungal strains used: Sporotrichum pruinosum, Candida albicans and Aspergillus niger.

The bacterial cultures were maintained on nutrient agar media (pH-7.0). They were sub-cultured weekly and subsequently stored at 40C. The fungal cultures were maintained on Czapek Dox Agar media (pH-5.6) and they were sub-cultured weekly and subsequently stored at 40C for another 24 hours. After complete drying, peels and shells were subjected to homogenization to make fine powder. These powders were sealed in air tight bottles. They were then used for solvent extraction.

Extraction Procedure: The dried powder of peels and shells were extracted individually by using solvents such as Acetone, Methanol, Hot water & Cold Water. The powder and solvent were mixed in 1:10 (Powder: Solvent) ratio in a conical flask and the flasks were plugged tightly. The temperature for the hot water extraction and cold water extraction were maintained at 500C and room temperature respectively. After 24 hours, these extracts were centrifuged at 4000rpm for 5 minutes and the supernatant was collected and this supernatant was subjected for lyophilization to evaporate the solvent. Then the resulting dried crude extract was used in a concentration of 3mg/ml by using sterile water for antimicrobial susceptibility testing.

Bacterial and Fungal Strains & Culture Preparation: The bacterial strains used in the study were Staphylococcus aureus, Proteus vulgaris, Lactococcus lactis, Bacillus subtilis, Escherichia coli, Klebsiella pneumonia. The fungal strains were Sporotrichum pruinosum, Candida albicans and Aspergillus niger.

The bacterial cultures were maintained on nutrient agar media (pH7.0). They were sub-cultured weekly and subsequently stored at 40C. The fungal cultures were preserved on Czapek Dox Agar media (pH 5.6) and were sub-cultured weekly and subsequently stored at 40C

ANTIMICROBIAL ACTIVITY ASSAY

The antimicrobial activity of the obtained crude samples was performed by Agar Well Diffusion method which is also known as Kirby Bauer or Cup Plate method.

Antibacterial Activity: Sterile nutrient agar media was poured onto the sterile petriplates and allowed to solidify. After solidification 20µl of each bacterial culture was spread with a sterile glass spreader on respective plate labeled earlier as Staphylococcus aureus, Proteus vulgaris, Lactococcus lactis, Bacillus subtilis, Escherichia coli, Klebsiella pneumonia. Using a sterile cup-borer, wells of diameter of 17.78mm were made and labeled on the back of the plate. To this, 20µl of each samples (3mg/ml) was dispensed in respective labeled wells. Bacterial plates were incubated at 370C for 24hours. After this the antibacterial activity of each extract was expressed in terms of mean of diameter, for the zone of inhibition (mm) formed by addition of each extract.

Antifungal Activity: Sterile Czapek Dox Agar media was transferred onto the sterile petri plates and allowed to solidify. After solidification, with a sterile glass spreader, 20µl of each fungal culture was spread on respective plate labeled earlier as Sporotrichum pruinosum, Candida albicans and Aspergillus niger. using a sterile cup-borer, wells of diameter of 17.78mm were prepared and labeled at the flipside of the plate. 20µl of each sample (3mg/ml) was dispensed in respective labeled wells.



The fungal plates were incubated at 300C for 48 hours. After 48 hours of incubation the antifungal activity of each extract expressed in terms of the mean of diameter of zone of inhibition (mm) produced by each extract.



RESULTS:

A.1-Orange Peel Methanol Extract, A.2- Orange Peel Acetone Extract, A.3- Orange Peel Hot Water Extract, A.4- Orange Peel Cold Water Extract; B.1- Bael Shell Methanol Extract, B.2- Bael Shell Acetone Extract, B.3- Bael Shell Hot Water Extract, B.4- Bael Shell Cold Water Extract; C.1- Pea peel Methanol Extract, , C.2- Pea peel Acetone Extract C.3- Pea peel Hot Water Extract, C.4- Pea peel Cold Water Extract; D.1- Coconut shell Methanol Extract, D.2- Coconut shell Acetone Extract, D.3- Coconut shell Hot Water Extract, D.4- Coconut shell Cold Water Extract; E.1- Jackfruit Peel Methanol Extract, E.2- Jackfruit Peel Acetone Extract, E.3- Jackfruit Peel Hot Water Extract, E.4- Jackfruit Peel cold Water Extract F.1- Banana Inflorescence Peel Methanol Extract; F.2- Banana Inflorescence Peel Acetone Extract, F.3- Banana Inflorescence Peel Hot Water Extract, F.4- Banana Inflorescence Cold Water Extract G.1- Pumpkin Peel Methanol Extract, G.2- Pumpkin Peel Acetone Extract, G.3- Pumpkin Peel Hot Water Extract, G.4- Pumpkin Peel Cold Water Extract; H.1- Watermelon Peel Methanol Extract, H.2- Watermelon Peel Acetone Extract, H.3- Watermelon Peel Hot Water Extract, H.4- Watermelon Peel Cold Water Extract; I.1- Black Cardamom Peel Methanol Extract, I.2- Black Cardamom Peel Acetone Extract, I.3- Black Cardamom Peel Hot Water Extract, I.4- Black Cardamom Peel Cold Water Extract; J.1- Nutmeg Peel Methanol Extract J.2- Nutmeg Peel Methanol Extract J.3- Nutmeg Peel Methanol Extract, J.4- Nutmeg Peel Methanol Extract



,



DISCUSSION

Amongst the samples used in previous work, only the orange peel had been used for antimicrobial substance extraction by several previous investigators12-16. Our studies which include a whole range of peels and shells indicate that, certain peels were found demonstrating higher zone of inhibition than the earlier studied orange peels. The above four solvents utilized in the studies, have been used earlier by Kumar et al.13, 15; Amandeep et al.14, Akhilesh et al.12. After lyophilization the extracts were subjected to antibacterial and antifungal susceptibility assay by agar well diffusion method.

In Fig.1 Effect of the extracts on Escherichia coli; The zone of inhibition for methanol and cold water extracts of orange peel were respectively 46.48mm and 33.78mm against Escherichia coli but other two extracts of orange peels were not effective against this bacteria. The inhibition zone for the methanol, hot water and cold water extracts of stone apple shell were respectively 55.58mm, 51.56mm and 33.78mm but the acetone extracts of this sample was not effective against E.coli. The methanol extracts of the stone apple shell demonstrated a maximum inhibition zone among all the samples used in this work. All the extracts i.e. methanol, acetone, hot and cold water extracts of pea peel were effective, their zone of inhibition demonstrated by these four extracts of the pea peels were respectively 33.02mm, 33.02mm, 28.70mm, 23.62mm. Amongst the four extracts of coconut shell, only methanol extracts was found effective (49.78mm). 37.08mm of inhibition zone was observed for cold water extract of jackfruit but no inhibition zone was observed for the three extracts of jackfruit peel. 30.48mm and 43.18mm of zone of inhibition was observed respectively for the hot and cold water extracts banana inflorescence peel and other two extracts were not effective against E.coli. The inhibition zone for the methanol and hot water extracts of pumpkin were respectively 27.94mm and 35.56mm but no zone was observed by the two extracts of pumpkin peel. Among four extracts of watermelon peel, only methanol extracts were capable to inhibiting the growth (32.04mm) of E.coli. The inhibition zone of the extracts of methanol and cold water extracts of black cardamom were 24.38mm and 38.86mm respectively and two other extract of this sample were not observed to be effective. In case of nutmeg, the acetone extract (43.18mm) and cold water extract (38.1mm) were effective but inhibition zone was not shown by other two extracts of this sample.

Fig.2 Effect of extracts on Klebsiella pneumonia: 43.94mm and 38.1mm of zone of inhibition was demonstrated by acetone and cold water extract of orange peel against K. pneumonia and two extracts of this extract were not effective against this bacteria. The inhibition zone observed for the two extracts i.e. methanol and cold water extracts of stone apple were respectively 43.18mm and 27.94mm. Out of four extracts of pea peel and coconut shell, the inhibition zone was observed only for methanol extracts (35.56mm and 48.26mm respectively). The maximum zone was produced by methanol extracts of coconut shell against K. pneumonia. The zone of inhibition which were observed for hot and cold water extracts of jackfruit peel, were 26.16mm and 21.84mm respectively but no inhibition zone was produced by other two extract of jack fruit. Among the four extracts of banana inflorescence peel, the inhibition zone was produced by hot water extract (27.94mm). Except cold water extracts of pumpkin peel the inhibition zones were observed for three extracts i.e. methanol extract (38.1mm), acetone extract (21.84mm) and hot water extract (40.64mm). No inhibition zone was observed for all four extracts of watermelon peel. Inhibition zone of 38.1mm and 34.54mm were observed respectively for hot and cold water extract of black cardamom and other two extracts did not demonstrate inhibition zone. Amongst the four extracts of nutmeg, the zone was observed for methanol extracts i.e. 23.62mm.

In Fig.3 Effect of extracts on Staphylococcus aureus: except the acetone extract of orange peel, the other zone of inhibition were produced by remaining three extracts i.e. methanol extracts (27.94mm), hot and cold extracts (38.86mm and 37.08mm respectively). Except the acetone extract of stone apple,



the zone of inhibition were 49.78mm, 43.18mm and 35.56mm respectively for methanol which demonstrated a maximum zone, hot and cold water extracts of stone apple against S. aureus. None of the four extracts of the pea peel produced inhibition zone. The inhibition zones were produced respectively by methanol extracts (47.75mm), acetone extracts (29.46mm) and hot water extracts (20.32mm) of coconut shell but no zone was observed for cold water extract of this sample. 30.48mm and 33.78mm of inhibition zone was observed respectively for the acetone and hot water extract of jackfruit peel and no zone of inhibition was seen for other two extract of this sample. Among the four extracts of banana inflorescence, the inhibition zone (32.04mm) was observed only for hot water extract of this sample against S. aureus. Apart from cold water extract of pumpkin peel, the inhibition zones were observed for methanol extracts (26.16mm), acetone extract (34.54mm) and hot water extract (38.1mm). Inhibition zone of 33.02mm and 24.38mm were observed respectively for methanol and hot water extract of watermelon peel but remaining two extracts of this sample did not produce any observable zone of inhibition. Among the four extracts of black cardamom, inhibition zone (36.32mm) was observed only for methanol extract of this sample. Among the four extracts of nutmeg, the zone was only observed for cold water extracts i.e. 42.16mm.

In Fig.4 Effect of extracts on Bacillus subtilis; the zone of inhibition was observed by all four extracts i.e. methanol extract (40.64mm), acetone extract (30.48mm), hot water extract (26.16mm) and cold water extract (45.72mm) of orange peel. Apart from acetone extract of stone apple shell, the zone of inhibition was observed as 48.26mm, 39.62mm and 38.86mm respectively by methanol extract, cold and hot water extract of this sample; the highest zone of inhibition was shown by the methanol extract of stone apple. Among the four extracts of pea peel, the inhibition zone (40.64mm) was only observed for methanol extract of this sample. Out of four extracts of coconut shell, only one extract i.e. acetone extract was effective against B.subtilis; giving a inhibition zone of 21.84mm.Among the four extracts of jackfruit peel, the inhibition zone (38.1mm) was only seen for cold water extract of this sample. The inhibition zone of 42.16mm and 43.18mm was detected respectively for acetone and cold water extract of banana inflorescence but no zone of inhibition for remaining two extract of this sample. Except cold water extracts of pumpkin peel, inhibition zone was observed for rest of the three extracts i.e. methanol extract (19.30mm), acetone extract (39.62mm) and hot water extract (38.86mm). Among the four extracts of watermelon peel, the inhibition zone (33.78mm) was only seen for hot water extract of this sample. The zone of inhibition of 47.24mm and 38.1mm was shown by two extracts i.e. hot and cold water extracts of black cardamom out of four extract and no zone was detected for remaining two extracts of the sample. Among the four extracts of nutmeg shell, the inhibition zone (27.94mm) was only seen for methanol extract of this sample.

Fig.5 Effect of extracts on Proteus vulgaris; the zone of inhibition of 23.62mm and 28.70mm were observed respectively for acetone and cold water extract of orange peel but the other two extracts were not to be effective against the organism. Except the acetone extract of the stone apple shell, the inhibition zone was observed as 41.40mm, 40.64mm, 36.32mm respectively for the methanol, hot and cold water extract of the sample. In case of pea peel, the inhibition zone (38.1mm) was only detected for methanol extract of pea peel. Apart from cold water extract of coconut shell remaining three extracts are effective against P. vulgaris; the zones were observed as 49.02mm, 28.70mm and 39.62mm respectively for methanol, acetone and hot water extract of the sample. Among the four extracts of jackfruit peel, the inhibition zone (84.58mm) was only observed for cold water extract of this sample. The inhibition zone was not observed for all extracts of banana inflorescence and water melon peel against P. vulgaris. In case of pumpkin peel, the inhibition zone (36.32mm) was only detected for methanol extract of pumpkin peel. Except the acetone extract of the black cardamom peel, the inhibition zone was observed as 75.18mm, 35.56mm, 48.26mm respectively for the methanol, hot and cold water extract of the sample. The zone of inhibition of 40.64mm and 38.1mm



were observed respectively for the methanol and cold water extract of nutmeg shell but the other two extracts were not found to be effective against the organism.

In Fig.6 Effect of extracts on Lactococcus lactis; among the four extracts of orange peel, the zone of inhibition was observed for hot and cold water extract (28.70mm and 54.86mm respectively); the maximum zone was given by cold water extracts of the orange peel. Except the acetone extract of the stone apple shell, the inhibition zone was observed as 43.18mm, 37.08mm, 47.24mm respectively for the methanol, hot and cold water extract of the sample. The inhibition zone for the methanol extract of stone apple was same against K. pneumonia and L. lactis. Among the four extracts of pea peel and coconut shell, the inhibition zone (30.48mm and 33.78mm) was only seen for methanol extract of these two samples. The inhibition zone (18.54mm) was observed only for the cold water extract of jackfruit peel sample. 29.46mm and 34.54mm inhibition zones are observed respectively for hot and cold water extracts of banana inflorescence peel but no zone of inhibition was seen for other two extracts of this sample. Among the four extracts of the pumpkin peel, the zones of inhibition were detected for two extracts i.e. methanol extract (35.56mm) and hot water extract (45.72mm). No zone of inhibition was produced by the four extracts of the watermelon peel. The inhibition zone by the hot water extracts of black cardamom peel and methanol extract of nutmeg shell were observed as 34.54mm and 27.94mm respectively but inhibition zone was not observed for the remaining extracts of these samples.

Fig.7: Effect of extracts on Aspergillus niger; the zone of inhibition of 41.40mm, 23.62mm, 21.84mm, 31.24mm, 49.02mm, 29.46mm, 30.48mm, 81.28mm (to note that highest zone of inhibition was observed against Aspergillus niger), 39.62mm were observed respectively for methanol and acetone extract of orange, methanol and acetone extract of stone apple, acetone and hot water extract of pea peel, hot water extract of coconut shell, cold water extracts of jackfruit peel and methanol extract of pumpkin peel.

In Fig.8 Effect of extracts on Candida albicans; among the four extracts of the orange peel and stone apple shell, the zone of inhibition were detected as 50.8mm, 54.86mm, 22.86mm, 55.88mm, 37.08mm respectively for the methanol extract of the orange peel, acetone extract of stone apple shell and pea peel, cold water extract of jackfruit peel and banana inflorescence peel and area of inhibition was not observed for other extracts of these five samples. The inhibition zone of 22.86mm and 41.40mm was detected respectively for methanol and acetone extracts of coconut shell but remaining two extracts were not effective against C.albicans. No zone of inhibition was observed for the pumpkin peel extracts against C. albicans. The inhibition zone of 74.42mm and 35.56mm was detected respectively for acetone and hot water extracts of watermelon peel but remaining two extracts were not effective against the organism. Except for the acetone extract of black cardamom, the remaining three extracts i.e. methanol extract (45.72mm), hot and cold water extract (46.48mm and 40.64mm respectively) were effective. Zone of inhibition of 48.26mm, 68.58mm, 25.4mm were observed respectively for methanol, acetone and hot water extracts of nutmeg shell. The highest zone was given by acetone extract of watermelon peel.

Fig.9 Effect of extracts on Sporotrichum pruinosum, the zone of inhibition of 34.54mm, 66.04mm and 56.64mm were observed respectively for the methanol, acetone and hot water extract of orange peel but cold water extract was not observed against the organism. The zone of inhibition of 36.32mm, 33.02mm and 66.04mm were observed respectively for the methanol, hot and cold water extract of stone apple shell but acetone extract was not found effective against the organism. The zone of inhibition of 39.62mm and 34.54mm were observed respectively for the hot and cold water extract of pea peel and no zone of inhibition was observed for remaining two extract. Apart from methanol extract of coconut shell, the area of inhibition was observed respectively by acetone extracts



(43.94mm), hot water extract (38.86mm) and cold water extract (33.78mm). In case of jackfruit peel, the zone of inhibition was observed only for two extracts i.e. methanol extract (26.16mm) and cold water extract (42.16mm). All the four extracts of banana inflorescence peel were not capable to inhibit the growth of S. pruinosum. 54.86mm and 43.94mm zone of inhibition was observed respectively for methanol extract of pumpkin peel and acetone extract of watermelon peel but no zone of inhibition for remaining extracts of these two samples. The zone of inhibition of 49.78mm and 34.54mm were detected for acetone and hot water extracts of black cardamom. Except the hot water extract of nutmeg shell, the zone of inhibition was detected for methanol extract (40.64mm), acetone extract (38.1mm), and cold water extracts (38.86mm). The highest zone of inhibition was observed for both acetone extract of orange peel and cold water extract of stone apple shell. From the above observations of the results, it is therefore evident that the peels, shells of vegetable, fruits, spice and shells have caused significant levels of antimicrobial effects on selected bacteria and fungi.

CONCLUSIONS

The experimental findings indicate that the peel extracts comprise imperative antibacterial and antifungal properties. Amongst the samples analyzed, the highest antibacterial and antifungal activity was found with cold water extract of jackfruit peel against Proteus vulgaris and Aspergillus niger, and also; methanol extract of black cardamom peel against Proteus vulgaris, methanol extract of stone apple or bael shell against E.coli., acetone extract of watermelon peel against C. albicans, acetone extract of nutmeg shell against C. albicans. Thus new aspects concerning the use of the wastes therapeutically are precisely interesting and attractive, as mentioned earlier, the present investigation focuses on the possibility of using the peels and shells waste as a source of low-cost natural antimicrobials. Except for the orange peel, antimicrobial activities of the other samples are thought to be reported in a new perspective, thus opening up new frontiers for drug exploration. Further studies are required to determine the nature of compounds responsible for the antimicrobial effects from these extracts. The chances are elevated, that the study may lead to the formulation of an antimicrobial drug after appropriate pharmacological evaluation and clinical trials. The above work on peels and shells, explore new therapeutic applications and also introduce an alternative method for agro-waste management.

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Corresponding author: Dr.John Barnabas, Post Graduate-Department of Microbiology, Centre for R&D in Life Sciences; Microbiology Research Laboratory, Dayananda Sagar Institutions, Dr.C.D.Sagar Centre for Life Sciences, Shavige Malleshwara Hills, Kumarswamy Layout Bangalore-560078, India. Email- johnbarnabas (at)gmail.com

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