6 December 2015 Volume 6
SI.No
1 A Facile Spectrophotometric Determination of Osmium(VIII) in very low 1 - 6 concentrations using DL-Pencillamine as Chromogenic Reagent Bincy Joseph and Abraham Joseph
2 Evaluation of insecticidal and larvicidal activity and the detection of lipase 7 - 14 from Eupatorium odoratum leaf extracts. Soniya P.A., Navia Sebastian and Dr. Deepa G Muricken
3 Biosynthesis of Gold Nanoparticles using Bacteria and Fungi 15 - 21 Nivea Wilson, Nileena PV, Dhanya KC
4 Comparative Study on Antibacterial profiles of Kanakasava 22 - 25 Elizabeth P Thomas, Uma V Menon P, Afeeda Beegum M K, Athira MR, Darsana NC, and KS Dhaneesha Sivadas
5 Antibacterial effect of citrus fruit juice against enteric and non enteric 26 - 33 pathogenic bacteria Geenat Paul , Rigi George A , Rahima N A
6 Hydrogen Peroxide Scavenging Ability of Scoparia Dulci&Piper Longum Linn 34 - 40 ABAS Geetha T December 2015 7 Effect of plant growth promoting Proteus spp.on fenugreek 41 - 49 Vol. 6, No.1 Jimtha John C, Mrudula M M, Anisha C, and Radhakrishnan EK
8 A study on the antibiotic sensitivity test of selected isolates from facial 50 - 60 microflora and their susceptibility to commercial face washes Femina K K, Finciya A T, Hanna T V, Jasira V, Jini Joy P & Mabel Merlen Jacob
9 Bioactive peptides derived from milk: a review 61 - 65 Reshma Chandran P 5 1 0 2 Annals of Basic and Applied Sciences December 2015, Volume 6, Number 1, (ISSN 2277-8756). (Official publication of St Mary's College, Trichur - 680020, Kerala, India)
Editor
Dr Meera C R Department of Microbiology, St. Mary's College, Trichur - 680020 Kerala, India.
Associate Editors
Dr. Dhanya KC Dr. Mabel Merlen Jacob Department of Microbiology, Department of Microbiology, St. Mary's College, Trichur - 680020, St. Mary's College, Trichur - 680020, Kerala, India. Kerala, India.
Editorial Board
Dr. Regi Raphael K Dr. Kezia Kuruvila Dr. Sheeja T Tharakan Dr. Manju Madhavan Department of Botany, Department of Zoology, Department of Botany, Department of Botany, St. Mary's College, Vimala College, Vimala College, Vimala College, Trichur-680020, Trichur-680009, Trichur - 680009, Trichur - 680020 Kerala, India. Kerala, India. Kerala, India. Kerala, India.
Dr Geetha T Dr. Manju Sebastian Dr. Rekha K Department of Chemistry, Department of Department of Botany, St. Mary's College, Chemistry, St. Mary's College, Trichur - 680020, St.Mary's College, Trichur - 20 Kerala, India. Trichur - 680020, Kerala, India. Kerala, India.
Scientific Advisory Board
Dr. Sr Jacintha C C Dr. K K Janardhanan Dr.C K K Nair Principal, Professor & Head, Director of Research, St. Mary's College, Department of Microbiology, St. Gregorios Dental Trichur - 680020, Amala Cancer Research Centre, College & Research Centre, Kerala, India. Trichur - 680555, Kerala, India. Kothamangalam – 686681, Kerala,India.
Dr. S G PrapuIla Dr.Valsa A K Department of Fermentation, Department of Microbiology, Technology & Bioengineering, Sree Sankara College. Kalady, CFTRI. Mysore - 570020, Emakulam-683574, Karnataka, India. Kerala,India.
Publishers St.Mary'sCollege,Trichur-680020.Kerala,India. A Facile Spectrophotometric Determination of Osmium(VIII) in very low concentrations using DL-Pencillamine as Chromogenic Reagent
Bincy Joseph and Abraham Joseph*
* Department of Chemistry ,University of Calicut, Calicut University Corresponding Author:Bincy Joseph,Dept.Of Chemistry,St.Mary’s College,Thrissur.Ph.No.9946355330 Email:[email protected].
ABSTRACT
A simple spectrophotometric technique of high sensitivity and selectivity has been developed for the determination of trace amount of osmium(VIII) using DL- Pencillamine(DL- Pen) as a chromogenic reagent. The method is based on the formation of a pale pink complex at a fast pace at room temperature by the reaction of osmium(VIII) with DL-Pen in strongly acidic solution of pH 1 which absorb at 540 nm. The decrease of absorbance is directly proportional to osmium concentration and obeys the Beer’s law in the range of 0.15-1.50 µg/ml. The optimum reaction condition and 4 - other analytical requirements were evaluated. The molar absorptivity(5.05 x 10 LMol 1 -1 -3 cm ), Sandell's sensitivity(3.8 x 10 -2 -1 -1 µgcm ), detection limit(0.066 µgml ) and quantitation limit(0.20 µgml ) of the method were computed.
Key words
Osmium, Spectrophotometry, DL-Pencillamine , Chromogenic reagent.
1 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
1.Introduction emission spectrometry, electron microprobe analysis, total reflection X- 6 2 Osmium(Z=76,5d 6s ) is a bluish ray fluorescence spectrometry, atomic white metal, the heaviest among the absorption spectrometry and neutron platinum metals, exhibits high specific activation analysis, the visible density(22.61g cm-3), high melting spectrophotometric methods seems to be 0 point(3050 C)and hardness(7.0Mhos the most appropriate analytical approach for the determination of toxic metals like scale) , occurs in ten different oxidation osmium, as it provides sensitive, precise states from Os(II) to Os(VIII). The and accurate measurements of suitable metal and its alloys are used for the analytes and offers practical and production of materials of extreme economical advantages over other hardness. methods. Most of these instruments are
highly expensive and their day to day The volatile osmium tetroxide, maintenance cost is high and are also OsO is formed by the oxidation of 4, not free from various types of inherent osmium, is one of the most important interferences (Somasiri LLWet al., compounds of the element and is easily 1991,Honegger K .,et al 1987 Ramesh et al., identified by its characteristic unpleasant 1992).Besides,visible spectrophotometric odour. Osmium tetroxide is a powerful detection is much more viable as a toxic agent, harmful to eyes and useful technique to develop a portable respiratory tracts. So special precautions on-line or at line system. are needed in sample collection, storage and other procedures due to its strong Most of the existing well tendency of oxidation to volatile established spectrphotometric methods tetroxide and possible losses of the using the sulfur containing molecules element. The volatility of OsO4 makes and ligands like thiourea, thiocynate and the basis of separation of osmium from 1,5-diphenylcarbazide lack adequate its mixtures with other noble metals by sensitivity and selectivity and necessitate the separation of osmium by distillation. The reduction of OsO4 to extraction or distillation before the the black osmium dioxide, OsO2, by determination. biological substances made the basis of its use as a staining reagent for the Many organic reagents have been microscopic examination of tissues. proposed for spectrophotometric determination of osmium, including Compared to the various modern Perphenazine (Gowda et al., 1984) optical methods of analysis including inductively coupled plasma atomic In the flotation methods, the molar
2 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
5 absorptivities are 2.2 x 10 at 655 nm ,2.0 containing ∼100 ml of water acidified 5 x 10 (with crystal violet and malachite with 3 ml of H2SO4 (1+1) wash, dry and green) (Balcerzak M. 1991) . Larger weigh the glass fragments of the amounts of osmium can be determined ampoule and calculate the weight of 2- 3 as osmate, OSO4 (ε = 2.75 x 10 at 340 OsO4 by difference. Dilute the osmium nm) (Ensafi et al 1991) .Other than direct solution with double distilled water till 1 spectrophotometric methods of ml contains precisely 1.5 mg of osmium. determination, certain reagents (Jamshid Perform all these operations in a fume et al 2000) are employed for the cupboard, and keep the osmium solution spectrophotometric determination of in a bottle with precision ground stopper osmium based on its catalytic activity. on account of the toxic properties and Derivative spectrophotometric methods offensive odour of the tetroxide( are also used for the determination of Marczenko Z 1986). osmium under different experimental conditions (Balcerzak M e t al., 1996, Pencillamine solution (1%): Balcerzak M et al.,1997, Kosiorek A et al., Dissolve 1g of reagent (HIMEDIA) in 2006) . 100 ml of double distilled water. Apparatus: The absorbance was In the present work, a reaction of measured and absorption spectra were Os(VIII) with DL-pencillamine have recorded using a systronics double beam been examined at various experimental uv-vis spectrophotometer 2201with conditions and the most suitable Quartz Cells (1cm). Digital pH meter analytical requirements have been (M.C. Dalal and Co. Madras) was used established for the quantitative for pH measurements. determination the element in pure Procedure:Aliquots of the stock solution aliquots and in the presence of various containing 0.15-1.5 µg of osmium(VIII) other metal ions. The proposed method were pipetted out into a series of 10 ml for the element offers many advantages standard flask and made up to the mark over the conventional using dil. H2SO4 (to maintain pH1). 3ml spectrophotometric methods. each of these solutions were taken and mixed with 0.1ml of 1% DL- 2.Experimental pencillamine solution and shaken well.
A pink colour is developed in the Reagents and chemicals:Standard solution and measure the absorbance at osmium solution (1.5 mg/ml) : carefully 540 nm against the reagent blank. The broken an accurately weighed glass calibration graphs were constructed by ampoule containing ∼1 g of OsO4 (J.M. plotting the absorbance against the Chemicals Ltd., London)in a beaker amount of metal ions.
3 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
3. Results and Discussion 0.75µg of osmium. It was noticed that an excess of the reagent does not have DL-pen reacts with osmium at any adverse effect on the sensitivity and room temperature to form a pink stability of the complex. coloured complex species having maximum absorbance at 540 nm. 3.3 Effect of time on absorbance Subsequent studies have been therefore carried out at this wavelength. The The absorbance of the complex influence of pH on the absorbance of was found to be stabilised within ten Os-DLpen chelate, effect of minutes and remained unaltered at least concentration of DL- pen and effect of for half an hour at room temperature, time has been investigated. indicates that the coloured complex species is having substantial stability. 3.1.Effect of pH 3.4.Analytical Data The variation of absorbance of known concentration of osmium with The Beer's law was obeyed in the pH of the medium was studied. A series range of 0.15 - 1.5 µg/ml with relative of buffer solutions differing by pH 0.5 standard deviation in the range of 1- was prepared, and using each of these 8%(Table 1). The molar absorptivity and buffers the experiment was carried out. sandell's sensitivity for the coloured 4 - The maximum absorbance was found at system was found to be 5.5 x 10 L mol pH 1. Hence, this pH was maintained 1 -1 -3 -2 cm and 3.8 x 10 µgcm throughout the investigation. respectively. The detection limit (DL 3.2. Effect of the concentration of DL- =3.3 σ/S) and quantitation limit (QL = pencillamine 10σ/S) (Where σ is the standard deviation of 1m reagent blank (n=5) and Varying amounts of DL- pen S is the slope of the calibration curve) were added to a fixed amount of for the osmium determination were osmium and the absorbance was found to be 0.066 µg/ml and 0.20 µg/ml measured according to the standard respectively. procedure. A volume of 0.1ml 1% solution of DL- pen was found to be optimum for the determination of
4 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
Table.1 Determination of Os(VIII) in pure aliquots
Osmium Osmium S.D (µg) R.S.D. (%) Relative error
µ µ %
taken ( g) found ( g)
0.15 0.14 0.03 7.14 -0.06
0.45 0.43 0.02 4.65 -0.04
0.75 0.74 0.01 1.35 -0.01
1.20 1.22 0.02 2.45 0.01
1.50 1.54 0.02 1.94 0.02
* Average of five determinations.
3.5.Interference Studies addition of a 2% solution of sodium The effect of mg amounts of fluoride and the interference due to several ions on the determination of 3+ 2+ 2+ 2+ 3- La , Co , Cu , Pd , VO4 and 0.75 µg of osmium was studied. No 3+ Ru was eliminated using0.1% interference was noticed in the - - - - solution of EDTA. presence of NO3 ,NO2 , Br ,SCN , 2- 3- 3- + 2+ 4. Conclusion SO3 ,AsSO3 ,AsO4 ,Li , Mg , 2+ 2+ 2+ 2+ 3+ + Ca , Ba , Cd , Hg , Al , Tl , DL-Pencillamine provide a 2+ 3+ 3+ 2+ 4+ 2+ Pb , Sb , Bi , Zn , Ce , UO2 , simple, rapid, sensitive and selective 4+ 3+ 2+ 2+ 3+ 2+ method for the spectrophotometric Se , Cr , Mn , Fe , Rb , Ni , determination of osmium. The major 4+ 2- 2 Pt , MoO4 and WO4 . The advantage of the proposed method is 3+ 3+ 3- presence of Fe , Ru , VO4 , its rapidity, as the colour development 2- 2+ 2+ 2+ is instantaneous at room temperature. CrO4 , Pd , Cu , and Co gave 4+ 4+ 4+ It does not require heating, cooling or high recoveries while Ir , Zr , Ti , long standing to record constant 3+ 2+ La and Be caused low recoveries. absorbance. The reagent is available The application of suitable in pure form and hence completely masking agents can sometimes free from multistage synthesis, as is effectively avoid the interference from the case with most of the reported different cations and anions. The methods .The proposed method does 3+ 4+ 4+ interference due to Fe , Zr , Ti , not require extraction with a solvent 2+ 4+ or addition of a surfactant to intensify Be and Ir was overcome by the the colour of the system which is an
5 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
inevitable part of quite a number of A case study on high purity coal “ reported methods.The metal salt and Mikrochim Acta, 1 : 225-230. the chromogenic reagent are highly Jamshid L. Manzoori, Mohammad soluble in water and hence the entire H. Sorouraddin and Mohammad experiment can be performed without Amjadi, ,(2000), “Spectrophotometric using any other solvent system. determination of osmium based on its catalytic effect on the oxidation of 5.References carminic acid by hydrogen peroxide” Balcerzak M. (1991) “Studies on Talanta 53,61-65. the separation of osmium by flotation Kosiorek A Swiecicka E and and spectrophotometric determination Balcerzak M (2006)” Speciation with Crystal Violet and Malachite Analysis of Osmium(VIII) and Green ,” Analytica Chimica Acta Osmium(IV) by UV‐VIS 242,185. Spectrophotometry Using Quercetin as the Reagent” , Analytical letters: 39, Balcerzak M and Swiecicka.E, 589-592. (1996), “Determination of ruthenium and osmium in each other's presence in Marczenko Z ,(1986) "Separation chloride solutions by direct and third- and spectrophotometric determination order derivative spectrophotometry”: of osmium" Ellis Horwood Ltd., Talanta, 43, 471-475. John Wiley & Sons, 429.
Balcerzak M and Swiecicka Ramesh A, Krishnamacharyulu J , E,(1997)” Rapid simultaneous Ravindranath kL and Brahmaji Rao S determination of ruthenium and osmium (1992 “Simultaneous determination of in aqueous solutions of their tetroxides uranium and thorium by second- by second-order derivative derivative spectrophotometry “ spectrophotometry” Analytica Chimica Analyst, 117, 1037-1039 . Acta : 349, 53-55. Somasiri LLW, Bimie A and. Ensafi A. A. and. Safavi A (1991) Edwards A C (1991)” Inductively “Sensitive spectrophotometric kinetic coupled plasma atomic emission determination of osmium by catalysis spectrometry for the analysis of soil of the pyrogallol red-bromate reaction” extracts prepared on ion-exchange Analytica Chimica Acta, 244, 231 resins “ The Analyst 116 : 601-605. Gowda A.T,. Gowda H.S and. Gowda N.M.M. (1984) The Analyst , “spectrophotometric determination of osmium with perphenazine dihydrochloride” 109, 651-655. Honegger K and Wintsch W , (1987) “Electron microprobe analysis of micro amount powder samples:
6 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
Evaluation of insecticidal and larvicidal activity and the detection of lipase from Eupatorium odoratum leaf extracts.
Soniya P.A.¹, Navia Sebastian2 and Dr. Deepa G Muricken3
1. Department of Microbiology, 2. Department of Biotechnology, 3. Department of Biochemistry, St .Mary’s College, Thrissur. Corresponding author: Deepa G Muricken Assistant Professor, Department of Biochemistry, St .Mary’s College, Thrissur. E mail:[email protected]
ABSTRACT
Eupatorium odoratum belongs to asteraceae family and is used by many people in the field of agriculture as a natural pest repellent. The preliminary phytochemical screening revealed the chemical composition of Eupatorium odoratum leaf extract containing tannins, phenols, phytosterols, flavonoids, saponins, coumarins, quinones, cardiac glycosides, terpenoids, anthraquinones, phytosteroids and steroids. This study aims at exploring the plant in various aspects like insecticidal property and larvicidal property against mosquito larvae. The larvicidal potential of Eupatorium odoratum leaf extract was tested against third instar mosquito larvae. The insecticidal activity was carried out using white mealy bugs and the mortality was observed at every 30 minutes. The present study also identified the presence of lipase enzyme from this plant. This enzyme has a variety of applications in industry. There are no reports of lipase enzyme from Eupatorium odoratum. The current investigation shows the Eupatorium odoratum leaf exhibited lipase activity.
Key words:
Lipase, larvicidal activity, insecticidal activity, Eupatorium odoratum.
7 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
1. Introduction leaves were washed thoroughly with tap water and shade dried at room temperature. Eupatorium odoratum is a fast-growing The dried plant leaves were finely perennial and invasive weed native to South powdered using an electric grinder and used and Central America. It has been introduced for aqueous and organic solvent extraction. into the tropical regions of Asia, Africa and other parts of the world. It is a species of 2.2 Mosquito larvae flowering shrub in the sunflower family, Asteraceae. It is also known as Third instar mosquito larvae were collected Chromolaena odorata. It is sometimes and used in the present study’ grown as a medicinal and ornamental plant. 2.3 Pests Common names of Eupatorium odoratum includes Siam weed, Christmas Bush and White mealy bugs were collected from the common Floss flower. infected plants from Thrissur area and is used for the study. Owolabi et al 2010 revealed that oil from Eupatorium odoratum showed 2.4 Larvicidal Activity antibacterial activity against Bacillus cereus and antifungal activity against Aspergillus Aqueous extract of Eupatorium niger. Ranjini and Nambiar 2015, studied odoratum leaves were used to analyze the that Eupatorium odoratum caused larvicidal activity. 10gms of leaf powder disruptive changes tolarvae of Orthaga was mixed in 100ml distilled water and exvinacea Hampson. Eupatorium odoratum kept for few hours. Filtered the mixture is used as a traditional medicine in using a muslin cloth and centrifuged at Indonesia. The young leaves are crushed 10000rpm for 10minutes. The supernatant and the resulting liquid can be used to treat was collected. Third instar larvae of skin wounds. (Panyaphu k., 2011). In mosquitos were used for this study. Five traditional medicine, a decoction of the leaf mosquito larvaes were placed in a petridish is used as a cough remedy and as an containing 10ml of leaf extract and a ingredient with lemon grass and guava control plate containing 10 ml water was leaves for the treatment of malaria. 17 also kept. The larval mortality was major and 26 minor compounds were monitored in every 10 minutes. identified in methanol and aqueous extracts of Eupatorium odoratum by GC-MS 2.5 Insecticidal Activity analysis showing significant antibacterial, The aqueous extract of Eupatorium antioxidant and other prophylactic activities odoratum was used to check the insecticidal (Raman et al, 2012). activity. To evaluate the insecticidal activity 2. Materials And Methods we used white mealy bug. 5-7 mealy bugs were placed in two petriplates. 10ml 2.1 Plant material aqueous extract was poured in one plate. One plate was kept as control. Water was The plant leaves were collected from added to the control plate. The insect various locations in Thrissur, Kerala. The
8 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
mortality was monitored in every 30 Lipase activity in Eupatorium minutes. odoratum leaf extract was analyzed using a simple method (Rajini Singh, 2011). Simple 2.6 Lipase Activity Of Eupatorium and few constituents are used for this test Odoratum Leaf Extract: (Table).
Table1. Composition of Lipase assay medium:
Components Quantity
Phenol red 0.01%
Substrate 1%
Calcium chloride 10mM
pH 7.3-7.4
Substrate: Gingily oil, Palm oil, Coconut oil, Tween 20.
Two main methods were used for the Chloroform extract, 4.Aqueous extract. detection of lipase activity. Each extracts were poured to the respective wells and incubated the plates 1. Plate method: at 37 ˚C overnight.
0.01% Phenol red, 10mM calcium 2. Tube method- modified: chloride and 2% agar mixed in 100ml Media composition was same as in distilled water. pH was adjusted with the plate method without 2% agar. After sodium hydroxide (7.3-7.4). Media was the substrate were added to the respective equally distributed to three beakers and tubes, mixed thoroughly/ vortexed. pH melted by heating. 250µl substrates was adjusted with sodium hydroxide. (Gingily oil, coconut oil, Palm oil, One tube considered as blank in which Tween 20) were added to respective no substrate was added. In case of beakers and mixed thoroughly/vortexed control tubes, only media was used, and poured to the respective plates and extract not added. All tubes were plates were solidified. After incubated at 50˚C for 10 minutes. After solidification, 4 wells were punched on incubation, the color difference was each plate and numbered as 1.DMSO monitored with respect to control and test (control), 2. Methanol Extract, 3. tubes
9 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
Eupatorium odoratum was analysed for larvicidal activity against mosquito 3. Results And Discussion larvae. The mortality of larvae was 3.1 Larvicidal Activity Of Eupatorium gradually increased after each time period. Odoratum The graph shows mortality of larvae in every 10 minutes.
Fig: 1. Larvicidal activity of Eupatorium odoratum leaf extract.
The above data shows the larvicidal can cause degenerative effects in the activity of Eupatorium odoratum leaf midgut epithelium. Due to these extract. After 60 minutes all mosquito disruptive changes they can be employed larvae were dead. for the management of this pest.
Ranjini and Nambiar 2015, studied the 3.2 Insecticidal Activity Of Eupatorium histomorphological changes due to the Odoratum effect of leaf extracts of Clerodendrum infortunatum and Eupatorium odoratum Eupatorium odoratum has on the midgut tissue of sixth instar larvae insecticidal activity also. We used white of Orthaga exvinacea. The studies mealy bug to check the insecticidal revealed the effect of extracts on the activity of the extract. The graph given morphometric and histological changes in below shows the mortality (%) of the the midgut tissue and showed that they insect at an interval of 30 minutes.
10 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
Fig: 2. Insecticidal activity of Eupatorium odoratum Recent research has proved that From the above data we got a clear effectiveness of plant derived compounds, idea about the insecticidal activity of such as saponine (Wiseman and Eupatorium odoratum leaf extract. All the Chapagain, 2008), steroids (Ghosh et al., insects were dead after 5 hours. 2008), isoflavonoids (Joseph et al., 2004), essential oils (Cavalcanti et al., 2004), Plants are rich source of alternative alkaloids and tannins (Khanna V G and agents for control of mosquitoes, because Kannabirank, 2007) are potential they possess bioactive chemicals, which mosquito larvicides. Plant secondary act against limited number of species metabolites and their synthetic derivatives including specific target-insects and are provide alternative source in the control eco-friendly (Sukumar et al., 1991). of mosquitoes. Traditionally plant based products have been used in human communities for 3.3 Lipase Activity Of Eupatorium many centuries for managing insects. Odoratum Leaf Extract Several secondary metabolites present in plants serve as a defense mechanism 1. Plate method: against insect attacks. These bioactive After the lipase assay, color chemicals may act as insecticides, anti- changes were produced around the wells. feedants, moulting hormones, oviposition Color changed from pink to yellow. All deterrents, repellents, juvenile hormone extracts shows the changes. This denoted mimics, growth inhibitors, antimoulting that the leaf extract have oil degrading hormones as well as attractants. Plant activity or lipase activity. This is the first based pesticides are less toxic, delay the report for the presence of lipase from this development of resistance because of its plant. new structure and easily biodegradable (Ignacimuthu, 2000). 2. Tube method: The results obtained Plant based products does not have colorimetrically showed the degradation any hazardous effect on ecosystem. of oils by lipase enzyme present in the
11 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
leaf extract of Eupatorium odoratum. the inability to quantitize the amount of The degrading activity shows more lipase present. Since the absorption effectively in coconut oil. Here we used maximums of phenol red at both colors different substrates to check the lipase (red and yellow) are at different activity. This method is a simple and cost wavelengths we are unable to quantify effective method to check for the the amount of lipase by this method. But presence of lipase. The presence of the the method is very effective in mass protein can be analyzed in a short period screening for the presence of lipses. of time. Only drawback of the method is
ABC
Fig: 3. Lipase activity of Eupatorium odoratum leaf extract with different substrates. (A) Coconut oil (B) gingili oil (C) tween20
A B
Fig: 4. Spectrum of Eupatorium odoratum leaf extract showing lipase activity.
A) Control tube without leaf extract. B) Lipase enzyme gains attention due to Leaf extract showing lipase activity. their wide variety of industrial applications. This enzyme is wide spread in occurrence, Phenol red shows absorption i.e., it is seen in unicellular life forms like maximum at 550 nm at alkaline pH. In the microbes to the highly evolved plant figure B, the shift in peak near yellow- species, both dicots and monocots and also orange range is visible due to lipase activity. seen in animals including primates like humans. Lipases can be used in cosmetics,
12 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
detergents, biodiesel preparation and variety Akah P.A and A.I Nwambie (1994). of pharmaceutical applications. Application Evaluation of Nigerial traditional medicine: of lipases in detergents marked a new era in Plants used for rheumatic (inflammatory) detergent industry. Traditional fossil fuels didorder: J. Ethanopharmacol. 42: 179-182. like petroleum products becoming a limited source made us to think in different way to Alisi CS, Ojiako OA, Osuagwu CG & create new fuels. This led to the Onyeze GOC. (2011) Free Radical development of bio-fuels which employs the Scavenging and In-vitro Antioxidant Effects potential of lipase in the field. Employing of Ethanol Extract of the Medicinal Herb this enzyme, bio-fuels can be developed Chromolaena odorata Linn, British Journal even from things we considered as waste. of Pharmaceutical Research. 1: 141. Thus this enzyme paves an efficient way in Apichart Suksamrarn, Apinya Chotipong, waste management which is the current Tananit Suavansri,Somnuk Boongird, major issue of the era. Puntip Timsuksai, Saovaluk Vimuttipong 4. Conclusion and Aporn Chuaynugul, (2004) Antimycobacterial Activity and Eupatorium odoratum (Asteraceae) is Cytotoxicity of Flavonoids from the a perennial scandent or semi-woody shrub. Flowers of Chromolaena odorata. Arch It has been distributed into the tropical Pharm Res., 27(5): 507-511. regions of Asia, Africa and other parts of the world. The present study reveals the Bartold, P.M., Wiebkin, O.W., Thonard, significant larvicidal and insecticidal J.C. (1984). The effect of oxygen-derived activity of Eupatorium odoratum leaf. free radicals on gingival proteoglycans and According to our results, we can suggest hyaluronic acid. J. Periodontol., 19: 390– that Eupatorium odoratum have potent 400. insecticidal and larvicidal activity. Due to B. Jeffery, D. Harborne, (2000) Taxonomy. its lipase activity and bio enhancer activity 49: 435-449. of leaf extract, it can provide newer leads and clues for modern drug design and for Bouda, H., L.A. Tapondjou, D.A. Fontem, industrial applications. In fact, waste and M.Y.D. Gumedzoe. 2001. “Effect of degradation a main problem in environment, Essential Oils from Leaves of Ageratum Eupatorium odoratum ensure its great role conyzoides, Lantana camara and in nature and in advanced activity. Chromolaena odorata on the Mortality of Sitophilus zeamais (Coleoptera, 5. References Curculionidae)”. Journal of Stored Products A. F´elicien, A. G. Alain,D. T. S´ebastien et Research. 37:103–109. al. (2012 ) Chemical composition and C. Franz and J. Novak, “Sources of essential Biological activities of the Essential oil oils,” in Handbook of Essential Oils extracted from the Fresh leaves of Science, Technology, and Applications, K. Chromolaena odorata (L. Robinson) H. C. Baser and G. Buchbauer, Eds., pp. growing in Benin. Journal of Biological 39–81, CRC Press, New York, NY, USA, Sciences. vol. 1: 7–13. 2010. 13 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
Compori, M. (1985). Lipid peroxidation and Panyaphu K, On TV, Sirisa-ard P, Srisa-nga cellular damage in toxic liver injury. P, ChansaKaow S & Nathakarnkitkul S, Laboratory Investigation, 53, 599–620. Medicinal plants of the Mien (Yao) in Northern Thailand and their potential value Coopoosamy RM, Naidoo KK, Buwa L, in the primary healthcare of postpartum Mayekiso B (2010). Screening of women, J Ethnopharmacol 2011; 135: 226. Siphonochilus aetiopicus (Schweinf.) B. L. Burtt for antibacterial and antifungal KR Ranjini, Jagadeesh G Nambiar (2015) properties. J. Med. Plant Res., 4(12), 1228- Histopathological effects of leaf extracts of 1231. Clerodendrum infortunatum and Eupatorium odoratum on the midgut tissue Cragg, G.M. and D.J. Newman, 2001. of sixth instar larvae of Orthaga exvinacea Natural products drug discovery in the next Hampson (Lepidoptera: Pyralidae Journal millennium. Pharm. Biol., 39:8-17 of Entomology and Zoology Studies 3:5 296- 301. Eastwood M.A, 2001, “A molecular biological basis for the nutritional and pharmacological benefits of dietary plants”, an international Journal of medicine. 94(1).
Ertürk O, Kati H, Yayli N, Demirbag Z (2006). Antimicrobial properties of Silene multifida (Adams) Rohrb. plant extracts. Turk. J. Biol. 30: 17-21.
G. Lamaty, C. Menut, P. H. A. Zollo et al., “Aromatic plants of tropical central Africa. IV. Essential oils of Eupatorium odoratum from Cameroon and Congo,” Journal of Essential Oil Research, vol. 4, pp. 101–105, 1992.
Hill AF (1952). Economic Botany. A textbook of useful plants and plant products. 2 nd edn. McGraw-Hill Book Company Inc, New York.
M S. Owolab, Akintayo Ogundajo , Kamil O. Yusuf , Labunmi Lajide , Heather E. Villanueva , Jessika A. Tuten , and William N. Setzer (2010) Chemical Composition and Bioactivity of the Essential Oil of Chromolaena odorata from Nigeria Moses. Rec. Nat. Prod. 4:1 72-78.
14 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
Biosynthesis of Gold Nanoparticles using Bacteria and Fungi
Nivea Wilson, Nileena PV, Dhanya KC
St. Mary’s College, Thrissur, Kerala Corresponding author: Dhanya KC Email id: [email protected], Phone No: 9947496077 Department of Microbiology, St Mary’s College, Thrissur-680020, Kerala, India.
Abstract
Nanotechnology is one of the most active areas of research in modern material sciences. This field is developing day by day, making an impact in all spheres of human life making vast implications in life sciences especially biomedical devices and biotechnology since it is expected to open new avenues to fight and prevent disease using atomic scale tailoring of materials. Gold nanoparticles are traditionally being synthesized by chemical and physical methods which needs severe reaction conditions, for example, aggressive agents like sodium borohydride, hydrazinium hydroxide, cetyl triethyl ammnonium bromide, harmful solvent system to environment and ecology, higher temperature and higher pressure etc. On the other hand, in green synthesis approach, both prokaryotic and eukaryotic organisms including bacteria, fungi and yeasts could synthesize nanoparticles by means of bioreduction. This biological route involving micro-organisms provides great advantages over traditional methods, as it has the potential to be cost-effective, simple and environmentally friendly and allow size and shape controlled nanoparticle synthesis.
Key words:
Nanoparticles, Nanotechnology, Green synthesis, Biosynthesis
15 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
Introduction Biosynthesis of nanoparticles by bacteria
Metal nanoparticles have been gaining Bacteria are well known to produce importance in the past years because of their inorganic materials either intracellularly or unique properties. Substantial research has extracellularly. Microorganisms are been directed towards their reliable concluded as a potential biofactory for the synthesis to explore their potential synthesis of nanoparticles like gold, silver applications. Amongst the noble metals and cadmium sulphide. There are several (silver, gold, and platinum), gold types of bacteria used to synthesize gold nanoparticles (GNPs) are of increasing nanoparticles from aqueous HAuCl4 interest due to their use in divergent fields of solution either intra or extracellularly. science. GNPs are being extensively used For the first time microbial synthesis of for colorimetric detection of DNA and AuNPs was reported in Bacillus subtilis 168 proteins, in form of biosensors, bioimaging, which revealed the presence of 5–25 nm diagnostics, and therapeutic agents. octahedral NPs inside the cell wall. In Biosynthesis or green synthesis can be Rhodopseudomonas capsulata, spherical successfully used for the production of AuNPs with 10–20 nm range have been smaller particles in large scale. Biological observed [He et al., 2007] at lower systems like bacteria, fungi, actinomycetes concentration and nanowires with network and plants have the ability to produce NPs. at higher concentration. Use of microorganisms as potential Nakajima [2003] screened 30 species of biofactories for synthesis of AuNPs is a various microorganisms for gold relatively new area of research and is accumulation. An extremely high ability to environmentally acceptable, economic, time accumulate gold from a solution containing saving and can be easily scaled up for large Au+3 was found in bacterial strains such as scale synthesis. E. coli and Pseudomonas maltophila. The Among the reported microorganisms, fungus accumulation was very rapid and was offers the advantage of easy handling and affected by the pH of the solution. fast downstream processing and allows The precipitation of gold by magnetotactic feasible large scale synthesis of cocci bacteria occurred in the Phosphorus– nanoparticles due to the high secretion of Sulfur–Iron (PSFe) granules which suggest enzymes and proteins. Cytosolic extracts of the association of gold with these elements. the fungi Candida albicans, Aspergillus The reduction and precipitation of gold by niger, Penicillium sp., and Aspergillus dissimilatory iron (III) reducing bacteria clavatus have been successfully used for were observed. Deplanche and Macaskie GNP synthesis. [2007] investigated the hydrogenase enzyme on the role of gold precipitation by
Escherichia coli and Desulfovibrio desulfuricans ATCC 29577 and suggested that the periplasmic hydrogenases were involved in the gold precipitation. 16 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
Feng et al [2008] discovered that solution of HAuCl4, monodispersed Rhodobacter capsulatus were able to spherical gold NPs capped with self- precipitate gold nanoparticles within and assembled monolayer (SAM) of thiol have outside the bacterial cells, forming spherical been synthesized, extracellularly. The gold and irregular gold with average size ranging NPs were stable without any aggregation from 10 to 48 nm. Bacillus licheniformis over a period of several weeks. precipitated gold nanocubes (10–100 nm) within 2 days at 250C [Kalishwaralal et al., Lactobacillus strains, when exposed to gold 2009]. Monodispersed gold nanoparticles ions, resulted in formation of gold NPs capped with dodecanethiol were within the bacterial cells [Nair and Pradeep, biosynthesized extracellularly by Bacillus 2002]. Escherichia coli DH5α can be used megatherium D01 at 260C. Nangia et al in synthesizing gold NPs. In another study, [2009] reported the biosynthesis of gold biorecovery of gold from jewellery wastes nanoparticles by Stenotrophomonas was obtained using E. coli MC4100 maltophilia forming ~40 nm spherical and (nonpathogenic strain) and Desulfovibrio irregular shapes at 250C. desulfuricans ATCC 29577 [Deplanche and Macaskie, 2007]. Rhodopseudomonas Growth conditions play an important role capsulata showed the ability to produce during the production of nanoparticles. gold NPs in different sizes, and the shape of When gold ions were incubated with the gold NPs was controlled by Ph [He et al., Trichothecium sp. Biomass, under stationary 2007]. R. capsulate was capable of conditions it led to the formation of producing gold NPs extracellularly and the extracellular gold nanoparticles. While gold NPs were quite stable in the solution under shaking conditions, this has resulted and formation of gold NPs might be due to in the formation of intracellular gold NADH dependent enzymes secreted by R. nanoparticles. Venkatesan et al [2011] capsulate. studied the synthesis and optimization of gold nanoparticles from Pseudomonas sp. Southam and Beveridge have demonstrated isolated from soil sample. They found the that gold particles of nanoscale dimensions production of gold nanoparticles with may readily be precipitated within bacterial controlled size, shape and composition by cells by incubation of the cells with Au3+ optimizing various parameters like pH, ions [Southam and Beveridge, 1996]. temperature and concentration of aurium Monodisperse gold nanoparticles have been chloride. synthesized by using alkalotolerant Rhodococcus sp. under extreme biological Satyanarayana et al [2010] reported that the conditions like alkaline and slightly elevated NPs were stabilized by the surface-active temperature conditions [Ahmad et al., molecules, that is, surfactin or other 2003]. Lengke [2006] claimed the synthesis biomolecules released into the solution by B. of gold nanostructures in different shapes subtilis. Bacillus megaterium D01 have (spherical, cubic, and octahedral) by shown the strong potential of Au3+ filamentous cyanobacteria from Au(I)- adsorption. When B. megaterium D01 thiosulfate and Au(III)-chloride complexes biomass was exposed to the aqueous and analyzed their formation mechanisms 17 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
[Lengke et al.,2006]. Nair and Pradeep • They produce more extracellular reported the growth of nanocrystals and secretions of reductive proteins and can nanoalloys using Lactobacillus [Nair and T. easily undergo downstream processing Pradeep, 2002]. [Musarrat et al., 2010].
Another example of gold deposition in AuNP formation can occur either in the prokaryotes is the demonstration by Kashefi intracellular or extracellular space. et al [2001]. They screened a range of Extracellular AuNP formation is commonly anaerobic Fe(III)-reducing bacteria reported for fungi when Au3+ ions are (mesophiles and thermophiles) for their trapped and reduced by proteins in the cell ability to reduce Au+3 and observed a wall. Previous work with the fungus complete reduction of Au+3 after 25 min Verticillium sp. ruled out the possibility that using the Pyrobaculum islandicum. The use reduced sugars in the cell wall are of various strains of Pseudomonas responsible for the reduction of Au3+ ions aeruginosa for extracellular biosynthesis of and suggested adsorption of AuCl4- ions on gold nanoparticles was described. In the cell-wall enzymes by electrostatic different microorganisms, various enzymes interaction with positively charged groups are believed to take part in the bioreduction (e.g. lysine) [Duran et al., 2005]. In the case process involving the transport of electrons of intracellular AuNP formation, Au3+ ions from certain electron donors to metal diffuse through the cell membrane and are electron acceptors. Some studies of reduced by cystolic redox mediators [Das et nonenzymatic reduction mechanism al., 2010]. suggested that some organic functional groups of microbial cell walls could be Another recent study reports the production responsible for the bioreduction process of intracellular AuNPs in metal-tolerant [Crookes-Goodson et al., 2008]. Aspergillus fumigatus and Aspergillus flavus. The intracellular AuNPs have an Biosynthesis of nanoparticles by fungi average diameter of 22 ± 2 nm, slightly larger than those observed in the Fungi appear to be more promising for large extracellular space. The enzymatic reduction scale production of NPs as they are simpler mechanism of Au3+ is essentially the same to grow both in the laboratory and at an for intracellular and extracellular AuNPs industrial scale as well as secrete large [Gupta and Bector, 2013]. In viable cells, amount of proteins. Fungi are regarded as Au ions are reduced by nicotinamide more advantageous for GNPs biosynthesis adenine dinucleotide(NADH)/nicotinamide because of the following features; adenine dinucleotide phosphate (NADPH) oxidoreductases either in the cell surface or • Fungal mycelial mesh can withstand flow in the cytoplasm. pressure, agitation, and other conditions in bioreactors compared to bacteria, Other mechanisms for fungal AuNP biosynthesis have been proposed. The • They are fastidious to grow and easy to fungal pathogen Candida albicans is handle capable of synthesizing phytochelatins,
18 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
which are made of chain links of glucose, the treatment of the biomass of F. cysteine and glycine ((c-GluCys) n-Gly) by oxysporum with silver or gold ions. the transpeptidation reaction of c-Glu-Cys dipeptide from a succession of glutathione An endophytic fungus Colletotrichum sp. molecules. In the presence of glutathione, growing in geranium leaves [Shankar et al., metal ions, including Au, trigger 2003] and alkalotorerant fungus Tricothecium sp., when exposed to aqueous phytochelatin synthesis, in which Au3+ ions get reduced to AuNPs, which are then chloroaurate ions, synthesized gold capped by glutathione [Chauhan et al., nanoparticles. Xie et al [2007] showed that 2011]. AuNP synthesis has been detected by Aspergillus niger may also be used for the UV-visible spectrophotometry in heat- synthesis of gold nanoparticles. The denatured cell-free filtrate of Sclerotium exposure of the mycelia-free spent medium rolfsii in the presence of co-enzyme or extract of A. niger to gold ions resulted in the growth of gold nanoparticles of various NADPH and 1 mM of Au3+ . This indicates the involvement of thermostable NADPH- shapes and sizes. dependent enzymes in the AuNP The rate of particle formation and the size of biosynthesis process. Cyclic voltammetry the nanoparticles could be manipulated by analysis showed that NADH produced from controlling parameters such as pH, the fermentation of lactate by Hansenula temperature, gold concentration and anomala reduces Au to AuNPs [Kumar et 3+ exposure time. The extracellular secretion of al., 2011]. The involvement of biosynthetic the microorganisms offers the advantage of redox mediators in the fungal biosynthesis obtaining large quantities in a relatively pure of AuNPs has been observed in Yarrowia state, free from other cellular proteins lipolytica. These fungi secrete melanin, associated with the organism with relatively which interestingly appears to reduce Au 3+ simpler downstream processing. to AuNPs. Gold nanoparticles are one of the most The extracellular synthesis of gold attractive nanomaterials for various nanoparticles was reported by fungus applications like antimicrobial, electronic, Fusarium oxysporum and Verticillium . catalytic, and various biomedical Shankar et al [2004] demonstrated that gold applications. The present review nanoplates can be synthesized by using summarises literature on synthesis of gold fungal extracts. The results obtained from nanoparticles using bacteria and fungi. This the Gericke and Pinches [2006] study green synthesis is useful since it can be used demonstrated the possibility to manipulate to produce large quantities of nanoparticles the size and shape of gold nanoparticles have advantage over the other physical synthesized by Verticillium luteoalbum. methods as it is safe, eco-friendly and Ahmad et al [2003] screened a number of simple to use for the production of gold species belonging to different genera of nanoparticles of desired shape and size. molds and observed the extracellular synthesis of silver and gold nanoparticles by References
19 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
1. Ahmad A, Mukherjee P, Senapati S, Fusarium oxysporum strains, J Mandal D, Khan MI, Kumar R, Nanobiotechnol, 3:1. Sastry M (2003). Extracellular 9. Feng Y, Lin X, Wang Y, Wang Y, synthesis of silver nanoparticles of Hua J (2008) Diversity of aurum silver nanoparticles using the fungus bioreduction by Rhodobacter Fusarium oxysporum, Colloids Surf capsulatus, Mater Lett, 62:4299. B: Biointerfaces, 28:313. 10. Gericke M, Pinches A (2006) 2. Bhambure R, Bule M, Shaligram N, Microbial production of gold Kamat M, Singhal R (2009). nanoparticles. Gold Bull, 39:22. Extracellular biosynthesis of gold 11. Gupta S, Bector S (2013). nanoparticles using Aspergillus niger Biosynthesis of extracellular and – its characterization and stability, intracellular gold nanoparticles by Chem Eng Technol, 32:1036. Aspergillus fumigatus and A. flavus, 3. Brayner R, Barberousse H, Hemadi Anton Leeuw, 103: 1113. M, Djedjat C, Yéprémian C, Coradin 12. He S, Guo Z, Zhang Y, Zhang S, T, et al.( 2007) J Nanosci Wang J, Gu N (2007). Biosynthesis Nanotechnol;7:2696. of gold nanoparticles using the 4. Chauhan A, Zubair S, Tufail S, bacteria Rhodopseudomonas Sherwani A, Sajid M, Raman SC capsulate, Mater Lett, 61: 3984. (2011). Fungus-mediated biological 13. Kalishwaralal K, Deepak V, Pandian synthesis of gold nanoparticles: SRK, Gurunathan S (2009). potential in detection of liver cancer, Biological synthesis of gold Int J Nanomedicine, 6: 2305. nanocubes from Bacillus 5. Crookes-Goodson WJ, Slocik JM, licheniformis, Biosource Technol, Naik RR (2008) Bio-directed 100:5356. synthesis and assembly of 14. Kashefi K, Tor JM, Nevin KP, nanomaterials, Chem Soc Rev, Lovley DR (2001). Reductive 37:2403. precipitation of gold by dissimilatory 6. Das SK, Marsili EA (2010). Green Fe(III)-reducing bacteria and chemical approach for the synthesis archaea, Appl Environ Microbiol, of gold nanoparticles: 67:3275. characterization and mechanistic 15. Kumar SK, Amutha R, Arumugam aspect, Rev Environ Sci Biotechnol, P, Berchmans S (2011). Synthesis of 9:199. gold nanoparticles: an ecofriendly 7. Deplanche K, Macaskie LE (2007). approach using Hansenula anomala, Biorecovery of gold by Escherichia ACS Appl Mater Interfaces, 3: 1418. coli and Desulfovibrio desulfuricans, 16. Lengke M F, Fleet ME, Southam G Biotechnol Bioeng, 99:1055. (2006). Morphology of gold 8. Duran N, Marcato PD, Alves OL, nanoparticles synthesized by DeSouza G, Esposito E (2005). filamentous cyanobacteria from Mechanistic aspects of biosynthesis gold(I)-Thiosulfate and gold(III)- of silver nanoparticles by several
20 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
chloride complexes, Langmuir, 22: chloroaurate ions by geranium leaves 2780. and its endophytic fungus yields gold 17. Musarrat J, Dwivedi S, Singh BR, nanoparticles of different shapes, J Al-Khedhairy AA, Naqvi AAA Mater Chem, 13:1822. (2010). Production of antimicrobial 23. Shankar SS, Ahmad A, Rai A, Sastry silver nanoparticles in water extracts M (2004). Rapid synthesis of Au, Ag of the fungus Amylomyces and bimetallic Au core- Ag shell rouxii strain KSU-09, Bioresour nanoparticles by using neem Techno,101:8772. (Azadirachta indica) leaf broth, J 18. Nair and Pradeep T (2002). Colloid Interface Sci, 275:496. Coalescence of nanoclusters and 24. Southam G. and Beveridge TJ formation of submicron crystallites (1996). The occurrence of sulphur assisted by lactobacillus strains, and phosphorus within bacterially Crystal Growth & Design, 2: 293. derived crystalline and 19. Nakajima A (2003). Accumulation pseudocrystalline octahedral gold of gold by microorganisms, World J formed in vitro, Geochimica et Microbiol Biotechnol, 19:369. Cosmochimica Acta, 60: 4369. 20. Nangia Y, Wangoo N, Sharma S, 25. Venkatesan P, Santhanalakshmi J Wu J, Dravid V, Shekhawat GS, Suri (2011). Core-Shell bimetallic Au-Pd CR (2009). Facile biosynthesis of nanoparticles: Synthesis, structure, phosphate capped gold nanoparticles optical and catalytic properties, J by a bacterial isolate Nanosci Nanotechnol, 1: 43. Stenotrophomonas maltophilia, Appl 26. Xie J, Lee JY, Wang DIC, Ting YP Phys Lett, 94:1. (2007). High-yield synthesis of 21. Satyanarayana RA. Chen C, Jean J complex gold nanostructures in a et al., (2010) “Biological synthesis of fungal system, J Phys Chem, gold and silver nanoparticles 111:16858. mediated by the bacteria Bacillus subtilis,” Journal of Nanoscience and Nanotechnology, 10: 6567. 22. Shankar SS, Ahmad A, Parischa S, Sastry M (2003). Bioreduction of
21 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
Comparative Study on Antibacterial profiles of Kanakasava
Elizabeth P Thomas* , Uma V Menon P, Afeeda Beegum M K, Athira MR, Darsana NC, and KS Dhaneesha Sivadas
Corresponding author: Elizabeth P Thomas, Department of Microbiology, St Mary’s College, Thrissur-680020, Kerala Email id: [email protected], Ph. No: 9746343500
ABSTRACT
Ayurveda is a traditional Indian medicinal system being practiced thousands of years. Considerable research on pharmacognosy, chemistry, pharmacology and clinical therapeutics has been carried out on ayurvedic medicinal plants. The present study was designed to observe antimicrobial activities of the ayurvedic medicine namely Kanakasava against four different common bacterial pathogens. The current study was structured for screening of in-vitro antimicrobial activities of collected samples by agar well diffusion method. The main objective of this study is to know how far the four bacterial species namely Streptococcus, Staphylococcus, Pseudomonas and Escherichia coli are sensitive to Kanakasava during different stages of production and also to know which among these will produce highest zone of clearance.
Keywords:
Ayurvedic medicines, Medicinal plants, Antimicrobial study
1.Introduction
22 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
1.Introduction contains 5 – 10 % self generated alcohol in it. This self generated alcohol and the There are many traditional systems of water present in the product act as a media medicine in the world, each with to deliver water and alcohol soluble the different associated philosophies and active herbal components to the body. It is cultural origins. Ayurveda is the most widely used in the treatment of respiratory widely practised of the Indian conditions such as asthma, cough, fever traditional medicine systems, but there and bleeding diseases. It is a natural are others such as Siddha and Unani mucolytic and bronchodilator. It is also which are also used in the Indian used in treatment of bleeding diseases, subcontinent (Gogtay et al., 2002). injuries and chronic fever. It helps to Although there is limited data on the use relieve chest mucous congestion. A very of Ayurvedic medicines specifically in high dosage of Kanakasava may cause other areas of the world, the use of stomach irritation and loose stool. The traditional medicines has increased main ingredients of Kanakasava are: significantly in recent years in North Kanaka (purified and processed Datura America, Europe. Ayurvedic medicines metel), Vrushamoola, Yashtimadhu, are of various type, so as to meet the pippali(long pepper), Vyahgri, Keshara, diverse requirements in the treatment of Vishvabheshaja(shunti-ginger), Bharngi, illness. They are herbal teas, infusions, Talisapatra, Dhataki, Draksha(raisins), decoctions, tinctures, capsules and water, Sharkara(sugar), Kshaudra (honey). powders, infused oils, ointments, creams, lotion (Kumar et al., 2010). Arishta and 2. Methodology asava are considered as unique and valuable therapeutics in Ayurveda. They 2.1 Sample Collection are self generated herbal fermentation of Kanakasava was prepared according to traditional ayuredic system. They are Sahasrayogha. Samples for analysis were alcoholic medicaments prepared by collected aseptically during 30 day allowing the herbal juices or their fermentation . The in process and quality decoctions to undergo fermentation with control for the preparation was strictly the addition of sugars. Arishtas are made controlled and monitored. with decoctions of herbs in boiling water while asavas are prepared by directly 2.2 Determination of antibacterial using fresh herbal juices. The method of activity preparation of arishta and asava is known as Sandhana kalpana (S.Sekar et al.,2006). The antibacterial activity of Kankasava Kanakasava is an ayurvedic medicine can be checked by measuring the zone of practiced now-a-days for mainly clearance on different bacterial culture menstrual irregularities. plates. Agarwell diffusion method is widely used to evaluate the antimicrobial Kanakasava is a liquid activity of plants or microbial extracts. ayurvedic medicine, widely used in The nutrient agar plate surface was treating asthma, cough, fever etc. It inoculated by spreading a volume of the
23 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
microbial inoculum over the entire agar 3. Result surface. Then, a hole with a diameter of 6 to 8 mm was punched aseptically with a Ayurvedic medicine Kanakasava was tip, and a volume 50µl of Kanakasava was tested (agar well diffusion method) for introduced into the well. Then,agar plates their antimicrobial activity against were incubated at 370C for 24 hours and Staphylococcus species, Streptococcus checked for the zone of inhibition. The species, Pseudomonas species and antimicrobial agent diffuses in the agar Escherichia coli. In this antibacterial medium and inhibits the growth of the screening of test product were used at a microbial strain tested. concentration of 50µl/well. All the tested samples showed antibacterial activity . 12 day,15 day, 18 day fermented samples showed highest zone of inhibition (25mm) against Streptococcus species.
Figure 1: Anbacterial activity of Kanakasava against Streptococcus species
4. Discussion and Conclusion helps to relieve chest mucous congestion. In this present study, Kanakasava showed Kanakasava is a liquid medicine widely effective antimicrobial activity against all used in treating asthma, cough, fever, etc. the test organisms. Samples of Datura is the main ingredient in Kanakasava showed the highest zone of Kanakasava that has both antimicrobial inhibition against Streptococcus species. and antioxidant activities. Kanakasava is a This study shows the most effective natural mucolytic and bronchodilator. It is antibacterial activity of Kanakasava also used in the treatment of bleeding against four different bacterial species diseases, injuries and chronic fever. It 24 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
during different fermentation periods (days).
5. Bibliography
1 Husain, A., Sofi, G. D., Tajuddin, Dang, R., Kumar, N., (2010) Unani system of medicine introduction and challenges. Medical Journal of Islamic world Academy of sciences, 1891): 27-30
2 Mishra, K., Ravi, B., Vinjamury, S., (2008) Ayurvedic medicine. Caring Ambassadors Program, Inc. 153-165.
3 National center for complementery and alternative medicine, (2005) 1-12 ( Web site: nccam.nih.gov).
4 Praveen Kumar S.V., Sandeep M., Kamal D., Nishanth B.C., Megharaj H.K., Prashith Kekuda T.R., Gurucharan D.N. (2010) Antibacterial and anthelmintic activity of selected fermented Ayurvedic herbal formulations. 2(7): 347-348.
5 Tiwari, P., Patel, R.K., (2011) Evaulation of antihyperlipidemic activity of Draksharishta prepared by traditional and modern methods in hyperlipidemic rats. Pharmacologyonline 2: 576-585.
25 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
Antibacterial effect of citrus fruit juice against enteric and non enteric pathogenic bacteria
Geenat Paul , Rigi George A , Rahima N A
email id: [email protected]
Department of Microbiology, St Mary’s College, Thrissur, Kerala – 680020
ABSTRACT
The present study was carried out to determine the antimicrobial effect of three different citrus fruit juices against four enteric and two non enteric pathogens named Escherichia coli , Salmonella , Proteus, Klebsiella ,Vibrio, and Staphylococcus species. The study was done by well diffusion and disc diffusion method. The use of different concentrations (100%, 75%, 50%, 25%) of citrus juice extracts had an effective antibacterial activity. Lemon juice was the most effective against the test organisms in both undiluted and diluted concentration. Pomelo juice showed antimicrobial activity only against Vibrio and Salmonella. Orange juice showed antimicrobial activity only against Staphylococcus and Escherichia coli .
Key Words :
citrus fruit,,well diffusion , disc diffusion , antimicrobial effect
26 Annals of Basic and Applied Sciences, Vol 6, No. 1, Dec 2015
1. Introductionriiiiiiiiiii of bioac-tive compounds close related to iiiindrr iiBacterial infections are among herbs, commonly referred as the important infectious diseases. Hence, phytochemicals such as tannins, over 50 years of extensive researches have carotenoids, polyphe-nols and been launched for achieving new anthocyanins (Khushwaha et al 2012). antimicrobial medicines isolated from The current research focuses on different sources. Despite progress in the extraction and assay of antibacterial development of antibacterial agents, there component from citrus fruit which are are still special needs to find new easily available at very low cost. antibacterial agents due to development of multidrug resistant bacteria (R.Wise,T. 2. Materials And Methods Hart, O.Cars et al. ,1998) 2.1 Bacterial cultures - Escherichia coli , The emergence of resistant organisms Salmonella , Proteus, Klebsiella ,Vibrio, presents a major challenge for the and Staphylococcus species. were kindly antimicrobial therapy of infectious provided from Poly Clinic Pvt Ltd, diseases and increases the incidence of Thrissur , Kerala , India. mortality and morbidity.The increase in 2.2 Citrus fruits – Fresh lemon antibiotic resistance bacteria is largely due ,Orange , Pomelo were obtained to the widespread use of antibiotics in from local market of Thrissur. medicine in animal care and agriculture (Bansode et al 2012). )Citrus fruits have 2.3 Taking of juice from fruits been of interest for extraction of Surface of the fruit was disinfected using antimicrobial metabolites by large number ethanol. Fruit was pierced and juice was of researchers (Kumar et al., 2011; Kumar aspirated and collected in beaker in sterile et al., 2010; Amandeep et al., 2009) but condition. the peels have been less studied. Lemon 2.4 Preparation of different concen- juice has been used in the treatment of trations of fruit juice oral thrush in HIV/AID patients (Wright Different concentrations were made by et al., 2009). The antioxidant activities of adding sterile physiological saline(0.85%) citrus flavonoids and phenolic into the juice. To prepare 1ml of 25% compounds exhibited a potent concentration of fruit juice,0.25 ml juice antibacterial activity which is probably was added to 0.75 ml of saline, 1ml of due to their ability to complex with 50% concentraton,0.5 ml juice was added bacterial cell walls and disrupt microbial to 0.5ml saline, for 1ml of 75% membrane . Studies have shown that concentration ,0.75 ml of juice was added concentrated or freshly squeezed lemon to 0.25ml of saline,and for 1 ml of 100% juice has antibacterial activity against concentration,1ml juice was used.The Vibrio species (Tomotake et al., 2006), physiological saline was used as control. Antibacterial properties of plant extract 2.5 Anti-bacterial activity testing have been a hot topic for the researchers. • Well diffusion method Besides plants, fruits also have been Antibacterial test were carried out by studied by the researchers for the presence the well diffusion method . Overnight 27 Annals of Basic and Applied Sciences, Vol 6, No. 1, Dec 2015
bacterial cultures were diluted in the The antibacterial activity was determined nutrient broth to obtain a bacterial by measuring the inhibition zone. suspension of 10^8 CFU/ml. (S.Sundar and koilpillai, 2015) Petriplates containing 20 ml of Muller-Hinton Agar media were 3. Result And Discussion inoculated with diluted cultures with a sterile cotton swab is dipped into From three citrus fruits used in the study standardized bacterial test suspension for evaluation of their antimicrobial and used to evenly inoculate the entire activity against four enteric bacteria and surface of the Muller-Hinton Agar two non enteric bacteria initially plates. After the agar surface has dried determined by Muller Hinton Agar well for about 5 minutes, five wells, all are diffusion method. All of the isolated have equal diameter were made in the species showed activity against citrus plate at equal distance. Citrus fruit fruit juices. Juice of Citrus limon juice of different concentrations showed highest inhibitory effect against (25,50,75,100µl)and a control (0.85%) Vibrio species with largest DIZ saline were poured on each well by (Diameter of Inhibition Zone) value using micropipette. Plates were followed by Staphylococcus species incubated at 37⁰C for 24h .The ,Salmonella species and Proteus species. antibacterial activity was determined Juice of Citrus maxima showed highest by measuring the inhibition zone. inhibitory effect against Vibrio species • Agar disc diffusion assay followed by Salmonella species. de Castillo etal (2000) also reported that The antibacterial activity of the citrus freshly squeezed lemon juice inhibited fruits extracts was determined by the disc the growth of V. Cholerae . Juices of diffusion method.Briefly ,overnight Citrus sinesis showed highest inhibitory bacterial culture were diluted in the effect against Staphylococcus species Muller-Hinton broth to obtain a bacterial followed by E.coli. suspension of 10^8 CFU/ml. Petriplates containing 20ml of Muller-Hinton Agar The antibacterial activity of these media.A sterile cotton swab is dipped into pathogens are further determined by disc a standardized bacterial test suspension diffusion method. All isolated bacteria and used to evenly inoculate the entire showed susceptibility against citrus fruit surface of the Muller-Hinton Agar juices. Juice of Citrus limon showed plate.After the agar surface has dried for highest inhibitory effect against Vibrio about 5 minutes , Citrus fruit juice of species with largest DIZ followed by different concentrations (25,50,75,100µl) Staphylococcus species ,salmonella were loaded on to the filter paper discs species and Proteus species. Juice (whatman No.1 ,6mm diameter) were of Citrus maxima showed highest placed on the inoculated agar surface were inhibitory effect against Vibrio species allowed to dry completely. Standard followed by Salmonella species. Citrus saline (0.85%) was placed as control. sinesis showed highest inhibitory effect Plates were incubated at for 37⁰C 24h. against Staphylococcus species followed
28 Annals of Basic and Applied Sciences, Vol 6, No. 1, Dec 2015
by E.coli . Citrus limon showed activity of different citrus fruits the highest inhibitory activity against results showed that Citrus limon>Citrus Vibrio species while lowest activity maxima>citrus sinensis. against Proteus species. The results The results of Table 1 and Table 2 showed in Table 1 and Table 2. (Kumar revealed that Citrus limon juice showed et al., 2011; Kumar et al., 2010; highest activity against almost all enteric Amandeep et al., 2009; Nurmahani et al., pathogens and non enteric pathogens. 2012) have also reported similar results From the results obtained, the highest for the various extracts from citrus inhibitory effect showed by 100µl fruits.On comparison of antimicrobial concentration and least effect by 25µl.
The Table 1. Showing Antibacterial Activity By Well Diffusion Method
Bacteria Diameter Of Inhibition Zone(DIZ) in mm.
Citrus limon Citrus maxima Citrus sinensis 0.85 % 100 75 50 25 100 75 50 25 100 75 50 25 Salin µl µl µl µl µl µl µl µl µl µl µl µl e as contr ol Enterobacteriacea
E.coli 15 10 11 7 ------7 4 2 ------Salmonella 21 19 17 14 8 14 10 ------Proteus 19 17 15 11 ------Klebsiella 13 11 10 3 ------Non-Enterobacteriacea
Vibrio 23 22 18 16 21 18 3 ------Staphyloco 22 19 17 14 ------2 10 8 7 --- ccus ------
29 Annals of Basic and Applied Sciences, Vol 6, No. 1, Dec 2015
The Table2. Showing Antibacterial Activity By Disc Diffusion Method
Bacteria Diameter Of Inhibition Zone(DIZ) in mm
Citrus limon Citrus maxima Citrus sinensis 0.85 % 100 75 50 25 100 75 50 25 100 75 50 25 Salin µl µl µl µl µl µl µl µl µl µl µl µl e as contr ol
Enterobacteriacea E.coli 10 7 4 3 ------4 2 ------
Salmonella 13 12 10 7 10 8 6 ------
Proteus 12 10 8 6 ------
Klebsiella 10 9 3 1 ------Non-Enterobacteriacea
Vibrio 18 14 12 10 18 10 8 5 ------
Staphylococc 15 12 10 7 ------10 7 6 2 ------us
30 Annals of Basic and Applied Sciences, Vol 6, No. 1, Dec 2015
Antibacterial Activity Of Fruit Juice Of Citrus Limon By Well Diffusion And Disc Diffusion Methods
Staphylococcus
50 100 25
25 75 50 75 100
Salmonella
50 25 50 25
75 100 75 100
31 Annals of Basic and Applied Sciences, Vol 6, No. 1, Dec 2015
4. Conclusion selected enteric pathogens. The study summarised here demonstrate IRJP.3(11):122-126. that the microbial load of human body 3. de Castillo M C, de Allori C constitute enteric and non-enteric G , de Gutierrez R C, de pathogens.The bacterial isolates Saab O A, de Fernandez N identified in this study are common P, de Ruiz C S et al.(2000) human enteric and non-enteric Bacterial activity of lemon pathogens.This study emphasizes the juice and lemon derivatives need of usage of citrus fruits in our against Vibrio cholera. diet.Because the results showed that the BioPharma Bull.(10):1235- citrus fruit juice have high antibacterial 1238. activity against almost all enteric and 4. Khushwaha A,Singh non-enteric pathogens .The well R.P,Gupta V, Singh diffusion and disc diffusion methods M.(2012) Antimicrobial using citrus fruit juice showed properties of peels of citrus antibacterial activity against Vibrio, fruits .IJPLS .2(2):24-32 Salmonella, Staphylococcus, E.coli, 5. Kumar K. Ashok, Subanthini Proteus and Klebsiella..The study points A., Jayakumar M. out that the infections with enteric and Antimicrobial Activity and non-enteric pathogens can be Phytochemical Analysis of successfully eliminated by the use of Citrus Fruit Peels -Utilization easily available citrus fruit juices. This of Fruit Waste. 2011; 3(6): approach however go a long way in 5414-5421. combining the rising tide of antibacterial 6. . Kumar Vivek R, Anti resistance. Typhoid Activity of Aqueous Extract of Fruit Peel Citrus sinensis (L.). International 5. Reference Journal of Pharm. Res. And Development. 2010; 2(9): 1. Amandeep Singh, Ahmed R. 217-221. Bilal, In vitro antibiotic 7. Nurmahani MM, Osman A, activity of isolated volatile Abdul Hamid A, Mohamad oil of citrus Sinensis. Ghazali F and Pak Dek MS. International journal of Antibacterial property of pharm. Res. And Hylocereuspolyrhizus and development. 2009; 7(1): 1- Hylocereusundatus peel 4. extracts. International Food Research Journal. 2012; 2. Bansode D. S,Chavan M 19(1): 77-84. D,(2012) Studies on 8. R.Wise,T.Hart,O.Cars et al. antimicrobial activity and ,(1998).Antimicrobial phytochemical analysis of resistance, is a major threat citrus fruit juices against
32 Annals of Basic and Applied Sciences, Vol 6, No. 1, Dec 2015
to public health. BMJ 317 (7159) 609-10 9. S.Sundhar and Justin koilpillai(2015) Antimicrobial potential of petroleum ether extract and active column fraction of the solanum incanum leaves. Journal of Pure & Applied Microbiology.9(4):49-54 10. .Tomotake H, Koga T, Yamato M, Kassu A and Ota F (2006). Antibacterial activity of citrus fruit juices against Vibrio species. J. Nutr. Sci. Vitaminol. (Tokyo), 52(2): 157-160.
11. Wright SC, Maree JE and Sibanyoni M (2009). Treatment of oral thrush in HIV/AIDS patients with lemon juice and lemon grass (Cymbopogon citratus) and gentian violet. Phytomedicine, 16(2-3):118- 124.
33 Annals of Basic and Applied Sciences, Vol 6, No. 1, Dec 2015
Hydrogen Peroxide Scavenging Ability of Scoparia Dulci&Piper Longum Linn
Geetha T
Dept. of Chemistry, St. Mary’s College, Thrissur Corresponding author : T. Geetha Phone No. 9495951805 E-mail: [email protected]
ABSTRACT
In this study the Hydrogen Peroxide Radical Scavenging (H2O2) Assay of various extractants of Scoparia Dulcis and Piper Longum Linn were studied. Based on phytochemical analysis aqueous methanol extract was found to be the most efficient extract, to evaluate radical scavenging property. Both Scoparia Dulcis and Piper Longum Linn show antioxidant property. In the case of Scoparia Dulcis, its whole plant shows more anti-oxidizing property, than its roots, stem seed and leaves. But in the case of Piper Longum Linn, its stem and leaf have no antioxidant properties, while its root shows higher antioxidising property than the whole plant.
Key Words :
phytochemistry, Scoparia Dulcis and Piper Longum Linn, Aqueous methanol extractant, chloroform, ethanol and water.
34 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
1.0 Introduction standard compounds such as gallic acid(Bendiabdellah, A et al., 2012). 1.1,Free radicals are chemically active atoms or molecular fragments that have a 1.2Hydrogen peroxide radical scavenging charge due to an excess or deficient (H2O2) assay number of electrons. Free radicals Hydrogen peroxide occurs naturally at containing oxygen known as reactive low concentration levels in the air, water, oxygen species (ROS) are the most human body, plants, microorganisms, biologically significant free radicals. ROS food and beverages. It is widely used as a include the radicals-superoxide and bleaching agent in the textile, paper and hydroxyl radical, plus derivatives of pulp industries. Human beings exposed to oxygen that do not contain unpaired H2O2 indirectly via the environment are electrons, such as hydrogen peroxide, estimated as 0.28 mg/kg/day with intake singlet oxygen and hypochlorous acid. from leaf crops contributing most to this Because they have one or more unpaired exposure. Hydrogen peroxide enters the electrons, free radicals are highly human body through inhalation of vapour unstable. They scavenge our body to grab or mist and through eye or skin contact. In or donate electrons, there by damaging the body, H2O2 is rapidly decomposed cells, proteins and DNA. It is impossible into oxygen and water and this may for us to avoid damage by free radicals. produce hydroxyl radicals (OH˙) that can Free radicals arise from the sources both initiate lipid peroxidation and cause DNA inside (endogenous) and outside damage. Hence Hydrogen peroxide
(exogenous) our bodies. Anti-oxidants are radical scavenging (H2O2) assay is an substances like nutrients (vitamins and important tool in assessing the antioxidant minerals) as well as enzymes that are property of plant (Chand S & Dave R capable of counteracting the damaging, 2009). but normal, effects of the physiological process of oxidation in animal tissue. 2.0 Materials& Methods They are believed to play a role in 2.1Collection and processing of plant preventing the development of chronic samples diseases such as cancer, heart diseases, stroke, Alzheimer’s disease, rheumatoid Scoparia dulcis and Piper longum linn arthritis and cataracts. were collected from Thrissur district. Plants were identified in the Botany Dept. Plants are one of the largest sources of of St. Mary’s College Thrissur. The plants antioxidants. Studies by Harshay Meena were washed with water to remove all et al.(2012), Latha B, et al. (2013), unwanted materials; the plant parts were PiseNavnath, et al. (2010), Arulmozhi, separated and dried in the dark. These S.et al.(2010), Rahmat Ali Khan, et were then grounded to a fine powder and al.(2012), Naima Saeed, et al.(2010), have stored in an airtight container. all concluded that the plant extracts show antioxidant property. Some plants like 2.2 Preparation of extracts Daucus crinitus Desf., had antioxidant The aqueous methanol extract of the potential comparable to those of the plants were prepared by shaking 5g of 35 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
powdered samples in 50ml of (50:50) buffer without hydrogen peroxide. The methanol and double distilled water for 1 percentage of hydrogen peroxide hr using a shaker. The extract was then scavenging is calculated as follows: filtered using double filter paper and stored in an airtight container. The % Scavenged (H2O2) = (A0 – A1 / A0) X chloroform, ethanol and aqueous extract 100 of the plants were prepared by shaking 5g Where A0 is the absorbance of control and of powdered samples in 50ml of A1 is the absorbance of test. chloroform, ethanol & water respectively for 1 hr using shaker. The extract was 3.0 Results& Conclusion then filtered using double filter paper and 3.1 Qualitative Analysis stored in an airtight container. Preliminary phytochemical screening
revealed the presence of tannins, phenols, 2.3 Phytochemical Screening Methods glycosides, carbohydrates, saponins, flavonoids and alkaloids in different The phytochemical tests to detect the extracts of Scoparia dulcis and Piper presence of alkaloids, carbohydrates, longum linn. The results show that these saponins, tannins, flavonoids, phenols and plants contain a number of chemical glycosides were performed according to ingredients, which may be responsible for the method described by Harborne JB the various pharmacological actions (1998). Alkaloids were detected by although their specific roles remain to be Hager’s test Carbohydrates by Molisch’s investigated. In the phytochemical test, glycosides by Salkowski’s test, qualitative analysis of Scoparia Dulcis, it tannins by ferric test, saponins by has been seen that alkaloids, frothing, Phenols by FeCl test and 3 carbohydrates, glycosides and tannins are Flavonoids by Lead acetate solution Test. present in ethanolic extract, while 2.4 Hydrogen Peroxide Radical saponin, phenol and flavonoid are absent.
Scavenging (H2O2) Assay In the case of aqueous methanol extract, The ability of plant extracts to scavenge alkaloid, carbohydrate, glycosides, hydrogen peroxide is determined flavanoid and tannin are present whereas according to the method of Ruch et al. saponin and phenol are absent. The (1989). A solution of hydrogen peroxide chloroform extract shows only the (40 mM) is prepared in phosphate buffer presence of carbohydrate, phenol and (50mMpH 7.4). The concentration of tannin and others are absent. But in the hydrogen peroxide is determined by case aqueous extract, only tannin, phenol absorption at 230 nm using a and flavonoid are absent and all others are spectrophotometer (UV-1800 present(Table 1). In the phytochemical SHIMADZU; UV Spectrophotometer). 1 qualitative analysis of Piper Longum ml of plant extract is added to2 ml Linn, carbohydrate, phenol, flavonoid and hydrogen peroxide and absorbance at 230 tannin are present in ethanolic extract, nm is determined after 10 min against a while alkaloid, glycosides and saponin are blank solution containing phosphate absent. The aqueous methanol extract
36 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
shows only the absence of saponin all 3.3. Conclusion others are present. But in the case of chloroform extract, only carbohydrate, Aqueous methanol extract was found to glycosides, and tannin are present, all the be the most efficient extract, for radical rest are absent. In aqueous extract, only scavenging property. Both Scoparia tannin and phenol are absent and all Dulcis and Piper Longum Linn show others are present(Table 2). antioxidant property. In the case of Scoparia Dulcis, its whole plant shows In the case of Scoparia dulcis, ethanol more anti-oxidizing property, than its extract shows the presence of four roots, stem seed and leaves. But in the constituents, aqueous extract shows the case of Piper Longum Linn, its stem and presence of four constituents, chloroform leaf have no antioxidant properties, while shows only the presence of three its root shows higher antioxidising constituents and aqueous methanol extract property than the whole plant. shows the presence of five constituent. In 4.0. Reference the case of Piper longum linn, ethanol Arulmozhi, Sinnathambi, et al. extract shows the presence of four "Antidiabetic and antihyperlipidemic constituents, aqueous extract shows the activity of leaves of Alstonia scholaris presence of five constituents, chloroform Linn. R. Br." European Journal of shows only the presence of three Integrative Medicine 2.1 (2010): 23-32. constituents and aqueous methanol extract shows the presence of six constituent. Bendiabdellah, Amel, et al. "Preliminary From the phytochemical screening, it is phytochemical screening andantioxidant understood that aqueous methanol extract activities of solvent extracts from Daucus is the good extract. Hence the Hydrogen crinitus Desf., from Algeria." Journal of peroxide radical scavenging (H2O2) assay Applied Pharmaceutical Science 2.7 was conducted using the aqueous (2012): 92. methanol extract. Chanda, S., & Dave, R. (2009). In vitro models for antioxidant activity evaluation 3.2 Hydrogen Peroxide Scavenging and some medicinal plants possessing Effects on Scoparia Dulcis & Piper antioxidant properties: An overview. Longum Linn African Journal of Microbiology In the case of Scoparia Dulcis, highest Research, 3(13), 981-996. anti-oxidizing property was shown by whole plant followed by root, then stem Harborne JB, (1998). ‘Phytochemical met and seed. Its leaf possessed least anti- hods: A guide to modern technique of pla oxidizing property (Table 3). In the case nt analysis, Champman and Hall, London. of Piper Longum Linn, root shows higher 288pp anti-oxidizing property than whole plant. Its stem and leaves have no anti-oxidizing Khan, Rahmat Ali, et al. "Evaluation of properties (Table 4). phenolic contents and antioxidant activity of various solvent extracts of Sonchus
37 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
asper (L.) Hill." Chemistry Central constituents of Porphyra species from Journal 6.1 (2012): 1. India. Ruch, R. J., Cheng, S. J., &Klaunig, J. E. Latha, B., Rumaisa, Y., Soumya, C. K., (1989). Prevention of cytotoxicity and Shahul, S., & Sadhiya, N. (2013). inhibition of intercellular communication Phytochemical studies on Leucas aspera. J by antioxidant catechins isolated from Chem Pharm Res, 5, 222-8. Chinese green tea. Carcinogenesis, 10(6), 1003-1008. Meena, H., Pandey, H. K., Pandey, P., Arya, M. C., & Ahmed, Z. (2012). Saeed, Naima, Muhammad R. Khan, and Evaluation of antioxidant activity of two Maria Shabbir. "Antioxidant activity, total important memory enhancing medicinal phenolic and total flavonoid contents of plants Baccopa monnieri and Centella whole plant extracts Torilis leptophylla asiatica. Indian Journal of Pharmacology, L." BMC Complementary and Alternative 44(1), 114. Medicine 12.1 (2012): 1.
Pise, N. M., Jena, K. B., Maharana, D., Gaikwad, D., & Jagtap, T. G. (2010). Free radical scavenging potential, reducing power, phenolic and biochemical
Table 1. Phytochemical qualitative analysis of ScopariaDulcis
Sl. Phytochemical test for Ethanol Aqueous Chloroform Water No. methanol
1 Alkaloid
2 Carbohydrate
3 Glycosides
4 Tannin
5 Saponin
6 Phenol
7 Flavonoid
“ indicates presence and “ ”- indicates absence
38 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
Table 2. Phytochemical qualitative analysis of Piper Longum Linn.
Sl. Phytochemical test for Ethanol Aqueous Chloroform Water No. methanol 1 Alkaloid
2 Carbohydrate
3 Glycosides
4 Tannin
5 Saponin
6 Phenol
7 Flavonoid
“ indicates presence and “ ”- indicates absence
Table 3. Hydrogen Peroxide Scavenging Effects on Scoparia Dulcis
% of H2O2 Scavenging Plant part/ control UV Asorption value Activity
Control 2.9
Root 2.2 24.1
Stem 2.4 17.2
Leaf 2.8 3.4
Seed 2.4 17.2
Whole plant 2.0 31.0
39 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
Table 4. Hydrogen Peroxide Scavenging Effects on Piper Longum Linn
Plant part/ control UV Asorption value % of H2O2 Scavenging Activity
Control 2.9
Root 2.2 24.1
Leaf 2.9 0
Stem 2.9 0
Whole plant 2.6 10.3
40 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
Effect of plant growth promoting Proteus spp.on fenugreek
Jimtha John C1#, Mrudula M M2#, Anisha C1#, and Radhakrishnan EK1*
1School of Biosciences, Mahatma Gandhi University, PD Hills PO, Kottayam, Kerala, India. 2Sreenarayana Arts and Science College, Kumarakom, Kottayam, Kerala, India. *Corresponding author email – [email protected] # authors contributed equally.
ABSTRACT
Rhizospheric microorganisms support the growth of host plants by production of phytohormones, N2 fixation, synthesis of ACC deaminase, phosphate solubilization and also by means of production of antimicrobial metabolites or siderophores. In the present study, effect of rhizospheric Proteus spp. was analysed on the growth enhancement of Fenugreek (Trigonella foenum-graecum L.), a well known spice with medicinal properties. A bacterial strain Proteus spp.was selected as plant growth promoting rhizobacteria on the basis of preliminary screening of plant growth promotion.The bacterial strain Proteus spp. was Gram negative, motile, and positive for catalase and oxidase. Strain was positive for IAA, Ammonia, ACC deamine, siderophore, can solubilize phosphate and fix nitrogen. The plantlets formed from the treatment were statistically analysed for leaf number, shoot length, root length, wet and dry weights. Application of Proteus spp. in Fenugreek seedlings significantly increased shoot length (3.2 fold), root length (2.6 fold) dry weights (1.19 fold) and wet weights (1.5 fold), when compared with the control. The obtained results are of great significance as it widens the exploration of rhizosperic bacteria on enhanced growth of medicinal plants.
Keywords
Plant growth promotion (PGP), Fenugreek, Rhizosphere, Indole 3 Acetic Acid (IAA).
41 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
Fenugreek (Trigonella foenum- graecum) is a self pollinated crop belongs 1. Introduction to family fabaceae. The major fenugreek growing states are Rajasthan, Gujarat, Bacterial strains isolated from the Madhya Pradesh, Tamil Nadu and Uttar rhizosphere hold great promise as seed Pradesh. More than 80 per cent area and inoculants in new agricultural systems to production of India is contributed by promote plant growth and yield. These Rajasthan state alone as fenugreek is bacteria are termed as plant growth fairly tolerant to salinity which makes it promoting rhizobacteria (PGPR), to suitable for cultivation in major parts of accentuate their intimate association with the state. The crop has immense medicinal roots. Studies with PGPR indicate that the value and is a good source of vitamins, root microflora can profitably be protein and essential oils. It is mainly used manipulated through use of bacteria which for culinary and medicinal purposes and live as epiphytes on roots and beneficially continues to be important winter season modulate the ecological niche. The direct legume spice mainly cultivated in India. application of microorganisms to seed or Being an important rabi spice crop, other plant parts give them a competitive farmers largely include it in their cropping advantage over pathogens that must plan. The seeds contain an alkaloid compete for nutrients and sites for “Trigonellin (0.12 to 0.38%) is thought to attachment prior to infection. Routine use reduce glycosuria in diabetes. Fenugreek of biological systems in controlling plant seed helps not only reducing blood sugar diseases and high yield have become more levels with its high concentration of attractive due to the added benefits of phytochemicals, but also reduced low enhanced plant growth. Keeping this in density cholesterols and triacylglycerols. view an experiment was conducted to The productivity of the crop is below study the effect of (PGPR) on growth and potential due to variety of reasons yield of fenugreek. Rhizospheric bacteria including environmental factors. have been reported to be equipped with Fenugreek (Trigonella foenum-graecum the potential for nutrient cycling, L.) is grown as a spice in most parts of the production of antiphytopathogenic world and is an important source of chemicals and plant growth regulators steroidal sapogenins such as diosgenin (Khalid et al. 2004). In other words they which are used extensively by both have direct effect on plants through the pharmaceutical and nutraceutical production of phytohormones, N2 fixation, industries. Diosgenin is often used as a synthesis of ACC (1-aminocyclopropane- raw precursor for the production of 1-carboxylate) deaminase, phosphate steroidal drugs and hormones such as solubilization and indirect effects by testosterone, glucocorticoids and means of production of antimicrobial progesterone. Natural diosgenin is mainly metabolites or siderophores. The extent to isolated from the tubers of certain wild which these bacteria can be exploited for species of Mexican yam (Dioscorea the growth modulation of medicinal plants species). However this process is both is least explored. time consuming and costly, requiring
42 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
several years for the yam tubers to grow hypochlorite for 15 minute and 70% to a size to possess significant ethanol for 10 minute(Jasim et al. 2014). concentration of diosgenin to be used for Then 6 times washed with sterile distilled commercial and pharmaceutical purposes water and soaked in sterile distilled water (McAnuff et al. 2005; Obour et al. 2015). for 24 hours. Hence fenugreek may be a viable 50 ml of 2% agar was prepared in alternative for production of diosgenin a wide mouth flask (250 ml) and because of its shorter growing cycle and sterilized. 10 sprouting seedlings were lower production costs. So enhanced placed on the flask. Then 200 µl of culture growth of T. foenum-graecum can have supernatant of Proteus spp. was applied to important applications. seedlings in triplicate to each seedlings in flask. Uninoculated media was taken as Proteus species with rhizospheric origin control and was used for treating the seeds can thus offer highly promising in control flask. After 4 days the seedlings agronomical application as plant were transferred to another set of wide probiotics and their long term survival in mouth flask containing 100g sterilized the host is of great significance to be soil. 200µl of Proteus spp. supernatant exploited. Due to their remarkable impact was added to each seed. Control was on plant physiology, this organism is well treated with 200µl uninoculated media. expected to have modulatory effect on These flasks were kept for 5 days. After plant growth. Hence rhizospheric Proteus each day, 200µl sterile distilled water was species isolated from our previous study added to each seedling. After 5 days of were investigated for the enhanced growth growth, the plantlets were removed from of T. foenum-graecum. flask and their shoot length, root length, leaf number, and fresh weight were 2. Materials and methods measured for statistical analysis. Then the Bacterial strains, culture conditions, plantlets were subjected to drying in hot media and treatment air oven at 650C for 2 hours. Dry weight Proteus species already isolated from of plantlets was measured. Western Ghats forest, Konni, Statistical analysis Pathanamthitta having many plant growth The results were analyzed by statistical promoting properties were used in this program IBM SPSS Statistics 20. One study. way ANOVA performed for comparison 50 ml nutrient broth supplemented among groups. By using Post hoc multiple with 0.2% tryptophan was prepared in a comparison test the significant difference conical flask. Proteus spp. was inoculated among groups were determined. into it and incubated at room temperature with continuous shaking. One flask with 3. Results and Discussion sterile media was taken as control. After 10 days of incubation the culture Effect of isolates on growth promotion supernatant of Proteus spp. was taken by Proteus spp. showed significant centrifugation.10 Fenugreek seeds were effect in increasing the biomass of surface sterilized with 2% sodium fenugreek plant. This isolate can produce
43 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
IAA, Siderophore, ACC Deaminase, shoot growth of inoculated plants (Gadagi Ammonia and can solubilize phosphate et al. 2004). Application of IAA to P- and fix nitrogen. The study was carried deficient plants increased the root surface, out on the basis of comparison of different carbohydrate release and acid-phosphatase parameters like leaf number, shoot length, activity (Wittenmayer and Merbach root length, wet weight and dry weight of 2005). Also, IAA secreted by a bacterium the seedlings after treatment(Figure 1). may promote root growth directly by Shoot length, root length and wet weight stimulating plant cell elongation or cell showed significant difference when division or indirectly by influencing compared with control(Figure 2 & 3). bacterial 1-aminocyclopropane-1- The highest mean shoot length, carboxylate (ACC) deaminase activity 3.239±1.95 cm, was showed by the plants (Patten and Glick 2002), which is the treated with Proteus spp. The root length immediate precursor of the phytohormone is higher in the case of Proteus spp. with a ethylene, and thereby prevents the mean root length of 1.0527±0.695 cm. reduction of plant growth-inhibiting levels The higher mean wet weight was also of ethylene (Penrose et al. 2001). Also, showed by Proteus spp. treated plants. IAA production contributes to the 0.0093±0.0017g was the highest mean dry colonization efficiency and to the growth weight, which was showed by the plants and survival of bacteria on its host plants treated with Proteus spp(Table1). (Vandeputte et al. 2005). The presence of The yield and plant growth enhancement high number of bacteria in the rhizosphere effects of bacteria used in this study on T. is undoubtedly also important, since they foenum-graecum could be explained with may convert organic and inorganic N2-fixing capacity, IAA production, substances into available plant nutrients. phosphate solubilization, ACC deaminase, IAA dependent growth promotion siderophore and ammonia. The positive mechanisms have previously been effects of PGPR on the yield and growth described in many cases (Jimtha et al. of crops such as apricot, tomatoes, sugar 2014). In the current study, Tryptophan beet, and barley were explained by N2 based IAA production analysis showed fixation ability and antimicrobial the isolate Proteus spp. observed to substance production (Esitken et al. 2006; influence the enhancement of growth of Esitken et al. 2010; Ghorbani et al. 2008; fenugreek seedlings. In addition to IAA Karlidag et al. 2007; Şahin et al. 2004) concentration and its synergistic effect and by IAA production in apricot. In the with other plant growth promoting factors present study, we have also found that the might have involved in growth promotion inoculation of Proteus spp. increased yield of fenugreek seedlings. and growth in fenugreek. PGPRs are getting more attraction Proposed roles for bacterial IAA synthesis in the development of methods for include the determination of rooting sustainable agriculture. Increased use of capacity(Foga�a and Fett-Neto 2005) , biofertilizers with PGPR demand the stimulation of the release of plant identification of more potent organisms metabolites (Lambrecht et al. 2000), and with adaptive mechanisms for various the promotion of root elongation and agro-ecological niches and with diverse
44 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
plant growth promoting and plant olive (Lange 2005). Thus, producing IAA protective properties. From this point of by Proteus spp. may have positive effects view, biodiversity richness of Western on fenugreek seedlings. Our study Ghat is highly promising as it is very demonstrates that Proteus spp. can reasonable to consider the evolution of promote the growth of fenugreek, plant beneficial microorganisms along specifically, it increases root length, with or before evolution of biodiversity. shoot length, dry weight and fresh weight Hence the potential of microorganisms with the bacterial inoculation against the from such sources will be highly control plants. The results of the present demanding. So the current study was study suggested Proteus spp. has a great directed towards the microbiome of potential to increase the yield, growth of Western Ghat. The comparatively higher fenugreek seedlings. The production of percentage of plant growth promoting plant growth hormones has been properties as identified in the study is suggested as one of the mechanisms by highly supportive to the presence and which PGPRs stimulate fenugreek distribution of various plant growth seedlings growth. The growth-promoting enhancement mechanisms in this effect appears to be direct, with possible organism. involvement of the plant growth The interactions between plants and regulators indole-3-acetic acid. In view of rhizobacteria are naturally based on environmental pollution due to excessive exchanges of information between the use of fertilizers and high costs of the plants and the bacteria; it has become one production of fertilizers, PGPR strains of the most demanding research topics tested in our study have potential to be during the last decade. It is well known used for the sustainable and that some auxins exert positive effects on environmentally benign horticultural fruit set and development in various plant production. species, as strawberry, fig, Citrus and
P. mirabilis Control
Fig. 1- Plantlets showing plant growth enhancement property when treated with the supernatant of Proteus spp. along with control.
45 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
Leaf Number Shoot length Root length Wet Weight (g) Dry Weight (g)
(cm) (cm)
Control 0.833± 1.0247±0.678a 0.4130±0.178a 0.0636±0.0161a 0.0078±0.0004a 0.543a
Proteus 0.767±0.274a 3.239±1.95 1.0527±0.695 0.0977±0.036 0.0093±0.0017 mirabilis
Table. 1 Comparative analysis of variation in shoot length, root length, wet and dry mass of fenugreek seedlings under the influence of Proteus spp. along with control. Data represented as mean ± SE. n = 10. Mean values followed by the same alphabet are not significantly different by Duncan’s multiple range test at *P≤0.05.
Fig. 2- Graph showing the effect of Proteus spp. on shoot and root length of plantlets used for plant growth promotion. Data represented as mean ± SE. n = 10. Mean values followed by the same alphabet are not significantly different by Duncan’s multiple range test at *P≤0.05.
46 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
Fig. 3- Graph showing the effect of Proteus spp. on wet and dry weight of plantlets used for plant growth promotion. Data represented as mean ± SE. n = 10. Mean values followed by the same alphabet are not significantly different by Duncan’s multiple range test at *P≤0.05.
REFERENCES flower Gaillardia pulchella. Scientia Esitken A, Pirlak L, Turan M,Sahin F (2006) Horticulturae100: 323-332. Effects of floral and foliar application Ghorbani R, Koocheki A, Jahan M,Asadi GA of plant growth promoting (2008) Impact of organic amendments rhizobacteria (PGPR) on yield, growth and compost extracts on tomato and nutrition of sweet cherry. Scientia production and storability in Horticulturae110: 324-327. agroecological systems. Agronomy for Esitken A, Yildiz HE, Ercisli S, Figen Donmez M, Sustainable Development28: 307-311. Turan M,Gunes A (2010) Effects of plant Jasim B, Jimtha John C, Shimil V, Jyothis growth promoting bacteria (PGPB) on M,Radhakrishnan EK (2014) Studies on the yield, growth and nutrient contents of factors modulating indole3acetic acid organically grown strawberry. Scientia production in endophytic bacterial Horticulturae124: 62-66. isolates fromPiper nigrumand Foga�a CuM,Fett-Neto AG (2005) Role of molecular analysis ofipdcgene. Journal auxin and its modulators in the of Applied Microbiology117: 786-799. adventitious rooting of Eucalyptus Jimtha JC, Smitha PV, Anisha C, Deepthi T, Meekha G, Radhakrishnan EK., et al. (2014) species differing in recalcitrance. Plant Isolation of endophytic bacteria from Growth Regulation45: 1-10. Gadagi RS, Krishnaraj PU, Kulkarni JH,Sa T embryogenic suspension culture of (2004) The effect of combined banana and assessment of their plant Azospirillum inoculation and nitrogen growth promoting properties. Plant Cell, fertilizer on plant growth promotion Tissue and Organ Culture (PCTOC)118: 57-66. and yield response of the blanket Karlidag H, Esitken A, Turan M,Sahin F (2007)
47 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
Effects of root inoculation of plant Patten CL,Glick BR (2002) Role of growth promoting rhizobacteria Pseudomonas putida Indoleacetic Acid (PGPR) on yield, growth and nutrient in Development of the Host Plant Root element contents of leaves of apple. System. Applied and Environmental Scientia Horticulturae114: 16-20. Microbiology68: 3795-3801. Khalid A, Arshad M,Zahir ZA (2004) Screening Penrose DM, Moffatt BA,Glick BR (2001) plant growthpromoting rhizobacteria Determination of 1aminocycopropane for improving growth and yield of 1carboxylic acid (ACC) to assess the wheat. J Appl Microbiol96: 473-80. effects of ACC deaminasecontaining Lambrecht M, Okon Y, Vande Broek bacteria on roots of canola seedlings. A,Vanderleyden J (2000) Indole3acetic Can J Microbiol47: 77-80. acid: a reciprocal signalling molecule Şahin F, Çakmakçi R,Kantar F (2004) Sugar in bacteriaplant interactions. Trends beet and barley yields in relation to Microbiol8: 298-300. inoculation with N2fixing and Lange T (2005) Gibberellin Biosynthesis in phosphate solubilizing bacteria. Plant Developing Pumpkin Seedlings. Plant and Soil265: 123-129. Physiology139: 213-223. Vandeputte O, Oden S, Mol A, Vereecke D, McAnuff MA, Omoruyi FO, Morrison Goethals K, El Jaziri M., et al. (2005) EYSA,Asemota HN (2005) Changes in Biosynthesis of Auxin by the Gram some liver enzymes in streptozotocin Positive Phytopathogen Rhodococcus induced diabetic rats fed sapogenin fascians Is Controlled by Compounds extract from bitter yam (Dioscorea Specific to Infected Plant Tissues. polygonoides) or commercial Applied and Environmental Microbiology71: diosgenin. West Indian Medical Journal54. 1169-1177. Obour A, Obeng E,Holman JD (2015) Influence Wittenmayer L,Merbach W (2005) Plant of Different Seeding Dates on responses to drought and phosphorus Fenugreek (Trigonella foenum deficiency: contribution of graecum L.) Forage Yield and Nutritive phytohormones in rootrelated Value. Kansas Agricultural Experiment processes. Journal of Plant Nutrition and Soil Station Research Reports1. Science168: 531-540.
48 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
A study on the antibiotic sensitivity test of selected isolates from facial microflora and their susceptibility to commercial face washes
Femina K K, Finciya A T, Hanna T V, Jasira V, Jini Joy P & Mabel Merlen Jacob*
Department of Microbiology, St. Mary’s College, Thrissur-680020, Kerala, India *Corresponding author: Mabel Merlen Jacob, Phone No: 8129442956, Email address: [email protected]
ABSTRACT
The skin is a milieu for controlled bacterial growth and it supports the growth of commensal bacteria, with specific genera populating various body regions during particular periods in an individual's life. The resident micro biota on the skin play a major role in the delicate balance which if disrupted can make the skin susceptible to pathogens. The present study was on isolation of facial micro flora followed by their susceptibility testing to some common antibiotics and face washes available in market. About 23 different strains were isolated from facial microflora and the major isolates were tentatively identified to be Staphylococci, Bacillus, Pseudomonas, E. coli and Micrococci. Antibiotic sensitivity tests showed that almost all the isolates were susceptible to the common antibiotics with a few exceptions. Antibacterial activity of five face washes viz., GARNIER, EVERYOUTH, HIMALAYA, PEARS and VIVEL against the isolated microflora showed that the VIVEL brand was the most potent among the five and could inhibit all the isolates at the dilution tested.
Keywords:
Facial microflora, Antibiotic sensitivity, Antibacterial activity, disc diffusion method
49 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
1. Introduction human. Some of the best-studied long-term and transient bacterial residents isolated Skin provides good example of from the skin include those from the genera various microenvironments with varying Staphylococcus, Corynebacterium, Propio - amounts of moisture, body temperatures, nibacterium, Streptococcus and and greater concentrations of surface lipids. Pseudomonas (Cogen et al., 2008). They are The skin is a milieu for controlled bacterial part of the natural environment of the skin growth and it supports the growth of and as such are generally benign. The commensal bacteria, which protect the host number of bacteria identified from human from pathogenic bacteria. The abundance skin has expanded significantly, and will and types of microbes on the skin (Roth and probably continue to increase as genotyping James, 1989; Fredricks et al., 2001; techniques advance (Gao et al., 2007; Dekio Hadaway, 2003), is attributed to the skin et al., 2005). being the primary external coating of the human body which is exposed to the Many commensal microbes inhabit environment. Human skin has a variety of our facial skin. The skin microflora acts as a mechanisms for interacting with protective mechanism by colonizing the skin microorganisms, which promote the and restricting the colonization by other, propagation of certain organisms while hostile microorganisms by competitive attacking others. The relationship between exclusion. The environment of the skin also microorganisms and human skin is complex predisposes the skin to selective with some microorganisms being friendly colonization. Glands of the skin secrete residents, while others are harmful compounds called fatty acids. Many pathogens. The number of bacteria on an organisms will not tolerate these fatty acids. individual's skin remains relatively constant; But, the normal microflora of the skin is able bacterial survival and the extent of to tolerate and grow in the presence of the colonization probably depend partly on the fatty acids. Also, sweat contains a natural exposure of skin to a particular environment antibiotic known as dermicidin. The normal and partly on the innate and species-specific flora appears to be more tolerant to bactericidal activity in skin. Also, a high dermicidin than the invading microbes. degree of specificity is involved in the Thus, the presence of a normal population of adherence of bacteria to epithelial surfaces. microorganisms on the skin is encouraged by the normal physiological conditions of Skin being the primary external the body. coating of the human body is exposed to the In contrast to the protection they environment and is inhabited by a number of bestow, skin microorganisms can cause bacteria. The majority of the skin microbes infections if they gain entry to other parts of are found in the first few layers of the the body, such as through cut or during a epidermis which is the outermost layer of surgical procedure, or because of a skin and in the upper regions of the hair malfunctioning immune system. Bacteria follicles. The bacteria found here are mostly and other microbes that are normal residents commensals whose association is beneficial of the skin cause some six to ten percent of for the microbe and not harmful to the common hospital-acquired infections. 50 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
Fortunately, these problematic bacteria can considered to be more effective than beauty be easily removed by washing with ordinary (plain) soaps and deodorant (Toshima et al., soap. However, washing using harsh soaps, 2001). or very frequent washing can increase the acidity of the skin, which can counteract In the present study, an attempt was some of the protective fatty acid secretions. made to isolate and characterize a few Also the physical act of washing will shed common members of facial microflora. skin cells. If washing is excessive, the Identification of bacteria was done by protective microflora will be removed, biochemical tests followed by examining leaving the newly exposed skin susceptible their susceptibility towards a few selected to colonization by another, potentially antibiotics. The study also attempted to harmful microorganism. Health care investigate the antibacterial efficiency of workers, who scrub their hands frequently, different brands of face washes obtained in are prone to skin infections and damage. the market. Many hand and face washes are now 2. Materials and Methods available with antimicrobial properties capable of acting against such infections. 2.1 Isolation of micro-organisms from face High quality cleansing products are designed to remove impurities, infective Sterile swab sticks damped with bacteria, make-up and sloughing skin cells sterile saline were used to collect samples without irritating or stripping the skin. Soaps from face, which were inoculated into 0 are the combination of fats and oils (of nutrient agar plates and incubated at 37 C animal or vegetable origin) and salt. for 24 hours. The most prominent isolates Although fats and oils are general ingredient were selected, Gram stained, and then of soaps but some detergents are added to inoculated onto slants containing nutrient enhance the antibacterial activities of soaps agar. The colony characters were recorded (Friedman and Wolf, 1996). Soaps play an and further identification of the organisms important role in removing and killing was carried out by biochemical bacteria and antibacterial soaps can remove characterization. 65 to 85% bacteria from human skin 2.2 Identification of isolated organisms (Osborne and Grube, 1982). Transient bacteria deposited on the skin surface from Identification of bacteria was done environmental sources and which cause skin by using different biochemical tests based on infections including Pseudomonas the gram stain reaction of bacterial strains. aeruginosa (Fluit et al., 2001), Different biochemical tests such as Indole Staphylococcus aureus (Higaki et al., 2000), Test, Methyl Red Test, Voges Proskauer have been shown to be more susceptible to Test, Citrate Utilization Test, Urease Test, soaps containing antimicrobial active Catalase Test etc. were performed to identify ingredients as compared to plain soap (Lucet the isolated microorganisms. Special media et al., 2002). With the use of antibacterial like Cetrimide agar and Mannitol Salt agar soaps, removal as well as killing of bacteria is effected and antibacterial soaps are thus 51 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
were employed for the identification of The single dilution was prepared by specific organisms. dissolving 1 ml of face wash in 10 ml sterile distilled water and used for the test. Nutrient 2.3 Antibiotic sensitivity test of selected agar plates were prepared and 0.1ml of isolates selected bacterial culture was uniformly Antibiotic sensitivity of the isolated spread on nutrient agar plates to prepare facial micro flora against five common lawn culture. After solidification, wells were antibiotics namely, penicillin, erythromycin, made and each well incorporated with 20 to tetracycline, chloramphenicol and bacitracin 30 μl of diluted face washes followed by o was performed using disc diffusion method. incubation at 37 C for 24 hours. The zone of 0.1ml of selected bacterial culture was inhibition was determined by measuring the uniformly spread on nutrient agar plates to diameter in millimetres of zone to which the prepare lawn culture. Sterile antibiotic discs face washes inhibited the growth of the were placed on the surface of nutrient agar organism. After incubation the plates were plates at a distance of 2 cm using a sterile observed and the results were recorded. 0C forceps. The plates were incubated at 37 3. Results and Discussion for 24 hrs and after incubation, zone diameter was measured. 3.1 Isolation and identification of micro- organisms from face 2.4 Antibacterial activity of Face Wash About 23 different strains were Face washes of different brands like isolated from facial microflora of different Garnier, Vivel, Ever youth, Himalaya and volunteers. They were cultured onto nutrient Pears were purchased from local super agar plates and after overnight incubation, market and used to compare the antibacterial the colony characters were noted followed activities according to the standard method. by gram staining reaction (Table 3.1.1).
Isolate Colony characters Gram staining reaction 1 pale, dry, flat Gram negative rod 2 mucoid, circular, raised Gram positive cocci 3 Yellow, flat Gram positive cocci 4 pale, dry, flat Gram positive rod 5 yellow, flat Gram positive rod 6 medium, raised Gram positive rod 7 yellow, flat Gram positive rod 8 raised, opaque Gram negative rod 9 transparent, flat Gram positive rod 10 opaque, irregular, raised Gram positive rod 11 opaque, irregular, flat Gram negative rod 12 transparent, dry, flat Gram positive rod 13 opaque, pin point, raised Gram positive cocci 14 Irregular, opaque, flat Gram negative rod 15 yellow, opaque, mucoid, raised Gram positive cocci 16 round, mucoid, opaque, raised Gram positive cocci
52 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
17 round, mucoid, yellow, raised Gram positive cocci 18 round, pin point, opaque, raised Gram positive cocci 19 large, irregular, opaque, dry, flat Gram negative rod 20 small, round, raised, opaque, dry Gram negative rod 21 small, round, white, opaque, dry Gram positive cocci 22 yellow, raised, small, mucoid Gram positive cocci 23 irregular, raised, opaque, dry Gram negative rod
Table 3.1 Colony characteristics of isolated facial microflora
The different strains were further characterized by specific biochemical tests and were tentatively identified as shown in Table 3.2.
BIOCHEMICAL TEST Isolate Gram Motility Indol MR VP Citrate Catalase Ureas Tentative stain e e identification 1 Gram *Pseudomonas negative rod motile - + - + + - 2 Gram *Micrococci non positive motile cocci - - + - + + 3 Gram *Staphylococci positive non cocci motile - + - - + - 4 Gram *Bacillus positive rod motile - + - + + - 5 Gram *Bacillus positive motile rod - - - + + + 6 Gram *Bacillus positive - - - + - + rod motile 7 Gram - + + - + + *Bacillus positive motile rod 8 + + - - + - E. coli Gram negative rod motile 9 Gram - + + - + - *Bacillus positive motile rod 10 - - + - + - *Bacillus Gram positive rod motile 53 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
11 *Pseudomonas Gram negative rod motile - + _ + + - 12 *Bacillus Gram positive motile rod - - - + + + 13 *Staphylococci Gram positive non cocci motile - + - - + - 14 + + - - + - E. coli Gram negative rod motile 15 Gram *Micrococci non positive motile cocci - - + - + + 16 *Staphylococci Gram - - - + + - positive non cocci motile 17 *Staphylococci Gram - - - + + - positive non cocci motile 18 Gram *Staphylococci positive non - - - + + - cocci motile 19 *Pseudomonas Gram negative rod motile - + - + + - 20 *Pseudomonas Gram negative rod motile - + - + + - 21 *Staphylococci Gram positive non cocci motile - + - - + - 22 *Staphylococci Gram positive non cocci motile - + - - + - 23 *Pseudomonas Gram negative rod motile - + - + + -
Table 3.2 Biochemical characteristics of isolated facial microflora
54 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
Special media like Cetrimide agar yellow zones, whereas non-mannitol and Mannitol Salt agar were employed for fermenting and coagulase-negative the identification of Pseudomonas and Staphylococci produced small pink or red Staphylococcus species respectively. colonies with no color change to the Cetrimide agar contains the selective medium. Among the isolates, it could be agent, cetrimide, which inhibits the identified that facial microflora comprised growth of other bacteria and enhances the of both mannitol fermenting production of Pseudomonas pigments Staphylococcus aureus and non-mannitol such as pyocyanin and so is used for the fermenting Staphylococcus epidermidis. selective isolation of Pseudomonas aeruginosa. However there was no characteristic blue-green or yellow-green 3.2 Antibiotic sensitivity test of selected colour which rules out the isolate being isolates Pseudomonas aeruginosa. The major isolates from facial Mannitol salt agar or MSA microflora tentatively identified by contains a high concentration(~7.5-10%) biochemical tests as well as use of of salt (NaCl), as well as mannitol with selective media were Staphylococci, phenyl red as indicator, thus being Bacillus, Pseudomonas, E. coli and employed as selective media for gram Micrococci. These were subjected to positive Staphylococci which can survive antibiotic sensitivity test against five at the high salt concentration unlike most common antibiotics namely, penicillin, other bacteria. Mannitol fermentation erythromycin, tetracycline, chloramphe- ability of Coagulase-positive Staphylo- nicol and bacitracin using disc diffusion cocci resulted in yellow colonies with method (Table 3.3).
Isolate ANTIBIOTIC DISC Erythromycin Tetracycline Chloramphenicol Bacitracin Penicillin Pseudomonas 1.2 1.8 2.3 1.7 1.5 Micrococci 0.6 1.6 2.9 R R Staphylococci 2.6 2.8 3 1.4 1.5 Bacillus 2.3 1.5 2.5 0.8 R Bacillus 1 2 2.2 1.6 0.9 Bacillus 2 1.9 2.4 1 R Bacillus 1.2 1.8 2.2 1 1 E. coli 2.9 1.8 2.2 1.5 R Bacillus 1.1 2.6 1.5 1.2 1.2 Bacillus 1.1 1.8 2.3 1 R Pseudomonas 0.7 1.8 2.2 R 1 Bacillus 1 1.8 2.1 1.8 R Staphylococci 1 2 2.2 1.2 1 E. coli 2 1.1 2.7 1.2 R Micrococci 1 1.3 2.1 R R Staphylococci R 2 1.8 1 R Staphylococci 2.8 2.4 2.9 1.9 4.5 55 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
Staphylococci 2.4 2.5 2.9 1.7 2.2 Pseudomonas 1.7 2.3 2 1.8 1.9 Pseudomonas 3.1 2.5 3 1.3 3.8 Staphylococci 3.1 2.5 4 1.2 3 Staphylococci 3 1.5 3.3 1.2 1.2 Pseudomonas 0.8 2.1 2.4 1 0.9
Table 3.3 Antibiotic sensitivity test of isolated facial microflora
Antibiotic sensitivity tests showed that needs special consideration while that almost all the isolates were studying them. susceptible to the common antibiotics with a few exceptions. These included 3.3 Antibacterial activity of Face Wash Micrococci which were resistant to against isolated facial microflora Bacitracin and Penicillin, Bacillus and E. coli which were resistant to Penicillin, The facial microflora isolates Pseudomonas which was resistant to tentatively identified by biochemical tests Bacitracin and Staphylococci which was as well as use of selective media resistant to Erythromycin and Penicillin. comprising Staphylococci, Bacillus, The facial micro flora being an easy target Pseudomonas, E. coli and Micrococci for becoming vulnerable opportunistic were subjected to antibacterial activity of pathogens, antibiotic resistance is a factor Face Washes and the results tabulated (Table 3.3.1).
Isolate FACEWASH GARNIER VIVEL EVERYOUTH HIMALAYA PEARS Pseudomonas 1.2 2.5 1.5 1.3 1.8 Micrococci R 2.3 R R 1.9 Staphylococci 1.1 2.3 1.1 1.2 1.5 Bacillus 1.2 2.8 2 2.5 1.8 Bacillus 1.2 2.5 1.5 1.3 1.8 Bacillus R 2.3 R R 1.5 Bacillus 1.5 1.4 1.5 1.5 1.5 E. coli 1.5 2.6 2 2.3 2.5 Bacillus 1.4 2.5 2 2.5 3 Bacillus 1.1 1.8 2.3 1 R Pseudomonas 0.7 1.8 2.2 R 1 Bacillus 1 1.8 2.1 1.8 R Staphylococci 1 2 2.2 1.2 1 E. coli 2 1.1 2.7 1.2 R Micrococci 1 1.3 2.1 R R Staphylococci 1 2.1 1.3 1.5 1.9 Staphylococci 1.2 2.1 1.3 1.9 2 Staphylococci R 1.7 R 1.5 1.2 Pseudomonas R 1.6 R 1.5 1.8 Pseudomonas 1.5 2.5 1.6 2.2 2.2
56 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
Staphylococci 2.9 2.8 2.9 2.9 3.6 Staphylococci 2.5 2.1 2.5 2.7 2.7 Pseudomonas 1.2 2.6 1.3 1.1 1.6
Table 3.4 Antibacterial activity of Face Wash against isolated facial microflora
The five antibacterial face washes viz., GARNIER, EVERYOUTH, HIMALAYA, PEARS and VIVEL which were tested, had varying activities against the isolated microflora. The single dilution prepared and added into the wells cut in the nutrient agar plates was found in most cases, to be potent against bacterial growth. In few instances, the isolates showed resistance which were as follows:
o Micrococci - Resistant against GARNIER, EVERYOUTH, HIMALAYA, PEARS o Bacillus - Resistant against GARNIER, EVERYOUTH, Fig 3. 1 Antibacterial activity of Face Wash against isolated facial HIMALAYA, PEARS microflora o Pseudomonas- Resistant Plate 1 – complete inhibition as against against HIMALAYA, water as control GARNIER, EVERYOUTH Plate 2 – 2 isolates resistant and 3 sensitive as against water as control o E. coli - Resistant against PEARS o Staphylococci - Resistant against GARNIER EVERYOUTH
57 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
The VIVEL brand was found to be Micrococci which were resistant to inhibiting all the isolates at the dilution bacitracin and penicillin, Bacillus and tested and could be selected as the most E. coli which were resistant to potent among the five. This could be an penicillin, Pseudomonas which was interesting observation which has to be resistant to Bacitracin and studied in detail with regard to its MIC, Staphylococci which was resistant to MBC etc. So also, similar to the results in erythromycin and penicillin. antibiotic sensitivity tests, Micrococci • Antibacterial activity of five face were found to be resistant to all the four washes viz., GARNIER, brands tested except VIVEL and this EVERYOUTH, HIMALAYA, could also be a potentially interesting PEARS and VIVEL showed that the observation for further studies. VIVEL brand was the most potent among the five and could inhibit all 4. Conclusions the isolates at the dilution tested. The complex host– microbe and • Some isolates showed resistance to microbe–microbe interactions that exist different face washes and they were on the surface of human skin illustrate Micrococci - Resistant against that the microbiota have a beneficial role GARNIER, EVERYOUTH, in resisting infections by pathogenic HIMALAYA, PEARS, Bacillus- microbes. The present studies on isolation Resistant against GARNIER, of facial micro flora followed by their EVERYOUTH, HIMALAYA, susceptibility testing to some common PEARS, Pseudomonas- Resistant antibiotics and face washes available in against HIMALAYA, GARNIER, market, is of considerable merit since an EVERYOUTH, E. coli - Resistant overuse of antimicrobial agents may against PEARS and Staphylococci - disrupt the delicate balance of the Resistant against GARNIER cutaneous microflora leaving the skin EVERYOUTH. susceptible to pathogens kept in control • However, the majority of isolates by the resident micro biota. could be found to be inhibited by one The major findings of the study face wash or the other and the included, antibacterial activity of these products • About 23 different strains were could be termed satisfactory. isolated from facial microflora of different volunteers and the major isolates were tentatively identified to be Staphylococci, Bacillus, Pseudomonas, E. coli and Micrococci. • Antibiotic sensitivity tests showed that almost all the isolates were susceptible to the common antibiotics with a few exceptions. These were
58 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
5. References 9. Higaki S, Kitagawa T, Kagoura, 1. Cogen AL, Nizet V & Gallo RL. M, Morohashi M, Yamagishi T. Skin microbiota: a source of Predominant Staphylococcus disease or defence? British J aureus isolated from various skin Derm 2008; 158, 442–455 diseases. J. Int. Med. Res. 2000; 28: 87- 190. 2. Dekio I, Hayashi H & Sakamoto 10. Lucet JC, Rigaud MP, Mentre F, M. Detection of potentially novel Kassis N, Deblangy C, Andremont bacterial components of the human A, & Bouvet E. Contamination skin microbiota using culture- before and after different hygine independent molecular profiling. J techniques: a randomized clinical Med Microbiol 2005; 54:1231–8. trial. J. Hosp. Infect. 2002; 50: 3. Fredricks DN. Microbial ecology 276-280. of human skin in health and 11. Osborne RC, Grube J Hand disease. J Investig Dermatol Symp disinfection in dental practice, Proc 2001; 6:167–9. J.Clin. Prev. Dent. 1982; 4: 11-15. 12. Toshima Y, Ojima M, Yamada H, 4. Gao Z, Tseng CH & Pei Z. Mori H, Tonomura M, Hioki Y & Molecular analysis of human Koya E Observation of everyday forearm superficial skin bacterial hand-washing behavior of biota. Proc Natl Acad Sci USA Japanese and effect of antibacterial 2007; 104:2927–32. soap, Int. J. Food Microbiol. 2001; 5. Hadaway LC. Skin flora and 68: 83-91. infection. J Infus Nurs 2003; 26:44–8. 6. Roth RR, James WD. Microbiology of the skin: resident flora, ecology, infection. J Am Acad Dermatol 1989; 20:367–90. 7. Friedman M &Wolf R Chemistry of soaps and detergents: various types of commercial products and their ingredients. Clin. Dermatol. 1996; 14: 7-13. 8. Fluit AC, Schmitz FJ & Verhoef J Frequency and isolation of pathogens from bloodstream, nosocomial pneumonia, skin and soft tissue, and urinary tract infections occurring in European patients. Eur. J. Clin. Microbiol. Infect. 2001; 20: 188-191. 59 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
Bioactive peptides derived from milk: a review
Reshma Chandran P
Department of Biochemistry, St.Mary’s College, Thrissur, Kerala Corresponding author: Reshma Chandran P, Phone No: 9526246453 Email ID: [email protected]
ABSTRACT
Milk is an excellent source of well-balanced nutrients and also exhibits a range of biological activities that influence digestion, metabolic responses to absorbed nutrients, growth and development of specific organs, and resistance to disease. These biological activities are mainly due to the peptides and proteins in milk. Bioactive peptides are produced during digestion of milk in the gastrointestinal tract, fermentation and food processing. The beneficial health effects may be classified as antimicrobial, antioxidative, antihypertensive and immunomodulary. The present review summarizes isolation of bioactive milk-derived peptides along with their physiological functions.
Key words:
Bioative peptide, antimicrobial, antioxidative, antihypertensive and immunomodulary
60 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
Introduction 2. Isolation of bioactive peptide from milk Milk and derived dairy products are considered as an important constituent of a Bioactive peptides are defined as specific balanced diet. Moreover, it is a source of protein fragments that have a positive many bioactive components, such as high- impact on the functioning or conditions of quality proteins, lipids, carbohydrates, living beings, thereby improving their lactose, vitamins, minerals, enzymes, health (Nagpal et al., 2011). These are hormones, immunoglobulins, and growth derived from milk proteins by different factors, These components help to meet methods like enzymatic breakdown by human nutritional requirements and also digestive enzymes or by the proteinase play a relevant role in preventing various enzymes produced by lactobacilli during disorders such as hypertension and the fermentation of milk. Milk-derived cardiovascular diseases, obesity, bioactive peptides are usually comprised osteoporosis, dental caries, poor of 2–20 amino acids and become active gastrointestinal health, colorectal cancer, after release from the precursor protein ageing, and others (Chia-Cheinet al., where they are encrypted either by 2015). digestion proteolysis both in vivo or in vitro (Mohanthy et al., 2016). Bioactive Milk is a rich source of protein. Bioactive peptides may be released in vivo during peptides derived from milk proteins, gastrointestinal digestion as a result of the especially caseins and whey proteins are degradation of casein with an important source of these compounds. severalproteases such as pepsin; trypsin or Caseins comprises about 80 percent of the chymotrypsin Regulatory peptides can be total protein content in bovine milk and are released by enzymatic proteolysis of food divided into α-, ß- and κ-caseins(Nagpalet proteins and may act as potential al., 2011). Whey protein is composed of ß- physiological modulators of metabolism lactoglobulin, α-lactalbumin, during the intestinal digestion of the diet. immunoglobulins, and minor proteins such The possible regulatory effects of peptides as lactoperoxidase, lysozyme and relate to nutrient uptake, immune defense, lactoferrin. opioid and antihypertensive activities (Kay Milk proteins can be degraded into 2012). numerous peptide fragments by enzymatic 2. Physiological effects of bioactive proteolysis and serve as source of peptide bioactive peptides, may affect the major body systems—namely, the 2.1. Effects on cardiovascular system cardiovascular, digestive, immune and nervous systems. During recent years, Acute and chronic cardiovascular events major whey protein components, α- may result from alterations in the activity lactalbumin and β-lactoglobulin, were also of the renin-angiotensin aldosterone shown to contain bioactive sequences system and activation of the coagulation (Srikantsharmaet al., 2011). cascade and of platelets. Medications that inhibit angiotensin converting enzyme
61 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
(ACE) are widely prescribed in the bioactive peptides work as anti- treatment and prevention of cardiovascular atherogenic agents through the inhibition disease. ACE inhibitor peptides are of of endothelial-dependent adhesive particular interest due to the presence of interactions with monocytes by inhibiting encrypted inhibitory peptide sequences. In the NF-κB pathway through a PPAR-γ particular, Ile-Pro-Pro and Val-Pro-Pro are dependent mechanism. fore runners in ACE inhibition, and have Peptide-rich milk protein hydrolysates been incorporated into commercial have shown anti-inflammatory effects in products (Fissesha T Welderufael et al., cultured endothelial cells and appear to 2012). inhibit leukocyte-endothelial interaction which may explain some of their 2.2. Bioactive peptide act as anti- beneficial vascular/endothelial roles in hypertensive addition to ACE inhibitor effects. While some bioactive peptides such as Ser-Ser- ACE is a multifunctional enzyme that is Ser, Glu-Glu-Glu and Val-Pro-Leu have located in many tissues and plays an all been shown to attenuate leukocyte- important role in blood pressure regulation endothelial interactions (SepidehJabbari et and in turn hypertension. Therefore, ACE al 2012). inhibition mainly results in a hypotensive effect. Recent research has shown that 2.4. Bioactive peptide act as anti- enzymatic digestion of casein and whey microbial proteins generate bioactive peptides that have the ability to inhibit ACE. The best Biologically active peptides produced known ACE-inhibitory peptides, Val-Pro- from milk and milk products have very Pro (VPP) and Ile-Pro-Pro (IPP) with IC healthy impact on human physiology and 50 values (concentration of peptides give protection against different type of mediating 50% inhibition of ACE activity) pathogens. The cell-free supernatants and of 9 and 5 µMoles respectively have been peptide of lactobacillus KSBT46 exhibited identified from a Japanese sour milk antibacterial activity. Lactic acid bacteria drink(Prasad patel et al., 2015). have an essential role in most food and beverage fermentation processes. The 2.3. Bioactive peptide act as anti- bacterial isolate have positive impact on inflammatory their use as starter cultures for traditional fermented foods, with a view to improve Bioactive peptides with known anti- the hygiene and safety (Mohanthy et al., inflammatory effects are potential 2014). candidates for improving endothelial dysfunction. Studies were carried out to 2.5. Bioactive peptide acts as anti-oxidant. check the effects of milk-derived bioactive peptides on the expression of the Bioactive peptides possess the potential inflammatory phenotype of human antioxidant property to be used in endothelial cells and their effects on physiologically functional foods or monocyte adherence to endothelial cells. pharmaceutical industries. Bovine casein These results suggested that milk-derived 62 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
hydrolysates were prepared by enzymatic the human body. Bioactive peptides are hydrolysis using trypsin alone and with a encrypted in milk proteins and are only combination of trypsin and pepsin at released by enzymatic hydrolysis in vivo different time intervals. Trypsin during gastrointestinal digestion, food hydrolysate showed the highest processing or by microbial enzymes in antioxidant activity (13.7 %). Crude casein fermented products. At present significant hydrolysate (CH) was further fractionated research is being undertaken on the health into 2 main types by using ultrafiltration. effects of Milk derived peptides. So these The combined effect of trypsin and pepsin Bioactive peptides have the potential to be did not prove to be effective in radical used in the formulation of health- scavenging. However, fractionation by enhancing nutraceuticals, and as potent ultrafiltration proved to be effective in drugs with well-defined pharmacological radical scavenging (SanlidereAloglu et al., effects. 2011) References Goat milk proteins have gained increasing attention especially the bioactive peptides 1. Ahmed S. Ahamed., Tawfik El released from the parent proteins by Bassiony., LailaM Ibrahim and digestive enzymes. Proteins of goat milk Hisham R. Ibrahim (2015). were fractionated into caseins (GCP) and Identification of potent antioxidant whey proteins (GWP), hydrolyzed by bioactive peptides from goat pepsin and the generated peptides were milk.Food and Research examined for radical scavenging activities. Inernational, 74, 80-85. The hydrolysates of whey (P-GWP) and 2. Chia-Chien Hsieh., Blanca casein (P-GCP) proteins exhibited potent Hernández-Ledesma., Samuel ･− Fernández-Tomé., Valerie superoxide anion (O2 ) scavenging activity in a dose-dependent manner, as Weinborn., Daniela Barile and investigated using the natural Juliana María (2015) Milk xanthine/xanthine oxidase (X/XOD) proteins,Peptides and Oligosaccharides: Effects against system. MALDI-TOF-MS allowed the st identification of several antioxidant the 21 Centuary disorders. BioMed peptides derived from both caseins and Research International,ID 146840. whey proteins, with β-casein and β- 3. Fissesha T Welderufael., Trevor lactoglobulin being the major contributors, Gibson and Paula jauregi (2012). respectively (Ahemadet al., 2015). Production of Angiotensin-1- Converting Enzyme Inhibitory 3. Conclusion Peptides from Lactoglobulin-and casein-Derived Peptides: Recent research has shown that bioactive Approach. Wiley Online Library. peptide have a positive impact on the 4. Kay J. Rutherfurd-Markwick functioning or conditions of living beings, (2012).Food proteins as asource of with antihypertensive, immunostimulating, bioactive peptides with diverse antimicrobial and antioxidative activity in
63 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
functions. British journal of Bioactive Peptdes Nutrition, 108, 149-157 :AReview.International journal of 5. Mohanty D P., Mohapatra S.,Misra BIO Automation, 15, 223-250. S and Sahu P S (2016). Milk derived bioactive peptides and their impact on human health- A review. Saudi journal of Biological Sciences, 23, 577-583 6. Mohanty D P., Mohapatra S.,Misra S and Sahu P S (2016). Milk derived bioactive peptides and their impact on human health- A review. Saudi journal of Biological Sciences, 23, 577-583 7. Nagpal R.,Behare P V and KumarM(2012) .Milk, milk products, and disease free health: an updated overview,Critical Reviews in Food Science and Nutrition, 52, 321–333.
8. Prasad Patil., AkankshaWadehra., VarshaGarg.,KanchanMunjal.,Kum ar Tomar andSurajitMandal (2015).Biofunctional properties of milk protein derived bioactive peptides-A review.Asian.J.Diary&Food Res, 34(4) , 203-258. 9. SanlidereAloglu H and Oner Z (2011). Determination of antioxidant activity of bioactive peptide fraction obtained from Yogurt. Journal of Dairy Science, 94, 5305-5314. 10. SepidehJabbari., RaheleHasani., FarshidKafilzadeh and Saharjanfeshan (2012).Antimicrobial peptides from milk proteins: A Prospectus. Annals of Biological Research, 3 (11), 5313-5318. 11. Srikantsharma.,Ragahvendar Singh and ShashankRana (2011). 64 Annals of Basic and Applied Sciences, Vol 6, No.1, Dec 2015
Annals of Basic and Applied Sciences
Guide for Authors
Annals of Basic and Applied Sciences (ABAS) (ISSN: 2277 – 8756), an official publication of St Mary’s College, Thrissur, Kerala, India is being published since 2010. The journal's aim is to advance and disseminate knowledge in all the latest developments of science and technology. The Journal welcomes the submission of manuscripts that meet the general criteria of significance and scientific excellence. ABAS consider all manuscripts on the strict condition that they have not been published already, nor are they under consideration for publication or in press elsewhere.
The Annals of Basic and Applied Sciences will only accept manuscripts submitted as e-mail attachments. The text, tables, and figures should be in cluded in a single Microsoft Word file, in Times New Roman font. Submit manuscripts as e-mail attachment to the Editorial Office at [email protected] .
Article Types
Two types of manuscripts may be submitted:
Original Research Papers: These should describe new and carefully confirmed findings on any aspects of Basic and Applied Sciences. The experimental procedures should be given in sufficient detail for others to verify the work. The length of a full paper should be the minimum required to describe and interpret the work clearly.
Mini-reviews: Submissions of mini-reviews and perspectives covering topics of current interest are welcome and encouraged. Mini-reviews should be concise and no longer than 4-6 printed pages.
Review Process
All manuscripts are reviewed by editors and members of the Editorial Board or qualified outside reviewers. Decisions will be made as rapidly as possible with a goal of publishing the manuscripts in the month of December every year.
Manuscript preparation
Original Research Papers
The manuscript must be typed in Times New Roman, font size 12, double-spaced and all pages should be numbered starting from the title page. Headings should be Times New Roman, small letter, bold. Font size 12. Sub headings should be Times New Roman, small letter, italics and without bold. Font size 12.
The main sections should be numbered 1, 2 etc., the sub-sections 1.1, 1.2, etc., and further subsections (if necessary) 1.1.1, 1.1.2, etc.
The Title should be a brief phrase describing the conte nts of the paper. The Title Page should include
• Concise and informative title. (Times New Roman. Text font size 16)
• Author names and affiliations. (Times New Roman. Text font size 12)
• Name of Corresponding author (Times New Roman. Text font size 12) with telephone, e- mail address and the complete postal address. (Times New Roman. Text font size 11)
Structured Abstracts are required for all papers and should include objectives, key findings and major conclusions. It should be a single paragraph with n ot more than 250 words. References should be avoided in abstract.
Following the abstract, about 3 to 6 key words should be listed.
A list of non-standard Abbreviations should be added. Each abbreviation should be spelled out and introduced in parentheses the first time it is used in the text. Recommended SI units only should be used.
The Introduction should provide a background to the study and should clearly state the specific aims of the study. It should be understandable to the audience from a broa d range of scientific disciplines. Approximate length is 500-1000 words.
Materials and methods should be complete enough to allow experiments to be reproduced. Methods in general use need not be described in detail. Subheadings should be used. Please include details of ethical approval in this section. Approximate length: 500- 1000 words.
Results should be clear and concise with Graphs or Tables, may be inserted along with the matter or may be given as separate with the detailed title and in that case t he places where the figures are to be inserted should be mentioned in the manuscript. Each figure and table should be numbered in Arabic numerals and mentioned in the text. Figures must be clearly lettered and suitable for reproduction to fit either one co lumn width (8.5 cm) or two columns width (17 cm). Black and white photographs only are acceptable. The lettering in the figures should be readable. In addition to the inserted version in the word document, the figures can be supplied in electronic format as JPEG or TIFF. Tables should be kept to a minimum and be designed to be as simple as possible. Each table should be on a separate page, numbered consecutively (Table 1, Table 2 etc) and supplied with a heading and a legend. Tables should be prepared in Mi crosoft Word. The same data should not be presented in both table and graph forms.
The Discussion should interpret the findings in view of the results obtained in this and in past studies on this topic. A combined Results and Discussion section is also encouraged. State the Conclusions in a few sentences at the end of the paper. The main conclusions of the study may be presented in a single paragraph.
The Acknowledgments of people, grants, funds, etc should be brief.
References: In the text, a reference identified by means of an author‘s name should be followed by the date of the reference in parentheses. When there are more than two authors, only the first author‘s name should be mentioned, followed by ’et al‘. In the event that an author cited has had t wo or more works published during the same year, the reference, both in the text and in the reference list, should be identified by a lower case letter like ’a‘ and ’b‘ after the date to distinguish the works.
Examples:
(Smith, 2000), (Chandra and Singh, 1992), (Blake et al., 2003), (Chege, 1998; Steddy, 1987a, b; Gold, 1993, 1995).
References should be listed at the end of the paper in alphabetical order. Authors are fully responsible for the accuracy of the references.
Reference to a journal publication:
Diaz E, Prieto MA (2000). Bacterial promoters triggering biodegradation of aromatic pollutants. Curr. Opin. Biotech. 11: 467-475.
Reference to a book:
Pitter P, Chudoba J (1990). Biodegradability of Organic Substances in the Aquatic Environment. CRC press, Boca Raton, Florida, USA.
Reference to a chapter in a book:
Mandell GL, Petri WA, 1996. Antimicrobial Agents: Penicillins, Cephalosporins, and other ß-Lactam Antibiotics, In: Goodman and Gilman`s. The Pharmacological Basis of Therapeutics. 9 th , Ed. J.G. Hardman and L.E. Limbird, McGraw-Hill: NY. Vol. 23; PP. 1073–1101.
Mini reviews
The format requirements for original research papers apply to reviews too.
Submission
Submission in electronic form of the final version of the manuscript to the email id [email protected] .