Mini Project Report Submitted. (Post-Graduate Student) Tamil Nadu State Council Fo r High er Educatio n, CHENNAI. S.SOWBACKYA, M.

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1 ASSAY OF ANTIBACTERIAL ACTIVITY OF CADMIUM OXIDE NANOPARTICLES, BARK EXTRACT OF CINNAMOMUM VERUM (FAMILY: LAURACEAE) AND CADMIUM OXIDE DOPEN WITH BARK EXTRACT ON THE SELECTED BACTERIAL BATHOGENS OF CATTLE Mini Project Report Submitted (Post-Graduate Student) To Tamil Nadu State Council Fo r High er Educatio n, CHENNAI Submitted By S.SOWBACKYA, M.Sc Under the guidance of Dr.V.RAMADAS, Ph.D P.G & RESEARCH DEPARTMENT OF ZOOLOGY RAJA DORAISINGAM GOVERNMENT ARTS COLLEGE, (Affiliated to Alagappa University, Karaikudi ) SIVAGANGAI , TAMIL NADU October

2 1.INITRODUCTION Cattle farmers have problems of poor production, cattle diseases and the publica is vulnerable to zoonoses. Indigenous practices such as the use of herbal medicines and concoctions have been a form of therapy for cattle among resource-poor small holder farmers. Indigenous practices are considered by scientists to be risky to both human and animal health. India is primarily an agricultural country with the majority of the population residing in rural areas. Their main occupation is agriculture in addition cattle are valuable assets of the rural poor and are critical in supporting their livelihoods particularly during unfavorable times. Cattle are raised as livestock for meat, as dairy animals for milk and other dairy products, and as draft animals (Obianwu, 1984). A few farmers use conventional veterinary drugs like antibiotics most times unnecessarily and this is making disease causing bacteria more resistant to the drugs and therefore becoming a threat to public health.to solve the problems facing the cattle industry, documentation of data about indigenous knowledge about the antibacaterial activity of nanoparticles and herbal plants were carried out many workers. Many infectious diseases have been known to be treated with herbal remedies throughout the history of mankind. Natural products, either as pure compounds or as standardized plant extracts, provide unlimited opportunities for new drug leads because of the unmatched availability of chemical diversity. There is a continuous and urgent need to discover new antimicrobial compounds with diverse chemical structures and novel mechanisms of action for new and re-emerging infectious diseases (Rojas et al., 2003). Treatment of cattle diseases with medicinal plants has been practiced in India over the centuries. Even with the advent of modern medicines for treatment of veterinary diseases, most farmers still relay to some extent on veterinary traditional medicinal healers for treatment of cattle disease. This is because of the higher price of modern medicines and also because of the lack of accessibility to a modern veterinarian in the rural areas. Successful use of herbal 2

3 remedies in modern health care systems is being achieved in China, India and the Soviet Union (Obianwu, The field of Nanotechnology is one of the most active areas of research in modern material science. Nanotechnology is the application of science and technology to control matter at the molecular level. Nanoparticles are viewed as the fundamental building blocks of nanotechnology. They are the starting points for preparing many nano structured materials and devices. Nanoparticles have been known to be used for numerous physical, biological, and pharmaceutical applications. (Mansoori et al., 2007).Three applications of nanotechnology are particularly suited to biomedicine: diagnostic techniques, drugs, and prostheses and implants. There are several previous reports on antibacterial activity of medicinal plants and nanoparticles against cattle pathogens. Maluventhan viji et al., (2010) and Akharaiyi, (2011) have investigated that, the acetone and ethanol extracts of some medicinal plants and nanoparticles, have antibacterial activity against pathogenic bacteria. There are several approaches are available for synthesize of Cadmium oxide nanoparticles for example, Cdo ions are reduced by radiation, chemicalreduction, electrochemical, photochemical methods, Langmuir Blodgett, sol-gel, and biological techniques. It is well known that metal nanoparticles have good antimicrobial properties. Many authors have suggested that, Cdo nanoparticles can find applications in food systems and can be used to inhibit growth of pathogenic bacteria. There are several mechanisms which have been proposed to explain the antibacterial activity of Cdo nanoparticles. The generation of hydrogen peroxide from the surface of Cdo is considered as an effective mean for the inhibition of bacterial growth. The main aim of the present investigation was to study the antimicrobial activity of streptomycin, Cdo Nps(dopen with bark extract Nanaoparticles, combined effect of Cadmium oxide NPs (dopen with bark extract) with acetone and ethanalic extract of bark of Cinnamomum Verum on selected ten cattle pathogenic bacteria such as, Bacillus subtilis, Staphylococcus aereus, Escherichia coli, 3

4 Proteus vulgaris, Pseudomonas aeruginosa, Salmonella typhi, Serratia marcescens, Klebisella pneumonia, Methicillin-resistant staphylococcus aureus (MRSA) and Pseudomonas putida which are the major livestock pathogenic bacterial species and an important economical factor in many countries. Several Indian medicinal plants have been extensively used in the Indian traditional system of medicine for the management of human organ disorders. Medicinal plants have been identified and used throughout human history. Plants have the ability to synthesize a wide variety of chemical compounds that are used to perform important biological functions, and to defend against attack from predators and pathogenic microorganisms. At least 12,000 such compounds have been isolated so far; a number estimated to be less than 10% of the total (Padulosi et al., 2002). So far many studies have been carried out on the assessment of efficacy of many Nanoparticles and crude extracts of medicinal plant parts but studies with regard to the efficacy of Cdo Nps(dopen with bark extract) and bark extract of Cinnamomum Verum against cattle pathogenic bacterial species are need to be studied and hence, in the present investigation Cdo Nps(dopen with bark extract) and bark extract of Cinnamomum Verum have been used antimicrobial agents against four different species of cattle pathogenic bacteria. 2.OBJECTIVES To synthesize CdO nanoparticles with two different concentrations (0.5% & 1%) for the control of selected bacterial pathogens of cattles. To test antibacterial efficiency of common antibiotic streptomycin on the selected bacterial pathogens of cattle as a positive control. To prepare and test the efficacy of acetonic and ethanolic extracts bark of Cinnamomum verum (medicinal plant) for control of selected 10 bacterial pathogens of cattle. To study the individual and combined effects of nanoparticles and plant extracts on the selected bacterial pathogens. 4

5 To elucidate the samples with highest efficiency of antibacterial efficiency against disease causing pathogenic bacterial species of cattle and other pathogenic species. 3.MATERIAL AND METHODS 3.1.Synthesis of CdO Nanoparticles: The CdO nanoparticles was prepared by mixing of 0.2M of Cadmium chloride dehydrates and 0.4M Sodium hydroxide in 25ml distilled water with the aid of magnetic stirrer for 30 minutes, with this 0.12gm of Poly Vinyl Pyrolidone (PVP) dissolved in 25 ml of distilled water was added drop wise to the above mixture and then followed by microwave irradiation for 20 sec at 720 Watt. The precipitates formed will be separated from the solution by filtration, washed several times with water and absolute ethanol, and then dried in oven at 60 0 C for 24 hrs to obtain CdO nanoparticles. PLATE - 1 (CINNAMOMUM VERUM, Family: Lauraceae) 5

6 3.2.Preparation of Plant extract: CHEMICAL CONSTITUENTS, CLINICAL IMPORTANCE AND ANTIMICROBIAL PROPERTIES OF C.VERUM Chemical constituents: Cinnamon consists of a variety of resinous compounds, including cinnamaldehyde, cinnamate, cinnamic acid, and numerous essential oils. Singh et al reported that the spicy taste and fragrance are due to the presence of cinnamaldehyde and occur dueto the absorption of oxygen. As cinnamon ages, it darkens in color,improving the resinous compounds. Sangal reported variousphysiochemical properties of cinnamon. The presence of a wide range of essential oils, such as trans-cinnamaldehyde, cinnamyl acetate, eugenol, L-borneol, caryophyllene oxide, b-caryophyllene, Lbornyl acetate, E-nerolidol, α-cubebene, α-terpineol, terpinolene, andα-thujene, has been reported.chemical constituents of different parts of cinnamon Antioxidant Activity Antioxidant compounds present in foodstuffs play a vital role in human life, acting as health-protecting agents. In addition to this role,antioxidants are one of the key additives used in fats and oils. Even in the food processing industry, antioxidants have been used to delay or prevent food spoilage. Spices and medicinal plants have received rapid consideration as sources of beneficial antioxidants against various diseases. Antioxidants have been considered the most important drivers in the progress and existence of humans, as they respond to free radicals and damage in metabolic diseases and agerelated syndromes of humans and other animals. Mancini- Filho et al. reported various extracts of cinnamon, such as ether, aqueous, and methanolic extracts that have shown considerable antioxidant activities. A study on rats reported that the administration of the bark powderof C. verum (10%) for 90 days produced antioxidant activities as indicated by cardiac and hepatic antioxidant enzymes, lipid conjugate dienes, and glutathione (GSH). 6

7 A research group reported that cinnamon oil potentially exhibits superoxide-dismutase- (SOD-) like activity as indicated by the inhibition of the inhibiting capacity of pyrogallol autoxidation. The aqueous and alcoholic extract (1 : 1) of cinnamon potentially significantly inhibits fatty acid oxidation and lipid peroxidation in vitro. Different flavonoids isolated from cinnamon have freeradical- scavenging activities and antioxidant properties. A study of the inhibitory effects of cinnamaldehyde and other compounds of cinnamon on nitric oxide production revealed that cinnamaldehyde possesses potential activity against the production of nitric oxide as well as the expression of inducible nitric oxide. The highest inhibitory activities were reported as 81.5%, 71.7%, and 41.2% at 1.0, 0.5, and 0.1 μg/μl, respectively. Lin et al. reported the in vivo antioxidant activity of two different extracts, the ethanolic and hot water extracts of the dry bark of C. cassia. The ethanolic extract of C.cassia exhibited significant inhibition (96.3%) compared to the natural antioxidant α-tocopherol (93.74%). Overall, cinnamon exhibited higher antioxidant activities compared to that of other dessert spices. The essential oils and some of the major compounds present in cinnamon, including (E)-cinnamaldehyde, eugenol, and linalool, were investigated in reference to peroxynitrite-induced nitration and lipid peroxidation. Eugenol and the essential oils were more effective than the other two compounds. In a comparative study among 26 spices, cinnamon showed the highest antioxidant activity, indicating that it can be applied as an antioxidant used in foods. Another study investigated the effectiveness of a mixture of spices on oxidative stress markers as well as the antioxidant activity in high fructose-fed insulin-resistant rats. The mixture, which consisted of 1g/100 g cinnamon bark, showed a significant antioxidant activitycompared to the fructose alone group. Volatile oils from C.zeylanicum showed significant biological activities. Forty-one different volatile compounds in the bark oil of cinnamon have been identified and were found to vary significantly in percentage composition depending on the growth stages and segments of the C. cassia tree. To extract essential oil for industrial use, the yields and compositions of bark oil during Cinnamomum cassia growth (1 3 7

8 years old for the branch bark and 5 12 years old for the stem bark) were determined. These researchers also found that the branch bark fraction tended to yield more essential oil compared to the entire branch, indicating that selecting the bark based on the tree growth stages as well as separating the stem barks into top, center, and lower sections within a tree can significantly improve the extraction efficiency of essentialoils. A preliminary study on C. malabatrum leaves was conducted in various types of extracts (n-hexane, alcoholic, and aqueous extracts) to determine the presence of phenolic compounds, which indicate antioxidant activity. All of the extracts had moderate amounts of phenolic compounds and showed potential activity against hydrogen peroxide, nitric oxide, and lipid peroxide free radicals. A recent study investigated the antioxidant properties of several parts (i.e., the leaves, barks, and buds) of C. cassia. The ethanolic extract of all of the plant parts had significant antioxidant properties compared with the extraction using the supercritical fluid. The supercritical extracts showed decreased activity compared to the ethanol extracts, indicating that the active components are constituents with high polarity. C. tamala has potential antioxidant activities in diabetic rats, while C. osmophloeum, a species from Taiwan, has significant in vitro and in vivo antioxidant activities under oxidative stress. The antioxidant activity of C. zeylanicum has been investigated using various methods. In addition to the antioxidant activity, cinnamon can be used as a preservative in cakes and other food products. A recent study reported that pectin film coated with cinnamon leaf extract yielded high antioxidant and antibacterial activities. Dong et al. reported that cinnamaldehyde (E) extracted from C. cassia is the main compound and is present in levels as high as 72.7% compared to other volatile components. Cinnamaldehyde (E) is well known for its antityrosinase activity. Currently, much attention is given to tyrosinase inhibitors due to their actions in suppressing hyperpigmentation as well as the unsightly browning effects observed in mushrooms, fruits, and vegetables when they are exposed to sunlight or air. Hence, 8

9 antityrosinase agents are associated with a wide range of applications, such as cosmetics,medicine, and food Anti-Inflammatory Activities Several studies on medicinal plants and their components have indicated the antiinflammatory activities of cinnamon.various studies reported the anti-inflammatory activity of cinnamon and its essential oils. To date, there are several flavonoid compounds (e.g., gossypin, gnaphalin, hesperidin, hibifolin, hypolaetin, oroxindin, and quercetin) that have been isolated and have anti-inflammatory activities. A recent study reported that 2 -hydroxycinnamaldehyde isolated from C. cassia bark exhibited an inhibitory effect on the production of nitric oxide by inhibiting the activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), indicating that this substance can potentially be used as an anti-inflammatory agent. The ethanolic extract of C. cassia showed significant anti-inflammatory effects by reducing the activation of Src/spleentyrosine- kinase- (Src/Syk-) mediated NF-κB. Various compounds contained in C. ramulus showed anti-inflammatory effects by suppressing the expression of inducible nitric oxide synthesis (inos), cyclooxygenase-2 (COX-2), and nitric oxide (NO) production in the central nervous system (CNS). By this mechanism, C. ramulus could be a potential source for the therapeutic treatment or prevention of inflammation-mediated neurodegenerative diseases. Furthermore, the aqueous extract of cinnamon decreases the lipopolysaccharide-induced tumor necrosis factor-α levels in the serum. The bark of the Cinnamomum verum will collected from shops and dried under shade (airdried). Then the air dried barks will be powdered, 30 grams of the powder of the plant will be taken in a conical flask containing 100 ml of acetone, plugged with cotton wool and kept on a rotary shaker at 150 rpm for 48 hrs. After 48 hours the supernatant will be collected and the solvent will be allowed to evaporate to make the final volume and the content will be stored at 4 0 C in airtight bottles (WHO, 1983). Similarly, ethanolic extract was also be prepared. 9

10 3.3.Bacterial pathogenic species will be used in the study Pathogenic bacterial species like Bacillus subtilis, Staphylococcus aereus, Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella typhi, Serratia marcescens, Klebisella pneumonia, Methicillin-resistant staphylococcus aureus (MRSA) and Pseudomonas putida were collected from Veterinary College & Research Institute, Namakkal, Tamil Nadu. 3.4.Assay of antimicrobial activity using disc diffusion method What man s No.3 filter paper was punched into 6 mm (diameter) disc form and they were sterilized. An amount of 15 l of all the experimental samples was added to the sterile disc (for ex., 15 l disc contains 1500 g concentration) separately and then dried in the hot air oven. Liquid nutrient agar media and the petriplates were sterilized by autoclaving at 120 C for 30 minutes. The sterilized petriplates were then labeled with the selected bacterial strains. Under aseptic conditions in the laminar flow hood, 20 ml of agar medium was dispersed into the petriplates. After solidification of the media, microbial strains (animal pathogenic bacterial species of cattle) were swabbed on the surface of the agar plates separately. Using sterile forceps, the Antibiotic discs already prepared were placed on the surface of the agar plates. The plates were then incubated at 37 C for 24 hrs. The bacterial culture containing the discs without the addition of the CdO nano-particles was also taken as the standard and the sterile discs loaded with 20ml of solvent (acetone and ethanol) were used as the control for this study. After incubation, all the plates were examined for the presence of evaluated zones of inhibition as a property of anti-microbial activity with intervals of 24hrs. The diameter of the zone was measured in mm and the results were interpreted based on the standard. 4. RESULTS The anti-microbial activity of streptomycin, Cdo Nanoparticles (dopen with bark extracts), bark extracts of C.verum (using acetone and ethanolic solvent) and mixture of plant extract and Cdo (dopen with bark extracts) were used in the present investigation was tested against some of the common cattle pathogenic bacteria Staphylococcus aureus (SA), Escherichia coli (E.coli), Proteus vulgaris (PV) and Klebisella pneumonia (KP), Bacillus subtilis (BS), Pseudomonas aeruginosa (PA), Salmonella typhi (ST), Serratia marcescens (SM), 10

11 Pseudomonas putida (PP), Methicillin resistant staphylococcus aureus (MRSA) and the values were tabulated. In the present investigation the solvent acetone, ethanol and distilled water were used as control in the experiments carried out for antibacterial assay MEASUREMENT OF ZONE OF INHIBITION In the present study 30µl of plant extracts and the mixture of Cdo (dopen with bark extracts) were taken in this study. The zone of inhibition (ZOI) was found to vary according to the plant extracts and compounds used. The diameter of the clear zone was measured in mm (millimeter) and the results were interpreted based on the standard. The solvents acetone, ethanol and distilled water used for the experimental sample preparation have been used as a control against cattle pathogenic bacterial species. In addition, the antibiotics streptomycin was used as a positive control ANTIBACTERIAL ACTIVITY OF Cdo( 0.5% & 1%) (DOPEN WITH BARK EXTRACTS) AND ACETONE AND ETHANOL EXTRACTS OF BARK EXTRACTS AND MIXTURE OF Cdo NPs (0.5% & 1%) WITH BARK EXTRACTS The impact of experimental samples on the pathogenic bacteria were (zone of inhibition) measured and recorded (Plate-1). The Cdo (0.5%) (dopen with bark extracts) was tested against all the ten selected cattle pathogenic bacteria. The Cdo (dopen with bark extracts) was found to have greater extent of anti-microbial activity on Proteus vulgaris (15) and Klebisella pneumoniae (14) followed by test of the bacterial species such as SA (13), E.coli (13), BS(12), PA (11), SM (11), ST (10), PP (10) and MRSA (9). The Cdo (1%) (dopen with bark extracts) was tested against all the ten selected cattle pathogenic bacteria and the zone of inhibition in all the petriplates were measured and recorded. The Cdo (dopen with bark extracts) was found to have greater extent of anti-microbial activity on Klebisella pneumoniae (19) and Proteus vulgaris (17) followed by rest of the bacterial species such as, E.coli (16), SA (15), BS (14), PA (13), SM (13), ST (12), PP (11) and MRSA (11). 11

12 The plant extract was tested against all the ten selected cattle pathogenic bacteria and the zone of inhibition in all the petriplates were measured and recorded. The acetone extract of bark of C.verum was found to have greater extent of anti-microbial activity on Klebisella pneumoniae (22), Methilin resistant staphylococcus aureus (20), followed by rest of the bacterial species such as E.coli (18), PV (14), PA (14), PP (14), SA (12),BS (12), ST (12) and SM (12). Whereas, the ethanol extract of bark of C.verum was found to have greater extent of anti-microbial activity on Methilin resistant staphylococcus aureus (12) and Escherichia coli (10) followed by rest of the bacterial species such as, BS (10), SA (8), PA (8), SM (8), PV (6), KP (6), ST (6) and PP (4). Similarly, the mixture of Cdo (0.5%) (dopen with bark extracts) and acetone extract of bark of C.verum was found to have greater extent of anti-microbial activity on Klebisella pnemoniae (23), Methilin resistant staphylococcus aureus (21) and followed by rest of the bacterial species such as E.coli (19), PA (16), PV (15), PP (15), SA (14), SM (14), BS (13), and ST(13). The mixture of Cdo (0.5%) (dopen with bark extracts) and the plant extract was tested against all the ten selected cattle pathogenic bacteria and the zone of inhibition in all the petriplates were measured and recorded. The mixture of Cdo (0.5%) (dopen with bark extracts) and ethanol extract of bark of C.verum was found to have greater extent of anti-microbial activity on Escherichia coli (12) and Bacillus subtilis (12) followed by rest of the bacterial species such as PP (12),MRSA (11), SA (10), PA (10), SM (10), KP (9), PV (8) and ST (8). The mixture of Cdo (1%) (dopen with bark extracts) of Cdo (1%) (dopen with bark extracts) and acetone extract of bark of C.verum was found to have greater extent of antimicrobial activity on Klebisella pneumoniae (24) and Methilin resistant Staphylococcus aureus (22) followed by rest of the bacterial species such as E.coli (20), PA (18), SA (16), PV (16), PP (16), ST (15), SM (15) and BS (14). 12

13 Zone of inhibition 24hrs(mm) The mixture of Cdo (1%) (dopen with bark extracts) and the plant extract was tested against all the ten selected cattle pathogenic bacteria and the zone of inhibition in all the petriplates were measured and recorded. The mixture of Cdo (1%) (dopen with bark extracts) and ethanol extract of bark of C.verum was found to have greater extent of anti-microbial activity on Pseudomonas putida (15) and Escherichia coli (14) followed by rest of the bacterial species such as BS (14),KP (13), PA (13), SM (13), MRSA (13), SA (12), PV (11) and ST (11). The rates of zone of inhibition by all the samples used in the present investigation have been given in the Figure SA E.coli PV KP BS PA ST SM PP MRSA STREPTOMYSIN AB EB Cdo(0.5%) Cdo(1%) C(0.5%)+AB C(0.5%)+EB C(1%)+AB C(1%)+EB Figure-1. Showing the rate of zone of inhibition by the experimental samples against the ten selected bacterial species. 13

14 Table-1. Zone of inhibition by the experimental samples used against ten different bacterial species S. NO Name of Bacteria Streptomycin BARK A E Cdo (0.5%) Cdo (1%) AB+Cdo (0.5%) AB+Cdo (1%) EB+Cdo (0.5%) EB+Cdo (1%) ZONE OF INIHIBITION (mm) 1 SA E.coli PV KP BS PA ST SM PP MRSA DISCUSSION Cinnamon consists of a variety of resinous compounds, including cinnamaldehyde, cinnamate, cinnamic acid, and numerous essential oils. Singh et al reported that the spicy taste and fragrance are due to the presence of cinnamaldehyde and occur dueto the absorption of oxygen. As cinnamon ages, it darkens in color,improving the resinous compounds. Sangal reported variousphysiochemical properties of cinnamon. The presence of a wide range of essential oils, such as trans-cinnamaldehyde, cinnamyl acetate, eugenol, L-borneol, caryophyllene oxide, b-caryophyllene, Lbornyl acetate, E-nerolidol, α-cubebene, α-terpineol, terpinolene, andα-thujene, has been reported.chemical constituents of different parts of cinnamon. In the recent study, along with the elevation in Cadmium Oxide nanoparticles concentration, the antimicrobial property augments and the bacteria growth rate declines being in line with the other researches about the nanoparticles effect on microorganisms. It can be concluded that at the presence of nanoparticles the cell destruction 14

15 prompts. It is proposed to use cadmium oxide nanoparticles in elimination of environmental bacteria resistant to traditional antibiotics. In the present investigation among the various experimental samples used against the ten different bacterial species, the acetonic and ethanolic extracts of bark of C.verum and 1% NiO NPs doppen with acetonic and ethanolic extracts of barks of C.verum have greatest impact on the Bacillus subtilis followed by Klebisella pneumonia, Whereas, the rest of the species have showed moderate impact with the experimental samples used in the study. These results were closely related with the reports of the previous study. Signature of student (S.SOWBACKYA) Signature of the Guide HOD/ Department of Zoology Signature of the Principal Place: Sivagangai Date: 15