ANTIMICROBIAL ACTIVITIES OF SILVER NANOPARTICLES SYNTHESIZED FROM CISSUS QUADRANGULARIS PLANT EXTRACT

* Sudha Rameshwari. K 1 and Maheshwari. V 2 . 1. Assistant Professor, Department of PG Biochemistry, V.V.Vanniaperumal College for Women, Virudhunagar626001, Tamilnadu, India. 2. Research Scholar, Department of Biochemistry, University of Madras, Chennai, Tamilnadu, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


ISSN: 2320-5407
Int. J. Adv. Res. 5(4), 1171-1177 1172 Vajravalli in Sanskrit, Kandvel in Marathi, Haddjor in Punjabi, Hadbhanga in Oria, Vedhari in Gujrati, Perandai in Tamil and Nalleru in Telugu in Indian languages and in English it is called as Edible-stemmed Vine. Based on morphological characters three different variants have been identified, they are squarestemmed, round-stemmed and flat stemmed. Frequently available varieties are square stemmed and round-stemmed. These are rich in source of ascorbic acid, carotene, anabolic steroid substance and calcium. The whole plant is considered to be edible while each part of the plant pharmacologically contributes to some activity. Cissus quadrangularis Linn has potent fracture healing property, antimicrobial, antiulcer, antioxidative, antiosteoporotic, cholinergic activity as well as beneficial effects on cardiovascular diseases (Jainu, M and Devi, CS, 2003). The efficacy against different activities varies for different extract (Jainu, M and Devi, CS, 2005). In Indian traditional medicine Cissus quadrangularis is used as a component of a plaster for treating swelling and bone fractures (Annie shirwackar et al., 2004). The biogenic synthesis of metal nanoparticles reduces the hazards through the elimination/minimization of generated waste and the implementation of sustainable processes. Biological methods of nanoparticles synthesis using microorganisms (Klaus, T et al., 1999;Konishi, Y et al., 2007) and plant or plant extracts have been studied as possible eco-friendly alternatives to chemical methods. Using plants for nanoparticle synthesis can be advantageous over other biological process of maintaining cell cultures and can be scaled up for large-scale nanoparticle synthesis (Shankar SS et al., 2004). In recent year, the biosynthesis method using plant extracts has received more attention than chemical and physical methods and even more than the use of microbes, for the nanoparticle metal synthesis due to the absence of any requirement to maintain an aseptic environment. Nanoparticles have attracted considerable attention because of their various applications. Silver nanoparticles are reported to possess anti-bacterial (Sathishkumar M et al., 2009). There is a widespread belief that green medicines are healthier and more harmless or safer than synthetic ones (Parimaladevi.B et al., 2004). Medicinal plants have been used to cure a number of diseases. Though the recovery is slow, the therapeutic use of medicinal plant is becoming popular because of its inability to cause side effects and antibiotic resistant microorganisms (Rawat,R.B.S. and Uniyal , 2003 ) Hence in the present investigation, much focus of attention was given by extracting Cissus quadrangularis to validate its medicinal importance by synthesizing nanoparticles of Cissus quadrangularis against antimicrobial activity.

Materials and methods:-
The present project was carried out in the department of PG Biochemistry at V.V.Vanniaperumal College for Women, Virudhunagar, Tamilnadu, India. The preliminary work (silver nanoparticle synthesis and antimicrobial activity) was done in V.V.Vanniaperumal College for Women, Virudhunagar, Tamilnadu, India. The pharmacological activity of mice study was carried out in the Venture Institute of Biotechnology and Bioinformatics Research, Madurai,Tamilnadu,India.

Collection of samples:-
The plant Cissus quadrangularis was collected from Madurai District, Tamil Nadu, India. The plant was identified and authenticated by a botanist Assistant Professor Dr.B.Karunaiselvi, Department of Botany, V.V.Vanniaperumal College for women, Virudhunagar, Tamilnadu, India. The stem part of the plant was washed several times with distilled water and kept for drying under shade. After drying, it was powdered using a domestic grinder. The plant powdered was then stored in an airtight container.
Preparation of the extract:-10 g of plant powder were weighed and was extracted with 80 ml of petroleum ether and 20 ml of chloroform using soxhlet apparatus with temperature maintained for extraction was 55 0 C. The duration of soxhlet extraction was 3 to 4 hours. The extracts were concentrated by pouring them into clean round bottom flask and were allowed for evaporation of solvents by using distillation apparatus. Then the concentrated crude extract was stored at 4 0 C in airtight bottle until further use.
Preparation of stock solution of plant extract:-400mg of extract was taken and dissolved in 40 ml of double distilled water and filtered using Whatmann filter paper No 1. It was stored at room temperature for further experiments as stock solution.

Synthesis of silver nanoparticles (AgNPs):-
In a typical synthesis of silver (Ag) nanoparticles, the stock solution (40 ml) was added to 10 ml of 10mM silver nitrate aqueous solution and kept at room temperature. The experiment was done in triplicate for reproducibility.

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After 1 hour the colour of the solution changed from colorless to honey brown [ Fig -1] indicating the formation of silver nanoparticles this is confirmed by UV-Visible spectroscopy.

UV-Vis Spectra Analysis:-
The bio reduction of pure Ag + ions was monitored by measuring the UV-Visible spectra of the reaction medium. UV-Visible spectral analysis was carried out with a SYSTRONICS UV-Vis Spectrophotometer 119. UV-Visible absorption spectrophotometer with a resolution of 1nm between 400nm and 520 nm. A small aliquot of 1ml of the nanoparticles is diluted 10 times with Millipore water to avoid errors due to high optical density of the solution.
Fourier Transform infrared (FT-IR) spectroscopy analysis:-FTIR measurements, the silver nanoparticles solution was centrifuged at 10,000rpm for 30 min. The pellet was washed three times with 20 ml of de-ionized water to get rid of the free proteins/enzymes that are not capping the silver nanoparticles. The samples were dried and grinded with potassium bromide pellets and analyzed. The pellet was immediately put into the sample holder and FT-IR spectra were recorded in the range 4000-400 cm -1 .

Preliminary Phytochemical Analysis of AgNps of Cissus quadrangularis:-
The sequentially extracted nanoparticles were used for the qualitative analysis for the identification of phytoconstituents (Harborne JB, 1998

Agar Disc Diffusion Method:-
The nanoparticles were screened against five bacterial pathogens. Antibacterial activity was carried out using disc diffusion method. The bacterial strains were inoculated in Nutrient broth and incubated for 24 hours before used in antibacterial assay. Sterile Muller Hinton Agar (Hi -Media) plates were prepared and allowed to set. The cultures to be screened were swabbed on tap of the solidified media. Disc impregnated with the nanoparticles were placed on the swabbed plate. Petroleum ether and Chloroform solvent combination was used as a control. Ampicillin (antibiotic) was used as standard. The plates were incubated at 37ºc for 24 hours. After incubation, the inhibition zone was measured. Assay was carried out in triplicates and control plates were maintained. Zone of inhibition was measured from the edge of the disc to the clear zone in millimeter (mm).

Result:-
In the present investigation, synthesis of AgNO 3 and plant mediated AgNPs was carried out. The biosynthesis of NPs was initially detected by the colour change of the solution from light yellow to honey brown. The synthesized NPs were well characterized.    Table-1. It revealed the presence of flavonoids, proteins, alkaloids, phenols, carbohydrates and terpenoids. Alkaloids and flavonoids have been associated with medicinal uses and it is one of the common biological properties with antibacterial activity. Anthraquinone Negative Antimicrobial Activity:-The antimicrobial activity was performed with five bacterial pathogens Escherichia coli, Pseudomonas aeruginosa, Shigella flexneri, Staphylococcus aureus and Pseudomonas fluorescence using well diffusion method [ Fig-4 and Table-2]. Cissus quadrangularis was found to be very effective against Stahylococcus aureus which exhibited a maximum zone of inhibition with 28 mm in diameter. Pseudomonas aeruginosa and Pseudomonas fluorescence both pathogens revealed a zone of inhibition with 26 mm in diameter.

Discussion:-
The bio-reduction of silver ions to silver nanoparticles in the solution was monitored by UV-Visibe spectroscopy. In the present study, UV-Visible spectra recorded the silver nanoparticles at 400 -450 nm. Our results coincide with the findings of Gowghami Marquis et al., 2016. FTIR is a valuable tool for measuring many chemical constituents in plants and it is used to reveal some qualitative aspects regarding the organic compounds. Several indicator bands that are pertained to functional groups represent chemical components or metabolic products (Kristin Lammers et al.,2009). In the present investigation, synthesized nanoparticles of the plant Cissus quadrangularis were characterized by FTIR analysis. The green chemistry approach towards the synthesis of silver nanoparticle was studied and it has many advantages such as, eco-friendly rapid approach and economic viability application of this eco-friendly nanoparticle antibacterial was performed. In the present study phytochemical analysis of the Cissus 1176 quadrangularis nanoparticles was carried out to investigate antimicrobial activity. The antibacterial activity tested with Cissus quadrangularis nanoparticles against Staphylococcus aureus revealed 28mm in diameter which is considered to be the best result with the plant extract. It is followed by Pseudomonas fluorescence and Pseudomonas aeruginosa exhibiting a zone of inhibition of 26 mm in diameter. The plant extract showed a remarkable activity with the above organisms. Our results coincides with the findings of Garima et al., 2009 where in they reported that Cissus quadrangularis extracts was found to be inhibitory against both gram positive and gram negative bacterial pathogens.Since ancient ages, plants have served human beings as a natural source of treatments and therapies. Amongst them, medicinal herbs have gain attention because of its wide use and less side effects. Previous study made by Raquel, 2007 had reported that steroids present in plant extracts have antibacterial activity. Alkaloids have been associated with medicinal uses for centuries and one of the common biological properties is their antibacterial property (Okwu, DE , 2004). Plants are important source of potentially useful structures for the development of new chemotherapeutic agents. The first step towards this goal is the invitro antibacterial assay (Tona, L et al., 1998). Many reports are available on the antibacterial properties of plants ( Rameshwari,2016 ). The minerals and phytochemical constituents may be responsible for the antimicrobial activities of the plants (Prema sampathkumar and Shalini S, 2012). In the present study, antibacterial activity of petroleum etherchloroform extracts of Cissus quadrangularis was studied. The extracts revealed remarkable inhibitory activity against gram positive and gram negative bacterial pathogens. Treatment of diseases possesses challenging problems due to emerging infectious disease and increasing number of multidrug resistance microbial pathogens. In spite of large number of antibiotics and drugs used a substantial need for new class of potential compound as drug is obtained from plant extract mandatory. Plant extract play an important group in designing a new class of structural antibiotics of medicinal importance with new mechanism of action. Similar results were reported in Chloroform extract of Glinus lotides linn showed good antibacterial activity against Staphylococcus aureus (Sudha Rameshwari.K et al.,2013).The silver nanoparticles obtained from Cissus quadrangularis exhibited significant antimicrobial activities without any serious side effects. These findings clearly demonstrated that the bioactive metabolites present in Cissus quadrangularis can be used for the treatment of disease.