Photoscopic characterization of green synthesized silver nanoparticles from Trichosanthes tricuspidata and its antibacterial potential

Accepted Manuscript Photoscopic characterization of green synthesized silver nanoparticles from Trichosanthes tricuspidata and its antibacterial potential Ragunathan Yuvarajan, Devarajan Natarajan, Chinnasamy Ragavendren, Ramasamy Jayavel PII: DOI: Reference:

S1011-1344(15)00158-X http://dx.doi.org/10.1016/j.jphotobiol.2015.04.032 JPB 10032

To appear in:

Journal of Photochemistry and Photobiology B: Biology

Received Date: Revised Date: Accepted Date:

13 October 2014 9 April 2015 27 April 2015

Please cite this article as: R. Yuvarajan, D. Natarajan, C. Ragavendren, R. Jayavel, Photoscopic characterization of green synthesized silver nanoparticles from Trichosanthes tricuspidata and its antibacterial potential, Journal of Photochemistry and Photobiology B: Biology (2015), doi: http://dx.doi.org/10.1016/j.jphotobiol.2015.04.032

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Photoscopic characterization of green synthesized silver nanoparticles from Trichosanthes tricuspidata and its antibacterial potential Ragunathan Yuvarajana, Devarajan Natarajana*, Chinnasamy Ragavendrena Ramasamy Jayavelb a

Natural Drug Research Laboratory, Department of Biotechnology, Periyar University,

Salem.636011, Tamil Nadu, India. b

Centre for Nanoscience and Technology, Anna University, Chennai 600025, Tamil Nadu, India

*Corresponding author. Tel.: +91 94438 57440; +91-0427 2345766 (Extn. 225). E-mail address: [email protected] (D. Natarajan) Research Highlights •

We synthesize silver nanoparticles from Trichosanthes tricuspidata leaf methanol extract

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We observed sharp peak of plant mediated AgNps under UV-Visible spectrum (at 430 nm).

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The PSA and X-ray diffraction analysis reflects the size and peak ranges of AgNPs.

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SEM and AFM images of AgNPs revealed the spherical shape in nature

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The antibacterial potential of AgNPs showed significant activity.

Abstract The present study focused on the finding of reducing agents for the formation of silver nanoparticles (AgNPs) from the plant, Trichosanthes tricuspidata. The synthesized AgNPs were characterized using UV-Visible spectroscopy, particle size analyzer (PSA), X-ray diffraction (XRD), scanning electron microscopy (SEM) and atomic force microscopy (AFM) analyses. The UV-Visible spectrum resulted a sharp peak (at 430 nm) represents the strong plasmon resonance of silver. The average size distributions of AgNPs were found to be 78.49 nm, through (PSA), and the silver ion with its crystalline nature was confirmed using intensity (2θ) peak value of 38.22o, 44.66 o, 64.61 o, and 77.49 o. The SEM micrograph revealed that the synthesized AgNPs have a spherical morphology with the size ranges from 20 to 28 nm. AFM showed the presence of polydispersed AgNPs with its size (20 to 60 nm in height). The gas chromatography-mass spectroscopy (GC-MS) study analyzed the responsible compounds present in the methanolic extracts for the bio-reduction of AgNPs and their antibacterial effect was studied. AgNPs exhibited preponderant activity than the methanolic extracts on clinical pathogens. Thus, the

synthesized AgNPs might act as an effective antibacterial agent. Further studies are required to isolate the specific compound responsible for the reduction capability and its their inhibitory mechanisms for target bacterial strains. Keywords: Silver nanoparticles, Spectral studies, Antibacterial activity, T. tricuspidata. 1. Introduction Medicinal plants are the main sources of chemical substances with potential therapeutic effects. Now-a-days many compounds have been characterized from different plants, which are in use for the treatment of many diseases. Naturally occurring compounds from the plant, fungi and microbes have been yet used in pharmaceutical preparation, there are about three hundred species were used in 7800 medicinal drug-manufacturing units in India, which consume 2000 tons of herbs annually [1-2]. Plants have been potent known as biochemical and components of phytomedicine. Since time immemorial, man has been able to obtain from them a marvelous assortment of industrial chemicals. Plant based natural constituents can be derived from any part of the plant (like bark, leaves, flowers, roots, fruits, seeds, etc.) [3]. Since, the last decade nanoparticle biosynthesis from natural sources are the active area of research in nanotechnology. Nanoparticles found vast applications in various areas ranged from medical to physical fields [79]. Nanoparticles can be produced on different methods among them; chemical approaches are the most popular [10-11]. However, these methods cannot avoid the use of toxic chemicals in the synthesized protocol. The most effective nanoparticles were recently made from the noble metals, especially silver [4], gold [5] and platinum [6]. Gold, silver and platinum nanoparticles were widely applied to human contact areas such as shampoos, soaps, detergents, shoes, cosmetic products and toothpaste as well as medical and pharmaceutical applications [12]. Silver nanoparticles are the core vision of research nowadays, as it is implementing new findings in various fields of pharmaceutics as antimicrobial agents due to their high specific surface to volume ratio, surface-enhanced Raman scattering and its optical properties cover a pathway to material sciences for developing biosensors, medical devices, electrical batteries, and solar cell production [13]. T. tricuspidata is a large climber, often attaining a height of 9-10meters belonging to the family, Cucurbitaceae, which is commonly known as ‘red ball snake gourd’ found (at an elevation of 1200 to 2300 m) throughout in India, China and tropical Australia. It is also known as T. palmata, T. bracteata, T. pubera or Modecca bracteata [14-15]. Traditionally, this plant

was used for the treatment of inflammatory, migraines, opthalmia, epilepsy, lung disease, diabetic carbuncles, stomatitis, cold, influenza and headaches [16]. In India, the leaf extract of T. tricuspidata is used for curing snake bite poisoning [17]. Recently, the leaf extract was found to have antipyretic activity [18] and strong free radical scavenging potential [19]. Nanoparticle synthesis can be advantageous by using plant over other biological processes such as microbial route, because it eliminates the elaborate process of maintaining the cell cultures and can also be suitably scaled up for large-scale synthesis of nanoparticles [20]. The present study was aimed to synthesis of silver nanoparticles from methanolic crude extracts of T. tricuspidata and test its antibacterial potential in vitro. 2. Materials and Methods 2.1. Plant material and preparation of extracts The leaves of T. tricuspidata were collected from Kalvarayan hills (Altitude of 1200MSL) at Salem (Dt), Tamilnadu, India. The study plant was authenticated by Dr. D. Natarajan, Assistant Professor, Department of Biotechnology, Periyar University, Salem and also checked with available books and herbarium records. The voucher specimen was deposited in the Natural Drug Research Laboratory for further reference. The collected leaves were washed with distilled water and shade-dried at room temperature for a week to make fine powder. The powdered material (10 g) was macerated with 100 ml of polar (methanol (Merck, Mumbai, India) and water) and non-polar (chloroform and petroleum ether (Merck, Mumbai, India)) solvents for the extraction of active residues. The mixture of solvents were filtered through Whatman No. 1 filter paper and the crude extracts were collected and stored at 4oC for future use [21]. 2.2. Phytochemical analysis The aqueous and other different solvent crude extracts of T. tricuspidata were examined for the presence of phytoconstituents like carbohydrate, alkaloids, amino acids, phenolic compounds, saponin, fats and oils by following the standard protocol [22]. 2.3. GC-MS analysis and identification of compounds The methanolic crude extracts of leaves of T. tricuspidata was analyzed using Perkin Elmer GC–MS (Model Perkin Elmer Clarus 500, USA) equipped with a fused silica capillary column (30 m × 0.25 i.d., film thickness 0.25 µm) coupled with Perkin Elmer Clarus 600C MS. An electron ionization system with ionization energy (70 eV) was used for the detection of

compounds. Inert gas, helium was used as a carrier gas at a constant flow rate of 1 ml/min. Mass transfer line and injector temperatures were set at 220 and 300oC, respectively. The oven temperature was initially programmed from 50 to 150oC at 3oC/min and then held for 10 min and finally raised to 300oC at 10oC/min. The crude methanolic extract of sample was diluted with appropriate solvent (1/100: v/v) and filtered. The particle-free diluted crude methanolic extract (1 µ l) was taken in a syringe and injected into the split mode (The split ratio was about 1:120). The percentage composition of the crude extract constituents was expressed as percentage of peak area. The chemical compounds were identified and characterized based on its retention time (RT). The obtained mass spectral data (GC-MS) was matched with those of standards available in the existing computer library (NIST) data base [23]. 2.4. Synthesis of silver nanoparticles The synthesis of silver nanoparticles from methanolic crude extract was done as per the modified method of Dubey et al. [24]. 10 ml of methanolic leaf extract was mixed with 1 mM silver nitrate (LOBA CHEMIE, Mumbai, India) solution (90 ml) and the reaction mixture was stirred overnight for constant mixing at room temperature using a magnetic stirrer. The reaction mixture was observed further for the precipitation (formation of NPs) at the bottom of the beaker. Then the synthesized nanoparticles were tested for the characterization and antibacterial potential. 2.5. Characterization of AgNPs The samples were preliminary characterized by a UV visible spectrophotometer at the wavelength of 200 to 700 nm interval. The process of AgNPs formation methanolic was monitored at regular intervals for the completion of bio-reduction. The OD value was measured by diluting suitable aliquot of sample to make a final volume of 2 ml using Millipore water. The particle size ranges of nanoparticles were determined using particle size analyzer (Malvern Zetasizer). X–ray diffraction pattern was measured using the instrument Rigaku Miniflex II Xray diffractometer (XRD) maintained at the scanning rate of 5°C/minute, operating voltage of 40 kV and the current of 15 mA was used in a coupled radiation system of Cu-Kα radiation (λ = 1.54060A˚) in 2θ range of 30-80°. The AgNPs suspension was kept in a hot air oven at 65ºC for 30min in order to extract powder samples. Totally, 65 mg of powdered material was obtained from 100ml of suspension. The suspension of nanoparticles was air-dried to make fine powder. The powder of sample (1 mg) was coated with gold (JEOL JSM 1600A), which is called

sputtering process. Then, SEM analysis was carried out to visualize particle size and shape (JEOL JSM 6360A model). AFM is an advanced method used to characterize the height and roughness of green synthesized silver nanoparticles. The sample solution (1µl) was dispersed on a mica-based substrate with a thin layer, dried using sonicator at 37ºC for 15 minutes and visualized under AFM (NT-MDT). 2.6. Organisms source The tested clinical pathogens of Proteus vulgaris, Vibrio cholerae, Salmonella typhimurium A, Serratia marsence, Shigella boydii, Klebsiella aerogenes, Escherichia coli and Enterococcus feacalis were collected from the clinical laboratory in and around Salem and Namakkal districts of Tamil Nadu. 2.6. Antibacterial assay The water, methanol, chloroform and petroleum ether crude extracts of T. tricuspidata were taken (at the concentration of 1 mg/ml) for antibacterial activity against clinical pathogens by agar well diffusion method. About 0.1 ml of testing bacterial cultures was swabbed on Muller Hinton Agar plates. The plates were kept for overnight incubation at 37oC. Six consecutive wells (5 mm in diameter) were made in each agar plate for the different extracts (each of about 50µ l per well) including positive (Amoxycilin-1mg/ml) and negative control (10% DMSO) respectively [25]. After the observation of maximum inhibitory zone in the methanolic crude extract, the silver nanoparticles synthesized using methanolic crude extract were subjected to their antibacterial study. Similarly, the synthesized silver nanoparticles (1mg/ml) also treated on S. typhi A, E. faecalis, S. marcense, C. diptheriae, S. sonii, S. boydii, K. aerogenes and V. cholerae, by following the modified method [26]. 3. Results and discussions 3.1. Phytochemical test The screening of phytochemical constituents of four different extracts result showed the presence or absence of carbohydrates, amino acids, saponins, alkaloids, phenolic compounds, fats and oils (Table.1.). The methanolic extract of T. tricuspidata showed the presence of carbohydrate, alkaloids, phenolic compounds, saponin, fats and oils. The similar findings were recorded from the methanolic extract of Coccinia cordifolia [27], which might possess some phytochemical constituents and support this study. Other plants, Cucurma longa, C. amada and C. caecia [28] reported as having major phytoconstituents, which are responsible of bioactivity.

3.2. Identification of compounds by GC-MS analysis A total of eighteen compounds was identified from the methanolic extracts of T. tricuspiadata by GCMS analysis(Table.2.). Among them, eight are major compounds (2furancarboxaldehyde, 5-(hydroxymethyl)- (13.842% and 5.678%), L-(+)-ascorbic acid 2,6dihexadecanoate (11.692%), acetic acid, 17-(1,5-dimethylhex-4-enyl)- 4, 4, 8, 10, 14pentamethyl-2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,14,15,16-(11.577%), 2-pyrrolidinone, 1-[3,7dimethyl-9-(2, 2, 6-, tri methyl cyclohexyl) nonyl]-(8.406%), 4, 4, 6A, 6B, 8A, 11,11,14 Boctamethyl-1, 4, 4A, 5, 6, 6A, 6B (8.170%),

3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-

trimethyltridecyl)- (7.691%), acetate, [2R-[, isothiazole, 3-methyl) (6.338%), methyl 11,14,17eicosatrienoate (5.627) and rest of them are come under minor compounds (Fig.1 & 2.). Pramod Kumar et al., [29] have identified 42 compounds from the ethanol extract of Momordica tuberosa, with Isopulegol, a monoterpene and a steroid, Androstane as main constituents. Similiarly, Ambethkar and Ananthalakshmi, [30] were reported twelve major compounds from Trichosanthes anguina, which supports the present investigation. 3.3. Silver Nanoparticles synthesis Synthesis of green based silver nanoparticles was observed due to color changes of silver nitrate solution. After addition of plant extract (10ml) into the silver nitrate solution (90ml), light yellow color has been changed as dark brownish (Fig 3), which indicates the presence of silver nanoparticles[31]. 3.4. Characterization 3.4.1. UV Visible spectroscopy UV Visible spectroscopy study was performed for the characterization of synthesized silver nanoparticles. The formation of synthesized nanoparticles was confirmed its OD value (in the wavelength range of 200-700nm) and the broad absorption band was noticed at422nm (Fig.4.). The previous report stated that absorbance at peak range of around 430nm is ideal for visualizing the silver nanoparticles [32]. Generally, the Surface Plasmon Resonance (SPR) bands are influenced by the size, shape, morphology, composition and dielectric environment of the prepared nanoparticles [33-34]. Previous studies have shown that the spherical Ag-NPs contribute to the absorption bands at around 400 nm in the UV–visible spectra [35]. According to

Kamyar et al., [36] SPR band characteristics of Ag-NPs were detected around 412–437 nm, which strongly suggests that the Ag-NPs were identified as spherical in shape. 3.4.2. Particle Size Distribution The green synthesized silver nanoparticles were analyzed for distribution of particle size and the results showed it was distributed in the various sizes in mode of poly-dispersible. DLS was used to measure the particle size in a colloidal solution. First and second peaks, revealed the size of the particles were found to be 67.49 and 1121nm with diameter of 26.48 and 522.2 nm and the peak intensity was found to be 74.4% and 25.6%respectively. The mean average size of silver nanoparticle was 78.93 nm. The polydispersity index was found to be 0.385 (Fig.5.). Polydispersity index represents the ratio of particles of different size to total number of particles [37]. 3.4.3. X-ray diffraction X-ray diffraction patterns were established different size dependent features of silver and leading to anomalous peak positions, height and width ranges of particles. The intensity peaks of XRD pattern obtained for the plant silver nanoparticles show its corresponding Bragg reflections to (111), (200) and (220) sets of lattice planes are indicated by various peak ranges of 38.22, 44.66, 64.61, and 77.49 which are confirmed silver peaks (Fig.6.). These data’s are matched with the standard joint committee for the powder diffraction set (JCPDS) (File No: 040783), confirming a face centered cubic lattices of silver nanoparticles. Silver nanocrystallites display, an optical absorption band peak approximately 3 keV, which is typical of the absorption of metallic silver nanocrystals due to surface plasmonresonance [38]. 3.4.4. Scanning Electron Microscopy The green synthesized silver nanoparticles were characterized for morphology and size by scanning electron microscopy. The SEM micrograph revealed that the synthesized silver nanoparticles have a spherical morphology (with size ranged from 20 to 28 nm) (Fig.7.). Similarly, morphological characteristics of silver nanoparticles were measured by SEM analysis in various plants i.e., Rhynchotechum ellipticum [39], Eclipta prostrata [40]. Trachyspermum ammi and Papaver somniferum [41], O. tenuiflorum, S. cumini, C. sinensis, S. tricobatum and C. asiatica [26] to determine the different size ranges of AgNPs.

3.4.5. Atomic Force Microscopy

The dimensions of nanoparticles (including the size, shape, dispersion) were observed by the atomic force microscopy. The AFM images showed the presence of bright spots on the surface indicates the presence of silver nanoparticles and the inverse showed the dark spots indicating size and dispersion (20 to 60 nm in height) of the nanoparticles. The 3D image shows the sharp peaks with lower to higher sizes of nanoparticles and the histogram of AFM provides the basic information for the average size of the nanoparticles (Fig.8.). Many researchers have confirmed AgNPs size and shape through AFM, i.e., 3-D image of AgNPs produced by Geranium water extract estimated size range was approximately 15.2 nm [42]. Pterocarpus santalinus leaf extract mediated Ag NPs possess spherical shape and have the calculated sizes in the range of 20 to 50 nm [43]. The AgNPs of lemon extract sizes measured approximately 12 nm height and 100 nm in width [44]. 3.2. Antibacterial activity The overall antibacterial activity assay (Table 2) resulted that the methanol extracts showed significant activity against all the tested organisms especially in S. boydii (12mm), P. vulgaris (10mm) and E. faecalis(10mm). Petroleum ether, chloroform and water extract exhibit moderate activity against S. boydii (11mm), K. aerogenes(9mm), E. coli (9mm) and E. faecalis (9mm). There is no antibacterial activity of petroleum ether, water and chloroform extracts against P. vulgaris, V. cholorae and S. aureus. Methanol, water, chloroform has no growth inhibition against S. typhimurium A and S. marcense (Fig.9 & Table 3.). The earlier study results revealed that the antibacterial potential of different plant extracts of Coccinia grandis (Cucurbitaceae) showed more significant antibacterial activity against all the tested microorganisms [45-48]. Hexane, chloroform and ethyl acetate extracts of Coccinia cordifolia exposed different biological activity, including antimicrobial, antioxidant and cytotoxicity effects [49]. The in vitro antimicrobial activity of methanolic and aqueous extracts of different parts of Luffa acutangula (fruits, leaves, roots and seeds) were evaluated against E. coli, S. aureus, K. pneumoniae, P. vulgaris, C. albicans, Aspergillus niger, and Fusarium species [50]. In this present findings, the methanolic leaf extract mediated AgNPs of T. tricupidata was compared with the crude methanolic extract, in which plant mediated AgNPs exhibits significant antibacterial potential (Fig 10 & Table 4.) against all the above tested organisms like Proteus vulgaris (15 mm), Vibrio cholerae (12 mm), Salmonella typhimurium A (21 mm), Serratia marcense (18 mm), Shigella boydii (21 mm), Klebsiella aerogenes (14 mm), Escherichia coli

(20 mm) and Enterococcus faecalis (13 mm). Previous report suggested that the synthesized AgNPs from the seeds of Cucumis melo showed a good antibacterial against E. coli, S. aureus and P. aeruginosa than the leaves [51]. The researchers reported that the novel AgNPs from Citrullus colocynthis exhibited a tremendous antibacterial activity against bioflim bacteria such as E. coli, V. paraheamolyticus, P. aeruginosa, P. vulgaris and L. monocytogens and also observed that it showed no activity against P. mirabilis, S. enteritidis, and S. aureus [52]. AgNPs-containing leaf extract showed a higher antioxidant and antimicrobial activity compared to C. murale leaf extract alone or silver nitrate [53]. The synthesized AgNPS from Securinega leucopyrus showed significant antibacterial activity against both Gram positive and Gram negative bacteria [54]. 4. Conclusion The green synthesized nanoparticles confirmed by the various technical aspects, like UVvisible spectroscopy, PSA, XRD, SEM and AFM. The potential active components for reduction agents of silver ions were identified by preliminary screening of phytoconstituents and GC-MS analysis. Our research concluded that methanol extracts of T. tricuspidata showed significant activity against most of the tested organisms and it possesses reduction of silver ions to form silver nanoparticles. Plant based silver nanoparticles have more potential for the clinical pathogens, while compare with crude extracts. References 1. J.S Kim, E. Kuk, Yu KN, J.H. Kim, S.J. Park, H.J. Lee, Antimicrobial effects of silver nanoparticles. Nanomedicine: Nanotechnology, Biology and Medicine. 3(2007) 795-101 2. O. Choi, K.K. Deng, N.J. Kim, L. Ross, R.Y. Surampalli, Z.Q. Hu, The inhibitory effects of silver nanoparticles, silver ions and silver chloride colloids on microbial growth,Water Res. 42 (2008) 3066–3074. 3. M. C. Gordon, J.N David, Natural product drug discovery in the next millennium, Pharm. Biol. 39 (2001)8-17. 4. N. Durán, P. D. Marcato, O. L Alves, G. H. D. Souza, E. Esposito, Mechanistic aspects of biosynthesis of silver nanoparticles by several Fusarium oxysporum strains, Journal of Nanobiotechnology, 3:8. 1-7, doi:10.1186/1477-3155-3-8. 5. B. Ankamwar, Biosynthesis of Gold Nanoparticles (Green-Gold) Using Leaf Extract of Terminalia catappa, E-Journal of Chemistry.7(2010),1334-1339.

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List of figures

Legend: METT-TT – Methanolic crude extracts of T. tricuspidata Fig. 1. GC-MS Chromatogram of methanolic extracts of T. tricuspidata

Legend: Structure of eighteen identified compounds Fig.2. Compounds in methanol extracts

Legend: Silver nitrate 90ml + plant extract 10ml Fig. 3. Formation of AgNPs of T. tricuspidata

Legend: The broad absorption peak 430nm Fig. 4. UV spectroscopy analysis of AgNPs of T.tricuspidata

Legend: Particle size distribution of silver nanoparticles Fig.5. Particle size distribution of AgNps

Legend: Crystalline nature of AgNps Fig. 6. X- ray diffraction of analysis of AgNps

Legend: Magnification of AgNPs under 2µm Scale Fig. 7. Morphology of AgNps by SEM analysis

Legend: Graphical visualization of Height and roughness of AgNps Fig. 8. AFM analysis of AgNp of T tricuspidata

Fig. 9. Screening of antibacterial activity of T. tricuspiadata

Fig. 10. Antibacterial activity of AgNPs of T. tricuspidata against clinical pathogens

Graphical Abstract

Table.1. Screening of phytochemical constituents of T. tricuspiadata

S. no

Test

Methanol

Chloroform

Petroleum

Water

ether 1

2

3

4 5 6

Test for Carbohydrate Fehling’s test + Barfoed’s test + Test for Alkaloids _ Wagner’s test + Mayer’s test Test for Phenolic compound Ferric chloride test Lead acetate + Test for Amino acid Ninhydrin test Test for Saponin Kokate 1999 + Test for Oil and fats Spot test +

Legend : ‘+’ - Presence

+ -

+ +

+ +

+ +

+ +

+ +

+

+

+

+

+

+

-

-

-

+

-

+

-

-

+

‘-’ Absence

Table.2. List of phytocomponents present in the methanolic leaf extract of T. tricuspidata S.No

RT - % Peak Compound name value area 6.390

8.406

1 2 3

6.515 8.191

6.338 1.842

Molecular weight

Formula

2-PYRROLIDINONE, 1-[3,7-DIMETHYL-9(2,2,6-TRIMETHYLCYCLOHEXYL)NONYL]-

363

C24H45ON

ISOTHIAZOLE, 3-METHYL-

99

C4H5NS

332

C17H32O6

126

C6H6O3

126

C6H6O3

2-METHOXY-4-VINYLPHENOL

150

C9H10O2

PHENOL, 3,5-BIS(1,1-DIMETHYLETHYL)-

206

C14H22O

TETRADECANOIC ACID

228

C14H28O2

Z,Z-6,28-HEPTATRIACTONTADIEN-2-ONE

530

C37H70O

L-(+)-ASCORBIC DIHEXADECANOATE

652

C38H68O8

HEXACOSANOL, ACETATE

424

C28H56O2

METHYL 11,14-EICOSADIENOATE

322

C21H38O2

METHYL 11,14,17-EICOSATRIENOATE

320

C21H36O2

OCTADECANOIC ACID

284

C18H36O2

458

C30H50O3

472

C31H52O3

4,4,6A,6B,8A,11,11,14B-OCTAMETHYL1,4,4A,5,6,6A,6B,7,8,8A,9,10,11,12,12A,14,14A,

424

C30H48O

ACETIC ACID, 17-(1,5-DIMETHYLHEX-4ENYL)-4,4,8,10,14-PENTAMETHYL-2,3,4,5,6,

468

C32H52O2

.BETA.-D-MANNOFURANOSIDE, UNDECENYL)-

1-O-(10-

13.842

4

9.701

2-FURANCARBOXALDEHYDE, (HYDROXYMETHYL)-

5-

2-FURANCARBOXALDEHYDE, (HYDROXYMETHYL)-

5-

5.678

5 6

9.901

10.447

3.102 1.970

7

12.893

8

15.794

9

16.449

10

17.815

11 12 13 14 15 16 17

18

18.005

1.585 3.739 11.692 1.829

19.390

2.593

19.450

5.627

19.640

1.582

26.238

2.734

26.878

7.691

29.514

30.029

8.170

11.577

ACID

2,6-

3,4-DIHYDRO-3,5,8-TRIMETHYL-3-(4,8,12TRIMETHYLTRIDECYL)-(2H)1-BENZOPYR 2H-1-BENZOPYRAN-6-OL, 3,4-DIHYDRO2,5,7,8-TETRAMETHYL-2-(4,8,12-TRIMETHY

Legend: RT – Retention time

S. No

Pathogen

Zone of inhibition (mm in diameter) of different extracts Methanol

Petroleum ether

Water

Chloroform

Positive control

Negative control

1 2 3

P. vulgaris V. cholorae S. typhimurium A

10 8 -

7

-

-

15 15 16

-

4 5 6

S. marcense S. boydii K. aerogenes

12 9

7 11 9

11 8

10 9

15 19 15

-

7 8

E. coli E. faecalis

9 10

9 9

8 8

8 9

15 16

-

Table.3. Screening of antibacterial activity of T. tricuspiadata

Legend: - Nil activity; mm- millimeter

Table.4. Antibacterial activity of AgNPs of T. tricuspidata Pathogen

Plant extract

AgNps

(Ciprofloxacin)

P. vulgaris V. cholorae

-

15 12

26 37

S. typhimurium A

-

21

28

S. marcense S. boydii K. aerogenes

-

18 21 14

25 23 22

E. coli

-

20

36

E. faecalis

-

13

38

Legend: Nil activity