Green functionalized silver nanoparticles With ... · Jivan R. Kote et. al. / International Journal...

Jivan R. Kote et. al. / International Journal of New Technologies in Science and Engineering

Vol. 3, Issue. 2,Feb 2016, ISSN 2349-0780

Available online @ www.ijntse.com 12

Green functionalized silver nanoparticles With significantly enhanced

Antimycobactericidal and Cytotoxicity Performances of Asparagus racemosus

Linn. Jivan R. Kotea , Ambadas S. Kadama*, Shyam S. Patila, Rajaram S. Maneb

aJivan Rameshrao Kote Research student School of Life Science Swami Ramanand Teerth Marathwada University Nanded [email protected]

a*Ambadas S. Kadam Associate Professor DSM Arts, Commerce, Science College Jintur Dist-Parbhani [email protected]

bShyam S. Patil Asistant Professor Sharad Chandra College Naigaon Dist-Nanded cRajaram S. Mane Professor School of Physical Science Swami Ramanand Teerth Marathwada

University Nanded

Abstract

In the present investigation, Ag NPs were synthesized by using leaf extract of aqueous Asparagus racemosus L., and the X-ray diffraction spectrum. As-synthesized Ag NPs were separated with fine channels. Fourier transform infrared spectrum has confirmed capping of the phyt-constituents; silver nanoparticles could be easily modulated by reacting the novel sundried biomass of Asparagus racemosus L. The results obtained from the antimycobacterial activity of potentiality of these Ag NPs against four Mycobacterium species like M. tuberculosis (MTCC-300), M. pheli (MTCC-1723) and M. avium (MTCC-1724), M. spegmatis (MTCC-994) where for all cases 99.9% cell death is achieved. Haemolytic assay of A Cytotoxic effect against in-vitro human blood cells (10μg/ml/24 h) by was remarkable where minimum inhibitory concentration level of the Ag NPs was within 10-20 μg/ml. Keywords: Asparagus racemosus L., silver nanoparticles; antimycobacterial activity, hemolytic assay, minimum inhibitory concentration

I. Introduction Tuberculosis (TB) is one of the commonly known disease occurred worldwide due to which about 9.4 million cases were newly registered and about 1.7 million were reported as dead in 2009. TB is a chronic infectious disease caused by several species of mycobacteria are deadly bacterial pathogen is M. tuberculosis is the causative agent. Due to multidrug resistant-strains of mycobacteria to a high prevalence of tuberculosis in patients who have Acquired Human Immunodeficiency Syndrome (AIDS), treatment of HIV-related TB is complex as there is overlapping of drug toxicities and drug-drug interactions between antiretroviral and anti-TB treatment. This causes the risk for development of immune reconstitution inflammatory disease. There is need to develop a targeted drug delivery system for tuberculosis for improving patient’s compliance. Functionalized silver nanoparticles (Ag NPs)-based drug delivery system which has high affinity towards infected tubercle cells than normal cells [1]. These NPs are being extensively applied in surgical instruments, wound dressings, bond

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prostheses and heart valves, electronics, and biosensing [2, 3]. Normally Ag NPs are used in textiles, laundry additives, room sprays, water cleaners, and food storage containers, cancer, catalysis, ambient temperature [4-7]. Colorimetric ensing [8] and endophytic fungus [9]-supported Ag NPs interact with the cell membrane of bacteria wherein Ag+ ions bind to sulfur, oxygen, and nitrogen of essential biological molecules thereby inhibit bacterial growth [10]. The literature survey revealed that the Ag NPs syntheszied by using environmentally clean i.e. green route produce better results as compared to that of synthesized from a chemical base [11, 12]. In the present investigation, using plant antitubercle agent source, synthesis of Ag NPs is reported using green approach. These Ag NPs were targeted on four Mycobacterium species to develop novel antituberculosis agents.

II. Materials and method

A. Plant materials

Asparagus racemosus L., belong in the family Asparagaceae family. The leaves of Asparagus

racemosus L, used in this study, were collected from the campus of Swami Ramanand Teerth

Marathawada University Nanded campus, Maharashtra India. These were primarily cleaned

with Millipore Deionised (DI) water, washed and dried by pressing with blotting paper and

then were shade-dried and chopped into small pieces and grinded into fine powder.

B. Synthesis of plant extract

Grinded powder of Leaves 10 g were poured into 100 ml of distilled water and irradiated for

5 min with microwaves using ultrasonics cleaner, obtained extract was filtered through

Whatman filter paper, and stored at 4ºC for further experiments.

C. The Biosynthesis of silver nanoparticles (Ag NPs)

A 100 ml, 0.1 M AgNO3 was kept in a conical flask wherein 10 ml aqueous Asparagus race

mosus L., extract was added while vigorous magnetic stirring. The reduction of Ag+ ions into

Ag NPs at room temperature was completed in about 30 min, as color of soltuion was

changed from greenish-yellow to dark brown indicating the formation of Ag NPs (Fig. 1).

For the purification of Ag NPs the reduced solution was centrifuged at 5000 rpm for 15 min

for two to three times. The supernatant liquid was discarded, and the residue was dispersed in

distilled water. The residue was collected, washed with distilled water, dried and stored in





vacuum tight chamber for further use. Before the final use, Ag NPs and distilled water was

mixed in appropriate proportions for desired Ag NPs concentration.

Fig1. Phytoreduction of Ag+ to Ag NPs, presents change of colour from tranparent to

brown after 24 h incubation [a) AgNO3, b) Asparagus racemosus L. leaf extract, c)

AgNO3 with leaf extract].

D. Structural and morphological studies

The X-ray diffraction (XRD) measurement was carried out using a Structural elucidation X-

ray diffraction spectrum obtained from X-ray Diffractometer (Rigaku/MAX 2500 V,

Cu Kα, λ = 1.5418 ºA). Morphological evolution of Ag NPs was confirmed from the field-

emission scanning electron microscopy (FE- SEM, Hitachi S-4200) image. The functional

groups present in the phyto-constituents in the leaves extract of Asparagus racemosus L., and

their involvement in the synthesis of Ag NPs were confirmed from the FT-IR plot on Nicolet

IS-10 (Thermo Fischer Scientific Instruments) for which the Ag NPs were mixed with KBr

for forming a pellet.

E. Collection of pathogen

The Mycobacterium tuberculosis (MTCC 300), Mycobacterium pheli (MTCC 1723),

Mycobacterium avim (MTCC 1724), Mycobacterium spegmatis (994) were obtained from

Microbial Type Culture Collection and Gene Bank, Institute of Microbial Technology,





Chandigarh (PB), India. These were subcultured and maintained into Lowenstein-Jensen

media.

F. Antimycobacterial activity of the test drug

As per experimental standardization, initially 176 mg/100 ml (1M) concentration of Ag NPs

was used for antimycobacterial analysis which was further taken up to only 20 μl/ml added

into the well, however, the clear zone of inhibition was observed under the experimental

condition. The sensitivity test of four different strains of M.tuberculosis, M.pheli, M.avium

and M.spegmatis was studied by agar diffusion method. In short, a sterile cork borer of 7mm

diameter was used to bore holes into the inoculums seeded solidified Lowenstein Jensen

medium. A 20 µl volume of AgNPs was loaded into the well using a sterile pipette. From the

AgNO3 solution (176 mg/100 ml), 20µl was added in agar well plates. The plates were

frizzed for 10 min, useful for an easy diffusion and then incubated at 37º C for 48 h. Growth

of M. tuberculosis, M. Pheli, M. avium and M. spegmatis was observed after the diameter of

the inhibition zone measured the well size. Rifampicin (10µg/ml) was used as reference

standard.

G. In-vitro stability studies

Ag NPs of 1 ml (176 mg) was incubated at 37ºC for 48 h with 0.5 ml of 2-4M saline and PBS

(pH 4.5-4.7). Dipersion was preserved for 2, 4, 6,12, 24, 48 and 72 h and then analyzed

spectrophotometrically.

H. Minimum inhibitory concentration (MIC)

The minimum inhibitory concentration (MIC) test was studied by employing a micro-dilution

method [13,27]. The Ag NPs of 20 µl against mycobacterium species, as starting stage, were

used during all measurements. The 96-well microtitre plates, filled with each of 20 µl

volume, were maintained along with a sterility control and growth control (containing culture

broth plus distilled water without antimicrobial substances). Each test (with growth control





well) was inoculated with 5 µl of a bacterial suspension (102 CFU (colony focusing unit) per

ml or 98 CFU per well). In the present study, equal and the micro-dilution trays were

incubated at 36ºC for 18 h and the bacterial growth was detected by measuring the optical

density (ELISA reader, Thermo Multiscan EX Ref:51118170) and by adding equal

concentration of Ag NPs and solvent i.e. water. The microtitre plates were again incubated at

36 ºC for 30 min, in those wells where bacterial growth was seen, indicating the change of Ag

NPs color from yellow to brown. [14]

J. Hemolytic assay

The cytotoxicity activity of Ag NPs study against the human red blood cells by Hemolytic

assay [14]. Red blood cells collected in tubes from healthy persons were preferred in tubes

containing ethylene-diamine tetra-acetic acid (1–2 mg/ml) as anti-coagulant. The red blood

cells were harvested by centrifugation (Heraeus Megafuge 40, Thermo Fisher Scientific Inc.,

MA ) for 10 min at 634 × g at 20 ºC and washed three times in PBS (Phosphate Buffer

Solution). Added PBS the pellet to yield a 10% (v/v) erythrocytes/PBS suspension. The 10%

suspension was diluted with PBS in 1:10 ratio. From each suspension, 100 μl was added in

triplicate to 100 μl of a different dilution series of test compounds (or Rifampicin is a

standard Antimycobacterial agent). Total hemolysis was achieved using 1% Triton X-100.

The tubes were incubated for 1 h at 48 ºC and centrifuged for 10 min at 634 × g at 20°C.

From the supernatant fluid, 150 μl was transferred to a flat-bottomed microtitre plate (Tarson

India Ltd., India), and the absorbance was studied at 450°C.

……..1

III. Results and discussion

A. Structure, bonding morphology studies





In present investigation, Ag NPs were successfully synthesized from green leaves. A change

of solution colour from white to brown, after biotransformation during the biosynthetic

process [16-18], Furthermore, during overnight incubation in dark room condition, the

transparent reaction mixture was turned into a dark brown solution, providing qualitative

evidence for the formation of Ag NPs (Fig. 1) [19]. The XRD pattern of the biosynthesized

Ag NPs produced by the leaf extract of Asparagus racemosus L., extract is presented in Fig.

2a. The 2ϴ values at 27.97º, 32.47º, 38.32º, 41.10º, 46.40º, 55.02º, 57.54º, 64.70º, 69.33º

were assigned tp 110, 120, 022, 132, 023, 151, 213, 240 reflection planes of Ag (JCPDF file

no.401054) which was nearly same as reported for Ag NPs obtained from Annonaa reticulata

leaf extract [20] (Fig. 2a). FTIR is a valuable tool for knowing the participation of functional

groups of metal particles and biomolecules interactions [20-22]. In the present study, FTIR

measurement was performed to identify the biomolecules responsible for capping and

stabilizing the Ag NPs. The FTIR spectrum (Fig. 2b) of Ag NPs showed a very strong peak at

3704.50cm−1, 3625.35cm−1, 674.11cm−1, 3643.12cm−1 corresponding to –OH stretching in

alcohols and phenolic compounds [20-22]. Peaks observed around 1735.35 cm−1, 1592.72

cm−1 were assigned to the C–H stretching vibration of methyl, methylene, and methoxy

groups. The medium intense peaks at 1394.13, 1072.60 cm−1, 1011.39 and 1239.53 cm−1

were due to the stretching vibrations of C-O and C-C, respectively [20, 23-25]. The

vibrational bands corresponding to bonds such as –C-C and C-O might be from the

compounds such as flavonoids in Asparagus racemosus L leaf hence, it was presumed that

these biomolecules were responsible for capping and stabilization. The absorption band at

2980.75 cm−1 was characteristics of amide II (N-H) from proteins, suggesting interaction of

proteins with the biosynthesized Ag NPs. This result also proved that Ag NPs inhibited the

growth of three Mycobacterium species. FESEM (Fig.2c) images, recorded for two

magnifications, clearly present Ag NPs. It was difficult to trace out an exact dimension of this





Ag NPs. Squre type nanocrystalline were seen by a normal view whereas irregular void

channels were found under high resolution inspection.

Fig 2. a) XRD, b) FTIR, c) low-magnified SEM images of Ag NPs.

B. The antimycobacterial activity of test drugs

The antimycobacterial activity of four Mycobacterium species was performed against

biosynthesized Ag NPs. The four Mycobacterium strains resistant to antibiotics were special

and could be a new direction for detection. This effect was dose-dependent and effective

against selected Mycobacterium strains [26, 27]. To assess the antimicrobial activities of the

synthesised NPs, only those who exhibited antibacterial activities compared standard TB

drug such as Rifampicin, are shown in [Fig. 4 a-d]. The antimycobacterial activity zones,

inhibition around the disk of approximately 6 mm each with Ag NPs aqueous extract against

the four Mycobacterium strains, are presented in Table 1. In fig.3a, activity of Ag NPs against

the four Mycobacterium species shows not more than standard Rifampicin but only to showd

that biofunctional nanoparticles are inhibited the growth of mycobacterium species (fig.3a

and table.1)





Table: 1 Table shows antimycobacterial activity of silver nanoparticles of Asparagus

racemosus L.

Sr

No.

Compounds Zone of inhibition in (mm)

M.tuberculosis M. avium M. pheli M.spegmatis

1. Ag NPs 09.11± 0.14 10.14±0.15 06.15±0.11 05.10±0.12

2. Rifampicin 31.14±0.22 31.23±0.17 32.16±0.25 31.23±0.17

Fig 3. Zone of inhibition shown by a) AsRAgNPs against M.tuberculosis, b) AsRAgNPs against M.avium, AsRAgNPs against M.pheli, and c) AsRAgNPs against M.spegmatis

The inhibition results showed that triterpenoid diterpenoid from Asparagus

racemosus L., plants leave was responsible for the increase of antimycobacterial activity of

Asparagus racemosus L., leaf extract capped Ag NPs [30]. The antimycobacterial study

results for Ag NPs and the standard drug Rifampicin when tested at two concentrations

against four different Mycobacterium species [Fig.3a-d], it is clear that Asparagus racemosus

L. Leaf with AgNO3 shows novel antituberculosis drugs. Standard drug Rifampicin showed

moderate activity against four species at concentration of 20µg/ml, Ag NPs, at concentration





inhibit the growth of four Mycobacterium species. These results proved that Ag NPs kill the

growth of Mycobacterium species 99.9% as compared to Standard drug Rifampicin. This

indicated that the surface area of the NPs could act more effectively on the bacterial cells

through the membrane and its charge-related aspects [31]. The MIC values of the Ag NPs and

the standard against test microorganisms are given in Table 2. The tested microorganisms

were completely inhibited at concentrations of 20 µg /ml of Ag NPs and the standard. Ag

NPs concentration at 20 µg /ml, and the standard Rifampicin showed inhibition at 10 µg /ml.

AgNPs were found to be most effective against M.tuberculosis, M.pheli, M.avim, and

M.spegmatis thus it is concluded from the result showed in fig.3a-d of this study that the

AgNPs showed potential activity against four mycobacterium species.

C. Hemolytic assay

The in-vitro of hemolytic assay for toxicity study towards host cells [14].Varying

concentrations like 1~10 µg/ml. Stock 1mg/ml for, Ag NPs dissolve distilled water 1, 2, 3, 4,

5, µg/ml concentration Ag NPs tested showed no significant toxicity to human erythrocytes at

low concentrations, The concentrations 6, 7, 8, 9, 10 µg/ml were maximum growth inhibitory

concentration (Fig-4 table). Whereas they had profound effects of 99.9% kill the

Mycobacterium species Ag NPs as well as Standered drug Rifampicin (Fig-4). Only 0–12%

hemolysis was observed at 1-5 µg/ml the tested concentrations.





Fig 4. Cytotoxicity study by hemolytic assay

Table 2. Cytotoxicity study of AgNPs by hemolytic assay

Fig3. Zone of inhibition shown by a) Ag NPs against M.tuberculosis, b) Ag NPs against M.avim c) Ag NPs against M.pheli d) Ag NPs against M. spegmatis and Standered drug Rifampicin against Mycobacterium species. Table3. Minimum inhibitory concentration (MIC) of Ag NPs and standard tested against four Mycobacterium species (µg / ml).





4. Conclusions

In present study we focus only the bio-functionalized synthesized AgNPs from AgNO3 and

Asparagus racemosus L., plant extract in the presence of sunlight are kill the four

Mycobacterium species. These synthesized Ag NPs are significant antimycobacterial agent.

Till date, there have been no report on the as antimycobacterial agent and cytotoxic study of

Ag NPs from Asparagus racemosus L. Four Mycobacterium species like M.pheli, M.avim,

and M.tuberculosis and M. spegmatis against Ag NPs cause the 99.9% cell death. A direct

connection between green approach Ag NPs stability and antimycobacterial effects were also

demonstrated. The present study reveals that usual biogreenery and biocompatible properties,

bio-aspects of green synthesized Ag NPs are also a ecofriendly, easily synthesized, less toxic,

target oriented. Potential TB agent.

Acknowledgement

The authors are thankful to the school of life Sciences Swami Ramanand Teerth Marathwada

University Nanded for providing laboratory facilities

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