Claims:We Claim:-

1. A silver-platinum bimetallic nanohybrids prepared by a process comprising steps of:

a. preparing Dioscorea bulbifera tuber extract by putting 5 gm of tuber powder into a 300 ml Erlenmeyer flask with 100 ml of sterile distilled water;

b. boiling the mixture for 5 minutes, then decanting it;

c. adding 5 ml of Dioscorea bulbifera tuber extract in 95 ml of an aqueous solution containing 10-3 M of both H2PtCl6.6H2O and AgNO3;

d. incubating the mixture of step c at 100°C for 5 h for the synthesis of silver-platinum bimetallic nanohybrids (Ag-PtNHs).

2. The silver-platinum bimetallic nanohybrids prepared by the process as claimed in claim1, wherein the inhibitory activity of Ag-PtNHs is more against all test pathogens in comparison to individual silver nanoparticles (AgNPs) or platinum nanoparticles (PtNPs).

3. The silver-platinum bimetallic nanohybrids prepared by the process as claimed in claim1, wherein the Ag-PtNHs having nano-cluster shape are mono-dispersed with an average diameter in range of 40-80 nm, preferably 55-65 nm and more preferably 59 nm.

4. The silver-platinum bimetallic nanohybrids prepared by the process as claimed in claim 2, wherein the test pathogens are Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus.

Dated this 26th September, 2020
, Description:

FORM 2
THE PATENTS ACT, 1970
(39 OF 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10; rule 13)

1. Title of the invention – A SILVER-PLATINUM BIMETALLIC NANOHYBRIDS USING DIOSCOREA BULBIFERA

2. APPLICANT(S)
RK University Indian RK University, Bhavnagar Highway, Kasturbadham Rajkot - 360020, Gujarat, India
3. PREAMBLE TO THE DESCRIPTION:
The following specification particularly describes the invention and the manner in which it is to be performed.

The biological material Dioscorea bulbifera used in the present invention is from the India. In particular, the source of biological material is Western Ghats of Jawahar region in Maharashtra, India.

FIELD OF THE INVENTION:

The present invention relates to a silver-platinum bimetallic nanohybrids using Dioscorea bulbifera. Particularly it relates to a synthesis of silver nano-particles and platinum nano-particles using Dioscorea bulbifera tuber extract for antimicrobial activity.

BACKGROUND OF THE INVENTION:

Recently, nano-biotechnology has got wide attention due to multiple applications in the fields of electronics, catalysis, textiles, food industries and therapeutics. Among various nanoparticles, silver nanoparticles (AgNPs) are used in bio-sensing, biomedical imaging and drug delivery. Further, AgNPs are also used for designing antimicrobial surfaces, cosmetics, paints and plastics. Because of the bactericidal and fungicidal properties, AgNPs are also used for fabrication of wound dressings. AgNPs inhibit HIV-1, Tacaribe virus, Hepatitis B virus, Recombinant respiratory syncytial virus, Monkey pox virus, Murine norovirus-1, and Influenza virus. Moreover, AgNPs are also reported for their anticancer properties against hepatocellular carcinoma, skin and oral carcinoma, lung cancer.

Similarly, platinum nanoparticles (PtNPs) are also reported for their excellent catalytic activity in fuel cell. Further, PtNPs are potent anticancer agents against ovarian, lung, breast and pancreatic cancer cell. Platinum based drugs like cisplatin, carboplatin, oxyplatin are used for treatment of cancer. PtNPs are antimicrobial agents with a strong ability of discouraging the growth of unwanted and harmful bacteria.

Various physical and chemical methods for synthesis of nanoparticles, like chemical reduction, template method, electrochemical or ultrasonic-assisted reduction, photo induced or photo catalytic reduction, microwave-assisted synthesis, irradiation reduction, micro emulsion and biochemical reduction involves toxic chemicals and hazardous conditions.

Drawback of such methods significantly compromise the biocompatibility of the resulting nanoparticles for the biomedical applications. Various prior arts discloses the synthesis of the nano particles.

US8057682B2 discloses a method of making and using and compositions of metal nanoparticles formed by green chemistry synthetic techniques. For example, the present invention relates to metal nanoparticles formed with solutions of plant extracts and use of these metal nanoparticles in removing contaminants from soil and groundwater and other contaminated sites. In some embodiments, the invention comprises methods of making and using compositions of metal nanoparticles formed using green chemistry techniques.

US20110110723A1 discloses a methods of making and using and compositions of metal nanoparticles formed by green chemistry synthetic techniques. For example, the present invention relates to metal nanoparticles formed with solutions of fruit extracts and use of these metal nanoparticles in removing contaminants from soil and groundwater and other contaminated sites.

Ghosh et al. discloses development of an environmentally benign process for the synthesis of silver nano materials as an important aspect of current nanotechnology research. Among the 600 species of the genus Dioscorea, Dioscorea bulbifera has profound therapeutic applications due to its unique phytochemistry. In this research , we report on the rapid synthesis of silver nanoparticles by reduction of aqueous Ag+ ions using D. bulbifera tuber extract. In this prior art, there is discloser of synthesis using D. bulbifera for the silver nanoparticles only. Also, the nanoparticles synthesized were poly-dispersed i.e. had different size and shape.

Ruiz et al. discloses novel silver-platinum nanoparticles for anticancer and antimicrobial applications and synthesized novel dendritic assembly of silver (Ag)-platinum (Pt) nanoparticles. It also discloses that the TEM analysis of silver-platinum (AgPt) nanoparticles showed dendrimer shape nanoparticles with a mean size of 42 ± 11nm. Elemental composition was analysed by EDX, confirming the presence of both Ag and Pt metals. The synthesized nanoparticles significantly inhibited the growth of medically important pathogenic, Gram-positive Staphylococcus aureus, Gram-negative Pseudomonas aeruginosa and Gram-negative multi-drug resistant Escherichia coli.

Ahmad et al. discloses green synthesis of silver nanoparticles. It discloses that the biosynthesis of AgNPs can be accomplished by physical, chemical, and green synthesis; however, synthesis via biological precursors has shown remarkable outcomes. In available reported data, these entities are used as reducing agents where the synthesized NPs are characterized by ultraviolet-visible and Fourier-transform infrared spectra and X-ray diffraction, scanning electron microscopy, and transmission electron microscopy.

The problems associated with available synthesis process of nanoparticles using microbes require tedious culturing process, optimization, aseptic condition, and downstream processing. Hereby, medicinal plants with numerous phytochemical diversity like terpenes, polyphenols, flavonoids, alkaloids, coumarin, and saponins have served as attractive materials for both reduction of metal ions to their corresponding nanoparticles and their stabilization. Several plants like Gnidia glauca, Plumbago zeylanica, Platanus orientalis, Litchi chinensis, Dioscorea oppositifolia, Barleria prionitis and Gloriosa superba are reported to synthesize metal nanoparticles with exotic shapes and sizes with significant biomedical applications in prior arts.

But microbial interaction with the biogenic nano scale metals are noteworthy as significant bactericidal efficacy is exhibited by metal nanoparticles in compared to their bulk counterparts. Further, synergistic antimicrobial action with multi-metal complexes can be of utmost significance as they might induce higher oxidative stress thereby killing the microbes. Also, the Dioscorea bulbifera bulbils are used in the treatment of piles, dysentery, syphilis, ulcers, cough, leprosy, diabetes, asthma, and cancer.

So, it is desperately needed to develop green and environmentally benign route which will help to synthesize nanoparticles with broad spectrum therapeutic potential with the help of Dioscorea bulbifera. The invention of the present invention is as described herein.

OBJECT OF THE INVENTION:

The principle object of the present invention is to provide a silver-platinum bimetallic nanohybrids using Dioscorea bulbifera.

Another object of the present invention is to provide a fabrication of novel bactericidal agents which can effectively combat the bacterial infections.

Another object of the present invention is to provide a nanohybrids having dual property of silver nanoparticles and platinum nano-particles to exhibits synergistic effect with antibiotics.

Another object of the present invention is to provide a nano medicine using Ag-PtNHs for treating bacterial infections.

Another object of the present invention is to provide a process that does not use any harmful toxic reducing agent and toxic stabilizing agent.

Yet another object of the present invention is to provide a biogenic silver-platinum bimetallic nanohybrids which is beneficial to design antimicrobial nano-formulation.

Yet another aspect of the present invention is to provide a process which is cost effective, more stable and based on one pot synthesis.

SUMMARY OF THE INVENTION:

The present invention is all about a silver-platinum bimetallic nanohybrids using Dioscorea bulbifera.

The main aspect of the present invention is to provide a synthesis of silver nanoparticles and platinum nano particles using the Dioscorea bulbifera tuber extract having antimicrobial activity.

The main aspect of the present invention is to provide a silver-platinum bimetallic nanohybrids prepared by a process comprising steps of:

preparing Dioscorea bulbifera tuber extract by putting 5 gm of tuber powder into a 300 ml Erlenmeyer flask with 100 ml of sterile distilled water;

boiling the mixture for 5 minutes, then decanting it;

adding 5 ml of Dioscorea bulbifera tuber extract in 95 ml of an aqueous solution containing 10-3 M of both H2PtCl6.6H2O and AgNO3;

incubating the mixture of step c at 100°C for 5 h for the synthesis of silver-platinum bimetallic nanohybrids (Ag-PtNHs).

Another aspect of the present invention is to provide the silver-platinum bimetallic nanohybrids having the inhibitory activity of Ag-PtNHs which is more against all test pathogens in comparison to individual silver nanoparticles (AgNPs) or platinum nanoparticles (PtNPs).

Yet another aspect of the present invention is to provide the fabrication of novel bactericidal agents which can effectively combat the biofilm associated bacterial infections.

BRIEF DESCRIPTION OF THE DRAWINGS:

The above and other objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with the accompanying drawings, in which:

Fig.1 is a result of UV-visible spectra for nanoparticles synthesized by DBTE with insets representing the visible colour change on complete synthesis after 5 h. (a) AgNPs; (b) PtNPs; (c) Ag-PtNHs.

Fig.2 is a result of Transmission electron micrographs of Ag-PtNHs synthesized by DBTE.(a) Images showing nano clusters with inset scale bar showing particle size distribution; (b) Discrete Ag-PtNHs with inset scale bar showing 200 nm; (c) Magnified images of with inset scale bar of 100 nm; (d) Granular nanoparticles assembling to form larger nano clusters.

Fig.3 is a result of representative spot Energy dispersive spectra (EDS) profile confirming the presence of Ag and Pt in the Ag-PtNHs synthesized by Dioscorea bulbifera tuber extract.

Fig. 4 is a result of Fourier transform infrared absorption spectra of dried Dioscorea bulbifera tuber extract (DBTE) after complete bio reduction of (a) AgNPs; (b) PtNPs; (c) Ag-PtNHs and (d) before bio reduction.

Fig.5 is a result of Antimicrobial activity of biogenic nanoparticles synthesized by Dioscorea bulbifera tuber extract.

DETAILED DESCRIPTION OF THE INVENTION:

Detailed embodiments of the present invention are disclosed herein, however, it is to be understood that the disclosed embodiments are merely exemplary of the invention, which may be embodied in various forms. Therefore, specific functional and structural details disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the present invention in virtually any appropriately detailed structure.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs.

The present invention overcomes the aforesaid drawbacks of the above and other objects, features, and advantages of the present invention will now be described in greater detail. Also, the following description includes various specific details and is to be regarded as merely exemplary. Accordingly, those of ordinary skill in the art will recognize that: without departing from the scope and spirit of the present disclosure and its various embodiments there may be any number of changes and modifications described herein.

"Optional" or "optionally' means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event occurs and instances where it does not.

It must also be noted that as used herein and in the appended claims, the singular forms "a", "an," and "the" include plural references unless the context clearly dictates otherwise. Although any systems and methods similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred, systems and methods are now described.

The main embodiment of the present invention is to provide a process of synthesis of silver nanoparticles and platinum nanoparticles using the Dioscorea bulbifera tuber extract having antimicrobial activity.

As per another main embodiment of the present invention, a silver-platinum bimetallic nanohybrids prepared by a process comprising steps of:

preparing Dioscorea bulbifera tuber extract by putting 5 gm of tuber powder into a 300 ml Erlenmeyer flask with 100 ml of sterile distilled water;

boiling the mixture for 5 minutes, then decanting it;

adding 5 ml of Dioscorea bulbifera tuber extract in 95 ml of an aqueous solution containing 10-3 M of both H2PtCl6.6H2O and AgNO3;

d. incubating the mixture of step c at 100°C for 5 h for the synthesis of silver-platinum bimetallic nanohybrids (Ag-PtNHs).

Dioscorea bulbifera

Dioscorea bulbifera is unique amongst 600 species of the genus Dioscorea due to its species-specific phytochemistry, supporting its widespread application in therapeutics. Dioscorea bulbifera tuber extract is rich in polyphenolic compounds, especially flavonoids and catechin, which have contributed to its pronounced antioxidant and anti-diabetic properties.

Dioscorea bulbifera were collected from the Western Ghats of Jawahar region in Maharashtra, India.

As per detailed embodiment of the present invention, the tuber extract was prepared by putting 5 g of thoroughly washed and finely ground tuber powder into a 300 ml Erlenmeyer flask with 100 ml of sterile distilled water, boiling the mixture for 5 minutes, then decanting it. The extract obtain was filtered through filter paper. The filtrate extract was collected and store at 4°C for further use.

As per the detailed embodiment, the properties of synthesized Ag-PtNHs compare with the synthesized silver nanohybrids (AgNHs) and platinum nanohybrids (PtNHs) to prove the highest antimicrobial activity among three of them.

As per detailed embodiment, addition of 5 ml of Dioscorea bulbifera tuber extract to 95 ml of 10-3 M aqueous AgNO3 solution to synthesis of silver nanoparticles. Reduction of the Ag+ ions was monitored by measuring the ultraviolet-visible spectrum of the solution at regular intervals on a spectrophotometer operating at a resolution of 1 nm. The effects of temperature on the rate of synthesis and size of the prepared silver nanoparticles were studied by carrying out the reaction in a water bath at 4°C–50°C with reflux. The concentration of AgNO3 solution was also varied from 0.3 to 5 mm.

As per detail embodiment, platinum nanoparticles are synthesized by the reduction of PtCl_6^(2-) ions on addition of 5ml of Dioscorea bulbifera tuber extract to 95ml of 10-3 M aqueous solution of H2PtCl6.6H2O which was incubated at 100°C for 5h. Synthesis of the material was confirmed by recording the UV-visible spectrum of the solution after 5h on a spectrophotometer operating at a resolution of 1 nm.

As per one embodiment, the silver-platinum bimetallic nanohybrids having inhibitory activity of Ag-PtNHs which was more against all test pathogens in comparison to individual silver nanoparticles (AgNPs) or platinum nanoparticles (PtNPs).

As per one embodiment, the silver-platinum bimetallic nanohybrids having nanocluster shape are mono-dispersed with an average diameter in range of 40-80 nm, preferably 55-65 nm and more preferably 59 nm.

As per one embodiment, the test pathogens are Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus.

As per another embodiment, the antibiotics used were streptomycin, rifampicin, chloramphenicol, novobiocin and ampicillin

The present invention was experimented and illustrated more in details in the following example. The example describes and demonstrates embodiments within the scope of the present invention. This example was given solely for the purpose of illustration and is not to be construed as limitations of the present invention, as many variations thereof are possible without departing from spirit and scope.

Example 1: Preparation of tuber extract
The tuber extract was prepared of Dioscorea bulbifera.

Procedure:
washing the tuber of Dioscorea bulbifera, cutting into thin slices followed by drying and fine grinding.
putting 5 gm of tuber powder into a 300 mL Erlenmeyer flask with 100 mL of sterile distilled water.
boiling the mixture for 5 minutes, then decanting it.
filtering the extract mixture through filter paper and store at 4°C for further use.

Example 2: synthesis of Ag-PtNHs
The synthesis of silver nanoparticles and platinum nano particles using the Dioscorea bulbifera tuber extract.

Procedure:
adding 5 ml of Dioscorea bulbifera tuber extract in 95 ml of an aqueous solution containing 10-3 M of both H2PtCl6.6H2O and AgNO3;
incubating the mixture at 100°C for 5 h for the synthesis of silver-platinum bimetallic nanohybrids (Ag-PtNHs).

Example 3: synthesis of AgNPs
The synthesis of silver nano particles using the Dioscorea bulbifera tuber extract.

Procedure:
adding 5 ml of Dioscorea bulbifera tuber extract to 95 ml of 10-3 M aqueous AgNO3 solution to synthesis of silver nanoparticles.
incubating the mixture at 100°C for 5 h for the synthesis of silver nanoparticles (AgNPs)
recording the UV-visible spectrum of the solution after 5h on a spectrophotometer operating at a resolution of 1 nm to confirmed the synthesis of the material

Example 4: synthesis of PtNPs
The synthesis of platinum nanoparticles using the Dioscorea bulbifera tuber extract.

Procedure:
adding 5ml of Dioscorea bulbifera tuber extract to 95 ml of 10-3 M aqueous solution of H2PtCl6.6H2O and incubating at 100°C for 5h for the reduction of PtCl_6^(2-) ions.

recording the UV-visible spectrum of the solution after 5h on a spectrophotometer operating at a resolution of 1 nm to confirmed the synthesis of the material

Example 5: Characterization of Ag-PtNHs, AgNPs and PtNPs of example 2, example 3 and example 4.
Preliminary confirmations of biosynthesized AgNPs, PtNPs and AgPtNPs were carried out through visual observation of colour change after the completion of the synthesis. The bio reduced nanoparticles were further characterized by using several standard techniques, such as FTIR, UV-vis spectroscopy, transmission electron microscopy (TEM), energy dispersive spectra (EDS) and dynamic light scattering (DLS).

Example 5.1 UV-vis spectroscopy

The synthesis of AgNPs, PtNPs and Ag-PtNHs were completed in 5 h. The appearance of brown colour confirmed the synthesis of the nanoparticles as shown in the inset of Figure 1. AgNPs with light brown colour were well dispersed in the colloidal solution while PtNPs were settled down as a loose mass with blackish brown colour. Ag-PtNHs on the other hand formed relatively well dispersed dark colloidal suspension. Figure 1 shows that AgNPs exhibited a prominent peak at 420 nm in the UV-visible spectra while PtNPs and Ag-PtNHs showed a formless peak.

Example 5.2 Transmission electron microscopy (TEM)

Figure 2 depicts the TEM micrographs of Ag-PtNHs which indicated that very small nanoparticles of ~ 2 nm assembled to form nano clusters with particle size distribution from 20 to 80 nm, average being 59 nm. The Ag-PtNHs were stable and discrete and formed spherical shaped nano clusters. Also, the nanoparticles were seen to be stabilized by the biological components of the plant extract.

Example 5.3 Energy dispersive spectra (EDS)

Figure 3 shows the EDS spectra confirming the presence of both elemental Ag and Pt in the Ag-PtNHs. Zeta potential measurements seen in the inset of Figure 3 further rationalize the observation where AgNPs showed more negative value (-14.46 mV) followed by Ag-PtNHs (-11.39 mV) and PtNPs (-1.09 mV).

Example 5.4 FTIR Analysis

FTIR spectroscopy was employed to understand the underlying mechanism of the synthesis of the nanoparticles using Dioscorea bulbifera tuber extract. In this method, Dioscorea bulbifera tuber extract after and before synthesis of Ag-PtNHs was subjected to Fourier-transform infrared spectroscopy measurement using the potassium bromide (KBr) pellet technique in diffuse reflection mode at a resolution of 4 cm-1. An infrared source of wavelength lying within 500–4000 cm-1 was used.

Fourier-transform infrared spectroscopic (FTIR) analyses of Dioscorea bulbifera tuber extract before and after synthesis of AgNPs, PtNPs, and Ag-PtNHs were shown in Figure 4.

Some typical peaks observed at 3400 cm-1, 2100 cm-1, 1636 cm-1, and 1215 cm-1 were present in all spectra. Those peaks are originated from the different vibration bands (i.e., ?avonoids, terpenoids, phenanthrenes, amino acids, proteins, and glycosides) present within the Dioscorea bulbifera tuber extract. The strong peak at ~3400 cm-1 is a characteristic of the hydroxyl group in polyphenolic compounds present in the plant extract. The other bands at 2100 cm-1, 1636 cm-1, and 1212 cm-1 are assigned to C=C stretching of the alkyne, C=C groups or conjugated C–C with a benzene ring phenolic groups There are some additional peaks which appeared after synthesis that include 1738 cm-1 and 1368 cm-1, which are originated from C=O carbonyl stretch from carboxylic acid and C–N stretching vibration of aromatic, respectively. It was also observed that the sharpness of the peak representing the O–H bond was reduced in the FTIR spectrum of Dioscorea bulbifera tuber extract after synthesis of the nanoparticles, which confirms the bio reduction efficacies of Dioscorea bulbifera tuber extract.

Therefore, the FTIR results demonstrated that the Dioscorea bulbifera tuber extract could perform dual functions of reduction and stabilization of AgNPs, PtNPs and Ag-PtNHs. The overall observation confirms that the presence of complex compounds in Dioscorea bulbifera tuber extract can bind to nanoparticles either through free amine groups via electrostatic attraction of negatively charged carboxylate groups and therefore stabilize the phytogenic nanoparticles.

Example 5.5 The antimicrobial activity of the Ag-PtNHs, AgNPs and PtNPs
The effects of AgNPs, PtNPs and Ag-PtNHs were tested against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus aureus, on Mueller Hinton Agar (MHA) plates using well diffusion assay. Overnight grown cultures of the test organism (OD600 = 0.05) were spread plated on MHA plates and wells were made on the surface with the help of sterile cork borer of diameter 5 mm. 30 µL of nanoparticles suspension (100µg/mL) was added in the wells followed by incubation at 37°C for 18 h, the zone of inhibition were measured.
The biogenic nanoparticles showed variable degrees of antimicrobial activity against the four test pathogens as represented in Figure 5 in terms of zone of inhibition. AgNPs showed the highest activity against P. aeruginosa while identical inhibition was observed against E. coli, B. subtilis and S. aureus. PtNPs also showed similar level of inhibition against all bacteria except B. subtilis where lower inhibition was observed. Interestingly, inhibitory activity of Ag-PtNHs was more against all test pathogens in compared to individual AgNPs or PtNPs. Ag-PtNPs showed the highest inhibitory zone of 10 mm against P. aeruginosa followed by E. coli (8 mm), S. aureus (8 mm) and B. subtilis (7 mm).

Example 5.5 The antimicrobial synergy of the Ag-PtNHs, AgNPs and PtNPs
Agar well diffusion technique was used for evaluating the antimicrobial synergy of the nanoparticles in presence of antibiotics. The antibiotics used were streptomycin, rifampicin, chloramphenicol, novobiocin and ampicillin. The test pathogens were inoculated in sterile Mueller Hinton Broth (MHB) and incubated in shaker for overnight at 37°C. 100 µl of the overnight grown culture were spread uniformly on MHA plates. Wells were made on the surface with the help of sterile cork borer of diameter 6 mm and 30µL of the nanoparticles, mixture of nanoparticles and antibiotic, and only antibiotics were added. The plates were then incubated at 37ºC for 18 h and observed for zone of inhibition around the well. The diameters of zone of inhibition were measured and the degree of antimicrobial synergy was evaluated.

In antimicrobial synergy the activity of antibiotics individually and in combination of the nanoparticles were checked and fold increase in terms of the zone of inhibition was evaluated. Table 1 shows the synergistic antimicrobial activity of various antibiotics in combination with AgNPs. The activity of streptomycin was found to get enhanced 8 folds against E. coli when supplemented with AgNPs. Rifampicin exhibited the highest antimicrobial synergy in combination with AgNPs against S. aureus (15 folds) followed by intermediate and identical activity against both E. coli (4.64 folds) and B. subtilis (4.90 folds). Chloramphenicol also showed most superior antimicrobial synergy in presence of AgNPs against B. subtilis (11.76 folds) while intermediate synergy in both E. coli and S. aureus. Novobiocin exhibited high synergistic inhibition of S. aureus (15 folds) followed by E. coli and B. subtilis. Interestingly, ampicillin exhibited selectivity towards Gram-positive bacteria during synergistic activity in combination with AgNPs. B. subtilis and S. aureus were inhibited up to 13.06 and 12.14 folds, respectively by combination of ampicillin and AgNPs.

Antibiotics E. coli P. aeruginosa B. subtilis S. aureus
A B C A B C A B C A B C
Streptomycin 7 21 8.00 8 14 2.06 8 14 2.06 9 15 1.78
Rifampicin 8 19 4.64 8 9 0.27 7 17 4.90 8 32 15.00
Chloramphenicol 8 22 6.56 7 9 0.65 7 25 11.76 7 20 7.16
Novobiocin 7 22 8.88 8 10 0.56 8 21 5.89 6 24 15.00
Ampicillin 8 14 2.06 7 8 0.31 8 30 13.06 8 29 12.14
Table 1. Zone of inhibition (mm) of different antibiotics against bacteria in absence and in presence of AgNPs (30 µg/well).

Notes: All experiments were done in triplicate, and standard deviations were negligible. Fold increases (C) for different antibiotics against four bacterial pathogens were calculated as (B2 - A2)/A2, where A &B are the inhibition zones in mm for antibiotic only and antibiotic in combination with AgNPs, respectively. In the absence of bacterial growth inhibition zones, the well diameters (6 mm) were used to calculate the fold increase (C).

Antimicrobial synergy of antibiotics in combination with PtNPs showed the selectivity and variability as observed in Table 2. Supplementation with PtNPs has significantly enhanced the inhibitory activity of streptomycin selectively against Gram-negative bacteria E. coli (10.76 folds) and P. aeruginosa (8 folds). In the presence of PtNPs antimicrobial activity of rifampicin was increased notably against Gram-positive bacteria particularly S. aureus (16.02 folds). Likewise, increment in the activity of chloramphenicol in presence of PtNPs was notable against Gram-positive pathogens, B. subtilis (12.8 folds) followed by S. aureus (8 folds). Highest antimicrobial synergy of novobiocin with PtNPs was seen against S. aureus (16.36 folds), followed by E. coli (10.76 folds) while intermediate and low synergy was evident against B. subtilis and P. aeruginosa, respectively. Ampicillin exhibited highest synergistic enhancement against B. subtilis up to 8.77 folds.

Table 2. Zone of inhibition (mm) of different antibiotics against bacteria in absence and in presence of PtNPs (30 µg/well).

Antibiotics E. coli P. aeruginosa B. subtilis S. aureus
A B C A B C A B C A B C
Streptomycin 7 24 10.76 8 24 8.00 8 11 0.89 9 13 1.09
Rifampicin 8 13 1.64 8 12 1.25 7 15 3.59 8 33 16.02
Chloramphenicol 8 19 4.64 7 11 1.47 7 26 12.80 7 21 8.00
Novobiocin 7 24 10.76 8 14 2.06 8 24 8.00 6 25 16.36
Ampicillin 8 18 4.06 7 12 1.94 8 25 8.77 8 15 2.52

Notes: All experiments were done in triplicate, and standard deviations were negligible. Fold increases (C) for different antibiotics against four bacterial pathogens were calculated as (B2 - A2)/A2, where A & B are the inhibition zones in mm for antibiotic only and antibiotic in combination with PtNPs, respectively. In the absence of bacterial growth inhibition zones, the well diameters (6 mm) were used to calculate the fold increase (C).

As observed in Table 3, antimicrobial activity of rifampicin and novobiocin increased significantly up to 15 and 13.69 folds, respectively in the presence of a combination of Ag-PtNHs against S. aureus. Similarly, activity of chloramphenicol and ampicillin against B. subtilis was enhanced up to 11.76 and 14.02 folds, respectively when combined with Ag-PtNHs. Thus, antimicrobial synergy of antibiotics in presence of Ag-PtNHs was found to be more selective against Gram-positive bacteria.

Table 3. Zone of inhibition (mm) of different antibiotics against bacteria in absence and in presence of AgPtNHs (30 µg/well)
Antibiotics E. coli P. aeruginosa B. subtilis S. aureus
A B C A B C A B C A B C
Streptomycin 7 20 7.16 8 12 1.25 8 11 0.89 9 15 1.78
Rifampicin 8 15 2.52 8 11 0.89 7 14 3.00 8 32 15.00
Chloramphenicol 8 20 5.25 7 10 1.04 7 25 11.76 7 18 5.61
Novobiocin 7 23 9.80 8 8 0.00 8 22 6.56 6 23 13.69
Ampicillin 8 13 1.64 7 13 2.45 8 31 14.02 8 15 2.52

Notes: All experiments were done in triplicate, and standard deviations were negligible. Fold increases (C) for different antibiotics against four bacterial pathogens were calculated as (B2 - A2)/A2, where A and B are the inhibition zones in mm for antibiotic only and antibiotic in combination with Ag-PtNHs, respectively. In the absence of bacterial growth inhibition zones, the well diameters (6 mm) were used to calculate the fold increase (C).

Conclusions
In summary, Ag-PtNHs were synthesized by using aqueous extract of Dioscorea bulbifera tuber to evaluate the antimicrobial synergy in combination of antibiotics against bacterial pathogens. The nano cluster shaped Ag-PtNHs were mono-dispersed with an average diameter in range of 40-80 nm, preferably 55-65 nm and more preferably 59 nm. Phytochemicals present in Dioscorea bulbifera tuber extract facilitated the synthesis of Ag-PtNHs and also their stabilization. Four test pathogens, E. coli, P. aeruginosa, B. subtilis and S. aureus were inhibited by Ag-PtNHs while combination of Ag-PtNHs with antibiotics like rifampicin and novobiocin showed high antimicrobial synergy against S. aureus among all test pathogens. The novel Ag-PtNHs can prove to be promising nanomedicine for treating bacterial infections.