Biogenic synthesis of Au, Ag and Au–Ag alloy nanoparticles using Cannabis sativa leaf extract

Abstract

Biogenic synthesis of gold (Au), silver (Ag) and bimetallic alloy Au–Ag nanoparticles (NPs) from aqueous solutions using Cannabis sativa as reducing and stabilising agent has been presented in this report. Formation of NPs was monitored using UV–visible spectroscopy. Morphology of the synthesised metallic and bimetallic NPs was investigated using X‐ray diffraction and scanning electron microscopy. Elemental composition and the surface chemical state of NPs were confirmed by energy dispersive X‐ray spectroscopy analysis. Fourier transform‐infrared spectroscopy was utilised to identify the possible biomolecules responsible for the reduction and stabilisation of the NPs. Biological applicability of biosynthesised NPs was tested against five bacterial strains namely Klebsiella pneumonia, Bacillus subtilis (B. subtilis), Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa (P. aeruginosa) and Leishmania major promastigotes. The results showed considerable antibacterial and anti‐leishmanial activity. The Au–Ag bimetallic NPs showed improved antibacterial activity against B. subtilis and P. aeruginosa as compared to Au and Ag alone, while maximum anti‐leishmanial activity was observed at 250 μg ml⁻¹ NP concentration. These results suggest that biosynthesised NPs can be used as potent antibiotic and anti‐leishmanial agents.

1 Introduction

In comparison with their bulk similitudes, metallic nanoparticles (NPs) study is a completely new dimension of research. This is because of the unique and tunable physical, chemical and biological properties of metal NPs. Currently, a plethora of techniques like chemical and physical vapour deposition, microwave aided synthesis, sol–gel synthesis, ultra‐sonication; electrochemical synthesis, precipitation method, and biological synthesis are being practiced for the synthesis of metallic NPs [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11]. Of all these techniques, the most promising method is to utilise biological beings and the extracts prepared from plants as reducing agents for the biological synthesis of metallic NPs. The biological method is an important alternative due to its environmentally friendly nature as compared to its counterpart techniques involving the use of noxious and hazardous chemicals [12, 13, 14].

Biosynthesised metallic NPs have been extensively utilised in medical research due to their non‐toxic attribute [15, 16]. Since the very early days of mankind, gold (Au) and silver (Ag) were found to have soaring ornamental value and have been known for biomedical applications. Recent research indicates that these biosynthesised metallic NPs can have a promising impact in diverse areas including antibacterial activities [17], wound dressing [18] anticancer drug delivery [19], medical imaging [20] and catalysis sensors [21] and so on.

Cannabis sativa L. (marijuana) is an important medicinal plant distributed worldwide. It is a dicotyledonous plant belonging to Cannabaceae family [22, 23]. The plant is annual dioecious which, depending upon its genetic and environmental factors, can grow from 1 to 5 m in height [24]. Cannabis has a highly complex and diverse phytochemistry [25]. It is regarded as a factory of secondary metabolites. These secondary metabolites include invaluable compounds including alkaloids, phenolics, lignins and so on [26]. Another important class of compounds found abundantly in Cannabis are terpenes. The characteristic odour/smell of the crop is because of the presence of terpenes [27].

As much as 421 chemical compounds have been isolated from Cannabis [28]. The most studied out of these compounds from cannabis are cannabinoids. Terpenophenolic compounds that have 22 carbons (21 carbons in the case of neutral form) are termed as cannabinoids. Owing to its rich phytochemistry, Cannabis is widely utilised as anti‐spasm, anti‐vomiting, anti‐emetic and for stimulating appetite [29].

A variety of methodologies [30, 31, 32, 33, 34] have been reported to synthesise alloy NPs. In order to control the composition and size of the NPs, these methods rely on stabilisers and capping agents. One of the examples includes the use of poly(vinyl pyrrolidone) and oleylamine as stabilising agents for synthesising Ag and Au alloy NPs [35, 36]. The major challenge in synthesising bimetallic alloy NPs is coming up with eco‐friendly and reliable protocols. A possible solution to this problem is the use of biological materials for synthesising a variety of highly complex inorganic materials [37, 38, 39, 40, 41, 42, 43, 44, 45]. It is unfortunate that the centre of contemplation in most of these studies is biosynthesising NPs that are monometallic in nature. The only few reports on the biosynthesis of bimetallic NPs were previously reported on Au/Ag [46] and Ti/Ni [47] systems, respectively.

A plethora of studies have been carried out on exploiting ‘natural factories’ that most commonly includes microbes and plants for NPs biosynthesis [48, 49, 50]. Scaling up the methods utilising plants for bulk synthesis of NPs is more convenient in comparison to other contemporary methods [51].

This study focuses on an eco‐friendly and rapid biosynthesis of Ag and Au NPs using the Cannabis sativa leaf extract and the characterisation of the synthesised NPs using UV–visible (UV–vis) spectroscopy, X‐ray diffraction (XRD) analysis, Fourier transform‐infrared (FTIR) spectroscopy and scanning electron microscopy (SEM). Here, we report for the first time an environmentally benign solution method to synthesise Au–Ag alloy NPs using a Cannabis sativa leaf extract as both reductant and stabiliser. The secondary metabolites, ubiquitously found in plants especially the terpenoid rich essential oils, have a significant role in AgNPs synthesis. Every organism contains terpenoids, the most abundantly found families of biological products. It has ample of consideration for synthesising AgNPs through green route [52]. Full scheme of the mechanism of synthesis, characterisation and biological applications of metallic and bimetallic NPs is illustrated in Fig. 1.

Fig. 1.

Full schematic diagram of biogenic metallic and bimetallic NPs synthesis, characterisation and potential biological applications

2 Materials and methods

All chemicals used in this investigation were of reagent grade and used as received.

2.1 Leaf extract preparation

Dried Cannabis sativa leaves (Quaid‐i‐Azam University Islamabad, Pakistan) were first milled and then screened using a 20 mesh sieve for the subsequent experiments. To obtain the leaf extract of Cannabis sativa, 10 g of the screened Cannabis sativa leaf powder was dispersed in 100 ml distilled water. The solution was then placed in an incubator overnight at 30°C. The mixture was filtered to remove the residual insoluble biomass, and the resulting filtrate was then used for the subsequent NP synthesis.

2.2 Synthesis of gold NPs (GNPs)

5 ml of the leaf extract was added to 50 ml of HAuCl₄ ·3H₂ O (10⁻³ M) aqueous solution. After 15 min, the colour of the mixed solution (G1) turned to vivid magenta indicating the formation of GNPs. Two more samples, namely, G2 and G3, were also prepared by varying the extract volume in 1:2 and 1:5.

2.3 Synthesis of silver NPs (SNPs)

5 ml of the leaf extract was added to 50 ml aqueous solution of AgNO₃ (10⁻³ M). After 20 min, the mixed solution (S1) turned to light brownish indicating the formation of SNPs. Two more samples, namely, S2 and S3, were also prepared by varying the extract volume in 1:2 and 1:5.

2.4 Synthesis of bimetallic NPs (BNPs)

Briefly, 10⁻³ M of metallic salt (HAuCl₄ ·3H₂ O) and 10⁻³ M of metallic salt (AgNO₃) were separately dissolved in 50 ml of distilled water, 5 ml of Cannabis sativa leaf extract was added into the 50 ml aqueous solution of gold(III) chloride trihydrate. The mixed dispersions were kept in a magnetic stirrer for 20 min. After observing the colour change, 50 ml aqueous solution of AgNO₃ was added to that solution followed by the addition of 5 ml leaf extract and stirring was continued for additional 10 min to complete the formation of Au–Ag BNPs. The synthesised Au–Ag BNPs were sonicated for 40 min to get fine dispersion of the NPs with a ‘fast‐clean’ ultra‐sonic cleaner at room temperature.

2.5 Characterisation of GNPs, SNPs and BNPs

2.5.1 UV–vis spectroscopy analysis

Formation and stability of metallic and bimetallic NPs were examined by recording UV–vis absorption spectra using Shimadzu UV‐1650 PC Spectrophotometer through a quartz cell with 10 mm optical path. The samples were filled in a quartz cuvette of 1 cm light path length, and the light absorption spectra were given in reference to distilled water.

2.5.2 FTIR spectroscopy

Functional groups bonded with the synthesised NPs were identified by FTIR spectroscope (Spectrum One, Perkin Elmer, Waltham, MA, USA) using KBr pellet at spectral range of 500–3500 cm⁻¹.

2.5.3 XRD analysis

XRD analysis was done to confirm the crystalline structure of NPs by using XRD instrument (Model‐D8 Advance, Germany) that have cathode ray that emits X‐rays in the direction of the sample. About 1 mg of powdered NPs was used for analysis. A Debye–Scherrer equation was used to calculate particle size of NPs

$$D=k\lambda /\beta \cos \theta$$

where k is the shape factor (0.94), λ is the X‐ray wavelength (1.5418 Å), β is the full width at half maximum (FWHM) in radians and θ is the Bragg's angle

2.5.4 Energy dispersive X‐ray (EDX) and SEM analysis

SEM (MIRA3 TESCAN model) operated at 10 kV was used for determination of the morphology and particle size of NPs. The small amount of the sample was dropped on a copper grid; coating was done by carbon and dried for 5 min under mercury lamp, samples were analysed and images were taken. For elemental analysis, EDX was used. Samples were carbon coated on the grid and dried then analysis was done by using an EDX detector that was attached to SEM.

2.6 Antibacterial activities

Antibacterial efficacy against five bacterial strains Klebsiella pneumonia (K. pneumonia), Bacillus subtilis (B. subtilis), Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and P. aeruginosa was analysed by using well diffusion method. 10 mg of NPs was dissolved in 1 ml of sterile distilled water. Petri plates containing media were swabbed with bacterial strains. About 15 μl of NPs, 0.02 M precursor salt as a positive control, Cefixime 10 mgml⁻¹ as standard and whole plant extract (WPE) as negative control were added into wells. The plates were kept in an incubator at 30°C for incubation for 24 h and zone of inhibition was measured in millimetre. Data were collected from three independent experiments for each strain.

2.7 Anti‐leishmanial activity

2.7.1 Parasite preparation

The RPMI 1640 medium containing 10% foetal bovine serum, 4.5 mgml⁻¹ glucose and 292 μg ml⁻¹ L‐glutamine (all supplied by Sigma) was used for culturing the parasites. After six days of culture, the parasite stationary phase of growth was obtained. The culture (incubated at 25°C) was utilised within two weeks of cultivation.

2.7.2 MTT assay for checking anti‐leishmanial efficacy of GNPs, SNPs and BNPs

The anti‐leishmanial activity of metallic and bimetallic NPs was evaluated in vitro by utilising MTT (3‐(4,5‐dimethylthiazol‐2yl)‐2,5‐diphenyltetrazolium bromide)‐based microassay as a marker of cell viability against the promastigote forms of Leishmania major (L. major).

An MTT (Sigma Chemical Co., St. Louis, Mo.) stock solution was prepared by dissolving MTT (1 mgml⁻¹) in phosphate‐buffered saline and stored it for two weeks in dark at 4°C prior to utilisation. Analysing anti‐leishmanial action of NPs, in 96‐well flat‐bottom plates, the culture (100 μl/well) was seeded that consist of 1 × 10⁵ cells ml⁻¹ promastigotes. 50 µl NPs was added and incubated for 24 h at 25 ± 1°C. The first well was kept as a blank, which consists of only 100 μl culture medium without any parasite, extracts, and drug. After the incubation period, to each well and plate, MTT (10 μl) was added and incubated at 25 ± 1°C for 3 h. DMSO (100 μl) was added to stop the enzymatic reaction and plates were again incubated at room temperature for an extra 30 min in vigorous shaking. Relative optical density was calculated using an ELISA reader at 540 nm wavelength.

For the reproducibility and concentration needed, the experiment was repeated twice, to kill the cells 50% and (IC₅₀) was calculated. The formazan absorbance, build up by the metabolically viable cells mitochondrial dehydrogenases activity is displayed to relate to the total number of viable cells.

3 Results and discussion

3.1 UV–vis spectroscopy analysis

Au and Ag NPs were successfully synthesised at room temperature. Colour change was the first indicator of synthesis of these NPs. In Ag NPs synthesis, yellow colour of Cannabis sativa leaf extract turned brownish and for AuNPs vibrant magenta colour appeared. However, in Au–Ag alloy NPs violet colour was appeared (Fig. 2). The UV–vis absorbance spectra of the Au and Ag mono‐metallic NPs and Au–Ag BNPs are shown in (Fig. 3). Au and Ag NPs are known to exhibit the surface plasmon resonance (SPR) band which depends on the type, size and shape of the NPs, and the surrounding environment [53, 54]. The absorption peak at 552 and 426 nm can be attributed to the SPR corresponding to the mono‐metallic Au and Ag NPs, respectively. The Au–Ag BNPs synthesised by successive reduction method at the Au³⁺ /Ag⁺² ratio 1:1 at a constant extract concentration exhibited SPR peak at 538 nm. Gopalakrishnan and Raghu [55] also reported this range of SPR. The synthesised monometallic and bimetallic NPs were used for further characterisation.

Fig. 2.

Colour change of metallic and bimetallic biosynthesised NPs

(A) Cannabis sativa leaves extract, (B) SNPs, (C) GNPs, (D) Comparison of colour of GNPs and BNPs

Fig. 3.

UV–vis spectra of biosynthesised GNPs, SNPs and BNPs

3.2 FTIR spectroscopic analysis

Figs. 4 a –c show the FTIR spectra of synthesised Au, Ag and Au–Ag BNPs, respectively. Comparing FTIR spectra of the biogenic NPs may help in identifying the possible biomolecules responsible for the reduction, capping and efficient stabilisation of Au–Ag BNPs. The results showed peaks at 3311.62, 2925.14, 2164.07, 2031.10, 1983.14, 1745.22, 1630.81, 1353.39, 1102.75, 1031.07 and 991.69 cm⁻¹ for AuNPs, 3376.14, 2933.26, 2494.28, 2159.28, 2032.54, 1973.3, 1743.7, 1565.72, 1348.36, 1104.85, 1045.39, 985.47, 927.73, 866.09, 823.76 and 703.90 cm⁻¹ for AgNPs, and 3300.44, 2927.65, 2157.44, 2029.00, 1974.96, 1741.96, 1623.74, 1353.38, 1035.88, 993.89 and 826.59 cm⁻¹ for Au–AgNPs.

Fig. 4.

FTIR spectra of biosynthesised NPs

(a) GNPs, (b) SNPs, (c) BNPs

Broad peaks at 3311.62, 3376.14 and 3300.44 cm⁻¹ are due to the O–H stretching in alcohols [56]. Peaks around 2925.14, 2933.26 and 2927.65 cm⁻¹ represent the C–H stretching in aromatic compounds [57]. Peaks at 1745.22, 1743.7 and 1741.96 cm⁻¹ show the C = O stretching vibrations in aldehydes, ketones and carboxylic acids, peaks at 1031.07, 1035.88 and 1045.39 cm⁻¹ can be attributed to the –C–O group while peaks at 866.09, 826.59 and 823.76 cm⁻¹ may be due to the –C–N stretching of amines [58]. The peak at 1630.81 cm⁻¹ may be due to the carbonyl stretching in amides and those at 1102.75 and 1104.85 cm⁻¹ are due to the C–O stretching vibration [59]. Peaks at 1353.39, 1353.38 and 1348.36 cm⁻¹ are due to the –C–O– like phenol groups [60]. This coating of several phytochemicals on the surface of NPs helps in the stabilisation of NPs and prevents their agglomeration. It can be conjectured in the light of FTIR results that proteins and other biomolecules with functional groups of aldehydic, carboxylates, ethers and alcoholic nature bind the metallic surface of NPs and stabilises them by preventing their agglomeration. The non‐toxicity and eco‐friendly nature of biogenic NPs can be attributed to the biomolecules responsible for reduction and stabilisation [61].

3.3 XRD pattern analysis

The crystalline nature of NPs was confirmed by XRD analysis. Figs. 5 a –c depict the XRD patterns of GNPs and SNPs and BNPs, respectively. The values of the diffraction angle (2θ), d ‐spacing, FWHM, and Miller indices for GNPs, SNPs and BNPs are compiled in Tables 1, 2, 3, respectively. For GNPs, the peaks at 2θ  = 37.9822°, 44.267°, 64.5725° and 77.58° were indexed to (1 1 1), (2 0 0), (220), and (3 1 1) sets of planes of the face centred cubic (fcc) structure (with reference to JCPDS File no. 04‐0784), respectively. The XRD patterns of AuNPs obtained were similar to the results reported earlier [62]. For SNPs, the peaks at 2θ  = 27.5962°, 32.067° and 46.1376° were indexed to (98), (101), (200) sets of lattice planes of the fcc structure (with reference to JCPDS File no. 04‐0783), respectively.

Fig. 5.

XRD pattern of NPs

(a) GNPs, (b) SNPs, (c) BNPs

Table 1.

Values of diffraction angle (2θ), d ‐spacing, FWHM, crystalline size and Miller indices for GNPs

Au	Pos. [°2Th.]	Miller indices	FWHM	d ‐spacing, Å	Crystalline size, nm
1	37.9822	111	0.3936	2.36904	38.94
2	44.267	200	0.6298	2.0462	24.85
3	64.5725	220	1.152	1.4421	15.45
4	77.58	311	0.4222	1.23061	44.03

Table 2.

Values of diffraction angle (2θ), d ‐spacing, FWHM, crystalline size and Miller indices for SNPs

Ag	Pos. [°2Th.]	Miller indices	FWHM	d ‐spacing, Å	Crystalline size, nm
1	27.5962	98	0.3296	3.23242	45.3
2	32.067	101	0.4723	2.79123	31.92
3	46.1376	200	1.152	1.96587	13.67

Table 3.

Values of diffraction angle (2θ), d ‐spacing, FWHM, crystalline size and Miller indices for BNPs

Bimetallic	Pos. [°2Th.]	Miller indices	FWHM	d ‐spacing, Å	Crystalline size, nm
1	38.7576	111	0.3149	2.32341	48.79
2	44.9621	200	0.9446	2.01617	16.605
3	65.2408	220	1.152	1.42894	14.936
4	78.3042	311	0.224	1.22103	83.44

Similar observations were reported by Ondari and Nalini for Ag NPs synthesised using Tridax procumbens [63]. The XRD pattern for the Au–Ag BNPs shows diffraction peaks at 38.7576°, 44.9621°, 65.2408° and 78.3042° and are indexed to (1 1 1), (2 0 0), (2 2 0) and (3 1 1) sets of lattice planes of the fcc structure [46], respectively. The peak corresponding to the (111) plane is more intense than that of other planes, suggesting that the (111) plane is the predominant orientation. These results also demonstrated that the nanoscale Au–Ag BNPs is highly crystalline. The mean size of BNPs was calculated using Debye–Scherer's equation

$$D=K\lambda /\beta \cos \theta$$

Here, D is the size of the particle, K is the shape dependent Scherer's constant, λ is the wavelength of the X‐ray (1.54060 A), is the full peak width and is the diffraction angle. Using the FWHM of peaks, the average size of Au–Ag BNPs was calculated to be nm.

3.4 EDX and SEM analysis

The presence of elements in Au, Ag and Au–Ag NPs was conducted through EDX analysis. The EDX elemental mapping of metallic and bimetallic NPs is shown in Fig. 6. The peak corresponding to Au at 2.2 keV was observed in elemental mapping. For Au–Ag, the peak appeared at 2.2, 3 and 3.25 keV proving that the Au and Ag–Au were homogeneously distributed over the entire NP structure. Furthermore, EDX elemental line scanning on a single NP confirms that the Au–Ag bimetallic structure was alloyed. Strong signals of Au and Ag were observed in EDX profile. Apart from this, weak chlorine and nitrogen peaks were also observed that might have originated due to the biomolecules bound to the NPs surface. Similar EDX profile for Au nanotriangles synthesized using Aloe vera extract was obtained by Chandran et al. [64].

Fig. 6.

EDX graphs of biosynthesised

(a) GNPs and (b) BNPs

SEM analysis was done for further characterisation of size, morphology and structure of GNPs, SNPs and BNPs. Fig. 7 shows the SEM images of the Au, Ag and Au–Ag NPs to confirm the morphology of the colloidal solution. The synthesised Au, Ag and Au–Ag NPs have a well‐defined triangular and spherical structure. Size calculation by SigmaScan Pro software attached with SEM showed that synthesised metallic and bimetallic NPs were clearly distinguishable owing to their size differences. The SEM images also showed that the large bioactive compounds are attached to the surfaces of small AgNPs, making them stable by preventing agglomeration.

Fig. 7.

SEM analysis graphs of biosynthesised

(a) GNPs, (b) SNPs and (c) BNPs

3.5 Antibacterial assay

Bimetallic Au–Ag NPs have strong antibacterial potential against several human pathogens such as Klebsiella aerogenes, E. coli, Plasmodium desmolyticum and S. aureus. Assessment of metallic and bimetallic‐NPs synthesised by green method was done by agar well diffusion method against five bacterial strains K. pneumonia, B. subtilis, E. coli, S. aureus and P. aeruginosa. Table 4 describes the antibacterial potential of monometallic and bimetallic NPs against aforementioned human pathogens while Fig. 8 shows their potency against P. aeruginosa and B. subtilis. Au–Ag NPs gave 3 and 2.5 mm zone of inhibition in a plate having B. subtilis and P. aeruginosa, respectively, which is somewhat close to the zone of inhibition of 4 mm given by Cefixime (an antibacterial drug used as a control). Leaf extract and precursor salts of Au and Ag have no significant antibacterial potential and showed very little activity. The facts about the accurate mechanism of NPs correspondence with the bacterial cell are yet inadequate [65]. Results showed that bimetallic‐NPs synthesised by a green method using plant extract have antibacterial potential. The antibacterial activity may be due to the following assumptions: microorganisms like bacteria have a small pore in the cell membrane. Reactive oxygen species (ROSs) are generated [66] from the bimetallic‐NPs actively penetrates the cell membrane using pores of the cell. The leakage of proteins, minerals and some matters from the cell because of the ROS penetrate the cell wall. The cell membrane is damaged, so the bacteria are killed or inhibited by cell growth.

Table 4.

Antibacterial activities of GNPs, SNPs and BNPs against different human pathogens

Bacterial strains	Diameter of inhibition zone, mm
	AgNPs,	AuNPs,	Au–Ag NPs,	Cefixime (10 μg ml−1),	WPE	Average value of Au and Ag precursor salts,
K. pneumonia	2	1.5	1.1	3.7	—	0.6
B. subtilis	1	2.5	3	4	—	1.1
E. coli	1	1.5	1	3	1.2	1.1
S. aureus	1.3	1.5	1.5	2	0.6	—
P. aeruginosa	1.5	2	2.5	4	—	0.5

In this table, represented values are an average of results of three different experiments.

Fig. 8.

Antibacterial activity of Au, Ag and Au–Ag NPs on P. aeruginosa and B. subtilis

3.6 Anti‐leishmanial efficacy of synthesised Ag, Au and ag–Au NPs

The anti‐leishmanial efficacy of NPs was determined quantitatively and microscopically by applying the MTT assay on metabolic activity of L. major promastigotes at various concentrations (25, 50, 100, 150, 200 and 250 μg ml⁻¹) of metallic and bimetallic NPs in the dark. Fig. 9 displays that under dark conditions the formazan crystals; the signature of metabolic activity, the formation was very intense in the AgNPs group. However, lesser formazan crystals were formed upon exposure to AuNPs in dark as compared to Ag and Au–Ag NPs groups. The efficacy of Au and Au‐Ag NPs at various concentrations in dark conditions is shown in Fig. 9. L. major parasites metabolic activity was inversely proportional to the concentrations of NPs.

Fig. 9.

Anti‐leishmanial activity of the GNPs, SNPs and BNPs against promastigote forms of L. major. Parasites (1 × 106) were exposed to various concentrations for 48 h, and promastigotes killing percentage was observed

Compared to AgNPs, the metabolic activity of L. major promastigotes was negatively influenced by each applied concentration of AuNPs. Based on the IC₅₀ against promastigotes, AuNPs induced a high leishmanicidal effect with IC₅₀ value 282.597 μg ml⁻¹ as compared to AgNPs with IC₅₀ 155.824 μgml⁻¹. Bimetallic NPs also showed a considerable effect with IC₅₀ value 227.277 μg ml⁻¹. Chemotherapy was the best option for the prevention of leishmaniasis. The pentavalent pentamidine, allopurinol, antimonials, miltefosine, amphotericin B, paromomycin and many others are well‐known anti‐leishmanial drugs applied at various developmental stages [67, 68]. Concerning to this, for enhancing intracellular drug assembly via efficient drug delivery, strategies were developed that lowers the dosage and its related toxicity. Under these conditions, biosynthesised bimetallic and AuNPs having a small size, large surface area and attachment capability to sulphur and phosphorus groups would be great alternative medicine to treat leishmaniasis and its co‐infections.

4 Conclusions

Au, Ag and bimetallic Au–Ag NPs were successfully synthesised by using Cannabis sativa leaf extract as reducing and stabilising agent. The reaction was rapid, green, eco‐friendly, and economical. Syntheses of GNPs, SNPs and BNPs were studied using UV–vis spectroscopy, XRD analyses, FTIR and SEM analysis. Characterisation techniques revealed that flavonoids, terpenoids and amides of proteins in the plant extract were involved in the reduction and capping of metal ions into NPs. It also showed that the bimetallic NPs were alloyed. Bimetallic NPs are an arrangement of two metal NPs which are made‐up by various synthesis methods including physical, chemical as well as biological processes. Greener means are chosen, as they are environmentally salubrious and safe. Synergistic effects of two metals in bimetallism carry out certain functions which were otherwise not possible with monometallic NPs alone. Bimetallic NPs are of great interest as they hold a number of applications in the field of drug delivery, in biosensing, for as catalysts, as anti‐microbials, in overcoming environmental pollution and several others, which are a topic of further study. Development of improved bimetallic NPs will surely prove to be a benefit to a society.