All the plant materials collected for the study are listed in Fig. 1.
Preparation of the stock solution—chloroauric acid
The stock solution of chloroauric acid was prepared in different concentrations and used for further synthesis of nanoparticles. A high molar concentration of 25.8 mM preparation has been reported for the synthesis of gold nanoparticles by Boruah et al. (2012). In this study, a successful synthesis of gold nanoparticles has been achieved at very minimal concentrations of 10–1, 10–2, 10–3 and 10–4. This minimal concentration of nanoparticles ensures a biologically much safer nanoparticle for in vivo applications.
Preparation of the plant extracts
The plant extracts were prepared through hot extraction, cold extraction, methanolic extraction and soxhlet extraction as shown in Fig. 2. The synthesis of nanoparticles was carried out using the extracts obtained through all the above-mentioned processes. The nanoparticles were successfully synthesized with all the above extracts. When the nanoparticles could be exploited for in vivo applications such as novel therapeutics and drug delivery molecules, the aqueous base nanoparticles would render less toxic or no toxicity effect.
Synthesis of the gold nanoparticles
The nanoparticles were synthesized using the 15 medicinal plant extracts on reduction with chloroauric acid. The synthesized nanoparticles were pale pink to deep wine red in color as observed in Fig. 3. In this study, the plant materials were exclusively selected based on their significant anti-HIV and anticancer activities. The various phytocompounds such as volatile oils, fatty oils, vitamins, tannins, phenols, flavonoids and others present in the plant extracts acted as the reducing and stabilizing agent for synthesis.
Rate of the synthesis of nanoparticles
The synthesis of nanoparticles took place in varying time durations as mentioned in Table 2. Lal and Nayak (2012) in their study reported that the minimum time of synthesis was recorded as 5 min and maximum time as 5 h. In the present study, the minimum duration was observed to be less than a minute and the maximum duration was between 3 and 4 h for synthesis. The synthesis of gold nanoparticles was achieved in a much faster rate than the previous study.
Intensity of the nanoparticles produced
The bioreduction of metallic gold into nanogold resulted in a distinctive shift in color of the colloidal solution. The precursor chloroauric solution was pale yellow in color. The nanoparticles synthesized were predominantly deep red in color, while few exhibited pink color as mentioned in Table 3 and the intensity of the color of the synthesized nanoparticles varied with different plant extracts as observed in Fig. 4. Based on the intensity of the nanoparticles solution, the nanoparticles were further quantified.
A novel attempt of nanoconjugate synthesis
Despite use of biological entities such as plant, bacteria or fungi as an agent for the synthesis of nanoparticles, plants have been exploited only as a reducing agent for nanomaterials. The present study focused on a novel pharmacognostic approach over the usage of plants source. The nanoparticle synthesized through medicinal plants will possess the potential therapeutic properties of the plants. The resultant nanoparticle probably carried the attributes of a nanomaterial alongside the phytoactivity. Initially, individual nanoparticle (NP) was synthesized through a single plant extract and studied for its effective anti-HIV and anticancer activity. Finally, a nanoconjugate (NC) comprising of the 3 extracts (trio extract) in one nanoparticle was synthesized as observed in Fig. 5 based on the results of anti-HIV and anticancer activity. The nanoconjugate (NC) showed a combined synergistic effect and increased anti-HIV/anticancer activity than the nanoparticle.
Synthesis of nanodrug conjugate
It was reported by Czeczuga et al. (2004) that doxorubicin in combination with estradiol, tamoxifen and retinoic acid showed the most effective and statistically significant decrease in the percentage of MCF-7 cells. In the present study, nanodrug conjugate (NDC) was prepared in combination of AuNPs and the chemotherapeutic drug, doxorubicin for cancer and AuNPs with antiretroviral drug azidothymidine (AZT) for HIV as shown in Fig. 6. A nanodrug conjugate helps to enhance the efficiency of the drug. The nanoparticle (AuNP) and nanoconjugate (AuNC) with anticancer and anti-HIV activity were conjugated with the drugs. Thus, the nanodrug conjugates bring about a combinatorial effect of the nanomaterials and the drug.
Synthesis of chitosan nanodrug conjugate
Chitosan has various characteristics which makes it ideal for in vivo applications. It is biocompatible, biodegradable, non-toxic, non-antigenic and has strong adsorption properties (Hettiarachchi et al. 2011). It was reported by Zambito (2013) that Chitosan nanoparticles conjugated with peptide drug insulin resulted in an enhanced transport of insulin compared to native unconjugated insulin. In the present study, a biocompatible nanocarrier was developed as a conjugate with chitosan and gold nanoparticles using STPP as gelating agent for the drug doxorubicin as represented in Fig. 7.
Optimization of pH on the synthesis
The pH has a crucial part in the successful reduction of bulk materials and in the synthesis of nanomaterials. The pH range was checked between pH 4, pH 6, pH 8 and pH 10. It was observed that at pH 8, there was a complete reduction of bulk metal into metallic nanoparticles. At the other tested pH gradients, a partial reduction of the bulk metal was observed with varying color changes of the precursor solution. The reaction between chloroauric acid and plant extracts was observed at various pH gradients of pH 4, pH 6, pH 8 and pH 10.
The observed results are represented in Fig. 8, which were in contrast to the reports stated by Pandey et al. (2012) and Sharon et al. (2012) where it has been stated that pH 10 was the optimum condition for the synthesis. In the present study, pH 8 was observed to be the optimum pH that resulted in successful synthesis of nanoparticles.
Optimization of temperature on the synthesis
The reaction was carried out in varying temperatures ranging from room temperature (28ºC) to 100ºC. It was found that a complete reduction that yielded the nanoparticles was observed at room temperature. In other temperatures, the reduction was incomplete or intermediate which was indicated by varied color changes.
Sterilization of the synthesized nanoparticles
The synthesized gold nanoparticles were subjected to sterilization in autoclave for 15 to 20 min, at 121 lbs. The nanoparticles were analyzed before and after the process under UV–visible spectrophotometer as represented in Fig. 9.
In vitro stability of the synthesized nanoparticles
The synthesized nanoparticles were checked for stability at different pH mimicking the physiological environment using phosphate-buffered saline (PBS). The various conditions included pH 5.7, pH 6.1, pH 7.0, pH 8.0 and normal saline. The synthesized gold nanoparticles were incubated in different buffers and analyzed in UV–visible spectrophotometer as represented in Fig. 10.
Storage conditions of synthesized nanoparticles
Upon storing the synthesized AuNPs at different storage conditions such as room temperature (± 28), 4 °C (refrigerator) and -20 °C (deep freezer), the nanoparticles were analyzed in UV–visible spectrophotometer as shown in Fig. 11 and compared with the standard spectrum. It was observed that the AuNPs were stable in all these 3 stored conditions without any significant difference in the state of the nanoparticles. Hence, the nanoparticles could be stored at any appropriate storage conditions.
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