Nanotechnology owing to its promising applications has received tremendous attention in the past decades. As building blocks in nanotechnology, various methods [1–3] have been developed to fabricate nanostructures of well-defined compositions. However, conventional physical and chemical methods either are energy intensive or impose environmental hazards due to toxic solvents or additives as well as hazardous by-products. Hence, it is of great interest to develop environmentally benign alternatives, among which biological systems arise as a typical instance. In 1999, Klaus et al.  initiated the biosynthesis of Ag nanoparticles (NPs) by Pseudomonas stutzeri AG259, and the shift from bacteria to fungus was leaded by Sastry et al. [5–7]. However, in addition to the delicate culture and storage, subsequent processing of NPs formed by intracellular biosynthesis is generally difficult, and microorganisms used for the extracellular biosynthesis of NPs must be extensively screened . In recent years, biosynthetic method employing plant extracts or biomass has appeared as a simple and viable alternative to microorganisms, e.g., plants such as coriander alfalfa , Aloe vera, Avena sativa biomass , lemongrass , Cinnamommum camphora etc. have been reported relatedly. Our group have demonstrated that a large number of plants possess the capability to convert Au(III) into Au(0) .
But there remains a significant challenge in understanding and predicting nanoparticle size and shape from a given set of biosynthetic conditions (e.g., choices of plants), which involves a full understanding of the bio-protocol. Even an accurate determination of the involved biocompounds that provides the premise for illustration of the bio-protocol could be tough. The diversity of biocompounds in the biomass makes individual purification and determination of all the biocompounds not viable. Synergic effects among these compounds might also add to the complexity. Moreover, even if for generation of the same kind of metal NPs, cases vary greatly among different bio-systems [9–13]. Consequently, a most universal explanation to account for generation of those NPs should cover as many cases as possible.
Currently, the Fourier transform infrared spectroscopy (FTIR) analyses by Huang et al.  revealed that polyols were responsible for the generation and stabilization of NPs. Among various polyols, the reducing sugars and/or the terpenoids were speculated to play a role in the bioreduction . Water-soluble heterocyclic biocompounds or proteins were considered as stabilizing ligands of the NPs [12, 13]. And the pH condition could also affect the process . There were also investigations that isolated individual biocompounds such as chitosan  and established possible mechanisms to illustrate the process.
The above studies were single organism based, focusing on individual organisms or biocompounds, and the specific information of which might not be applicable to other various cases. As well, currently the FTIR spectroscopy that mainly renders local information about related functional groups has dominated the existing methods of research, but the involved biocompounds could not be accurately determined only by FTIR since the same functional group could exist in a variety of different biocompounds. Therefore, it is imperative to explore complementary methods to illustrate the mechanism underlying biosynthesis of metal NPs.
To contribute to the determination of biocompounds involved in biosynthesis of gold nanoparticles (GNPs) by foliar broths, a statistical analysis is proposed in this work to investigate the influences of five immanent parameters of the foliar broths, i.e., the original pH value, the content of reducing sugars, flavonoids and proteins and the anti-oxidant capability, upon the Au(III) conversion and the size/shape distribution of the biosynthesized GNPs. As the parameters of the foliar broths are, respectively, evaluated, the pertinence of the research is enhanced. Moreover, due to its statistical characteristics the present research tends to be systematic. To our knowledge, this is the first report using a statistical method attempting to view bio-protocol of GNPs in a systematic perspective.