The organoleptic and quality parameters such as solubility, taste, color, and consistency were checked as per standards and found to be insoluble in water and other organic acids, tasteless, black, fine particles, respectively. The quantitative analysis of the formulation showed that coumarins, tannin and resins present.

Characterization of chemical composition of the formulation

The spectroscopic techniques were used to characterize the elemental, surface, and organic property of the formulation (Fig. 1). The absorption spectrum of sample is depicted in Fig. 1a. The observed resonance spectrum around 286–296 nm which may correlated to the optical property of Hg, and confirm the presence of Hg2+ (Fig. 1a). The spectral distribution was compared with NIST database and corroborate to Hg (II), As (III) and S. The spectra maximum at 210 and around 285 nm is bound to HgS and blue shift was observed compared to the bulk material (Maynard 2007). The report on core-shell Ag2S-HgS, demonstrated the slight red shift in peaks at 284 nm (for Ag2S/ 0.1 M Hg2+) and at 290 nm (for Ag2S /0.2 M Hg2+) due to increased concentration of Hg2+ and increase in size (Bednarek et al. 2000). The formulation is free from such excitation, and this ensure the purity and quality of the preparation. The comparative study on HgO, HgS and Makardhwaj had similar results and reported that the formulation is completely free of HgO (Mukherjee et al. 2010). The extended tail observed in the spectrum may due to agglomeration and aggregation of HgS (Sathyavathi et al. 2013) (Fig. 1a). Moreover, this elucidate that inhomogeneous size distribution and aggregation of particles (Yu et al. 1997). The calculated bandgap energy of the formulation had good agreement with cinnabar is in the range of 2.2–3.4 eV (hexagonal unit cell). The higher bandgap energy is may be due to the blue shift and increased particle size of the formulation (Selvaraj et al. 2014). These may also increase the stability of the formulation (Sin et al. 1983). The peaks at fingerprint region of FTIR spectra at 460, 620, 771 and 1020.38 (cm− 1) (Fig. 1b) may attribute to interaction of Hg and S, and represents the metal-sulphur linkage in the formulation. These peaks had good agreement with reported works on HgS based formulation, such as Linga chenduram (Sudha et al. 2009), Arumuga chenduram (Shibi et al. 2012) and Poorna chendrodayam (Austin 2012). The observed peaks at 2359.02, 2864.39, 2929.97 and 3373.61 are reveals fraction of organic moieties in the sample (Shibi et al. 2012). Similar formulation, Rasa chenduram was reported to have organo-metallic complex, with legitimately sharp peaks in functional group region (2724 and 2870 cm− 1) (Singh et al. 2009). The peak at 2927 and 2854 cm−1 are matched with nC-H bonds of aliphatic and aromatic hydrogens, respectively, and no broad peaks around 3200–3400 cm− 1 (vibrational bands due to adsorbed water). This indicate that the loss of H-O-H molecules by crystallization (Kamath et al. 2014).

Fig. 1

(a) UV absorbance spectrum of KK (inset, Tauc plot for direct transitions. The bandgap energy is calculated by extrapolation of the tangent line); (b) the FTIR spectrum of KK; (c) XPS wide spectrum of KK

The formulation is prepared with combination of mercury and arsenic along with the herbal and animal origin materials. Moreover, both elements are classified as class I toxic to human. It is essential to quantify the nature and chemical form of Hg and As on the basis of their efficacy, disposition, and toxicity. Because, the organic and oxide form both were known for its toxicity against the environment and human. The chemical composition of the formulation is characterized with XPS. The sample had spectral similarity with the higher intensities to Hg and S. The background of the spectrum was corrected with the C1s peak at 285.2 Ev (Kannan et al. 2021). The excited binding energy of two distinct peaks at 100.4 and 104.65 eV are assigned to Hg(4f) (Fig. S1a) (Kannan et al. 2018, 2021). The peaks at 396.63 (Hg4d) and 532.64 (Hg4p) were assigned to Hg transitions (Fig. S1a). The S2p (S2p3/2 and S2p1/2) transitions were observed in the S region measured at 160.62 and 162.54 eV (Fig. S1b) (Kannan et al. 2018, 2021). The C peak around 285 eV is may be due to the penetration of organic matter in the initial stage of purification and/or due to the calcination process at later stage of preparation. These values had good agreement with the reported data of Zuotai, a Tibetan mercury based medicine (Li et al. 2016). The results of XPS is more consistent with the ICP-OES analysis. These results corroborate that the sample is rich of Hg and S and calculated (atomic percentage) to be approximately 1:1.2. The peaks at 164.14, 165.56 and 224.56 eV were matched to S2s of sulphur alone (Fig. S1b). The deviation in the expected ratio may be due to the excess amount of sulphur in the sample, which was observed to be consistent with the EDX elemental mapping of Hg and S (Fig. 2b).

Fig. 2

The XRD (a), EDS (b) and Zeta potential (c) profile of the formulation

Size, elemental mapping and stability of KK formulation

The XRD spectrum of the formulation shown in Fig. 2a. The most intense peaks are predominantly similar to HgS (JCPDS file No: 75-1538; 73-1593). The peaks (2θ) at 21.51, 25.10, 34.52, 40.72, 50.87 and 54.6024 had good agreement with β-HgS of (111), (111), (200), (102), (311), and (222) (Maynard 2007). The low intense peak (2θ) at 22.79 and 45.70 are matched with free sulphur (JCPDS file No: 08-0247) (Maynard 2007). There is no standalone identical peak for arsenic or arsenic sulphide. Moreover, there is no prominent peaks other than HgS in XRD, these endorse that the heat treatment is significant and further confirms the crystallinity of HgS. The surface oxidation is expected to occur on HgS/amalgam due to the pudam (heat treatment) process. These are confirmed with the low intensity peaks in XRD. The mercury based medicine, Rasa chenduram showed identical peak to Hg (JCPDS File No. 89-3711) and confirm the preparation is composed of Hg and HgS (Paul and Sharma 2011). The study on Rasa chenduram (mercury-based ayurvedic medicine) were reported that the peaks are matching with hexagonal, HgS with a calculated crystallite size range of 16–22 nm (Kamath et al. 2014). The obtained results had good agreement with β-HgS (meta cinnabar) nanoparticles (Marimuthu et al. 2015). The crystalline size of the sample has been estimated for FWHM of planes, (111), (200), (102), (311) and (222) using the Scherrer equation and is found to be around 35–70 nm.

The EDS is referred as spot or selected area elemental analysis, the sample was characterized using EDS (Fig. 2b). The EDS spectrum of the sample had peak corresponds to S (2.30 keV), Hg (2.2 and 10 keV) and As (1.28 kev). The atomic percentage of the sample is 1:1(Hg: S) and 1:2(As: S). The proportion and distribution of elements was shown in Fig. 2b. The sample had higher percentage of S and complexed with Hg and As. This is in agreement with the results of XPS (Fig. 1c). The occurrence of C peak in the formulation may be due to the extent of purification process. This may tend to bind with the metal and may be converted into herbo-metal form. The study on other traditional mercury-based preparation such as Linga chenduram (Hg, 33.75% and 7.10%, S) (Sudha et al. 2009), Rasa chenduram (84.80% of Hg and 14.13% of S) (Singh et al. 2009), Poorna chendrodayam (Hg, 78.11%, Au, 9.78% and 11.95% of S) (Sathyavathi et al. 2013) were in agreement with our findings. The sample was analysed for metal content by ICP-OES. The Hg, As, and S was quantified and had agreement with the XPS and EDS results. Thus, the results of XPS, EDX, ICP-OES confirm the absence of toxic oxide form of Hg. Nevertheless, the atomic percentage of the sample had potential match with the the synthesized cinnabar (HgS). The sample was analyzed in an aqueous dispersion medium and the zeta potential (Fig. 2c) is found to be -36.0mV, for 100% area under the curve with the width of 7.72mV. The range of -23 to -37mV were reported for Rasa chenduram (HgS based formulation) (Paul and Sharma 2011). Highly dispersed and heterogeneity of the particles was observed (Fig. 2d) with intensity of 78.1%. The hydrodynamic diameter of the sample found to be 186.9 nm and width of 42.68 nm with low intensity of 18.1%. The reported particle size for β-HgS aggregates is 296.2 ± 22.5 nm; the larger aggregates may be due to the sample drying and preparation (Sin et al. 1983).

Morphological characterization of the formulation

The morphological characterization of the formulation was observed using TEM and FESEM (Figs. 3 and 4). The presence of solid tubes, individual grains of spherical and plate like structure were observed (Fig. 3a&b). It is found that there is grain growth assembly and arranged into layer-by-layer (uniform grains) with bipyramid structures (Fig. 3d). The formulation is charging inside the chamber this could be because of HgS complex annealed at high temperature (Kannan et al. 2021). The images shows hexagonal cells, which allows the particles to grow different shapes (polydisperse particles) (Fig. 3c and d). The size of an individual grains were of 10–180 nm (Fig. 3b&c) and particle distribution were found to be 0–110 nm (Fig. 3b, inset) (calculated using image J software) is in correlation with XRD result. The polydispersed particles with the rough edged surface were reported for the commercial product of Makardhwaj (Mukherjee et al. 2010). The large chunks present in the samples is due to the agglomeration. The images represents that area coverage is appreciated and has very few void space in the formulation.

Fig. 3

The FESEM monograph of the samples, (a) solid tubes; (b) aggregated particles; (c) individual grain growth and grain size; (d) hexagonal cells

Fig. 4

The TEM monograph of the samples, a-d, different magnification of distributed particles

Cytotoxicity of formulation in cell line

The embryonic kidney-derived (Hek 293) and breast cancer (MCF-7) cell lines was selected to evaluate the cytotoxicity of formulation. The selected cell lines were treated with sample and viability was determined by MTT assay over a period of 72 h (Fig. 5). The sample showed relatively moderate effect on both cell lines, Hek293 (IC50 of 65.64) and MCF-7 (IC50 of 45.30 µg/mL) at 24 h (Fig. 5).

Fig. 5

The in vitro analysis of formulation, (a) MTT assay and (b) FACS analysis

The lethal concentration results that the formulation is not affecting the normal cells even at 60 µg/mL (Fig. S2 a-f). This is in agreement with the reports on HgS and As2S2 (Pastorek et al. 2014). This is supported by the characterization results as less toxic form of Hg and As (Kannan et al. 2017). The sample induced necrosis and early apoptosis in MCF-7 cells. The Hek 293 showed only late apoptotic cells. About 55–70% of cell viability was retained even after treating with higher concentrations (Fig. 5c). Based on the results, the formulation is cyto-compatible to the normal cell and impaired toxic to the MCF7 cells (Li et al. 2016; Geng et al. 2017). The different concentrations used to treat the cells and the results had agreement with the MTT. The treated and stained cells were compared with control, for Hek293 (Fig. 6a-d). The acridine orange dye is used to visualize both viable and dead cells, whereas ethidium bromide stain used to visualize only non-viable cells. The dual staining (AO-EB) resulted that the cell membrane was damaged and apoptosis (early and late) of on Hek 293 cells at toxicological end point (72 h). The untreated cells (control) showed green fluorescence emitted by acridine orange indicates the live cells. The control group showed a homogeneous monolayer (Fig. 6e&f).

Fig. 6

The Trypan blue staining (a-d), and AO-EB staining (e &f) of Hek 293

Biological effects in zebrafish model

The Zebrafish (Danio rerio) model has 70% of protein-coding genes are related to normal and disease associated human genes (Santoriello et al. 2012). Moreover, the short fertilization time, transparent embryo, heart phenotype is analogous to human cardiomyopathies and ease of genetic manipulation endorses Zebrafish embryos as a successful model to evaluate toxicity, especially in screening nanomaterials (Kalueff et al. 2013; Penberthy et al. 2002). The embryos were treated with the different concentrations to evaluate safe dosage of the formulations (Fig. 7a&b). The number of viable embryos at endpoint was shown in Fig. 7c. The maximum non-lethal concentration of the formulation was measured to be 100 µg/mL. The Zebrafish embryos were assessed for mortality, hatching time and rate. The heartbeat rate (HBR) of embryos was measured at 72 hpf for tested concentration. The cardiac cycle of control and test samples were assessed and captured with 20 frames to complete one cycle for control, while the treated (MNLC) embryos had only 11 frames and 6 frames for LC50. The HBR of 46 ± 2 beats/15sec for control and 45 ± 2 beats/15sec for test sample was observed. When the concentration increased to 1 mg/mL, the HBR decreased to 30 beats/15sec (Fig. 7d). Similarly, the morphological and developmental toxicity also observed. The LC50 of the sample is 533 µg/mL. These confirm the biocompatibility and low toxicity of the formulation in Zebrafish models (Fig. 7c). The lethal concentration (> 1 mg/mL) of the formulation showed 100% embryo lethality.

Fig. 7

Assessment of developmental toxicity of the formulation in Zebrafish model, a. Zebrafish embryo; b, malformation in embryo; c. viability of embryos; d. heart beat rate of embryos