Therapeutic potential of biogenic and optimized silver nanoparticles using Rubia cordifolia L. leaf extract

Rubia cordifolia L. is a widely used traditional medicine in the Indian sub-continent and Eastern Asia. In the present study, the aqueous leaf extract of the R. Cordifolia was used to fabricate silver nanoparticles (RC@AgNPs), following a green synthesis approach. Effect of temperature (60 °C), pH (8), as well the concentration of leaf extract (2 ml) and silver nitrate (2 mM) were optimized for the synthesis of stable RC@AgNPs. The phytofabrication of nanosilver was validated by UV–visible spectral analysis, which displayed a distinctive surface plasmon resonance peak at 432 nm. The effective functional molecules as capping and stabilizing agents, and responsible for the conversion of Ag+ to nanosilver (Ag0) were identified using the FTIR spectra. The spherical RC@AgNPs with an average size of ~ 20.98 nm, crystalline nature, and 61% elemental composition were revealed by TEM, SEM, XRD, and. EDX. Biogenic RC@AgNPs displayed a remarkable anticancer activity against B16F10 (melanoma) and A431 (carcinoma) cell lines with respective IC50 of 36.63 and 54.09 µg/mL, respectively. Besides, RC@AgNPs showed strong antifungal activity against aflatoxigenic Aspergillus flavus, DNA-binding properties, and DPPH and ABTS free radical inhibition. The presented research provides a potential therapeutic agent to be utilized in various biomedical applications.


Material and methods
Plant materials. R. cordifolia leaves were collected in January 2019 from the forest area of Amarkantak, Madhya Pradesh, India. The identification of R. cordifolia was certified by a subject expert, and a voucher specimen (DOB/07/RC/120/2019) was deposited in the Botany department, IGNTU, Amarkantak, MP, India. The collection of the plant material and related studies complies with relevant institutional, national, and international guidelines and legislation.
Preparation of leaf extract. Approximately, 10 g of leaves of R. cordifolia (Fig. 1) were weighed through the analytical balance, washed twice with DDW, and dried at room temperature. The chopped leaves were placed in a 250 mL conical flask with 100 mL of DDW, boiled for half an hour at 70 0 C, and filtered twice using Whatman No. 1 filter paper. The filtrate was kept at 4 0 C for further use.
Phytochemical analysis. The principal phytochemicals in R. cordifolia leaf extract (RCLE) were examined following the procedure of Chandraker et al. 22 . Furthermore, for the detection of free and combined anthraquinones, Borntrager's test was adopted following Ukwubile et al. 23 .
Optimized synthesis of AgNPs. Several factors, including temperature, pH, AgNO 3 concentration, and RCLE, were optimized to synthesize saturated and stable R. cordifolia-mediated RC@AgNPs. To optimize the suitable temperature, four reaction mixtures of 1 mL of RCLE and 9 mL of AgNO 3 (1.0 mM) were mixed and incubated at four different temperatures, viz., 20, 40, 60, and 80 °C for 60 min. To optimize the pH, the above experimental process was repeated at different pHs (2, 4, 6, 7, and 8). Four different volumes of RCLE (9.5, 9, 8.5, and 8 mL) were treated with corresponding volumes (0.5, 1.0, 1.5, and 2.0 mL) of 1 mM AgNO 3 solution to find the optimum concentration of RCLE. Similarly, to optimize AgNO 3 concentration, 0.2, 0.5, 1.0, and 2.0 mM of Applications of RC@AgNPs. Anticancer activity. The culture and maintenance of cells were done in Dulbecco's Modified Eagle's Medium (DMEM) with 10% Fetal Bovine Serum (FBS) and 1% antibiotic and antimitotic solution at 37 °C with 5% CO 2 . For the experiment, NPs were suspended in dimethylsulfoxide (DMSO) with the concentration of DMSO in the medium not exceeding 0.1% in all the treatment groups. The standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to assess the anticancer efficacy of RC@AgNPs against A431 (squamous cell carcinoma cell line) and B16F10 (melanoma cell line). Both the cell types were seeded at the concentration of 5000 cells/well in 96 well plates. After incubating the cells for 24 h, treatments were given to cells for 24 and 48 h with different concentrations (10,25,50, and 100 µg/mL) of RC@AgNPs. MTT was added to the wells, and the plate was further incubated for 4 h. The added MTT was replaced gently by 100 µl of DMSO and incubated briefly for ten minutes at 37 0 C, with the absorption measured in a microplate reader at 570 nm. The percentage of cell viability was estimated using the following formula: DNA interaction activity. Calf-thymus DNA (CT-DNA) solution was prepared with Tris buffer (pH = 7.2). Different concentrations of RC@AgNPs (0.010-0.090 nM) were treated with a concentration of CT-DNA (250 µM). The interaction between RC@AgNPs and CT-DNA has been carefully monitored in a UV-visible spectrophotometer by absorption-titration experiments.
Antifungal activity. For antifungal activity, the RC@AgNPs were evaluated against a food-spoiling, saprophytic, pathogenic, aflatoxigenic, and ubiquitous fungi Aspergillus flavus. The fungal strain (No. 277) was procured from the Microbial Type Culture Collection and the Gene Bank (MTCC) of the Institute of Microbial Technology (IMTECH), Chandigarh, India. Contact assay was adopted to determine antifungal activity, following Shukla et al. 24 . Five different doses of RC@AgNPs (1.25, 2.5, 5.0, 10.0, and 20.0 mg) were taken in separate Petri dishes (90 mm) and 20 mL PDA was poured into each petri dish. Finally, 5 mm fungal discs taken from the A. flavus colony (One week old) were inoculated at the center of RC@AgNPs and PDA-containing plates. The inoculated Petri dishes were incubated for seven days in the dark at 27 °C to determine the inhibition of the mycelial growth of the fungi. The negative control plates without RC@AgNPs, and positive control plates with RCLE and 1 mM silver nitrate solution, separately were also incubated under the same growth conditions. The effect of RC@ AgNPs, RCLE and 1 mM silver nitrate on the growth of A. flavus was tested in triplicates. After seven days, the radial colony's growth and photographic record were established.
The percentage of fungal inhibition was calculated based on the rate of radial growth inhibition, as follows: where IRG = Inhibition of the radial growth, R1 = Control's radial growth, and R2 = Radial growth in treatments, IRG was estimated using the mean ± standard error (SE) of the triplicate data.
Antioxidant capability. The antioxidant properties of RC@AgNPs were determined using the DPPH and ABTS free radical scavenging tests described by Chandraker et al. 25 . Using UV-visible spectroscopy, the antioxidant potential of RC@AgNPs was tested in the presence of DPPH and ABTS free radicals, and ascorbic acid was used as a standard reference. The percentage of scavenging activity was calculated using the following formula.
Biogenic synthesis of RC@AgNPs. Silver nanoparticles were synthesized following an eco-friendly method 32 . RCLE was used as a capping, stabilizing, and a reducing agent to fabricate RC@AgNPs from AgNO 3 . The schematic illustration of nanosilver synthesis from two major anthraquinones of RCLE is shown in Fig. 2.
The change of color from light-green to reddish-brown has been considered as RC@AgNPs synthesis. Changes in the color of the reaction mixture are due to the conversion of Ag + to Ag 0 , and they were easily monitored by Ultraviolet-visible spectroscopy. Figure 3 represents the UV-visible analysis of RC@AgNPs, RCLE, and silver nitrate solution. In Fig. 3 it is clearly shown that RC@AgNPs have a signature AgNPs' absorbance peak at 432 nm, whereas, RCLE and AgNO 3 do not. Another peak shown around 380 nm may originate from an interband transition 33 . RC@AgNPs displayed a negligible shift in λ max from 432 to 435 nm, after 5 months of synthesis, thus found to be stable. Due to surface plasmon resonance (SPR), AgNPs tend to exhibit a characteristic absorption maximum (λ max ) in the 400-500 nm range 34 . The absorption maximum of Justicia adhatoda mediated AgNPs was 463 nm 35 , Sonchus arvensis mediated AgNPs was 440 nm 36 , Equisetum arvense mediated AgNPs was 488 nm 37 , and Withania coagulans mediated AgNPs were 445 nm 38 , SPR is a cumulative oscillation of conduction electrons triggered by incoming light at the interface between (-)ive and ( +)ive materials. The condition is regulated by the capping agents on Ag-surface. Plants have variations in their phytochemical constituents, and thus, varied functional groups act as capping, reducing, and stabilizing agents. Therefore, different plant-mediated AgNPs show variable λ max , size, and stability.
Optimization of biogenic RC@AgNPs. The yield of NPs is influenced by the reaction conditions of the biogenic synthesis process. Various reaction parameters explicitly alter the size distribution and reaction rate of the NPs synthesis. Figure 4 shows the alterations in the color of the reaction mixture under several experimental conditions 39 .
The reaction temperature was indeed a vital aspect that expressively related to the rate of biogenic synthesis of RC@AgNP. To investigate the effect of temperature on the phytosynthesis of RC@AgNPs, 1 mL of RCLE was treated with 9 mL of 2 mM AgNO 3 in 4 different vials and kept at temperatures ranging from 20 °C to 80 °C. According to the findings, increasing the temperature in the reaction mixture increased the biogenic synthesis of RC@AgNPs (Fig. 4a). The considerable RC@AgNP synthesis was observed at 60 °C, even though absolute RC@AgNP synthesis was reported at 80 °C. It shows that the entire synthesis of RC@AgNPs may be achieved at temperatures of 60 °C and 80 °C, where RCLE phytochemicals conduct optimal reduction and stabilisation 40 . The absorbance peak at 80 °C seems distorted due to the accumulation of RC@AgNPs generated by high-temperature biogenic production.
The pH value is always crucial for any reaction. In the biogenic synthesis of AgNPs, pH is essential. The color of the reaction medium, the strength of the SPR peak, and the shape and size of the NPs were investigated to be pH-dependent. RC@AgNPs absorbance peak rises with increasing pH, and the maximum fabrication of RC@ AgNPs occurred at pH 8. In our investigation, the acidic media with pH 2 and 4 had a lower absorption peak than pH 6 and 8. As a result, we concluded that an alkaline pH of 8 is ideal for the synthesis of RC@AgNPs (Fig. 4b). According to our observations, absorption increases with rising pH, indicating that an alkaline condition is preferable to an acidic one for the synthesis of NPs. During the synthesis of NPs, the change in the pH of     Figure 4c indicates no absorption spectrum at 0.5 RCLE concentrations. On increasing the concentration of RCLE, absorption peaks were observed, and at 2.0 mL RCLE concentration, maximum absorbance was found. The increasing concentration of biomolecules involved in metal reduction has enhanced the synthesis of environmental-friendly NPs. Similar results were also found with the Pongamia pinnata leaf extract 45 and Citrullus lanatus fruit rind extracts 46 .
The concentration of AgNO 3 also affects the phytosynthesis of AgNPs, significantly. Multiple concentrations (0.2, 0.5, 1, and 2 mM) of AgNO 3 solution were used to determine their effect on RC@AgNPs synthesis. AgNO 3 concentration of 2.0 mM results in maximum RC@AgNPs synthesis, with an absorbance peak at 432 nm (Fig. 4d). Poor peaks at lower concentrations of 0.2 and 0.5, and 1.0 mM AgNO 3 could be due to the deficient availability of Ag + ions in the reaction mixture.
Characterization of RC@AgNPs. XRD analysis. The crystalline nature of the particles was confirmed with XRD. Figure 5a shows an XRD pattern of the AgNPs synthesized by using RCLE. The XRD pattern showed many Bragg reflections based on Ag's face-centered cubic (fcc) structure. The nanosize of RC@AgNPs was confirmed after matching the obtained XRD spectrum with the standard (  SEM and EDX analysis. SEM image (Fig. 5b) shows the granular appearance of RC@AgNPs. Different elements' composition in RC@AgNPs is confirmed by EDX analysis at 3 keV. In Fig. 6a (Fig. 6b).
TEM analysis. Figure 7a shows TEM images of RC@AgNPs that illustrate the formation of isotropic spherical AgNPs. The particle size distribution histogram of RC@AgNPs using TEM images revealed an average particle size of 20.98 nm (Fig. 7b). This particle size spectrum was obtained using spectroscopy-based SPR and XRD.
FT-IR spectral analysis. FT-IR analysis of RCLE and RC@AgNPs was done to reveal functional groups present in the extract of R. cordifolia and present on the surface of AgNPs. Table 2 shows the wavenumber and interpretation of possible functional groups of RC@AgNPs and RCLE. Figure 8a-b shows the FT-IR spectrum of RC@AgNPs and RCLE. The leaf extract contains various phytochemicals, which might be responsible for the reduction and stabilization of Ag + to Ag 0 and form RC@AgNPs. A similar pattern of the result was found with Ageratum houstonianum extracts and resulting NPs 22 .
Zeta particle size and zeta potential of RC@AgNPs. Dynamic light scattering (DLS) and Laser Doppler Electrophoresis (LDE) measurements were carried out to determine the particle size distribution and zeta potential of RC@AgNPs, respectively in an aqueous solution. The resulting average zeta particle size is 183.76 nm, with a polydispersity index (PDI) of 26.2, as shown in Fig. 9a. Because DLS measurements were dependent on the

Applications of RC@AgNPs
Anticancer activity. MTT assay was performed on A431 and B16F10 cells to determine the cytotoxic activity of RC@AgNPs. In this assay, yellow color dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) is reduced by the mitochondrial enzyme succinate dehydrogenase, which leads to the formation of purple-blue formazan crystals. Since it is a colorimetric measurement, the absorbance is recorded for the results.
Higher the cytotoxicity of NPs lower will be the viability of cancer cells (human squamous cell carcinoma: A431 and mouse melanoma: B16F10). The results of the in vitro assays suggest the cytotoxic activity of RC@AgNPs against the A431 and B16F10 cells (Fig. 10). In the results, it was found that there was a concentration-dependent decrease in the cell viability of both A431 and B16F10 cells at both time points. In the case of A431 cells, after 24 h of treatment, there was a 10%, 20%, 50%, and 80% decrease in cell viability at 10, 20, 50, and 100 µg/mL of concentration, respectively (Fig. 10a,i). After 48 h, the decrease was around 50 and 80% at higher concentrations of 50 and 100 µg/mL (Fig. 10b,i). In B16F10 cells after 24 h of treatment the decrease in cell viability was 20%, 30%, 70% and 90% at 10, 20, 50 and 100 µg/mL of concentration respectively (Fig. 10a,ii). Treatment with RC@AgNPs for 48 h caused a 50% decrease at a lower concentration of 10 µg/mL and a higher concentration of 100 µg/mL, causing a remarkable ~ 80% decrease in the cell viability of B16F10 cells (Fig. 10b,ii). The IC 50 value of RC@AgNPs against A431 and B16F10 cells were 54.09 and 36.63 µg/mL, respectively. There are some reports on the cytotoxic activity of AgNPs against different cancer cell lines. Mainly, the activity is attributed to the oxidative stress induced by AgNPs and induction of apoptosis via caspase-dependent pathway 20 . In a previous investigation, Bryophyllum pinnatum mediated BP-AgNPs also displayed cytotoxicity against the same cell lines 49     Few reports are published on the antiproliferative activity of R. cordifolia. Cytotoxic activity of ZnO and CeO 2 NPs from R. cordifolia leaf extract has been reported against MG-63, a human osteosarcoma cell line 52 . Cytotoxic activity of methanolic plant extracts of R. cordifolia is also reported against Hela and Hep-2 cell lines, with respective IC 50 values of 23.12 and 11.92 µg/Ml 53 . R. cordifolia has shown antiproliferative activity against a wide range of cancer cells, such as human colon carcinoma (HT-29), human breast carcinoma (MCF-7), and human liver carcinoma (HepG2) cell lines, and human colon carcinoma (HT-29) 54 . The probable mechanism for the antiproliferative activity might be inhibition of the DNA synthesis. R. cordiofolia can inhibit the incorporation of [3H] thymidine and c-fos gene expression, and the c-fos gene is responsible for the proliferation and differentiation of cells 55 .
Further, in a study done by Adwankar and Chitnis, they isolated a pure compound RC-18 from R. cordifolia, which was found to have antitumor activity against different types of in vivo solid tumor models (B16 melanoma) 53 . The cyclic hexapeptides of R. cordifolia were also reported for their anticancer activity by inhibiting the process of protein synthesis. It does so by binding to the 80 s subunit of the ribosome and thus inhibiting the binding of aminoacyl-tRNA and translocation of peptidyl-tRNA. Secondary metabolites found in R. cordifolia L., such as purpurin and munjistin have antitumor activity 20 . They may be contributing to the anticancer activity of R. cordifolia against different types of cancer 18-20 . DNA interaction capability. This is one of the most frequently used techniques to investigate the interactions between CT-DNA and NPs. This study relies on interaction to change or shift the maximum absorption of AgNPs. According to Topală et al., the hypochromic, hypsochromic (blue shift), and bathochromic (redshift) effects are caused by intercalative binding mode, whereas hyperchromism is caused by groove binding (minor and major), electrostatic interactions, and hydrogen bonding 56 . Chandraker et al. proposed that the stability of CT-DNA be tested at 15-min intervals before introducing NPs, and the absorption peak be studied for 1 hour 25 . Therefore, UV-Visible titration analyses were performed to examine the interaction between CR@AgNPs and CT-DNA.
To study the interactive effect of CR@AgNPs with CT-DNA, the varied concentration of CR@AgNPs (0.010 to 0.090 nM) was used. UV-visible spectra of CT-DNA at increasing concentrations of CR@AgNPs and constant CT-DNA 250 µM are shown in Fig. 11. The results indicate that when RC@AgNPs are introduced, the absorption at 253 nm increases without a detectable change in the value of λ max . According to Rahban et al. alteration in the secondary structure of DNA is due to hyperchromic effects 57 . The RC@AgNPs absorption spectra also showed a bathochromic effect from 453 to 463 nm. The hyperchromic effect in CT-DNA absorption spectra and the bathochromic effect in CR@AgNPs absorption spectra indicate a strong interaction between both. Only a few research on the interaction between NPs and DNA have been reported 58,59 . According to a prior study, the N7 atoms of guanine and adenine bases are likely contact sites in CT-DNA with NPs, while the N3 atoms of cytosine and thymine bases are involved in hydrogen bonding 25 . Antifungal activity of CR@AgNPs. Aspergillus flavus is a serious pathogen causing preharvest and postharvest infections of nuts, cereals, and legumes. Aflatoxins, produced by certain strains of A. flavus can lead to neutropenia, immunosuppression, acute hepatitis, and hepatocellular carcinona 60 . The antifungal activity of the phytofabricated RC@AgNPs was assessed by observing the radial growth of the mycelial colony in all the www.nature.com/scientificreports/ treatments. Figure 12 depicts the effect of RC@AgNP inhibition at various doses against A. flavus over a six-day incubation period. In our research, we found that the concentration of RC@AgNPs had a direct impact on A. flavus mycelia growth. When the concentrations of RC@AgNPs increased, the inhibition of radial growth (IRG) increased as well (Fig. 13)   assays. Both the assays are comparatively quick and sensitive for analyzing the antioxidant activity of a particular substance compared to others 25 . The scavenging impact of RC@AgNPs on DPPH (Fig. 14a) and ABTS (Fig. 14b) free radicals was noticed in a dose-dependent manner. DPPH is a strong oxidant with an absorption wavelength range of 515-520 nm. between 515-520 nm. DPPH has a purple-blue color that changes into a bright yellow or colorless when it reacts with a substance having strong; stable free radical scavenging property 65 . The intensity of color transition is dependent on the total dose and nature of the sample. The DPPH radical inhibition of RC@ AgNPs was 59.43 ± 0.296 to 89.58 ± 0.221% in the concentration range of 31.25 to 500 µg/mL. The same concentration of the standard reference (ascorbic acid) showed 75.64 ± 0.08 to 98.94 ± 0.03% inhibition (Fig. 14a).
Similarly, at concentrations of 31.25 to 500 mg/mL of RC@AgNPs, ABTS free radical inhibition ranged from 26.54 ± 0.05 to 85.96 ± 0.10%. Ascorbic acid, on the other hand, was inhibited from 38.39 ± 0.05 to 88.09 ± 0.1% (Fig. 14b). AgNPs mediated by Prosopis farcta fruit extract, Cucumis prophetarum leaf extract, and blackcurrant pomace extract have similar DPPH free radical scavenging capabilities [66][67][68] . The capacity of the NPs to quench neutral and cationic radicals was examined, indicating that the RC@AgNPs may generate stable neutral radicals by DPPH and free cation radicals from ABTS. The antioxidant mechanism was distinct in both of the assays used. The DPPH test demonstrates AgNPs' ability to transfer electrons and neutralize reactive DPPH free radicals in the reaction medium 69 . The ABTS assay identifies cationic-free radical scavenging activities utilizing both eland H + transport mechanisms 70 .

Conclusions
R. cordifolia-mediated AgNPs were synthesized following a green synthesis and eco-friendly approach. The biogenic AgNPs are formed during the reduction of Ag + ions by RCLE. The phytosynthesized RC@AgNPs were spherical with an average size of ~ 20.98 nm. Different biomedical activities (DNA-binding, antifungal, and antioxidant) of RC@AgNPs were explored, and it was found to have significant cytotoxic activity against skin cancer cell lines, A431 and B16F10. This is the first report on R. Cordifolia leaf-mediated biocompatible and bio-fabricated AgNPs. Based on the remarkable biological activities, RC@AgNPs is hereby recommended for its uses in biomedical applications with elaborated research. The study motivates further therapeutic research in the fields of cancer and antifungal agents.  License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.