Biological reaction mediated engineered AuNPs facilitated delivery encore the anticancer, antiglycation, and antidiabetic potential of garcinol

2.1 Materials

Garcinol (G) was obtained from Cayman Chemical, Michigan, U.S.A., Chloroauric acid H[AuCl₄] was purchased from Sigma-Aldrich Chemicals Private Limited, Bangalore-India. Bovine serum albumin (BSA) along with 2′-Deoxyribose sugar, nitro blue tetrazolium (NBT), trichloroacetic acid (TCA), oxalic acid, sodium bicarbonate, sodium carbonate, disodium hydrogen phosphate, sodium chloride, sodium hydroxide was obtained from HiMedia Laboratories Pvt ltd, Mumbai, Maharashtra (India). Ethanol and sodium azide were obtained from Merck, and 2, 4, 6 trinitrobenzene 1 sulphonic acid (TNBS) was obtained from G-Biosciences (St. Louis, U.S.A). Phenanthrenequinone and triphenylphosphine were purchased from Sigma Aldrich. Dinitrophenylhydrazine (DNPH) and ethyl acetate were obtained from Rankem (VWR Lab Products Pvt. ltd. Bengaluru-India). Guanidine hydrochloride was obtained from S.D. Fine-chem limited, hydrochloric acid (HCL) from Thermo Fisher Scientific India Pvt. ltd, Maharashtra-India. NBT solution was prepared.

2.2 Glycation reaction mediated synthesis of AuNPs

The process was carried out in two steps, firstly glycation assay has been prepared, afterwards H[AuCl₄] was supplied to the reaction mixture for the synthesis of AuNPs.

For glycation assay, a reaction having BSA (6 µM) and 2′-Deoxyribose (100 mM) in 0.1 M phosphate buffer saline (PBS), pH 7.2 with 0.05 % sodium azide was set for 19 days at 37 °C and this reaction mixture was utilized as control, additionally in another similar reaction mixture,1 mM (H[AuCl₄]) was supplied at the third day to the reaction mixture. The colour change in in the reaction mixture containing (H[AuCl4]) indicated the synthesis of gold nanoparticles (AuNPs) which was further confirmed by UV–Visible spectroscopy (Rafi et al., 2020). Further, a similar reaction except the addition of H[AuCl₄] was used as a control (Rafi et al., 2020). The extraction of AuNPs from the reaction mixture was extracted/recovered through centrifugation (10,000 g) for 30 min. All unbound components of AuNPs were removed by flushing them with 50 % ethanol and twice with Milli Q water.

2.3 Bioconjugation of synthesized AuNPs with G

In-vitro glycation reaction mediated synthesized AuNPs were coupled with the herbal drug G utilising a coupling agent or activator, 1-ethyl-3-(3-dimethyl) carbodiimide (EDC). A reaction volume of 5 ml had been made including 250 μg G, 250 μg AuNPs, and 50 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer and incubated at 40 °C for 3–4 h. The trace amounts of EDC were added dropwise at regular intervals during the aforesaid incubation time period.

2.4 Loading efficiency (LE) of G on G-AuNPs

By analysing the difference in wavelength at 276 nm, before and after the bioconjugation, the total loading percentage of G on G-AuNPs was determined (Pan et al., 2002). Finally, the overall loading percentage on G-AuNPs is calculated by adding these absorbance intensities together. By entering the values of A and B into the following equation, the loading efficiency of G was calculated. $$\mathrm{P}\mathrm{e}\mathrm{r}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{l}\mathrm{o}\mathrm{a}\mathrm{d}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{o}\mathrm{f}\mathrm{G}\mathrm{o}\mathrm{n}\mathrm{G}-\mathrm{A}\mathrm{u}\mathrm{N}\mathrm{P}\mathrm{s}=\frac{\mathrm{A}-\mathrm{B}}{\mathrm{A}}\times 100$$

Where A is the total quantity of drug G added for bioconjugation with AuNPs, and

B is the absorbance intensity of G in the supernatant post bioconjugation process.

Both A and B were measured at 276 nm, which resembles the pure G's characteristic peak. (Pan et al., 2002). The sum of the unbound drug during bioconjugation with AuNPs was measured using the standard curve of the native drug G, established at the wavelength of 276 nm. The amount of bioconjugated drug was determined by subtracting the cumulative amount of drug added in the reaction mixture from the amount of unbound drug. The following equation was used to quantify the precise quantity of the bioconjugated drug. $$\mathrm{P}\mathrm{e}\mathrm{r}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{G}\mathrm{b}\mathrm{i}\mathrm{o}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{j}\mathrm{u}\mathrm{g}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d}=\frac{\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{a}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{G}\mathrm{b}\mathrm{i}\mathrm{o}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{j}\mathrm{u}\mathrm{g}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d}}{\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{a}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{G}\mathrm{a}\mathrm{d}\mathrm{d}\mathrm{e}\mathrm{d}}\times 100$$

2.5 Characterization of synthesized AuNPs and G-AuNPs by various physical techniques

For spectroscopy analysis, a Shimadzu dual-beam spectrophotometer (model UV-1601 PC) with a resolution of 1 nm was used. TEM (Transmission Electron Microscopy) was performed on a Tecnai G2 Spirit TEM instrument assembled with BioTwin lens configuration (Hillsboro, OR, USA) with an accelerating voltage of 80 kV. The sample was made by drying a drop of AuNP and G-AuNP solution on carbon-coated TEM copper grids and then measuring it under the microscope. A dynamic light scattering (DLS) particle size analyzer was used to determine the mean particle size of G-AuNPs (Zeta Sizer Nano-ZS, Model ZEN3600, Malvern Instrument ltd, Malvern, UK). A disposable sizing cuvette of 1.5 ml (DTS0112 small volume) was used to collect the sample. In deionized water, the sample powder was dissolved to a concentration of 0.5 percent (w/v). Sonic & Material Inc., New Town, CT, USA, was used to sonicate the particles for 1 min at 30 W (20 sec on and 5 sec off). The sum of triplicate measurements for a single sample was used to calculate the mean particle size. A Zeta Sizer Nano-ZS, Model ZEN3600, was used to calculate the surface charge of G-AuNPs (Malvern Instrument ltd, Malvern, UK). Fourier transform infrared spectroscopy (FTIR) was used to analyze the conformational shifts and binding in G-AuNPs which is performed on a Shimadzu FTIR-8201 PC instrument in the diffuse reflectance mode with a resolution of 4 cm-1. Two hundred fifty-six scans of the bioconjugate film were taken in the range 400–4000 cm-1 to achieve good signal-to-noise ratios. By coupling the inductively coupled plasma mass spectroscopy (ICP-MS) analysis reading with drug loading efficiency values, we also resolved the total number of drug molecules attached to each AuNP. After the successful bioconjugation of AuNPs with drug G, the secondary structural changes were determined by (Far-UV CD) through spectropolarimeter (Jasco J1500). Different secondary forms are represented by the spectrum between 200 and 260 nm using a 1 mm path length cuvette(Rafi et al., 2020)(Ashraf et al., 2015).

2.6 Antiglycation potential of G and G-AuNPs

For antiglycation experiments, six different reactions (total volume − 3 ml) were formulated in separate test tubes containing BSA (0.4 mg/ml), 2′-Deoxyribose sugar (100 mM) in phosphate buffer saline (PBS) at pH 7.4 comprising 0.05 % sodium azide with Pure G (concentrations − 1,2 and 4 mM) and G-AuNPs (Concentrations − 16.25, 32.55, and 65.1 µM) at different concentrations to analyse the potential of inhibitors. Additionally, the above reaction mixture without any inhibitor(s) was treated as a control. The reaction mixtures were incubated for sixteen days at 37 °C and dialysis was performed to remove unbound constituents.

2.7 Determination of keto-amine content

Protein glycation generates Amadori products/ketoamines, which further transform into AGEs, even more stable compounds. Therefore, the inhibition of keto-amine formation via pure G, AuNPs and G-AuNPs was examined by nitroblue tetrazolium assay (NBT) (Ravindran et al., 2010). For NBT assay, 20 µl modified BSA (control) was mixed with NBT (0.25 mM) and different concentrations of pure G and G-AuNPs in separate reactions in 180 µl sodium carbonate-bicarbonate buffer (100 mM; pH 10.8) at 31 °C for 40 min. Subsequently, the absorption intensity was measured on daily basis at 525 nm until a decline in the absorbance intensity was observed and finally the keto-amine content was calculated using the formula below (Ahmad et al., 2012) using the extension coefficient of 12,640 M⁻¹cm⁻¹. $$\mathrm{K}\mathrm{e}\mathrm{t}\mathrm{o}\mathrm{a}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{t}=\frac{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{s}\mathrm{i}\mathrm{t}\mathrm{y}\mathrm{a}\mathrm{t}525\mathrm{n}\mathrm{m}}{12640}\times {10}^6$$

2.8 Quantification of hydroxymethylfurfural (HMF) content

Thiobarbituric assay (TBA) was used to quantify the HMF content in unmodified BSA (native), glycated BSA (control), glycated BSA treated with G and G-AuNPs (test reactions) (Ansari et al., 2009). HMF is a glycation intermediate generated during the development of early glycation end products. The HMF concentration was determined by combining the aforementioned samples with 400 µl of 1 M oxalic acid, keeping the reaction mixture at 100 °C for 1 h and then cooling to room temperature. The samples were then precipitated with 500 µl of 40 % trichloroacetic acid at 24 °C for 10 min followed by centrifugation at 8,000 rpm at 24 °C. The supernatant was removed and 500 µl thiobarbituric acid (0.05 M) was added, followed by 30 min of incubation at 40 °C. At 443 nm, the absorption was measured against thiobarbituric acid, which served as a blank, and the concentration of HMF (nmol/ml) was determined utilizing molar extinction coefficient of 4x10⁴ M⁻¹cm⁻¹.

2.9 Determination of carbonyl content

Carbonyls attached to proteins are intermediates in the formation of AGEs, and their concentration increases with the degree of glycation. Therefore, the amount of carbonyl content in native samples containing unmodified BSA (native), glycated BSA (control), glycated BSA treated with G and G-AuNPs (test reactions) was determined. 400 µl of DNPH solution was mixed with 100 µl of each of the above-mentioned samples, and the mixture was incubated in the dark for 60 min in order to precipitate the specimens. The reaction was completed by adding 500 µl of trichloroacetic acid to the mixture. Following that, a 5-min incubation on ice was performed, followed by centrifugation at 4 °C and 10,000 rpm. Subsequently, the precipitates were washed three times with ethanol-ethyl acetate in a ratio of 1:1(v/v), and the precipitate was dissolved using 6 M guanidine hydrochloride. At 360 nm, the absorbance was measured against guanidine hydrochloride, which was used as a blank. Carbonyl content was calculated using an extinction coefficient of 22000 M⁻¹cm⁻¹, as previously published (Ansari et al., 2009).

2.10 Determination of unbound lysine content

Lysine residues are glycation-prone; therefore, the level of unbound lysine residues tends to decrease during glycation. Thus, the proportion of free lysine was measured using the 2, 4, 6 trinitrobenzenes-1-sulphonic acid (TNBS) method in unmodified BSA (native), glycated BSA (control), glycated BSA treated with G and G-AuNPs (test reactions). During the experiment, 500 µl of each of the above-mentioned samples was thoroughly mixed with 500 µl of 4 % (w/v) sodium bicarbonate buffer and 500 µl of 0.1 % aqueous TNBS solution. After 2 h of incubation at 40 °C, 25 ml of concentrated HCL was added, afterwards, the samples were incubated for another 90 min at 110 °C. Subsequently, after cooling the reaction mixture to room temperature, the samples were centrifuged for 10 min at 3000 rpm and the supernatant was added with ether (5 ml) to eliminate the TNP α amino complex. The resultant solution was maintained in boiling water to allow the leftover ether to evaporate. Finally, the absorbance of the reaction mixture was measured at 346 nm against a blank using a quartz cuvette with a path length of 1 cm on a bio spectrum - kinetics spectrophotometer (Eppendorf). (S. D. Sharma et al., 2002). the percentage of unmodified lysine residues was calculated using the following equation: Percent of unmodified lysine residues = [(Absorbance of glycated BSA- Absorbance of unmodified or inhibitor treated BSA)/ (Absorbance of glycated BSA)] × 100

2.11 Quantification of unbound arginine residues

Along with occurrence at lysine residues, the glycation reaction also tends to occur at the arginine residues making it a prone site for glycation. These arginine residues produce fluorescence when interact with phenanthrenequinone, therefore we assessed the percentage of unreacted arginine in unmodified BSA (native), glycated BSA (control), glycated BSA treated with G and G-AuNPs (test reactions) using standard method (Smith & MacQuarrie, 1978). For the quantification procedure, 500 µl of 200 µM phenanthrenequinone was added to each sample and properly mixed, followed by the addition of 0.5 ml of 2 N NaOH and incubation at 30 °C for 60 min. Following incubation, the samples were treated with 0.5 ml HCl (1.2 M), and the fluorescence spectra of all the above-mentioned samples were recorded in the wavelength range of 350–450 nm using a 312 nm excitation wavelength (Smith & MacQuarrie, 1978).

2.12 Fluorescence studies for determination of AGEs in G and G-AuNPs treated samples

As mentioned in a prior study, fluorescence spectra were recorded (Rafi et al., 2020)(Jairajpuri et al., 2015) on carrying eclipse fluorescence spectrophotometer G9550-64000 by Agilent technologies has a temperature controller with a steady temperature keeper with a precision of 0.1 °C and a quartz cell with a 1 cm path length. The unmodified BSA (native), glycated-BSA (control) and G plus G-AuNPs treated samples were excited at wavelength of 340 nm, and emission intensities were measured between 330 and 550 nm. (Sattarahmady et al., 2007). The following equation calculated percent change in the fluorescence Intensities (% FI): Increase in fluorescence intensity = [(FI of modified BSA - FI of unmodified or inhibitor treated BSA)/ (FI of modified BSA)] × 100

2.13 α-amylase inhibition assay of G and G-AuNPs

The slightly modified standard procedure was adopted (Bernfeld, 1955) to investigate the in vitro inhibitory potential of G, AuNPs and G-AuNPs against porcine pancreatic α-amylase. The enzyme was dissolved in the ice-cold phosphate buffer (20 mM, pH 6.7) in order to achieve the final concentration of 0.15 unit/mL. Except the blank, 25 μl of α-amylase was mixed with 10 μl of samples (glycated BSA as a control, G, AuNPs and G-AuNPs) in separate test tubes. The mixtures were pre-incubated for 20 min at 37 °C. After incubation, 25 μl substrate (0.5 % w/v starch in 20 mM phosphate buffer; pH 6.7) was added to each test tube in order to start the reaction. Further, the vortexed mixture was incubated for 15 min at 37 °C. 200 µl of DNS color reagent (40 mM DNS, 1 M K-Na tartarate and 0.4 M NaOH) was added, vortexed and boiled in a water bath at 100 °C for 10 min. Subsequently, the mixture was cooled down, and the absorbance was read at 540 nm. Each experiment was performed in triplicates including the blank. Inhibition was calculated using the formula:

Percent inhibition = 100 − (mean product in sample/mean product in control) × 100.

2.14 In-vitro cytotoxicity analysis & anticancer potential of G & G-AuNPs

3.1 Characterization of synthesized AuNPs and G-AuNPs

A variety of bionanotechnological applications have been made possible by the unique properties and surface activities of gold nanoparticles (AuNPs) (Al Hagbani et al., 2022)(Alshahrani et al., 2021)(Khan et al., 2021). It is possible to synthesize AuNPs by using a wide range of techniques. All of these methods make use of reducing and encapsulating drugs or biomolecules. It is also possible to add ligands, pharmaceutical chemicals and other moieties to the GNP framework. By minimizing the necessity for a reducing/capping agent, this approach also reduces the danger of residual contamination caused by an external chemical or biomolecule(Khan et al., 2021). The synthesis of AuNPs was validated in this study by a steady colour shift of the reaction solution to ruby red after incubation. Therefore, surface plasmon resonance (SPR) in AuNPs explains the colour change. The formation of early glycation end products (EGEPs) during the glycation of BSA aided in the synthesis of AuNPs in this investigation. According to NBT data as represented in our previous study (Rafi et al., 2020), AuNPs of a significant size were generated on the day when EGEP creation was at its peak (Day 8 of the reaction), suggesting that EGEPs are responsible for reducing the gold salt into AuNPs and that, after reduction, they may act as a cap on AuNPs.

3.2 UV–Visible spectroscopy

The UV–Visible absorption spectra of G-AuNPs displayed a surface plasmon resonance (SPR) band centred at 536 nm including the characteristic peak of the drug centred at 276 nm. Compared to the SPR band of synthesized AuNPs, an 8 nm redshift is observed resulting from bioconjugation of the AuNPs with G, as shown in Fig. 2 A. The aforesaid redshift is observed due to the capping of G over the surface of the AuNPs resulting by bioconjugation procedure.

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Fig. 2

(A) SPR band of AuNPs (527 nm) & G-AuNPs (536 nm) (B) TEM micrograph of G-AuNPs (26.5 d. nm) at 50 nm and 100 nm resolution(inset), (C) Size distribution profile of G-AuNPs, DLS (∼79.5 d. nm) (D) Zeta potential (-18.6 mV) of G-AuNPs (E) FTIR measurements of AuNPs (F) FTIR measurements of G-AuNPs performed in the range 4000–450 cm⁻¹ (G) Far UV CD spectra of modified BSA sample (control), AuNPs and G-AuNPs (H) Standard curve of pure drug garcinol plotted at varying concentrations at its characteristic peak (276 nm). The investigations are the average of three independent experiments/scans performed under identical experimental conditions.

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3.3 TEM, DLS and zeta potential

The core size of G-AuNPs has been validated by TEM micrographs and found to be ∼ 29 d. nm (Fig. 2 B). The findings of the TEM micrograph indicated an increase in the size of the AuNPs post bioconjugation with G. Also, the blurr image of TEM indicated the bioconjugation. Further, the UV visible spectroscopy showed the characteristic peak (276 nm) of the drug G demonstrated that G is successfully bioconjugated over the surface of the AuNPs (Fig. 2 A). TEM computes the size of nanoparticles by direct transmitting electrons that have only inorganic core details and that do not contain a hydration layer.

To examine the elevation in hydrodynamic radius of G-AuNPs in contrast to AuNPs, DLS was also performed. In an aqueous solution, the surface of the nanoparticles is bound by a thin electrical dipole film in the liquid. Therefore, when the DLS of the AuNPs is done, the hydrodynamic particle layer and the coating materials (solvent layer that is covered by the nanoparticle) is analyzed (Berne & Pecora, 2000). DLS of the synthesized AuNPs was found to be 71 d. nm, whereas, after the bioconjugation with G, G-AuNPs showed the size of 79.5 d. nm including the hydrodynamic layer (Fig. 2 C). Besides, zeta potential is responsible for propagating, aggregation, ionization, exposure, or shielding of charged moieties and nanoparticle adsorption. (Rabinovich-Guilatt et al., 2004). The synthesized nanoparticles zeta potential was found to be −15.9 mV, and of G-AuNPs were observed to be −18.4 mV (Fig. 2 D). The functional group of lysine-rich early glycation end products that capped the AuNPs is responsible for the nanoparticle’s negative charge and attributed to the high stability of the AuNPs and G-AuNPs(Ansari & Dash, 2013). The differences in AuNPs and G-AuNPs zeta potential is attributed to the bioconjugation of G to AuNPs.

3.4 Circular dichroism analysis

CD was performed to assess if lysine-rich(Ansari & Dash, 2013) early glycation end products have undergone conformational changes after successful bioconjugation with AuNPs. Two negative bands at 208 and 222 nm distinguish the far-UV CD signal. These bands resulted by amide group π- π* and n- π* transitions, are typical in proteins with a high helical content(Greenfield, 1999). CD of the modified BSA sample (control), AuNPs and G-AuNPs were performed. The observed results portrayed a loss of 88.19 % in negative ellipticity at 208 nm in G-AuNPs against negative ellipticity of AuNPs at 208 nm. However, 56.13 % loss in negative ellipticity at 222 nm has been observed in G-AuNPs comparative to the negative ellipticity of AuNPs (222 nm) (Fig. 2 G). Conformational modifications that occurred after the bioconjugation of G with AuNPs culminated in a decreased percentage of negative ellipticity. Our findings are well justified based on the previous studies which addressed that ellipticity transition after bioconjugation with gold nanoparticles are resulted by structural adjustment (Matei et al., 2019).

3.5 Fourier transform infra-red spectroscopy

FTIR spectra of AuNPs and G-AuNPs are shown in Fig. 2 E & F. The AuNPs spectrum revealed distinct frequencies at 3429.1, 2102.86, 1,641.26, 1081.77 cm⁻¹ with a transmittance of 3.78, 31.62, 12.17, and 30.74 % respectively while the G-AuNP spectrum obtained after the bioconjugation of drug G with AuNPs represented distinct frequencies at 3405.29, 2107.67, 16.42.46 and 1083.26 with a transmittance of 0.1, 25.06, 2.8 and 28.39 % respectively. These altered frequencies and transmittance are attributed to the structural modifications that occurred after the possible capping of G over AuNPs (Yamaguchi et al., 2000)(Fernando et al., 2019a). The existence of asymmetric C—H stretching vibrations in the range 3100–3000 cm⁻¹, which is the characteristic region for detection of C—H stretching vibrations, can be seen in aromatic organic compounds; derivatives of benzene(Vinod et al., 2015)(Bellamy, 2013)(Puviarasan et al., 2002). A broad peak at 3405.29 cm⁻¹ (Krishnaprabha & Pattabi, 2016) allocated to C—H stretching vibrations observed in our result confirms the presence of G over the surface of AuNPs after efficient bioconjugation with AuNPs comparative to the FTIR of the sample containing only synthesized AuNPs(Fernando et al., 2019b).

3.6 Analysis of drug loading efficiency

The loading percentage of G over AuNPs was calculated and found to be 78.6 % attributed to efficient capping of G over AuNPs. The recorded values of A and B were 2.035 and 0.554 respectively. The standard curve of pure drug G curve was plotted at 276 nm (Fig. 2 H), and the unbound drug was calculated from the standard curve. The amount of the bioconjugated drug was computed by excluding unbound drugs from the total amount of drug used in the reaction mixture. The exact quantity of G bioconjugated with AuNPs was found to be 39.3 µg/ml of the reaction mixture.

3.7 Inductively coupled plasma mass spectrometry (ICP-MS) analysis

The elemental analysis of AuNPs and G- AuNPs revealed that an ample amount of drug G is loaded over the surface of AuNPs. The number of AuNPs under ICP-MS was found to be 3.485 X 10¹⁶ against 38746 ppb of gold in AuNPs. Similarly, 18149 ppb correspond to 7.08 G molecules/AuNPs was established with ICP-MS analysis (Table 1).

3.8 Antiglycation studies of G and G-AuNPs

3.8.1

3.8.1 Early glycation adducts, keto-amine inhibition exerted by G and G-AuNPs

Glycation reaction is initiated by the establishment of Schiff's base, followed by the formation of keto-amines or amadori products which are known to be early markers of the glycation process. To further suppress the establishment of AGEs, the inhibitory effect of G and G-AuNPs at different concentrations was evaluated by measurement of the keto-amines produced through NBT reduction assay. As shown in the Fig. 3, the keto-amine content examined in glycated BSA was found to be 44.02 ± 0.22 nmol/mg in comparison to unmodified BSA (native), which was found 1.92 ± 0.04. Furthermore, it has been observed that test inhibitors G and G-AuNPs and standard inhibitor AG inhibited amadori products formation to different degrees. Under experimental conditions, it has been observed that the number of keto-amines produced in the presence of 4 mM AG is 32.79 ± 0.03, while in the presence of 1 mM, 2 mM & 4 mM G, the keto-amines produced were 34.95 ± 0.05, 28.87 ± 0.09 and 17.97 ± 0.10 nmol/mg respectively (Fig. 3 A & B). However, in the presence of different concentrations (16.25, 35.55 and 65.1 µM) of G-AuNPs the keto-amines produced were found to be 39.45 ± 0.05, 35.79 ± 0.12 and 32.50 ± 0.14 nmol/mg, respectively. Nevertheless, in the comparison to G-AuNPs, the AuNPs at a concentration of 0.5 mM were observed to produce 41.77 ± 0.19 nmol/mg of keto-amine (Fig. 3 C & D). The results of the NBT assay indicated that G is a significant inhibitor of amadori product development but the results also justified that G-AuNPs have also inhibited the keto-amine formation at much lower concentration than pure G. Several inhibitors of AGEs are known to function at the late stage of glycation, while others play their inhibitory role in the early stages of glycation. (Mesías et al., 2013). The NBT reduction assay test confirmed the inhibitory role of G and G-AuNPs in the early stages of glycation by inhibiting the formation of keto-amines.

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Fig. 3

(A) NBT reduction assay for unmodified BSA (native), glycated BSA (control) and glycated samples treated with varying concentrations of G and AG (standard inhibitor) (B) The bar diagram represents keto-amine contents of unmodified BSA (native), glycated BSA (control) and glycated samples treated with varying concentrations with G and AG (C) NBT reduction assay for unmodified BSA (native), glycated BSA (control) and glycated samples treated with varying concentrations of AuNPs, G-AuNPs and AG (standard inhibitor) (D) The bar diagram represents keto-amine contents of unmodified BSA (native), glycated BSA (control) and glycated BSA samples treated with AG, AuNPs and G-AuNPs. The data represented are the mean ± SD of three determinations under identical experimental conditions. Significantly different from control at *** p < 0.001, significantly different from control at ** p < 0.01, non-significantly different from control at ns p > 0.05.

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3.8.2

3.8.2 Inhibition of hydroxymethyl furfural content by G and G-AuNPs

Similar to keto-amine moieties, HMF is an intermediate product of the glycation process. HMF is a thiobarbituric reactive substance resulting from hydrolysis in the earliest stages of glycation. The HMF content in the glycated BSA sample (control) was found to be increased by 89.65 % (9.09 ± 0.01 nmol/ml) compared with the unmodified native BSA (0.94 ± 0.04 nmol). However, in the presence of different concentrations of test inhibitor G (1 mM, 2 mM & 4 mM), the HMF content was reduced to 6.92 ± 0.02, 5.71 ± 0.03 and 3.63 ± 0.02 nmol/ml, respectively, representing 23.91 ± 0.23, 37.18 ± 0.29 and 60.10 ± 0.23 % reduction in HMF content. Whereas AG (4 mM) was used as a standard inhibitor, showed 6.45 ± 0.03 nmol/ml of HMF justifying a 29.08 ± 0.28 % decrease in the HMF comparatively with the control reaction (Fig. 4 A).

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Fig. 4

(A) Bar diagram showing percent decrease in HMF/CC content in glycated BSA samples treated with varying concentrations of G (B) Percent decrease in HMF/CC content in glycated BSA samples treated with varying concentrations of G-AuNPs. AG is used as a standard inhibitor (C) Bar diagram represents a percent of unreacted lysine residues in glycated BSA (control) and samples treated with varying concentrations of G (D) Percent of unreacted lysine residues in glycated BSA (control) and samples treated with varying concentrations of G-AuNPs. The data represented are the mean ± SD of three determinations under identical experimental conditions. Significantly different from control at *** p < 0.001, significantly different from control at ** p < 0.01, non-significantly different from control at ns p > 0.05.

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However, in the presence of test inhibitor G-AuNPs at concentrations of 16.25, 35.55, and 65.1 µM, the amount of HMF recorded to 7.91 ± 0.04, 7.10 ± 0.02 and 6.59 ± 0.03 nmol/ml, respectively justifying a 12.98 ± 0.48, 21.89 ± 0.19 and 27.51 ± 0.38 % decrease in the HMF comparative to the HMF content observed in the control sample. However, AuNPs (0.5 mM) have shown a slight reduction of 9.90 ± 0.11 % in HMF compared with the control sample (Fig. 4 B). These decline in HMF content levels in the samples, which contain inhibitors in various concentrations, align with the NBT findings. The decrease in HMF levels suggests that the inhibitors prevent the structural changes in BSA caused by 2′-Deoxyribose (Siddiqui et al., 2018). In contrast to the pure G and aminoguanidine, G-AuNPs were found to be substantially strong inhibitor at much lower concentrations.

3.8.5

3.8.5 Inhibitory effect of G & G-AuNPs via unreacted arginine estimation

Glycation and the development of AGEs both are resulted from the binding of arginine residue to reducing sugar molecules. The percentage of unreacted arginine residues was calculated using phenanthrenequinone, which, when reacts with the arginine residues in the sample mixture, produces fluorescence in the range of 300–500 nm at the emission wavelength of 395 nm. During the estimation process, a substantial reduction of 61.1 % in the fluorescence intensity of glycated BSA (control) was observed in comparison to unmodified native BSA, implying that only 39.64 ± 0.85 % of (unreacted) arginine was not involved in the glycation process in the glycated BSA (control). In contrast, others are involved in the glycation reaction. Our findings showed that the samples treated with 1,2 and 4 mM of test inhibitor G showed 53.63 ± 0.68, 60.97 ± 0.60, and 77.92 ± 0.20 %, respectively of unreacted arginine residues, whereas the standard inhibitor AG (4 mM) treated samples showed 57.46 ± 0.66 % of unreacted arginine residues (Fig. 5 A & B).

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Fig. 5

Fluorescence spectroscopy of (A) unmodified BSA (native), glycated BSA (control) and samples treated with varying concentrations of G (B)The bar diagram represents the percent of free arginine residues in unmodified BSA (native), glycated BSA (control) and samples treated with varying concentrations of G. (C) Fluorescence spectroscopy of unmodified BSA (native), glycated BSA (control) and samples treated with varying concentrations of G-AuNPs, AG is used as a standard inhibitor. The spectra are the average of three determinations under identical experimental conditions (D). The bar diagram illustrates the percent of free arginine residues in unmodified BSA (native), glycated BSA (control) and samples treated with varying concentrations of G-AuNPs; the data represented are the mean ± SD of three determinations under identical experimental conditions. Significantly different from control at *** p < 0.001, significantly different from control at ** p < 0.01, non-significantly different from control at ns p > 0.05.

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Similarly, samples treated with 16.25, 35.55, and 65.1 µM of test inhibitor G-AuNP have shown 47.53 ± 0.46, 52.56 ± 0.50, 56.20 ± 0.38 % of unreacted arginine residues, respectively. Moreover, AuNPs treated samples showed 45.16 ± 0.73 % of unreacted arginine residues (Fig. 5 C & D) compared to unmodified BSA samples (native), the percentage of reacted arginine residues was determined by a decrease in fluorescence intensity (at the emission wavelength of 395 nm) after the glycation reaction. Because BSA contains a total of 23 arginine residues, it is more prone to glycation; thus, the percent reduction in unbound arginine residues is attributable to the BSA being modified by 2′-Deoxyribose. This is consistent with the fact that arginine residues had a significant role in the generation of AGEs during the reaction. Whereas the inhibitor-containing reactions contained significantly more unreacted arginine residues than the control reaction. It was deduced that inhibitors hindered the development of AGEs, hence minimising the role of lysine residues in the process.

3.8.6

3.8.6 Inhibition of AGEs via G and G-AuNPs

Glycation reaction leads to the development of fluorogenic AGEs. (Kessel et al., 2002), these fluorescent AGEs have heterocyclic structures that allow glycation adducts to exhibit fluorescence (Akhter et al., 2013). By using fluorescence spectroscopy, inhibition of fluorogenic AGEs in the samples was determined. The unmodified BSA (native), glycated BSA (control) and samples containing test inhibitors G and G-AuNPs and standard inhibitor AG at varying concentrations were all excited at 340 nm, with an emission wavelength of 433 nm (Fig. 6). It has been observed that the fluorescence intensity (FI) of glycated BSA (control) containing no inhibitor increased by 87.54 ± 0.20, which is a significant predictor of AGE formation after glycation. However, comparative to the glycated BSA (control) the samples treated with 1-, 2- and 4-mM G samples portrayed 24.77 ± 0.85, 43.00 ± 0.69 and 64.07 ± 0.06 %, respectively decrease in the FI, while the standard inhibitor AG(4 mM) showed 43.89 ± 0.47 % decrease in FI indicating the reduced formation of AGEs (Fig. 6 A & B).

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Fig. 6

Fluorescence spectra of (A) unmodified BSA (native), glycated BSA (control) and samples treated with varying concentrations of G (B)The bar diagram represents the keto-amine contents of unmodified BSA (native), glycated BSA (control) and samples treated with varying concentrations of G (C) Fluorescence spectra of unmodified BSA (native), glycated BSA (control) and samples treated with varying concentrations of G-AuNPs, AG is used as a standard inhibitor. The spectra are the average of three determinations under identical experimental conditions (D) The bar diagram represents a percent decrease in fluorescence intensity of unmodified BSA (native), glycated BSA (control) and samples treated with varying concentrations of G-AuNPs the data represented are the mean ± SD of three determinations under identical experimental conditions. Significantly different from control at *** p < 0.001, significantly different from control at ** p < 0.01, non-significantly different from control at ns p > 0.05.

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Additionally, the samples treated with 16.25, 35.55, and 65.1 µM of G-AuNP have shown 15.76 ± 0.72, 26.47 ± 0.71 and 27.25 ± 0.79 %, respectively decreases in FI. However, the sample containing AuNPs showed an 11.41 ± 0.80 % reduction in FI (Fig. 6 C & D). The antiglycation effects of G and G-AuNPs were found to be positively associated with free radical scavenging activity and significant antioxidant activity as observed previously (Hirata et al., 2005)(X. Wang et al., 2011).

3.9 α-amylase inhibition potential of G and G-AuNPs

The G and G-AuNPs were examined for the α-amylase inhibitory property to investigate the anti-diabetic potential. The hydrolysis of complex starches to maltose is derived by pancreas α-amylase. The inhibition of α-amylase results in delayed carbohydrate digestion and absorption, reducing the amount of postprandial glucose (Apostolidis et al., 2007; Shim et al., 2003). In the current analysis, G showed concentration dependent inhibition of α-amylase production with an IC₅₀ value of 8.9 µM (Fig. 7), which is significantly higher compared to the IC₅₀ value of standard inhibitor acarbose (0.118 mM). However, owing to confirmative shifts in α-amylase caused by the association of the AuNPs in AuNPs and G-AuNPs, there was no inhibition in the α-amylase activity upto the concentration of 65.1 µM (Deka et al., 2012). Furthermore, our findings showed that G is an effective α-amylase inhibitor and may be used for therapeutic use in humans due to significant inhibitory activity compared to acarbose. G intake from processed foods and beverages may help prevent diabetes and obesity in patients as well as treat diabetes.

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Fig. 7

Bar diagram representing percent inhibition of α-amylase exerted by varying concentrations of (A) G and (B) Standard inhibitor acarbose. The data represented are the mean ± SD of three determinations under identical experimental conditions. Significantly different from control at *** p < 0.001, significantly different from control at ** p < 0.01, non-significantly different from control at ns p > 0.05.

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3.10 In-vitro cytotoxic effect of G & G-AuNPs on HaCat and A549 cells

Using the MTT assay, the in-vitro cytotoxic effects of G and G-AuNPs on HaCat and A549cells were assessed at different concentrations (10, 20, 40,60, 80, 100, 120, 140 160 and 180 µM) of pure G and G-AuNPs with untreated corresponding cells serving as control. Similarly, the cytotoxicity analyses studies against same cell lines were calculated at various concentrations (5, 10, 15, 20, 25 and 30 µM) of G and G-AuNPs. It was found that cytotoxicity was dose-dependent. Pure G was found to inhibit cell fifty percent propagations of HaCat cells at 43.63 µM (Fig. 9B) whereas it halted half of the growth of A549cells at 28.7 µM (Fig. 9 C). However, no cytotoxicity was observed in case of AuNPs when tested against HaCat cells upto the concentration of 480 µM, but the IC 50 of AuNPs against A549cells were depicted to be 200.16 µM. Interestingly, G-AuNPs were found to produce IC 50 s against HaCat and A549cells at 86.05 µM (Fig. 9 B) and 13.3 µM (Fig. 9 C) concentrations, respectively. Our findings indicated that G-AuNPs had much lower IC₅₀ values than pure G, implying that the anti-cancer potential of pure G has increased enormously whereas cytotoxic effect minimised after bioconjugation with AuNPs.

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Fig. 8

Cytomorphological images of normal human keratinocyte HaCat cells after treatment with G and G AuNPs at a concentration of IC₂₅ (21.81 µM), IC₅₀ (43.63 µM) and IC₇₅(65.44 µM) of pure G and IC₂₅ (43.02 µM), IC₅₀ (86.05 µM) and IC₇₅(129.07 µM) of G-AuNPs.

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Fig. 9

(A) The cytotoxicity (dose-dependent) study of AuNPs on A549 cells (no cytotoxicity observed against Human keratinocyte HaCat cells up to the concentration of 480 µM) (B) The cytotoxicity (dose-dependent) study of pure G & G-AuNPs on Human keratinocyte HaCat cells (C) The cytotoxicity (dose-dependent) study of pure G & G-AuNPs on human lung adenocarcinoma A549 cells. The data represented are the mean ± SD of three determinations under identical experimental conditions. Significantly different from control at *** p < 0.001, significantly different from control at ** p < 0.01, non-significantly different from control at ns p > 0.05.

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4.1 Assessment of nuclear condensation and internalization of G and G-AuNPs

Using the fluorescent stain 4′, 6-diamidino-2-phenylindole (DAPI), potential mechanistic insights into G and G-AuNP internalization and their cellular uptake were explored. G-AuNPs could be taken up by cancerous cells via an endocytosis process, followed by drug release of G-AuNPs from the endosome/lysosome into the cancerous cells, where the interaction between herbal drug G and DNA occur (Hande, 1998). A549 cells were incubated with IC₅₀ concentrations of G and G-AuNPs (determined previously by MTT assay) for 24 h at 37 °C until being stained with DAPI. In contrast to the untreated cells serving as control, G and G-AuNPs exposure caused an apoptotic impact in cells (Fig. 10), including improved permeability, which reflected in condensed chromatin and a bright blue fluorescent condensed nucleus in A549 cells (Fig. 10). Our results showed that A549 cells treated with G and G-AuNPs have shown 69.23 % (Fig. 10 B) and 87.36 % (Fig. 10 C) in increase in fluorescence intensity respectively. This nuclear condensation observed in the G and G-AuNPs treated cells might be the result of the cytotoxic reaction caused by stress in the cells (Cutts et al., 2005).

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Fig. 10

Images of DAPI, DCFDA, and mitrotracker red-stained A549 cells treated with G and G-AuNPs.

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4.2 Detection of reactive oxygen species

Since DCFH-DA is an oxidation-sensitive fluorogenic marker for ROS in viable cells, it is used to calculate the extent of intracellular ROS produced in A549 cells due to interactions with G and G-AuNPs. The amount of fluorescent light detected was proportional to the number of reactive oxygen species generated by the cells (Fig. 10). The G treated cells showed a 65.2 % increase in fluorescence intensity (Fig. 10 E); however, G-AuNPs treated cells showed an increase of 86.1 % in fluorescence intensity (Fig. 10 F). The fluorescent intensity observed was directly proportional to the ROS production in the cells. A549 cells treated with G & G-AuNPs emitted bright fluorescence with distorted morphology due to plasma integrity changes resulting from ROS generation, but no noticeable fluorescence was observed in untreated cells.

4.3 Mitochondrial membrane potential estimation

Mitotracker Red CMX Ros is used to prove that G and G-AuNPs caused depolarization of the mitochondrial membrane in A549 lung cancer cells. Using mitochondrial potential, the dye was able to bind to the cells. The diminished strength of Mitotracker Red CMX Ros in G and G-AuNPs treated A549 cells was observed, suggesting that the mitochondrial membrane potential (ΔΨm) had been disturbed (Fig. 10). Observed results indicated a significant reduction of 48.63 % and 72.11 % in the intensity of Mitotracker Red CMX Ros in the G (Fig. 10 H) and G-AuNPs (Fig. 10 I) treated A549 cells respectively, justifying the disruption of the mitochondrial membrane potential (ΔΨm) (Fig. 10) of the A549 cells after the treatment. Our study portrayed that at G-AuNPs (13.3 µM) at much lower concentrations than pure G (28.7 µM) exerted more disruption in mitochondrial membrane potential.