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Received : 12-10-2024

Accepted : 06-11-2024



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Get Permission Mali, Bharti, Yadav, and Kansotiya: An in vitro antioxidant potential analysis of herbal silver nanoparticles synthesized from methanolic leaf extract of Cassia siamea


Introduction

Medicinal plants and natural products have been a rich source of medicinally active compounds. Plants include polyphenolic alkaloid compounds known as flavonoids such as resveratrol, curcumin, ginsenosides, triptolide, and others that have antioxidant activity. Antioxidant substances have antibacterial, anti-atherosclerotic, anti-tumour, antimutagenic, anti-carcinogenic and antiviral properties. 1 A study done by Mali et al, 2013, suggests oral administration of a 50% ethanol extract of Withania sominifera resulted in reduced fertility in male rats. 2 Several mushroom species was found useful as a source of nutrients for health benefits and having high nutritional value. It shows antioxidant, antibacterial, DNA protecting, anti-inflammatory, cytotoxic and hepatoprotective activities. Researches show that antioxidant molecules can help manage ageing and other conditions. The WHO also supports phytotherapy and promotes fundamental pharmacological validation methods, which are part of plant origin.3

Cassia siamea, (Fabaceae), A small to medium size tree, that is widely planted in Southeast Asia. It is often used to treat fever, skin diseases, hypertension, sleeplessness, diabetes, and asthma. This plant contains several phytochemicals, including β-sitosterol and flavonoids. The compounds responsible for pharmacological effects are glycosides, anthraquinones, bianthraquinones, alkaloids, and kaempferol.4 A study indicates that the alcoholic extract of C. siamea flowers has substantial antioxidant activity against free radicals, prevents oxidative damages and provides significant protection in the liver.5 Another previous study suggested by Musa et al, 2021, AgNPs of Senna siamea (C. siamea) flower extract was synthesized, characterized and their results shows great antibacterial properties.6 Following these important activities and phytochemical potential, In vitro antioxidant activity of AgNPs of C. siamea is comprehended further in this research.

Nanotechnology has emerged as a new interdisciplinary field of study, that blends the science of materials, biotechnology and bionanosciences. Due to their unique potential in the realms of electrical, magnetic, information storage and drug delivery, metal nanoparticle production is a major area of research in current biomaterial science.7 Nanoparticles are small particles ranging from 1-100 nm in size. As they are small in size and their high surface area to volume ratio increase and so their physical and chemical applications.8 There has been great development in the field of nanoscience from past decades, with an array of technologies developed to synthesize nanoparticles that have precise morphological (i.e., shape and size) and distribution features. Even though there are a number of techniques for creating specific and pure nanoparticles, they are highly costly and involve the use of dangerous chemicals that are harmful to humans, the environment and biological systems. This issue was resolved by the ecologically friendly synthesis of nanoparticles employing components that are safe for the environment, such as plants, fungi, seaweed, bacteria and enzymes. It has many benefits, including being non-toxic, time and money-efficient.9 Among many types of nanoparticles, the green synthesis of plant mediated nanoparticles has made an entirely different space, because of their wide variety of applications which includes, in biomedicine, wound healing, waste processing and food packaging. It has various advantages over other synthesis methods, since it is a simple and Straight forward strategy that uses extracts from plants, avoiding the requirement for sterile surroundings and the tedious processes required by other methods for nanoparticles synthesis.10 The use of AgNPs for biological applications has grown due to their stabilization by plant-derived bioactive substances. Nanoparticles derived from medicinal plants have considerable antioxidant activity. 11, 12 Thus, this study's objectives were to create and evaluate antioxidant capabilities of herbal AgNPs using methanolic extract of C. siamea leaf.

Materials and Methods

Collection of raw material

The fresh and healthy leaves of C. siamea was collectedFrom University of Rajasthan Campus and In and around Jaipur district of Rajasthan, in a fresh polythene packet. Specimen was submitted to the herbarium for verification, in, Department of Botany, at University of Rajasthan and got the voucher specimen RUBL21231 (Figure 2).

Preparation of leaf extract

The collected leaves samples were washed with double distilled water, shade dried and then ground into fine powder. 250 gm of the powder is weighed and then used for 50% v/v methanolic extract preparation using methanol and milli-Q water in Soxhlet apparatus at 40-45 ̊C for 48 hours (according to CG-04 protocol, WHO,1983). Extract prepared was then filtered, and kept for drying at room temperature (25 ̊C - 30 ̊C) in oven, for further use. 4

Synthesis of silver nanoparticles

5 ml of fresh aqueous leaf extract was prepared using dried extract powder. In a conical flask 55 ml of 1 mM (0.017 gm) silver nitrate (Aq.) solution was prepared using AgNO3 dissolved in Milli-Q water. Both solutions were mixed together and heated on a hot plate to a temperature of 45 ± 2.5 ̊C, following continuous stirring by magnetic stirrer for 15 minutes. 11

Characterization of AgNPs

UV- Vis Spectrophotometry

UV-vis spectrophotometry is a sensitive, rapid, and simple equipment, that is frequently used to examine the initial quality of biologically produced metal nanoparticles. Secondary metabolites in metal-reducing organic compounds of plants can absorb light in the UV and UV-Vis spectrums. Phytometabolites can mediate the reduction of silver ions, act as electron donors, and generate AgNPs. 13

The absorption behavior of synthesized AgNPs were identified using Thermoscientifics multiskan Go spectrophotometer, present in the Central Instrumentation Facility (CIF), in the Department of Zoology, University of Rajasthan, Jaipur. According to the protocol suggested by Chaudhari et al, 2016 in the range of 300-700 nm with sample in quartz cuvette. 7

FTIR spectroscopy

The FTIR (Fourier transformation infrared) study gives important details about AgNPs functional groups and chemical bonds. It depicts the vibrational bands of the existing chemical bonds and functional groups that cap and stabilize the nanoparticles, and shows various absorbance in infra-red range. Bands can be formed in between wavenumber (cm-1) of 2850-3300,1680-1750,1000-1300,3230-3550,2500-3300 showing functional group C-H (alkanes), C=O (Carbonyl group), C-O*, O-H (alcohols), O-H (acids), respectively. The FTIR spectrum was formed using Perkin Elmer Infra-red spectrum 10.4.00, facility was sourced by MRC-MNIT, Jaipur. FTIR was performed on KBr pellet mode in the wavenumber 4000-450 cm.

X-ray Diffraction

To detect the distribution of molecules within any crystalline structure, XRD technique is used. Dried sample was prepared for detection of structure of AgNPs according to the process mentioned by Ravichandaran et al, 2016.14 The crystal nature of AgNPs were analyzed using the PANalytical X’pert PRO X-ray diffractometer system, for the detection of crystalline lattice of the material. Using Cu-Kα radiation system, at a setting of 40ma/40kv, from a range of 2 theta 20̊- 80̊. 15

Field Emission-Scanning Electron Microscopy

The surface topography of nanoparticles was detected facilitated by FE-SEM (Nova nano SEM 450) at magnification of 25000x and HV (High Vacuum) at voltage of 15.0 Kv, facility was sourced by MRC-MNIT, Jaipur. The sample was provided as a thin film on a small glass slide. 15

Zeta-potential

In order to determine the stability of the composition of nanoparticles and surface charge, zeta potential studies are typically performed. This analysis measures the velocity of the nanoparticles, allowing one to assess the colloidal stability of the green synthesised AgNPs. 16 The surface electric charge of AgNPs was identified by zeta potential analyzer, using water as dispersant, with 12 zeta runs in a zetasizer ver. 7.11 Malvern instruments Ltd. (MRC-MNIT, Jaipur), when electric repulsion occurs between the particles.17

In- vitro Antioxidant Activity

To understand the total In-vitro antioxidant potential of leaf extract and synthesized AgNPs of C. siamea, it was identified by the, two assays, DPPH and FRAP assay. Various concentrations of the samples were prepared by dissolving extracts/AgNPs into Dimethyl sulphide (DMSO). Nanoparticles were sonicated before use.

DPPH radical % scavenging assay

The antioxidant activities that are related to the ability of the extract and AgNPs, to transfer electrons or hydrogen atoms by bleaching the purple color of the DPPH (1,1, diphenyl 1-2 picrylhydrazyl) into yellow solution.18

The scavenging potential of the leaf extracts and AgNPs from methanolic leaf extract was determined using the method of Akintola et al, 2020.19 Concentration was taken as 200, 400, 600, 800 and 1000 µg/ml. The samples were subjected for DPPH free radical scavenging activity by adding 100 µl of leaf extract/AgNPs of each concentration and 3.9 ml of DPPH solution (1mM solution: prepared by dissolving in methanol). For each sample, 5 test tubes were kept in a set. Besides samples, one blank/negative control (methanol only) and one positive control (DPPH only) were taken. As standard drug, similar set for ascorbic acid was also prepared. All tubes were kept in dark for 30 minutes. After that absorbance were measured at 517 nm.

Free radical scavenging activity or the percent antioxidant activity was measured by following formula- % activity = (Ac-At)/Ac*100 Ac, is the absorbance of control (DPPH radicle +methanol); At- Absorbance of test (Cs-E/Cs-AgNPs) + DPPH radicle.

FRAP % reducing ability assay

The Ferric Reducing Ability of Plasma (FRAP) assay, developed by Benzie and Strain in 1996, is a simple and automated method used to assess the "antioxidant power" of a sample by measuring its ability to reduce ferric ions. At a low pH, ferric ions (Fe³⁺) are reduced to ferrous ions (Fe²⁺), forming a blue-coloured ferrous-tripyridyl triazine complex from an initially colourless solution. The FRAP value is determined by comparing the change in absorbance at 593 nm between the test samples and standard solutions of ferrous ions at known concentrations. 20

The FRAP assay was slightly modified to assess the overall antioxidant potential of leaf extracts and AgNPs. Stock solutions included 300 mM acetate buffer (0.3 M acetic acid and sodium acetate, pH 3.6), 10 mM TPTZ (2,4,6-tripyridyl-s-triazine in 40 mM HCl), and 20 mM FeCl3·6H2O. A fresh working solution was prepared by combining 25 ml of acetate buffer with 2.5 ml of FeCl3·6H2O and TPTZ solutions, with the temperature increased to 37°C just before use. Leaf extracts and nanoparticles, prepared in 100 µl of DMSO, were reacted with 290 µl of FRAP solution for 30 minutes in dark conditions. The absorbance of the resulting ferrous-tripyridyl triazine complex was measured at 593 nm. In the negative control (blank), only FRAP solution was used, while ascorbic acid served as the positive control (standard).

The catalase enzyme concentration was found out using following equation- % catalase activity = 100- ((Control-test/control) *100)

Table 1

Average crystal size of C. siamea- AgNPs was calculated using parameters mention in table below.

Sample

2

hkl

FWHM

Crystal size D (nm)

C. siamea-

AgNPs

27.97

(100)

0.1378

59.38

32.39

(122)

0.1574

21.00

46.38

(231)

0.1378

62.69

54.99

(142)

0.2755

32.49

57.64

(241)

0.2755

53.85

64.68

(220)

0.3936

23.88

76.84

(311)

0.1574

64.39

D average Size

45.38 nm

Table 2

DPPH free radical scavenging activity of extract and AgNPs of C. siamea

Concentration (μg/ml)

DPPH Scavenging activity (%)

Extract

Cs/AgNP

Ascorbic acid

200

32.73±0.27

47.96±0.30

71.30±3.02

400

35.90±0.27

59.84±0.23

81.65±0.30

600

36.98±0.23

68.40±0.27

85.83±0.31

800

37.97±0.27

76.49±0.27

88.47±0.34

1000

38.17±0.34

79.76±0.23

94.93±3.72

IC50

2711.60±56.6

188.88±7.01

[i] (Data are represented in Triplicates (n=3) as Mean±S.D. at p<0.05)

Table 3

FRAP % scavenging activity of extract and AgNPs of C. siamea

Concentration (μg/ml)

FRAP Scavenging activity (%)

Extract

Cs/AgNP

Ascorbic acid

20

8.46±0.20

29.13±0.26

46.05±0.20

40

9.25±0.33

31.26±0.36

56.50±0.36

60

10.40±0.33

33.60±0.28

60.15±0.24

80

11.13±0.24

36.62±0.41

74.80±0.28

100

11.87±0.24

39.59±0.28

80.96±0.24

IC50

975.98±19.99

181.40±2.51

28.90±0.66

[i] (Dataare represented in Triplicates (n=3) as Mean±S.D. at p<0.05)

Figure 1

Graphical representation of synthesis, characterization and in vitro antioxidant activity evaluation of Cassia siamea mediated AgNPs.

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Figure 2

RUBL21231 Cassia siamea

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Figure 3

A- Colour change of the solution observed, during the synthesis of AgNPs. B- UV-visible spectra of AgNPs at different concentrations. C-Comparative UV-Visible spectrums of plant extract and AgNPs. D- Zeta Potential analysis. E- X-ray Diffractogram of synthesized AgNPs

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Figure 4

(A): and (B)

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Figure 5

SEM images of C. siamea silver nanoparticles.

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Figure 6

(A): DPPH % radical Scavenging activity and (B): FRAP reducing activity of AgNPs synthesized by C. siamea.

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Results

UV- Vis spectral and color change analysis

After keeping the reaction mixture for some time of incubation, color change of the solution was observed (Figure 3A) from white to light brown and then turns darker with respect to time. Change of the color is also the primary indication of Ag+ ion has been reduced into Ag0 and synthesis of AgNPs.

After the color change was observed, the absorbance of AgNPs was measured with UV- spectrophotometer, between the range of 350nm-700nm in a quartz cuvette, and they show a range between 410nm- 450 nm, best peaks were observed at 434 nm, as shown in figure 3B. This absorbance shows conformation that AgNPs has been synthesize. Figure 3C is showing comparison and difference in absorbance of UV-vis light between crude leaf extract and synthesized AgNPs of plant leaf extract.

FTIR analysis

The FTIR analysis aims to identify and study interaction of functional group and chemical bonds present between plant secondary metabolite and Ag+ to form AgNPs. The biomolecules present in leaf extract were responsible for reducing silver nitrate. Figure 4A (C. siamea-extract) and figure 4B, AgNPs, depicting various bands at different % transmittance, for both the plants extract and AgNPs, respectively. Presence of different peaks suggest the various bond types and functional groups (alkane, alkene, hydroxyl, carbonyl, ketone) present in AgNPs, which absorbs at different wavelength. 21

Various bands are prominently visible in FTIR spectra of C. siamea leaf extract (Figure 4A) at which corresponds to different vibrations of alcoholic O-H, Alkanes C-H, Phenolic O-H and C-O stretches. Analysis of C. siamea- AgNPs (Figure 4B) shows a strong and broad round band, O-H stretching at 3431.79, strong N=C=S Isothiocyanate band at 2076.24, a medium C=C (Conjugated alkene) band at 1637.11, a medium C-H alkane band at 1384.09 and a strong C=C alkene band at 695.63, variation in these band from leaf extract bands shows vibrational pattern of the molecules present and they form chemical bonds.

AgNPs synthesized using methanolic leaf extract of C. siamea, shows an average negative charge with potential of -8.28±5.81 mV (Mean±S.D.), at conductivity 0.191 mS/cm, in zeta potential report. Generally higher the negative charge, shows higher stability of formed nanoparticles (Figure 3D). Concentration of AgNO3 and other factors may affect the stability of synthesized AgNPs. Here AgNPs are showing good negative charge which confirmed that synthesized nanoparticles are stable, for a longer period of time and can be used for further analysis.16

XRD analysis

X-ray diffraction was used to analyse the crystalline nature of the nanoparticles, as illustrated in the figure 3E, The measured spectrum displays eight primary peaks at varied 2θ, in the range of 20̊ - 80̊, corresponding to various planes as (100), (122), (111), (220), and (311).

Sharpening of the peaks indicated their presence in the nano area, and this XRD pattern demonstrates the presence of synthesised AgNPs in the FCC (Face cubic centre) crystalline structure, according to Helfish et al, 2021. 22 These results shows that AgNPs has been synthesized. The crystalline size of AgNPs was calculated using full width half maximum, Bragg reflection by Debye-Scherrer equation-

D = 0.9λ/βCosθ

where D is the crystallite size, λ ­ = 1.5406 Å is the wavelength of X-ray, β is the full width half maximums (FWHM) of the peak in radians, and θ is the Bragg angle. The crystallite size of the AgNPs, calculated by the above-mentioned equation using hkl value and FWHM value are 45.35 nm.23

FE-SEM analysis

SEM analysis (Figure 5) helps in direct visualization of nanoparticles by analyzing their surface morphology. From surface morphology it has been observed that nanoparticles are spherical in shape and shows uniform aggregation. The aggregation of AgNPs shows that there is free static charge at their surface. They produce crystalline structure. The particle diameter of AgNPs were ranging between 15.0 nm- 51.91nm, thus nanoparticles are below 100 nm in size.

DPPH assay analysis

The antioxidant potential of the methanolic leaf extract and synthesized AgNPs was evaluated using the DPPH radical scavenging assay. The IC50 values and percentage scavenging activities are presented in Table 2, while the dose-dependent scavenging effects are illustrated in figure 6A, the leaf extract and AgNPs were tested at concentrations of 200 µg/ml, 400 µg/ml, 600 µg/ml, 800 µg/ml, and 1000 µg/ml, demonstrating a significant increase in DPPH scavenging activity with increasing concentrations. At the lowest concentration (200 µg/ml), the leaf extract exhibited a minimum scavenging activity of 32.73±0.27% (Mean±S.D.), which gradually increased to a maximum of 38.17±0.34% at 1000 µg/ml. In comparison, the synthesized AgNPs demonstrated superior scavenging potential, with a minimum activity of 47.96±0.30% at 200 µg/ml and a maximum of 79.76±0.23% at 1000 µg/ml. However, both the leaf extract and AgNPs displayed lower DPPH scavenging activity than reference standard, ascorbic acid. Ascorbic acid, was utilized as a positive control, exhibited significantly higher scavenging activity, with 71.30±3.02% at 200 µg/ml and a maximum activity of 94.93±3.72% at 1000 µg/ml. Notably, while the AgNPs demonstrated greater scavenging potential compared to the leaf extract at all tested concentrations, their activity remained lower than that of ascorbic acid across the concentration range.

The IC50 value represents that concentration of a compound, which is required to achieve 50% of its maximal effect, which, in this context, reflects the scavenging potential of the sample. A lower IC50 value indicates a higher antioxidant capacity, as it signifies that a lower concentration is sufficient to achieve 50% DPPH radical inhibition. Thus, IC50 values are inversely related to antioxidant potential.23, 24

In Table 2, the results of the DPPH analysis show no IC50 value for ascorbic acid (standard), as the 50% inhibition threshold was reached at a concentration lower than the minimum concentration tested. The AgNPs exhibited a concentration-dependent increase in DPPH scavenging activity, as demonstrated by their corresponding IC50 values. The gradual increase in scavenging activity with increasing concentrations of AgNPs shows their antioxidant potential.

FRAP assay analysis

The results of the FRAP assay in figure 6B, presented in Table 3, demonstrate a clear concentration-dependent increase in the antioxidant capacity of AgNPs synthesized from C. siamea leaf extract. The AgNPs, leaf extract, and standard ascorbic acid were tested at concentrations of 20, 40, 60, 80, and 100 µg/ml. Among these, AgNPs consistently exhibited superior ferrous reducing activity compared to the leaf extract alone, the exhibiting enhanced antioxidant potential of the nanoparticles.

At a concentration of 20 µg/ml, the leaf extract displayed the lowest reducing power, with a ferrous reducing activity of 8.46±0.20% (Mean±S.D.), this lower activity is likely due to the lower concentration of bioactive compounds in the crude extract. In contrast, the AgNPs showed a significantly higher reducing activity at the same concentration, indicating that the incorporation of bioactive compounds onto the nanoparticle surface enhances their antioxidant capacity.25, 26 The enhanced activity of AgNPs is likely attributed to the surface-bound phytochemicals acting synergistically, amplifying the antioxidant effects. The results also highlight the benchmark antioxidant activity of ascorbic acid, which showed the highest reducing power across all concentrations. However, while the AgNPs did not match the reducing potential of ascorbic acid, they demonstrated a considerable antioxidant capacity, particularly at higher concentrations.

As the concentration increased, both the AgNPs and the leaf extract demonstrated improved FRAP activity; however, the AgNPs consistently outperformed the leaf extract. At the highest concentration tested (100 µg/ml), the AgNPs exhibited a maximum ferrous reducing activity of 39.59±0.28%, which was significantly higher than the leaf extract’s activity of 11.87±0.24%, but significantly lower than the standard ascorbic acid 80.96±0.24.

Statistical analysis

GraphPad PRISM 8.3 software (GraphPad, La Jolla, CA, USA) and Microsoft Xl was used for statistical analysis. Data is calculated in Triplicates (n=3) as Mean±SD. Tukey’s one-way ANOVA test was used to determine significant differences between control and different concentrations of samples for their antioxidant activity (p<0.05).

Discussion

The present study successfully synthesized and characterized AgNPs using UV-vis, FTIR, XRD, Zeta potential analysis and SEM Techniques, using methanolic leaf extract of C. siamea. The use of plant-based extracts for the biosynthesis of nanoparticles is an eco-friendly and cost-effective alternative to traditional chemical synthesis methods. 27

One of the study's important finds was the antioxidant activity of the synthesized AgNPs, when compared to methanolic leaf extracts, AgNPs from C. siamea displayed greater free radical scavenging capacity. The DPPH assay showed that AgNPs could donate electrons or hydrogen atoms to neutralize DPPH free radicals, demonstrating their potential antioxidant capacity and the FRAP assay confirmed AgNPs' ability to reduce ferric ions (Fe3+) to ferrous ions (Fe2+), providing additional evidence of their reducing power. The increased antioxidant activity of the AgNPs may be related to the synergistic effects of the silver and bioactive chemicals contained in C. siamea, though it was found lower than the standard. 28, 29 This shows that the phytochemicals used in the production of AgNPs retained their antioxidant capabilities within the nanoparticles. 30, 12, 31 sThe capacity of AgNPs to scavenge free radicals suggests that they could have therapeutic implications in reducing oxidative damage in biological systems. Future research should focus on characterizing the produced AgNPs, notably their size, shape, and surface charge, as these features can have a substantial impact on their biological activities. Furthermore, in vivo investigations are required to assess the safeguarding, biological compatibility, and therapeutic efficacy of the produced AgNPs in biological environments.

Conclusions

By the above investigation, we can conclude that synthesis of AgNPs from C. siamea methanolic leaf extract has been successfully done. AgNPs from C. siamea exhibited better antioxidant activity compared to the extract. This suggests that the phytochemicals acting as reducing agents during AgNPs synthesis retained their antioxidant properties in the nanoparticles. The nanoparticles effectively scavenge free radicals, as evidenced by their performance in DPPH and FRAP assays. In conclusion, AgNPs of C. siamea demonstrate promising antioxidant potential. Further studies are suggested to explore their biocompatibility and potential applications in antioxidant therapy and other biomedical fields, with beneficial biological activities, contributing to the development of eco-friendly and sustainable nanotechnologies.

Ethical Committee Approval

Not applicable.

Clinical Trial Registry

Not applicable for current study.

Author Contributions

Pratap Chand Mali- Supervision, investigation and Validation. Neha Bharti- Conceptualization, Data Curation, Funding acquisition, Methodology, Writing- Original Draft. Prity Yadav Formal Analysis, Resources, Writing and editing and Ashish Kumar Kansotiya- Visualization.

Data Availability Statements

The datasets utilized and analyzed in this study are accessible from the corresponding author upon reasonable request.

Source of Funding

We acknowledge CSIR-UGC, New Delhi, for financial assistance by providing Junior research fellowship (NTA ref. No. 221610083539, Dated 29/11/2022).Top of Form Bottom of Form

Conflict of Interest

None.

Acknowledgements

Authors are thankful to the Head Department of Zoology, University of Rajasthan for providing necessary facilities in Central instrument facility (CIF). To RUSA 2.0 Project No.5 for providing glassware and necessary chemicals, to Biomitra Pvt. Ltd. and to MNIT-MRC for characterization of AgNPs.

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